Journal Pre-proofs

Original Research Article

Antihyperglycemic and hepatoprotective properties of miracle fruit (Synsepa‐

lum dulcificum) compared to aspartame in alloxan-induced diabetic mice

Suzan G. Haddad, Mariam Mohammad, Karim Raafat, Fatima A. Saleh

PII: S2095-4964(20)30101-1
DOI: https://doi.org/10.1016/j.joim.2020.09.001
Reference: JOIM 185

To appear in: Journal of Integrative Medicine

Received Date: 21 January 2020

Accepted Date: 1 April 2020

Please cite this article as: S.G. Haddad, M. Mohammad, K. Raafat, F.A. Saleh, Antihyperglycemic and
hepatoprotective properties of miracle fruit (Synsepalum dulcificum) compared to aspartame in alloxan-induced
diabetic mice, Journal of Integrative Medicine (2020), doi: https://doi.org/10.1016/j.joim.2020.09.001

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© 2020 Shanghai Changhai Hospital. Published by ELSEVIER B.V.

 JIM-01-2020-OA-ER-0017
Original Research Article

Antihyperglycemic and hepatoprotective properties of miracle

fruit (Synsepalum dulcificum) compared to aspartame in alloxan-
induced diabetic mice

Suzan G. Haddad1, Mariam Mohammad1, Karim Raafat2, Fatima A. Saleh1

1. Department of Medical Laboratory Sciences, Faculty of Health Sciences, Beirut Arab
University, Beirut 115020, Lebanon
2. Department of Pharmaceutical Sciences, Faculty of Pharmacy, Beirut Arab University,
Beirut 115020, Lebanon

ABSTRACT
Objective: This study was undertaken to investigate the antihyperglycemic potential of
miracle fruit (MF) as well as its hepatic safety as compared to aspartame in alloxan-induced
diabetic mice.
Methods: MF extracts were prepared and screened for their phytochemical composition using
high-performance liquid chromatography (HPLC). Total phenolic, flavonoid and tannin
contents and antioxidant potential were also determined. Additionally, MF was evaluated for
sensory attributes. For in vivo work, MF ethanol extract at high (MFH: 500 mg/kg body
weight [BW]) and low (MFL: 250 mg/kg BW) doses as well as aspartame were injected
intraperitoneally into alloxan-induced diabetic mice. Blood glucose levels were determined
following acute and subchronic treatment. At the end of the study, animals were sacrificed,
serum was collected for biochemical analysis and liver tissues were obtained for
histopathological examination.
Results: MF ethanol extract contained more flavonoids and tannins, and had higher 1,1-
diphenyl-1-picrylhydrazyl radical-scavenging activity (79.61%) compared to MF aqueous
extract (P < 0.05). HPLC analysis of MF ethanol extract also revealed the presence of 10
antioxidants with quercetin comprising the major polyphenol. Additionally, sensory analysis
of MF showed that its intake is effective in masking undesirable sourness. Subchronic
administration of MFH proved amelioration of hyperglycemia in mice as compared to
aspartame. Moreover, aspartame treatment significantly elevated (P < 0.05) the level of
alanine aminotransferase and had destructive effects on the liver histopathology; however,
hepatic architecture was restored by low and high doses of MF.
Conclusion: MF is an effective antihyperglycemic with hepatoprotective properties that can
be used as a healthier alternative sweetening agent in place of aspartame for sour beverages.

Please cite this article as: Haddad SG, Mohammad M, Raafat K, Saleh FA.
Antihyperglycemic and hepatoprotective properties of miracle fruit (Synsepalum dulcificum)
compared to aspartame in alloxan-induced diabetic mice. J Integr Med. 2020; Epub ahead of
print.

Received Jan 21, 2020; accepted Apr 1, 2020.

Keywords: Miracle fruit; Synsepalum dulcificum; Aspartame; Histopathology;

Phytochemical analysis; Sensory evaluation

Correspondence: Fatima A. Saleh; E-mail address: f.saleh@bau.edu.lb

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1. Introduction

The intake of sugar has skyrocketed globally in the last few decades [1], but there is now
increasing public awareness that excessive consumption of sugar can have adverse health
effects [2,3]. For the many people who want to have the taste of sweetness without the sugar,
artificial sweeteners, whether natural or artificial, have become preferred alternatives. Natural
sweeteners are naturally occurring compounds that are extracted from plants or natural
substances with no chemicals added [2,3]. On the other hand, artificial sweeteners are
synthetic sugar substitutes or derivatives of naturally occurring compounds, which are
recognized as intense sweeteners since they are much sweeter than regular sugar [3]. There
are various types of both natural and artificial sweeteners, such as miracle fruit (MF) and
aspartame, respectively [4,5].
Aspartame, a commonly known artificial sweetener, is widely used as a nonnutritive
sweetener, yet there are still concerns regarding its safety [6]. Aspartame consumption has
been associated with damage and toxicity to the liver and kidneys [7]. Some studies also
showed an association between artificial sweeteners and diseases such as obesity, diabetes
and cancer [8]. These concerns related to the safety of artificial sweeteners coupled with
increased consumers’ interest in pursuing a healthy lifestyle have caused a dramatic increase
worldwide in the demand for natural sweeteners [9].
MF, also called miracle berry or sweet berry, is the plant Synsepalum dulcificum. It is an
evergreen bush or tree that grows up to 18 feet, with fruit that contains one big seed
surrounded by a thin layer of flesh characterized by a faint cherry-like taste [10–12]. This
plant is widely propagated in West Africa and was first harvested by local tribes, who eat the
berries before meals and use them as natural sweeteners [12,13]. MF contains a glycoprotein
in its pulp called miraculin, which turns any sour or bitter food into a sweet one by binding to
the tongue’s taste buds and activating the taste receptors (hT1R2–hT1R3) in response to
acidic pH [14–16]. Miraculin is tasteless by itself; however, once it triggers the sweet
sensation, it is valued to be 400,000 times sweeter than sucrose [14].
The main objective of this study was to determine the concentrations of total phenolics,
flavonoids and tannins in MF extract. The phytochemical composition was also investigated
using high-performance liquid chromatography (HPLC). 1,1-diphenyl-1-picrylhydrazyl free
radical (DPPH) was also used to determine MF antioxidant activity. Additionally, the sensory
properties of MF were examined in order to determine its suitability as an alternative natural
sweetener in sour beverages. The study also sought to determine the MF antihyperglycemic
potential in alloxan-induced diabetic mice compared to aspartame and whether its
consumption has any effect on body weight (BW). The safety of MF compared to aspartame
was also investigated by measuring serum liver enzymes and examining liver histopathology.

2. Materials and methods

2.1. Chemicals
HPLC-grade formic acid, trifluoroacetic acid, acetonitrile, alloxan and reference standards
were purchased from Sigma-Aldrich (Germany). DPPH, Folin-Ciocalteu’s (FC) reagent,
gallic acid, catechin and absolute ethanol were also obtained from Sigma-Aldrich (Germany).
In addition, sodium carbonate anhydrous (Na2CO3) and aluminum chloride salt (AlCl3) were
attained from Merck Company (USA). MF (S. dulcificum) powder (MFP) was transported
from SweetFreaks in USA and aspartame powder was purchased from local commercial
stores.

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2.2. Preparation of MF powder extract
Aqueous and ethanol extracts of MFP were prepared. In brief, 7 g of MFP was weighed and
added to 70 mL of distilled water or 80% ethanol, followed by incubation at 40 °C for 24 h,
and then filtration. For in vivo work, the ethanol extract filtrate was covered with a gauze and
left at room temperature (RT) for 3 d for further evaporation until it turned into a powder.
2.3. Dry matter content determination
The dry matter content of the raw material was determined by weighing 3 g of MFP sample
initially and drying it for 24 h using a ventilated oven at 105 °C.
2.4. Quantification of total phenolic content by FC method
Total phenolic content of each extract was examined by the FC method with some
modifications as described previously [17]. Briefly, an aliquot of 0.2 mL of the MF extract
was added to 0.1 mL of FC agent. After stirring using a magnetic stirrer, the blend was kept
in the dark for 5 min. Then, 0.8 mL of 7.5% Na2CO3 solution was mixed with the blend and
incubated for 10 min at 60 °C. Then, the absorbance was read at 750 nm by ultraviolet-visible
spectrophotometer (Gold S54T UV-VIS, China). Gallic acid was used as a standard for the
calibration curve, which was plotted between concentrations of 0–1 mg/mL of gallic acid.
2.5. Determination of total flavonoid content
Total flavonoid content (TFC) was determined by applying the following method with a few
modifications [17]. One milliliter of the MF extract was mixed with 4 mL of distilled water
and kept in the dark for 5 min. Then, 0.3 mL of sodium nitrite (NaNO2: 5%) and 1.5 mL of
AlCl3 (2%) were mixed with the previous blend. The mixture was then stirred and left for 5
min in the dark. Finally, 1 mL of sodium hydroxide (NaOH: 1 mol/L) was added to the
mixture and the absorbance was read at 510 nm. TFC content was determined by using a
calibration curve obtained from the catechin standard calibration curve.
2.6. Determination of tannin concentration
The total tannin content was established with some modifications as described by Hosu et al.
[18]. Two tubes were used, each containing 1 mL of the MF extract, 0.5 mL distilled water,
and 1.5 mL of hydrogen chloride (HCl; 12 mol/L). The first tube was held at RT for 30 min,
while the other was heated in the incubator at 100 °C for the same duration. Rapid cooling
was applied for each tube, and then 0.25 mL of ethanol was added to each tube and the
absorbance was measured at 520 nm.
2.7. Determination of the antiradical activity using DPPH
The free radical-scavenging activity was inspected by the DPPH assay according to a
previous study by Zhang and Hamauzu [19] with minor modifications. Four milliliter of 0.1
mmol/L methanolic solution of DPPH was combined with 0.2 mL of the MF extract. This
blend was stirred and left in the dark at ambient temperature for a period of 30 min to prevent
degradation. A control was treated with 4 mL of DPPH. Afterwards, the absorbance was read
at 517 nm and converted into the percentage of antioxidant activity using the following
equation: percentage of antioxidant activity = 100 – (Asample – Ablank) × 100)/Acontrol..
2.8. Reversed phase HPLC analysis
Phytochemical composition of MF ethanol extract was assessed using HPLC. HPLC was
carried out under gradient conditions using C18 column (4.6 mm × 250 mm) equipped with 5
μm diameter particles. The mobile phase was formed of solvent A, formic acid:water (1:9
v/v), and solvent B, acetonitrile:water:formic acid (5:4:1 v/v/v), at a flow rate of 0.8 mL/min,
and injection volume of 50 μL, using ultraviolet-detector at wavelength of 215–370 nm.
Chromatographic peaks were verified by comparing them to the corresponding standards
[20].
2.9. Animals and experimental design
Fifteen Swiss-Webster male mice (22–32 g, aged 12–16 weeks) were obtained from the
Animal House Facility at the Faculty of Pharmacy, Beirut Arab University (BAU). One week

3
before undergoing the experimental work, the mice were housed in a 12-hour dark/light
cycle, and had water free access and standard tablets (5% fats, 20% proteins and 1%
multivitamins). However, the animals had no food access for 16 h prior to the experiment
[21]. This experimental study as summarized in Table 1 was conducted in the Biomedical
Laboratory at BAU after authorization of BAU-Institutional Review Board with approval
number 2019H-0067-HS-M-0322.
MF ethanol extract powder was suspended in 0.9% w/v normal saline, with intraperitoneal
(IP) administration at the doses of 250 and 500 mg/kg BW for a 10-day period. The
aspartame was freshly prepared in saline every time prior to IP injection at a dose of 250
mg/kg BW.

Table 1. Protocol of experimental design to study acute (0, 0.5, 2 and 6 h) and subchronic (1, 3, 5 and 10
d) effects of aspartame, MFL and MFH doses in diabetic and nondiabetic mice.
Group n Tested substance Description
I 3 Diabetic control Diabetic mice: vehicle (0.9% sterile cold saline, IP)
II 3 Nondiabetic control Nondiabetic mice: vehicle (0.9% sterile cold saline, IP)
III 3 Aspartame Diabetic mice: aspartame 250 mg/kg BW, IP
IV 3 Miracle fruit (low dose) Diabetic mice: MFL 250 mg/kg BW, IP
V 3 Miracle fruit (high dose) Diabetic mice: MFH 500 mg/kg BW, IP
Mice were randomly divided into 5 groups (3 mice per group). MFL: miracle fruit extract at low dose;
MFH: miracle fruit extract at high dose; BW: body weight; IP: intraperitoneal.

2.10. Induction of diabetes

Alloxan-induced diabetes is one of the widely used models to induce type I diabetes mellitus
in experimental animals [22]. Five milligrams of alloxan was freshly dissolved in 5 mL cold
saline solution (0.9%), followed by IP injection into the mice three times every 48 h at a 180
mg/kg BW dosage to induce type I diabetes mellitus [21,23]. Seventy-two hours after the last
alloxan injection, blood glucose level (BGL) was examined for each animal using Roche Test
Meter and Accu-chek Active™ glucose strips (USA) [21]. Mice having BGL ≥ 200 mg/dL
were considered diabetic and thus included in the in vivo studies. The antihyperglycemic
properties of low and high doses of MF were then examined compared to aspartame after
acute (0, 0.5, 2 and 6 h) and subchronic (1, 3, 5 and 10 d) treatment [24]. Furthermore, the
BWs of the mice were monitored using a top loader weighing balance. At the end of the
study, animals were sacrificed, serum was collected for biochemical analysis and liver tissues
were obtained for histopathological examination.
2.11. Biochemical assessment
Serum liver enzymes such as alanine aminotransferase (ALT) and total cholesterol were
determined according to manufacturer’s instructions using diagnostic kits (Spinreact, Spain).
Results were expressed as U/L for ALT and mg/dL for total cholesterol.
2.12. Histological examination
After mice were sacrificed, liver tissue samples were obtained and fixed in 10% formal
saline. Fixed tissue samples were then treated by paraffin embedding, and then cut using a
microtome to obtain 5 μm-thick paraffin sections, followed by staining using hematoxylin
and eosin. Stained samples were then analyzed under a light microscope (Zeiss) and
photographed using Olympus digital camera (Optiphot 2; Nikon, Tokyo, Japan).
2.13. Sensory evaluation of sweeteners
Ten panelists of both genders and different age groups were selected randomly to participate
in the sensory evaluation of four different lemonade samples with or without sweeteners.
Lemonade was freshly prepared in a ratio of 3:1 (water:lemon) [4]. Four samples of
lemonade were used in this experiment (Table 2) as previously described by Rodrigues and
colleagues [4]. Each of the lemonade samples was poured in a plastic cup, labeled with a

4
 three-digit code and placed in random order for each panelist. Prior to tasting, specific
instructions were given to the panelists. Every panelist had to taste each sample from left to
right and then rate it with the line scale provided for each attribute (color, turbidity, fresh
lemon, sweetness, sourness, tartness, lemon taste, astringent, presence of particles,
astringency, lemon flavor, sweetness, and sourness). Subsequently, a web of the sensory
attributes was constructed. Note that water and crackers were used between tasting the
samples. Afterwards, each panelist had to choose which sample he/she most preferred.

Table 2. Lemonade samples and method of preparation [4].

Sample Method of preparation
Unsweetened lemonade Water and lemon mixed together with no added sugar
Sweetened lemonade with sugar Water and lemon mixed with sugar (134 g/L)
Sweetened lemonade with aspartame Water and lemon mixed with aspartame (4
tablespoon/L)
Unsweetened lemonade consumed after the intake Water and lemon mixed together with no added sugar
of MFP
MFP: miracle fruit powder extract.

2.14. Statistical analysis

Each determination was repeated in triplicate. The results were expressed as mean ± standard
error. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey
Honestly Significant Difference (HSD) test for statistical comparisons against control using
SPSS statistical software version 24 (SPSS, Inc., Chicago, IL, USA). A P-value
corresponding to the F-statistic of one-way ANOVA lower than 0.05 suggests that the one or
more treatments are significantly different, and the Tukey HSD test of multiple comparisons
follows. These posthoc tests would likely identify which of the pairs of treatments are
significantly different from each other.

3. Results

3.1. Phytochemical assessment

The phytochemical analysis of aqueous and ethanol extracts of MF powder was investigated
and summarized in Table 3. Greater content (P < 0.05) of total flavanoid compounds and
tannins was found in the ethanol extract when compared to the aqueous extract. The DPPH
scavenging method was used to evaluate the antioxidant capacity of the samples. The
percentage of antioxidant activity was found to be significantly higher (P < 0.05) in the MFP
ethanol extract compared to the aqueous extract with values of 79.61% ± 1.29% and 67.96%
± 1.80%, respectively.

Table 3. Quantitative phytochemical assessment of aqueous and ethanolic extracts of miracle fruit powder.
Sample Extraction TPC (mg GAE/g TFC (mg CE/g dry Tannin concentration DPPH (%)
type dry matter) matter) (mg/L)
Miracle fruit Aqueous 64.600 ± 0.116a 7.825 ± 0.014b 1.469 ± 0.287d 67.96 ± 1.80e
Miracle fruit Ethanol 63.070 ± 0.003a 13.672 ± 0.005c 5.890 ± 0.468e 79.61 ± 1.30f
Results were done in triplicates and values were expressed as mean ± standard error. Similar letters
indicate nonsignificance; while different letters indicate significance (P < 0.05). TPC: total phenolic
content; GAE: gallic acid equivalent; TFC: total flavonoid content; CE: catechin equivalent; DPPH: 1,1-
diphenyl-1-picrylhydrazyl.

3.2. HPLC analysis of MF ethanol extract

Ethanol extract of MF was subjected to reversed phase HPLC as described above. Major

5
peaks with retention time up to 52 min were observed. As shown in Fig. 1, the highest
percentage was that of the antioxidant quercetin (16.3%) followed by rutin (14%), myricetin
(12.4%), ascorbic acid (9.1%), ellagic acid (8.4%), syringic acid (8.1%), gallic acid (7.8%),
kaempferol (7.4%), ferulic acid (7.3%) and epicatechin (7.1%).

Fig. 1. Reversed phase HPLC chromatogram of miracle fruit ethanolic extract. 1: ascorbic acid; 2: gallic
acid; 3: epicatechin; 4: syringic acid; 5: ellagic acid; 6: rutin; 7: ferulic acid; 8: myricetin; 9: quercetin; 10:
kaempferol. The mobile phase consisted of formic acid (1%) in Milli-Q water:methanol (57:43, v/v) at 254
nm and 370 nm and the flow rate was 0.8 mL/min at 40 °C. HPLC: high-performance liquid
chromatography.

3.3. Effects on BW
Aspartame significantly increased (P < 0.05) the BW of test mice as compared to the diabetic
control after 10 d. However, no significant increase (P > 0.05) in BW was shown with the
group treated with low and high doses of MF extract as compared to the diabetic control after
10 d as shown in Fig. 2.

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Fig. 2. Effects of aspartame and miracle fruit on body weight in diabetic and nondiabetic mice (n = 3). *P <
0.05, vs diabetic control.

3.4. Acute and subchronic antihyperglycemic effects

After inducing diabetes using alloxan, BGL increased indicating diabetic mice being ready
for experimentation. As Fig. 3 reveals, at time zero, the BGL was considered high for the
diabetic control, aspartame, MF extract at low dose (MFL) and MF extract at high dose
(MFH) groups as compared to the BGL of the control nondiabetic group. The BGL of
diabetic mice treated with MFH dropped after 6 h as compared to that with diabetic control,
aspartame (mg/dL) and MFL; however, this was not significant (P > 0.05) (Fig. 3A).
Subchronic assessment of BGL was evaluated over a 10-day period. As Fig. 3B displays, the
BGL of diabetic mice treated with MFH dropped greatly after 10 d as compared to that of
diabetic control, aspartame and MFL. However, this was not significant (P > 0.05).

Fig. 3. Acute (A) and subchronic (B) effects of aspartame and miracle fruit on blood glucose of diabetic
and nondiabetic mice (n = 3).

3.5. Liver function test

As shown in Fig. 4, aspartame treatment significantly increased (P < 0.05) ALT level as
compared to control and untreated diabetic mice. However, diabetic mice treated with MFH

7
showed similar serum ALT level ([23.08 ± 12.42] U/L) to control ([25.31 ± 6.60] U/L) and
diabetic control mice ([24.28 ± 6.16] U/L). The marked increase in ALT enzyme after
aspartame treatment for 10 d indicates that aspartame negatively affected the liver function,
causing toxicity.

Fig. 4. ALT level among different treatment groups (n = 3). *P < 0.05, vs diabetic control. ALT: alanine
aminotransferase.

3.6. Total cholesterol test

Diabetic mice treated with MFH showed a significant decrease (P < 0.05) in total cholesterol
as compared to that of diabetic control as shown in Fig. 5.

Fig. 5. Total cholesterol among different treatment groups (n = 3). *P < 0.05, vs diabetic control.

3.7. Histological examination of mice liver tissues

The induction of diabetes by alloxan showed changes in the histological architecture of the
liver with microvesicular fatty changes and some hepatocyte vacuolization as shown in Fig.
6b. However, there was increased hepatocellular injury with more pronounced vacuolated
hepatocytes and cell degeneration after aspartame treatment (Fig. 6c). MF treatment at low
and high doses (Fig. 6d and 6e) dramatically alleviated these histological changes and
restored the normal architecture of the liver with hepatocytes radiating from the central vein.

8
Fig. 6. Representative photomicrographs of mouse liver sections after hematoxylin and eosin staining. a:
nondiabetic control; b: diabetic control group; c: aspartame-treated diabetic group; d: miracle fruit low
dose-treated diabetic group; e: miracle fruit high dose-treated diabetic group. CV: central vein; H:
hepatocytes; S: sinusoids; VC: vacuolated cytoplasm. Magnification: 400×.

3.8. Sensory analysis

Unsweetened lemonade with no sweeteners recorded the highest scores for both sourness
(10.53) and lemon taste (11.82). However, the unsweetened lemonade after ingestion of MF
recorded the highest score for the attribute sweetness (11.63) as shown in Fig. 7. Moreover,
seven out of ten panelists preferred the unsweetened lemonade with MF.

Fig. 7. Spider web chart of sensory analysis.

4. Discussion

The main purpose of this study was to determine whether MF can be used as a safer sugar

9
substitute or taste modifier in sour beverages as compared to artificial sweeteners such as
aspartame, and whether MF has any potential antihyperglycemic activity in alloxan-induced
diabetic mice. The safety of different doses of MF was also investigated by biochemical
assays and histopathological examination of the liver.
Moreover, MF was screened for its phytochemical composition and antioxidant activity. The
ethanol extract of MF exhibited higher concentrations of total flavonoids ([13.672 ± 0.005]
mg CE/g dry matter), tannin ([5.890 ± 0.468] mg/L) and DPPH (79.61% ± 1.29%) than the
aqueous extract. Similarly, a study showed that the ethanol extraction of Tetrapleura
tetraptera fruit was more efficient than the aqueous extraction [25]. Such type of extraction is
more effective in isolating certain components in the material being studied. Our results
showed that MF is a rich source of antioxidants and phytochemical compounds, which were
best distinguished with ethanol extraction.
In this study, the antioxidant activities of MF extract were evaluated by measuring DPPH
radical-scavenging ability. The DPPH of the ethanolic extract of MF was as high as 79.6%
which was superior to that of other berry fruits such as strawberry (11%–15%), raspberry
(8.27%–23.3%) and blackcurrant (42%–70%) [26]. However, this activity was different from
that reported by Du et al. [27] as they studied miracle berry seeds and flesh separately with
DPPH values of 54.3% and 96.3%, respectively, indicating that the flesh had significantly
higher antioxidant capability than the seed extract.
HPLC analysis showed quercetin to be the most abundant antioxidant in the MF. Quercetin is
a known polyphenol that exhibits anti-inflammatory, antihypertensive and anticarcinogenic
properties [11]. The human body cannot synthesize quercetin; instead, we must ingest it from
many plant sources such as berries, vegetables, tea, nuts and seeds [28]. The high quercetin
content shows that MF can be considered a rich source of antioxidants and disease-fighting
substances.
Diabetic mice receiving aspartame showed significant increase in BW after 10 days of
treatment. According to a study done on two groups of mice, the first group fed a high-fat
diet with aspartame showed increase in weight compared to the second group fed a high-fat
diet with plain water [29]. This is in accordance with our findings, in which aspartame also
increased the BW of mice.
In our study, both acute and subchronic antihyperglycemic effects of aspartame and MF were
assessed. Results showed that subchronic treatment with MFH (500 mg/kg BW) was found to
be more effective in lowering the BGL. This might be due to the antioxidants and
phytochemicals like flavonoids and the glycoprotein (miraculin) in the MF, which are known
to exhibit antidiabetic potential [30].
The liver enzyme (ALT) and total cholesterol levels were assessed. It was noticeable that
ALT serum level was significantly elevated in the diabetic mice treated with aspartame as
compared to controls and diabetic controls; however, it dropped when treated with low and
high doses of MF. Similar findings by another study displayed elevated ALT levels ([123.1
±13.1] U/L) for diabetic-rodents treated with aspartame, which was slightly higher than that
of the diabetic control that was (119.1 ± 11.7) U/L [31]. In agreement with our results,
treatment with Stevia rebaudiana showed a great drop in the ALT [31]. As for cholesterol
test, our results showed a significant decrease in the total cholesterol levels after
administration of the MFH as compared to that of diabetic control. This was in agreement
with another study showing that diabetic mice treated with methanol extract of MF had
cholesterol concentration of (73.87 ± 5.92) mg/dL, which was lower than that of diabetic
control that was (206.84 ± 3.68) mg/dL [32].
In the histopathological analysis of liver tissues, it was obvious that there was an architectural
change in the liver tissues of the diabetic groups. Diabetes is known to cause several
complications, specifically liver damage [33]. However, diabetic mice treated with aspartame

10
showed dramatic deterioration in the liver tissues, which was parallel to the elevated ALT
during biochemical assessment. Such findings were supported by other studies [34,35].
However, the liver histopathological architecture was greatly restored after treatment with
low and high doses of MF. These results demonstrated that MF has hepatoprotective effects
on diabetic mice.
Sensory analysis of MF showed that its intake is effective in masking the sourness of
unsweetened beverages while leaving a sweet sensation in the mouth. Findings by Rodrigues
and colleagues [4] supported that MF consumption prior to lemonade reduced the sour taste
and increased sweetness. This could be explained by the glycoprotein miraculin which is
tasteless by itself, but once ingested, it binds and stimulates the sweet taste receptors as the
pH decreases (acidic medium) [11]. Persistence of the sweet taste remained the highest (9.69)
for the unsweetened lemonade after ingestion of MF, making it a useful natural sweetener for
consumers.

5. Conclusion

Our study suggests that MF could be used as a flavor enhancer and sugar substitute in several
sour foods and beverages. Moreover, it revealed that the ethanol extract of MF exhibited
antioxidant potential as well as antihyperglycemic activity, especially when administered at
high doses. Additionally, MF can be used as a healthier alternative sweetening agent for
artificial sweeteners such as aspartame, especially with the histological data showing the
significant hepatoprotective effects of MF versus the marked deterioration of hepatic function
with aspartame treatment. Further studies should be undertaken to determine the constituents
behind the medicinal properties of MF and the corresponding mechanism of action.

Funding

This research received no external funding.

Authors’ contribution

FS contributed to experimental design, developing manuscript and critical discussion, and

performed the histological experiments and analysis; SH conceived the study and contributed
to writing the manuscript as well as data collection and statistical analysis; KR performed
HPLC experiments and its analysis and helped to draft the manuscript; MM contributed to
animal handling and blood collection.

Conflicts of interest

The authors declare no competing interests.

References

[1] Schmidt LA. The toxic truth about sugar. Nature 2012; 452: 7–9.
[2] Saad A, Khan FA, Hayee A, Nazir MS. A review on potential toxicity of artificial
sweetners vs. safety of stevia: a natural bio-sweetner. J Biol Agr Health 2014; 4(15): 1–12.
[3] Neacşu NA, Madar A. Artificial sweeteners vs. natural sweeteners. Bulletin Transilvania
University Braşov 2014; 7(56): 59–64.
[4] Rodrigues JF, Andrade RDS, Bastos SC, Coelho SB, Pinheiro ACM. Miracle fruit: an

11
alternative sugar substitute in sour beverages. Appetite 2016; 107: 645–53.
[5] Choudhary AK, Pretorius E. Revisiting the safety of aspartame. Nutr Rev 2017; 75: 718–
30.
[6] Choudhary AK. Aspartame: should individuals with type II diabetes be taking it? Curr
Diabetes Rev 2018; 14: 350–62.
[7] Zafar T, Shrivastava V. Aspartame: effects and awareness. MOJ Toxicol 2017; 3(2): 23–
6.
[8] Marinovich M, Galli CL, Bosetti C, Gallus S, La C. Aspartame, low-calorie sweeteners
and disease: regulatory safety and epidemiological issues. Food Chem Toxicol 2013; 60:
109–15.
[9] Sánchez-Tapia M, Martínez-Medina J, Tovar AR, Torres N. Natural and artificial
sweeteners and high fat diet modify differential taste receptors, insulin, and TLR4-mediated
inflammatory pathways in adipose tissues of rats. Nutrients 2019; 11 (4): 880.
[10] Chen CC, Liu IM, Cheng JT. Improvement of insulin resistance by miracle fruit
(Synsepalum dulcificum) in fructose-rich chow-fed rats. Phytother Res 2006; 20: 987–92.
[11] Misaka T. Molecular mechanisms of the action of miraculin, a taste-modifying protein.
Semin Cell Dev Biol 2013; 24: 222–5.
[12] Shi YC, Lin KS, Jhai YF, Lee BH, Han Y, Cui Z, et al. Miracle fruit (Synsepalum
dulcificum) exhibits as a novel anti-hyperuricaemia agent. Molecules 2016; 21(2): 140.
[13] Njoku NE, Ubbaonu CN, Alagbaoso SO, Eluchie CN, Umelo MC. Amino acid profile
and oxidizable vitamin content of Synsepalum dulcificum berry (miracle fruit ) pulp. Food Sci
Nutr 2015; 3: 252–6.
[14] Wong JM, Kern M. Miracle fruit improves sweetness of a low-calorie dessert without
promoting subsequent energy compensation. Appetite 2011; 56: 163–6.
[15] Koizumi A, Tsuchiya A, Nakajima K, Ito K, Terada T, Shimizu-Ibuka A, et al. Human
sweet taste receptor mediates acid-induced sweetness of miraculin. Proc Natl Acad Sci U S A
2011; 108: 16819–24.
[16] Chinelo N, Obi N, Florence E. Phytochemical, antinutrient and amino acid composition
of Synsepalum dulcificum Pulp. J Pharma Bio Sci 2014; 9(2): 25–9.
[17] Kim DO, Chun OK, Kim YJ, Moon HY, Lee CY. Quantification of polyphenolics and
their antioxidant capacity in fresh plums. J Agric Food Chem 2003; 51: 6509–15.
[18] Hosu A, Cristea V, Cimpoiu C. Analysis of total phenolic, flavonoids , anthocyanins and
tannins content in Romanian red wines: prediction of antioxidant activities and classification
of wines using artificial neural networks. Food Chem 2014; 150: 113–8.
[19] Zhang D, Hamauzu Y. Phenolics, ascorbic acid, carotenoids and antioxidant activity of
broccoli and their changes during conventional and microwave cooking. Food Chem 2004;
88: 503–9.
[20] Obafemi TO, Akinmoladun AC, Olaleye MT, Onasanya A, Komolafe KC, Falode JA, et
al. High-performance liquid chromatography (HPLC) fingerprinting, mineral composition
and in vitro antioxidant activity of methanol leaf extract of Synsepalum dulcificum
(Sapotaceae). J Appl Pharm Sci 2017;7:110–8.
[21] Saleh FA, El-Darra N, Raafat K. Hypoglycemic effects of Prunus cerasus L. pulp and
seed extracts on alloxan-induced diabetic mice with histopathological evaluation. Biomed
Pharmacother 2017; 88: 870–7.
[22] Rohilla A, Ali S. Alloxan-induced diabetes: mechanisms and effects. Int J Res Pharma
Biomed Sci 2012; 3: 819–23.
[23] Ighodaro OM, Adeosun AM, Akinloye OA. Alloxan-induced diabetes, a common model
for evaluating the glycemic-control potential of therapeutic compounds and plants extracts in
experimental studies. Medicina 2018:1–10.
[24] Raafat K, El-darra N, Saleh FA, Rajha HN, Maroun RG, Louka N. Infrared-assisted

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extraction and HPLC-analysis of Prunus armeniaca L. Pomace and Detoxified-Kernel and
their antidiabetic effects. Phytochem Anal 2017; 29: 156–67.
[25] Gberikon G, Adeoti I, Aondoackaa AD. Effect of ethanol and aqueous solutions as
extraction solvents on phytochemical screening and antibacterial activity of fruit and stem
bark extracts of Tetrapleura tetrapteraon, Streptococcus salivarus and Streptococcus mutans.
Int J Curr Microbiol Appl Sci 2015; 4: 404–10.
[26] Balogh E, Hegedűs A, Stefanovits-Bányai E. Application of and correlation among
antioxidant and antiradical assays for characterizing antioxidant capacity of berries. Scientia
Horticulturae 2010; 125: 332–6.
[27] Du L, Shen Y, Zhang X, Prinyawiwatkul W, Xu Z. Antioxidant-rich phytochemicals in
miracle berry (Synsepalum dulcificum) and antioxidant activity of its extracts. Food Chem
2013;153:279–84.
[28] Li Y, Yao J, Han C, Yang J, Chaudhry MT, Wang S, et al. Quercetin, inflammation and
immunity. Nutrients 2016; 8:167.
[29] Gul SS, Hamilton AR, Munoz AR, Phupitakphol T, Liu W, Hyoju SK, et al. Inhibition
of the gut enzyme intestinal alkaline phosphatase may explain how aspartame promotes
glucose intolerance and obesity in mice. Appl Physiol Nutr Metab 2017; 42(1):77–83.
[30] Dioso MKM, Satsatin DCA, Ching JA. Hypoglycemic effects of Synsepalum dulcificum
(Schumach. & Thonn.) Daniell (miracle berry) fruit and leaf extracts on the blood glucose
level of albino rats. Der Pharmacia Lettre 2016; 8: 104–8.
[31] Abdelwahab AH, Yousuf AF, Ramadan BK, Elimam H. Comparative effects of Stevia
rebaudiana and aspartame on hepato-renal function of diabetic rats: biochemical and
histological approaches. J Appl Pharm Sci 2017; 7(8): 34–42.
[32] Obafemi OT, Akinmoladun CA, Olaleye TM, Agboade SO, Onasanya AA. Antidiabetic
potential of methanolic and flavonoid-rich leaf extracts of Synsepalum dulcificum in type 2
diabetic rats. J Ayurveda Integr Med 2017; 8: 238–46.
[33] Mohamed J, Nafizah AHN, Zariyantey AH, Budin SB. Mechanisms of diabetes-induced
liver damage: the role of oxidative stress and inflammation. Sultan Qaboos Univ Med J 2016;
16: 132–41.
[34] Alkafafy ME, Ibrahim ZS, Ahmed M, El-shazly SA. Impact of aspartame and saccharin
on the rat liver: Biochemical, molecular, and histological approach. Int J Immunopathol
Pharmacol 2015; 28: 247–55.
[35] Khidr BM, El-sokkary GH, Saleh SMM. Study on morphological changes induced by
aspartame on liver of normal and diabetic male albino rats. Histol Histopathol 2017; 4: 1–7.

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