Vaccinium Extracts as Modulators in Experimental Type 1 Diabetes

Abstract

Antihyperglycemic effects of four extracts obtained from leaves and fruits of Vaccinium myrtillus and Vaccinium corymbosum were assessed in diabetic rats. In addition, the effects of extracts on diabetic-related complications such as the development of diabetic cataract and oxidative stress were evaluated. Type 1 diabetes was induced with a single dose of streptozotocin in Wistar rats. The rats were randomly divided into seven equal groups: NC—normal control, DC—diabetic control, PC—positive control treated with metformin, VML—received V. myrtillus leaf extract, VMLF—received VML and fruit extract, VCL—received V. corymbosum leaf extract, and VCLF—received VCL and fruit extract. Body weight and glucose levels were monitored every second week. After 8 weeks of treatment, serum glucose, insulin, and malondialdehyde were measured. Lenses were removed after sacrifice and eight lenses from each group were randomly selected for evaluation of cataract development. A decrease in body weight was observed in all diabetic groups in the first weeks. In the VML group, no significant decrease was observed. Glucose levels during the experiment were high in DC, PC, and VCL groups, with no improvement during the 8 weeks. In VML, VMLF, and VCLF groups, a decrease in blood glucose levels was observed. Similar results regarding serum insulin and glucose levels at the end of the experiment were observed within groups. V. myrtillus extracts prevented the development of cataract compared with the DC group (P < .05).

Introduction

Diabetes is a metabolic disorder that leads to multiple complications over time. Type 1 diabetes mellitus is an autoimmune disease characterized by insufficient secretion of insulin. This leads, in addition, to hyperglycemia because insulin normally induces the translocation of specific proteins, thus stimulating glucose uptake and storage.¹ During the last years, many types of researches have been conducted to identify or isolate natural compounds that have beneficial effects in the management of diabetes with lesser side effects than synthetic drugs. Vaccinium species have been reported to have antihyperglycemic and antihyperlipidemic effects and could prevent the development of diabetic retinopathy, nephropathy, and neuropathy.^2,3

Vaccinium myrtillus leaves (VMLs) have a long history of empiric use in diabetes, which started in the early 20th century. Despite this long use as an antidiabetic agent, there is a lack of recent data to support this use.⁴ In view of the large number of species of the genus Vaccinium, scientific literature frequently encounters confusion about the name of the plant when the Latin name is not mentioned. Vaccinium myrtillus, also called bilberry, is a wild species native to Europe. Vaccinium corymbosum, also called blueberry or high-bush blueberry, is a cultivated species native from North America.⁵ Anthocyanins isolated from the fruits are responsible for antioxidant activity, and recent studies suggest that this class of compounds may activate AMP kinase in the liver, white adipose tissue, and muscle, similar to biguanides developed after isolation of guanidine and galegine from Galega officinalis.⁶ Hydroxycinnamic acids, flavonoid glycosides, and proanthocyanidins could be responsible for the antihyperglycemic effect of the leaves.^7,8

Due to influences of seasonal variations and geographical characteristics, the phytochemical profile and possible pharmacological actions of each species could be unique.⁹ The aim of this study was to investigate the effects of four types of extracts obtained from Vaccinium sp. in chemically induced diabetes in rats. It is the first reported study of the antihyperglycemic action of leaves and fruits obtained from Vaccinium sp. collected from Mureş County, Romania.

Materials and Methods

Chemicals

Streptozotocin (STZ) was purchased from Sigma. Glucometer strips and metformin were commercial products available in the Romanian pharmaceutical market. Reference substances were cyanin chloride, isoquercitrin, rutin (Carl Roth GmbH), kuromanin, kaempferol, caffeic acid, and chlorogenic acid (Cayman Chemical Company). Other chemicals used (methanol, acetonitrile, acetone, chloroform, and trifluoroacetic acid) were of analytical or high-performance liquid chromatography (HPLC) grade.

Plant material

V. myrtillus L. leaves and fruits were harvested during the ripening season from a natural habitat in Lunca Bradului, Mureş County, Romania. V. corymbosum fruits were harvested in July 2015 from a local culture near Trei Sate, Mureş County, Romania, and the red leaves were harvested in October 2015 from the same culture. The fruits were immediately frozen and kept in a freezer at −20°C and leaves were air-dried and preserved in laboratory conditions.

Sample extraction and LC-MS/MS analysis

Two methods were tested for phytochemical profile assessment. Both types of fruits and both types of leaves underwent the same procedure.

1. Plant material was mixed with quartz sand in a glass mortar at a plant–sand ratio of 1:1 for fresh fruits or 1:2 for dried leaves, and the material was then introduced in a glass column with methanol. The column was eluted with methanol and then with 70% acetone. Elution was stopped when no more compounds were extracted (thin-layer chromatography [TLC] examination).

2. Plant material was extracted with methanol in an ultrasonic water bath at a temperature of 40°C in a plant–solvent ratio of 2.5:10 for fruits and 1:10 for leaves. The extract was then concentrated in a rotary evaporator. The concentrated extract was introduced in a glass column packed with Sephadex LH-20 and was eluted with methanol and 70% acetone.

For LC-MS/MS analysis, methanolic fractions were selected according to TLC analysis (data not shown). The samples were analyzed on an LC-MS/MS system that consisted of an 1100 series model HPLC-UV equipment coupled with a mass spectrometer QQQ 6410 (Agilent). Chromatographic separation was performed at 35°C on an Inertsil ODS, 150 × 4.6 mm, 3-μm column (GL Sciences, Inc.). The mobile phase consisted of a mixture of trifluoroacetic acid 0.1% (Phase A), acetonitrile (Phase B), and methanol (Phase C). The elution gradient used was as follows: 0–15 min, 87–73% A, 8–22% B; 15–20 min, 73–72% A, 22–23% B; 20–25 min, 72–15% A, 23–80% B; 25–30 min, 15% A, 80% B; 30–31 min, 15–87% A, 80–8% B; and 31–35 min, 87% A, 8% B. Phase C had a constant concentration of 5%. Twenty microliters of analytes was injected, with a flow rate of 0.8 mL/min and a constant column temperature of 35°C. Chromatograms were acquired at 280 nm. Conditions used in ESI+ analysis were as follows: 35°C, gas flow 10 L/min, nebulizer 50 psi, 3500 V, scan mode, scan time 100, and fragmentor 180. The following fragments were scanned: m/z 633, 611.5, 472, 449.5, 287, 355, 377, 163, 181, 493, 331, 303, 465, 242, 487, and 463. Identification was carried out based on retention times and mass spectra compared with those of standard substances.

Experiment on animals

Female Wistar rats (24–28 weeks) were provided by the Department of Experimental Animal Facilities of the University of Medicine and Pharmacy from Târgu Mureş, Romania. The animals were housed (two or three) in a cage with free access to water in a room with a 12-h light–12-h dark cycle (7:00 am–7:00 pm) and temperature of 24°C ± 1°C, and the animals were weighed weekly. During the acclimatization period, each animal was fed a regular diet ad libitum. The rats were fed a conventional diet for 1 week and they were divided into two groups: a nondiabetic control (DC) group and a diabetic group. The experimental protocol was approved by The Ethics Committee of the University of Medicine and Pharmacy of Târgu Mureş, Romania (Registration number 66/2016).

Preparation of extracts for experiments on animals

Grounded leaves were extracted for 30 min with 50% ethanol in an ultrasonic water bath (VMLs) or in a boiling water bath (V. corymbosum leaves [VCLs]) in a plant–solvent ratio of 1:20. Grounded fruits were extracted for 30 min with 50% ethanol in an ultrasonic water bath at a temperature of 40°C in a plant–solvent ratio of 3:20. After the extraction process, all extracts were introduced into a rotary evaporator, at 40°C, under reduced pressure to remove ethanol.

Induction of experimental diabetes

STZ was freshly dissolved in saline water and it was injected (60 mg/kg) intraperitoneally into overnight fasted rats. To overcome hypoglycemia, which could appear during the first 24 h following STZ administration, diabetic rats were immediately allowed to eat.¹⁰

Blood samples were taken from the tail vein 48 h after STZ injection to measure glucose levels with a portable glucometer. Only animals with a 12-h fasting blood glucose above 250 mg/dL were considered diabetic and used in the present study.

Experimental procedure

The rats were divided into seven groups, each containing eight animals, as presented in Table 1.

Table 1.

Treatments Applied to Each Experimental Group

Group	Abbreviation	Treatment
Non-DC	NC	Healthy untreated rats
DC	DC	Diabetic untreated rats
PC	PC	Metformin (500 mg/kg)-treated diabetic rats
VML diabetic	VML	Diabetic rats treated with VML extract equivalent to 870 mg dried V. myrtillus leaves/kg
VMLF diabetic	VMLF	Diabetic rats treated with VMLF extract equivalent to 430 mg dried V. myrtillus leaves/kg +1300 mg fresh V. myrtillus fruits/kg
VCL diabetic	VCL	Diabetic rats treated with VCL extract equivalent to 870 mg dried VCL/kg
VCLF diabetic	VCLF	Diabetic rats treated with VCLF extract equivalent to 430 mg dried VCL/kg +1300 mg fresh V. corymbosum fruits/kg

DC, diabetic control; NC, normal control; PC, positive control; VCLFs, V. corymbosum leaves and fruits; VMLFs, Vaccinium myrtillus leaves and fruits.

The extracts were administered with a gastric gavage needle. Diabetes development was then checked every other week by determination of glucose concentration in the blood. Fasting blood glucose concentration was determined using a commercial glucometer test (Bayer). After 8 weeks of treatment, the rats were sacrificed by exsanguination after isoflurane anesthesia. Blood was collected in plain vials for serum. The blood was allowed to clot and then centrifuged at 280 × g for 10 min at 4°C to obtain clear serum.

Biochemical assays

The glucose in serum was determined with the rat glucose assay kit (#55433; Crystal Chem) and insulin was determined with the rat/mouse insulin ELISA kit, (#EZRMI-13K; Merck). Malondialdehyde (MDA) was determined by using an HPLC method described by Fogarasi et al. ¹¹

Morphological examination of lenses

Four rats from each group were randomly chosen for evaluation of cataract development. In the sixth week of the experiment, 1% tropicamide was instilled in the eyes and an ophthalmologist carried out the evaluation with an ophthalmoscope. Morphological changes were observed, but the degree of cataract could not be assessed. After sacrifice, lenses from both eyes of each rat were excised. Eight lenses from each group were randomly selected and analyzed under a microscope. The degree of opacification was graded according to Geraldine et al. ¹²

Statistical analysis

Results are expressed as means ± standard deviations. Data were analyzed by one-way or two-way analysis of variance (ANOVA) with Tukey's post hoc test, depending on the type of data. Cataract stage between groups was analyzed with Kruskal–Wallis nonparametric analysis, followed by Dunn's test. Statistical significance was set at the 5% level.

Results

LC-MS/MS analysis

In all samples obtained from V. corymbosum fruits, the following were identified: chlorogenic acid, malvidin-3-O-glucoside, quercetin-3-rutinoside, and quercetin-3-glucoside. All samples from V. myrtillus fruits contained chlorogenic acid, cyanidin-3-O-glucoside, malvidin-3-O-glucoside, and quercetin-3-glucoside. In VCLs, chlorogenic acid, cyanidin-3-O-glucoside, quercetin-3-rutinoside, and quercetin-3-glucoside were identified. In VMLs, chlorogenic acid and quercetin-3-glucoside were identified. No differences in composition were observed between the two methods used for extraction and fractionation.

Mortality

During the 8 weeks of treatment, mortalities occurred in every group, except the normal control (NC) group. The rates of survival were 75% for the DC group, 87.5% for the positive control (PC) group, 87.5% for the VML group, 75% for the VML and fruit (VMLF) group, 75% for the VCL group, and 75% for the VCL and fruit (VCLF) group.

Effects of extracts on body weight and glucose levels during the study

As shown in Table 2, blood glucose levels remained constant during the experiment for the NC group. In the DC group, after STZ injection, blood glucose levels increased significantly (P < .05), with no improvement until the end of the study. The same response was observed in the PC group treated with metformin. In the VML group, blood glucose levels decreased, and after 1 week of treatment, the glucose level was significantly lower compared with the initial level after induction of diabetes. Beginning from the fourth week, blood glucose increased slightly until the end of the study. In the VMLF group, a small improvement in blood glucose has been observed starting with the sixth week, but with no statistical significance compared with the initial blood glucose in diabetic rats. No positive effects were seen after administration of VCL extract in the VCL group. In the VCLF group, an improvement was observed in blood glucose, but with no significant difference compared with the initial glycemic level, after the STZ injection (Fig. 1).

FIG. 1.

Glucose level variations during the experiment. Color images available online at www.liebertpub.com/jmf

Table 2.

Glucose Levels (mg/dl) During 8 Weeks of Treatment

	Week
Group	0	1	2	4	6	8
NC	109.9 ± 7.8	105.4 ± 8.6	90.9 ± 10.7	91.7 ± 7.4	92.5 ± 9.4	96.6 ± 15.1
DC	96.2 ± 5.6^a	377.4 ± 57.5^b	313.2 ± 94.2^b	413.1 ± 90.9^* ^b	413.7 ± 80.7^** ^b	409.2 ± 128.6^** ^b
PC	106.4 ± 3.8^a	330.6 ± 66.1^b	367.2 ± 118.6^b	397.4 ± 140.7^* ^b	391.4 ± 150.8^* ^b	422.8 ± 149.7^* ^b
VML	109.13 ± 6.6^a	342.4 ± 118.2^b	209.6 ± 122.7^a ^b ^#	248.9 ± 174.1^ab ♮	291.8 ± 130.4^* ^b	241 ± 122.1^* ^b
VMLF	96.88 ± 8.6^a	364.6 ± 36.3^b	372.4 ± 164.8^b	381.3 ± 129.9^b	314.3 ± 132.9^* ^b	293.5 ± 129.6^** ^b
VCL	103.8 ± 5^a	378.9 ± 56.6^b	405.2 ± 75.4^b	358.1 ± 98.3^b	433 ± 138.1^b	460.3 ± 148.7^** ^b
VCLF	106.4 ± 7^a	329.9 ± 45.6^b	269.9 ± 128.3^b	335.1 ± 154.8^b	262.7 ± 151.9^** ^b	263 ± 140.1^** ^b

Data are expressed as means ± SD (n = 8 in each group, if not otherwise stated, ^* n = 7, ^** n = 6). Values in the same row with different superscript letters are significantly different (P < 0.05).

Significantly different compared to PC group.

♮

Significantly different compared to DC group. DC, diabetic control group; NC, non-diabetic control group; PC, positive control, treated with metformin; VCL, Vaccinium corymbosum leaves group; VCLF, Vaccinium corymbosum leaves and fruits group VML, Vaccinium myrtillus leaves group; VMLF, Vaccinium myrtillus leaves and fruits group.

The weights of rats from the NC group had increased until the end of the experiment. After STZ injection, weights of diabetic rats started to decrease in all groups (Table 3). In DC and PC groups, weight loss was significantly different compared with the initial weight. The weight of rats treated with V. myrtillus leaf extract from the VML group started to decrease after STZ injection, but after 4 weeks of treatment, weight remained constant until the end of the study. However, no significant differences in body weight were observed in this group over the 8 weeks of treatment. In VMLF, VCL, and VCLF groups, a notable decrease in weight was observed during the study. In the VCLF group, after a high weight loss during the first weeks of treatment, an improvement was observed, but it overlaps with the deaths recorded in this group (Fig. 2).

FIG. 2.

Body weight variations during the experiment. Color images available online at www.liebertpub.com/jmf

Table 3.

Body Weight Variations (g) During the Experiment

	Week
Group	0	1	2	4	6	8
NC	237.5 ± 11.6	233.7 ± 13	243.7 ± 15.7	256.2 ± 17.3	266.9 ± 21.2	267.5 ± 16.5
DC	232.5 ± 10.3^a	216.2 ± 20^ab	200 ± 29.9^ab	185 ± 37.9^* ^b	183.3 ± 38.7^** ^b	173.3 ± 42.5^** ^b
PC	231.2 ± 18.1^a	216.9 ± 24.3^ab	214.4 ± 24.6^ab	201.4 ± 31^* ^a ^b	187.1 ± 21.8^* ^b	176.4 ± 43.1^* ^b
VML	239.4 ± 7.8	218.9 ± 15.6	208.7 ± 25.5	200 ± 31.1	205 ± 32.1^*	208.6 ± 39.8^*
VMLF	230 ± 18.5^a	213.1 ± 24^ab	196.1 ± 37.3^ab	193.7 ± 37.4^ab	183.3 ± 59.5^* ^b	188 ± 53^** ^ab
VCL	233.7 ± 14.1^a	219.4 ± 13.5^ab	208.2 ± 14.9^abc	184.4 ± 24.7^bc	180.6 ± 22.7^bc	174.2 ± 27.3^** ^c
VCLF	230.6 ± 19.3^a	211.9 ± 21^ab	186.9 ± 25.2^b	182.5 ± 38.2^b	188.3 ± 37.1^** ^ab	199 ± 46^** ^ab

Effects of extracts on serum glucose, serum insulin, and MDA levels after 8 weeks of treatment

The serum insulin level in the DC group was significantly lower than in the NC group, so was the insulin level in the PC group (Table 4). The VML group had the highest level of insulin among the diabetic groups and it was not significantly different compared with the NC group. VMLF and VCL groups had a similar level of insulin to the PC group. In the VCLF group, the insulin level was higher compared with the DC group and PC group, but with no significant difference. Serum glucose and MDA levels showed a similar trend, the group with higher insulin levels had a lower glucose level and a lower MDA level.

Table 4.

Serum Glucose, Insulin, and Malondialdehyde Levels at the End of the Experiment

Group	Insulin (ng/mL)	Glucose (mg/dL)	MDA (ng/mL)
NC (n = 8)	2.39 ± 0.87^a	158.79 ± 21.26^a	20.55 ± 8
DC (n = 6)	0.65 ± 0.45^b	266.52 ± 67.75^b	29.29 ± 11.18
PC (n = 7)	0.9 ± 0.66^b	227.01 ± 27.93^ab	27.92 ± 10.89
VML (n = 7)	1.46 ± 0.87^ab	189.83 ± 52.47^ab	26.92 ± 6.83
VMLF (n = 6)	1.06 ± 1.04^b	227.24 ± 41.75^ab	30.32 ± 9.79
VCL (n = 6)	1.02 ± 0.91^b	237.13 ± 68.51^ab	30.63 ± 5.2
VCLF (n = 6)	1.27 ± 0.29^ab	217.7 ± 77.63^ab	27.89 ± 5.12

Data are expressed as mean ± SD. n, number of animals in each group at the moment of analysis. Values in the same column with different superscript letters are significantly different (P < 0.05).

MDA, malondialdehyde.

Lens morphological evaluation

Results obtained after lens morphological evaluation are presented in Table 5. Figure 3 represents examples for each grade of lens opacification.

FIG. 3.

Stages of cataract formation. Color images available online at www.liebertpub.com/jmf

Table 5.

Cataract Development

	Number of lenses
Group	Grade 0	Grade 1	Grade 2	Grade 3
NC #	8	0	0	0
DC	0	0	1	7
PC	0	0	2	6
VML #	0	5	2	1
VMLF #	1	5	1	1
VCL	1	3	3	1
VCLF	0	3	3	2

Significantly different compared to DC group (p < 0.05).

Discussion

LC-MS/MS analysis was performed to identify the main compounds from our plant materials. Two fractionation methods were used to assess the difference between low heat or no heat extraction, knowing that temperature is a critical parameter in the stability of phenolic compounds, especially anthocyanins.¹³ No differences were observed from the qualitative perspective, although the compounds present in these plant materials have been described and geographical variation can occur, as we noticed in this case. Quercetin-3-rutinoside is a characteristic compound for a specific genotype of V. corymbosum; therefore, many researchers have noticed the absence of this compound in blueberry fruits collected from different cultivars.¹⁴ In addition, scientific literature regarding the phytochemical characterization of this kind of plant from Romania reports the presence of cyanidin-3-O-glucoside in the fruits.^15,16

The extraction preparation method was chosen based on our previous studies, which showed a greater concentration of polyphenolic compounds in extracts prepared with 50% ethanol.⁹ Ethanol was removed by distillation under reduced pressure to avoid alcohol toxicity in rats.

STZ-induced diabetes in rats is a widely used method for creating a type 1 diabetic model. The majority of rodent experiments are done on males, but females are more sensible (see the rate of mortality) and that is why we have chosen to conduct this experiment on female rats. Another reason for choosing females was that among the human population, diabetes is more prevalent in women.¹⁷ STZ produces an irreversible necrosis of Langerhans islets in the pancreas, which results in high glycemic levels and low insulin levels. As seen in Table 2, after STZ injection, the glucose level increased significantly and diabetic symptoms such as polydipsia and polyuria were noticed. The gavage method was used for administration of extracts to control the dosage. Although many articles have reported that gavage induces an extra stress on rats, we considered the right dosage to be very important for the final outcome. Because of the high tannin content, which is assumed to have antinutritional effects, higher and lower doses were tested.¹⁸ For the same reason, and to reduce the stress on rats, administration of extracts was performed every other day. The lower doses of leaf extracts were supplemented with fruit extracts to compensate for reduction in polyphenolic content.

Compared with other studies, we observed a 17.85% mortality rate (counting deaths in all seven groups). This average is in accordance with the few data found from scientific papers.¹⁹ The interpretation of this percentage is, as yet, difficult because these data are rarely reported.

It has been shown that in STZ-induced diabetes, the level of insulin decreases with the progress of diabetes. We noticed, in the groups that received extracts, a higher insulin level than in the DC group. This could be explained by the capacity of extracts to restore the beta-cell function and could have a presumed secretagogue effect. A higher rise in insulin level was noticed in the VML group, which received a concentrated extract from VMLs, followed by the group that received the VCLF extract.

V. myrtillus extracts prevented weight loss, characteristic in type 1 diabetes, and decreased the glucose level. A great majority of studies have focused on the effect of Vaccinium extracts in type 2 diabetes, but our results suggest that these extracts also have beneficial effects in type 1 diabetes. The beneficial effects in type 2 diabetes are attributed to the inhibitory effects of the extracts on α-glucosidase and maybe on α-amylase.²⁰ Previous experiments on diabetic rats have reached contradictory results, for example, bilberry extracts introduced in the drinking water of diabetic rats proved to have little effect on blood glucose, but in a more recent study on aloxan-induced diabetes, bilberry powder proved to reduce the blood glucose level and increased serum insulin.^21,22 These results indicate that the effect of bilberry fruits in diabetes could be dose dependent. Therefore, our protocol of administration—every second day—may be insufficient for the treatment with anthocyanins; daily doses could be required for a better outcome.

In the VMLF group that received a lower dose of V. myrtillus leaf extract combined with fruit extract, a decrease in the glucose level was observed during the 8 weeks of treatment, but at a lower percentage than in the VML group. Other studies have shown that bilberry fruits are more effective in preventing diabetic retinopathy than modulating glucose levels.²³ Previous studies have shown that blueberry anthocyanins inhibit carbohydrate-hydrolyzing enzymes, but this capacity is of modest importance in type 1 diabetes when insulin therapy is used; moreover, the risk of hypoglycemia increases.^24,25 No improvement has been observed in glucose, insulin, and malondialdehyde levels in the VCL group.

Oxidative stress, another complication of metabolic syndrome, is induced by hyperglycemia. Therefore, supplementation of diet with antioxidants is the latest trend in managing oxidative stress and its effects on diabetes. Polyphenols are a class of compounds with high antioxidant capacities. Flavonoids, anthocyanins, and carboxylic acids are known for their individual antioxidant activity. In an extract, where multiple classes of compounds exist, a synergistic effect could appear.

Diabetes triggers a set of specific complications in time, and cataract is one of the frequent ones. The development of cataract is induced by accumulation of sorbitol due to reduction of glucose under the action of aldose reductase, an NADPH-dependent enzyme. Sorbitol accumulation induces osmotic and oxidative stresses in the lens. Aldose reductase is usually found in tissues that are not insulin sensitive such as the lens, peripheric nerves, and glomerulus.²⁶ Therefore, aldose reductase inhibition could prevent the development of diabetic complications induced by hyperosmosis such as cataract, nephropathy, neuropathy, and retinopathy. Hyperglycemia caused by administration of STZ has led to development of cataract in the DC group. In both VML and VMLF groups, significant differences in cataract formation were observed compared with the DC group. The prevention of cataract development by V. myrtillus extracts could be explained by their high percentage of chlorogenic acid in leaves as it was previously determined that chlorogenic acid is an aldose reductase inhibitor.²⁷ Although the degree of opacification was lower in VCL and VCLF groups compared with the DC group, no significant differences were observed.

This study indicated that administration of bilberry leaf extracts had the potential to lower blood glucose and to improve insulin secretion. In addition, both leaf extracts and the combination of leaf and fruit extracts were capable of preventing the development of diabetic-induced cataract in vivo. A comparable effect has been shown with the combination of leaf and fruit extracts for the same species, but with a lower outcome. The results obtained after administration of VCLFs show promising effects, but further studies are needed, with fractions obtained from these extracts, for a better understanding of the possible mechanisms of action. To our knowledge, this is the first study to compare the effects of V. myrtillus and V. corymbosum extracts on metabolic changes occurring in experimental type 1 diabetes.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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