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IJDS#1674996, VOL 0, ISS 0
Effect of Raspberry Ketone on Normal, Obese and
Health-Compromised Obese Mice: A Preliminary
Study
Tahir Maqbool Mir, Guoyi Ma, Zulfiqar Ali, Ikhlas A. Khan, and Mohammad
K. Ashfaq
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ARTICLE
Effect of Raspberry Ketone on Normal, Obese and
Health-Compromised Obese Mice: A Preliminary Study
Tahir Maqbool Mir
a
, Guoyi Ma
b
, Zulfiqar Ali
a
, Ikhlas A. Khan
a
, and
Mohammad K. Ashfaq
a
Q1
a
National Center for Natural Product Research, School of Pharmacy, University of Mississippi, University,
MS, USA;
b
Drug Discovery Division, Southern Research, Birmingham, AL, UK
ABSTRACT
Background: Raspberry ketone (RK)—an aromatic compound found
mostly in red raspberries (Rubus idaeus) is widely used as an over
the counter product for weight loss. Aim: The present study was
conducted to determine adverse effects associated with RK in obese
and health-compromised obese mice. Methodology: Two sets of
experiments were conducted on normal obese and health-compro-
mised obese mice treated with RK for a duration of 10 days. Obese
conditions were induced by feeding mice a high fat diet for
10 weeks, while the health compromised obese mouse model was
developed by a single intraperitoneal injection of a nontoxic dose of
lipopolysaccharide (LPS) (6 mg/kg) to obese mice. Results: Results
showed that RK (165, 330, and 500 mg/kg) under obese as well as
health-compromised condition retarded the gain in body weights as
compared to the control groups. RK at doses 330 and 500mg/kg
resulted in 67.6 and 50% mortality, respectively in normal obese
mice and 70% mortality was observed in health-compromised obese
mice treated with RK at 500 mg/kg. At higher doses deaths were
observed earlier than those given lower doses of RK. Significant ele-
vations in blood alanine transaminase (ALT) were also observed with
RK treatment in obese mice. Blood glucose levels were significantly
elevated in all groups of mice treated with RK. Conclusion: This study
suggests that higher doses of RK may cause adverse effects in health
compromised conditions. Under these conditions, prolonged use of
RK, especially in high doses, may pose a health hazard.
Q2
KEYWORDS
Health compromised
conditions; high fat diet;
mortality; obese;
Raspberry ketone
Introduction
Since ancient times, plants have served as the primary and invaluable sources of new
drugs. Renewed interests in plant-derived drugs over conventional medicines have
emerged, likely due to consumer perception pertaining to side effects associated with
conventional drugs. Till now, natural products in the form of herbal remedies, medi-
cinal plants, functional foods, and their constituents are effective in treating many
diseases including cancer (Kim et al. 2018). Tremendous research efforts on natural
products are being carried out to develop alternatives to synthetic drugs. As per
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CONTACT Mohammad K. Ashfaq mkashfaq@olemiss.edu National Center for Natural Product Research, School of
Pharmacy, University of Mississippi, 2047 TCRC, University, MS 38677.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ijds.
ß2019 Taylor & Francis Group, LLC
JOURNAL OF DIETARY SUPPLEMENTS
https://doi.org/10.1080/19390211.2019.1674996
Editorial in Lancet, Gastroenterology and hepatology (2018), the sales of natural supple-
ments in the United States alone, reached $37 billion in 2014, an increase from $7 bil-
lion in 1994. In Australia, spending on complementary medicines increased by more
than 100% between 1996 and 2004 (Bailey et al. 2013). Hundreds of natural products
are being sold in stores as over the counter herbal products or online with tremen-
dously conflated statements pertaining to their abilities to positively affect weight loss,
immune health, anti-diabetics, hair growth as well as dietary supplements. However, the
health benefits associated with natural products are substantiated with little clinical evi-
dence to prove their mechanistic aspects. Experimental studies have demonstrated that
several natural products have been associated with harmful side effects including liver
toxicity (Abdel-Bakky et al. 2010; Chan et al. 2010; Wang et al. 2015). The severity of
side effects associated with natural products depends on the dose, consumption duration
of the product and most importantly on the general health of the individual consumer.
The general health of the individual plays an important role in herbal product associ-
ated toxicities. It has been reported that several natural products can contribute to liver
toxicity when consumed during health compromised conditions (Maddox et al. 2010). It
has been found that a nontoxic dose of lipopolysaccharide (LPS) reduces the toxic
threshold of xenobiotics by inducing mild inflammatory conditions in the body (Ganey
et al. 2004; Deng et al. 2009; Tu et al. 2015). To this end, we have already established a
mouse model for hepatotoxicity, with co-administration of pyrrolizidine alkaloid
(monocrotaline) and a nontoxic dose of LPS (6 mg/kg) (Abdel-Bakky et al. 2010).
Similarly, our previous studies demonstrated that administration of epigallocatechin-3-
gallate (EGCG), a major constituent of green tea to mice sensitized with nontoxic dose
of LPS caused liver damage (Saleh et al. 2013). Raspberry ketone (4-(4-hydroxyphenyl)
butan-2-one, RK) is a major aromatic compound of red raspberries (Rubus idaeus)
(Schinz and Seidel 1957; Gallois 1982; Patel et al. 2004). Raspberry ketone is used as fla-
voring substance in a variety of processed foods such as soft drinks, puddings, yogurt,
baked goods, sweets and ice cream (Beekwilder et al. 2007; Crispim et al. 2010).
Initially, it was introduced as a fragrance and flavoring agent (Opdyke 1978). The
Flavor and Extract Manufacturers Association of the United States placed Raspberry
ketone on generally recognized as safe (GRAS) status (Opdyke 1978; Lin et al. 2011).
However, since 2012 the whole scenario about RK changed when claims of its anti-
obesity (weight reducing) property were made in the US media as “No. 1”miraculous
weight-loss supplement. This resulted in the dramatic increase in sales of dietary supple-
ments containing RK (Bootsman et al. 2014). RK is believed to affect fat metabolism by
enhancing lipolysis and fatty acid oxidation (Park 2010), resulting in its use in a wide
array of anti-obesity or weight loss supplements. In addition to its role as a weight
reducing natural supplement, RK is claimed to possess other pharmacological activities
including hepatoprotective, anti-androgenic activity (Ogawa et al. 2010), anti-inflamma-
tory activity by blocking the nuclear transcription factor-kB (NF-kB) activation (Jeong
and Jeong 2010), depigmenting activity (Lin et al. 2011), anti-obese activity (Park 2010;
Lopez et al. 2013), antihypertensive (Jia et al. 2011) and cardio-protective (Khan et al.
2018) activities.
Several reports revealed that the amount of RK sold in US markets in dietary supple-
ments is well above the maximum recommended concentration for food products.
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2 T. M. MIR ET AL.
Further studies have been recommended to ensure its safety at such concentrations
(Bredsdorff et al. 2015; Lee 2016). As the weight reducing products are usually con-
sumed by overweight individuals, it is imperative that a safe dose be determined.
Considering that some of the consumers of such weight reduction products tend to
become over ambitious and may use higher doses than those prescribed by the manu-
facturer. Thus, studies need to be undertaken to define the safe levels of such products
under healthy and health-compromised conditions. To this end, we conducted a prelim-
inary study to determine the adverse effects of RK at different doses in obese and
health-compromised obese mice.
Materials and methods
Materials
Raspberry ketone and lipopolysaccharide (E. Coli O55:B5) were purchased from Sigma-
Aldrich (St. Louis, MO) Blood ALT level was measured using an automated VetScan
dry chemistry analyzer with V-DPP rotors (Abaxis, Union City, CA). Normal mouse
chow (TekLad 57001) and High Fat Diet (HFD) (TD. 06414) was purchased from
Envigo (Indianapolis, Indiana, USA).
Animals
Male ND-4 mice were obtained from Envigo (Indianapolis, Indiana, USA) at 5 weeks of
age and 18–20 g body weight; housed in micro isolator cages with corn cob bedding
with 12 h light/12 h dark cycle at 72F (22 C) with 35–50% relative humidity. Mice
were fed on laboratory chow and High Fat Diet (HFD) and water ad libitum as per the
protocol. All animal studies were approved by the Institutional Animal Care and Use
Committee (IACUC), University of Mississippi, USA.
Preparation of LPS and Raspberry ketone
Lipopolysaccharide (6 mg/kg in saline). RK was dissolved in propylene glycol and water
(1:1) to prepare doses of 165 mg/kg, 330 mg/kg and 500 mg/kg body weight.
Treatment protocol and study design
Experiment # 1
Mice were divided into five groups (n¼4–7) as shown in Table 1. Group I was
fed a normal diet and served as the control. Groups II to V were fed HFD for ten
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Table 1. Table showing experimental regimen for different dosing groups of HFD fed mice.
Mice ND4 Diet up to 10 weeks Treatments Day 1 onwards Day 10
th
after RK treatment
Group 1 (n55) Normal Diet Water Animals were sacrificed
Group 2 (n55) HFD Vehicle (Oral)
Group 3 (n56) HFD RK (500 mg/kg) (Oral)
Group 4 (n57) HFD RK (330 mg/kg) (Oral)
Group 5 (n54) HFD RK (165 mg/kg) (Oral)
JOURNAL OF DIETARY SUPPLEMENTS 3
weeks. HFD was continued throughout the experimental procedure as shown in
Table 1. Thereafter, group II was administered vehicle (propylene glycol and water
1:1) orally for 10 days and served as negative control (Obese group), Groups III, IV
and V were given different doses (500 mg/kg, 330 mg/kg, and 165 mg/kg, respect-
ively) of RK for 10 days. On day 10, one hour after RK administration, all mice
were sacrificed. Body weights before and after RK treatment were recorded. ALT
and fasting blood glucose levels were determined (Relion confirm glucometer) at the
end of the experiment. Mortality of mice throughout the dosing period
was recorded.
Experiment # 2
Mice (ND4) were divided into 4 groups (n¼3–5) (Table 2). Group I was fed on nor-
mal diet, and served as the control group. Mice in groups II, III, and IV were given
HFD for 10 weeks. Thereafter, group II mice were given vehicle only. Group III and
group IV mice were given single i.p injection of LPS (6 mg/kg) on day 0 (Hammad
et al. 2013). Group IV mice were given RK at a dose of 500 mg/kg body weight,
orally from day 1 to day 10. After 10
th
dose of RK, blood was collected by cardiac
puncture. Fasting blood glucose and ALT levels were determined at the end of the
experiment. Body weights and animal mortality were recorded throughout the dos-
ing period.
Histopathology
The liver tissues were fixed in 10% buffered formalin for at least 24 h, thereafter, the
specimens were dehydrated in ascending grades of ethanol, cleared in xylene, and
embedded in paraffin wax. Blocks were made and 5 lm thick sections were cut. The
paraffin embedded liver tissue sections were deparaffinized using xylene and ethanol.
The slides were washed with phosphate buffered saline (PBS). The sections were stained
with hematoxylin and eosin (H & E). These stained liver sections were observed under
light microscope at 40magnifications to investigate the histological architecture of
the liver.
Statistical analysis
The data from individual groups were presented as the mean ± SEM. Differences
between groups were analyzed using analysis of variance (ANOVA) followed by Tukey’s
comparisons test and minimum criterion for statistical significance was set at p<0.05
for all comparisons.
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Table 2. Table showing dosing pattern of raspberry ketone and LPS in HFD fed mice.
Mice ND4 Diet up to 10 weeks Treatments Day 0 Day 1 onwards Day 10
th
After RK treatment
Group I (n55) Normal diet Saline (i.p) Water Animals were sacrificed
Group II (n55) HFD Saline (i.p) Vehicle (Oral)
Group III (n53) HFD LPS (6 mg/kg; i.p) Vehicle (Oral)
Group IV (n54) HFD LPS (6 mg/kg; i.p) RK (500 mg/kg) (Oral)
4 T. M. MIR ET AL.
Results
Experiment # 1
Effect of RK on body weights
Administration of RK (500 and 165 mg/kg) did not cause any appreciable reduction in
the body weight of obese mice. Treatment of HFD fed mouse with RK (330 mg/kg)
resulted in a reduced body weight as compared with the normal control and obese
mice. Normal control animals exhibited an almost linear increase in body weight over
time. (Figure 1). Values represent mean ± SEM
Effect of RK on daily mortality of animals
Figure 2 represents the daily mortality of animals treated with RK (500 mg/kg, 330 mg/
kg and 165 mg/kg). In the present study, it was observed that 5 out of 10 mice died
within 24 h after administration of the first dose of RK (500 mg/kg). After three doses,
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Figure 1. Effect of Raspberry ketone treatment on Body weight of Mice. Normal Control: Mice group
fed on normal diet. HFD: High Fat Diet. Vehicle: propylene glycol and water (1:1); RK 500, RK 330,
and RK 165: Raspberry ketone at doses 500 mg/kg, 330 mg/kg and 165 mg/kg, respectively. No signifi-
cant change in body weight gain were observed in mice treated with RK 500 and RK 330mg/kg. A
slight reduction in body weight gain was observed in mice group treated with RK 165 mg/kg.
Figure 2. Daily mortality observed in mice treated with Raspberry ketone at doses 500, 330, and
165 mg/kg orally for 10days. Normal Control: Mice group fed on normal diet. HFD: High Fat Diet.
Vehicle: propylene glycol and water (1:1); RK 500, RK 330, and RK 165: Raspberry ketone at doses
500 mg/kg, 330 mg/kg and 165 mg/kg, respectively.
JOURNAL OF DIETARY SUPPLEMENTS 5
another mortality occurred in this group. RK treatment of mice at a dose of 330 mg/kg,
caused mortality in 4 out of 10 mice after the first dose; and another mouse died after
administration of the third dose. No mortality was observed in any of the animals
treated with RK (165 mg/kg), normal control and vehicle control animals.
Effect of RK treatment on cumulative deaths of mice
Obese mice treated with RK at doses of 500 mg/kg and 330 mg/kg showed mortality of
67.6 and 50%, respectively. While as RK (165 mg/kg), normal control and vehicle con-
trol mice showed no mortality (Figure 3).
Effect of RK on blood glucose levels
Figure 4 shows the blood glucose levels of animals before and after RK (500 mg/kg,
330 mg/kg and 165 mg/kg) treatments. We observed that mice fed with HFD and treated
with RK (500 mg/kg (p>0.05), 330 mg/kg (p>0.001) and 165 mg/kg (p>0.01) showed
elevated levels of blood glucose levels after RK treatment. While mice in the normal
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Figure 3. Percent cumulative deaths observed in mice groups treated with Raspberry ketone at doses
500, 330, and 165 mg/kg orally for 10days. Normal Control: Mice group fed on normal diet. HFD:
High Fat Diet. Vehicle: propylene glycol and water (1:1); RK 500, RK 330, and RK 165: Raspberry
ketone at doses 500 mg/kg, 330 mg/kg and 165 mg/kg, respectively.
Figure 4. Effect of Raspberry ketone treatment on blood glucose levels. Normal Control: Mice group
fed on normal diet. HFD: High Fat Diet. Vehicle: propylene glycol and water (1:1); RK 500, RK 330,
and RK 165: Raspberry ketone at doses 500 mg/kg, 330 mg/kg, and 165 mg/kg, respectively.
6 T. M. MIR ET AL.
and vehicle control groups did not show any significant elevations in blood glu-
cose levels.
Effect of RK treatment on blood ALT levels
Mice treated with RK at doses of 500 and 330 mg/kg showed significant (p<0.01 and
p<0.05, respectively) elevation in blood ALT levels as compared to vehicle control
group. While the mice treated with RK (165 mg/kg) group showed ALT levels in the
normal range (Figure 5). Values are shown as Mean ± SEM.
Effect of RK on histopathological morphology of liver
Histopathological architecture of the liver samples from normal and HFD treated
groups exhibit fatty changes compared with each other. Liver micrographs from the
normal group mice showed normal cellular architecture and did not exhibit any vacu-
olar changes, whereas group II animals (HFD treated) showed occurrence of hepatocel-
lular vacuolar change and steatosis that was consistent with fatty liver alterations
(mostly microvesicular steatosis occasional with macrovesicular steatosis or lipidosis)
(Figure 6B). RK treatments showed apparent reduction in the size of fat vacuoles
(Figure 6).
Experiment # 2
Effect of RK on body weight of mice under health compromised condition
Under health compromised conditions, the 500 mg/kg dose of RK did not affect the
body weight of mice as compared to the vehicle control group. Both vehicle control
group and RK þLPS showed a similar trend in body weights of mice. Mice in the nor-
mal control group exhibited a linear increase in body weight over time (Figure 7).
Values are shown as Mean ± SEM.
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Figure 5. Effect of Raspberry ketone treatment on blood ALT levels. ALT: Alanine transaminase.
Normal Control: Mice group fed on normal diet. HFD: High Fat Diet. Vehicle: propylene glycol and
water (1:1); RK 500, RK 330, and RK 165: Raspberry ketone at doses 500 mg/kg, 330mg/kg, and
165 mg/kg, respectively.
JOURNAL OF DIETARY SUPPLEMENTS 7
Effect of RK on daily mortality of animals under health compromised conditions
No mortalities were observed in normal and vehicle control groups of mice. However,
in only LPS treated group (single i.p injection of LPS on day 0), one out of three mice
died on day 3 post LPS injection. Whereas, in HFD þLPS þRK(500 mg/kg) and
HFD þRK(500 mg/kg) groups two out of four mice and three out of six mice died on
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Figure 6. Effect of RK on HFD induced Liver histopathology changes. Representative photomicro-
graphs (magnification 40). Normal Control: Mice group fed on normal diet. HFD: High Fat Diet.
Vehicle: propylene glycol and water (1:1); RK 500, RK 330, and RK 165: Raspberry ketone at doses
500 mg/kg, 330 mg/kg, and 165mg/kg, respectively. (A) Normal diet (Group I), (B) HFD þVehicle
(Group II), (C) HFD þRK-500 (Group III), (D) HFD þRK-330 (Group IV), and (E) HFD þRK-165 (Group V).
(Black arrow: microvesicles; yellow arrow: macrovesicles).
Figure 7. Effect of RK on body weight of mice under health compromised condition. Normal control:
Mice group fed on normal diet. HFD: High fat diet. Vehicle: propylene glycol and water (1:1); LPS:
Lipopolysaccharide; RK 500: Raspberry ketone at dose 500 mg/kg.
8 T. M. MIR ET AL.
day 1 of the study, respectively. On days 2 and three of the study, one mouse from
each group (HFD þLPS þRK-500 mg/kg and HFD þRK-500 mg/kg) died (Figure 8).
Effect of RK treatment on cumulative deaths of mice under health compro-
mised conditions
Obese mice sensitized with LPS resulted in the 40% mortality, while mice treated with
HFD þLPS þRK-500 mg/kg and HFD þRK-500 mg/kg showed mortalities of 75% and
67%, respectively (Figure 9).
Effect of RK on blood glucose levels of mice under health compromised conditions
Figure 10, shows the blood glucose levels of mice treated with RK at a dose of 500 mg/
kg under health compromised condition. Increased blood glucose levels were observed
in the groups of mice given HFD þLPS or HFD þRK-500 mg/kg. As there was 75%,
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Figure 8. Daily mortality observed in obese mice treated with RK plus LPS. Normal control: Mice
group fed on normal diet. HFD: High fat diet. Vehicle: propylene glycol and water (1:1); LPS:
Lipopolysaccharide; RK 500: Raspberry ketone at dose 500 mg/kg.
Figure 9. Effect of RK treatment on cumulative deaths of mice under health compromised conditions.
High fat diet (HFD); Lipopolysaccharide (LPS); Raspberry ketone at dose 500mg/kg (RK 500).
JOURNAL OF DIETARY SUPPLEMENTS 9
mortality observed in group of mice treated with HFD þLPS þRK-500 mg/kg, blood
samples could not be collected from all animals, therefore glucose estimation was done
in the remaining mice in this group (Figure 10). Values are shown as Mean ± SEM
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Figure 10. Effect of RK on blood glucose levels of mice under health compromised conditions. High
fat diet (HFD); Lipopolysaccharide (LPS); Raspberry ketone at dose 500mg/kg (RK 500).
Figure 11. Effect of RK on HFD induced liver histopathology changes under health compromised con-
ditions. High fat diet (HFD); Lipopolysaccharide (LPS); Raspberry ketone at dose 500 mg/kg (RK 500).
Representative photomicrographs (magnification 40), CV: Central vein; micro and macro vesicles
(arrows). (A) Normal diet (Group I), (B) HFDþVehicle (Group II), (C) HFD þLPS (Group III), and (D)
HFD þLPS þRK-500 (Group IV).
10 T. M. MIR ET AL.
Effect of RK on histopathological morphology of liver under health compro-
mised conditions
Under health compromised conditions, it was observed that RK at high dose (500 mg/
kg) reduced the effects exhibited by HFD on liver (Figure 11A). Liver architecture of
mice treated with HFD showed accumulation of fat vesicles. The livers from mice
treated with HFD þLPS showed less degree of lipidosis (Figure 11B) as compared with
HFD group (Figure 11C).
Discussion
Fruits of Rubus idaeus L. commonly known as red raspberries are widely available
and possess both nutritional and medicinal properties. They possess several essential
micronutrients, dietary fibers, and phenolic components, especially raspberry ketone
ellagitannins, and anthocyanins. Scientific reports indicate that raspberries are being
used in the treatment of diabetes mellitus, cancer, cardiovascular disorders, obesity,
and neurodegeneration (Burton-Freeman et al. 2016; Kristo et al. 2016; Noratto et al.
2016). Raspberry ketone is an aromatic compound predominantly found in raspberries
and also in other fruits, including black raspberries and kiwifruit (Ulbricht et al.
2011). It is widely used as a fragrance in cosmetics and flavoring agent in foodstuff.
Several reports have demonstrated its lipolytic and anti-obesity activities (Morimoto
et al. 2005; Leu et al. 2018; Mehanna et al. 2018). In the present study, we induced
obesity in mice by feeding them for 10 weeks on a high-fat diet. Increases in body
weight and fat storage was noted in accordance with the previously published reports
(Morimoto et al. 2005; Mukai et al. 2016). In addition, we explored the possible
adverse effects of consumption of RK during normal obese and health compromised
obese condition in mice.
We observed a slight trend of decreased gain in body weights in mice treated with
RK at 330 mg/kg; however, this may be attributed to reduced intake of feed as has been
hypothesized by others (Cotten et al. 2017). At other doses (500 and 165 mg/kg), no
appreciable change in body weight was observed. At the higher dose (500 mg/kg), RK
may be toxic and cause deaths within the 2 days post RK treatment, reducing the num-
ber of animals for statistical comparison of body weights at the termination of the
experiment. At the lower dose (165 m g/kg), RK may not be as effective in lowering the
body weight gain especially under this short duration (10 days) of RK treatment. A simi-
lar pattern of deaths and reduction in body weights was observed in the LPS sensitized
obese mice treated with RK at 500 mg/kg.
Dietary fat is digested by pancreatic lipase and absorbed from the small intestine
(Hernell et al. 1990). RK decreased the body weight and this may be due to inhibition
of pancreatic lipase enzyme activity and inhibition of intestinal absorption. Thus, the
weight lowering effect observed in this study conforms with other reports that demon-
strated weight reducing property of RK (Morimoto et al. 2005; Park 2010; Lopez et al.
2013). However, the retardation in weight gain during the 10-day study could also be
due to loss of appetite in the groups given LPS and RK. This point needs to be further
evaluated as in this study food intake was not measured.
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In the present study, our major objective was to assess the potential adverse effects
that may be associated with RK, if it was consumed at high doses or by individuals
with health compromised conditions. The LPS sensitized mouse (health compromised)
model used in this study has been used in other studies to demonstrate the hepatotoxic
potential of natural products (Abdel-Bakky et al. 2010; Saleh et al. 2013) as well as
pharmaceutical drugs (Shaw et al. 2007; Poulsen et al. 2014). The results of the current
preliminary study showed dose-related mortality in obese mice treated with RK. In add-
ition, LPS sensitized obese mice treated with high dose of RK showed higher mortality;
however, no mortality was observed in mice treated with RK at 165 mg/kg. These results
indicate the narrow spectrum of effective weight reducing dose of RK. Alanine amino-
transferase is a cytoplasmic enzyme of hepatocytes, and its elevation in blood is consid-
ered one of the key indicators of liver injury (McGill and Jaeschke 2013). During any
hepatotoxic insult, this enzyme is released from the hepatocytes into the blood stream
resulting in its higher levels in the blood (Kim et al. 2008). We have also observed sig-
nificant (p<0.05 and p<0.01 at 330 and 500 mg/kg, respectively) elevation of blood
ALT levels in mice treated with higher doses of RK as compared to those fed on normal
or high fat diet only. Our results are similar and support the ones reported earlier by
Joint FAO/WHO Expert Committee on Food Additives (JECFA) report. (study by
Hoffman 2004 cited from Joint FAO WHO Expert Committee on Food Additives,
World Health Organization 2011) (Hoffman 2004; Joint FAO WHO Expert Committee
on Food Additives, World Health Organization 2011). At lower doses, a rising trend in
the blood ALT levels was observed; however, it was not statistically significant, perhaps,
due to short duration of the study. Therefore, further studies of longer duration should
be undertaken to assess the toxic and safety potential of RK in both obese and health
compromised obese mice.
In this study, we observed a significant increase in blood glucose levels in mice
treated with different doses of RK compared to those of normal control and obese
mice. Even in health-compromised obese mice, elevated blood glucose levels were
observed. None of the published work on RK has earlier reported this observation. This
is the first report on elevated blood glucose level by RK treatment in obese and health
compromised obese mice. This increase in blood glucose levels implies RK’s indirect
effect on glucose metabolism, as RK is involved in lipolysis and fatty acid oxidation in
the cells (Park 2010; Tsai et al. 2017), resulting in the production of ketone bodies that
are used as a source of fuel in the cells instead of glucose. This may result in the non-
utilization of glucose by cells resulting in higher glucose levels in blood (Collier
et al. 1993).
The extent of lipid accumulation in liver parenchymal cells is the direct indices of
liver steatosis (Mezey 1999). Several reports indicate HFD administration to mice over a
period, resulted in fat accumulation in the liver (Choi et al. 2018; Kim et al. 2016) lead-
ing to steatosis. To assess the effect of RK on hepatic steatosis, we performed H & E
staining. Histology revealed that liver from mice fed on HFD exhibited hepatic lipid
vesicles compared to none seen in mice fed on normal diet. Although, RK treatments
resulted in the reduction in the size of lipid vesicles in liver tissue, but it did not com-
pletely eliminate the effects of HFD in this short period of study. Similar results were
observed under health-compromised conditions.
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Conclusions
In conclusion, the results of the present study showed that while RK may have weight-
reducing properties at higher doses and longer durations, it also has the potential to be
hepatotoxic. Weight-reducing agents are often consumed for long durations to achieve
the desired level of effect. Therefore, long-term experiments are essential at more
physiologically relevant doses to determine a safe dose of RK, under obese and health
compromised-obese conditions.
Acknowledgments
The authors thank Dr. Jon F. Parcher for editing the manuscript for English language. The
authors sincerely thank Ms. Penny Bolton for assistance in maintenance of animals used in this
study and biological work.
Conflicts of interest
The authors declare no conflicts of interest.
Funding
This work was supported in part by the United States Department of Agriculture, Agricultural
Research Service, Specific Cooperative agreement 58-6408-1-603-04 and US Food and Drug
Administration 1U01FD004246-04.
Q3
References
Abdel-Bakky MS, Hammad MA, Walker LA, Ashfaq MK. 2010. Developing and characterizing a
mouse model of hepatotoxicity using oral pyrrolizidine alkaloid (monocrotaline) administra-
tion, with potentiation of the liver injury by co-administration of LPS. Nat Prod Commun.
5(9):1457–62.
Bailey RL, Gahche JJ, Miller PE, Thomas PR, Dwyer JT. 2013. Why US adults use dietary supple-
ments. JAMA Intern Med. 173(5):355–61. doi:10.1001/jamainternmed.2013.2299/.
Beekwilder J, van der Meer IM, Sibbesen O, Broekgaarden M, Qvist I, Mikkelsen JD, Hall RD.
2007. Microbial production of natural raspberry ketone. Biotechnol J. 2(10):1270–1279. doi:10.
1002/biot.200700076.
Bootsman N, Blackburn DF, Taylor J. 2014. The Oz craze. The effect of pop culture media on
health Care. Can Pharm J/Rev Pharm Can. 147(2):2–4. doi:10.1177/1715163514521965.
Bredsdorff L, Wedebye EB, Nikolov NG, Hallas-Møller T, Pilegaard K. 2015. Raspberry ketone in
food supplements–high intake, few toxicity data–a cause for safety concern? Regul Toxicol
Pharmacol. 73(1):196–200. doi:10.1016/j.yrtph.2015.06.022.
Burton-Freeman BM, Sandhu AK, Edirisinghe I. 2016. Red raspberries and their bioactive poly-
phenols: cardiometabolic and neuronal health links. Adv Nutr. 7(1):44–65. doi:10.3945/an.115.
009639.
Chan PC, Ramot Y, Malarkey DE, Blackshear P, Kissling GE, Travlos G, Nyska A. 2010.
Fourteen-week toxicity study of green tea extract in rats and mice. Toxicol Pathol. 38(7):
1070–1084. doi:10.1177/0192623310382437.
Choi BH, Jin Z, Yi CO, Oh J, Jeong EA, Lee JY, Park KA, Kim KE, Lee JE, Kim HJ, Hahm JR,
et al. 2018. Effects of lobeglitazone on insulin resistance and hepatic steatosis in high-fat diet-
fed mice. PLoS One. 13(7):e0200336. doi:10.1371/journal.pone.0200336.
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
JOURNAL OF DIETARY SUPPLEMENTS 13
Collier G, Traianedes K, Macaulay S, O’Dea K. 1993. Effect of fatty acid oxidation inhibition on
glucose metabolism in diabetic rats. Horm Metab Res. 25(01):9–12. doi:10.1055/s-2007-
1002035.
Cotten BM, Diamond SA, Banh T, Hsiao YH, Cole RM, Li J, Simons CT, Bruno RS, Belury MA,
Vodovotz Y. 2017. Raspberry ketone fails to reduce adiposity beyond decreasing food intake in
C57BL/6 mice fed a high-fat diet. Food Funct. 8(4):1512–8. doi:10.1039/C6FO01831A.
Crispim SP, Geelen A, Le Donne C. 2010. Dietary exposure to flavouring substances: from
screening methods to detailed assessments using food consumption data collected with EPIC-
Soft software. Food Addit Contam Part A. 27 (4):433–46. http://dx.doi.org/10.1080/
19440040903420614.
Deng X, Luyendyk JP, Ganey PE, Roth RA. 2009. Inflammatory stress and idiosyncratic hepato-
toxicity: hints from animal models. Pharmacol Rev. 61(3):262–282. doi:10.1124/pr.109.001727.
Editorial. 2018. Herbal assault: liver toxicity of herbal and dietary supplements. Lancet
Gastroenterol Hepatol. 3(2):141. DOI:https://doi.org/10.1016/S2468-1253(18)30011-6.
Gallois A. 1982. Quantitative evaluation of raspberry ketone using thin-layer chromatography. Sci
Aliments.]
Q4 Ganey PE, Luyendyk JP, Maddox JF, Roth RA. 2004. Adverse hepatic drug reactions: inflamma-
tory episodes as consequence and contributor. Chem Biol Interact. 150(1):35–51. doi:10.1016/j.
cbi.2004.09.002.
Hammad MA, Abdel-Bakky MS, Walker LA, Ashfaq MK. 2013. Tissue factor antisense deoxyoli-
gonucleotide prevents monocrotaline/LPS hepatotoxicity in mice. J Appl Toxicol. 33(8):774–83.
doi:10.1002/jat.2728.
Hernell O, Staggers JE, Carey MC. 1990. Physical-chemical behavior of dietary and biliary lipids
during intestinal digestion and absorption. 2. Phase analysis and aggregation states of luminal
lipids during duodenal fat digestion in healthy adult human beings. Biochemistry. 29(8):
2041–56. doi:10.1021/bi00460a012.
Hoffman GM. 2004. A 90-day dietary toxicity study of 4-(p-hydroxyphenyl)-2-butanone (rasp-
berry ketone) in rats. Unpublished report to the Research Institute of Fragrance Materials,
Woodcliff Lake, NJ, USA. Submitted to WHO by the International Organization of the Flavor
Industry, Brussels, Belgium.
Jeong JB, Jeong HJ. 2010. Rheosmin, a naturally occurring phenolic compound inhibits LPS-
induced iNOS and COX-2 expression in RAW264.7 cells by blocking NF- jB activation path-
way. Food Chem Toxicol. 48(8-9):2148–53. doi:10.1016/j.fct.2010.05.020.
Jia H, Liu JW, Ufur H, He GS, Liqian H, Chen P. 2011. The antihypertensive effect of ethyl acet-
ate extract from red raspberry fruit in hypertensive rats. Phcog Mag. 7(25):19–24. doi:10.4103/
0973-1296.75885.
Joint FAO WHO Expert Committee on Food Additives, World Health Organization. 2011. Safety
evaluation of certain food additives and contaminants: prepared by the Seventy fourth meeting
of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). https://apps.who.int/
iris/bitstream/handle/10665/171781/9789240693982_eng.pdf;jsessionid=79EFFBD803B75293027
EDA351F998A18?sequence=3.
Q5 Khan V, Sharma S, Bhandari U, Mansoor S, Ehtaishamul S. 2018. Raspberry ketone protects
against isoproterenol-induced myocardial infarction in rats. Life Sci. 194:205–12. doi:10.1016/j.
lfs.2017.12.013.
Kim JH, Kismali G, Gupta SC. 2018. Natural products for the prevention and treatment of
chronic inflammatory diseases: integrating traditional medicine into modern chronic diseases
care. Evidence Based Complement Alternat Med. ;2018:1–2. doi:10.1155/2018/9837863.
Kim KE, Jung Y, Min S, Nam M, Heo RW, Jeon BT, Song DH, Yi C-O, Jeong EA, Kim H, et al.
2016. Caloric restriction of db/db mice reverts hepatic steatosis and body weight with diver-
gent hepatic metabolism. Sci Rep. 6(1):30111. doi:10.1038/srep30111.
Kim WR, Flamm SL, Di Bisceglie AM, Bodenheimer HC. 2008. Serum activity of alanine amino-
transferase (ALT) as an indicator of health and disease. Hepatology. 47(4):1363–70. doi:10.
1002/hep.22109.
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
14 T. M. MIR ET AL.
Kristo A, Klimis-Zacas D, Sikalidis A. 2016. Protective role of dietary berries in cancer.
Antioxidants. 5(4):37. doi:10.3390/antiox5040037.
Lee J. 2016. Further research on the biological activities and the safety of raspberry ketone is
needed. NFS J. 2:15–18. doi:10.1016/j.nfs.2015.12.001.
Leu SY, Tsai YC, Chen WC, Hsu CH, Lee YM, Cheng PY. 2018. Raspberry ketone induces
brown-like adipocyte formation through suppression of autophagy in adipocytes and adipose
tissue. J Nutr Biochem. 56:116–25. doi:10.1016/j.jnutbio.2018.01.017.
Lin VCH, Ding HY, Kuo SY, Chin LW, Wu JY, Chang TS. 2011. Evaluation of in vitro and in
vivo depigmenting activity of raspberry ketone from Rheum officinale. Int J Mol Sci. 12(8):
4819–35. doi:10.3390/ijms12084819.
Lopez HL, Ziegenfuss TN, Hofheins JE, Habowski SM, Arent SM, Weir JP, Ferrando AA. 2013.
Eight weeks of supplementation with a multi-ingredient weight loss product enhances body
composition, reduces hip and waist girth, and increases energy levels in overweight men and
women. J Int Soc Sports Nutr. 10(1):22. doi:10.1186/1550-2783-10-22.
Maddox JF, Amuzie CJ, Li M, Newport SW, Sparkenbaugh E, Cuff CF, Pestka JJ, Cantor GH,
Roth RA, Ganey PE. 2010. Bacterial- and viral-induced inflammation increases sensitivity to
acetaminophen hepatotoxicity. J Toxicol Environ Health A. 73(1):58–73. doi:10.1080/
15287390903249057.
McGill MR, Jaeschke H. 2013. Metabolism and disposition of acetaminophen: recent advances in
relation to hepatotoxicity and diagnosis. Pharm Res. 30(9):2174–2187. doi:10.1007/s11095-013-
1007-6.
Mehanna ET, Barakat BM, ElSayed MH, Tawfik MK. 2018. An optimized dose of raspberry
ketones controls hyperlipidemia and insulin resistance in male obese rats: effect on adipose tis-
sue expression of adipocytokines and Aquaporin 7. Eur J Pharmacol. 832:81–89. doi:10.1016/j.
ejphar.2018.05.028.
Mezey E. 1999. Fatty liver. In: Schiff ER, Sorrell MF, Maddrey WC, editors. Schiff’s diseases of
the liver. Philadelphia (PA): Lippincott-Raven. p. 1185–97.
Morimoto C, Satoh Y, Hara M, Inoue S, Tsujita T, Okuda H. 2005. Anti-obese action of rasp-
berry ketone. Life Sci. 77(2):194–204. doi:10.1016/j.lfs.2004.12.029.
Mukai K, Miyagi T, Nishio K, Yokoyama Y, Yoshioka T, Saito Y, Tanaka S, Shigekawa M, Nawa
T, Hikita H, et al. 2016. S100A8 production in CXCR2-expressing CD11b þGr-1 high cells
aggravates hepatitis in mice fed a high-fat and high-cholesterol diet. J Immunol. 196(1):
395–406. doi:10.4049/jimmunol.1402709.
Noratto G, Chew BP, Ivanov I. 2016. Red raspberry decreases heart biomarkers of cardiac remod-
eling associated with oxidative and inflammatory stress in obese diabetic db/db mice. Food
Funct. 7(12):4944–4955. doi:10.1039/C6FO01330A.
Ogawa Y, Akamatsu M, Hotta Y, Hosoda A, Tamura H. 2010. Effect of essential oils, such as
raspberry ketone and its derivatives, on antiandrogenic activity based on in vitro reporter gene
assay. Bioorg Med Chem Lett. 20(7):2111–2114. doi:10.1016/j.bmcl.2010.02.059.
Opdyke D. 1978. 4-(p-Hydroxyphenyl)-2-butanone. Food Cosmet Toxicol. 16:781–2. doi:10.1016/
0015-6264(78)90079-2.
Park KS. 2010. Raspberry ketone increases both lipolysis and fatty acid oxidation in 3T3-L1 adi-
pocytes. Planta Med. 76(15):1654–8. doi:10.1055/s-0030-1249860.
Patel A, Rojas-Vera J, Dacke C. 2004. Therapeutic constituents and actions of rubus species. Curr
Med Chem. 11(11):1501–12. doi:10.2174/0929867043365143.
Poulsen KL, Olivero-Verbel J, Beggs KM, Ganey PE, Roth RA. 2014. Trovafloxacin enhances lipo-
polysaccharide-stimulated production of tumor necrosis factor- by macrophages: role of the
DNA damage response. J Pharmacol Exp Ther. 350(1):164–70. doi:10.1124/jpet.114.214189.
Saleh IG, Ali Z, Abe N, Wilson FD, Hamada FM, Abd-Ellah MF, Walker LA, Khan IA, Ashfaq
MK. 2013. Effect of green tea and its polyphenols on mouse liver. Fitoterapia 90:151–9. doi:10.
1016/j.fitote.2013.07.014.
Schinz H, Seidel CF. 1957. Untersuchungen €
uber Aromastoffe. 1. Mitteilung.
€
Uber das
Himbeeraroma. Helv Chim Acta. 40(6):1839–59. doi:10.1002/hlca.19570400635.
645
646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
JOURNAL OF DIETARY SUPPLEMENTS 15
Shaw PJ, Hopfensperger MJ, Ganey PE, Roth RA. 2007. Lipopolysaccharide and trovafloxacin
coexposure in mice causes idiosyncrasy-like liver injury dependent on tumor necrosis factor-
alpha. Toxicol Sci. 100(1):259–266. doi:10.1093/toxsci/kfm218.
Tsai YC, Yang BC, Peng WH, Lee YM, Yen MH, Cheng PY. 2017. Heme oxygenase-1 mediates
anti-adipogenesis effect of raspberry ketone in 3T3-L1 cells. Phytomedicine. 31:11–17. doi:10.
1016/j.phymed.2017.05.005.
Tu C, Gao D, Li XF, Li CY, Li RS, Zhao YL, Li N, Jia GL, Pang JY, Cui HR, Ma ZJ, et al. 2015.
Inflammatory stress potentiates emodin-induced liver injury in rats. Front Pharmacol. 6:
233.doi:10.3389/fphar.2015.00233.
Ulbricht C, Seamon E, Windsor RC, Armbruester N, Bryan JK, Costa D, Giese N, Gruenwald J,
Iovin R, Isaac R, et al. 2011. An evidence-based systematic review of cinnamon (Cinnamomum
spp.) by the natural standard research collaboration. J Diet Suppl. 8(4):378–454. doi:10.3109/
19390211.2011.627783.
Wang D, Wang Y, Wan X, Yang CS, Zhang J. 2015. Green tea polyphenol ()-epigallocatechin-
3-gallate triggered hepatotoxicity in mice: responses of major antioxidant enzymes and the
Nrf2 rescue pathway. Toxicol Appl Pharmacol. 283(1):65–74. doi:10.1016/j.taap.2014.12.018.
691
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728
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