Journal of Ethnopharmacology 296 (2022) 115473

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Loquat (Eriobotrya japonica (Thunb) Lindl.): Evaluation of nutritional value,

polyphenol composition, antidiabetic effect, and toxicity of leaf
aqueous extract
Tarik Khouya a, *, Mhamed Ramchoun a, b, Hamza Elbouny a, Abdelbassat Hmidani a,
Eimad dine Tariq Bouhlali a, c, Chakib Alem a
a
Biochemistry and Natural Substances Team, Department of Biology, Faculty of Sciences & Techniques, University Moulay Ismail, Errachidia, 52000, Morocco
b
Laboratory of Biotechnology & Sustainable Development of Natural Resources, Polydisciplinary Faculty, Beni Mellal, 23000, Morocco
c
National Institute of Agronomic Research Regional Center, Errachidia, 52000, Morocco

A R T I C L E I N F O A B S T R A C T

Keywords: Ethnopharmacological relevance: Loquat (Eriobotrya japonica (Thunb.) Lindl.) is an evergreen tree native to China,
Eriobotrya japonica which is introduced in many Mediterranean countries. As in many ancient medical systems, loquat leaves have
Micronutrients been used in Moroccan traditional medicine to treat diabetes and its complications.
Polyphenols
Aim of the study: This study aims to determine the nutritional and polyphenol composition and to evaluate the in
Diabetes
vivo antidiabetic, and antihyperlipidemic properties and oral toxicity of a leaf aqueous extract (LLE) derived from
Hyperlipidemia
Mice loquat grown in Morocco.
Materials and methods: Energy value and fiber, fatty acids, minerals, vitamins, total carbohydrate, sugar, lipid,
and protein contents were determined according to international methods committee guidelines. Polyphenol
profiling was carried out using the HPLC-DAD method. Mice fed a high-fat and high-glucose (HFG) diet for 10
weeks were used as a model to assess the antidiabetic and antihyperlipidemic effects of a daily administration of
LLE at three different doses (150, 200, 250 mg/kg body weight (BW)/day), in comparison with metformin (50
mg/kg BW/day) and pravastatin (20 mg/kg BW/day). The oral toxicity was determined following OECD 425
Guideline.
Results: Loquat leaves were found to be rich in fiber, minerals (potassium, calcium, magnesium, iron, and so­
dium), and vitamins (B2, B6, and B12) and lower in energy, sugar, and fat. Ten different phenolic compounds
were characterized. Naringenin, procyanidin C1, epicatechin, and rutin were the more abundant compounds in
LLE. The administration of the LLE dose-dependently ameliorated hyperglycemia, insulin resistance, oxidative
stress, and hyperlipidemia in HFG diet-fed mice. The median lethal dose of LLE was higher than 5000 mg/kg BW.
Conclusions: Loquat leaves are a potential source of micronutrients and polyphenols with beneficial effects on
diabetes and its complications.

Parhofer, 2014). High blood glucose is often associated with several

1. Introduction
Diabetes mellitus, commonly known as diabetes, is a group of
chronic physiological disorders mainly manifested by high blood sugar
due to insulin resistance or insulin deficiency. Untreated diabetes results
in serious complications especially cardiovascular diseases (Caussy
et al., 2021). Hyperlipidemia is known as the main link between diabetes
and increased cardiovascular risks in diabetic patients (Wu and
lipid abnormalities, including elevated levels of plasma total cholesterol
(TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C),
and decreased levels of high-density lipoprotein cholesterol (HDL-C)
(Wu and Parhofer, 2014).
Physiologically, insulin regulates lipid and glucose metabolism as
well as lipid transport. Moreover, insulin resistance led to lipid and
sugar accumulation and thereby promotes obesity, a serious risk factor
for heart diseases (Caussy et al., 2021). Excess lipid accumulation

* Corresponding author. Laboratory of Biochemistry, Department of Biology, Faculty of Sciences & Techniques, University Moulay Ismail, 52000, Errachidia,
Morocco.
E-mail addresses: tarikkhouya@yahoo.com (T. Khouya), ramchoun_10@yahoo.fr (M. Ramchoun), elbouny.hamza@gmail.com (H. Elbouny),
abdelbassathmidani@gmail.com (A. Hmidani), bouhlali.eimad@gmail.com (E.T. Bouhlali), alem04@yahoo.fr (C. Alem).

https://doi.org/10.1016/j.jep.2022.115473
Received 2 February 2022; Received in revised form 5 June 2022; Accepted 13 June 2022
Available online 17 June 2022
0378-8741/© 2022 Elsevier B.V. All rights reserved.
T. Khouya et al.
Abbreviations
DE dried extract
HDL-C high-density lipoprotein cholesterol
HFG high fat/high-glucose
LDL-C low-density lipoprotein cholesterol
LLE loquat leaf aqueous extract
MDA malondialdehyde
TG triglycerides
VLDL very-low-density lipoprotein
increases oxidative metabolism, which results in an overproduction of
reactive oxygen species (ROS). ROS can either induce insulin resistance
by disrupting insulin receptor signal transduction or cause insulin
deficiency by attacking insulin-producing cells (Geisler and Renquist,
2017). Lipids and lipoproteins are highly prone to oxidation resulting in
toxic products such as oxidized-LDL (ox-LDL) and malondialdehydes
(MDA) (Wang et al., 2019).
Controlling diet, lowering blood glucose, and correcting lipid
metabolic disorders are the main therapeutic strategies to manage dia­
betes and its complications (Namazi et al., 2021; Wu and Parhofer,
2014). High-energy foods are associated with body weight (BW) gain,
hyperlipidemia, and diabetes (Moreno-Fernández et al., 2018). How­
ever, micronutrient-rich and low-energy foods have been recommended
for subjects suffering from glucose and lipid metabolic disorders (Mar­
tini et al., 2010). Micronutrients play an important role in the
improvement of diabetes and hyperlipidemia as they are essential ele­
ments in glucose and lipid metabolism (Martini et al., 2010).
Despite significant advances in the management of diabetes with oral
antihyperglycemic drugs, the search for effective active herbal phar­
maceutical ingredients continues as the available synthetic drugs show
various limitations. Hundreds of plants have been recognized as anti­
diabetic remedies and are still used in traditional medicine (Naza­
rian-Samani et al., 2018). The antidiabetic effects of plants are
attributed to their bioactive phytochemicals, especially polyphenols.
Several polyphenols have been found to correct metabolic disturbances
related to diabetes (Qin et al., 2021). In addition, the plants are rich in
micronutrients (Shergill-Bonner, 2017). Thus, plants are a potential
source of safe and effective compounds as alternatives or supplements to
conventional antidiabetic and hypolipidemic drugs (Nazarian-Samani
et al., 2018).
Loquat (Eriobotrya japonica (Thunb.) Lindl.), which is a subtropical
evergreen tree native to China, is cultivated in different regions around
the globe for its delicious and nutrient-rich fruit. Loquat was considered
an ancient remedial plant in many traditional systems of medicine to
potentially treat diabetes mellitus, heart diseases, and various inflam­
matory diseases (Häkkinen, 2007). In Morocco, drinks prepared by
infusion or decoction from loquat (locally name Mzah) leaves have been
used as hypoglycemic remedies for subjects with diabetes and its related
cardiovascular complications (Ziyyat et al., 1997).
Loquat leaves have been reported to have several biological activities
including antioxidant, antitumoral, antinociceptive, antiinflammatory,
antarthritic, and antibacterial properties (Cha et al., 2011; Kur­
aoka-Oliveira et al., 2020). The studies reporting the antidiabetic effect
of loquat leaves were more focused on sesquiterpene-rich extracts. It has
been shown that the sesquiterpene glycoside-rich extracts from loquat
showed a hypoglycemic effect in alloxan-induced diabetic rats (Wu
et al., 2021).
Moreover, there are few studies on the polyphenol-rich extract from
loquat leaves and their pharmacological effects. Loquat was introduced
in Morocco in the sixties and is today intensively cultivated in many
regions of the country for the economic importance of its fruit (Marouani
et al., 2021). However, there is very little available information about
2
Journal of Ethnopharmacology 296 (2022) 115473
the chemical composition and the pharmacological value of the loquat
cultivar growing in Morocco.
The present study was designed to determine the nutritional and
polyphenol composition and to evaluate the in vivo antidiabetic, and
antihyperlipidemic properties and oral toxicity of a leaf aqueous extract
(LLE) derived from a loquat cultivar grown in Morocco.
2. Materials and methods
2.1. Reagents and drugs
LDL-C and HDL-C quantification kit (cat. no. Z5030057) and mouse
MDA ELISA kit (cat. no. AE33973MO-480) were purchased from Hex­
aBiogen (Marrakech, Maroc). The RTU (cat. no. 61218) and PAP 150
(cat. no. 61236) enzymatic kits were purchased from BioMérieux
(Charbonnières-les-Bains, France). Mouse insulin Elisa kit (cat. no.
A05105) was supplied by SpiBio (Montigny-le-Bretonneux, France). All
other chemical compounds were purchased from Sigma Aldrich
(Verpillière, France).
2.2. Plant material
The leaves of loquat (Eriobotrya japonica (Thunb.) Lindl.) were
collected from Zegzel valley located in the Beni Snassen Mountains,
Berkane Province, Oriental Region, Morocco (34 87′ 73.3 ′′ N; 2 35′ 26.1′′
W) in January 2018. The leaves were randomly collected from different
trees. Healthy leaves were selected, and cleaned with distilled water.
Then, they were dried under shade and ground into a fine powder (100
mesh) using a crushing machine. A total of 2 kg of leaf powder was
obtained.
The plant has been authenticated by Pr. Rejdali Mouh and a voucher
specimen (ZL3) has been deposited at the Botany Laboratory of the
Agronomic and Veterinary Institute of Hassan II at Rabat.
The name of the plant has been checked with http://www.theplantl
ist.org/tpl1.1/record/rjp-135.
2.3. Preparation of leaf aqueous extract
The powdered leaf material was submitted to extraction by infusion
(1:10 w/v) with hot distilled water (80–85 ◦ C). After 6 h, the extract
solution was filtered and concentrated using an evaporator under
reduced pressure. The obtained loquat leaf extract (LLE) was kept and
stored at 4 ◦ C until use. The percentage extraction yield (three repeti­
tions) was 10.76 ± 1.63% (w/w).
2.4. Determination of total phenolic and flavonoid contents
Total phenolic and flavonoid contents were determined using Folin-
Ciocalteu and aluminum chloride reagents, respectively (Khouya et al.,
2015). The total phenolic content was expressed as mg caffeic acid
equivalents per g of dried extract (DE). The results of the flavonoid
content were expressed as rutin equivalents mg/g of DE. Total phenolic
and flavonoid contents of a thyme leaf extract reported in a previous
study were used as reference values (Khouya et al., 2015).
2.5. HPLC analysis
A method coupling HPLC with a diode-array detector (DAD) was
used to separate and quantify LLE polyphenols. Chromatographic sep­
arations were carried out using an Agilent Zorbax Eclipse XDB C18
column (4.6 mm × 150 mm, 5 μm) kept at 40 ◦ C. The test samples were
prepared by dissolving 1 g of extract in 5 mL of slightly acidified
methanol (1 N HCl/methanol/water, 1/80/19, v/v/v). A volume of 20
μL of the sample was injected into the HPLC-DAD system. A binary
mobile phase consisted of 6% acetic acid (pH 2.55) in 2 mM sodium
acetate (6v/94v, solvent A) and acetonitrile (solvent B). The system was
T. Khouya et al.
run with a gradient program: 0–8% of B between 0 and 5 min (linear
gradient), 8–25% of B between 5 and 25 min (linear gradient), 25% of B
between 25 and 30 min (isocratic elution), and 25–90% of B between 30
and 50 min (linear gradient). The flow rate of the mobile phase was kept
at 0.5 mL/min and the absorbance changes were monitored at 215, 250,
and 280 nm. The standards used to identify polyphenol compounds
were: chlorogenic acid, gallic acid, protocatechuic acid,
epigallocatechin-3-gallate, naringenin, epicatechin, syringic acid, rutin,
procyanidin C1, quercetin, kaempferol-rhamnose, and kaempferol. The
peaks were identified based on phenolic standard retention time and UV
spectra, and their quantity was determined using a calibration curve.
The results were expressed as mg of phenolic compound per g of DE.
2.6. Determination of nutritional composition
The nutritional composition of fresh leaves was analyzed in the SGS
Multilab Laboratory in Lisbon, Portugal. All analyses were carried out
following international methods committee guidelines. The analysis was
started on January 19, 2018 and completed on February 21, 2018.
2.6.1. Energy
The energy was calculated according to the Regulation European
Parliament and Council of the European Union No 1169, 2011 as
follows:
Energy = (% fat x 37 kJ/g) + (% protein x 17 kJ/g) + (% carbohydrate x 17 kJ/
g)
2.6.2. Moisture, sugar, carbohydrates, proteins, and fiber
The moisture content was determined using a moisture analyzer
(Kern DAB 100–3; Kern & Sohn GmbH, Balingen, Germany). The sugar,
fiber, protein, carbohydrate, and caffeine contents were determined
according to Norma Portuguesa-1420 1987, AOAC 985.29 1986, AOAC
990.03 2006, AOAC 979.06 1980, and AOAC 968.08 1981, respectively.
2.6.3. Total fat and fatty acids
The total fat was determined according to International Standard
Organisation (ISO) 6492 1999 method. Gas chromatography with flame
ionization detection method was used to analyze saturated, mono­
unsaturated, and polyunsaturated fatty acids (AOAC 996.01 2000).
2.6.4. Minerals
Minerals including potassium, calcium, magnesium, iron, and so­
dium were determined using atomic absorption spectroscopy (AOAC
968.08 1996).
2.6.5. Vitamins
The determination of vitamin A by HPLC was carried out according
to EN 12823-2, 2000 method. Vitamins D2 and D3 were analyzed using
HPLC-MS according to EN 12821 2009. Riboflavin (B2) was extracted
and analyzed with a UHPLC method (EN 14152, 2014). Vitamin B6,
which is the sum of pyridoxine, pyridoxal, pyridoxamine, and their
phosphorylated derivatives, was determined by HPLC-DAD-FLD
following EN 14164, 2014. Cobalamin (B12) was tested using a spe­
cific AOAC microbiological method (AOAC 952.20 1960).
2.7. In vivo assay
2.7.1. Animal
Swiss albino mice, weighing around 23–30 g, were obtained from the
animal facility in the Biology Department (Faculty of Sciences and
Techniques, Errachidia, Morocco). The mice were housed in standard
cages with a 12/12-h light/dark cycle and controlled temperature (22 ±
3 ◦ C) and humidity (50–60%). The animals were acclimatized for 14
3
Journal of Ethnopharmacology 296 (2022) 115473
days before the in vivo study. Food and water were made available ad
libitum. All animal experiments were approved by the institutional ani­
mal ethics committee of the Faculty of Medicine and Pharmacy in
Morocco (FMPR 187/2017) and were performed following the Care and
Use of Laboratory Animals Guidelines, published by the US National
Institutes of Health (NIH Publication No. 85–23, revised 1996). All
surgery was performed under sodium pentobarbital anesthesia, and all
efforts were made to minimize suffering.
2.7.2. Experimental diet
A standard chow diet (SCD) and a high-fat and high-glucose (HFG)
diet were used in this study. The SCD is composed of 8% rice bran, 51%
maize, 30% soybean powder, 3% bone powder, and 8 % micronutrients
(multivitamins and minerals). The HFG diet consists of 76.5% SCD, 4%
glucose, 2% cholesterol, 7% lard, 10% yolk powder, and 0.5% bile salt.
2.7.3. Experimental design
The mice were randomly divided into six groups of ten animals (n =
10). The control group was fed SCD and orally received distilled water.
The HFG control was fed the HFG diet and administered distilled water.
The positive controls received the HFG diet and were treated with pra­
vastatin (20 mg/kg BW/day) or metformin (50 mg/kg BW/day),
respectively. Loquat-treated groups were fed the HFG diet and orally
administered LLE at 150, 200, or 250 mg/kg BW/day. All groups were
studied for 10 weeks. The BW was recorded at the beginning and the end
of the study. At the end of the treatment, after 12 h fasting, the blood was
collected and immediately divided for the preparation of plasma and
serum. Then, the animals were sacrificed with an intraperitoneal in­
jection of an overdose of sodium pentobarbital, and the liver was
explanted for lipid analysis. The used doses of LLE were chosen based on
preliminary tests. All samples were dissolved in distilled water and
administered once a day at 8 a.m.
2.7.4. Biochemical analysis
2.7.4.1. Hepatic lipids. The livers were conserved at − 80 ◦ C immedi­
ately after collecting. To extract cholesterol and TG, about 0.5 g of the
liver was homogenized in 5 mL of a mixture of chloroform/methanol
(2:1, v/v). The mixture was allowed to stand for 30 h at 4 ◦ C and then
centrifuged for 10 min at 1000 g. Cholesterol and TG contents in the
supernatant were determined using RTU (ready to use) and PAP
(peroxidase anti-peroxidase) 150 enzymatic kits (BioMérieux, France),
according to the manufacturer’s instructions.
2.7.4.2. Blood parameters. The serum was prepared from coagulated
blood by centrifugation at 2500g for 15 min at 4 ◦ C and stored at − 20 ◦ C
until use. The TC and TG levels were determined with the same enzy­
matic kits used for hepatic lipid analysis. Serum MDA was measured
according to the manufacturer’s protocol of the mouse MDA ELISA kit
(cat. no. AE33973MO-480, HexaBiogen, Morocco). The separation and
quantification of LDL-C and HDL-C were carried out by an enzymatic
colorimetric method using a commercial BioChain kit according to the
manufacturer’s instructions.
Blood samples collected in heparinized tubes were centrifugated
(1000 g, 15 min, 4 ◦ C) to obtain plasma. Plasma was stored at − 20 ◦ C
until analysis. The plasma glucose was determined using a glucometer
(Roche Diagnostics, France). The determination of the plasma insulin
levels was carried out using a mouse ELISA kit (SpiBio, France).
2.8. Oral toxicity
The acute toxicity of LLE was determined using the organization for
economic cooperation and development (OECD)-Guideline 425 (OECD,
2008). Mice fasted for 4h were randomly divided into six groups (n =
10), weighted, and then received different doses of LLE (0, 500, 1000,
T. Khouya et al. Journal of Ethnopharmacology 296 (2022) 115473

1500, 2000, and 5000 mg/kg BW). All groups were kept under obser­ Table 2
vation for 48 h. For groups treated with 0 (distilled water, control), Characterized phenolic compounds and their contents.
2000, and 5000 mg/kg BW, mice were observed, for 14 days, for their Compound Peak tr Content % in Content (mg/
onset and duration of behavioral changes, toxicity, mortality, and BW No (min) (mg/g of DE g of fresh leaf)
changes. DE)

Protocatechuic 1 2.75 5.34 ± 0.02 7.65 0.57 ± 0.01

acid
2.9. Statistical analysis
Epigallocatechin 2 3.69 6.23 ± 0.06 8.93 0.67 ± 0.01
gallate
The results were expressed as mean ± standard error (SEM). The Chlorogenic acid 3 8.95 5.94 ± 0.08 8.51 0.64 ± 0.02
differences among groups were analyzed by one-way ANOVA followed Naringenin 4 10.01 10.93 ± 15.66 1.18 ± 0.00
by Tukey’s post hoc test using GraphPad Prism 8.0 software. p < 0.05 0.19
Epicatechin 5 11.04 8.43 ± 0.04 12.08 0.91 ± 0.01
was considered representative of significant differences.
Rutin 6 14.20 7.55 ± 0.12 10.82 0.81 ± 0.02
Procyanidin C1 7 16.05 9.33 ± 0.16 13.37 1.00 ± 0.01
3. Results Quercetin 8 17.88 5.93 ± 0.13 8.50 0.64 ± 0.01
Kaempferol- 9 18.42 5.98 ± 0.10 8.57 0.64 ± 0.01
rhamnose
3.1. Total polyphenols and flavonoids
Kaempferol 10 24.55 4.13 ± 0.10 5.92 0.44 ± 0.01
Total - - 69.79 100 7.51
The total polyphenol and flavonoid contents in LLE were about
tr: retention time. DE: dried extract. Results are expressed as the mean ± SEM,
240.65 ± 12.56 mg of caffeic acid equivalents per g of DE and 95.53 ±
(n = 3).
9.04 mg of rutin equivalents per g of DE, respectively (Table 1). The total
polyphenol and flavonoid contents in LLE were lower than those in a
thyme aqueous extract used as reference.

3.2. HPLC analysis

HPLC analysis revealed ten compounds consisting of two phenolic

acids (protocatechuic acid, and chlorogenic acid), seven flavonoids
(rutin, quercetin, naringenin, epicatechin, epigallocatechin-3-gallate,
kaempferol-rhamnose, and kaempferol), and a condensed tannin, pro­
cyanidin C1 (Table 2 and Fig. 1). The more abundant compounds were
naringenin (10.93 ± 0.19 mg/g), procyanidin C1 (9.33 ± 0.16 mg/g),
epicatechin (8.43 ± 0.04 mg/g) and rutin (7.55 ± 0.12 mg/g). The other
compounds had relatively similar content values, which ranged from
4.13 ± 0.10 mg/g for kaempferol to 6.23 ± 0.06 mg/g for epi­
gallocatechin-3-gallate.
Fig. 1. HPLC profile of polyphenols detected in loquat leaf aqueous
3.3. Nutritional composition extract (LLE).

Table 3 shows the results of energy value and nutritional composi­

tion of loquat fresh leaves. The energy value was 205.00 Kcal/100 g Table 3
(856.00 Kj/100 g). The moisture content was 9.75%. The leaves had a Energy value and nutritional composition of loquat fresh leaves.
high amount of carbohydrates (71.60%) and fiber (63.80%), while the Parameter Value Unit
total fat content was only 1.00%. Total fat seemed to be composed of Energy 205.00 Kcal/100g
saturated (0.50 g/100 g), monounsaturated (0.40 g/100 g), and poly­ 856.00 Kj/100g
unsaturated (0.10 g/100 g) fatty acids. Moisture 9.75 %
Total sugar and proteins in loquat leaves were about 0.8% and Carbohydrates 71.60 %
Fiber 63.80 %
9.30%, respectively. Among the analyzed vitamins, vitamin B6 was Proteins 9.30 %
higher (2.42 μg/100 g) in fresh leaves compared to other vitamins. The Total sugars 0.80 %
content of vitamin A, B2, B12, D2 and D3 were <0.50, 0.45, 0.28, <0.05, Fatty acids
and <0.05 μg/100 g, respectively. Total lipids 1.00 %
Saturated 0.50 g/100g
The leaves contained a high amount of calcium, potassium, and
Monounsaturated 0.40 g/100g
magnesium with values of 267.50, 953.80, and 279.60 mg/100 g, a Polyunsaturated 0.10 g/100g
moderate level of sodium (40 mg/100 g), and a relatively low amount of Mineral elements
Potassium 953.80 mg/100g
Calcium 267.50 mg/100g
Table 1
Magnesium 279.60 mg/100g
Total polyphenol and flavonoid contents of the loquat leaf aqueous extract Iron 0.50 mg/100g
(LLE). Sodium 40.00 mg/100g
Total polyphenol content a Flavonoid content b Vitamins
A <0.50 μg/100g
Loquat leaf aqueous extract 240.65 ± 12.56 95.53 ± 9.04 B2 0.454 μg/100g
Thyme aqueous extract c 495.47 ± 6.10 155.11 ± 3.90 B6 2.42 μg/100g
a B12 0.278 μg/100g
Total polyphenol content was expressed as mg of caffeic acid equivalents/g
D2 <0.05 μg/100g
of dried extract. D3 <0.05 μg/100g
b
Flavonoid content was expressed as mg of rutin equivalents/g of dried Caffeine <0.005 g/100g
extract.
c
Thyme aqueous extract was used as a reference.

4
T. Khouya et al. Journal of Ethnopharmacology 296 (2022) 115473

iron (0.5 mg/100 g). The amount of caffeine was less than 0.005 g/100 pravastatin-treated group was found (p ˂ 0.01). Hepatic cholesterol
g. levels were similar in pravastatin and LLE (250 mg/kg) groups (p >
0.05).

3.4. Effect of LLE on HFG diet-fed mice 3.4.4. Serum MDA

MDA is the end product of lipid peroxidation. It is widely used as a
3.4.1. Body weight and glycemic parameters marker of peroxidation. MDA levels were seen to be increased 2-fold in
Initial and final BW, plasma glucose, and insulin values are given in serum of the group feeding the HFG diet in comparison with control (p <
Table 4. Compared with the control, the HFG group significantly (p ˂ 0.001, Fig. 2D). LLE-treated groups showed a significant decrease in
0.01) gained more BW and had higher (p ˂ 0.01) levels of fasting blood MDA levels when compared to the HFG diet (p < 0.01). Both metformin
glucose and insulin. and pravastatin significantly reduced serum MDA levels (p ˂ 0.01). The
LLE dose-dependently prevented elevated BW gain and decreased level of serum MDA in groups treated with LLE at 200 or 250 mg/kg BW/
glucose and insulin levels when compared to the HFG group (p ˂ 0.01). day was similar to that in control (p > 0.05).
Mice treated with metformin, which served as a standard antidiabetic
drug, or pravastatin had similar glucose and insulin levels and main­ 3.5. Oral toxicity
tained similar weight as control mice feeding SCD (p > 0.05).
There were no significant differences (p > 0.05) between the body
3.4.2. Serum lipid parameters weights of control mice and those treated orally with 2000 or 5000 mg/
The levels of TC and TG, LDL-C, and HDL-C in the serum of all groups kg for 15 days (data not shown). No signs of ptosis, tremors, locomotor
are given in Fig. 2A and B. Compared with control, HFG showed sig­ activity reduction, or any other symptoms of acute and clinical oral
nificant increases (p ˂ 0.001) in serum TC, TG, and LDL-C levels, along toxicity were observed in any animal treated with LEE at 2000 or 5000
with a slight but significant decrease in HDL-C level (p ˂ 0.01). The g/kg BW. Thus, the median lethal dose (LD50) was more than 5000 mg/
administration of LLE to HFG diet-fed mice significantly reduced TC, TG, kg BW.
and LDL-C levels in a dose-dependent manner when compared to the
HFG group (p ˂ 0.01). Pravastatin completely prevented the perturba­ 4. Discussion
tion of lipid parameters induced by the HFG diet. No significant differ­
ences in TC, TG, LDL-C, and HDL-C levels between the control group and The main findings of the present study are that the oral adminis­
the pravastatin group were noted (p > 0.05). LLE at 250 mg/kg seemed tration of LLE dose-dependently prevented weight gain, hyperglycemia,
to have a similar effect as pravastatin on all analyzed parameters (p > hyperinsulinemia, hyperlipidemia, and oxidative stress induced by an
0.05). As compared to the HFG group, HDL-C levels were significantly (p HFG diet in mice. In addition, we revealed that loquat leaves are rich in
˂ 0.01) higher in the group receiving LLE at 250 mg/kg or pravastatin. phenolic acids, flavonoids, and micronutrients.
Metformin significantly (p ˂ 0.01) reduced serum TC, TG, and LDL-C but Diabetes is often associated with hyperlipidemia, which constitutes
did not affect HDL-C levels (p > 0.05). the main link between diabetes and cardiovascular diseases (Wu and
Parhofer, 2014). Treating hyperlipidemia in diabetic subjects is an
3.4.3. Hepatic cholesterol and triglycerides essential strategy to alleviate diabetes and prevent cardiovascular
Fig. 2C gives the levels of hepatic cholesterol and TG. The levels of complications (Artasensi et al., 2020). Moreover, plant extracts always
hepatic cholesterol and TG were seen to be significantly (p ˂ 0.001) contain a wide range of bioactive molecules that can act additively or
higher in mice fed the HFG diet compared to mice in the group receiving synergically on different therapeutic targets implicated in the develop­
SCD. The accumulation of lipids in the liver was significantly reduced by ment of multifactorial diseases. The use of multi-target drugs and their
LLE in a dose-dependent manner (p ˂ 0.01). Metformin effectively (p ˂ effectiveness in the treatment of diabetes are intensively discussed in
0.01) reduced hepatic cholesterol levels but did not affect hepatic TG (p recent years (Artasensi et al., 2020).
> 0.05). A significant decrease in both hepatic cholesterol and TG in the In the current study, we observed that feeding mice with the HFG
diet for 10 weeks induced BW gain, an increase in blood TC, TG, and
Table 4 MDA, hepatic lipid accumulation, and an increase in plasma glucose and
Body weight, blood glucose, and insulin in the studied groups. insulin levels. The administration of LLE improved HFG-induced dia­
Weight Glucose (mg/ Insulin (ng/ betes, which was evidenced by a decrease in fasting plasma glucose and
dL) mL) insulin levels. Many previous studies have reported the antidiabetic ef­
Initial Final
fects of the extracts from different parts of the loquat tree mainly fruits,
Control 25.45 ± 30.40 ± 101.37 ± 1.16 ±
1.5 2.40* 1.00* 0.03*
seeds, and flowers (Liu et al., 2016). Most studies reported that the
HFG group 25.63 ± 39.43 ± 142.69 ± 2.26 ± 0.04 antidiabetic effect of loquat was related to the existence of triterpene
1.17 2.85 1.65 acids and polysaccharides. Few studies have been conducted on poly­
HFG + LLE (150 25.75 ± 30.00 ± 139.27 ± 1.90 ± phenolic extracts from loquat leaves (Liu et al., 2016). Moreover, it has
mg/kg) 1.00 1.55* 1.02* 0.05*
been shown that 300 mg/kg BW of flavonoid-rich fraction significantly
HFG + LLE (200 24.95 ± 31.00 ± 133.40 ± 1.65 ±
mg/kg) 1.5 1.50* 1.03* 0.06* decreased the plasma glucose and serum insulin in the STZ-induced
HFG + LLE (250 25.00 ± 33.04 ± 125.33 ± 1.49 ± diabetic mice (Lü et al., 2009). By using a high-fat and fructose diet,
mg/kg) 1.82 1.50* 1.08* 0.07* the ethanol extract from the loquat leaf prevented the increase of fasting
Metformin (50 mg/ 25.00 ± 32.30 ± 106.88 ± 1.20 ± blood glucose in rats after four weeks of treatment by a dose of 500
kg) 1.00 2.34* 0.98* 0.06*
Pravastatin (20 mg/ 25.3 ± 31.24 ± 116.04 ± 1.24 ±
mg/kg BW (Chen et al., 2017). In our study, a daily dose of 200 mg/kg of
kg) 0.95 1.50* 1.06* 0.05* LLE completely reversed HFG-induced diabetes and exhibited an anti­
diabetic effect similar to metformin administered orally at 50 mg/kg
The mice in the HFG group were fed a high-fat/high-glucose (HFG) diet and
BW/day after 10 weeks of treatment. The possible mechanisms of action
orally received distilled water. Plant-treated groups were fed the HFG diet and
orally administered loquat leaf aqueous extract (LLE) at 150, 200, or 250 mg/kg for the antihyperglycemic effect of LLE may be through the inhibition of
BW/day for 10 weeks. Positive controls received the HFG diet and a daily dose of α-amylase and α-glucosidase (Chen et al., 2017) and improvement of
pravastatin (20 mg/kg BW) or metformin (50 mg/kg BW). The results are insulin secretion or insulin sensitivity (Dhiman et al., 2021).
expressed as mean ± SEM and analyzed using the ANOVA test followed by We also observed that the daily administration of LLE prevented lipid
Tukey’s post hoc test. n = 10; *p ˂ 0.01. vs. HFG group. disorders in a dose-dependent manner in HFG diet-induced mice. We

5
T. Khouya et al.
showed that LLE decreased serum TC, TG, and LDL-C and prevented the
accumulation of hepatic cholesterol. Excess lipid accumulation within
the liver is the main characteristic of non-alcoholic fatty liver disease,
which is positively correlated with a higher prevalence of diabetes
mellitus (Caussy et al., 2021). Interestingly, LLE at 250 mg/kg BW
increased HDL-C levels compared to the group feeding the HFG diet. The
HDL particles have been reported to have antioxidant capacities,
contribute to LDL stabilization and protect from diabetes, hyperlipid­
emia, and cardiovascular diseases (Hewing and Landmesser, 2015).
It is well established that oxidative stress is increased in obese mice
(Matsuda and Shimomura, 2013). The high-fat diet is associated with
augmented lipid accumulation in adipose tissue and excessive produc­
tion of ROS due to increased lipid oxidative metabolism (Mor­
eno-Fernández et al., 2018). Oxidative stress is one of the mechanisms
that link obesity to insulin resistance and diabetes (Matsuda and Shi­
momura, 2013). Treating oxidative stress and obesity attenuates the
development of diabetes and its complications (Matsuda and Shimo­
mura, 2013). In this context, our findings indicate that LLE alleviated
oxidative stress evidenced by decreased serum MDA levels, and pre­
vented BW gain and, thus, obesity. This may be an additional benefit of
the administration of LLE in diabetic mice.
Additionally, we showed that LLE displayed a lipid-lowering effect
similar to pravastatin. We also found that pravastatin exerted an anti­
diabetic effect. Pravastatin is an inhibitor of the rate-limiting step in the
de novo cholesterol synthesis (Trivedi et al., 2021). It has been demon­
strated that pharmacological inhibition of cholesterol biosynthesis is
accompanied by enhanced LDL particle uptake by LDL receptors, sup­
pression of synthesis and hepatic accumulation of TG, reduced glucose
levels, and improved insulin sensitivity (Han, 2018). Polyphenol-rich
extracts and isolated polyphenols from plants have been reported to
potently inhibit cholesterol biosynthesis, which may partly explain the
antihyperlipidemic and antihyperglycemic activity of LLE polyphenols
(Ramchoun et al., 2020).
Phytochemical analysis of LLE allowed us to characterize and
6
Journal of Ethnopharmacology 296 (2022) 115473
Fig. 2. Serum and liver parameters in different
groups. (A) Serum total cholesterol and triglycerides.
(B) Serum LDL cholesterol and HDL cholesterol. (C)
Hepatic cholesterol and triglycerides. (D) Serum
Malondialdehydes. Control mice were fed a standard
chow diet and orally received distilled water. The
mice in the HFG group were fed a high-fat/high-
glucose (HFG) diet and orally received distilled
water. Plant-treated groups were fed HFG diet and
orally administered loquat leaf aqueous extract (LLE)
at 150, 200, or 250 mg/kg BW/day for 10 weeks.
Positive controls received HFG diet and a daily dose
of pravastatin (20 mg/kg BW/day) or metformin (50
mg/kg BW/day). The results are expressed as mean
± SEM (n = 10) and analyzed using the ANOVA test
followed by Tukey’s post hoc test; ns: not significant,
*p ˂ 0.01. **p ˂ 0.001. vs. HFG group.
quantify ten different phenolic compounds: protocatechuic acid,
epigallocatechin-3-gallate, chlorogenic acid, naringenin, epicatechin,
rutin, procyanidin C1, quercetin, kaempferol-rhamnose, and kaemp­
ferol. Naringenin was the most abundant compound in LEE followed by
procyanidin C1 and epicatechin. A previous study conducted using LC-
DAD-MS/MS identified thirteen polyphenols with the main com­
pounds including catechin and procyanidins (Chen et al., 2017).
Many compounds characterized in LLE are known for their antioxi­
dant, antidiabetic and lipid-lowering properties. For example, nar­
ingenin is a potent antioxidant and was found to improve insulin
sensibility and oxidative stress by suppressing nuclear factor kappa B
(NF-κB) activation in a mouse model of diabetes (Nguyen-Ngo et al.,
2019).
Likely the antidiabetic and antihyperlipidemic effects of LLE are due
to the presence of high content of phenolic acids and flavonoids. The
mechanisms by which LLE polyphenols improved hyperlipidemia and
hyperglycemia may include: upregulation of the expression of receptors
and enzymes involved in glucose and lipid catabolism pathways, inhi­
bition of α-amylase and α-glucosidase, and suppression of oxidative
stress (Bhattacharjee et al., 2017; Li et al., 2018; Nguyen-Ngo et al.,
2019). However, additional studies are needed to determine the active
compounds and precise mechanisms of LLE action.
Apart from their polyphenol content, we found that loquat leaves
have a high amount of minerals, especially potassium, calcium, mag­
nesium, iron, and sodium, and a considerable quantity of riboflavin
(B2), pyridoxine (B6), and cobalamins (B12). Vitamins and minerals
play a crucial role in carbohydrate and lipid metabolism (Shergill-Bon­
ner, 2017). The deficiency of micronutrients has been observed in pa­
tients with metabolic disorders (Dubey et al., 2020). The maintenance of
an adequate amount of micronutrients is recommended especially in
subjects with diabetes and hyperlipidemia (Shah et al., 2021). We also
found that the leaves had a low energy value due to their lower total fat
and sugar, and showed a high amount of fiber.
Previous studies suggest that LLE is a safe traditional medicine for
T. Khouya et al.
therapeutic use at the right dose (Seong et al., 2018). It has been re­
ported that the no-observed-adverse-effect level of a leaf extract derived
from loquat leaves is more than 1000 mg/kg/day and the LD50 of a
loquat leaf triterpene acid is higher than 3000 mg/kg BW (Li et al., 2017;
Seong et al., 2018). Here, the acute toxicity was conducted in mice
following OECD guidelines. The oral treatment of mice with LLE at 2000
and 5000 mg/kg BW produced no deaths and no signs of acute oral
toxicity. It appeared that the LD50 of LLE was higher than 5000 mg/kg.
Thus, LLE can be classified in the category of non-toxic substances ac­
cording to OECD, 2008.
5. Conclusion
The aqueous extract derived from loquat leaves ameliorated hyper­
glycemia and hyperlipidemia by correcting blood glucose, insulin, and
lipid levels and preventing lipid oxidation in mice fed a diet rich in fat
and glucose. The leaves from loquat cultivated in Morocco are a rich
source of nutritional elements mainly vitamins and minerals, and
bioactive compounds (phenolic acids and flavonoids), which seem to
have beneficial effects on diabetes and hyperlipidemia.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
List of studied compounds
Metformin and pravastatin.
CRediT authorship contribution statement
Tarik Khouya: Conceptualization, and design of study, Data cura­
tion, Formal analysis, Interpretation of data, Writing – original draft,
Approval of the version of the manuscript to be published. Mhamed
Ramchoun: Conceptualization, and design of study, Data curation,
Formal analysis, Interpretation of data, Writing – original draft, Writing
– original draft, critically for important intellectual content, Approval of
the version of the manuscript to be published. Hamza Elbouny: Data
curation, Formal analysis, Interpretation of data, Writing – original
draft, critically for important intellectual content, Approval of the
version of the manuscript to be published. Abdelbassat Hmidani: Data
curation, Formal analysis, interpretation of data, Approval of the version
of the manuscript to be published. Eimad dine Tariq Bouhlali: Data
curation, Writing – original draft, critically for important intellectual
content, Approval of the version of the manuscript to be published.
Chakib Alem: Conceptualization, and design of study, Approval of the
version of the manuscript to be published.
Declaration of interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors wish to express their gratitude to Mr. Alshehri Hassan
Mohammed, director of First Dream company (Berkane, Morocco), for
his assistance in the collection of plant material and his valuable help.
The authors thank the director Mrs. Naima Laktati and all members of
My species cooperation (Temara, Morocco) for their help in plant extract
preparation.
7
Journal of Ethnopharmacology 296 (2022) 115473
References
AOAC, 1960. Official methods of analysis (952.20). Cobalamin (vitamin B12 activity. In:
Helrich, K. (Ed.), Vitamin Preparation, fifteenth ed. Association of Official Analytical
Chemists, Inc., Arlington, VA, USA. 1990.
AOAC, 1980. Official methods of analysis (979.06). Carbohydrate content in beer.
Gaithersbung, MD, 2007. In: sixteenth ed.Hortwitz, W., Latimer, C.W. (Eds.),
Association of Official Analytical Chemists 27.1.21.
AOAC, 1981. Official Methods of Analysis (968.08). Theobromine and Caffeine in Cacao
Products, 1981. AOAC International, Rockville, MD, USA.
AOAC, 1986. Official methods of analysis (985.29. In: Total Dietary Fiber in Foods,
Enzymatic-Gravimetric Method. AOAC International, Gaithersburg, MD, USA, 1986.
AOAC, 1996. Official methods of analysis (968.08. In: Minerals in Animal Feed and Pet
Food. Atomic Absorption Spectrophotometric Method, sixteenth ed., vol. 1. AOAC
Int. Inc., Gaithersburg, MD.
AOAC, 2000. Official methods of analysis. (996.01) Fat (total, saturated, unsaturated,
and monounsaturated. In: Cereal Products, seventeenth ed. AOAC International,
USA.
AOAC, 2006. Official Methods of Analysis (990.03). Protein (Crude) in Animal Feed,
Combustion Method. International, eighteenth ed. ASA-SSA Inc, Gaithersburg.
Artasensi, A., Pedretti, A., Vistoli, G., Fumagalli, L., 2020. Type 2 diabetes mellitus: a
review of multi-target drugs. Molecules 25, 1–20. https://doi.org/10.3390/
molecules25081987.
Bhattacharjee, N., Dua, T.K., Khanra, R., Joardar, S., Nandy, A., Saha, A., De Feo, V.,
Dewanjee, S., 2017. Protocatechuic acid, a phenolic from Sansevieria roxburghiana
leaves, suppresses diabetic cardiomyopathy via stimulating glucose metabolism,
ameliorating oxidative stress, and inhibiting inflammation. Front. Pharmacol. 8,
251. https://doi.org/10.3389/FPHAR.2017.00251/BIBTEX.
Caussy, C., Aubin, A., Loomba, R., 2021. The relationship between type 2 diabetes,
NAFLD, and cardiovascular risk. Curr. Diabetes Rep. 21, 15. https://doi.org/
10.1007/s11892-021-01383-7.
Cha, D.S., Eun, J.S., Jeon, H., 2011. Anti-inflammatory and antinociceptive properties of
the leaves of Eriobotrya japonica. J. Ethnopharmacol. 134, 305–312. https://doi.org/
10.1016/j.jep.2010.12.017.
Chen, B., Long, P., Sun, Y., Meng, Q., Liu, X., Cui, H., Lv, Q., Zhang, L., 2017. The
chemical profiling of loquat leaf extract by HPLC-DAD-ESI-MS and its effects on
hyperlipidemia and hyperglycemia in rats induced by a high-fat and fructose diet.
Food Funct. 8, 687–694. https://doi.org/10.1039/c6fo01578f.
Dhiman, A., Suhag, R., Thakur, D., Gupta, V., Prabhakar, P.K., 2021. Current status of
loquat (Eriobotrya Japonica lindl.): bioactive functions, preservation approaches, and
processed products. Food Rev. Int. 1–31. https://doi.org/10.1080/
87559129.2020.1866007.
Dubey, P., Thakur, V., Chattopadhyay, M., 2020. Role of minerals and trace elements in
diabetes and insulin resistance. Nutrients 12, 1–17. https://doi.org/10.3390/
nu12061864.
EN 12823-2, 2000. Foodstuffs – Determination of Vitamin A by High-Performance Liquid
Chromatography – Part 2: Measurement of β-carotene. European Committee for
Standardization, Brussels (Belgium).
EN 12821, 2009. Foodstuffs - Determination of vitamin D by high performance liquid
chromatography - Measurement of cholecalciferol (D3) or ergocalciferol (D2).
Available on . https://standards.iteh.ai/catalog/standards/cen/dbb81bab-588d
-4232-8fa2-3d2fa15eec14/en-12821-2009. (Accessed 22 January 2022), 2014.
EN 14152, 2014. Foodstuffs - Determination of vitamin B2 by high-performance liquid
chromatography. Available on: https://standards.iteh.ai/catalog/standards/cen/0
472ce56-5fec-49bb-ac22-9ab85768ef94/en-14152-2014. accessed 1.22.22.
EN 14164, 2014. Foodstuffs - determination of vitamin B6 by high-performance
chromatography. available on: https://standards.iteh.ai/catalog/standards/cen/9
bbe46f9-914d-4b65-8deb-78fa776680dd/en-14164-2014. accessed 1.23.22.
Geisler, C.E., Renquist, B.J., 2017. Hepatic lipid accumulation: cause and consequence of
dysregulated glucoregulatory hormones. J. Endocrinol. 234 (1), 1–21. https://doi.
org/10.1530/JOE-16-0513.
Häkkinen, M., 2007. Loquat: an ancient fruit crop with a promising future. Chron. Hortic.
47, 2.
Han, K.H., 2018. Functional implications of HMG-CoA reductase inhibition on glucose
metabolism. Korean Circ. J. 48, 951–963. https://doi.org/10.4070/kcj.2018.0307.
Hewing, B., Landmesser, U., 2015. LDL, HDL, VLDL, and CVD prevention: lessons from
genetics? Curr. Cardiol. Rep. 17, 56. https://doi.org/10.1007/s11886-015-0610-z.
International Standard Organisation. (1999). ISO 6492:1999: Animal feeding stuffs -
Determination of fat content.
Khouya, T., Ramchoun, M., Hmidani, A., Amrani, S., Harnafi, H., Benlyas, M.,
Zegzouti, Y.F., Alem, C., 2015. Anti-inflammatory, anticoagulant and antioxidant
effects of aqueous extracts from Moroccan thyme varieties. Asian Pac. J. Trop.
Biomed. 5 (8), 636–644. https://doi.org/10.1016/j.apjtb.2015.05.011.
Kuraoka-Oliveira, Â.M., Radai, J.A.S., Leitão, M.M., Lima Cardoso, C.A., Silva-Filho, S.E.,
Leite Kassuya, C.A., 2020. Anti-inflammatory and anti-arthritic activity in extract
from the leaves of Eriobotrya japonica. J. Ethnopharmacol. 249, 112418 https://doi.
org/10.1016/j.jep.2019.112418.
Li, F., Li, Y., Li, Q., Shi, X., Guo, Y., 2017. Acute and subacute oral toxicity evaluation of
Eriobotrya japonica leaf triterpene acids in ICR mice. Evidence-based complement.
Altern. Med., 4837839 https://doi.org/10.1155/2017/4837839, 2017.
Li, X., Li, S., Chen, M., Wang, J., Xie, B., Sun, Z., 2018. (-)-Epigallocatechin-3-gallate
(EGCG) inhibits starch digestion and improves glucose homeostasis through direct or
indirect activation of PXR/CAR-mediated phase II metabolism in diabetic mice. Food
Funct. 9, 4651–4663. https://doi.org/10.1039/c8fo01293h.
T. Khouya et al.
Liu, Y., Zhang, W., Xu, C., Li, X., 2016. Biological activities of extracts from Loquat
(Eriobotrya japonica Lindl.): a review. Int. J. Mol. Sci. 17, 1983. https://doi.org/
10.3390/ijms17121983.
Lü, H., Chen, J., Li, W.L., Ren, B.R., Wu, J.L., Zhang, H.Q., 2009. Hypoglycemic effect of
the total flavonoid fraction from Folium Eriobotryae. Phytomedicine 16, 967–971.
https://doi.org/10.1016/j.phymed.2009.03.024.
Marouani, M. El, Bouzbib, M., Hamdaoui, L. El, Pienaar, A., Trif, L., Tagne, M.S., Kifani-
Sahban, F., 2021. Eriobotrya japonica lindl. Kernels: kinetics of thermal degradation
under inert atmosphere using model-free and fitting methods. Biointerface Res. Appl.
Chem. 11, 11357–11379. https://doi.org/10.33263/BRIAC114.1135711379.
Martini, L.A., Catania, A.S., Ferreira, S.R.G., 2010. Role of vitamins and minerals in
prevention and management of type 2 diabetes mellitus. Nutr. Rev. 68, 341–354.
https://doi.org/10.1111/j.1753-4887.2010.00296.x.
Matsuda, M., Shimomura, I., 2013. Increased oxidative stress in obesity: implications for
metabolic syndrome, diabetes, hypertension, dyslipidemia, atherosclerosis, and
cancer. Obes. Res. Clin. Pract. 7, 1–12. https://doi.org/10.1016/j.orcp.2013.05.004.
Moreno-Fernández, S., Garcés-Rimón, M., Vera, G., Astier, J., Landrier, J.F., Miguel, M.,
2018. High fat/high glucose diet induces metabolic syndrome in an experimental rat
model. Nutrients 10, 1–15. https://doi.org/10.3390/nu10101502.
Namazi, N., Esmaeili, S., Ahmadikhatir, S., Razi, F., Nasli-Esfahani, E., Larijani, B., 2021.
Nutrition and diet therapy in diabetes mellitus: a roadmap based on available
evidence. J. Diabetes Metab. Disord. 20, 1913–1918. https://doi.org/10.1007/
s40200-021-00876-2.
Nazarian-Samani, Z., Sewell, R.D.E., Lorigooini, Z., Rafieian-Kopaei, M., 2018. Medicinal
plants with multiple effects on diabetes mellitus and its complications: a systematic
review. Curr. Diabetes Rep. 18, 72. https://doi.org/10.1007/s11892-018-1042-0.
Nguyen-Ngo, C., Willcox, J.C., Lappas, M., 2019. Anti-diabetic, anti-inflammatory, and
anti-oxidant effects of naringenin in an in Vitro human model and an in vivo murine
model of gestational diabetes mellitus. Mol. Nutr. Food Res. 63, 1–12. https://doi.
org/10.1002/mnfr.201900224.
Norma Portuguesa, 1987. Method 1420. Frutos, Prod. Hortícolas e Seus Deriv. Determ.
dos açúcares totais, dos açúcares redutores e não redutores (Sacarose). Técnica Luff-
Schoorl. Method routine’. Inst. Port. da Qualidade. Lisbon, Portugal.
OECD, 2008. Guidelines for Testing of Chemical, Guideline 425. Acute Oral Toxicity-up-
and-Down procedure (UDP), Paris, France.
Qin, L., Zang, M., Xu, Y., Zhao, R., Wang, Y., Mi, Y., Mei, Y., 2021. Chlorogenic acid
alleviates hyperglycemia-induced cardiac fibrosis through activation of the NO/
Journal of Ethnopharmacology 296 (2022) 115473
cGMP/PKG pathway in cardiac fibroblasts. Mol. Nutr. Food Res. 65, 1–28. https://
doi.org/10.1002/mnfr.202000810.
Ramchoun, M., Khouya, T., Alibrahim, E.A., Hmidani, A., Sellam, K., Amrani, S., Harnafi,
H., Benlyas, M., Chadli, F.K., Ouguerram, K., Alem, C., 2020. Thymus atlanticus
polyphenol-rich extract regulates cholesterol metabolism by inhibiting its
biosynthesis without affecting its excretion in hamsters fed a high-fat diet. https://
doi.org/10.1080/13813455.2020.1854308..
Regulation European Parliament and Council of the European Union. (2011). Regulation
(EU) No. 1169/2011 of the European Parliament and of the Council of 25 October
2011 on the provision of food information to consumers..
Seong, N.W., Seo, H.S., Kim, J.H., Kim, Y.J., Kim, E., Lee, J.Y., Ko, J.W., Kim, J.C., 2018.
A 13-week subchronic toxicity study of an Eriobotrya japonica leaf extract in rats.
J. Ethnopharmacol. 226, 1–10. https://doi.org/10.1016/J.JEP.2018.07.024.
Shah, I.U., Sameen, A., Manzoor, M.F., Ahmed, Z., Gao, J., Farooq, U., Siddiqi, S.M.,
Siddique, R., Habib, A., Sun, C., Siddeeg, A., 2021. Association of dietary calcium,
magnesium, and vitamin D with type 2 diabetes among US adults: National health
and nutrition examination survey 2007–2014—a cross-sectional study. Food Sci.
Nutr. 9, 1480–1490. https://doi.org/10.1002/fsn3.2118.
Shergill-Bonner, R., 2017. Micronutrients. Paediatr. Child Heal. (United Kingdom) 27,
357–362. https://doi.org/10.1016/j.paed.2017.04.002.
Trivedi, L.U., Femnou Mbuntum, L., Halm, E.A., Mansi, I., 2021. Is statin use associated
with risk of thyroid diseases? Results of a retrospective cohort study. Ann.
Pharmacother. 55, 1110–1119. https://doi.org/10.1177/1060028020986552.
Wang, Z., He, Z., Emara, A.M., Gan, X., Li, H., 2019. Effects of malondialdehyde as a
byproduct of lipid oxidation on protein oxidation in rabbit meat. Food Chem. 288,
405–412. https://doi.org/10.1016/j.foodchem.2019.02.126.
Wu, L., Parhofer, K.G., 2014. Diabetic dyslipidemia. Metabolism 63, 1469–1479. https://
doi.org/10.1016/j.metabol.2014.08.010.
Wu, R., Jian, T., Ding, X., Lv, H., Meng, X., Ren, B., Li, J., Chen, J., Li, W., 2021. Total
sesquiterpene glycosides from loquat leaves ameliorate HFD-induced insulin
resistance by modulating IRS-1/GLUT4, TRPV1, and SIRT6/Nrf2 signaling
pathways, 2021 Oxid. Med. Cell. Longev., 4706410. https://doi.org/10.1155/2021/
4706410.
Ziyyat, A., Legssyer, A., Mekhfi, H., Dassouli, A., Serhrouchni, M., Benjelloun, W., 1997.
Phytotherapy of hypertension and diabetes in oriental Morocco. J. Ethnopharmacol.
58, 45–54. https://doi.org/10.1016/S0378-8741(97)00077-9.