Volume 78, April 2015, Pages 122–129
Safety and toxicological evaluation of Meratrim®: An herbal formulation for weight management
- Under a Creative Commons license
Open Access
Highlights
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- Meratrim is an herbal formulation for weight management.
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- Safety of Meratrim was assessed in a battery of in vitro and in vivo toxicity studies.
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- Genotoxicity studies showed that Meratrim is non-mutagenic.
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- Acute oral LD50 of Meratrim was determined to be >5000 mg/kg in rats.
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- NOAEL for Meratrim was determined to be 1000 mg/kg/day in rats.
Abstract
Meratrim is a unique dietary ingredient consisting of extracts from Sphaeranthus indicus flower heads and Garcinia mangostana
fruit rind. Clinical studies have demonstrated that Meratrim is
effective and well-tolerated in weight management. Herein we assessed
the broad spectrum safety of Meratrim in a battery of in vitro
and animal toxicological studies including a sub-chronic repeated-dose
13-week oral toxicity study to determine the
no-observable-adverse-effect-level (NOAEL). The LD50 levels
of Meratrim in Sprague-Dawley (SD) rats, as determined by the acute oral
and dermal toxicity studies, were >5000 and >2000 mg/kg body
weight, respectively. The primary skin and eye irritation tests
classified Meratrim as non-irritating to the skin and mildly irritating
to the eye. Genotoxicity studies showed that Meratrim is non-mutagenic.
In the repeated-dose 13-week oral toxicity study, SD rats were orally
gavaged with Meratrim at 0, 250, 500 or 1000 mg/kg/day. No morbidity,
mortality, or significant adverse events were observed either during the
course of the study or on the 13th week. The NOAEL of Meratrim was
concluded to be 1000 mg/kg of body weight/day in male and female SD
rats. These results, combined with the tolerability of Meratrim in the
human clinical trials, demonstrate the broad spectrum safety of
Meratrim.
Keywords
- Meratrim;
- Acute oral and dermal toxicity;
- Primary skin and eye irritation;
- Ames' bacterial reverse mutation assay;
- Mammalian erythrocyte micronucleus test;
- Repeated dose 13-week oral toxicity study
1. Introduction
Obesity
has grown into a worldwide epidemic in recent years. Accumulating
evidence indicates that obesity is a risk factor for other diseases such
as type 2 diabetes, cardiovascular diseases and certain cancers
including colon cancer and breast cancer (Haslam, James, 2005 and Shaw et al, 2005). In fact, it is estimated that obesity may reduce life expectancy by 7 years at age 40 (Peeters et al., 2003).
Accordingly, the global socioeconomic burden for obesity and its
related disorders is tremendous and is expected to continue increasing.
In two clinical trials an herbal formulation (Meratrim) has been proven to be effective in weight management (Stern et al, 2013a and Stern et al, 2013b). Meratrim is a blend of extracts from the flower heads of Sphaeranthus indicus (S. indicus) and the fruit rinds of Garcinia mangostana (G. mangostana). The final formulation is standardized to contain at least 3% 7-hydroxyfrullanolide and 2% α-mangostin ( Stern et al., 2013b).
S. indicus,
a member of the aster and daisy family (Asteraceae), is widely used in
Ayurvedic system of medicine to treat various ailments including
diabetes, epilepsy, hepatopathy, and many others ( Galani et al., 2010).
A variety of secondary plant metabolites including sesquiterpenoids,
eudesmenolides, flavonoids, glycosides and essential oils have been
isolated from S. indicus ( Galani et al, 2010 and Ramachandran, 2013). G. mangostana,
commonly known as mangosteen, belongs to the family of Clusiaceae. The
trees are cultivated in the tropical rainforests of Southeast Asia ( Pedraza-Chaverri et al., 2008).
The pericarp (rind) of mangosteen-fruit is used as an Ayurvedic
medicine to treat inflammation, diarrhea, cholera and dysentery ( Al-Massarani et al, 2013, Balunas et al, 2008, Chen et al, 2008, Ee et al, 2006, Gopalakrishnan et al, 1980 and Obolskiy et al, 2009). Preclinical studies suggest that mangosteen pericarp extracts possess a wide range of biological activities ( Al-Massarani et al, 2013, Balunas et al, 2008, Chen et al, 2008, Ee et al, 2006, Gopalakrishnan et al, 1980, Kosem et al, 2013, Obolskiy et al, 2009, Pedraza-Chaverri et al, 2008, Sundaram et al, 1983, Tewtrakul et al, 2009 and Yoshikawa et al, 1994).
The objective of the present studies was to determine the safety profile of Meratrim by conducting a battery of in vitro and in vivo toxicity tests including a 13-week sub-chronic oral toxicity study.
2. Materials and methods
2.1. Test substance
Meratrim is an herbal blend containing the extracts of the flower heads of S. indicus and the fruit rinds of G. mangostana. Briefly, S. indicus
flower heads were pulverized and extracted with 6 volumes of methanol,
and then concentrated under vacuum followed by further extraction using
ethyl acetate. The resultant product from S. indicus extraction was a thick paste. Separately, G. mangostana fruit rinds were pulverized and extracted with 6 volumes of 80:20 ratio of methanol to water. The G. mangostana extract thus obtained was concentrated, washed with water, and then dried. The resulting flakes were then milled. The S. indicus paste and G. mangostana
powder extract prepared above were blended together in a 3:1 extract
ratio along with approximately 55% excipients to obtain Meratrim. The S. indicus extract contains a minimum of 12% 7-hydroxyfrullanolide while the G. mangostana
extract contains a minimum of 18% α-mangostin prior to blending to
ensure that the final blend contains at least 3% 7-hydroxyfrullanolide
and 2% α-mangostin ( Stern et al., 2013b).
In addition to 7-hydroxyfrullanolide and α-mangostin, the final blend
contains other minor constituents that include sphaeranthanolide and
frullanolide derived from S. indicus, and γ-mangostin, garcinone C and garcinone D derived from G. mangostana
(each at levels below 0.5%). The test substance was stored at room
temperature until use, and was provided by InterHealth Nutraceuticals
(Benicia, CA) under a license agreement with Laila Nutraceuticals
(Vijayawada, India).
2.2. Animals and treatment
Acute
oral toxicity, acute dermal toxicity, primary dermal toxicity and
primary eye irritation were conducted at Laila Impex Research Centre
(Vijayawada, India). A repeated dose 13-week oral toxicity study was
conducted at a certified GLP facility (Bioneeds, Bangalore, India). The
mammalian chromosome aberration and erythrocyte micronucleus tests were
conducted at Shriram Institute for Industrial Research (Delhi, India).
The Ames reverse mutation assay was performed at RCC Laboratories India
Pvt. Ltd. (Hyderabad, India). All tests complied with Good Laboratory
Practice (GLP) regulations as defined by 21CFR58 (US Food and Drug
Administration) and the specified testing procedures set by the
Organisation for Economic Co-operation and Development (OECD) guidelines
for the testing of chemicals. All animals used for toxicological
assessments were cared for in accordance with the Guide for the Care and
Use of Laboratory Animals DHEW (NIH). Animals were allowed free access
to standard feed and water. The animals were acclimated to facility
conditions 7 or 21 days prior to use. Animal rooms were kept at 22 to
25 °C, 40–70% humidity, and a 12 h light–dark cycle.
2.3. Acute oral toxicity
The
single dose acute oral toxicity evaluation (up-and-down procedure) was
conducted in rats. Three 10-week old Sprague-Dawley (SD) nulliparous and
non-pregnant female rats (180–195 g) were used for this study.
Meratrim
was administered in a single dose of 5000 mg/kg using an infant feeding
tube attached to a syringe. Following administration, feed was replaced
approximately 4 h after dosing. On the day of dosing, all the animals
were observed for mortality, signs of gross toxicity, and behavioral
changes for several hours following dosing then at least once daily for
14 days. Individual rat body weights were recorded before dosing (Day 0)
then at weekly intervals. Animals were euthanized using ether at the
end of 14 days. Gross necropsy was performed on all animals.
2.4. Acute dermal toxicity
Ten
healthy young adult SD rats (8–10 weeks old) were used in this test.
Individual doses of the Meratrim were calculated based on the initial
body weights obtained prior to dosing at 2000 mg/kg of body weight (bw).
On the day prior to application, the hair was removed by clipping the
dorsal area and the trunk. After clipping and prior to application, the
animals were examined for health, weighed (initial) and the skin checked
for any abnormalities. Meratrim was moistened with distilled water to
achieve a dry paste by preparing a 50% w/w mixture. Meratrim (2000 mg/kg
bw) was then applied to a 2 in × 3 in, 4-ply gauze pad and placed on
the dorsal area of the animal (approximately 10% of the body surface).
The gauze pad and the entire trunk of each animal were wrapped with
3-inch Durapore tape to avoid dislocation of the pad and to minimize
loss of the test substance. The rats were then returned to their
designated cages. The day of application was considered day 0 of the
study. After 24 h of exposure to the test substance, the pads were
removed and the test sites were gently cleansed of any residual test
substance.
The body weight of
each animal was recorded prior to test substance application and again
on days 7 and 14. Animals were observed for mortality, signs of gross
toxicity, and behavioral changes for several hours after application and
at least once daily for 14 days. Observations included evaluation of
skin and fur, eyes and mucous membranes, respiratory, circulatory, plus
autonomic and central nervous systems, somatomotor activity, and
behavior. Attention was paid to the occurrence of tremors, convulsions,
salivation, diarrhea and coma. Rats were euthanized using anesthetic
ether on day 14. Gross necropsies were performed on all animals. Tissues
and organs of the thoracic and abdominal cavities were examined.
2.5. Primary dermal irritation study in rabbits
Three
New Zealand albino young adult rabbits (two male and one nulliparous,
non-pregnant female) were obtained from the animal facility at the Laila
Impex R&D Centre for this test.
The
route of Meratrim administration was through a direct application of
test substance to clipped intact skin. On the day before application,
hair was removed by clipping the dorsal and the trunk area. On the day
of dosing, but prior to application, the animals were examined for
health and the skin abnormalities. No pre-existing skin irritations were
observed. To apply material, 500 mg of the test substance was moistened
with water then applied to a small area of the clipped skin
(approximately 6 cm2) and covered with a gauze patch. The
patch was loosely held in contact with the skin by means of a suitable
semi-occlusive dressing for the duration of the exposure period. Access
by the animal to the patch and ingestion or inhalation of Meratrim was
prevented.
Individual dose sites were scored according to the Draize scoring system (Draize, 1944)
at approximately 1, 24, 48, and 72 h after removal of Meratrim patch.
The classification of irritancy was obtained by adding the average
erythema and edema scores for 1, 24, 48, and 72 h scoring intervals and
dividing by the number of evaluation intervals. The resulting Primary
Dermal Irritation Index (PDII) was classified according to the
descriptive rating (Sreejayan et al., 2010).
Animals were also observed for signs of gross toxicity and behavioral
changes at least once daily during the test period. Observations
included gross evaluation of skin and fur, eyes and mucous membranes,
respiratory, circulatory, autonomic and central nervous systems,
somatomotor activity and behavior pattern. Occurrence of tremors,
convulsions, salivation, diarrhea, and coma was closely monitored.
2.6. Primary eye irritation study in rabbits
Three
healthy young adult New Zealand albino (2 male and 1 nulliparous,
non-pregnant female) rabbits, without pre-existing ocular irritation,
were selected from the animal facility of Laila Impex R&D Centre for
this study.
The route of
Meratrim administration was direct conjunctival instillation, standard
for assessment of local ocular irritative potential. Prior to
instillation, both eyes of each animal were examined for gross
abnormalities according to the Draize scale for scoring eye lesions (Draize, 1944 and Sreejayan et al, 2010).
Meratrim was used after thorough grinding using mortar and pestle.
Meratrim (100 mg) was instilled into the conjunctival sac of the right
(test) eye of each rabbit by gently pulling the lower lid away from the
eyeball. The upper and lower lids were then gently held together for
about few seconds before releasing to minimize loss of the test
material. The left (control) eye of each animal remained untreated.
Following
treatment, ocular irritation was evaluated macroscopically using a
high-intensity white light (Maglite, Ontario, CA) in accordance with Draize (1944)
at 1, 24, 48, and 72 h and daily from 4 to 10 days post-instillation.
Individual eye irritation scores were recorded for each animal.
Classification of eye irritation scores for all rabbits was determined
at each time point with the maximum mean total score (MMTS) by the
descriptive primary eye irritation scores system of Kay and Calandra (1962). Ocular lesions were scored according to the Draize scale for scoring eye lesions (Draize, 1944).
The average score for all rabbits at each scoring period was calculated
to aid in data interpretation. The rabbits were also observed at least
once daily for signs of gross toxicity and behavioral changes during the
test period. Observations included gross evaluation of skin and fur,
eyes and mucous membranes, respiratory, circulatory, plus autonomic and
central nervous systems, somatomotor activity, and changes in behavior
patterns. Occurrence of tremors, convulsions, salivation, diarrhea and
coma was closely monitored.
2.7. Bacterial reverse mutation assay
The Salmonella typhimurium
reverse mutation test was conducted to determine Meratrim's potential
to induce reverse mutation at selected histidine loci in five tester
strains of S. typhimurium viz. TA 98, TA 100, TA 102, TA 1535
and TA 1537 (Xenometrix GmbH, Allschwil, Switzerland) in the presence
and absence of metabolic activation system ( Ames et al, 1975 and Maron, Ames, 1983).
Suspensions of bacterial cells were exposed to Meratrim in triplicate
at concentrations of 313, 625, 1250, 2500 and 5000 µg/plate. The
suspensions were mixed with an overlay agar and plated immediately onto
minimal medium. After 48 h incubation at 37 ± 2 °C, revertant colonies
were counted manually and compared to the number of spontaneous
revertant colonies on vehicle and control plates.
2.8. Mammalian erythrocyte micronucleus test
Thirty
male and thirty female healthy young (8–12 weeks old) Swiss albino mice
from the animal facility of Shriram Institute for Industrial Research
(Delhi, India) were randomized into three groups (n = 20/group; 10/sex).
After randomization, the animals were housed in polypropylene cages
with each cage containing five animals per sex per group. Corn oil was
used as the vehicle for oral gavage of Meratrim. Oral administration was
chosen as it is the recommended route for human supplementation.
After
acclimation, mice were orally administered either corn oil (negative
group), Meratrim (test group) at 2000 mg/kg bw (20% corn oil
suspension), or cyclophosphamide (positive control) at 40 mg/kg bw. The
animals were sacrificed by cervical dislocation at 24 (n = 10/group;
5/sex) and 48 (n = 10/group; 5/sex) hours after dose administration. Two
hundred erythrocytes in the bone marrow cells of each animal were used
to score the total number of mature and immature erythrocytes. The
number of micronuclei per 2000 immature erythrocytes was also recorded.
The number of immature erythrocytes and mature erythrocytes, percent of
immature erythrocytes, and the number of micronuclei in immature
erythrocytes were all analyzed by ANOVA. A p-value <0.05 was
considered statistically significant.
