Genotoxicity testing of a Salacia oblonga extract.

Abstract

Salacia oblonga has been used for thousands of years in Ayurvedic medicine for the oral treatment of diabetes. The root extract has been shown to inhibit the activity of intestinal alpha-glucosidases, therefore S. oblonga holds potential as a natural method to mitigate the blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the potential genotoxicity of a S. oblonga root extract (SOE) was evaluated using the standard battery of tests (reverse mutation assay; chromosomal aberrations assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredients. SOE was determined not to be genotoxic under the conditions of the reverse mutation assay and mouse micronucleus assay, and weakly positive for the chromosomal aberrations assay. A reproducible, although weak, positive chromosomal aberrations response in human lymphocytes is of concern and further toxicity research is recommended. Use of SOE is presently expected to be safe, as anticipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, may prove be a useful ingredient in foodstuffs.

Full text

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Genotoxicity testing of a Salacia oblonga extract
A.M. Flammanga,*, G.L. Erexsonb, M.S. Mecchib, H. Murlib
aRoss Products Division Abbott Laboratories, 3300 Stelzer Road, 625 Cleveland Avenue, Columbus, OH 43215-1724, USA
bCovance Laboratories Inc., 9200 Leesburg Pike, Vienna, VA 22182, USA
Received 13 December 2005; accepted 6 June 2006
Abstract
Salacia oblonga has been used for thousands of years in Ayurvedic medicine for the oral treatment of diabetes. The root extract has
been shown to inhibit the activity of intestinal a-glucosidases, therefore S. oblonga holds potential as a natural method to mitigate the
blood glucose response for people with diabetes. As part of a safety evaluation of novel ingredients for use in blood glucose control, the
potential genotoxicity of a S. oblonga root extract (SOE) was evaluated using the standard battery of tests (reverse mutation assay; chro-
mosomal aberrations assay; mouse micronucleus assay) recommended by US Food and Drug Administration (FDA) for food ingredi-
ents. SOE was determined not to be genotoxic under the conditions of the reverse mutation assay and mouse micronucleus assay, and
weakly positive for the chromosomal aberrations assay. A reproducible, although weak, positive chromosomal aberrations response in
human lymphocytes is of concern and further toxicity research is recommended. Use of SOE is presently expected to be safe, as antic-
ipated intake is small compared to the doses administered in the genotoxicity assays and may, after further toxicity research, may prove
be a useful ingredient in foodstuffs.
? 2006 Elsevier Ltd. All rights reserved.
Keywords: Salacia oblonga; a-Glucosidase inhibitor; Toxicology; Safety; Reverse mutation assay; Chromosomal aberration assay; Mouse micronucleus
assay
1. Introduction
Salacia oblonga is a perennial wild, woody, climbing
vine native to India and Sri Lanka. A member of the
Celastaceae family, it is commonly known as ‘‘ponkoranti’’
due to its golden colored root bark. S. oblonga has been
used for thousands of years in Ayurvedic medicine for
the oral treatment of diabetes (Grover et al., 2002). Root
extracts of various Salacia species have been shown to inhi-
bit the activity of intestinal a-glucosidases (Matsuda et al.,
2005). Inhibitionofthe
enzymes results in delayed breakdown of oligosaccharides
and inhibits glucose absorption into the bloodstream.
carbohydratemetabolizing
Decreased glucose absorption results in a lower postpran-
dial glycemic response and improved overall glycemic
control. Extract constituents salacinol, kotalanol, and
mangiferin have been characterized as the active ingredi-
ents for blood glucose control by inhibition of enzymes
involved in glycemic response (Ghavami et al., 2001;
Matsuda et al., 1999; Matsuda et al., 2002; Miura et al.,
2001). Therefore, S. oblonga holds potential as a natural
method to mitigate the blood glucose response for people
with diabetes. As an ingredient incorporated into foodstuff,
S. oblonga may provide people with diabetes a convenient
way to help manage their blood glucose levels.
As part of a safety evaluation of novel ingredients for
use in blood glucose control, an evaluation of the potential
genotoxicity of a S. oblonga extract (SOE) was conducted
using the standard battery of tests recommended by FDA
for food ingredients. The tests included the bacterial
reverse mutation assay, the chromosomal aberrations
assay, and the mouse micronucleus assay.
0278-6915/$ - see front matter ? 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2006.06.005
Abbreviations: FDA, US Food and Drug Administration; ICH, Inter-
national Conference on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use; SOE, Salacia oblonga
extract.
*Corresponding author. Tel.: +1 614 899 9725; fax: +1 614 727 4168.
E-mail address: ann.flammang@abbott.com (A.M. Flammang).
www.elsevier.com/locate/foodchemtox
Food and Chemical Toxicology 44 (2006) 1868–1874

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2. Materials and methods
2.1. Chemicals, culture medium and S9 activation system
Cell culture grade water used in the mouse micronucleus assay was
purchased from BioWhittaker. Benzo[a]pyrene, cyclophosphamide (CP),
mitomycin C, sodium azide, and ICR-191 were purchased from Sigma
Chemical Co. 2-aminoanthracene and 2-nitrofluorene were obtained from
Aldrich Chemical Co. 4 nitroquinoline N oxide (4NQO) was obtained
from Supleco. Dimethylsulfoxide was obtained from Acros Organics. Cell
culture grade water used in the chromosomal aberration assay and the
reverse mutation assay, culture medium, antibiotics and L-glutamine were
obtained from Mediatech.
S9 liver homogenate was purchased from Molecular Toxicology, Inc.
The homogenate was prepared from male Sprague–Dawley rats pretreated
with AroclorTM1254 as described by Ames et al. (1975). For the reverse
mutation assay, the S9 mix consisted of 10% S9, 4 mM NADP, 5 mM
glucose-6-phosphate, 8 mM MgCl2, 33 mM KCl, and 100 mM sodium
phosphate, pH 7.4.
Top (overlay) agar for the reverse mutation assay was prepared with
0.7% (w/v) agar and 0.5% (w/v) NaCl and was supplemented with 10 mL
of (1) 0.5 mM histidine/biotin solution per 100 mL agar for selection of
histidine revertants, or (2) 0.5 mM tryptophan solution per 100 mL of
agar for selection of tryptophan revertants.
S. oblonga extract D (Lot# 050203) was obtained in powder form from
Tanabe USA, Inc.
2.2. Cells
Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535
and TA1537 were obtained from Dr. Bruce Ames, University of Califor-
nia, Berkeley. The Escherichia coli tryptophan auxotroph WP2uvrA was
received from the National Collection of Industrial Bacteria, Torrey
Research Station, Scotland (United Kingdom). The tester strains were
checked for retention of their characteristic phenotypic markers at the
time of use.
2.3. Salmonella–E. coli/mammalian-microsome assay
The bacterial reverse mutation assay, to evaluate the ability to induce
reverse mutations at the histidine loci in four strains of S. typhimurium
(TA98, TA100, TA1535 and TA1537) and at the tryptophan locus in
E. coli tester strain WP2uvrA, was conducted according to standard
procedures (Ames et al., 1975; Green and Muriel, 1976). Briefly, the tester
strains were exposed to SOE via the plate incorporation method in the
presence and absence of an exogenous metabolic activation system (S9).
SOE, tester strain, and S9 mix (when required) were added to molten top
agar supplemented with histidine and biotin or tryptophan. The mixture
was vortexed and overlaid onto the surface of 25 mL of minimal bottom
agar contained in a 15 · 100 mm petri dish. After the overlay solidified,
the plates were inverted and incubated for 52 ± 4 h at 37 ± 2 ?C. All doses
of the test article, the vehicle controls and the positive controls were plated
in triplicate. After incubation, the revertant colonies were counted. A
mutagenic response was characterized by at least a 2-fold (tester strains
TA98, TA100 and WP2uvrA) or 3-fold (TA1535 and TA1537) dose-
dependent increase in the mean revertants per plate of at least one of these
tester strains as compared to the concurrent vehicle control.
2.4. Chromosomal aberrations assay
Human venous blood from healthy, adult donors (nonsmokers with-
out a history of radiotherapy, chemotherapy or drug usage, and lacking
current viral infections) was used in the chromosomal aberrations assay,
conducted to evaluate the ability to cause structural chromosomal aber-
rations with and without exogenous metabolic activation system. Whole
blood cultures containing fresh heparinized blood, culture medium and
test article were incubated at 37 ?C ± 2 ?C in a humidified atmosphere of
5% ± 1.5% CO2in air. The medium was RPMI 1640 supplemented with
HEPES buffer (25 mM), ?20% heat-inactivated fetal bovine serum (FBS),
penicillin (100 mL), streptomycin (100 lg/mL), L-glutamine (2 mM) and
2% phytohemagglutinin M (PHA-M). Negative (untreated controls) and
vehicle controls (cultures treated with 10.0 lL of DMSO/mL) were used.
The positive control agents were mitomycin C (MMC) for the nonacti-
vation series and CP in the metabolic activation series.
The in vitro metabolic activation system (Maron and Ames, 1983)
consisted of a rat liver post-mitochondrial fraction (S9) and an energy-
producing system (NADP at 1.5 mg/mL (1.8 mM) and isocitric acid at
2.7 mg/mL (10.5 mM)). S9 was prepared five days after a single dose of
500 mg/kg of AroclorTM1254.
In the initial trial, cultures were treated for ?3 h with and without S9
and harvested ?22 h after initiation of treatment. In the second trial,
cultures were treated for ?22 h without S9 and ?3 h with S9 and har-
vested ?22 h after initiation of treatment, corresponding to 1.5 times a cell
cycle time of approximately 15 h after the lymphocytes are induced to
divide by the addition of PHA-M (Galloway et al., 1994). At harvest, cells
were swollen with 75 mM KCl hypotonic solution and fixed with absolute
methanol:glacial acetic acid (3:1, v/v) fixative. One hundred cells from
each duplicate culture were analyzed for the different types of chromo-
somal aberrations (Evans, 1962, 1976). Mitotic index, percent polyploidy
and endoreduplication were evaluated from the negative control, vehicle
control and a range of test article concentrations and used for measure-
ment of toxicity and selection of doses for analysis. Statistical analysis
employed a Cochran–Armitage test for linear trend and Fisher’s Exact
Test (Thakur et al., 1985).
2.5. In vivo mouse micronucleus assay
Healthy male Crl:CD -1(ICR)BR mice (8–10 weeks of age; Harlan
Sprague-Dawley, Inc.) were used in the micronucleus assay to evaluate the
ability to induce in vivo clastogenic activity and/or disruption of the
mitotic apparatus by detecting micronuclei in polychromatic erythrocyte
(PCE) cells in mouse bone marrow. Mice were dosed by oral gavage for
three consecutive days at 500, 1000 or 2000 mg/kg/day SOE, or the vehicle
control (cell culture grade water; five/group, approximately 24 h between
doses). A similar group was treated once with the positive control (80 mg/
kg cyclophosphamide) approximately 24 h prior to sacrifice.
Bone marrow was harvested from five mice per group approximately
24 h after the last dose. At least 2000 PCEs per animal were analyzed for
the frequency of micronuclei. Cytotoxicity was assessed by scoring the
number of PCEs and normochromatic erythrocytes (NCEs) in at least the
first 500 erythrocytes for each animal. The criteria for the identification of
micronuclei were those of Schmid (1976). Data analysis was performed
using an analysis of variance (Winer, 1971) on untransformed proportions
micronucleated cells per animal, and on untransformed PCE:NCE ratios
when the variances were homogeneous.
3. Results
3.1. Salmonella–E. coli/mammalian-microsome assay
The mutagenicity of SOE in bacteria was evaluated up
to a maximal dose of 5000 lg/plate. Normal growth was
observed in all five tester strains, and the test article was
freely soluble at all doses evaluated with and without S9.
No increases in revertant frequencies were observed at
any dose of SOE in any tester strains with or without S9
compared to the concurrent vehicle control cultures (Table
1). The test article was re-evaluated in an independent
confirmatory experiment under identical conditions, and
similar results were observed (Table 2).
A.M. Flammang et al. / Food and Chemical Toxicology 44 (2006) 1868–1874
1869

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Table 1
Bacterial reverse mutation assay initial results
Dose lg/PlateMean revertants per plate with standard deviation
TA98TA100TA1535TA1537 WP2uvrA
Mean SDMean SDMean SDMean SDMeanSD
+S9
Vehicle Controla
SOE
28
22
21
31
14
17
17
275
7
6
3
3
7
97
112
86
104
99
84
87
15
17
10
5
10
10
7
11
17
11
9
11
8
7
4
1
5
1
6
1
4
6
4
8
9
8
6
4
1
3
2
4
1
3
2
14
14
8
11
14
7
10
3
5
3
3
4
1
5
33.3
100
333
1000
3330
5000
2.5
2.5
25.0
10
2
48 Benzo[a]pyrene
2-Aminoanthracene
2-Aminoanthracene
?S9
Vehicle Controla
SOE
75689 11016142 27
292 101
8
7
2
2
2
4
0
2
3
6
70
77
80
69
63
64
64
3
6
5
6
4
91
3
4
4
1
6
7
5
5
5
5
4
6
4
3
3
2
3
2
2
5
14
15
12
14
13
9
9
3
1
3
3
7
1
1
33.3
100
333
1000
3330
5000
1.0
2.0
2.0
1.0
11
12
12
9
15
16
10
12
10
9
11
339
10
7
2-Nitrofluorene
Sodium azide
ICR-191
4-Nitroquinoline-N-oxide
1404395 856 30
904 87
346 70
aVehicle control = DMSO, 100 lL aliquot.
Table 2
Bacterial reverse mutation assay confirmatory results
Dose lg /Plate Mean revertants per plate with standard deviation
TA98 TA100TA1535 TA1537WP2uvrA
Mean SDMean SDMean SDMeanSD MeanSD
+S9
Vehicle Controla
SOE
23
21
23
22
24
20
17
3
7
3
4
5
5
6
98
95
107
95
96
89
90
8 13
12
13
9
10
11
10
4
4
3
1
2
4
1
12
15
13
13
10
9
9
5
4
4
3
5
2
1
16
15
17
11
14
13
9
2
4
1
3
3
2
2
33.3
100
333
1000
3330
5000
2.5
2.5
25.0
11
6
4
8
12
19
Benzo[a]pyrene
2-Aminoanthracene
2-Aminoanthracene
?S9
Vehicle Control
SOE
30225
571 6715881 9711
70480
12
14
12
17
14
10
11
3
6
7
5
5
3
5
90
88
87
82
85
89
81
211
10
7
10
13
7
9
2
2
4
5
5
2
5
7
8
8
6
2
2
2
4
3
2
2
15
16
18
21
12
9
10
4
3
7
6
6
3
6
33.3
100
333
1000
3330
5000
1.0
2.0
2.0
1.0
15
8
16
10
13
11
11
11
9
2-Nitrofluorene
Sodium azide
ICR-191
4-Nitroquinoline-N-oxide
29260
946 76 780111
80322
33940
aVehicle control = DMSO, 100 lL aliquot.
1870
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Table 3
Chromosomal aberration assay results without metabolic activation
Dose
(lg/mL)
# Cells scored
for aberrations
# Cells scored
for pp and er
% of
pp Cells
% of er
Cells
Judgement
(+/?)a
Percentages of cells showing structural chromosome aberrationsJudgement
(+/?)c
GapsSimple breakschtechre Mab Totalsb
?g
+g
Initial trial without metabolic activation
Controls
Negative:
Vehicle:
Positive:
SOE
RPMI 1640
DMSO
MMC
200
200
100
200
200
200
200
200
200
200
200
200
200
200
0.5
0.0
0.0
0.5
0.5
6.5
3.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.5
3.5
7.0
2.5
2.5
5.0
6.0
0.5
2.0
45.0
3.5
3.0
5.0
6.0
0.5
2.0
58.0
3.5
3.5
5.0
6.0
1.0
5.5
60.0
6.0
5.5
9.0
10.0
10.0d
1.00
353
504
1030
1470
?
?
?
+
?
23.0 +
?
?
?
?
0.5
Confirmatory trial without metabolic activation
Controls
Negative:
Vehicle:
Positive:
SOE
RPMI 1640
DMSO
MMC
200
200
100
200
300
300
300
200
200
200
200
200
200
200
0.0
0.0
0.0
1.5
0.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1.0
0.5
4.0
2.0
7.7
10.3
8.7
0.0
0.5
41.0
0.5
4.7
10.0
4.0
1.0
1.0
44.0
2.5
11.0
18.7
11.3
10.0d
0.300
150
225
300
450
0.5
35.0
0.5
4.3
9.7
4.0
?
?
?
?
?
8.0+
?
+
+
?
0.3
0.3
chte: chromatid exchange chre: chromosome exchange mab: multiple aberrations, greater than 4 aberrations pp: polyploidy er: endoreduplication.
aSignificantly greater in % polyploidy than the vehicle control, p 6 0.01.
b?g = # or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.
cSignificantly greater in ?g than the vehicle control, p 6 0.01.
dlL/mL RPMI 1640 = culture medium; DMSO = dimethylsulfoxide; MMC = Mitomycin C.
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1871

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Table 4
Chromosomal aberration assay results with metabolic activation
Dose (lg/mL) # Cells scored
for aberrations
# Cells scored
for pp and er
% of
pp Cells
% of er
cells
Judgement
(+/?)a
Percentages of cells showing structural chromosome
aberrations
Judgement
(+/?)c
Gaps Simple breaks chtechreMab Totalsb
?g
+g
Initial trial with metabolic activation
Controls
Negative:
Vehicle:
Positive:
SOE
RPMI 1640
DMSO
CP
200
200
100
200
200
200
200
200
200
200
200
200
200
200
0.0
0.0
0.0
0.0
0.5
1.5
7.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
1.0
1.0
7.0
2.0
2.0
2.5
4.5
1.0
1.5
37.0
2.5
1.5
3.0
4.5
1.0
1.5
47.0
2.5
1.5
3.0
4.5
2.0
2.5
52.0
4.5
3.5
5.0
8.5
10.0d
25.0
504
720
1030
1470
?
?
?
?
+
9.0 3.0+
?
?
?
?
0.5
Confirmatory trial with metabolic activation
Controls
Negative:
Vehicle:
Positive:
SOE
RPMI 1640
DMSO
CP
200
200
100
200
300
300
300
200
200
200
200
200
200
200
0.0
0.0
0.0
0.0
0.0
1.5
5.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
2.0
0.5
3.0
3.0
1.0
7.8
4.5
0.0
0.0
44.0
0.5
2.5
5.8
3.0
2.0
0.5
46.0
3.5
3.5
12.3
6.5
10.0d
25.0
300
600
1000
1500
?
?
?
?
+
42.0
0.5
2.5
5.3
3.0
8.0+
?
?
+
?
0.5
chte: chromatid exchange chre: chromosome exchange mab: multiple aberrations, greater than 4 aberrations pp: polyploidy er: endoreduplication.
aSignificantly greater in % polyploidy than the vehicle control, p 6 0.01.
b?g = # or % of cells with chromosome aberrations; +g = # or % of cells with chromosome aberrations + # or % of cells with gaps.
cSignificantly greater in ?g than the vehicle control, p 6 0.01.
dlL/mL RPMI 1640 = culture medium; DMSO = dimethylsulfoxide; CP = Cyclophosphamide.
1872
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3.2. Chromosomal aberrations assay
The highest concentration tested in the chromosomal
aberrations assay, 3000 lg/mL, was above the solubility
limit of the test article after dosing into culture medium.
In the initial chromosomal aberrations assay, the treatment
period was for ?3 h with and without metabolic activation,
and in the confirmatory chromosomal aberrations assay,
the treatment period was ?22 h without metabolic activa-
tion and ?3 h with metabolic activation. Cultures were
harvested ?22 h from the initiation of treatment. SOE
induced slight increases in structural and numerical chro-
mosomal aberrations (Tables 3 and 4).
3.3. In vivo mouse micronucleus assay
SOE did not induce signs of clinical toxicity, or evidence
of bone marrow cytotoxicity (i.e., no statistically significant
decrease in the PCE:NCE ratios), at doses up to 2000 mg/
kg/day. In addition, SOE did not induce any statistically
significant increases in micronucleated PCEs at any dose
examined (Table 5).
4. Discussion
Genotoxicity testing conducted on SOE followed the
standard 3-test battery recommended by the International
Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use
(ICH) and by FDA. Negative results in the bacterial
reverse mutation and mouse micronucleus assays demon-
strate that SOE is devoid of any significant genotoxic activ-
ity under the conditions of the assays. Weakly positive
results were observed in the human peripheral blood chro-
mosomal aberration assay at SOE concentrations with and
without precipitate. However, there was no clear or consis-
tent dose-related response observed. Therefore, the possi-
ble biological relevance of the positive response cannot
be assessed without additional work.
The existence of a reproducible, although weak, positive
chromosomal aberrations response in human peripheral
blood lymphocytes is of concern. Potential next steps
include, but are not limited to: a Syrian Hamster Embryo
Cell Transformation Assay, a mouse p53 transgenic assay,
an analysis of peripheral blood smears for micronuclei in
normochromatic erythrocytes from a repeat dose mouse
toxicity study, a DNA adduct analysis, or a rat or monkey
peripheral blood lymphocyte chromosomal aberrations
assay added on at the termination of a subchronic or
chronic repeat dose toxicity study. For SOE, the latter is
recommended. The possible assays to further investigate
the in vitro positive result observed with SOE are detailed
in the FDA Guidance for Industry and Review Staff
Document (January, 2006).
Use of SOE is presently expected to be safe, as antici-
pated intake is small compared to the doses administered
in the genotoxicity assays. The S. oblonga extract SOE,
after further toxicity research, may prove be a useful ingre-
dient in foodstuffs to help mitigate the blood glucose
response for people with diabetes.
References
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carcinogens and mutagens with the Salmonella/Mammalian-micro-
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Evans, H.J., 1962. Chromosomal aberrations produced by ionizing
radiation. International Review of Cytology 13, 221–321.
Evans, H.J., 1976. Cytological methods for detecting chemical mutagens.
In: Hollaender, A. (Ed.), Chemical Mutagens: Principles and Methods
for their Detection, vol. 4. Plenum Press, New York and London, pp.
1–29.
Galloway, S.M., Aardema, M.J., Ishidate Jr., M., Ivett, J.L., Kirkland,
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Table 5
In vivo Micronucleus assay results
Treatment Dosea
% Micronucleated PCEs mean
of 2000 per animal ± S.E.
Males
Ratio PCE:NCEbMean ± S.E.
Males
Controls
Water10 mL/kg/day
80 mg/kg
0.08 ± 0.02
3.03 ± 0.42c
0.62 ± 0.08
0.38 ± 0.07
Cyclophosphamide
SOE 500 mg/kg/day
1000 mg/kg/day
2000 mg/kg/day
0.09 ± 0.02
0.07 ± 0.02
0.05 ± 0.02
0.59 ± 0.10
0.59 ± 0.08
0.69 ± 0.10
aAnimals were treated for three consecutive days (water and SOE) or once (cyclophosphamide) and sacrificed 24 h after the last dose. Five males per
group were analyzed.
bAt least 2000 PCEs per animal were analyzed for the frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and
normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal.
cSignificantly greater than the corresponding vehicle control, p 6 0.01.
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