Herbal Supplement Extends Life Span Under Some Environmental Conditions and Boosts Stress Resistance

Abstract

Genetic studies indicate that aging is modulated by a great number of genetic pathways. We have used Drosophila longevity and stress assays to test a multipath intervention strategy. To carry out this strategy, we supplemented the flies with herbal extracts (SC100) that are predicted to modulate the expression of many genes involved in aging and stress resistance, such as mTOR, NOS, NF-KappaB, and VEGF. When flies were housed in large cages with SC100 added, daily mortality rates of both male and female flies were greatly diminished in mid to late life. Surprisingly, SC100 also stabilized midlife mortality rate increases so as to extend the maximum life span substantially beyond the limits previously reported for D. melanogaster. Under these conditions, SC100 also promoted robust resistance to partial starvation stress and to heat stress. Fertility was the same initially in both treated and control flies, but it became significantly higher in treated flies at older ages as the fertility of control flies declined. Mean and maximum life spans of flies in vials at the same test site were also extended by SC100, but the life spans were short in absolute terms. In contrast, at an independent test site where stress was minimized, the flies exhibited much longer mean life spans, but the survival curves became highly rectangular and the effects of SC100 on both mean and maximum life spans declined greatly or were abolished. The data indicate that SC100 is a novel herbal mix with striking effects on enhancing Drosophila stress resistance and life span in some environments, while minimizing mid to late life mortality rates. They also show that the environment and other factors can have transformative effects on both the length and distribution of survivorship, and on the ability of SC100 to extend the life span.

Full text

RESEARCH ARTICLE
Herbal Supplement Extends Life Span Under
Some Environmental Conditions and Boosts
Stress Resistance
Bryant Villeponteau
1
*, Kennedy Matsagas
1
, Amber C. Nobles
3
, Cristina Rizza
1
,
Marc Horwitz
1
, Gregory Benford
1,2
, Robin J. Mockett
3
1Genescient Inc., Fountain Valley, California, United States of America, 2University of California, Irvine,
California, United States of America, 3University of South Alabama, Mobile, Alabama, United States of
America
*bvillepo@san.rr.com
Abstract
Genetic studies indicate that aging is modulated by a great number of genetic pathways.
We have used Drosophila longevity and stress assays to test a multipath intervention strate-
gy. To carry out this strategy, we supplemented the flies with herbal extracts (SC100) that
are predicted to modulate the expression of many genes involved in aging and stress resis-
tance, such as mTOR, NOS, NF-KappaB, and VEGF. When flies were housed in large
cages with SC100 added, daily mortality rates of both male and female flies were greatly di-
minished in mid to late life. Surprisingly, SC100 also stabilized midlife mortality rate in-
creases so as to extend the maximum life span substantially beyond the limits previously
reported for D. melanogaster. Under these conditions, SC100 also promoted robust resis-
tance to partial starvation stress and to heat stress. Fertility was the same initially in both
treated and control flies, but it became significantly higher in treated flies at older ages as
the fertility of control flies declined. Mean and maximum life spans of flies in vials at the
same test site were also extended by SC100, but the life spans were short in absolute
terms. In contrast, at an independent test site where stress was minimized, the flies exhib-
ited much longer mean life spans, but the survival curves became highly rectangular and
the effects of SC100 on both mean and maximum life spans declined greatly or were abol-
ished. The data indicate that SC100 is a novel herbal mix with striking effects on enhancing
Drosophila stress resistance and life span in some environments, while minimizing mid to
late life mortality rates. They also show that the environment and other factors can have
transformative effects on both the length and distribution of survivorship, and on the ability
of SC100 to extend the life span.
Introduction
Many studies indicate that aging is modulated by a large number of aging-related genes that
often boost stress resistance [1–7]. This multipath nature of aging suggests that many genetic
PLOS ONE | DOI:10.1371/journal.pone.0119068 April 16, 2015 1/28
OPEN ACCESS
Citation: Villeponteau B, Matsagas K, Nobles AC,
Rizza C, Horwitz M, Benford G, et al. (2015) Herbal
Supplement Extends Life Span Under Some
Environmental Conditions and Boosts Stress
Resistance. PLoS ONE 10(4): e0119068.
doi:10.1371/journal.pone.0119068
Academic Editor: Aamir Nazir, CSIR-Central Drug
Research Institute, INDIA
Received: April 16, 2014
Accepted: January 21, 2015
Published: April 16, 2015
Copyright: © 2015 Villeponteau et al. This is an
open access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper.
Funding: The California part of this study was
funded by Genescient Corporation. The authors
recommended and received approval for publication
from the Genescient Board of Directors. Gregory
Benford is on the Board. The funder provided support
in the form of salaries for all authors, but did not have
any additional role in the study design, data collection
and analysis, or preparation of the manuscript.
Studies conducted at the University of South
Alabama were funded by donations from Mr. Douglas

patterns must be altered simultaneously for a successful intervention into the aging process. In
devising our longevity experiments, our overall aim was to test the hypothesis that aging in
Drosophila can be significantly slowed using an environmental intervention that acts on multi-
ple longevity genes and boosts stress resistance. We have tried to target several of the known
longevity genes, as well as stress resistance pathways such as oxidative stress, heat stress, starva-
tion stress, environmental stress, and excessive neuronal activation. As we detail below, the
four herbal extracts in SC100 provide a diverse set of bioactive compounds, which appear to
act on various pathways such as activators of telomerase, inhibitors of mTOR, polyphenolic an-
tioxidants, insulin sensitizers, stem cell activators, parasympathetic agonists, and stress
response genes.
Our initial stress and longevity experiments were done in large cages with one strain of flies,
as this allowed large numbers of flies (500 per cage) to be followed with the minimum of han-
dling. Nevertheless, we repeated the longevity experiments in vials using different fly strains,
laboratories, and other conditions. The wide range of conditions used permitted us to explore
the considerable role of environment in affecting life span.
To develop potential oral antiaging supplements, we initially set out to identify nutraceutical
or drug compounds that would target as many of the complementary longevity and stress path-
ways as possible and thereby extend Drosophila life span. Unfortunately, with the exception of
the drug lithium, none of the single compounds that we initially tested appeared to significantly
extend fly life span in our longevity or stress resistance screens. The lithium results and some
of our negative single substance data were published [8]. The typically poor longevity effects of
single oral compounds suggested that many single substance therapeutics directed to a single
target may not significantly extend life span.
We began testing mixtures of medicinal herbal extracts, as these have had a long history of
clinical success in Chinese and Indian traditional medicine and are known to have a wide spec-
trum of positive effects in humans [9–13]. To affect as many longevity genes as possible, we fo-
cused on complementary herbal extracts that have antioxidant, anti-inflammatory, and
metabolic properties (known factors in driving aging and stress) along with a positive effect on
longevity genes and a history of use in traditional herbal medicine to treat a wide spectrum of
diseases. A combination of four herbal extracts (SC100) containing Astragalus membranaceus
root, Pterocarpus marsupium bark, pine bark oligo-proanthocyanidins, and L-theanine was
more effective than any single substance or other combination of substances that we tested in
our preliminary stress resistance screens in Drosophila.
Aside from our longevity screening results, some of the published data on longevity genes,
as detailed below, also suggest that the 4-herb mix of SC100 might work synergistically to ex-
tend fly longevity. For example, stem cells are important for maintaining Drosophila intestines
and for high fertility rates. The major herbal extract in SC100 comes from the Chinese medici-
nal herb Astragalus membranaceus and Astragalus extracts are well known to promote stem
cells in several model systems [14–18]. Moreover Astragalus extracts are known to have favor-
able cardioprotective and angiogenic effects in rats, activate the expression of VEGF (Vascular
Epithelial Growth Factor), and inhibit mTOR (mammalian Target Of Rapamycin) expression
[19,20]. Low VEGF levels are linked to dementia [21–23] and aging [24–26], while the mTOR
gene is a well-known modulator of mammalian life span [27]. The stem cell, VEGF, and TOR
altering components of Astragalus may explain some of the SC100-induced effects on life span.
The second major herbal extract in SC100 is Pterocarpus marsupium bark (also called Indi-
an Kino Tree), which has been standardized for a high content of the resveratrol analog pteros-
tilbene. Pterostilbene is thought to be the key active ingredient in Pterocarpus marsupium
and is much more efficacious than resveratrol in reversing cognitive deficits in aged rats [28].
Pterostilbene has also been shown to reduce colon tumors, pro-inflammation cytokines, and
Herbal Supplement Extends Life Span and Boosts Stress Resistance
PLOS ONE | DOI:10.1371/journal.pone.0119068 April 16, 2015 2/28
R. Arends and an anonymous donor. The authors are
not aware of any competing interests with the
anonymous donor. The specific roles of these authors
are articulated in the ‘author contributions’section.
Competing Interests: The authors have the
following interests. This study was funded in part by
Genescient Corporation. The authors recommended
and received approval for publication from the
Genescient Board of Directors. Gregory Benford is on
the Board. B. Villeponteau received personal income
for consulting, and has been granted stock options
from Genescient Corporation; he is also an inventor
on a patent filing which Genescient has licensed
(Pending Patent #14521877). K. Matsagas received
compensation from, and has equity in, Genescient
Corporation. C. Rizza has equity in Genescient
Corporation; she is also an inventor on a patent filing
which Genescient has licensed (Pending Patent
#14521877). M. Horwitz has equity in Genescient
Corporation. G. Benford has equity in Genescient
Corporation and serves on the Board of Directors of
Genescient Corporation. L.D. Mueller received
personal income for consulting from, and has equity
in Genescient Corporation. A. Nobles and R. Mockett
have no competing interests. The SC100 formulation
described herein provides the essentialherbal
components for several nutritional supplements (e.g.
Stem Cell 100) that are currently commercially
available and may eventually provide financial
benefits to Genescient and its employees and
shareholders. There are no further patents, products
in development or marketed products to declare. This
does not alter the authors’adherence to all the PLOS
ONE policies on sharing data and materials, as
detailed online in the guide for authors.

Alzheimer pathology in rodents [29,30]. Pterocarpus marsupium bark also contains the resver-
atrol analog marsupsin, which significantly lowered blood glucose in hyperglycemic rats at a
level comparable to the antidiabetic drug metformin [31]. Another resveratrol analog in Ptero-
carpus bark is pterosupin, which lowers serum triglyceride and LDL cholesterol in hyperlipi-
demic rats [32].
The third herbal extract in SC100 is Pine Bark Extract (PBE), which was purified to 85%
proanthocyanidins. PBE improves endothelial function via activation of endothelial nitric
oxide synthesis [33] and is reported to help chronic venous insufficiency [34,35]. PBE also has
some anti-inflammatory potential, as it inhibits matrix metalloproteinase 9 (MMP-9) and
NF-Kappa B activation [36].
The fourth herbal extract in SC100 is L-Theanine, which is a unique neuroprotective amino
acid found in green tea that crosses the blood-brain barrier. L-Theanine has a structure similar
to glutamate, which is a neurotransmitter related to memory, and binds to the GABA receptors
in neurons. Even doses as low as 1 mg of L-Theanine per kg of body weight reduce the sizes of
cerebral infarcts following middle cerebral artery occlusion in mice [37]. L-Theanine also at-
tenuated neurotoxicity of rotenone and dieldrin-induced DNA fragmentation and apoptotic
death in cultured neural cells [38], as well as L-glutamate-induced amyloid beta neurotoxicity
[39]. Moreover, L-Theanine is reported to facilitate neurogenesis in the hippocampus of rats
following treatment, leading to enhanced memory [40]. As to its antiaging effects, L-Theanine
can suppress the shortened life span and learning impairment of senescence accelerated mice
under stress [41].
Results
SC100 Extended Drosophila Mean and Maximum Life Span in Cages:
CA Site
In our large cage experiments, we used three cages with 250 males and 250 females per cage for
each longevity screen, so that the longevity experiments were all done in triplicate cages. The
three treatment conditions were: C) Control untreated flies; T) SC100 treated flies; and CT)
Control/Treated flies that were switched from control conditions to SC100 treatment on day
36 of the longevity assay. Fig 1 shows female (Fig 1A–1C) and male (Fig 1D–1F) fly survivor-
ship for each of the three replicate treatment conditions (Cages A, B, and C).
As can be observed in Fig 1, SC100 extended Drosophila life span and generated significant-
ly lower mortality at later population ages. Similar life span extension occurred in both females
and males. Fig 1 shows that untreated Control flies (green solid triangles) were all dead before
60 d, while the SC100 treated samples (blue open squares) did not die off completely until
around 134 d. Surprisingly, SC100 worked nearly as well when the flies were on the control reg-
imen for 36 d and then shifted to SC100 at 36 d (red solid diamonds).
Another way to look at these longevity data for flies in cages is to look at the daily mortality
rate (Fig 2). The control flies (green triangles) had an increase in their daily mortality rate at
around 28 d and their mortality rate gradually increased until 54–58 d, when the daily mortali-
ty rate spiked as the last flies died. In both the lifetime SC100 treated flies (T, open blue
squares) and the mid-life SC100 treated flies (CT, red diamonds), the mortality rate did not in-
crease substantially until around 120 d, ending in a spike at 120–134 d. The midlife SC100
treated flies actually decreased their mortality rate several days after SC100 treatment com-
menced at 36 d and continued with a low stable mortality rate until the mortality spike at 120–
134 d (Fig 2B and 2C). Thus, SC100 stabilized the midlife mortality rate in the flies and that
may play a role in how SC100 extended maximum life span of a major fraction of the fly
population.
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SC100 had a highly significant effect on mean longevity. Table 1 gives a summary of mean
longevities for B4 flies in cages in days for the Control (C), SC100-treated (T), and Control
shifted to SC100-treated (CT) flies, along with their 95% confidence intervals. From Table 1 we
can conclude that lifelong SC100-treated and midlife SC100-treated flies both yielded signifi-
cantly higher mean longevities than the Control flies. In comparison to the untreated Control,
the lifelong SC100-treated flies had a 49% increase in mean life span, while the midlife
SC100-treated flies had a 27% increase in mean life span. Males and females did not have a sig-
nificant difference in longevity.
SC100 had particularly strong effects on maximum longevity, which is measured as the
number of days lived by the top 10% or 5% of survivors. Table 2 gives the data for the C, T, and
CT flies. Based on the confidence intervals, there was a highly significant doubling of maximum
life span on SC100 treatment and this was true even in the case where the treatment started at
midlife (36 days).
Fig 1. Life span of Drosophila in cages with or without SC100 treatment. Nine independent cages were used in 9 simultaneous longevity assays, with
each cage having 250 male and 250 female B4 flies at 25°C. Female or male sets of 3 cages each (Cages A, B, or C) are shown with the Survivorship
(percent survival) on the Y axis and the Adult Age in days on the X axis. These data show Survivorship for: C) Control Untreated flies (green triangles), T)
SC100 Treated flies (blue open squares), and CT) Control/Treated flies that were switched to SC100 treatment on day 36 of the assay (red diamonds). For
each treatment, females in Fig 1A, 1B, and 1C were housed together with males in Fig 1D, 1E, and 1F, respectively.
doi:10.1371/journal.pone.0119068.g001
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Fig 2. Male and female mortality rate changes in cages with or without SC100 treatment. (A) These
data are from the same longevity assays shown in Fig 1, wherein nine independent cages were used, with
each cage having 250 males and 250 female B4 flies. To correlate mortality rates with the longevity assays,
the male longevity data in the 3 sets of cages of Fig 1D, 1E, and 1F were pooled and composite average
values were taken on each day for: C) Control untreated male flies (green triangles), T) SC100 treated male
flies (blue squares), and CT) Control/Treated flies that were switched to SC100 treatment on day 36 of the
assay (red diamonds). (B) and (C) show composite daily mortality rates for male flies (B) and female flies (C),
which were determined by averaging the daily mortality rates from the three sets of cages for each treatment
or control.
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Gompertz Parameters Aand α
The effect of these treatments on the age-specific mortality (or survival) patterns can be exam-
ined in more detail by looking at the effect of each treatment and sex on the two parameters of
the Gompertz equation, which is determined by the rate of aging parameter αand the age-inde-
pendent parameter A.Table 3 gives the estimated Aand αparameter values. From this table it
is clear that the Control flies had one order of magnitude lower age-independent mortality pa-
rameter Athan either the lifelong SC100-treated flies or the midlife-SC100-treated flies. Con-
versely, the rate of aging αparameter was much higher in the Control than it was for the two
SC100 treatments.
Table 4 compares the C, T and CT treatments to see whether any of the differences in
Table 3 were significant. In every case, the C group had a significantly smaller Aparameter
than either the T or TC flies. Conversely, the αparameter was significantly greater in the C
than in either T or CT flies for both male and female flies (P<10
–16
). These are unique observa-
tions, as most environmental treatments that increase longevity do so by decreasing the age-in-
dependent parameter Aand have little effect on α. No treatment has been published previously
that has such a pronounced effect on α[42–44].
SC100 Extended the Short Life Span of Drosophila Housed in Vials: CA
Site
The 500 fly cage longevity screening assay described above is not used as commonly as small
test vial experiments where the Drosophila are tightly confined as to space and unable to fly. In
most test vial fly assays, male and female flies are separated into different vials, as it is well
known that sexual activity shortens fly life span. Since the test tube-like vial environment is
very different from the cage environment, we tested SC100 on flies in vials combined with or
separated from the opposite sex. Fig 3A and 3B show the survivorship curves for females and
males respectively, where 4 flies of the same sex or combined sexes were housed in each test
vial with 100 flies for each sex in each control and 100 flies for each sex in each of the
SC100-treated samples.
As expected, Fig 3A shows that female control flies had longer mean or maximum life spans
if housed in separate vials from males (solid green circles) versus maintaining females with
males (solid green triangles). SC100 treatment extended mean and maximum life span of the
females, whether they were housed separate from males (open blue circles) or combined with
males (open blue triangles). Fig 3B shows nearly the same results for males housed with only
other males or in combination with females, except the gains in longevity were not as
Table 1. Mean Drosophila life spans in cages and contrasts with control or differing SC100
treatments.
Population/Contrast Longevity (days) ±95% CI
Control (C) 37.6 ±2.5
Control SC100 (CT) 47.6 ±3.1
SC100 (T) 56.1 ±3.1
CT-C Increase 10.0 ±3.1
T-C Increase 18.6 ±3.1
T-CT Increase 8.6 ±3.1
Mean life spans for Control, Control switched to SC100 treated at 36 days, and SC100 treatments
throughout the life span are given in days. The ±95% CI is the Confidence Interval (CI) in days.
doi:10.1371/journal.pone.0119068.t001
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pronounced in the males that were housed separate from females. Neither females nor males
housed in vials had any hint of a mortality plateau, as was observed with T and CT flies housed
in cages.
Tables 5and 6summarize the vial fly data from Fig 3.Table 5 shows the mean life span of
Drosophila in days for flies housed in vials with single sex or combined sexes and with or with-
out SC100. All SC100 treated flies have significantly different life spans from non-treated Con-
trols (P<0.05%). Both males and females have significant longer life spans if they are housed in
same sex vials than when housed with the opposite sex of 2 males with 2 females (P<0.05%).
Table 6 analyses the data in Table 5 and shows the increases in fly life span for various com-
parisons. As indicated from viewing the raw data in Fig 3,Table 6 shows that SC100 signifi-
cantly enhanced the mean life span of flies housed in single sex vials (41% average increase on
second row) and with both sexes combined (38% average increase on first row (P<0.05%). Fe-
males appeared to benefit more from SC100 than males, especially when both sexes were com-
bined. The last two rows of Table 6 demonstrate that housing flies in single sex vials led to
higher life spans than vials that combined the sexes (P<0.05%). Moreover, females had more
improvement in life span in single sex vials than did males.
We have also done a Gompertz analysis of the effects of SC100 treatment on life span of
flies housed in vials. Averaged over both sexes and both housing treatments (combined and
separate), SC100 significantly increased the Aparameter of the Gompertz model by 15%
(P= 0.03). At the same time SC100 lowered the rate of aging parameters, α, by 36% (P<10
–8
).
Overall, the more substantial decrease in αresults in the observed increase in longevity.
Table 2. Maximum Drosophila life spans in cages for the top 10% and 5% of surviving flies for control and differing SC100 treatments.
Population Mean Top 10% Survival (days) ±95% Cl Mean Top 5% Survival (days) ±95% Cl
Control (C)—Male 46.9 ±2.0 50.1 ±3.7
Control (C)—Female 47.5 ±2.2 50.5 ±4.2
Control SC100 (CT)—Male 97.2 ±2.0 121 ±5.0
Control SC100 (CT)—Female 94.6 ±6.4 118 ±9.2
SC100 (T)—Male 114 ±17.8 125 ±6.3
SC100 (T)—Female 106 ±2.1 122 ±5.9
The ±95% CI is the Confidence Interval (CI) in days.
doi:10.1371/journal.pone.0119068.t002
Table 3. Estimated Gompertz parameter values in cages from a non-linear mixed effects model.
Parameter Sex Treatment Estimate
AMale Control 0.000484
Control->SC100 0.004720
SC100 0.008280
Female Control 0.000481
Control->SC100 0.006340
SC100 0.005920
αMale Control 0.1430
Control->SC100 0.0529
SC100 0.0195
Female Control 0.1400
Control->SC100 0.0369
SC100 0.0294
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Table 4. Treatment contrasts in cages for the two Gompertz parameters are highly significant.
Parameter Sex Contrast Estimate ±95% CI
AMale CT-C 0.00423 ±0.00069
T-C 0.00780 ±0.00069
Female CT-C 0.00586 ±0.00073
T-C 0.00540 ±0.00061
αMale CT-C -0.0902 ±0.013
T-C -0.1240 ±0.013
Female CT-C -0.1030 ±0.014
T-C -0.1100 ±0.012
The ±95% CI is the Confidence Interval (CI). All results are highly significant with P<10
–16
for all contrasts in cages.
doi:10.1371/journal.pone.0119068.t004
Fig 3. Life span of male and female Drosophila in single sex or combined sex vials with or without
SC100 treatment: CA site. 3A (Females) and 3B (Males) show the survivorship curves for female and male
B4 flies, respectively, where 4 flies of the same sex or 2 flies of each sex were housed at 25°C in each test
vial, with 100 flies for each sex in the control or SC100-treated samples. In (A), the data for females are
shown: Control females were housed in separate vials from males (solid green circles) versus housing of
females with males in combined vials (solid green triangles); SC100 treated female flies are housed separate
from males (open blue circles) or combined with males (open blue triangles). In (B), the data for males are
shown: Control males were housed in separate vials from females (solid green circles) versus housing of
males with females in combined vials (solid green triangles); SC100 treated male flies were housed separate
from females (open blue circles) or combined with females (open blue triangles).
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SC100 Did Not Extend the Long Life Span of Drosophila Housed in Vials
under Low-Stress Conditions: AL Site
To establish the consistency of effects of SC100 under different strain, food, and housing condi-
tions, the supplement was tested by a separate group of investigators at an independent test site
in Alabama (AL site) using low stress (single sex and 12 h on/12 h off lighting) vial conditions.
SC100 was administered simultaneously to both female and male flies of three different geno-
types provided with two different media, at three doses spanning a 9-fold range (1/3×, 1× and
3×). Flies housed in vials at the AL site exhibited highly rectangular survival curves, with no
hint of a mortality plateau at advanced ages (Figs 4–6). Despite the 6 fold higher population
density of flies in AL, the mean life spans of untreated (0×) flies were substantially longer for all
groups in AL than CA, by factors of 2.7 for B females (68.7 vs. 25.2 d) and 2.6 for B males (68.1
vs. 25.9 d) on the same BM medium used in vials in CA. On the other hand, the maximum life
span (90–100% mortality) was substantially shorter for supplemented flies in vials in AL than
in cages in CA.
A factorial analysis of variance for mean life spans of all flies revealed significant differences
based on sex, strain and food medium, but little or no difference among SC100 supplementa-
tion groups (Table 7). Significant interactions were observed for sex × strain, sex × medium,
strain × medium and medium × SC100 dosage, but supplementation did not interact signifi-
cantly with either sex or strain and none of the higher-order interactions were significant. Pool-
ing across dosages of SC100 (0–3×), female w
1118
flies lived 16% longer than males on BM and
28% longer on CM. The sex differences were much smaller for the other strains: 0% on BM and
7% on CM for B4 females, and -3% on BM and 7% on CM for ywflies. Mean life spans of yw
males were 20–27% longer than those of w
1118
males and 13–21% longer than B males on both
media. ywfemales lived 14–18% longer than B females, but only 1–6% longer than w
1118
Table 5. Mean Drosophila life span in days for flies housed in vials with the same or opposite sex: CA
site.
Fly Sample Male Female
Control Combined 21.4 17.4
SC100 Combined 27.6 25.6
Control Single Sex 25.9 25.2
SC100 Single Sex 35.4 36.6
All SC100 treated flies had significantly different life spans from untreated Controls (P<0.05). Both males
and females had significantly longer life spans if they were housed in same sex vials than when housed
with the opposite sex (2 males with 2 females) (P<0.05). Each life span mean is based on the mean
survival of 100 flies.
doi:10.1371/journal.pone.0119068.t005
Table 6. Percentage Increase in Mean Drosophila Life span for Table 5 data samples compared.
Samples Compared Male Female Average
SC100/Control Combined 29% 47% 38%*
SC100/Control Single 37% 45% 41%*
Control Single/Control Combined 21% 45% 33%*
SC100 Single/SC100 Combined 28% 43% 35%*
*All differences are significant (P<0.05) and are based on the mean survival of 100 flies.
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Fig 4. Life span of female and male B4 Drosophila in single sex vials with or without SC treatment: AL
site. Flies were separated by sex 1 d after eclosion, housed in groups of 25/vial at 25°C on a 12 h light: 12 h
dark cycle, and supplemented with SC100 at doses of 0× (solid green triangles), 1/3× (solid blue squares), 1×
(open black squares), and 3× (solid red squares) beginning 2 d after collection. Results are presented
separately for females (A,B) and males (C,D) on banana medium (BM; A,C) and cornmeal (CM; B,D). For
each medium and dosage, n = 3–4 vials (72–98 surviving flies) for females and 4 vials (89–100 survivors) for
males. Results presented in Figs 4–6were obtained concurrently.
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Fig 5. Life span of female and male w
1118
Drosophila in single sex vials with or without SC treatment:
AL site. Flies were separated by sex 1 d after eclosion, housed in groups of 25/vial at 25°C on a 12 h light: 12
h dark cycle, and supplemented with SC100 at doses of 0× (solid green triangles), 1/3× (solid blue squares),
1× (open black squares), and 3× (solid red squares) beginning 2 d after collection. Results are presented
separately for females (A,B) and males (C,D) on banana medium (BM; A,C) and cornmeal (CM; B,D). For
each medium and dosage, n = 4 vials (96–102 surviving flies for females and 95–100 for males). Results
presented in Figs 4–6were obtained concurrently.
doi:10.1371/journal.pone.0119068.g005
Herbal Supplement Extends Life Span and Boosts Stress Resistance
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Fig 6. Life span of female and male y w Drosophila in single sex vials with or without SC treatment: AL
site. Flies were separated by sex 1 d after eclosion, housed in groups of 25/vial at 25°C on a 12 h light: 12 h
dark cycle, and supplemented with SC100 at doses of 0× (solid green triangles), 1/3× (solid blue squares), 1×
(open black squares), and 3× (solid red squares) beginning 2 d after collection. Results are presented
separately for females (A,B) and males (C,D) on banana medium (BM; A,C) and cornmeal (CM; B,D). For
each medium and dosage, n = 3–4 vials (75–102 surviving flies) for females and n = 4 vials (91–99 survivors)
for males, except n = 2 vials (46 survivors) for females on 1/3× BM (A). Results presented in Figs 4–6 were
obtained concurrently.
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Herbal Supplement Extends Life Span and Boosts Stress Resistance
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females. BM also yielded differences in life span ranging from 5% shorter to 15% longer than
CM, depending on sex and strain. Pooling across all levels of sex, strain and medium, the over-
all effects of 1/3×, 1× and 3× SC100 on mean life span (relative to 0× controls) trended slightly
upward at +0.8%, +1.2% and +2.4%, respectively, but these differences were not significant
(P= 0.2, Table 7). Distributions of survivorship suggest that similar conclusions would be
reached in comparisons of mean or maximum life spans.
To establish whether the interaction of medium × SC100 dosage might reveal any effect of
the supplement on longevity on one medium but not the other, a separate 3-way ANOVA was
performed for each medium. For BM, differences were observed between the sexes and among
strains and SC100 dosages (Table 8). Sex × strain interaction was also significant. Therefore, re-
sults for females and males were compared separately. In females on BM, there were differences
among strains and dosages, but no interaction (Table 9); however, a 1-way ANOVA with strain
as a covariate showed no difference among dosages (Table 10). In males on BM, there were
Table 8. Effects of Sex, Strain and SC100 Dosage—BM Food: AL site.
Source of Variation Sum of Squares df*Mean Squares F Ratio PValue
Sex 155.489 1 155.489 8.967 0.004
Strain 2,560.046 2 1,280.023 73.817 <0.0005
Dosage of SC100 185.111 3 61.704 3.558 0.019
Sex × Strain 829.741 2 414.870 23.925 <0.0005
Sex × Dosage 69.737 3 23.246 1.341 0.269
Strain × Dosage 103.094 6 17.182 0.991 0.439
Sex × Strain × Dosage 123.843 6 20.640 1.190 0.322
Error 1,161.815 67 17.341
*df = degrees of freedom.
doi:10.1371/journal.pone.0119068.t008
Table 7. Effects of Sex, Strain, Food Medium and SC100 Supplementation: AL site.
Source of Variation SS*df*Mean Squares F Ratio PValue
Sex 1,637.405 1 1,637.405 97.793 <0.0005
Strain 4,257.391 2 2,128.695 127.135 <0.0005
Medium 195.453 1 195.453 11.673 0.001
Dosage of SC100 79.951 3 26.650 1.592 0.194
Sex × Strain 1,590.953 2 795.477 47.509 <0.0005
Sex × Medium 510.750 1 510.750 30.504 <0.0005
Sex × Dosage 99.736 3 33.245 1.986 0.119
Strain × Medium 469.642 2 234.821 14.025 <0.0005
Strain × Dosage 190.139 6 31.690 1.893 0.086
Medium × Dosage 204.352 3 68.117 4.068 0.008
Sex × Strain × Medium 15.656 2 7.828 0.468 0.628
Sex × Strain × Dosage 169.038 6 28.173 1.683 0.130
Sex × Medium × Dosage 69.309 3 23.103 1.380 0.252
Strain × Medium × Dosage 125.357 6 20.893 1.248 0.286
Sex × Strain × Medium × Dosage 156.206 6 26.034 1.555 0.165
Error 2,293.864 137 16.744
*SS = sum of squares, df = degrees of freedom.
doi:10.1371/journal.pone.0119068.t007
Herbal Supplement Extends Life Span and Boosts Stress Resistance
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differences among strains, but SC100 dosage had neither a main effect on life span nor an inter-
action with strain (Table 11). For CM, sex, strain and sex × strain interaction were significant;
SC100 dosage had no effect on life span, but strain × SC100 interaction was significant
(Table 12). Testing within each strain on CM, SC100 dosage had no main effect on life span or
interaction with sex in w
1118
or ywflies, but SC100 dosage × sex interaction was significant for
B flies (Table 13). Testing within each sex revealed differences among dosages for B4 females
(P= 0.025), but not males (P= 0.8). Pairwise comparisons among dosages showed that supple-
mentation with 1× SC100 extended life span by 20% in relation to unsupplemented controls
(P= 0.040; Table 14).
Fecundity: CA Site
Dietary restriction and many genetic changes that extend life span typically have the unwanted
side effect of depressing fecundity. To check whether this was the case with SC100 treatment,
we carried out fecundity experiments with control and SC100-treated flies (Table 15). Two
ages were tested: 2 weeks (young) and 4 weeks (old). The mean fecundity from a linear model
analysis is shown in Table 15 for young and older Control and SC100-treated flies. There was
no significant difference in fecundity for young females between Control and SC100-treated
flies (P= 0.74). However, for older flies, SC100-treated flies had significantly higher fecundity
than did Control flies (P<0.001). Thus, rather than inhibiting reproduction, these results indi-
cate SC100 may actually preserve higher fecundity over control levels in later life.
Heat and Starvation Stress Resistance on B4 and stress resistant O
strains: CA Site
Genetic or environmental treatments that enhance life span also typically enhance resistance to
environmental stress [1–3,5,6]. We first tested whether SC100 treatment also enhances stress
resistance to partial starvation (Table 16) using B4 flies. Two hundred flies (100 flies of each
sex) were placed in cages that had 10% of the normal food concentration at 25°C, but were oth-
erwise unrestricted in food availability. This condition puts a starvation stress on the flies that
leads to higher daily mortality rates. In two separate experiments (Table 16) the 50% median
Table 10. Effect of SC100 Dosage with Strain as Covariate—Females on BM Food: AL site.
Source of Variation Sum of Squares df*Mean Squares F Ratio PValue
Dosage of SC100 143.702 3 47.901 1.781 0.167
Strain 1,023.200 1 1,023.200 38.039 <0.0005
Error 1,022.158 38 26.899
*df = degrees of freedom.
doi:10.1371/journal.pone.0119068.t010
Table 9. Effects of Strain and SC100 Dosage—Females on BM Food: AL site.
Source of Variation Sum of Squares df*Mean Squares F Ratio PValue
Strain 1,380.741 2 690.371 42.622 <0.0005
Dosage of SC100 179.438 3 59.813 3.693 0.022
Strain × Dosage 161.840 6 26.973 1.665 0.163
Error 502.122 31 16.197
*df = degrees of freedom.
doi:10.1371/journal.pone.0119068.t009
Herbal Supplement Extends Life Span and Boosts Stress Resistance
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B4 fly survival fell to around 9–10 days with partial starvation. SC100 treatment enhanced
stress resistance to starvation and led to flies with a 50% median survival of 15 to 17 days or
about a 62% increase in median survival (P<0.001).
We have also tested whether SC100 treatment enhances stress resistance to a combination
of heat and partial starvation using both B4 flies and the stress resistant Methuselah O1 fly
strain (Fig 7). Female and male 20% B4 fly survival fell to less than 1 day with 29°C heat and
partial starvation (green filled triangles). SC100 treatment dramatically enhanced stress resis-
tance to the heat and starvation stressed B4 flies and led to a 20% survival of 4.7 days for fe-
males and 4 days for males (open blue squares).
With the much more stress resistant Methuselah O1 strain of Drosophila, the 20% survival
time for the untreated O1 flies was 6.0 days for females and 6.6 days for males (Fig 7, orange
solid circles). SC100 treatment of the O1 flies led to a 20% survival of 11 days for females and 8
days for males (Fig 7, open red diamonds). The SC100 treated O1 flies also had a dramatic in-
crease in survival of the last 15–20% of the survivors (Fig 7, see long tail of open red diamonds
in both males and females).
Discussion
Results of this study show that an herbal supplement, SC100, can have strikingly divergent ef-
fects on the life span of Drosophila under different test conditions. In relatively high stress envi-
ronments associated with short mean life spans, SC100 increased mean and maximum survival
times in both cages and vials and increased resistance to partial starvation stress without de-
creasing fecundity. In cages, it was also associated with a highly distinctive distribution of survi-
vorship, with only a slight effect on early mortality and mean longevity but a vast impact on
late mortality and maximum longevity. In vials, SC100 was beneficial to the entire population
Table 12. Effects of Sex, Strain and SC100 Dosage—CM Food: AL site.
Source of Variation Sum of Squares df*Mean Squares F ratio Pvalue
Sex 2,042.319 1 2,042.319 126.286 <0.0005
Strain 2,153.364 2 1,076.682 66.576 <0.0005
Dosage of SC100 98.841 3 32.947 2.037 0.117
Sex × Strain 772.630 2 386.315 23.888 <0.0005
Sex × Dosage 98.997 3 32.999 2.040 0.116
Strain × Dosage 218.356 6 36.393 2.250 0.048
Sex × Strain × Dosage 205.130 6 34.188 2.114 0.062
Error 1,132.049 70 16.172
*df = degrees of freedom.
doi:10.1371/journal.pone.0119068.t012
Table 11. Effects of Strain and SC100 Dosage—Males on BM Food: AL site.
Source of Variation Sum of Squares df*Mean Squares F Ratio PValue
Strain 2,148.484 2 1,074.242 58.622 <0.0005
Dosage of SC100 62.106 3 20.702 1.130 0.350
Strain × Dosage 60.388 6 10.065 0.549 0.767
Error 659.693 36 18.325
*df = degrees of freedom.
doi:10.1371/journal.pone.0119068.t011
Herbal Supplement Extends Life Span and Boosts Stress Resistance
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under high stress conditions, postponing both early and late mortality. In a lower stress envi-
ronment, it had no overall effect on survivorship, but it was associated with a longer mean life
span in female flies of the B4 strain on one food medium.
In comparing the vial experiments in CA to the vial experiments in AL, the mean life span
for the untreated B4 flies in single sex vials in CA (25.9 d) was much shorter than the untreated
single sex vials in AL (68.7 d). These very large increases in the life spans of untreated flies at
the AL site are likely due to environmental differences between the CA and AL sites, resulting
from a low stress environment at the AL site. However, housing flies in cages (CA site) strongly
promoted longevity in a subfraction of the SC100-treated flies, leading to flies that had a maxi-
mum life span that exceeded the maximum life span found for any of the treated or untreated
flies at both the CA and AL sites.
Altering the Survival Curve
While the ultimate causes of aging are still in dispute, aging can be simply defined as the bio-
logical changes that lead to progressive age-related increases in mortality rates. As an animal
cohort ages, survival has often been expressed by the well-known Gompertz curve, wherein the
number of individuals alive in the population declines due to an exponential increase in mor-
tality rates with age. For example, the Gompertz survival curve fits human populations quite
well for ages of 25 to 85 years [45] and is driven by the exponential increase in the annual mor-
tality rate or risk of dying with age. In humans, the annual mortality rate doubles every 8 years.
As human survivors over age 85 have increased in recent years, the Gompertz curve has be-
come a little less accurate in predicting mortality rates at extreme ages. Specifically, older hu-
mans are living slightly longer than predicted by the Gompertz curve. Moreover, the very rare
humans that survive to the supercentenarian status of 110 years reach an apparent plateau in
annual mortality rate of some 50 to 65% per year [46], suggesting that this highly selected
group of supercentenarians may deviate from the standard Gompertz curve dynamics. One ex-
planation of this is that the highly selected supercentenarians have a more favorable genetic
background for longevity and therefore have different Gompertz parameters (especially alpha).
Table 14. Tukey’s Honestly Significant Difference Test—B4 Females on CM Food: AL site.
Dose (i) Dose (j) Difference PValue
0× SC100 1/3× SC100 -11.070 0.139
0× SC100 1× SC100 -13.534 0.040
0× SC100 3× SC100 -1.910 0.969
1/3× SC100 1× SC100 -2.464 0.950
1/3× SC100 3× SC100 9.160 0.256
1× SC100 3× SC100 11.624 0.083
doi:10.1371/journal.pone.0119068.t014
Table 13. Effects of Sex and SC100 Dosage—B4 Flies on CM Food: AL site.
Source of Variation Sum of Squares df*Mean Squares F ratio Pvalue
Sex 186.277 1 186.277 5.972 0.023
Dosage of SC100 262.206 3 87.402 2.802 0.063
Sex × Dosage 285.597 3 95.199 3.052 0.049
Error 717.434 23 31.193
*df = degrees of freedom.
doi:10.1371/journal.pone.0119068.t013
Herbal Supplement Extends Life Span and Boosts Stress Resistance
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While the Gompertz curve with its exponential increases in mortality rates with age was
originally proposed in 1825 for human populations, it has also been highly successfully in
modeling mortality rates in aging populations of many animals [47]. Similar to the case with
human survival curves, data from several groups began appearing in the 1990s indicating that
fruit flies, medflies, nematodes, wasps, and yeast may hit a mortality rate plateau at later ages,
with only a very small subfraction of 1% of the population cohort still alive [48]. Thus, like hu-
mans, it appears that the parameters of the Gompertz curve may be altered in a small subfrac-
tion of the oldest members of an animal cohort.
Of the successful interventions to change animal survival curves (i.e. life span), transgenic
animal mutants and dietary restriction have typically generated the best results [49,50]. How-
ever, many genetic mutations and even dietary restriction have now been shown to extend life
span to various extents depending on the variable experimental conditions [51–57]. This vari-
ability in life extension is consistent with our results in the present paper: the life span exten-
sion of SC100 depends on the exact genotype, diet, and laboratory conditions. Stress resistance
or a low stress environment appears to be an underlying factor, as those conditions that lower
stress favor longer life spans [3].
Successful interventions into aging have been detected as increases in mean and/or maxi-
mum life span. Interventions that largely affect mean rather than maximum life span are often
observed to “square the survival curve”and to affect healthspan more than life span or aging.
Indeed, some researchers believe that only treatments that increase maximum life span are ac-
tually impacting the rate of aging. However, we are not aware of any interventions, whether ge-
netic or environmental, that double fly maximum life span and effect such significant changes
in the alpha parameter of the fly Gompertz curve. We clearly show in this article that treatment
of caged flies with SC100 alters the alpha parameter of Gompertz survival curve in a fundamen-
tal way.
Decline in Late-Life Mortality Rate
While some previous published articles have reported a decline in late-life mortality rate in a
small fraction of 1% of cohort survivors [48,58], our fly data indicate that SC100 induces a
greatly reduced mortality rate in late life at around 33% fly survival. Unlike worms and yeast,
genetic and dietary interventions have been limited in their capacity to extend mean and maxi-
mum life span in Drosophila by more than 50%. The elongation of maximum life span of caged
flies by SC100 appears to outperform the previously published Drosophila life span enhancing
Table 15. Mean fecundity estimated from a linear model of fecundity with two fixed effects: adult age
and treatment.
Age/Treatment Fecundity
2–4 week Control 23.8
2–4 week SC100-Treated 24.6*
4–6 week Control 13.3
4–6 week SC100-Treated 19.1*
Young Control and SC100-treated flies were tested for fecundity starting at 2 weeks old, whereas older
Control and SC100-treated flies were fecundity tested at 4 weeks of adult age. Fecundity value is shown as
average eggs per fly over a 2 week test period under optimal conditions.
*The 2–4 week SC100 Treated flies were not significantly different from Control flies (P= 0.74), whereas
the 4–6 week treated flies were significantly more fertile than 4–6 week Control flies (P<0.001), but not
significantly different from the younger 2–4 week Control flies in fertility.
doi:10.1371/journal.pone.0119068.t015
Herbal Supplement Extends Life Span and Boosts Stress Resistance
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treatments using transgenics and dietary restriction methods. We have also shown that SC100
treatment generates robust resistance to starvation and heat stress (Table 16, and Fig 7).
In the case of dietary restriction and many treatments that extend life span, there are often
trade-offs such as reductions in overall egg production and fertility [59,60]. It appears that re-
ducing egg production promotes longer life span. In the present longevity experiments, fecun-
dity was the same initially in SC100 treated and control flies, but became significantly higher in
treated flies compared to controls as the control flies aged. Thus, SC100 appears to have
Fig 7. Survival curves for female and male B4 and O Drosophila in cages during heat and partial
starvation treatment: CA site. 7A (Females) and 7B (Males) show the survivorship curves for B4 and O1
flies under heat (29°C) and partial starvation (10% dilution of normal food supply) stress. B4 Controls (solid
green triangles) and stress-resistant O1 Control (solid red circles) flies were each housed in separate cages
with both sexes combined (250 females and 250 males). Survivorship curves for the SC100 treated flies are
shown for B4 treated (blue open squares) and for stress resistant O1 (open red diamonds) flies.
doi:10.1371/journal.pone.0119068.g007
Table 16. Effects of SC100 on resistance to starvation stress.
Treatment Male 50% Survival Female 50% Survival Male Change Female Change
Exp. 1: Control 9.0 days 9.1 days
Exp. 1: SC100-treated 15.0 days 15.3 days 67%*68%*
Exp. 2: Control 10.4 days 10.7 days
Exp. 2: SC100-treated 16.7 days 16.7 days 61%*56%*
100 males and 100 females in each cage were subjected to 10% of normal food in Control or SC100-treated cages. The number of dead flies was
monitored daily and the 50% median survival in days is given for both males and females. The effects of SC100 are shown as the increase in survival
(Male or Female Change).
*In Experiments 1 and 2, survival times of treated flies were significantly different from those of Control flies (P<0.001).
doi:10.1371/journal.pone.0119068.t016
Herbal Supplement Extends Life Span and Boosts Stress Resistance
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striking effects on reducing both aging and mortality rates without the typical negative effect
on fecundity. Measurements of walking speed, duration of flight, spontaneous locomotor activ-
ity, oxygen consumption or carbon dioxide production would be needed to rule out trade-offs
entirely, but none were apparent to visual inspection of the caged flies.
The main cause of the large increase in maximum life span of caged flies produced by
SC100 appears to be the generation of a large decline in the late-life mortality rate after some
two-thirds of the population has died. In previous experiments where major declines in the late
life mortality rate have been reported, they occurred in only a vanishingly small fraction of 1%
of survivors from very large populations [48,58,61]. In our current Drosophila experiments
using cages, it is striking that SC100 treated flies reach a much reduced mortality rate when
roughly one third of the flies are still alive and the daily mortality remains fairly low. Even
when we started treating flies at midlife, when about 40% of the cohort had died, the remaining
flies reached a much reduced mortality rate when roughly a third of the flies were still alive.
Note that under these conditions with the cohort population of 1500 flies, we only see a ragged
mortality rate decline in the control flies among females beginning when fewer than 10% of the
flies were still alive and only lasting for a few days (Fig 2C). Thus, the SC100 treatment dramat-
ically altered the aging of Drosophila and apparently did so by preventing significant increases
in the mortality rate for an extended period of time in a substantial fraction of the population.
If a treatment could generate a significant decline in the mortality in mammals while a large
part of the population was still alive, that could be a novel way of extending longevity in the
surviving fraction of the population.
Effect of SC100 on Longevity Depends on Environmental Conditions
Vast phylogenetic, anatomical and physiological gaps separate D.melanogaster and Homo sapi-
ens. Therefore, the consistency of effects of SC100 on fly longevity was assessed under distinctly
different environmental conditions as a minimal precondition for any speculative inferences
regarding human aging. Experiments were performed independently in cages and in vials at
different population densities, with males and females segregated or housed together. A differ-
ent rationale underpinned each set of conditions. First, while most Drosophila longevity experi-
ments in the literature are still carried out using only one sex in either vials or bottles, large
Plexiglas cages with 250 flies of each sex provide a more natural environment that does not pre-
vent reproduction or exercise in the form of sustained flight. On the other hand, flies may differ
from humans in that both mating and exercise (increased metabolic activity) typically shorten
the life span. Nevertheless, a surprising finding in our results was that the cage experiments
containing both sexes where flies were frequently flying nevertheless had the longest maximum
life spans.
As has been found in many other laboratories, mean longevity was markedly lower in this
study when males and females were mixed in large cages than when they were housed separate-
ly in small vials at the CA or AL sites. SC100 was associated with a strong enhancement of the
short mean life spans in vials and cages at the CA site and almost no effects on long life spans
in vials at the AL site. Surprisingly, even though the population density was lowest in vials at
the CA site, these flies exhibited even shorter mean and maximum life spans than the control
flies in cages. Assuming that the length of life in control flies is indicative of the intensity of
stress, SC100 supplementation was beneficial to the entire population under conditions of
maximum stress (vials at the CA site), a large subpopulation at intermediate stress (cages at the
CA site) but not to any of the flies under low stress (vials at the AL site). Of course, this inter-
pretation does not identify the source of stress in the vials at the CA site. However, we do note
that a 12 h light: 12 h dark cycle was used at the AL site, whereas the lights were on 24 h every
Herbal Supplement Extends Life Span and Boosts Stress Resistance
PLOS ONE | DOI:10.1371/journal.pone.0119068 April 16, 2015 19 / 28

day for all experiments done at the CA site. Disruption of the circadian clock has been shown
to significantly shorten life span in Drosophila [62], so this is one stress factor that we can iden-
tify that could account for some of the difference between mean life spans in vials at the AL ver-
sus CA sites.
SC100 as a Multipath Approach to Enhancing Stress Resistance and
Slowing Aging
How is SC100 acting and do the fly housing conditions matter? SC100 could be extending life
span at the CA site by inducing more repair, autophagy, or regenerative capacity. These path-
ways were targeted, but we do not have direct evidence that there were significant changes in
these genetic pathways to explain the life extension at this site. We also had differences in the
survival curves depending on whether we used large cages with 500 flies or small vials with 4
flies in each vial (compare Fig 1 to Fig 3) or very low stress vials under near ideal conditions.
This shows that major differences in housing conditions for the flies can lead to major differ-
ences in survival curves. Of course, it has been known for some time that housing flies in a
same sex environment extends life span, but we have now shown that differences in survival
are also observed if one uses cages with large populations versus small vials. We have also ob-
tained very long mean life spans of over 70 days with untreated wild-type same-sex B4 Dro-
sophila in vials housing 25 flies per vial under ideal low stress conditions. In those cases where
the controls were in low stress housing conditions with normal circadian rhythm, SC100 had
little or no life-extending effect. These experiments suggest that fly housing can greatly alter life
span survival curves and the effects of supplements can vary depending on housing. Our hy-
pothesis is that a favorable low stress environment strongly promotes longevity in control flies.
Is SC100 acting only on a genetic subset of the flies (the cohort heterogeneity model)? While
we cannot completely rule out this possibility, it seems highly improbable that one third of an
interbreeding Drosophila population could have a strikingly different genotype or gene expres-
sion pattern, as this B4 population of flies has been interbreeding for some 30 years. The evolu-
tionary theory of aging could provide an explanation for our results if we assume that SC100
induces an alternative pattern of gene expression that counteracts dysfunctional gene expres-
sion patterns that develop at later fly ages, leading to an alteration in the Gompertz age-related
alpha parameter and an elongated survival curve.
It is safe to say that genetics, living environment, and diet are all factors determining longev-
ity. Indeed, longevity appears to be quite plastic and responsive to many factors. We have fo-
cused mainly on changing the diet using multiple dietary supplements. The many active
components in SC100 may provide a multipath approach to slowing aging and to altering the
basic age-related alpha parameter of the Gompertz curve. Our data in Drosophila housed in
cages indicate that such an approach may provide a practical way to dramatically slow the
aging process in this important model animal. We are not aware of any treatment with a mix of
substances or any of the many single-gene transgenics that has had more than a 50% increase
in Drosophila maximum life span, so the effects of SC100 on Drosophila maximum life span in
our cage experiments are impressive. Drosophila treated with SC100 underwent a significant al-
teration in their alpha parameter and thus slowed their rate of aging. However, these results
should be taken with the caveat that housing environment and stress can also be a big factor in
life span. The ability of housing environment alone to generate long-lived flies (mean life span
of >70 days for control flies, as was the case in the low-stress, same-sex vial experiments) sug-
gests that longevity gains can be promoted by alternative methods in non-synergistic ways.
Concerning ramifications for humans, two opposite interpretations can be entertained. (1)
Reducing stress and promoting stress resistance both appear to be valuable interventions to
Herbal Supplement Extends Life Span and Boosts Stress Resistance
PLOS ONE | DOI:10.1371/journal.pone.0119068 April 16, 2015 20 / 28

extend life span. With modern life apparently becoming more stressful, environmental inter-
ventions appear to be an effective approach to promoting health span and life span. (2) For
readers in developed nations with historically unprecedented rectangularization of survivor-
ship, where many bacterial and viral illnesses have been eradicated, where safe water, food,
waste disposal, and medications are readily available, and where life can be spent in thermally
controlled indoor environments, escaping strenuous physical activity, the results from the AL
site suggest that SC100 might not lead to further extension of life, unless the individual had a
stressful job, did not get enough sleep, or made unhealthy lifestyle choices. For those people
with stressful lives, the results from cages suggest that a long-lived subpopulation could benefit
substantially from SC100 supplementation. Under exceptionally stressful conditions, the re-
sults from vials at the CA site suggest that supplementation would assist the whole population.
A broader point is that realistic inferences about human supplementation should await rep-
lication of the results in mice and/or data from human clinical trials. A version of SC100 is cur-
rently being testing in a double-blind, placebo controlled clinical trial.
Materials and Methods
Testing Sites and Data Availability
Longevity experiments were performed at two sites: Genescient Inc., Fountain Valley, CA (des-
ignated ‘CA’) and the University of South Alabama, Mobile, AL (designated ‘AL’). Fertility and
stress resistance were measured at the CA site. All relevant data is within the paper.
Fly Strains and Media
All studies at the CA site employed stocks of the wild-type B4 line or the late-fertility-selected
O1 line of D.melanogaster (originally from the Michael Rose laboratory at the University of
California, Irvine), which were maintained in vials containing a banana medium at about 25°C.
The banana medium (BM) is a low protein diet containing a boiled mixture of (i) 100 g agar in
6.6 L distilled water, (ii) 900 g peeled bananas, 165 mL Eden barley malt syrup, 110 mL light
and 110 mL dark Karo corn syrup blended into 400 mL distilled water, and (iii) 240 g Fleisch-
mann’s instant dry yeast and 160 mL 95% ethanol blended into 460 mL water. After cooling to
48°C, 15.6 g methyl-4-hydroxybenzoic acid (10% w/v in 95% ethanol) was added as an antifun-
gal agent. Life spans on this medium are nearly as long as on dietary restriction diets [63]. Stud-
ies at the AL site employed the B4 line, which was shipped from the CA site and allowed
several weeks to acclimate, and parallel groups of flies from ywand w
1118
strains. Stocks were
maintained on a sucrose/yeast/cornmeal diet described previously [55].
Drug/Food Preparation
The SC100 herbal drug is a proprietary 4 herb blend comprising: 1) medicinal Astragalus mem-
branaceus extract; 2) Pterocarpus marsupium standardized for 25% pterostilbene; 3) Pine Bark
extract standardized for 85% Oligomeric-Proanthocyanidins, and 4) 98% pure L-Theanine.
SC100 was obtained from Life Code, Inc. (contact info@LifeCodeRx.com for SC100 research
samples or purchase the commercial version of SC100 online at www.lifecoderx.com).
For studies in CA, the SC100 dosage was calculated and prepared by pharmacist Marc Hor-
witz and independently verified. The standard dilution of SC100 (5% w/w) has a human dose
equivalent equal to 1 mL of the sample. The food plates were prepared using a syringe to mea-
sure out the appropriate SC100 dosage and transferred to a small “salsa dish”on a Denver In-
strument XL-410D fine scale with an accuracy of +/- 0.001 g. The SC100 was then diluted with
BM at 40–60°C up to 10.00 g (+/- 0.01 g). The food plates containing SC100 were labeled “T”
Herbal Supplement Extends Life Span and Boosts Stress Resistance
PLOS ONE | DOI:10.1371/journal.pone.0119068 April 16, 2015 21 / 28

or “CT”and the control plates were labeled “C”. Food was prepared every 2–3 d and was never
kept for more than 3 d to ensure freshness. The dilution was re-made weekly and stored at 4°C.
For studies in AL, the dosage was calculated based on a 10
8
fold difference in mass between
flies and humans (0.7 mg vs. 70 kg) and ingestion of 2 μL food per fly per day [64]. Accordingly,
a concentration of 4.5 mg/L SC100 (1X) corresponded to 9 ng per fly per day, equivalent to a
human dose to 900 mg or 2 capsules per day. SC100 was diluted 1:10 from stock solutions into
both BM and cornmeal medium (CM) at doses of 0 (control), 1/3X, 1X and 3X during cooling
(40–60°C). Fresh food was prepared every 2–3 d and stored at 4°C. Fresh stock solutions were
prepared weekly and stored at 4°C.
Fly Longevity Assays—Cages
All assays in cages were performed at the CA site. Flies were separated into 9 groups of 250
males and 250 females per cage. Thus, there were a total of 9 cages in the assay. Three cages of
B4 population flies (T) were treated with 1 mL of SC100 solution (5% w/w) in 10 mL banana-
agar media daily from day 1 after eclosion. Three cages of B4 population flies (CT) were treated
without SC100 for 36 days before starting SC100 treatment with 1 mL of SC100 solution.
Three control cages of B4 population flies (C) were given 10 mL banana-agar media daily with-
out SC100.
Fly Longevity Assays—Vials
As an alternative longevity assay, flies at the CA site were also housed in small vials with 4 flies/
vial and enough vials to have 100 flies of each sex. In this assay, B4 flies were housed with 4
flies of the same sex in each vial or with 2 females plus 2 males per vial. The vials were changed
every 2–3 d or 3 times per week, with flies combined into new vials to preserve the 4 flies per
vial as flies died. Sexual selection (2 of each sex or all of one sex) was preserved until all the flies
were dead.
At the AL site, male and female flies were housed separately in 4 groups of 25/vial. Vials
were exchanged and mortality was recorded every 2 d initially and daily after age 40 d. In this
case, the mean survival time of the original 25 flies was determined, so dead flies were not re-
placed to preserve the population density.
Mortality Assay
At the CA site, mortality counts were conducted between 1–4pm every day. Dead flies were re-
moved from the cages and scored along with gender data. Stocks of Drosophila melanogaster
were cultured in polystyrene vials one quarter full of banana agar medium at an ambient tem-
perature of 25°C. At 14 days of age from the egg (eclosion occurs 7 to 9 days from egg), the flies
were sexed after being anesthetized and then transferred to Plexiglas cages and kept throughout
adulthood at the same ambient temperature. The cages were custom made by Plastic Sales, Inc.
in Costa Mesa, CA. The cages were made from 0.5 cm thick Plexiglas sheets to form a box 25
cm long, 20 cm wide, and 14 cm high. Cages were designed so that the flies cannot escape with
daily feeding and the gathering of dead flies. Once in the cages, the flies were fed 15×100 mm
Petri dishes full of banana agar with 5% dry yeast. In the SC100 cages, the yeast paste contained
the assigned dosage (equivalent to a weight adjusted human dose of 820 mg) of SC100. The
food dishes were changed daily until all the flies were dead.
When fly cohorts are maintained in population cages of the design that we used, eggs laid
away from the food medium fail to develop successfully, while the food medium is removed
sufficiently often that development cannot be completed when the eggs are laid there. Further-
more, the pupal stage required to complete development is of sufficient duration to make the
Herbal Supplement Extends Life Span and Boosts Stress Resistance
PLOS ONE | DOI:10.1371/journal.pone.0119068 April 16, 2015 22 / 28

development of offspring easy to detect; no such pupal development was observed in the course
of these experiments.
We set up three cages for Control, lifelong SC100-treated, and midlife SC100-treated flies,
including two sets of four control cages. Each cage contained 500 young, mature fruit flies of
the species D.melanogaster. Every day we supplied the cages with new Petri dishes of banana-
yeast agar media. We counted, sexed and recorded the number of dead flies in each cage daily.
The dead flies were removed from the cages using an aspirator. Male flies were identified by
the presence of sex combs on the forelegs and female flies were identified by their larger, striped
abdomens and the absence of the sex combs. The few flies that died or escaped as a result of
handling procedures were not recorded because they did not die as a result of ingestion of their
assigned substance. The above procedure continued until all of the flies in all of the cages
had died.
Fecundity
For each of the lifelong and midlife SC100 treated samples, and the controls, we set up 60 vials,
one quarter full of charcoal agar media, containing one treated female and one treated male (or
untreated in the case of the controls.
We counted and recorded the number of eggs laid by the flies in each of 20 vials for each
cage, 60 for each dose, after one day spent in the charcoal vials. We also conducted fecundity
assays at 14 days and again at 28 days of cage life.
Heat and Partial Starvation Stress Experiments
For the stress experiments we used survivorship curves for B4 and O1 flies under heat (29°C)
and partial starvation conditions. Partial starvation used a 10% dilution of the normal banana-
agar medium. For each of the 4 samples (Control and SC100-Treated B4 flies along with Con-
trol and SC100-Treated O1 flies), 250 flies of each sex were housed in cages as described above
and survival was followed as before.
Statistical Analysis
All statistical analyses of life spans at the AL site were performed as described previously [55].
In summary, the mean life span was calculated for each vial and the 4 means for each group
were compared by analysis of variance, with sex, strain, medium and supplement dose as fac-
tors. Previous studies under similar conditions have shown that raw survivorship data do not
violate parametric modelling assumptions (normal distribution and equality of variances) in
most cases in this laboratory, and mean survivorship almost never violate the assumptions
[55].
Data from the CA site were analyzed with R (R Development Core Team, 2011, version
2.13.1; see http://www.R-project.org/), as follows:
Mean Longevity. For each replicate cage and each sex the mean longevity was computed.
Mean longevity (l
ijk
) was then modeled with the linear equation,
lijk ¼aþdibiþdjgjþbk
where δ
i
=0ifi= 1 and 1 otherwise, β
i
measures the effect of treatment (control, CT, or T), γ
j
measures the effect of sex (male or female) and b
k
is the random contribution of block k, which
is assumed to have a zero mean and variance of σ
2
. The parameters were estimated by the lme
function in R—see Chapter 2 in reference [65] for a description of maximum likelihood meth-
ods used in the lme function.
Herbal Supplement Extends Life Span and Boosts Stress Resistance
PLOS ONE | DOI:10.1371/journal.pone.0119068 April 16, 2015 23 / 28

Age-Specific Survival. In this formulation, we let the index iindicate one of the treatments
(control, CT, T), jindicate sex (1 = female, 2 = male), kindicate a replicate cage, and tindicate
age. Then the predicted probability of survival from the start of the experiment to age twas
y
ijkt
. The basic nonlinear model is given by,
yijkt ¼fðCijk;tÞþijkt ;1
where C
ijk
is the vector of parameters, tis the age, and 
ijkt
is the replicate cage variation. The
function fis the Gompertz model,
fðCijk;tÞ¼exp Aijk
aijk
½1expðaijktÞ
()
:2
The parameter Ais sometimes called the age-independent parameter of the Gompertz
model and is a reflection of background mortality that does not change with age. On the other
hand, αis called the age-dependent parameter and measures the rate at which mortality in-
creases with age, e.g. senescence. We assume that the parameters of the Gompertz equation
may be affected by the fixed effects, sex and drug treatment and the random cage environment.
These assumptions translate into a system of equations,
Aijk ¼b1þg1idiþ1djþb1k
aijk ¼b2þg2idiþ2djþb2k
;3
where δ
i
=0ifi= 1, or 1 otherwise. To test for significant effects of SC100 treatment on Aand
α, we determined whether γ
1i
or γ
2i
was significantly different from zero, respectively. Likewise
a test for the effects of sex on Aand αcorresponds to a test for whether ϕ
1
or ϕ
2
is significantly
different from zero.
The variance of mortality is expected to change with the mean value of mortality. The gener-
al formulation for the variance of 
ijkt
is
Varðijkt Þffis2g2ð^
uijkt ;tÞ;4
where ^
uijkt ¼Eðyijkt jbiÞ. In this analysis we used g(.) = | y
ijkt
|
δ
. The b
i
were distributed as,
biN0;s10
0s2
"# ! 5
The parameters in Equations (3–5) were estimated from a nonlinear mixed effects model as
implemented by the nlme package of R—see Chapter 7 in reference [65].
Female Fecundity
Fecundity was measured in females at two ages in all three SC100 treatments. We used a simple
linear model to estimate the effects of age, treatment, and their interaction with the R linear
model (lm) function. This linear model provides estimates of the magnitude of the effects of
each drug and their statistical significance.
Software
All statistical analyses in CA were carried out with R (version 2.7.0 and 2.7.1, The R Founda-
tion for Statistical Computing). The Gompertz analysis of the supplements used the nonlinear,
Herbal Supplement Extends Life Span and Boosts Stress Resistance
PLOS ONE | DOI:10.1371/journal.pone.0119068 April 16, 2015 24 / 28

mixed effects R program (nlme R-package). The fecundity results were analyzed with the linear
model function (lm) of R. The Gompertz utilized R-code used the optim R-function for finding
maxima of the likelihood function. Analyses in AL were carried out using SYSTAT 12
software.
Acknowledgments
We thank Dr. Laurence D. Mueller (UCI) for his Gompertz statistical analysis of our data.
Author Contributions
Conceived and designed the experiments: BV CR GB RM. Performed the experiments: KM AN
RM. Analyzed the data: BV KM RM. Contributed reagents/materials/analysis tools: MH BV
RM. Wrote the paper: BV KM RM.
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Herbal Supplement Extends Life Span and Boosts Stress Resistance
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