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Regulatory Toxicology and Pharmacology 90 (2017) 318e327

Contents lists available at ScienceDirect

Regulatory Toxicology and Pharmacology


journal homepage: www.elsevier.com/locate/yrtph

Citrus aurantium (bitter orange) extract: Safety assessment by acute


and 14-day oral toxicity studies in rats and the Ames Test for
mutagenicity
N.S. Deshmukh a, S.J. Stohs b, *, C.C. Magar a, S.B. Kadam a
a
INTOX Private LTD., 375, Urawade, Tal. Mulshi, Maharashtra, India
b
Creighton University Medical Center, Omaha, NE 68178, USA

a r t i c l e i n f o a b s t r a c t

Article history: The primary active constituent in bitter orange extract (BOE) is p-synephrine. This study assessed the
Received 10 June 2017 safety of a BOE standardized to 50% p-synephrine following short-term exposure to rats and by the Ames
Received in revised form Test. Following 5000 mg/kg of the extract orally to female rats all animals survived. Administration at
21 September 2017
2000 mg/kg to female rats for four days yielded no signs of toxicity. Five male and ve female rats were
Accepted 27 September 2017
Available online 28 September 2017
administered the BOE at 0, 250, 500, 1000 and 2000 mg/kg/day for 14 days. No signicant effects were
observed at any dose with respect to body weights, food intake, absolute and relative organ weights,
hematology, clinical chemistry, and pathology. Two male rats died after 2000 mg/kg with gastrointestinal
Keywords:
Citrus aurantium
impaction at necropsy. During week two of 1000 mg/kg and 2000 mg/kg/day, rats exhibited transient
Bitter orange signs of repetitive burrowing of heads in the bedding material (hypoactivity) for about 15 and 45 min,
p-synephrine respectively. The no-observed-effect-level (NOEL) was 500 mg/kg/day. The mutagenic potential was
Safety assessed at and up to the limit dose of 5000 mg/plate in a Salmonella typhimurium reverse mutation
LD50 (Ames) test, performed in duplicate as a pre-incubation assay in the presence and absence of metabolic
Ames Test activation (S9). The BOE did not induce an increase in the frequency of revertant colonies at any dose in
Salmonella typhimurium the ve tester strains, and was therefore non-mutagenic.
Reverse mutation assay
2017 Elsevier Inc. All rights reserved.

1. Introduction or [R-()] enantiomeric form, whereas synthetic p-synephrine is a


racemic mixture of the l- and d-enantiomeric forms and contains
Standardized aqueous-alcoholic extracts of the immature fruits only about half of the pharmacological activity of the naturally
of Citrus aurantium (bitter orange) are widely consumed in dietary occurring p-synephrine because the d-enantiomer exhibits about
supplements for appetite control, weight management, sports one-hundredth the adrenergic receptor binding activity of the l-
performance and energy, and bitter orange products are exten- enantiomer (Stohs and Preuss, 2011b; Stohs et al., 2011b).
sively consumed in the form of foods as juices and marmalades Relatively few animal studies have assessed the safety of bitter
(Stohs and Shara, 2013; Ratamess et al., 2015; Stohs, 2017). The orange extracts, particularly at high doses, primarily because bitter
primary active ingredient in bitter orange extract is the phenyl- orange has been used extensively in Traditional Chinese Medicine
ethylamine protoalkaloid p-synephrine which comprises about 90% (Chen and Chen, 2004; Fang et al., 2009; Stohs and Shara, 2013) and
or more of the total protoalkaloids (Pellati and Benvenuti, 2007), is used in food products as marmalades, avoring agents and juices
and to which products as the patented Advantra Z are standard- (Stohs and Preuss, 2011a; Stohs, 2017). Arbo et al. (2008) examined
ized (Stohs and Preuss, 2011a; Stohs et al., 2011a, 2012; Stohs and the acute toxicity of a bitter orange extract and p-synephrine in
Shara, 2013). mice. The acute oral administration of 300e2000 mg/kg of p-syn-
p-Synephrine has a hydroxyl group in the para position on the ephrine produced reduced locomotion, piloerection, salivation,
benzene ring of the molecule (Fig. 1). p-Synephrine exists in the l- gasping, and exophthalmia. All effects were reversible and resolved
in 3e4 h. The authors concluded that these effects were due to
adrenergic agonist activity. p-Synephrine is a poor adrenergic
* Corresponding author.
agonist, as a consequence high doses are required to produce these
E-mail address: sid.stohs9@gmail.com (S.J. Stohs). effects which may result from direct and/or indirect adrenergic

https://doi.org/10.1016/j.yrtph.2017.09.027
0273-2300/ 2017 Elsevier Inc. All rights reserved.
N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327 319

Test) using ve tester strains of Salmonella typhimurium. In addi-


tion, the study aimed at identifying markers for any toxicity which
could be assessed in future studies on this extract.

2. Materials and methods

2.1. Test material


Fig. 1. Structure of p-Synephrine.
Bitter orange extract (Advantra Z) standardized to 50.1% p-
synephrine was provided by Novel Ingredients, East Hanover, NJ
activity (Stohs et al., 2011b; Stohs and Badmaev, 2016).
07936 USA. The dried, powdered immature fruits are soaked in
In a study in mice, bitter orange extract (7.5% p-synephrine) at
water, and 0.5% HCl is added to convert p-synephrine to the soluble
doses of 400, 2000 or 4000 mg/kg (corresponding to 30, 150 and
hydrochloride. The aqueous acidied fraction is collected by
300 mg p-synephrine/kg) or p-synephrine at 30 mg or 300 mg/kg
ltration and absorbed onto a cation exchange resin. The p-syn-
were administered orally per day for 28 days (Arbo et al., 2009). A
ephrine binds to the resin, and impurities are removed by washing
reduction in body weight gain was observed at both doses of p-
the resin with puried water. The p-synephrine base is eluted using
synephrine relative to controls. No adverse effects were observed
dilute 5% aqueous ammonia, and the resulting solution is concen-
regarding organ weights, biochemical parameters, blood pressure
trated by evaporation which results in a crude p-synephrine
or heart rate in the treated mice at any of the doses.
product. This product is further puried by crystallization using
In addition, both doses (30 and 300 mg/kg) of p-synephrine and
food grade ethanol.
the high dose (4000 mg/kg) of the bitter orange extract which
The amount of p-synephrine (50.1%) present in the extract was
contained 300 mg p-synephrine resulted in increases in the anti-
conrmed by an independent testing laboratory (Intertek Labora-
oxidant and tissue protectant reduced glutathione (GSH), while the
tories, Champaign, IL) by HPLC with UV detection using a Hewlett
bitter orange extract decreased malondialdehyde content (an in-
Packard 1100 system with an Agilent 1200 gradient series, agreeing
dicator of lipid peroxidation and lipid damage), and p-synephrine
with the original HPLC analytical results of the manufacturer. Further
increased catalase which neutralizes hydrogen peroxide. The two
chemical analysis indicated that the remainder of the extract was
doses of p-synephrine as well as 400 mg/kg and 2000 mg/kg of the
composed of approximately 45% carbohydrate and 5% ash (minerals)
bitter orange extract which contained 30 and 150 mg of p-syn-
(Eurons Scientic, Inc., Petaluma, CA USA). The same batch of test
ephrine, respectively, also signicantly inhibited glutathione
material was used throughout the study. The extract (test item) was
peroxidase activity (Arbo et al., 2009). The results indicated a
suspended in analytical grade water which contained 0.50% w/v
benecial effect with respect to weight loss without adverse effects
carboxymethyl cellulose as a suspending agent to prepare dosing
while also providing an antioxidant and tissue protective effect.
formulations with the desired test concentrations. The visually ho-
Several animal studies have been conducted by the National
mogenous suspension was prepared freshly daily, shortly prior to
Center for Toxicological Research in conjunction with the US FDA
dose administration, and kept under constant stirring throughout
regarding the safety of bitter orange extract and p-synephrine
dosing administration by gavage. This is a standard practice for non-
(Hansen et al., 2011, 2012; 2013). In a study which examined the
clinical studies, and ensures homogeneity of distribution of sus-
developmental toxicity of Citrus aurantium in rats, the authors
pended test materials. Control animals received the vehicle
concluded that doses of up to 100 mg p-synephrine/kg body weight
composed of 0.50% carboxymethyl cellulose in water.
did not induce developmental toxicity (Hansen et al., 2011). At this
dose there were no adverse effects with respect to embryo lethality,
2.2. Animals
fetal weight, or incidence of gross, skeletal or visceral abnormal-
ities. No developmental effects were observed under the dosing
Male and female Sprague Dawley rats (NTac:SD) were obtained
conditions used in this study.
from Taconic Biosciences, Inc., USA through its vendor Vivo Bio Tech
The physiological effects were examined after administering p-
Ltd., Telangana, India. Female rats were nulliparous and non-
synephrine in the form of bitter orange extract as well as isolated p-
pregnant. The animals were subjected to veterinary examination
synephrine to rats for 28 days at doses of up to 50 mg/kg with and
prior to experimentation to ensure that the rats were in good
without caffeine at 25 mg/kg (Hansen et al., 2012). Minimal, clini-
health. Only female animals at 11 weeks of age were used for the
cally insignicant effects were produced by these high doses of p-
acute toxicity study while the rats used for the 14-day study were
synephrine with respect to heart rate and blood pressure. As ex-
between the ages of 6e7 weeks at the beginning of treatment.
pected, caffeine alone and in combination with p-synephrine pro-
Animals were housed singly for the acute study and for the 14-day
duced more pronounced but small increases in heart rate and blood
study they were housed in groups of two or three of similar sex per
pressure.
cage in sterilized, solid bottom, suspended, polypropylene cages
The potential cardiovascular effects of bitter orange extract and
with stainless steel grill tops and clean and sterilized corn-cob
p-synephrine were also examined in exercised rats given up to
bedding.
50 mg/kg p-synephrine in the presence and absence of 25 mg/kg
The animal facility was supplied with 100% fresh, ltered air
caffeine for 28 days (Hansen et al., 2013). Small increases in heart
with 10e15 air changes per hour. The room was maintained at a
rate and body temperature were reported due to caffeine, while p-
temperature between 19 and 25  C with the relative humidity be-
synephrine exhibited small, clinically insignicant effects on blood
tween 30 and 70%, and a 12 h light/dark cycle. The animals were fed
pressure at 50 mg/kg.
ad libitum Altromin brand extruded pelleted rat chow manufac-
The purpose of the current study was the assessment of the
tured by M/s Altromin Spezialfutter GmbH & Co. KG, Germany.
safety of a bitter orange extract standardized to 50% p-synephrine
Drinking water which had been passed through an Aquaguard
administered to rats via oral gavage as an acute dose and also when
water lter and subjected to ultra violet irradiation was provided ad
administered daily for 14 days at doses up to 2000 mg/kg (1000 mg/
libitum. The animals were allowed to acclimate for at least ve days
kg p-synephrine). The study also assessed the potential mutage-
prior to experimentation, assigned an individual tail number,
nicity of the extract in a bacterial reverse mutation assay (Ames
weighed and randomized.
320 N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327

2.3. Dosing for acute and 14-day repeated dose studies of rm steam extruded food pellets which are subject to minimal
spillage, the food consumption was not corrected for spillage.
The acute oral toxicity study was performed as per OECD Body weights of all animals were recorded individually on the
Guideline No. 425 Acute Oral Toxicity-Up and Down Procedure as a rst day of treatment (day-1), on days 4, 8, 11 and 14, and at nec-
limit test. Three female rats, fasted overnight, were dosed in a ropsy on day-15 (fasting body weights). At terminal necropsy, the
step-wise manner with 5000 mg/kg body weight of the 50% p- following organs from all surviving animals were carefully
synephrine containing bitter orange extract, and observed for 24 h. dissected and trimmed to remove fat and other contiguous tissue,
The software AOT425StatPgm, issued by US-EPA for performing the and immediately weighed: liver, heart, spleen, lungs, kidneys, ad-
test and calculating the results, was used for the test. Initially, a renals and brain. The relative organ weights as a percent of nec-
single animal was dosed with 5000 mg/kg body weight. When the ropsy body weights were calculated.
dosed animal survived, two additional animals were dosed On completion of the 14 days of treatment, all the surviving rats
sequentially so that the three animals were tested at minimum of were sacriced by exsanguination from the abdominal aorta under
48-h intervals. CO2 asphyxiation and subjected to a gross necropsy which included
This was followed by an exploratory repeated dose experiment the examination of the external surfaces, orices, cranial, thoracic
in which three female Sprague Dawley rats were daily given the and abdominal cavities, and their contents. Gross necropsy exam-
bitter orange extract at a dose of 2000 mg/kg body weight by oral inations were also carried out on all animals which died during the
gavage for four consecutive days. Based on the results of these study. The following tissues were preserved in 10% buffered
studies, groups of ve male and ve female rats were administered formalin, imbedded in parafn, sectioned, stained with hematox-
the 50% p-synephrine-containing bitter orange extract by oral ylin and eosin, and subjected to histopathological examination:
gavage for 14 days at doses of 250, 500, 1000 or 2000 mg/kg/day, lungs, heart kidneys, adrenals, liver, brain, duodenum, jejunum,
with concurrent vehicle control groups of ve male and ve female stomach, pancreas, spleen, and thyroid gland.
rats. The study initially began at doses of 500, 1000 and 2000 mg/ Blood samples were collected on day-15 prior to necropsy from
kg. To ensure a dose without effects, the dose of 250 mg/kg was all surviving animals. The animals were fasted overnight prior to
added at a later time due to behavioral effects beginning on day ve sampling. Sampling of blood was conducted under light CO2
following dosing with 1000 mg/kg. The dosage volumes were 5 ml/ asphyxiation, through the orbital sinus of the animals and the
kg for all dose levels except at the high dose of 2000 mg/kg and the samples were collected separately in tubes containing EDTA
vehicle control group, where it was 10 ml/kg, administered as a (dipotassium salt) for hematology, and heparin for clinical chem-
single daily gavage. However, from day 6 onwards, the control and istry, as anticoagulants.
high dose group rats received the test item in two divided doses The following hematological parameters were determined on
each of 5 ml/kg volume that were gavaged about 2e3 h apart. blood samples using an Abbott Cell Dyn 3700 Hematology Analyser
(Abbott Park, IL 60,064, USA): hemoglobin (Hb), hematocrit (PCV),
2.4. Assessments for acute and 14-day repeated dose studies total erythrocyte count (Total RBC), total leukocyte count (Total
WBC), total platelet count (platelets) and differential leukocyte
Following the acute administration of the extract at 5000 mg/kg, (WBC) count. Leucocytes were differentiated as neutrophils (N),
the animals were observed for death or abnormal clinical signs over lymphocytes (L), eosinophils (E), monocytes (M) and basophils (B).
a period of 14 days and were then terminated. Their body weights The following erythrocyte indices were calculated: mean corpus-
were recorded at one day prior to dosing (day 0), on the day of cular volume (MCV), mean corpuscular hemoglobin (MCH), and
dosing (day-1, fasting body weight), on day-7, and at termination mean corpuscular hemoglobin concentration (MCHC).
on day-15 when they were subjected to complete necropsy. The Plasma samples were analyzed individually for clinical chem-
rats in the exploratory four-day repeated dose experiment were istry parameters using a Dimension Xpand Plus Clinical Chemistry
subjected to cage-side clinical examinations and observed for System (Siemens Healthcare Diagnostics Inc. Newark, U.S.A.). The
survival. analyses were performed using commercially available diagnostic
Throughout the 14-day repeated dose study, all animals were kits manufactured by Siemens Healthcare Diagnostics Inc. The pa-
checked twice daily for death or moribundity. All rats that were rameters that were measured included: alanine amino-
found dead in the cage were subjected to detailed necropsy ex- transaminase (ALT), aspartate aminotransaminase (AST), alkaline
amination and tissue samples were preserved in 10% neutral buff- phosphatase (ALP), glucose, albumin, total protein, creatinine,
ered formalin. All signs of ill health, together with any behavioral globulin (calculated), albumin/globulin ratio (calculated), and urea
changes or reaction to treatment were recorded on clinical history nitrogen (BUN).
sheets for individual animals.
After dosing, the animals were subjected to general cage-side 2.5. Ames Test
clinical examination to determine the peak period and nature of
any effects. The rats were subjected to detailed clinical examina- The bacterial Salmonella typhimurium reverse mutation assay
tions weekly during the treatment period which were made (Ames Test) was performed by the pre-incubation method using
outside the home cage. The signs that were checked included, but the tester strains TA1535, TA97a, TA98, TA100 and TA102, each of
were not be limited to, changes in skin, fur, eyes, mucous mem- which contains a different kind of mutation in the histidine operon,
branes, occurrence of secretions and excretions and autonomic in the presence and absence of a rat liver S9 metabolic activation
activity such as lacrimation, piloerection, pupil size, and unusual system. The assay was conducted according to OECD guideline No.
respiratory pattern. Changes in gait, posture and response to 471 on conduct of Bacterial Reverse Mutation Test. The study also
handling as well as the presence of clonic or tonic movements, and followed in general, the recommendations on conduct and in-
sterotypies or bizarre behavior were recorded, if any. terpretations of this test made by the International Council on
The quantity of food consumed by rats in each cage was recor- Harmonization of Technical Requirements for Registration of
ded weekly. Food intake per rat per day was calculated using the Pharmaceuticals for Human Use S2 (R21).
amount of food offered to each cage during the period of mea- The bacterial strains were grown in oxoid nutrient broth No. 2
surement, left in each cage at end of this period, and the number of and pre-incubated at 37  C for about 20 min in a reaction mixture
rats in each cage. Because the chow offered to animals is in the form with concentrations of the bitter orange extract corresponding to
N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327 321

the test doses of 5000, 1500, 500, 150 or 50 mg/plate. Liver post- dose of the extract daily was found dead in the cage on day 5. A
mitochondrial S9 fraction, induced in rats by phenobarbital and male rat treated daily with 2000 mg/kg of the extract were found
b-naphthoavone, was used as the metabolic activation system. dead in the cage on day-9 of the study, while another male rat was
The exposed bacterial strains were plated in triplicate onto minimal found dead in the cage on day-12 of the study. No remarkable
glucose agar medium supplemented with L-histidine. The plates necropsy ndings were noted. All other male and female rats sur-
were incubated at 37  C for about 68 h after which the histidine vived the 14 day treatment until their termination on day-15.
revertant colonies were counted and their frequencies were Because rats died at the 2000 mg/kg dose of the extract during
compared with that in vehicle control groups. Sodium azide, 3- the treatment, additional groups of ve male and ve female rats
methylmethane sulfonate, ICR 191, and 4-nitroquinoline-N-oxide were also included in the study and were treated at the lower daily
were used as positive mutagenic controls without metabolic acti- dose of 250 mg/kg for 14 days. No abnormal clinical signs and no
vation while 2-aminouorene, 2-aminoanthracene and danthrone, incidence of mortality were observed in either male or female rats
were used as positive mutagenic controls with metabolic activa- treated with the extract at the doses of 250 and 500 mg/kg body
tion. In order to conrm the reproducibility of the results, the entire weight (Table 1). However, treatment at doses of 1000 mg/kg and
study was carried out twice. 2000 mg/kg body weight resulted in transient signs of discomfort
The mean number of histidine revertant colonies for all the in rats immediately after gavage administration, as described
treatment groups was compared with the number of revertants in below. Although the male and female rats treated with the extract
the respective vehicle control group. The mutagenic activity of the at 1000 mg/kg body weight did not exhibit any abnormalities
bitter orange extract was assessed by applying the following during rst week of the treatment, from 9th day following dose
criteria. The bitter orange extract was considered to be positive administration, they exhibited signs of repetitive burrowing of their
(mutagenic) if it induced a concentration dependent increase and/ heads in the bedding material and staying hypoactive. This
or an increase at one or more concentrations, in revertant fre- behavior was observed for about 15e30 min after dose
quency which was at least 2-fold (3-fold for TA1535) of that administration.
observed in the corresponding concurrent vehicle control. If the Male and female rats that received 2000 mg/kg of the extract
results for the bitter orange extract did not meet the above criteria, per day did not exhibit any clinical abnormalities for the initial 5
it was considered non-mutagenic in this test. days of treatment. However, from the 6th day of dose administra-
tion, they exhibited similar signs of repetitive burrowing of their
2.6. Statistical analysis heads in the bedding material and remaining hypoactive over a
slightly longer period of 30e45 min after dose administration.
Statistical analyses for the 14-day repeated dose study were With respect to food consumption, the food intake values were
performed using Statistical Package for the Social Sciences (vali- comparable to the control groups during the rst week of the study.
dated SPSS Version 23.0). Body weight, organ weight, hematology However, during the second week of the study male rats treated
and clinical chemistry data of the different groups were subjected with 2000 mg/kg per day of the bitter orange extract consumed
to Levene's test for homogeneity, and then to one-way analysis of approximately 8% less chow than the control group, while the fe-
variance (ANOVA). When an F value was signicant, Dunnett's male rats treated with 1000 mg and 2000 mg/kg per day consumed
multiple comparison of means of treated groups with control mean respectively 13% and 5% less chow than the corresponding control
was performed individually. The variance was evaluated at a 5% group.
level of signicance. The group mean values of body weights of the male (Fig. 2) and
female (Fig. 3) rats treated with 250, 500, 1000 and 2000 mg/kg of
3. Results the bitter orange extract per day for 14 days did not differ signi-
cantly from those of the control groups during the study (Figs. 2 and
Single doses of 5000 mg/kg body weight of the 50% p-syn- 3). As previously noted, the starting weights of the groups of rats
ephrine bitter orange extract given to female rats during the acute dosed with 250 mg/kg were higher since the animals were started
toxicity study did not cause the death of any of the rats. However, one week after the other dosing groups. No alterations or adverse
the test item induced signs of hypoactivity in all the rats with onset effects in growth were observed for animals in any of the dosing
at 30 min following their treatment and disappearing by 3rd day. groups.
No other overt signs of toxicity were evident. The body weight Daily treatment with the bitter orange extract at and up to the
change by treated rats was not adversely affected during the 14 dose of 2000 mg/kg body weight did not induce any signicant
days observation period and no gross pathological alterations were alterations in the absolute weights of liver, heart, spleen, kidneys,
encountered in tissues/organs of any of the female rats in this study testes and adrenals at the termination of the 14-day treatment
as evidenced during necropsy conducted at termination of the period. Small increases in relative liver weights were observed in
study. Therefore, the acute oral LD50 value of the test item was male rats at the 2000 mg/kg dose and at the 1000 mg/kg dose in
estimated to be greater than 5000 mg/kg body weight. In the female rats. All other values of relative weights of organs of the
subsequent exploratory repeated dose experiment in which three treated rats did not differ signicantly from those of the respective
female Sprague Dawley rats were treated orally by gavage with the vehicle control group values at all doses (P > 0.05). The weights of
bitter orange extract at a dose of 2000 mg/kg body weight per day the organs relative to body weights are presented in Table 2.
for four consecutive days, the treatment was well tolerated, and the Necropsy examinations of the two male rats treated with
rats did not demonstrate any overt signs of toxicity. 2000 mg/kg body weight of the bitter orange extract which died on
Based on these results, groups of ve male and ve female rats days 9 and 12 during the study revealed that their stomachs were
were initially treated with 0 (control vehicle), 500, 1000 and impacted with ingesta and residues of the administered test item.
2000 mg/kg per day of the 50% p-synephrine bitter orange extract Slight bleeding from the nostrils was observed for both male ani-
for 14 days. The extract at and up to the dose of 1000 mg/kg body mals, while slight scrotal ulceration was noted for one animal, and
weight did not have any effect on the survival of the male and fe- dark patches on the lungs were noted for one of the two male
male rats in the study. Daily treatment with the extract at a dose of animals. Necropsy of the female rat from the high dose group which
2000 mg/kg body weight resulted in the deaths of two male rats was found dead on day-5 of treatment only revealed an intestine
and one female rat. One female rat treated with the 2000 mg/kg that contained dark colored contents that appeared to be residue of
322 N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327

Table 1
Summary of clinical signs during treatment period (days 1e15).

Group G1 G5 G2 G3 G4

Vehicle Control Citrus aurantium 50%

Dose (mg/kg) 0 250 500 1000 2000


No. of Animals 5 5 5 5 5
per Group per Sex
Clinical Finding Incidence
(No. of animals with ndings)
Male Rats
No abnormality detected 5 5 5 e e
Transient signs of discomforta post-gavage, from day 9 e e 5 e
Transient signs of discomforta post-gavage, from day 6 e e 5
Female Rats
No abnormality detected 5 5 5 e e
Transient signs of discomforta post-gavage, from day 9 e e 5 e
Transient signs of discomforta post-gavage, from day 6 e e 5

Vehicle: Analytical Grade Water with 0.5% w/v of CM.


a
Transient signs of discomfort exhibited by the rats were signs of repetitive burrowing of their heads in the bedding material (corn-cob) and staying hypoactive during such
period; such behavior lasted for about 15e30 min post-gavage in G3 rats and for 30e45 min in G4 rats.

Fig. 2. Body weights of male rats.

Fig. 3. Body weights of female rats.

the test item. No other ndings were observed. It may be noted that 10 ml/kg body weight but the male rats, from day 6 onwards, had
the female rat died after being treated at the dosage volume of received the test item in two divided doses each of 5 ml/kg volume
N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327 323

Table 2
Summary of relative organ weights (%) at termination of treatment period (day 15).

MALE RATS

Group Dose mg/kg/bw/day Fasting Body Weight Kidneys Liver Adrenals Testes Spleen Heart Lungs Brain

G1 0 Mean 274.20 0.96 3.64 0.021 1.39 0.32 0.49 0.61 0.84
S. D. 12.21 0.06 0.25 0.004 0.57 0.04 0.03 0.10 0.25
n 5 5 5 5 5 5 5 5 5
G5 250 Mean 309.80 0.87 3.36 0.01 1.03 0.23 0.46 0.58 0.65
S. D. 32.41 0.06 0.12 0.00 0.05 0.04 0.03 0.06 0.06
n 5 5 5 5 5 5 5 5 5
G2 500 Mean 275.40 0.90 3.71 0.021 1.20 0.27 0.43 0.64 0.70
S. D. 13.35 0.05 0.21 0.006 0.12 0.04 0.05 0.10 0.03
n 5 5 5 5 5 5 5 5 5
G3 1000 Mean 273.80 0.92 3.75 0.021 1.16 0.26 0.47 0.62 0.74
S. D. 7.73 0.12 0.32 0.004 0.13 0.03 0.06 0.24 0.07
n 5 5 5 5 5 5 5 5 5
G4 2000 Mean 282.33 0.93 4.10S 0.017 1.13 0.27 0.48 0.68 0.67
S. D. 22.81 0.05 0.16 0.002 0.10 0.03 0.01 0.06 0.02
n 3 3 3 3 3 3 3 3 3

FEMALE RATS

Group Dose mg/kg/bw/day Body Wt. Kidneys Liver Adrenals Spleen Heart Lungs Brain

G1 0 Mean 211.80 0.95 3.51 0.029 0.300 0.456 0.771 0.946


S. D. 37.57 0.13 0.51 0.006 0.058 0.078 0.072 0.142
n 5 5 5 5 5 5 5 5
G5 250 Mean 205.60 0.89 3.53 0.03 0.32 0.48 0.81 0.97
S. D. 7.77 0.10 0.31 0.00 0.04 0.03 0.18 0.03
n 5 5 5 5 5 5 5 5
G2 500 Mean 199.20 0.95 4.11 0.031 0.294 0.497 0.766 1.029
S. D. 6.76 0.16 0.47 0.006 0.039 0.040 0.106 0.064
n 5 5 5 5 5 5 5 5
S
G3 1000 Mean 198.40 0.97 4.32 0.023 0.320 0.485 0.757 0.986
S. D. 9.10 0.11 0.34 0.011 0.038 0.019 0.079 0.022
n 5 5 5 5 5 5 5 5
G4 2000 Mean 197.50 0.89 4.00 0.030 0.279 0.469 0.713 0.982
S. D. 22.35 0.13 0.31 0.008 0.015 0.030 0.063 0.096
n 4 4 4 4 4 4 4 4

S: Mean values signicantly higher than control with P < 0.05.

that were gavaged about 2e3 h apart. (P < 0.05) were observed. In male rats, one such instance was noted
No gross pathological ndings were encountered in any of the for glucose at 500 mg/kg (lower than the control group). In female
rats subjected to necropsy at their termination on day 15 of the rats values higher than control mean values were observed for total
study, including the group of rats treated with the bitter orange protein at 500 mg/kg, for albumin and ALT at 250 mg/kg, and for
extract at the dose of 2000 mg/kg. Thus, It can be inferred that the AST at 250 and 500 mg/kg (Table 4). These increases did not occur
daily treatment with the bitter orange extract containing 50% p- in a dose dependent manner, and the values were comparable to
synephrine at and up to the dose of 2000 mg/kg body weight for 14 the historical control data for these parameters for Sprague-Dawley
days did not induce any remarkable gross abnormalities in organs/ rats of this age in the test facility. Therefore, these observations
tissues of either sex of rats. However, the test item at the dose of were considered to be incidental and of no toxicological
2000 mg/kg body weight did induce effects on the gastrointestinal signicance.
system in several rats that resulted in events leading to their In the absence of any remarkable necropsy ndings in any or-
deaths. gans/tissues in this study, and in the absence of any remarkable
Daily treatment with the 50% p-synephrine-containing bitter ndings in other study parameters as organ weights and clinical
orange extract at all doses up to and including 2000 mg/kg body hematology and chemistry which suggested an absence of systemic
weight did not induce any alterations in any hematological pa- or organ specic toxicity, histopathological examinations were not
rameters in either male or female rats (Table 3). The values of all performed on tissues/organs collected during necropsy.
hematological parameters for treated male and female rats did not With respect to the results for the Ames Test, frequencies of
differ signicantly from the values of the respective control group histidine revertant colonies in the ve tester strains treated with
rats (P > 0.05). the bitter orange extract at and up to a dose of 5000 mg/plate, both
Daily treatment with the bitter orange extract at doses of 250, in the presence and absence of metabolic activation systems in the
500, 1000 and 2000 mg/kg body weight did not induce any duplicate experiments, were comparable to the frequencies
remarkable alterations in the clinical chemistry parameters of male observed in the respective vehicle control groups. The data for both
and female rats at end of the 14-day treatment period (Table 4). experiments are presented in Table 5. Furthermore, frequencies of
With a few exceptions, as noted below and considered to be inci- histidine revertant colonies observed in the concurrent positive
dental in nature, the values of all clinical chemistry parameters of control plates for all ve tester strains, in the presence and absence
rats treated with the extract did not differ signicantly from the of metabolic activation systems, were signicantly higher than
values of the respective control groups (P > 0.05). those observed in the respective vehicle control groups. Therefore,
A few instances of mean values of clinical chemistry parameters under the conditions described for this study, it can be concluded
differing from the control means with statistical signicance that the bitter orange extract containing 50% p-synephrine is non-
324 N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327

Table 3
Summary of hematology data at termination of treatment period (day 15).

MALE RATS

Group Dose Hb PCV Total RBC RBC Indices Total WBC Differential WBC (%) Platelets
mg/kg/bw/day
MCH MCV MCHC N L M E B

(g/dl) (%) (X106/cmm) (pg) () (g/dl) (X103/cmm) (X103/cmm)

G1 0 Mean 14.94 42.80 7.04 21.23 60.83 34.90 5.73 13.52 83.72 0.68 2.04 0.00 945.40
S. D. 0.47 0.96 0.15 0.33 0.50 0.34 0.98 0.77 0.93 0.50 1.36 0.00 177.60
n 5 5 5 5 5 5 5 5 5 5 5 5 5
G5 250 Mean 16.64 47.46 8.74 19.05 54.34 35.06 5.30 18.32 77.98 0.44 3.22 0.03 607.80
Phase-2 S. D. 0.63 1.44 0.45 0.33 1.47 0.46 1.10 3.52 2.86 0.15 1.27 0.04 307.95
n 5 5 5 5 5 5 5 5 5 5 5 5 5
G2 500 Mean 14.86 42.76 7.19 20.67 59.50 34.73 5.65 16.02 81.22 0.77 1.93 0.05 1019.20
S. D. 0.97 2.15 0.32 0.61 1.13 0.62 1.83 2.61 2.91 0.23 0.59 0.04 91.64
n 5 5 5 5 5 5 5 5 5 5 5 5 5
G3 1000 Mean 15.16 43.82 7.30 20.76 60.02 34.60 5.63 16.72 81.38 0.44 1.45 0.02 987.60
S. D. 0.43 1.35 0.26 0.32 1.83 0.56 1.59 4.75 4.81 0.06 0.70 0.03 174.39
n 5 5 5 5 5 5 5 5 5 5 5 5 5
G4 2000 Mean 14.33 42.33 6.41 22.64 67.25 33.81 5.64 14.93 82.97 1.00 0.96 0.10 743.67
S. D. 1.46 2.75 1.20 2.19 9.51 1.45 1.47 0.74 1.53 0.73 0.19 0.16 200.61
n 3 3 3 3 3 3 3 3 3 3 3 3 3

FEMALE RATS

Group Dose Hb PCV Total RBC RBC Indices Total WBC Differential WBC (%) Platelets
mg/kg/ bw/day
MCH MCV MCHC N L M E B

(g/dl) (%) (X106/cmm) (pg) () (g/dl) (X103/cmm) (X103/cmm)

G1 0 Mean 14.94 43.54 7.19 20.78 60.55 34.32 5.57 12.13 83.82 0.87 3.16 0.02 1040.80
S. D. 0.43 1.62 0.30 0.30 0.36 0.35 1.57 3.89 5.07 0.29 1.53 0.02 100.44
n 5 5 5 5 5 5 5 5 5 5 5 5 5
G5 250 Mean 15.40 43.54 8.13 18.95 53.57 35.37 5.82 16.08 80.54 0.73 2.56 0.09 1041.20
Phase-2 S. D. 0.71 2.12 0.34 0.22 0.70 0.28 1.22 8.06 6.61 0.47 1.57 0.09
n 5 5 5 5 5 5 5 5 5 5 5 5 5
G2 500 Mean 14.86 42.96 7.40 20.09 58.07 34.59 3.65 18.82 72.44 4.86 3.83 0.00 1016.00
S. D. 0.23 0.32 0.24 0.68 2.03 0.33 0.84 5.35 8.34 7.57 1.71 0.01 176.76
n 5 5 5 5 5 5 5 5 5 5 5 5 5
G3 1000 Mean 14.86 42.78 7.45 20.04 57.71 34.74 4.87 13.31 82.70 1.15 2.81 0.02 983.20
S. D. 0.64 1.80 0.73 1.35 4.12 0.38 1.66 3.70 6.21 0.39 2.82 0.03 128.06
n 5 5 5 5 5 5 5 5 5 5 5 5 5
G4 2000 Mean 14.75 42.38 7.44 19.81 56.90 34.81 5.32 13.30 82.15 0.95 3.63 0.01 868.50
S. D. 1.16 3.34 0.43 0.56 1.31 0.57 1.14 1.80 1.66 0.25 0.89 0.02 213.76
n 4 4 4 4 4 4 4 4 4 4 4 4 4

Values of treated rats do not differ signicantly (P > 0.05) from those of the control group.

mutagenic in Salmonella typhimurium Reverse Mutation Assay 50% p-synephrine in the current study which permitted much
(Ames Test). higher doses of p-synephrine to be administered. As a consequence,
a more in depth safety assessment was possible.
No clinically signicant effects were observed at any dose with
4. Discussion respect to body weights, absolute and relative organ weights, gross
pathological ndings, and hematological or clinical chemistry pa-
The study examined the effects of treating male and female rats rameters in rats of either sex. Small decreases were observed with
for 14 days with 250, 500, 1000 and 2000 mg/kg/day of a bitter respect to food consumption in both male and female rats at doses
orange extract that contained 50% p-synephrine. Because this was a of 500 mg/kg and above during the second week of the study. In a
dose range nding study and based on OECD guideline No. 407 the study in which the human subjects consumed 103 mg p-synephr-
number of animals per group and sex were adequate for the pur- ine daily in the form of chocolateeavored chews, statistically
poses of the study. No clinical abnormalities were observed in the signicant increases in appetite/eating control and energy were
male and female rats treated at and up to 500 mg/kg/day, and observed relative to the placebo groups (Kaats and Stohs, 2017).
therefore 500 mg/kg of the bitter orange extract can be considered At 1000 and 2000 mg/kg of the extract, the rats exhibited
the no-observed-effect-level (NOEL). This dose of the extract cor- transient and fully reversible abnormal clinical signs from 6th day
responds to 250 mg/kg of p-synephrine. These results agree with a to the end of the study which involved burrowing of their heads in
previous study in which mice were given up to 300 mg/kg p-syn- the bedding material and staying hypoactive for about 15e45 min
ephrine per day for 28 days with no adverse effects being observed following oral administration of the extract. The reason for the
regarding organ weights, biochemical parameters, blood pressure burrowing activity is not known. Previous studies in mice have
or heart rate (Arbo et al., 2009). demonstrated a reduction in locomotor activity (hypoactivity)
Most previous studies in rodents have used doses of p-syn- (Arbo et al., 2008). These authors demonstrated this decrease in
ephrine in the range of 100e300 mg/kg (Arbo et al., 2009; Hansen activity using a spontaneous locomotor test with doses of bitter
et al., 2011, 2012; 2013) as compared to doses of up to 1000 mg used orange extract of 5000 and 10,000 mg/kg and p-synephrine at
in the current study. Furthermore, prior studies were conducted 300 mg/kg in mice.
with extracts that contained 6.0e7.5% p-synephrine as opposed to
N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327 325

Table 4
Summary of clinical chemistry data at termination of treatment period (day 15).

MALE RATS

Group Dose mg/kg/bw/day Total Albumin ALT AST ALP Glucose BUN CRE Globulin A/G
Protein

(g/dl) (g/dl) (IU/L) (IU/L) (IU/L) (mg/dl) (mg/dl) (mg/dl) (g/dl)

G1 0 Mean 6.08 1.20 44.00 76.00 210.60 88.40 15.00 0.35 4.88 0.25
S. D. 0.30 0.07 9.49 25.60 31.20 15.18 2.83 0.04 0.26 0.01
n 5 5 5 5 5 5 5 5 5 5
G5 250 Mean 6.58 1.36 40.60 75.75 157.00 88.60 13.60 0.36 5.22 0.26
Phase-2 S. D. 0.22 0.09 6.11 15.92 15.38 9.29 0.55 0.04 0.22 0.02
n 5 5 5 4 5 5 5 5 5 5
G2 500 Mean 6.14 1.28 38.20 80.20 176.00 71.00 14.40 0.39 4.86 0.26
S. D. 0.31 0.16 5.22 8.07 22.97 5.29 1.52 0.03 0.22 0.03
n 5 5 5 5 5 5 5 5 5 5
G3 1000 Mean 6.08 1.26 48.60 90.80 213.40 90.20 12.40 0.37 4.82 0.26
S. D. 0.33 0.09 5.59 8.98 26.50 7.73 2.30 0.02 0.24 0.01
n 5 5 5 5 5 5 5 5 5 5
G4 2000 Mean 6.33 1.33 44.00 84.67 193.00 86.67 13.67 0.30 5.00 0.27
S. D. 0.35 0.15 7.00 13.61 37.99 5.13 1.53 0.10 0.20 0.02
n 3 3 3 3 3 3 3 3 3 3

FEMALE RATS

Group Dose mg/kg/bw/day Total Albumin ALT AST ALP Glucose BUN CRE Globulin A/G
Protein

(g/dl) (g/dl) (IU/L) (IU/L) (IU/L) (mg/dl) (mg/dl) (mg/dl) (g/dl)

G1 0 Mean 6.24 1.24 31.40 73.40 129.40 95.00 16.60 0.34 5.00 0.25
S. D. 0.17 0.09 5.41 7.54 26.20 6.04 2.70 0.05 0.19 0.02
n 5 5 5 5 5 5 5 5 5 5
G5 250 Mean 6.56 1.32 31.80 76.40 98.80 95.20 14.00 0.36 5.24 0.25
Phase-2 S. D. 0.29 0.11 4.32 7.02 10.69 5.17 2.12 0.07 0.34 0.33
n 5 5 5 5 5 5 5 5 5 5
G2 500 Mean 6.64 1.50S 41.80S 99.60S 126.00 95.80 14.80 0.40 5.14 0.29
S. D. 0.30 0.07 9.81 4.83 25.86 4.55 1.92 0.08 0.24 0.01
n 5 5 5 5 5 5 5 5 5 5
G3 1000 Mean 6.72S 1.40 36.40 99.40S 147.40 91.00 15.80 0.37 5.32 0.26
S. D. 0.26 0.10 3.36 6.27 35.58 5.24 3.77 0.05 0.31 0.03
n 5 5 5 5 5 5 5 5 5 5
G4 2000 Mean 6.58 1.35 40.25 94.25 150.25 97.25 15.75 0.41 5.23 0.26
S. D. 0.25 0.13 2.87 12.15 21.72 6.34 3.10 0.04 0.17 0.02
n 4 4 4 4 4 4 4 4 4 4

Values of treated rats do not differ signicantly (P > 0.05) from those of the control group.
S: Values signicantly higher (P < 0.05) than the control group.

Deaths of two male rats and one female rat occurred during the subchronic toxicity study. The current study did not specically
14 day study at a dose of 2000 mg/kg body weight of the bitter address effects on the cardiovascular system, although no gross
orange extract (1000 mg/kg p-synephrine) with no incidence of pathological ndings or hyperactivity were observed. p-Synephrine
deaths at and up to the dose of 1000 mg/kg. Necropsy ndings is widely regarded as a stimulant, although this is not supported by
indicated impaction of stomach with ingesta and the test item, or its binding to adrenergic receptors (Stohs et al., 2012), or its effects
the presence of excess residues of extract in the intestine. Bitter on heart rate and blood pressure in animal studies (Hansen et al.,
orange has been used for hundreds of years in Traditional Chinese 2011, 2012; 2013; Arbo et al., 2009) and in numerous human clin-
Medicine for gastro-intestinal disorders including diarrhea and ical studies (Stohs and Shara, 2013; Stohs and Shara, 2013; Shara
dysentery (Chen and Chen, 2004; Fang et al., 2009; Stohs and Shara, et al., 2017). Based on these experimental considerations, p-syn-
2013), and the results of the present study suggests a general lack of ephrine has been referred to as a natural non-stimulant thermo-
detrimental effects in mammals from oral exposure. Furthermore, genic agent since it increases metabolism without cardiovascular
in a study involving a gastric emptying assay in rats, the authors effects at the doses that are commonly used (Stohs and Badmaev,
concluded that p-synephrine inhibited gastrointestinal movement 2016).
while motility was stimulated by hesperidin (Fang et al., 2009). The The bitter orange extract containing 50% p-synephrine was
bitter orange extract (Advantra Z) used in the current study was found to be non-mutagenic under the conditions of the Salmonella
devoid of hesperidin, and therefore the decrease in peristalsis was typhimurium Reverse Mutation Assay (Ames Test). Several other
due to the very high dose of p-synephrine. Previous studies in studies also support this conclusion. A mutagenicity screening of
humans has shown that the addition of bioavonoids including the aqueous and methanolic extracts of 104 crude drugs, including
hesperidin to p-synephrine results in an increase in resting meta- C. aurantium (bitter orange) was conducted by Morimoto et al.
bolic rate without an increase in heart rate or blood pressure (Stohs (1982). The assays involved the Salmonella typhimurium micro-
et al., 2011c), demonstrating an interaction between the two. somal reversion assay using strains TA98 and TA100 and the Ba-
This study indicates a high degree of safety for a bitter orange cillus subtilis rec-assay using Bacillus subtilis strains H17 REC and
extract (Advantra Z) containing 50% p-synephrine, and provides M45 REC. Bitter orange extracts were show to be negative in both
the information required for dosing in conjunction with a 90-day assays, while various other crude drugs were positive in both
326 N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327

Table 5
Summary of Salmonella typhimurium reverse mutation assay results (Ames test) for bitter orange extract containing 50% P-SYNEPHRINE using tester strains TA1535, TA97a,
TA98, TA100, and TA102.

EXPERIMENT NO.: 1 SUMMARY DATA ON HISTIDINE REVERTANT COLONIES (PRE-INCUBATION TEST)

Treatment Concentration TA1535 TA97a TA98 TA100 TA102

(mg/plate) S9 Mean S.D. Mean S.D. Mean S.D. Mean S.D. Mean S.D.

Citrus aurantium 50% 5000 _ 12.67 3.51 118.00 9.17 30.33 2.89 123.33 4.16 268.00 17.44
1500 14.00 5.29 118.00 4.00 33.00 4.36 130.00 6.00 250.67 12.22
500 6.00 2.00 122.67 6.11 29.67 3.06 125.33 6.11 258.67 12.22
150 13.33 4.16 114.00 6.00 30.33 3.06 134.67 5.03 277.33 10.07
50 9.67 1.15 123.33 11.72 35.33 2.31 125.33 4.16 186.67 116.78
Vehicle Control e 1.00 124.67 8.08 31.33 4.16 134.00 7.21 269.33 14.05
(Analytical grade water)

Citrus aurantium 50% 5000 17.33 1.15 112.67 11.02 34.33 2.08 126.00 6.00 254.67 8.33
1500 12.00 2.00 114.00 6.00 36.33 2.08 134.67 8.08 281.33 6.11
500 11.00 5.57 113.33 8.33 35.67 2.52 135.33 10.26 250.67 10.07
150 13.67 1.53 116.67 8.33 30.00 4.58 127.33 5.03 264.00 8.00
50 15.00 3.00 115.33 9.87 31.00 2.65 131.33 6.11 278.67 8.33
Vehicle Control 0 e 0.00 127.33 11.02 35.00 6.24 136.00 7.21 278.67 6.11
(Analytical grade water)

Positive Controls
Sodium azide 2 _ 482.67 56.19 e e e e e e e e
ICR 191 1 e e 685.33 56.76 e e e e e e
4-Nitroquinoline-N-oxide 0.5 e e e e 464.00 44.54 e e e e
3-Methylmethane Sulphonate 1 ml e e e e e e 981.33 52.05 1240.00 631.49

2-Aminoanthracene 10 488.00 40.00 e e e e e e e e


2-Aminouorene 20 e e 677.33 92.72 538.67 33.31 968.00 94.32 e e
30 e e e e e e e e 1573.00 16.17

EXPERIMENT NO.: 2 SUMMARY DATA ON HISTIDINE REVERTANT COLONIES (PRE-INCUBATION TEST)

Treatment Concentration TA1535 TA97a TA98 TA100 TA102

(mg/plate) S9 Mean S. D. Mean S. D. Mean S. D. Mean S. D. Mean S. D.

Citrus aurantium 50% 5000 _ 14.00 2.65 116.00 7.21 31.33 3.51 124.00 4.00 249.33 10.07
1500 13.67 3.51 121.33 4.16 30.33 1.53 125.33 4.16 254.67 12.22
500 12.67 3.51 123.33 5.03 32.33 1.53 126.00 4.00 265.33 8.33
150 15.00 3.00 119.33 9.45 30.00 3.00 125.33 6.11 250.67 12.22
50 11.33 1.53 120.00 9.17 31.67 5.86 130.67 5.03 258.67 14.05
Vehicle Control 100 ml 19.33 1.15 125.33 5.03 29.33 3.06 133.33 6.11 265.33 6.11
Analytical grade water

Citrus aurantium 50% 5000 11.67 1.53 124.00 8.00 29.33 4.04 123.33 3.06 253.33 16.65
1500 14.00 2.65 129.33 8.08 29.00 2.65 128.00 6.00 262.67 12.22
500 12.00 2.00 118.00 7.21 30.33 4.73 126.00 4.00 269.33 14.05
150 12.00 4.00 119.33 5.03 27.00 2.65 136.00 4.00 250.67 12.22
50 15.00 3.00 124.67 3.06 30.33 3.51 125.33 4.16 260.00 10.58
Vehicle Control 100 ml 16.33 1.53 119.33 8.33 29.33 2.52 134.67 4.16 270.67 12.22
Analytical grade water

Positive Controls
Sodium azide 2 _ 461.33 32.33 e e e e e e e e
ICR 191 1 e e 688.00 36.66 e e e e e e
4-Nitroquinoline-N-oxide 0.5 e e e e 488.00 21.17 e e e e
3-Methylmethane Sulphonate 1 ml e e e e e e 1298.67 44.06 2018.67 44.06

2-Aminoanthracene 10 472.00 32.00 e e e e e e e e


2-Aminouorene 20 e e 688.00 49.96 456.00 36.66 1336.00 60.40 e e
Danthron 30 e e e e e e e e 2024.00 64.00

S9: () Without S9, () With S9.

assays. The extracts employed in this study were not chemically viability or proliferation of leukocytes. Under the experimental
characterized. conditions employed and using the DNA comet tail assay, a small
More recently, p-synephrine and its forced degradation prod- increase in DNA damage was observed at 100 ng/mL, a concentra-
ucts were studied for their potential mutagenic effects (Kaefer, tion that is about 10 times greater than the blood levels observed in
2014). This study was published only in thesis form. Degradation humans two hours after a 49 mg dose of p-synephrine (Shara et al.,
of p-synephrine was produced under basic, acidic, proteolytic and 2016). No effect on DNA was observed at a p-synephrine concen-
oxidative conditions. The authors concluded that p-synephrine and tration of 50 ng/mL.
its degradation products were non-mutagenic. Up to the highest The results of this 14 day study indicate that the NOEL for a
tested concentration of 100 ng/ml p-synephrine did not result in bitter orange extract containing 50% p-synephrine is 500 mg/kg in
micronucleus production of cultured cells nor did it affect the both male and female Sprague-Dawley rats. The concentration of p-
N.S. Deshmukh et al. / Regulatory Toxicology and Pharmacology 90 (2017) 318e327 327

synephrine in the extract permitted higher doses of the extract to Herbology and Pharmacology. Art of Medicine Press, City of industry, CA, p. 485.
Fang, Y.S., Shan, D.M., Liu, J.W., Xu, W., Li, C.L., Wu, H.Z., Ji, G., 2009. Effect of con-
be administered, and demonstrated that the LD50 of the extract
stituents from Fructus aurantium immaturus and Radix praeoniae alba on
exceeded 5000 mg/kg (greater than 2500 mg/kg for p-synephrine). gastrointestinal movement. Planta Med. 75, 24e31.
No clinically signicant effects were observed at any dose with Hansen, D.K., Juliar, B.E., White, G.E., Pellicore, L.S., 2011. Developmental toxicity of
respect to gross pathological ndings, body weights, absolute and Citrus aurantium in rats. Birth Defects Res. Part B. Devel. Reprod. Toxicol. 92,
216e223.
relative organ weights, hematological parameters or clinical Hansen, D.K., George, N.I., White, G.E., Abdel-Rahman, A., Pellicore, L.S.,
chemistry parameters in the animals. The results afrmed the lack Fabricant, D., 2013. Cardiovascular toxicity of Citrus aurantium in exercised rats.
of clinically signicant effects of previous studies conducted with Cardiovasc. Toxicol. 13, 208e219.
Hansen, D.K., George, N.I., White, G.E., Pellicore, L.S., Abdel-Rahman, A.,
lower doses for longer periods of time. A weakness of the study was Fabricant, D., 2012. Physiological effects following administration of Citrus
the fact that the study was conducted for 14 days and not longer. aurantium for 28 days in rats. Toxicol. Appl. Pharmacol. 261, 236e247.
However, the results of this study enabled dosing parameters to be Kaats, G.R., Stohs, S.J., 2017. Increased eating control and energy levels associated
with consumption of a bitter orange (p-synephrine) extract chewda random-
established for a 90 day subchronic toxicity study. Finally, based on ized placebo controlled study. Nutr. Diet. Suppl. 9, 29e35.
the results of the Ames Test, p-synephrine and bitter orange extract Kaefer, C.L., 2014. Syephrine Development and Validation of a Stability Indicating
are non-mutagenic. Method and Preliminary Stability Study of Citrus aurantium L. Dry Extract. A
Dissertation. Post-Graduate Program in the Department of Pharmaceutics,
In summary, results of the study demonstrate an exceedingly Federal University of Pampa, Pampa, Uruguay, p. 55.
high LD50 for bitter orange extract and p-synephyrine, provide an Morimoto, I., Watanabe, F., Osawa, T., Okitsu, T., 1982. Mutagenicity screening of
initial NOEL under the conditions of the study, demonstrate a lack crude drugs with Bacillus subtilis rec-assay and Salmonella/microsomal rever-
sion assay. Mutat. Res. 97, 81e102.
of adverse events and pathological effects at very high doses, and
Pellati, F., Benvenuti, S., 2007. Chromatographic and electrophoretic methods for
indicate a lack of mutagenicity for the bitter orange extract and p- the analysis of phenethylamine alkaloids in Citrus aurantium. J. Chromatog A
synephrine. Taken together, these results afrm and extend previ- 1171, 71e88.
ous knowledge regarding the safety of bitter orange extract and p- Ratamess, N.A., Bush, J.A., Kang, J., Kraemer, W.J., Stohs, S.J., Nocera, V.G., Leise, M.D.,
Diamond, K.B., Fagenbaum, A.D., 2015. The effects of supplementation with p-
synephrine, and form the basis for future safety studies. synephrine alone and in combination with caffeine on acute resistances exer-
cise performance. J. Int. Soc. Sports Nutr. 12, 35. https://doi.org/10.1186/s12970-
Conicts of interest 015-0096-5.
Shara, M., Stohs, S.J., Mukattash, T.L., 2016. Cardiovascular safety of oral p-syn-
ephrine (bitter orange) in human subjects: a randomized placebo-controlled
The study was conducted at INTOX Private LTD., 375, Urawade, cross-over clinical trial. Phytother. Res. 30, 842e847.
Tal. Mulshi, Maharashtra India. SJS is a consultant for Novel In- Shara, M., Stohs, S.J., Smadi, M.M., 2017. Safety evaluation of bitter orange (p-syn-
ephrine) extract following oral administration for 15 days to healthy human
gredients. NSD, CMC and SBK are all associated with INTOX Private subjects a clinical trial. Phytother. Res. https://doi.org/10.1002/ptr.5953.
LTD. Stohs, S.J., 2017. Safety, efcacy and molecular studies regarding Citrus aurantium
(bitter orange) extract and p-synephrine. Phytother. Res. https://doi.org/
10.1002/ptr.5879.
Acknowledgements Stohs, S.J., Badmaev, V., 2016. A review of natural stimulant and non-stimulant
thermogenic agents. Phytother. Res. 30, 732e740.
The study was conducted under a grant from Novel Ingredients, Stohs, S.J., Preuss, H.G., 2011a. The safety of bitter orange (Citrus aurantium) and its
primary protoalkaloid p-synephrine. HerbalGram 89, 34e39.
LLC, East Hanover, NJ 07936 USA which also provided the test
Stohs, S.J., Preuss, H.G., 2011b. Stereochemical and pharmacological differences
product. between naturally occurring p-synephrine and synthetic p-synephrine. J. Funct.
Foods 2011, 9. https://doi.org/10.1016/j.jff.2011.09.004.
Transparency document Stohs, S.J., Preuss, H.G., Shara, M., 2011a. The safety of Citrus aurantium (bitter or-
ange) and its primary protoalkaloid p-synephrine. Phytother. Res. 25,
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