Diabetes mellitus is a chronic metabolic disease that is characterised by chronic impaired blood glucose levels and hyperglycaemia as a result of compromised insulin secretion or impaired insulin action (1). Physiologically and biochemically, in various tissues, the chronic hyperglycaemia can induce oxidative stress that terminate normal biological activities and cause a cascade of chronic diabetic complications (2).

The usage of plants and their extracts in many treatments of chronic diseases is widely known in traditional medicine, but it also has a big potential in the treatment of hyperglycaemia and diabetic complications, especially by reducing the levels of oxidative stress on a cellular cytoplasmic level (1, 3-6). Recent scientific data support the fact that polyphenols including flavonoids, phenolic acids, lignans and stilbenes found in most plants can have a positive effect on many chronic diseases, and act like potent antioxidants and anti-inflammatory agents (4, 5, 7, 8).

Blackthorn (Prunus spinosa L.) is a rich source of phytochemicals, polyphenols, including phenolic acids and flavonoids, A-type proanthocyanidins, anthocyanins, flavanols and flavones, flavan-3-ols and has many anti-inflammatory, diuretic, blood purifying, spasmolytic and antitumour activities (3, 9-11). All these plant phenolic compounds have potent antioxidant capacity (11) and there is evidence that they can regulate hyperglycaemic levels, adipocytokine gene expression and enhance some metabolic activities (12). The hypoglycaemic effects of medicinal herbs on hyperglycaemia include physiological mechanisms such as enhancing peripheral tissue insulin sensitivity, inhibition of digestive enzymes involved in carbohydrate breakdown and inhibition of glucose absorption in gastrointestinal tract (13, 14). The aim of this study is to determine the effect of 10-day consumption of blackthorn flower extract on blood glucose, glycaemic load and glycaemic parameters, serum α-amlyase activity and serum insulin concentration in alloxan-induced hyperglycaemic mice. Despite the traditional use of blackthorn as medicinal herb, there are no studies that investigated the effect of its flower extract on glycaemic homeostasis. The obtained results might serve as a guide towards the design of nutraceutical polyphenol mixture as supportive therapy in the hyperglycaemia treatment.

The blackthorn (Prunus spinosa L.) flower extract is a rich source of polyphenols, the most abundant being quercetin and kaemferol glycosides, represented by kaempferol-3-O-pentoside and rhamnoside and quercetin-3-O-pentosyl-pentoside, and also phenolic compounds belonging to the classes of hydroxycinnamic acids and flavonol gycosides (18).

The extract contains a significant concentration of sugar (Fig. 2), accounting for the total of 59.8 mg/L. The analysis of individual sugar types within the extract revealed that the most dominant sugar in blackthorn flower extract was fructose at 55.7% (33.3 mg/L), followed by glucose at 40.3% (24.3 mg/L) and sucrose, with only 3.8% (2.3 mg/L) of total sugar concentration. Therefore, the blackthorn flower extract contained 96% monosaccharides and only 3.8% disaccharides.

The results in Fig. 3 show serum blood glucose levels in the experimental groups of C57BL/6 mice and the effects of blackthorn flower extract intake on blood glucose in normoglycaemic and hyperglycaemic (alloxan-induced) mice after a single (1st day) or five (5th day) and ten (10th day) doses. The flower extract intake resulted in slightly but significantly (p≤0.05) increased blood glucose concentration in normoglycaemic mice treated with the extract compared to the untreated control mice, but only on the 1st (Fig. 3a) and 5th (Fig. 3b) experimental day. At the end of the experiment (Fig. 3b) and ten received doses, despite the treatment with the extract, the blood glucose concentrations were the same as in control group without any significant differences between the groups (Fig. 3c).

When the blackthorn flower extract was administered to the hyperglycaemic mice, there was a significant (p≤0.05) reduction of blood sugar concentration (compared to alloxan-treated hyperglycaemic group) after five (Fig. 3b) and ten (Fig. 3c) received extract doses (5th and 10th experimental day, Fig. 3b and Fig. 3c, respectively). This signifies that the consumption of the flower extract in alloxan-treated hyperglycaemic group decreased hyperglycaemia after at least 5 or 10 repeated daily consumptions. Interestingly, the single dose (1st experimental day, Fig. 3a) was not sufficient to reduce the hyperglycaemia significantly (comparison of alloxan-induced group and alloxan-induced hyperglycaemic group treated with the flower extract).

The oral glucose tolerance test (OGTT) in Fig. 4 shows the 2-hour dynamics of blood glucose adaptation. The blood glucose concentrations are shown as the pharmacokinetic curves (Figs. 4a-e) in all treatment groups after the consumption of glucose solution. The effects of parallel blackthorn flower extract and glucose intake in normoglycaemic (control and blackthorn flower extract group) and hyperglycaemic mice (treated with alloxan only or alloxan and flower extract) are shown separately on different experimental days. Fig. 4a and Fig. 4f depict the baseline blood glucose concentration without glucose addition in all groups but with a single extract dose in normoglycaemic and hyperglycaemic groups. Fig. 4b and Fig. 4g show treated groups challenged with oral glucose ingestion prior to blackthorn extract treatment. Fig. 4c and Fig. 4h show the groups receiving a single (1st day) dose, Fig. 4d and Fig. 4i those receiving five (5th day) and Fig. 4e and Fig. 4j ten (10th day) flower extract doses. It was noticed (Figs. 4a-e) that combined treatment with alloxan and flower extract improved glucose homeostasis and decreased blood glucose concentration compared to alloxan treatment alone. The differences in the areas under the pharmacokinetic curves were analysed (Fig. 4). For analysis simplicity, statistical significance was calculated only for the values of areas under the curve (Figs. 4f-j), which indicate glucose dynamics after glucose addition, over a period of 120 min for each group. There was a significant difference between the hyperglycaemic group treated with the blackthorn flower extract and the alloxan-treated hyperglycaemic group (p=0.0002) on day 0 (Fig. 4g) and day 5 (Fig. 4i). A significant difference in baseline treatment (p=0.0405) was between the group treated with the extract and the control on days 1 (Fig. 4h) (p=0.0007) and 10 (Fig. 4j) (p=0.004). Cumulatively, the blood glucose concentration over the period of 120 min presented in the graphs of the areas under the curve shows that in flower extract group, glucose homeostasis is achieved faster, even after 10 days of treatment.

The results in Figs. 5a-c show concentrations of insulin and activity of α-amylase in the serum of normoglycaemic (control) and hyperglycaemic (alloxan-induced) mouse experimental groups and the effects of blackthorn extract intake (in normoglycaemic and hyperglycaemic group treated with the extract) after single (1st day) or five (5th day) and ten (10th day) repeated doses.

The intake of the flower extract did not affect amylase activity until the 5th experimental day but resulted in significantly lower serum α-amylase activity (p<0.0001) in flower extract group than in the control group until the 10th (Fig. 5c) experimental day. This means that ten repeated doses of daily consumption of blackthorn flower extract had the potential to inhibit amylase activity. Alloxan significantly increased the α-amylase activity in hyperglycaemic group compared to normoglycaemic control group. However, when the extract was administrated to the hyperglycaemic mice, there was a significant reduction in α-amylase activity (p<0.0001) compared to the alloxan-treated group on the 1st (Fig. 5a) and 10th (Fig. 5c) experimental days. The results in Figs. 5d-f show that the concentrations of blood insulin were significantly higher (p=0.0017) on day 5 when the extract was administrated to normoglycaemic mice than in the control group. Alloxan-treated group had a significantly lower insulin concentrations than the control group on the 1st (p=0.0004) (Fig. 5d) and 10th (p=0.013) (Fig. 5f) experimental days. However, when the flower extract was administrated to hyperglycaemic mice, there was a significant increase of serum insulin concentration compared to the alloxan-treated group on the 1st (Fig. 5d) (p=0.0005) and 10th (Fig. 5f) (p=0.018) experimental days.

The consumption of the flower extract, shown with oral glucose tolerance test, both short term (in min) or long term repeatedly during 10 days can modulate blood glucose concentrations and in long term might influence the α-amylase activity and insulin concentrations.

When taken under normoglycaemic conditions (healthy non-hyperglycaemic individuals), the extract shows the ability to slightly but significantly raise blood sugar concentration, as shown in the results for the flower extract group compared to the control group (Fig. 4). In available literature, there are examples of certain plant extracts with similar properties. For example, Tourkey (25) reported significantly higher concentrations of glucose in mice treated with Moringa oleifera aqueous extract. The observed short-term glucose elevation in normoglycaemia could be explained by significant presence of sugars in the extract. On the other hand, relative normalisation of blood glucose balance in hyperglycaemia could be attributed to richness in polyphenol molecules and to polyphenolic content in the flower extract. Flavonoids are characterised by their basic skeleton arranged in the form C6-C3-C6, two aromatic rings A and B linked by a unit of three carbon atoms. Hydroxyl group and sugars are very common flavonoid skeleton substituents, and they increase the water solubility of flavonoids. Flavonoids exist naturally as glycosides (26, 27). The sugar concentration analysis showed that the flower extract contains a significant concentration of simple sugars, monosaccharides, up to approx. 97% of total sugars. Nevertheless, the results show that in the groups treated with the extract on the 1st and 5th day glucose concentrations tended to be slightly higher than in the control group, although they never reached pathologic hyperglycaemia as in alloxan-induced group. From metabolic and physiologic points of view, nutritional feature of food and beverages that are rich in available carbohydrates is that their consumption induces postprandial hyperglycaemia. The liberated glucose is absorbed within the intestinal enterocytes by the specific transporters (27). The fast absorption of glucose activates the regulatory mechanisms of glucose homeostasis. Besides free sugars present in the extract (Fig. 2), some of the sugars that covalently bind to phenolic compounds might be liberated during digestion and absorption, which overall led to the rise in blood serum glucose concentration. It was observed that despite the slight increase of blood glucose concentration under normal metabolic conditions, the extract in hyperglycaemic mice promotes faster recovery and improved glucose homeostasis, as shown in alloxan-induced hyperglycaemic group treated with flower extract, than alloxan treatment alone. Furman et al. (28) presented a review on medicinal plant extracts that express similar acute hypoglycaemic effect in diabetic rodents, for example Pycnanthus angolensis (rich in terpenoids), Phyllanthus sellowianus (rich in flavonoids), Gentiana olivieri (rich in flavons) and Cinnamonum zeylanicum (rich in cinnamaldehyde). Most researchers used polyphenol-rich plant extracts along with other bioactive substances to address their numerous antidiabetic mechanisms, which may include lowering blood glucose, inhibition of polysaccharide digestion, inhibition of hepatic gluconeogenesis, stimulation of peripheral glucose uptake, modulation of intestinal microbiome, antioxidant effect and stimulation of glucagon-like peptide-1 (GLP-1) secretion (4, 28, 29). Such protective effects are usually ascribed to the richness of phenolic molecules in plant extracts. It is known that blackthorn flowers contain flavonoid complex and flavonol derivates: kaempferol and quercetin with their glycosides bound with arabinose, rhamnose and xylose (30). In the flower extract, individual polyphenols like kaempferol and quercetin are glycosylated (10). Olszewska and Wolbiś (10) found in plant extracts compounds such as kaempferol 3-O-α-l-arabinofuranoside, kaempferol 3-O-(2’’-E-p-coumaroyl)-α-l-arabinofuranoside, kaempferol 3-O-β-d-xylopyranoside, kaempferol 7-O-α-l-rhamnopyranoside and kaempferol 3-O-α-l-rhamnopyranoside. In the flower extract we used in this work the composition and concentrations of 28 polyphenols were determined by UPLC-MS/MS (19). Flavonol glycosides were found to be the major polyphenolic class in blackthorn flowers, especially quercetin and kaempferol as kaempferol rhamnosyl-hexoside, quercetin-pentosyl-hexoside, kaempferol-pentosyl-hexoside, kaempferol-pentoside, quercetin-rhamnoside, kaempferol-rhamnoside, quercetin acetylhexoside and kaempferol acetylhexoside (19). In a previous experiment with a single dose, we show that a significant number of these glycosides can be traced by UPLC-MS/MS method in the plasma of mice (19) and in organs such as intestine, liver and kidney (11).

Besides the long-term effect of blackthorn flower extract on blood glucose, it is very important to know the short-term dynamics of adaptation of blood glucose after the consumption of glucose solution. The oral glucose tolerance test (OGTT) was used here to evaluate blood glucose homeostasis, intake of blood glucose inside tissues and also indirectly evaluate glucose absorption in 2 h. Fig. 4 shows the blood glucose levels during 2 h. The OGTT evaluates the ability to respond appropriately to a glucose change. For the OGTT, mice are typically fasted overnight, which provokes a catabolic state of metabolism, and reduction of liver glycogen stores. The longer the fast lasts, it decreases the metabolic rate and enhances glucose usage in mice, which is in contrast to humans. As the feeding patterns in mice also do not mimic human behaviour, it may be more physiological to perform an OGTT after a short fasting (31). Our results suggest that increased levels of glucose tolerance may be due to increased secretion of insulin, and therefore indicates that the flower extract possesses a hypoglycaemic effect.

In long term hyperglycaemia regulation, the flower extract supplementation in diabetic mice decreased fasting blood glucose concentration and postprandial glucose tolerance, which showed improvement exhibiting similar pattern as normal mice with peak increase at 30 min. Results in vivo indicate that the extract displayed a good concentration-dependent inhibitory effect on serum α-amylase activity. Inhibition of α-amylase activities has been demonstrated with polyphenols from plants in many research papers (4, 5, 14, 32). The elevated insulin secreting in hyperglycaemic group treated with the extract agrees with in vivo results, shown in Fig. 5, and confirms the evidence that the extract acts as a stimulator of insulin secretion.

Some studies suggest that polyphenols from the flower extract reduce hyperglycaemia through various mechanisms. Polyphenols from plants can inhibit α-amylase and α-glucosidase activity, stimulate insulin secretion from the β-cells, balance glucose release from the liver and promote glucose uptake in the insulin-sensitive tissue (29). Yang and Kang (33) reported that combined treatment of quercetin and resveratrol in streptozotocin-induced diabetic rats maintained the activities of hepatic glucose metabolic enzymes and structure of pancreatic β-cells, and significantly decreased glucose concentration. Consumption of tea (Camellia sinesis L.) and coffee (Coffea arabica L.) has been related to a lower risk of hyperglycaemia by improvement of glucose tolerance, insulin sensitivity and insulin secretion, reduction of glucose intestinal uptake and regulation of glycaemic homeostasis (4). Furman et al. (28) reported the antihyperglycaemic effect of caffeic acid in diabetic mice by reduction of blood glucose and increased plasma insulin. Chukwuma et al. (5) reported that combined acute treatment in vivo with chlorogenic and caffeic acids improved glucose tolerance as well as plant extracts that contain equal amounts of both phenolic acids, suggesting their synergistic effect. Previous studies of other plants used similar therapeutic approaches for lowering blood glucose and enhancing pancreatic β-cell function, peripheral tissue insulin sensitivity and inhibition of digestive enzymes involved in carbohydrate metabolism.

Our results confirm that the blackthorn flower extract has similar antihyperglycaemic effects as described by Ben Salem et al. (6), who suggest that ethanol extract of Cynara scolymus significantly decreased (p<0.001) the α-amylase activity in serum of diabetic rats, reduced blood glucose rate in the treated group compared to diabetic rats after 28 days of treatment. Similar results were obtained by Tang et al. (34), who showed that polyphenols from Punica granatum L. flower improved insulin sensitivity in diabetic rats, confirmed by OGTT and insulin tolerance tests.

Varshney et al. (35) showed in vivo that tested flavonoids quercetin, rutin, myricetin and kaempferol (all applied in doses of 25 mg per kg of body mass per day) significantly enhanced insulin sensitivity in streptozotocin-induced diabetic mice by decreasing glucose concentration with increase in time. Kaempferol has a highest influence on improving blood glucose level, glucose tolerance and insulin sensitivity. Alkhalidy et al. (36) reported that after four weeks of oral administration of kaempferol fasting blood glucose concentrations were reduced and insulin sensitivity improved in diet-induced obese C57BL/6 mice.

The obtained results show that consumption of blackthorn (Prunus spinosa L.) flower extract, measured as oral glucose tolerance test, both short term (in min) or long term (during 10 days) has the ability to slightly but significantly increase blood sugar concentration in metabolically healthy (normoglycaemic) mice. The conclusion of this study is that the intake of blackthorn flower extract for 10 days has a potentially protective effect against hyperglycaemia in C57BL/6 mice by lowering blood glucose concentration, improving glucose tolerance, enhancing insulin secretion and inhibiting serum α-amylase activity. Based on the current investigations, blackthorn flower extract may be a useful support in hyperglycaemia management, including its complications.