JOURNAL OF MEDICINAL FOOD

J Med Food 00 (0) 2019, 1–7

# Mary Ann Liebert, Inc., and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2019.0189

Eryngium carlinae Ethanol Extract Corrects Lipid Abnormalities

in Wistar Rats with Experimental Diabetes
Ruth Noriega-Cisneros,1 Donovan J. Peña-Montes,2,3 Maribel Huerta-Cervantes,2 Rafael Torres-Martı́nez,2
Miguel Huerta,3 Salvador Manzo-Avalos,2 Rafael Salgado-Garciglia,2 and Alfredo Saavedra-Molina2
1
Faculty of Nursing; 2Institute of Chemistry and Biological Research;
Michoacan University of Saint Nicholas of Hidalgo, Morelia, México.
3
University Center for Biomedical Research, University of Colima, Colima, México.

ABSTRACT Abnormalities in lipid metabolism, associated with increased risk of cardiovascular disease (CVD), frequently
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occur in people with diabetes. Eryngium carlinae is a plant used in traditional medicine to treat lipid abnormalities. The
chemical composition and hypolipidemic activity of the ethanolic extract of E. carlinae were analyzed to broaden our
knowledge of its mechanism of action. The ethanolic extract of E. carlinae was tested for hypolipidemic activity by oral
administration for 40 days. Atorvastatin, a widely used statin, was also administered to compare its effect with that of the
extract. Serum was used for analysis of the lipid profile and liver microsomes to assess 3-hydroxy-3-methylglutaryl coenzyme
A (HMG-CoA) reductase activity and low-density lipoprotein receptor (LDL-r) levels. The extract was able to reduce total
cholesterol and non-high-density lipoprotein cholesterol (C-HDL) levels and increase the C-HDL levels reduced in diabetes,
decreasing the atherogenic index and therefore the risk of suffering CVD at the same level as atorvastatin. The HMG-CoA
reductase activity and LDL-r levels were not modified by the administration of E. carlinae. The results demonstrate the
hypolipidemic potential of ethanol extract of E. carlinae and support its use in traditional medicine as a hypolipidemic agent.

KEYWORDS:  atorvastatin  cholesterol  diabetes  lipids  medicinal plant

INTRODUCTION correct them.4 Atorvastatin, a statin, inhibits 3-hydroxy-3-

methylglutaryl coenzyme A (HMG-CoA) reductase, a key
D iabetes mellitus (DM) is a chronic and progressive
metabolic disorder characterized by hyperglycemia due
to absolute or relative deficiency of insulin.1 It is estimated
enzyme in cholesterol biosynthesis, lowering circulating
cholesterol levels.5 However, in chronic consumption of
statins, the appearance of drug interactions and adverse
that there are *425 million adults living with diabetes in the
effects is frequent.6
world, a figure that could reach 629 million by 2045. DM is
Although a wide variety of medicines are currently
a risk factor for the development of cardiovascular disease
available to treat dyslipidemia and other conditions, people
(CVD), which in turn is the main cause of death of people
around the world maintain a strong attachment to the use of
with diabetes.2 Abnormalities in lipoprotein metabolism,
medicinal plants to cure diseases. Their use is based on
associated with increased risk of CVD, are frequent in
knowledge acquired with their consumption and transmitted
people with diabetes. Among the characteristics of diabetic
from generation to generation, becoming part of the culture
dyslipidemia are low levels of high-density lipoprotein
of a population.7 Therefore, knowing that the plants are used
cholesterol (C-HDL) and high levels of triglyceride (TG)
without any prescription, we cannot neglect to consider
and low-density lipoprotein cholesterol (C-LDL).3 Among
the effect they may have when consumed either alone or in
the actions that contribute to preventing or delaying CVD
conjunction with conventional medicine. Hence, it is im-
in people with diabetes is controlling glucose and dyslipi-
portant to study medicinal plants to ensure their effective-
demias.2 In addition to diet and exercise, medication to
ness and safety in consumption. Many plants are used in
treat lipid disorders is necessary for most patients with
traditional medicine to treat diabetes; however, few plants
diabetes, and statins are the drugs most commonly used to
have proven their effectiveness, such as Ocimum tenui-
florum L. and Trigonella foenum-graecum L., whose use are
Manuscript received 21 August 2019. Revision accepted 23 October 2019. supported by clinical data.8 The purpose of this study was
to contribute to the evidence on the beneficial effects of
Address correspondence to: Alfredo Saavedra-Molina, PhD, Instituto de Investigaciones
Quı´mico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-3.
consuming the ethanolic extract of Eryngium carlinae
C.U., Morelia 58030, México, E-mail: saavedra@umich.mx F. Delaroche (EC) to improve lipid alterations in diabetes.

1
 2 NORIEGA-CISNEROS ET AL.
E. carlinae is a plant used in traditional medicine in Mexico;
it is commonly known as ‘‘frog herb,’’ and decoctions of
the aerial part of the plant are consumed as ‘‘use water.’’
Previous research has shown that the hexanic extract of
inflorescences of E. carlinae has an antioxidant effect in
vitro and hypoglycemic and antioxidant effects in diabetic
rats.9,10 In another study, a hydroalcoholic extract of the
aerial part of E. carlinae was administered to hypercho-
lesterolemic mice, and a decrease in total cholesterol (TC)
and non-C-HDL levels was observed, without altering
C-HDL levels. Toxicity tests showed no damage to hepa-
tocytes.11 Regarding the ethanolic extract of the aerial part
of E. carlinae, we previously reported that it decreased
levels of creatinine, uric acid, TC, and TG.12 However, we
consider it necessary to deepen the analysis of the lipid
profile, since it is considered more relevant when assessing
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the risk of a cardiovascular event to examine the levels of
C-HDL and non-C-HDL than only TC.13 This is because
some patients with diabetes may have normal TC levels but
low C-HDL levels, which increase their atherogenic index
(AI) and cardiovascular risk. In this study we also per-
formed an analysis of the composition of the ethanolic
extract of E. carlinae, with the aim of identifying com-
pounds responsible for its action. Along with administra-
tion of E. carlinae, atorvastatin was administered to
compare its effect with that of the extract.
MATERIALS AND METHODS
Plant material
Aerial parts (stems, leaves, and flowers) from Eryngium
carlinae plants were collected in Nuevo San Juan Para-
ngaricutiro, Michoacán, México (1934’45’’N, 10217’30’’W,
at an altitude of 1885 meters above sea level). The species
was identified by Miguel Angel Bello González, PhD, of the
Agrobiology Faculty of Universidad Michoacana de San
Nicolás de Hidalgo (UMSNH). A voucher specimen was
registered with no. 15214 at the Biology Faculty Herbarium
of UMSNH.
Preparation of the ethanol extract
Aerial parts (stems, leaves, and flowers) from E. carlinae
plants were collected and dried at room temperature in the
absence of light. Subsequently, the aerial parts were pulver-
ized to obtain a fine powder, and the extract was obtained
with ethanol maceration (10 mL of absolute ethanol per gram
of plant powder) at 5C for 5 days. The extract was then
filtered (Whatman no. 1 filter paper), and ethanol was vacuum
evaporated to dryness at 45C. The residue was dissolved in
ethanol 96% at 1 g/mL and stored in the dark at 5C.
Gas chromatography/mass spectrometry analysis
The whole extract was analyzed by gas chromatogra-
phy/mass spectrometry (GC/MS). GC/MS analysis was
performed on an Agilent 6890 gas chromatograph coupled
with an Agilent 5973 mass spectrometer using an Equity-5
capillary column 30 m · 0.25 mm · 0.25 lm (Supelco). The
temperature programmed for gas chromatography was as
follows: initial temperature of 50C held for 13 min, linear
gradient of 5C/min to 300C, isocratic gradient, and a final
hold time of 30 min. The injector temperature was 250C,
and injection was performed in a split ratio of 1/30. The
carrier gas was helium (99.99% purity, 1.0 mL/min). The
injection volume of each sample was 1 lL. Retention indi-
ces for all compounds were determined using n-alkanes (C8
to C20) as standards. Compounds were identified by com-
paring their MS spectra with the NIST 98 Mass Spectral
Library (National Institute of Standards and Technology)
and by comparing their retention indices with those de-
scribed by Adams.14 Quantification of the peaks was per-
formed automatically with environmental ChemStation
software (Agilent Technologies) using the method area. The
major compounds detected by GC/MS are shown as per-
centage of abundance.
Animals
Male Wistar rats weighing 290–400 g were used. The
animals were housed and maintained at room temperature
with 12-h day/12-h night cycles. They were fed with
standard rodent diet and water ad libitum. To conduct this
study, we considered the recommendations of the regula-
tory standard for the use of animals issued by the Mexican
Ministry of Agriculture in the paragraph of the Federal
Regulations for the Use and Care of Animals (NOM-062-
ZOO-1999). The Institutional Committee for Use of An-
imals of UMSNH approved this study as well. Diabetes
was induced by intraperitoneal administration of strepto-
zotocin (STZ) (45 mg/kg body weight). STZ was dissolved
in citrate buffer (pH 4.5). Control rats were injected with
citrate alone. Five days after STZ administration, glucose
levels were determined to confirm hyperglycemia, and
ranking blood glucose levels >300 mg/dL were considered
for the study.12
Experimental protocol
Rats were randomly divided into six groups: Group I,
control (during the experimental period received only ve-
hicle, ethanol 50%); Group II, control+EC30 (received
30 mg/kg body weight of ethanol extract [E. carlinae]);
Group III, control+ator (received atorvastatin 10 mg/kg
body weight); Group IV, diabetic (received only vehicle,
ethanol 50%); Group V, diabetic+EC30 (received 30 mg/kg
body weight of E. carlinae); and Group VI, diabetic+ator
(received 10 mg/kg body weight of atorvastatin). Vehicle,
ethanol extract, and atorvastatin (Pfizer) were administered
orally once per day in the morning for 40 days. All animals
were weighed every 5 days throughout the experiment to
determine changes in body weight. Water and food con-
sumption were recorded daily. At the end of treatment,
night-fasted rats were euthanized by decapitation. The blood
was collected, and the serum was separated and used for
biochemical estimations. The liver was quickly removed,
 ERYNGIUM CARLINAE EXTRACT IN DIABETES 3

weighed, and placed in medium 1 (220 mM mannitol,
70 mM sucrose, 2 mM MOPS, 1 mM EGTA; pH 7.4) to
obtain microsomes.
Determination of glucose, lipid profile, and AI
Glucose, TC, TG, and C-HDL in serum were measured
spectrophotometrically using a Commercial Assay Kit
(BioSystems, Spain). Determination of glucose is based
on glucose oxidase catalyzing the oxidation of glucose to
gluconic acid. Then, the formed hydrogen peroxide reacts
with phenol and 4-aminophenazone in the presence of per-
oxidase. The intensity of the color formed is proportional to
the glucose concentration in the sample. To determine TC,
first esterified cholesterol is hydrolyzed by cholesterol es-
terase. Then, free cholesterol is oxidized by cholesterol ox-
idase. As in the previous method, the hydrogen peroxide
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EDTA, 10 mM DTT; pH 6.8) with 0.2 mM NADPH and
0.1 mM RS-HMG-CoA. One unit of enzymatic activity is
considered equivalent to the synthesis of 1 nmol of meva-
lonate per minute.
Immunodetection of hepatic LDL receptor
For immunodetection of hepatic low-density lipoprotein
receptor (LDL-r), the microsomal pellet was suspended in
SDS-lysis medium 4 (1% [w/v] SDS, 100 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 10 mM Tris-HCl; pH 6.8). One aliquot
was used to estimate the protein concentration. Samples
containing 45 lg protein were mixed with an equal volume
of medium 5 (15% SDS, 8 M urea, 10% [v/v] glycerol,
100 mM DTT, Tris-HCl; pH 6.8) and resolved on 10% SDS-
PAGE, then blotted on polyvinylidene difluoride membranes
(Amersham Biosciences). The membranes were blocked with

formed reacts with phenol and 4-aminoantipyrine in the 5% nonfat milk-TBS-T for 1 h at room temperature and then
presence of peroxidase. The intensity of the color formed is incubated with an anti-LDLr antibody (goat-polyclonal-IgG,
proportional to the cholesterol concentration in the sample. Santa Cruz Biotechnology) diluted at 1:1000. The membranes
In TG determination, the sample reacts with enzyme lipase were incubated with peroxidase-labeled secondary antibody
to obtain free glycerol. Glycerol is subjected to the action (donkey anti-goat IgG-HRP, Santa Cruz Biotechnology) di-
of two enzymes, glycerol kinase and glycerol-3-phosphate luted at 1:2000. Immunoreactive proteins were detected with a
oxidase, producing hydrogen peroxide. The glycerol is then chemiluminescence assay (Western blotting Luminol reagent,
determined by the colored complex formed by the reaction Santa Cruz Biotechnology). Specific bands were quantified by
between hydrogen peroxide and 4-aminoantipyrine and 4- densitometry using ImageJ software v. 1.46h.
chlorophenol. To determine C-HDL, very low-density lipo-
proteins (VLDLs) and low-density lipoproteins in the sample Statistical analysis
are precipitated with phosphotungstate and magnesium ions.
The results were expressed as mean – standard error. The
The supernatant that contains high-density lipoproteins is
data were statistically analyzed by applying Student’s t-test
used for determination. C-HDL is then measured by means of
to assess significant differences between two groups using
the coupled reactions, similar to the cholesterol determina-
GraphPad Prism v.5 software, and values of P < .05 were
tion described above. Non-C-HDL and AI were calculated,
considered to be significant.
the former by subtracting C-HDL from TC and the latter by
calculating the ratio of TC and C-HDL.15,16
RESULTS
Preparation of microsomes Composition of ethanolic extract of E. carlinae
Each rat liver was homogenized at 4C in medium 1 with Analysis of the chemical composition of the ethanol
a motor-driven glass Teflon homogenizer. The microsomes extract of E. carlinae was made by GC/MS, and the results
were prepared according to the method described by Honda are shown in Table 1. The most abundant compound was
et al.17 with some modifications. The homogenate was b-selinene (26.04%), followed by a-selinene (17.54%),
centrifuged for 10 min at 700 g, and the supernatant was stearic acid (14.54%), and palmitic acid (14.43%).
centrifuged at 7000 g for 10 min. The supernatant was then
centrifuged at 105,000 g for 90 min at 4C, the pellet was Table 1. Separation and Identification of the Most
suspended in medium 2 (100 mM sucrose, 50 mM KCl, Abundant Compounds in Ethanol Extract of Eryngium
40 mM KH2PO4, 30 mM EDTA; pH 7.2), and DTT was carlinae by Gas Chromatography/Mass Spectrometry
added to a final concentration of 10 mM. The microsomal
suspension was divided into aliquots and stored at -20C No. Ia Compounds % of abundance
until use. Protein concentrations of the microsomal sus- 1 1322.14 Elemene 1.61
pension were determined by the Lowry method.18 2 1387.28 Humulene 2.81
3 1419.60 b-Selinene 26.04
4 1426.97 a-Selinene 17.54
Assay of solubilized HMG-CoA reductase 5 1432.42 a-Cedrene 1.10
The assay was carried out using a spectrophotometric 6 1473.13 Elemol 1.78
method described by Edwards et al.19 with several modifi- 7 1857.16 Palmitic acid 14.43
8 2056.88 Stearic acid 14.54
cations for optimization. Previous solubilization of the en- 9 3113.35 Stigmasterol 2.12
zyme and activity of HMG-CoA reductase were assayed
using medium 3 (20 mM KCl, 160 mM KH2PO4, 4 mM a
Kovats retention index.
 4 NORIEGA-CISNEROS ET AL.

Table 2. Effect of Ethanolic Extract of Eryngium carlinae and Atorvastatin on Water and Food Intake,
Food Efficiency Ratio, and Percentage of Gained Weight at the End of 40 Days Administration

Group Initial weight (g) Final weight (g) Gained weight (%) Food intake (g/rat/day) Water intake (mL/rat/day) FER
Control 343 – 16 451 – 17 31 25.2 – 0.3 42 – 1 0.095
Control+EC30 338 – 14 437 – 26 29 25.0 – 0.3 33 – 1+ 0.088
Control+ator 332 – 19 415 – 25 23 26.4 – 0.6 51 – 2+ 0.070
Diabetic 304 – 12 297 – 09 -2 46.3 – 0.8+ 180 – 4+ -0.004
Diabetic+EC30 292 – 11 276 – 11 -4 48.6 – 0.6* 194 – 4* -0.006
Diabetic+ator 335 – 15 314 – 24 -6 55.2 – 0.8* 239 – 4* -0.009

Values represent the mean – SE. Control and diabetic groups n = 9–12, control+EC30 group n = 5–7, diabetic+EC30 group n = 9–14, control+ator group n = 4, and
diabetic+ator group n = 3–4. EC30: represents ethanol extract of E. carlinae in dose of 30 mg/kg weight. Ator: represents atorvastatin given in dose of 10 mg/kg
weight. FER represents food intake (g)/gained weight (g). +P < .05 versus control group. *P < .05 versus diabetic group.
FER, food efficiency ratio; SE, standard error.

Effect of ethanolic extract of E. carlinae on weight diabetic rats (Table 2), which suggests that there was no
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gain, water and food intake, and food efficiency index
Control groups presented weight gain that was higher
than groups without treatment (31%), and lower gain was
observed in the group that received atorvastatin (23%)
(Table 2). A similar trend occurred in the diabetic groups,
except in the majority; there was no gain but loss of weight.
The untreated diabetic group had a weight loss of 2%, and
the diabetic group that received atorvastatin had greater
weight loss (6%). With these results, we can assume that
the weight loss observed in the diabetic groups reflects
good establishment of the diabetes model. Water con-
sumption was similar among control and control+EC30
groups. The control+ator group had increased water con-
sumption compared to the control group without treatment
(P < .05). In the diabetic groups, water consumption was
higher compared with control groups. In the diabetic
groups receiving EC30 or atorvastatin, consumption was
significantly increased (P < .05) compared to the untreated
diabetic group. The same trend was observed in the con-
sumption of food; the diabetic groups consumed more food
than the control groups. These data also support the es-
tablishment of the diabetic condition. Both treatments,
EC30 and ator, increased food consumption (P < .05).
However, although diabetic rats consumed more food, the
food efficiency ratio (FER) was low and was negative in
absorption of the large amount of food they consumed
because of the diabetic condition; furthermore, this dif-
ference in food consumption in diabetic rats may be re-
sponsible for the observed increase in serum lipids.
Effect of ethanolic extract of E. carlinae
on glucose, lipid profile, and AI
High glucose levels in the diabetic groups confirmed the
induction of experimental diabetes (Table 3). The treatment
for 40 days with E. carlinae extract did not alter blood sugar
levels as reported previously.12 In the diabetic group that
received atorvastatin, blood glucose levels were increased
(P < .05). The effects of EC30 and atorvastatin administra-
tion on the lipid profile and AI are shown in Table 3. The
increased glucose levels in the diabetic group were ac-
companied by increased TG levels and decreased C-HDL
(P < .05), which are reflected in a high AI (6) and therefore
a greater risk of suffering a cardiovascular event.20 As pre-
viously reported, treatment with E. carlinae extract lowers
TC and TG levels.12 This study shows that it can also reduce
non-C-HDL and increase C-HDL (P < .05). We were able to
observe a decreased AI (3) to desirable levels and, therefore,
also decreased odds of suffering a cardiovascular event.
These effects were comparable with those produced by the
drug atorvastatin, which also presented a desirable AI (3) in

Table 3. Effect of the Ethanolic Extract of Eryngium carlinae and Atorvastatin on Glucose and Lipid Profile
at the End of 40 Days Oral Administration

Concentration (mg/dL)
Group Glucose TC TGs C-HDL Non-C-HDL AI
Control 76 – 6 68 – 3 63 – 3 22 – 1 47 – 6 3
Control+EC30 78 – 4 66 – 6 46 – 6+ 20 – 1 45 – 7 3
Control+ator 74 – 18 30 – 18+ 112 – 11+ 15 – 2+ 16 – 9+ 2
Diabetic 397 – 30 74 – 7 224 – 40+ 13 – 2+ 61 – 7 6
Diabetic+EC30 373 – 27 55 – 4* 123 – 23* 20 – 2* 35 – 4* 3
Diabetic+ator 564 – 69* 47 – 14 141 – 5 15 – 2 32 – 8* 3

Values represent the mean – SE. Control and diabetic groups n = 9–12, control+EC30 group n = 5–7, diabetic+EC30 group n = 9–14, control+ator group n = 4, and
diabetic+ator group n = 3–4. EC30: represents ethanol extract of E. carlinae in dose of 30 mg/kg weight. Ator: represents atorvastatin administered at a dose of
10 mg/kg weight. AI: TC/C-HDL. +P < .05 versus control group, *P < .05 versus diabetic group.
AI, atherogenic index; C-HDL, high-density lipoprotein cholesterol; TC, total cholesterol; TGs, triglycerides.
 ERYNGIUM CARLINAE EXTRACT IN DIABETES
the group that received it. Control groups with or without
therapy showed a low AI (between 2 and 3) since their TC
and non-C-HDL levels were low; therefore, they were not at
risk for a cardiovascular event (Table 3).
Effect of ethanolic extract of EC30 on HMG-CoA
reductase activity
Atorvastatin was administered to a diabetic group and a
control group to compare its effect on HMG-CoA reductase
with the ethanolic extract of E. carlinae. The results are
shown in Figure 1. Both groups, control and diabetic, that
received the ethanolic extract of E. carlinae for 40 days had
no significant changes in the activity of the enzyme HMG-
CoA reductase, which suggests that the hypolipidemic effect
of ethanolic extract of E. carlinae is not through the re-
duction of activity of this enzyme and therefore of the
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synthesis of cholesterol. In contrast, it should be noted that
both the control group and diabetic group that received
atorvastatin for 40 days had a significant decrease (P < .05)
in the activity of the enzyme (Fig. 1), which is the mecha-
nism of action of statins, lowering cholesterol levels, a
phenomenon observed in our work. It has been reported that
atorvastatin has a prolonged effect by inhibiting the bio-
synthesis of cholesterol compared to other statins.21
Effect of ethanolic extract of EC30 on LDL-r
protein level
The results of Figure 2 show that hepatic LDL-r levels did
not vary significantly in rats in either control or diabetic
groups that received the ethanolic extract of E. carlinae for
40 days. This result suggests that the ethanolic extract of
E. carlinae does not exert its effect through variation of the
number of receptors that capture the LDL, reducing circu-
lating cholesterol. It is therefore necessary to explore more
metabolic points that could be modified as a consequence of
FIG. 1. Effect of ethanolic extract of Eryngium carlinae on HMG-
CoA reductase activity in rat liver microsomes. Different rat groups
were treated with ethanol extract of E. carlinae or with atorvastatin
for 40 days. At the end of the treatment, rats were sacrificed, and liver
used to obtain microsomes used on enzymatic activity determination.
Values are mean – SE. Control groups n = 5–7, diabetic groups n = 3–
6. EC30: ethanol extract of E. carlinae (30 mg/kg weight). Ator:
atorvastatin (10 mg/kg weight). *P < .05 versus control without
treatment. +P < .05 versus diabetic without treatment. HMG-CoA, 3-
hydroxy-3-methylglutaryl coenzyme A; SE, standard error.
5
FIG. 2. Effect of ethanolic extract of E. carlinae on LDL-r levels in
rat liver microsomes. Densitometric analysis and Western blot rep-
resentative of hepatic LDL-r in control and diabetic groups treated
with ethanol extract of E. carlinae (EC30) or with atorvastatin (ator)
for 40 days. At the end of the treatment, rats were sacrificed, and liver
used to obtain microsomes to determine LDL-r levels. Values are
mean – SE of at least three independent experiments. EC30: ethanolic
extract of E. carlinae (30 mg/kg weight). Ator: atorvastatin (10 mg/kg
weight). *P < .05 versus control without treatment. Receptor protein
quantification was calculated by densitometric analysis and normal-
ized with actin as internal standard. LDL-r, low-density lipoprotein
receptor.
the extract. In the control groups that received atorvastatin,
we could observe a significant decrease in the number of
receptors (P < .05), and although the trend was similar in the
diabetic group, the decline was not significant (Fig. 2).
DISCUSSION
Phytochemical analysis of the ethanolic extract of
E. carlinae reveals as major components sesquiterpenes (b-
selinene, a-selinene, humulene, elemene, and a-cedrene)
reported to have anti-inflammatory,22 antiproliferative,23
hypolipidemic,24 and antimicrobial activity.25–27 Two sat-
urated fatty acids were found, palmitic acid and stearic acid.
The presence of saturated fatty acids is associated with el-
evated levels of cholesterol in serum.28 However, stearic
acid, as opposed to other saturated fatty acids such as pal-
mitic acid, does not increase TC serum or C-LDL levels.
Stigmasterol, a phytosterol, was also identified. Stigmas-
terol reduces plasma cholesterol levels through inhibition of
hepatic synthesis and its intestinal absorption29 making it,
along with elemene, a compound that is probably related
to the hypolipidemic effect found in the ethanolic extract of
E. carlinae.
Weight gain, blood glucose, and food and water con-
sumption were recorded during treatment of the rats, and
FER was calculated (Table 1). Groups of control rats gained
weight throughout the treatment as part of their normal
growth. In contrast, groups of diabetic rats did not register
 6 NORIEGA-CISNEROS ET AL.
weight gain; on the contrary, some weight loss was ob-
served, which is characteristic of the diabetes model. The
greater weight loss (or lower gain) observed in the groups
that received atorvastatin coincides with other reports where
atorvastatin caused weight reduction when administered to
normal rabbits.30 However, the effect of atorvastatin can be
complex, as it has been reported to prevent weight loss
caused by diabetes.31 The comparison between the records
of water and food consumption of control and diabetic
groups highlights the characteristic symptoms of diabetes,
polyphagia, and polydipsia, corroborating the establishment
of the diabetes model. Such symptoms were increased in the
groups that received ethanolic extract of E. carlinae or
atorvastatin. However, despite the higher consumption of
food, they had weight loss and lower FER due to impaired
food absorption or inability to metabolize carbohydrates due
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to the diabetic condition.
The effect of ethanolic extract of E. carlinae adminis-
tration on the lipid profile and AI is shown in Table 2. As
reported in a previous work,12 consumption of the ethanolic
extract of the aerial part of E. carlinae does not modify
glucose levels; however, other studies conducted with a
hexane extract obtained from inflorescences of E. carli-
nae9,10 showed that it is capable of lowering glucose levels
when administered to diabetic rats in the same concentration
as the ethanolic extract (30 mg/kg body weight). This is
possibly due to the different compounds that are extracted
using different solvents (ethanol or hexane); however, it is
not yet attributed to a specific compound. Although it is
generally considered that the C-LDL level must be the
therapeutic target to control dyslipidemia and reduce car-
diovascular risk, epidemiological studies have shown that
non-C-HDL and Apo B may be better predictors of car-
diovascular events.32,33 In our study, the ethanolic extract of
E. carlinae was able to reduce not only TC but also non-C-
HDL, and also increase C-HDL, contributing to a reduction
in the AI at the same level as atorvastatin, one of the most
used statins. The TG elevation observed in the diabetic
group without treatment (Table 3) may be due to the diabetic
condition. This generates an inability to take carbohydrates
and causes increased lipolysis in adipose tissue, therefore
increased circulating TG. The reduction of TG observed
in the diabetic group that received the ethanolic extract of
E. carlinae may be favored if the amount of Apo B, essential
for the formation of VLDL, is compromised, but this re-
mains to be clarified.
The microsomal enzyme HMG-CoA reductase catalyzes
the synthesis of mevalonate, a metabolic precursor of cho-
lesterol. This is a limiting step in the biosynthesis of cho-
lesterol34; hence it has been used as a therapeutic target.
Statins are inhibitors of HMG-CoA reductase, and they are
currently widely used to lower cholesterol levels by reduc-
ing their synthesis and increasing the numbers of hepatic
and extrahepatic LDL receptors, which leads to increased
uptake and catabolism of LDL. It is noteworthy that for a
time it was considered that the efficacy of statins in lowering
cholesterol levels was due to their ability to remove LDL
from serum by increasing the function of hepatic LDL-r.35
The mechanism of action of the extract differs from that of
atorvastatin, because it does not act on HMG-CoA reductase
(Fig. 1). The beneficial effects are attributed to the content
of stigmasterol in the extract, which has been seen to
compete with cholesterol for intestinal absorption.29
CONCLUSIONS
Ethanol extract of E. carlinae administered for 40 days
to STZ-induced diabetic rats was able to reduce the levels
of TC, TG, and non-C-HDL and increase serum levels of
C-HDL in similar or even greater magnitude than atorvas-
tatin, which was reflected as a decrease in the AI, therefore
representing a lower CVD risk in diabetes.
ACKNOWLEDGMENT
The authors express their gratitude to Yolanda Garcı́a for
her GC-MS analytical support.
AUTHOR DISCLOSURE STATEMENT
No competing financial interests exist.
FUNDING INFORMATION
Mexican grants supported this work: Coordinación de
Investigación Cientı́fica. UMSNH (2.10 to R.S.-G.; 2.16 to
A.S.-M.). R.N.-C. was CONACYT fellow.
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