Introduction

Diabetes mellitus is a major metabolic disorder affecting a huge population all over the world. It is a chronic metabolic disorder which is characterized by elevated level of glucose due to deficiency of insulin or insulin receptors [1]. Diabetes also leads to hyperlipidemia which is the leading cause of atherosclerosis and cardiovascular diseases [2]. In addition, diabetic patients experience nephropathy and neuropathy. Currently, the most common therapies used for diabetes are insulin, sulfonylureas, biguanides and thiazolidinediones which have many side effects. The prevalence of diabetes increased with the passage of time, but an effective treatment with no side effects is still lacking and needs to develop [3].

Plants have been employed in treating various diseases from the very beginning of human civilization [4]. Rosacea is one of the most economically important plant family in temperate regions and comprises of more than 100 genera and about 3000 species [5]. Phytochemicals like flavonoids, cyanogenic glycosides, phytoestrogens and phenolic compounds present in fruits of Rosaceae are major sources of human nutrition that could potentially promote health and have disease fighting advantages [6,7,, 7]. The genus Rubus comprises of almost 740 species, which have been divided into 12–15 subgenera, which makes it the largest genus of the Rosacea family and one of the most diverse among the plant kingdom [8]. Different plant parts of this genus have been used for the treatment for diabetes mellitus, rheumatism, sore throat, hemorrhoid, diarrhea and similar enteric disorders [9, 10]. Many species of this genus such as ellipticus and fructicosus were used for the treatment for fever, gastric troubles, diarrhea, dysentery wounds, colic, cough, sore throat, carminative, bleeding gums, anemia, kidneys stone, tonic and aphrodisiac [11, 12]. It has been reported previously that some of these species possessed compounds that act as antibacterial, antiallergic, antiasthmatic and antihyperglycemic agents [13, 14]. Different species of Rubus contain high contents of tannins and numerous other polyphenols including flavonoids (quercetin and kaempferol derivatives), anthocyanins and phenolic acids, particularly gallic and ellagic acid [15, 16].

Rubus ulmifolius Schott is a perennial thorny shrub and belongs to family of Rosacea. Fruits of R. ulmifolius are edible and have carminative properties. The unripe fruits are used as tonic and aphrodisiac [3, 17]. Leaves and roots are used to treat the skin diseases [11]. Rubus ulmifolius in Italian folk medicine has been used for abscesses and ulcers, externally for vaginal lavages and diarrhea [18]. Rubus ulmifolius is used in Chilean folk medicine due to its hypoglycemic actions [19]. Previously, many studies were performed on mice/rats to check the antidiabetic effects of different herbs. Mice and rats are widely accepted animal models, and they have resemblance to human diabetes mellitus [20, 21]. The albino mice are used as a diabetic model to conduct many studies [17, 22]. Albino mice were used to conduct in vivo antidiabetic activity tests of Swertia koutichensis extracts [23]. A similar, study was conducted on Piper longum roots, aerial parts of Artemisia indica and Drynaria quercifolia Linn rhizome and mung to evaluate the antihyperlipidemic and hyperglycemic effects in diabetic mice and rats [1, 20]. A low concentration of streptozotocin (50 mg/kg b.w) has been reported to induce type II diabetes in mice [3, 20]. Therefore, the current study aims to evaluate the antihyperglycemic and antihyperlipidemic effects of aerial parts of R. ulmifolius Schott in streptozotocin (STZ)-induced diabetic albino mice.

Materials and methods

Plant collection and preparation of extracts

Fresh aerial plant parts were collected from near hilly area of Gaddar, Chakdara, Dir lower Khyber Pakhtunkhwa, Pakistan, in the month of July 2016. The plant specimen was deposited in the Pharmacy department, University of Malakand, Khyber Pakhtunkhwa, Pakistan. The aerial parts of R. ulmifolius plants were washed with distilled water and dried at 65 °C for 48 h and grinded. 2.5 g of grinding powder was mixed with 500 ml of commercial methanol and kept at room temperature for 3 weeks at room temperature. During soaking the mixture was frequently shaken and filtered by two layers of cheesecloth and Whatman filter paper. The filtrate was concentrated to a semisolid mass using a rotary evaporator under reduced pressure at 45 °C for a final extract. The semisolid mass (789 g) was suspended in 500 ml distilled water. The extract was given orally as it was mixed with water as per body weight (150–300 mg/kg body weight). The same technique was used for ethyl acetate extract (RUE), chloroform extract (RUC) and butanol extract (RUB). These all extracts’ combined name is (RUCrd).

Phytochemicals analysis

Preliminary phytochemicals analysis of R. ulmifolius Schott (RUCrd) extract indicated the presence of saponins, flavonoids, glycosides, cardiac anthraquinone glycosides, carbohydrates, terpenoids, alkaloids, phenolic compounds, tannins, amino acids and proteins as shown in Table 1.

Table 1 Detection of phytochemicals in R. ulmifolius
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Chemicals

Streptozotocin was supplied by Sigma-Aldrich, glibenclamide was supplied by Sanofi Aventis Pharma (Pvt.) Ltd, Pakistan, glucose estimation kits were supplied by S.D Chek Gold Korea, normal saline was supplied by Utsoka pharma (Pvt) Ltd., Pakistan, and Tween-80 (2%) was supplied by Daejung Korea, according to the manufacturer’s instructions. The different organic solvents and chemicals used for the extraction were purchased from local suppliers of Merck, Germany.

Experimental animals

Standard experimental protocols were followed as per the guidelines of ethical committee of Department of Pharmacy, University of Malakand, Khyber Pakhtunkhwa, Pakistan. A total 48-, 60-day-old either sex (male and female) albino mice, weight range of 18–25 g, were used to evaluate the antidiabetic activity. Albino mice were purchased from the National Institute of Health, Islamabad. After one week of adaptation, the albino mice were divided into eight groups of 6 and treated as follows: normal control; 2% Tween-80 suspension (Diabetic control); glibenclamide (500 μg/kg/orally) (C); RU methanol extract (150 mg/kg/orally) (RUCrd1); RU methanol extract 300 mg/kg/orally) (RUCrd2); RU chloroform extract (150 mg/kg/orally) (RUC); RU ethyl acetate extract (150 mg/kg/orally) (RUE) and RU butanol extract (150 mg/kg/orally) (RUB). The albino mice were maintained in light and dark cycles for 12 h at room temperature (22–25 °C) in laboratory. The albino mice were provided feed and water ad libitum. Blood glucose level and body weight were measured at 1st, 4th, 7th, 10th and 15th day of periods.

Biochemical analysis

Acute toxicity of the RUCrd was determined using either sex (male and female) of albino mice in the weight range of (20–27 g). Acute toxicity study was conducted in two phases, each phase divided into three groups having 6 albino mice. In the first phase, G1 was served as a control, G2 was administered Tween-80 (2%), an oral dose of 250, 500 and 1000 mg/kg/b/w, the animal were constantly observed for any behavioral alteration or death toll with follow-up for 10 days. In the second phase, G3, G4, G5, G6, G7 and G8 received 10, 100, 1000, 1500, 2000 and 2500 mg/kg/b/w an oral dose of RUCrd, respectively. The animals were observed for behavioral changes, physical and pharmacological effects as shown in Fig. 1.

Fig. 1
figure 1

Acute toxicity analysis

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Induction of diabetes

Diabetes was induced in albino mice by STZ, 50 mg/kg b.w, through a single intraperitoneal injection reconstituted in 0.01 M citrate buffer (pH 4.5) after overnight fasting [24]. Blood glucose level was measured after 72 h of STZ administration to check the hyperglycemia condition, with one-touch glucometer strips using SD glucometer (Korea). Mice having fasting blood glucose level more than 300 mg/dl were selected. All the animals were allowed to access free food and water and maintained at room temperature in cages.

Blood collection

Blood samples were taken from the tip of the tail, and the blood glucose level was measured on the 1st, 4th, 7th, 10th and 15th day, with one touch of glucometer strips using SD glucometer (Korea). After completion of antidiabetic assay on 15th day, all animals were anesthetized by pentobarbital sodium (35 mg/kg) and samples of blood were collected through cardiac puncture for the analysis of biochemical parameters including serum alkaline phosphatase (ALP), high-density lipoprotein (HDL), low-density lipoproteins (LDL), total cholesterol (TC), triglycerides (TG) and serum creatinine levels [25, 26].

Statistical analysis

The experiment was a completely randomized design. The data were analyzed using the GLM procedure of SPSS (version 17.0). Means of the significantly affected traits were separated by the Duncan multiple range test [27]. A P value less than 0.05 was considered statistically significant.

Results

Results from the toxicity studies of RUCrd have revealed no mortality when a dose maximum up to 2500 mg/kg/b.w was orally administered. No substantial changes were seen in the animal behaviors in all of the test groups. Hence, 150 and 300 mg/kg doses of RUCrd were selected then to evaluate antidiabetic activities.

The daily oral effects of administered R. ulmifolius on blood glucose levels (hypoglycemic and antihyperlipidemic) in STZ-induced diabetic albino mice are shown in Table 2. The fasting blood glucose level in diabetic treated and untreated (G3-G8) albino mice was significantly high. After oral administration of the RUCrd1, RUCrd2, RUC, RUE and RUB at the rate of 150–300 mg/kg significantly (P < 0.05) decreased the blood glucose level as observed on the day of 1, 4, 7, 10 and 15 as compared to diabetic control.

Table 2 Effect of daily oral administered of RUCrd on blood glucose levels (hypoglycemic and antihyperlipidemic) in diabetic albino mice
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The effect of R. ulmifolius on body weight in STZ-induced diabetic albino mice is illustrated in Table 3. Changes in body weight of control and experimental diabetic albino mice treated with extracts of R. ulmifolius and glibenclamide were observed. A significant (P < 0.05) reduction in body weight was observed in STZ-induced diabetic albino mice compared to control during the 15 days. After the administration of RUCrd1, RUCrd2, RUC, RUE and RUB extracts, slightly increased body weight was observed on 1st, 4th, 7th, 10th and 15th day on diabetes albino mice.

Table 3 Effect of RUCrd on the body weight of diabetic albino mice
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The effect of RUCrd extracts on lipid profiles in control and experimental STZ-induced diabetes albino mice is shown in Table 4. The TC, TG and LDL increased after induction of STZ as compared to normal control. In addition, there was a substantial decrease (P < 0.05) in HDL cholesterol in diabetic control albino mice as compare to normal control. The administration of the RUCrd1, RUCrd2, RUC, RUE and RUB caused a significant decrease in TC, TG, LDL on the 4th, 7th, 10th and 15th day on diabetes control albino mice. The effect was comparable to that of glibenclamide (500 μg/kg) used as a standard drug. Furthermore, the oral administration of RUCrd1, RUCrd2, RUC, RUE, RUB (150–300 mg/kg) and glibenclamide (500 μg/kg) significantly (P < 0.05) increased HDL cholesterol level in diabetic albino mice.

Table 4 Effects of RUCrd on lipid profile in STZ-induced diabetic albino mice
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The effects of RuCrd on the activity of hepatic marker enzymes in STZ-induced diabetic albino mice are shown in Table 5. Level of ALP, serum glutamate pyruvate transaminase and serum glutamate oxaloacetate transaminase were significantly decreased in RUCrd1, RUCrd2, RUC, RUE and RUB groups as compare to standard drug glibenclamide and diabetic control albino mice. The serum creatinine was significantly decreased in RUCrd1, RUCrd2, RUC, RUE and RUB groups as compare to standard drug glibenclamide-treated albino mice.

Table 5 Effect of RUCrd and fractions on serum liver profile in STZ-induced diabetic albino mice
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Discussion

Rubus ulmifolius Schott has been used for therapeutic purposes and was considered an important component of herbal medicines. It has also been reported that leaves of R. ulmifolius Schott showed hypoglycemic activity and used as an important component of Chilean popular medicines [3, 28]. Previously, no study was reported on aerial parts of R. ulmifolius Schott in Pakistan as a potential antidiabetic herb. In the current study, aerial parts of R. ulmifolius were used to evaluate the antidiabetic and antihyperlipidemic activities in STZ-induced diabetic albino mice [3, 20]. A similar study reported on Piper longum roots and leaves used to evaluate antihyperlipidemic and hyperglycemic effects in diabetic rats [28, 29]. A low concentration of streptozotocin (50 mg/kg b.w) has been reported to induce type II diabetes in mice [20, 24, 26]. RU crude methanolic extracts, chloroform and ethyl acetate fractions showed significant reduction in FBG in STZ-induced diabetic mice (treated) compared to diabetic control (untreated) at a dosage of 150 mg/kg b.w. RUE fraction showed significantly high antidiabetic activity which was similar to standard drug glibenclamide-treated mice. RUB showed comparatively low antidiabetic activity, and this may be due to lack of phytochemicals involved in lowering blood glucose.

Preliminary findings on methanolic extracts of R. ulmifolius Schott in the current study of revealed the presence of phenolic compounds, flavonoids, alkaloids and terpenoids. Phytochemicals such as phenolic compounds have been reported as potential agents involved in the prevention of diabetes and cardiovascular disease [29, 30]. Flavonoids are one of the most common polyphenolic compounds which have been used for the treatment for diabetes mellitus since ancient times [31]. It was previously reported that phytochemicals like phenolic, flavonoids, alkaloids and glycosides from various plants such as Catharanthus roseus, Psidium guajava, Ephedra sinica and Rhizoma pinelliae showed antidiabetic activity [8, 32]. There are many mechanisms to control the blood sugar level. Inhibition of carbohydrate digestive enzymes especially of α-glucosidase and pancreatic α-amylase is one of the common mechanisms by which these compounds showed blood glucose lowering activity [33]. α-Glucosidase and α-amylase are important enzymes involved in intestinal glucose absorption and the breakdown of starch. The passage of carbohydrates into the blood stream could be slow down due to the inhibition of these enzymes and notably lowering the blood glucose level and play a key role in the management of diabetes [34].

The loss of body weight was observed in diabetic untreated mice (after 15-day treatment) which may be due to muscle wasting and loss of tissue proteins and insufficient utilization of carbohydrates [35]. RU crude methanolic extracts and fractions specifically chloroform and ethyl acetate-treated diabetic mice showed an increase in body weight, but no significant increase in body weight was observed in mice treated with RU butanol fraction. It was also observed that RU butanol fraction possessed very low antidiabetic activity which indicated that this fraction contained no antidiabetic agents.

It was previously reported that some local plants like neem, nayantara, fenugreek and tulsi are traditionally used to treat diabetes mellitus [32]. Similarly, due to common use and medicinal importance of R. ulmifolius in Chilean folk medicines the current study was conducted to observe the antidiabetic activity of R. ulmifolius (RUCrd) and different solvent fractions in STZ-induced diabetic mice and found very effective in the control of glucose level. Both crude extracts and different solvent fractions including RUE, RUC and RUB of R. ulmifolius leaves showed a significant decrease in blood glucose level and hyperlipidemic profile of diabetic in the current study. Since the preliminary phytochemicals analysis showed that R. ulmifolius leaves are rich in flavonoids, alkaloids, tannins, phenylpropanoids, terpenoids, carbohydrates and proteins. The antidiabetic and antihyperlipidemic activities of R. ulmifolius may be attributed to presence of these phytochemicals, but the exact mechanism is not known yet.

In additions, superoxide, hydrogen peroxide and hydroxyl radicals are reactive oxygen species (ROS), which can cause oxidative damage to cellular structures and functional molecules [36, 37]. There are many reports which showed that oxidative stress is the leading cause of many diseases, including diabetes [38]. Recently, phenolics and flavonoids have been proved to be more effective than vitamin C, E and carotenoids due to stability in structure and hence considered as great antioxidants [36, 39]. Mechanisms responsible for the antioxidant properties of flavonoid and phenolic compounds included scavenging radical species, reactive nitrogen species, suppressing ROS/RNS formation by inhibiting some enzymes or chelating trace metals concerned with free radical production and protecting antioxidant defense [40]. Plants rich in flavonoids and phenylpropanoid (antioxidants) may ameliorate the effects of these ROS due to their scavenging properties, and their common use may protect from many diseases.

Present study also reported that RUCrd and different solvent extracts of R. ulmifolius leaves to be very effective in normalizing liver and kidney profile of mice. This may be due to some potential constituent present in the extracts which may be useful in the treatment for liver- and kidney-related disorders. Further investigation is needed on the potential of effects of R. ulmifolius on kidney and liver profiles.