*Corresponding Author:
Z. Faezizadeh
Department of Laboratory Sciences, Iran
E-mail: [email protected]
Date of Submission 24 May 2016
Date of Revision 12 August 2016
Date of Acceptance 15 August 2016
Indian J Pharm Sci 2016;78(4):486-491  

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Abstract

Non-enzymatic glycation of proteins is the major cause of diabetic complications and therefore, inhibition of this reaction could reduce the morbidity of this disease. The aim of this study was to evaluate the in vitro effects of aqueous and hydroethanol extracts of Echium italicum L., Anchusa arvensis L. and Trichodesma incanum (Bunge) A. DC. of Boraginaceae on albumin, hemoglobin, and crystalline glycation reaction. The results showed that the hydroethanol extract of Echium italicum in the concentrations of 100, 50 and 10 mg/dl could reduce the rate of albumin glycation to 85.10±1.85, 64.14±1.12 and 40.13±1.17%, respectively, and also in the same conditions, rates of hemoglobin glycation were reduced to 69.20±1.71, 46.13±1.93 and 27.73±0.90%, respectively. Moreover, extracts of three tested plants have antiglycation effects on crystalline glycation reaction. Under the identical conditions, the inhibitory effects of hydroethanol extracts of Echium italicum on the albumin, hemoglobin, and crystalline glycation reaction were higher than other extracts. The findings showed that the extracts of tested species, particularly in the hydroethanol form, could be used to prevent and treat complications of diabetes mellitus.be used to prevent and treat complications of diabetes mellitus.

 

Keywords

Echium italicum L., Anchusa arvensis L., Trichodesma incanum (Bunge) A. DC., albumin, hemoglobin, glycation

Members of the family Boraginaceae include herbs, shrubs and trees totaling about 146 genera and 2000 species in the worldwide[1]. Some of these plants grow in Iran and used widely in Iran's folk medicine[2,3]. It has been reported that Borage (Borago officinalis L.) was useful for its antipyretic, antihypertensive, antispasmodic, aphrodisiac and diuretic properties[4,5]. Previous findings showed that some species of Boraginaceae were used for the treatment of hemorrhoids[6]. Furthermore, it has also been observed that various parts of Echium species could be used for their antiinflammatory, antibacterial, antidepressant, antiproliferative, antiviral, antioxidant, cytotoxic and anxiolytic properties[2,3]. Cyanidin, a natural phenolic compound is present in many kinds of plants, such as grapes, pomegranate and some species of Boraginaceae and according to the previous reports, this effective compound has antioxidant and antidiabetic potential[7-10].

Diabetes mellitus is a common endocrine disease and a leading cause of morbidity and mortality in the world[11]. In this disease, the macro and microvascular complications lead to severe effects on the eyes, central nervous systems and kidneys[12]. Evidence from in vitro and in vivo experiments indicated that diabetic hyperglycemia could increase the risk of complications[13,14]. It seems that non-enzymatic glycation plays an important role in progression of diabetic complications[11]. Due to prolonged hyperglycemia, the non-enzymatic glycation of proteins could significantly change their structural and functional properties[12]. Previous studies demonstrated that the protection of proteins from glycation could reduce the diabetic complications as well[15,16]. The use of glycation inhibitors is considered due to their therapeutic potential in patients with diabetes[17,18]. The investigation of new drugs with antiglycation activity has attracted the interest of various researchers[19,20]. Herbal medicines are nowadays gaining importance for treating diabetes mellitus due to their less toxicity, significant results as compared to allopathic medicine[21,22]. It is reported that some medicinal plants and their derivatives could inhibit the glycation of proteins as well[17,18].

Several species of Boraginaceae family such as E. italicum L. (fig. 1A) , Anchusa arvensis L. (fig. 1B) and Trichodesma incanum (Bunge) A. DC (fig. 1C) are used widely in Iran's folk medicine for their antidiabetic properties. According to our knowledge, there is no scientific research about antidiabetic mechanism of these plants and therefore, in this study, we investigated the antiprotein glycation efficacy of these selected species.

Materials and Methods

All the chemicals used were of analytical grade obtained from either Merck (Darmstadt, Germany) or Sigma (St. Louis, MO, USA). Human glycated albumin ELISA kit was obtained from Nacalai Tesque (Kyoto, Japan). Human glycated hemoglobin ELISA kit was obtained from MyBioSource, Inc. (San Diego, California, USA).

The three species of Boraginaceae family, including E. italicum, A. arvensis and T. incanum were collected during the spring from different regions of Borujerd (Lorestan Province, Iran). These regions have mild climate with mean temperature between 21-25° in the spring. Plant samples were identified and confirmed by comparison with voucher specimens deposited at the Department of Biology, Faculty of Basic Sciences, Borujerd Branch, Islamic Azad University, Borujerd, Iran.

Glycation of proteins

The glycated human serum albumin, hemoglobin, and crystalline were prepared according to Gharib et al. method[17]. In brief, 50 mg/ml of tested proteins were incubated with 40 mmol glucose, 100 μg/ml penicillin, 100 U/ml streptomycin, 2 mg/ml aprotinin, 5 mmol phenylmethylsulfonyl fluoride and 0.5 mg/ml leupeptin in PBS (10 mmol, pH 7.4) at 37° for 8 weeks. Subsequently, the sample was dialyzed extensively against PBS at 4°.

Measurement of glycated albumin, hemoglobin

The rate of glycated albumin was measured using the human glycated albumin ELISA kit (Nacalai Tesque Company), according to the manufacturerʼs protocol. In brief, 50 μl of phosphate buffer (10 mmol, pH 7.4) containing 20 μl of the test samples and 0.5 ml/l Tween 20 were added to each well of a 96-well micro titer plate that was coated with antihuman serum albumin antibody. Thereafter, wells were incubated for 20 min and washed three times. Subsequently, the boronate- horseradish peroxidase conjugate in 50 μl of glycine- NaOH (100 mmol, pH 9.0) containing MgCl2 (20 g/l), bovine hemoglobin (3 g/l), and Tween 20 (0.5 ml/l) was added to wells. Again, wells were incubated for 20 min and the reaction was terminated by adding 50 μl of sulfuric acid (2 mol), and the absorbance at 492 nm was measured using a microplate reader (MTP- 120; Corona Electronic). Finally, the percentage rate of glycated albumin in the samples was measured. The rate of glycated hemoglobin was measured using the human glycated hemoglobin ELISA kit (MyBioSource, Inc.), according to the manufacturer’s protocol. Furthermore, the rate of crystalline non- enzymatic glycation was measured by the TBA method as described previously[23].

Preparation of plants extracts

The hydroethanol and aqueous extracts of E. italicum, A. arvensis and T. incanum were prepared according to the previously reported method[24]. In brief, the air- dried plants were ground into powder using grinder (Moulinex, France), and then 1 g of sample powders was blended with 60 ml of 45% ethanol or water solvents. Subsequently, the blended samples were mixed on a sample stirrer at 900 rpm for 1 min, and then the liquid volumes were increased to 100 ml by solvents. These mixtures were placed in a bath by stirring and cooled in a refrigerator. Subsequently, the mixtures were centrifuged (Shimadzu, Japan) at 2500 rpm for 10 min and vacuum filtered, and the loss solvents were replaced. Finally, the prepared extracts were stored at 4° until used for analysis.

In vitro antiglycation studies

The percentages of glycated albumin, hemoglobin, and crystalline glycation in the samples were determined after incubation of these tested proteins and glucose in the presence of different concentrations (0, 10, 50, and 100 mg/dl) of each extract for 8 weeks. As positive control, 20 mg/ml aminoguanidine bicarbonate, as an antiglycation drug, was used.

Statistical analysis

The results were expressed as mean±SD. The statistical analysis was performed using one-way ANOVA followed by Tukey's test and P-value less than 0.05 was considered significant. Data were analyzed using SPSS software (version 19.0, SPSS, Inc., IL, USA). All experiments were done in triplicate.

Results and Discussion

In this research, the effects of the various concentrations of tested plant extracts on rates of albumin, hemoglobin, and crystalline glycation were studied. Under the identical conditions, the inhibitory effects of hydroethanol extracts of tested plants on glycation rates of three tested proteins were higher than those of aqueous extracts (Tables 1, 2 and 3). In all conditions, the albumin, hemoglobin, and crystalline glycation rates were significantly inhibited compared to the negative control group. Moreover, treatment with 100 mg/ml hydroethanol extracts of E. italicum, A. arvensis and T. incanum could reduce the rate of hemoglobin glycation to 69.20±1.71 and 41.90±1.25 and 27.85±1.30%, respectively (Table 2). As positive control aminoguanidine bicarbonate could decrease the rate of albumin, hemoglobin, and crystalline glycation to 56.12±1.39, 39.15±0.50 and 34.10±1.35%, respectively. Compared to control group, the rate of albumin glycation with 100 mg/ml hydroethanol extracts E. italicum, A. arvensis and T. incanum were reduced to 85.10±1.85, 53.10±0.69 and 38.13±1.81% at 8 weeks, respectively (Table 1). Under the identical conditions, the inhibitory effects of E. italicum extracts on albumin, hemoglobin, and crystalline glycation were significantly higher than those of other tested plants. Furthermore, the antiglycation rates of extracts on crystalline were lower than those of other tested proteins (Table 3).

Plant scientific name Extracts % Inhibition of albumin glycation
Concentrations of extracts (mg/dl)
10 50 100
.EchiumitalicumL Hydroethanol 40.13±1.17* 64.14±1.16* 85.10±1.85*
Aqueous 34.15±1.35* 57.10±1.56* 73.74±1.24*
.AnchusaarvensisL Hydroethanol 32.50±2.85* 38.08±1.30* 53.10±0.69*
Aqueous 25.58±0.90* 31.30±1.37* 48.20±1.35*
Trichodesmaincanum(Bunge) A. DC. Hydroethanol 28.18±1.90* 30.33±1.60* 38.13±1.81*
Aqueous 21.60±1.80* 24.17±1.21* 34.28±1.65*

Table 1: Effect of Hydroethanol and Aqueous Extracts on In Vitro Albumin Glycation Reaction

Plant scientific name Extracts % Inhibition of hemoglobin glycation
Concentrations of extracts (mg/dl)
10 50 100
.EchiumitalicumL Hydroethanol 27.73±0.90* 46.13±1.93* 69.20±1.71*
Aqueous 25.28±0.65* 30.90±1.10* 61.93±1.10*
.AnchusaarvensisL Hydroethanol 19.28±0.65* 30.90±1.10* 41.90±1.25*
Aqueous 17.23±0.51* 22.14±0.90* 32.10±1.13*
Trichodesmaincanum(Bunge) A. DC. Hydroethanol 15.13±0.54* 19.93±1.10* 27.85±1.30*
Aqueous 10.38±0.43* 13.19±0.75* 21.15±0.83*

Table 2: Effect of Hydroethanol and Aqueous Extracts on In Vitro Hemoglobin Glycation Reaction

Plant scientific name Extracts % Inhibition of crystalline glycation
Concentrations of extracts (mg/dl)
10 50 100
.EchiumitalicumL Hydroethanol 24.13±1.10* 47.25±0.86* 64.75±0.43*
Aqueous 21.35±0.70* 43.68±1.20* 58.77±0.25*
.AnchusaarvensisL Hydroethanol 15.83±0.53* 26.53±1.80* 39.87±1.45*
Aqueous 13.12±0.70* 19.78±0.72* 26.18±1.20*
Trichodesmaincanum(Bunge) A. DC. Hydroethanol 15.18±0.84* 15.18±0.80* 23.35±1.10*
Aqueous 7.18±0.32* 9.98±0.38* 18.13±0.75*

Table 3: Effect of Hydroethanol and Aqueous on In Vitro Crystalline Glycation Reaction

In diabetes mellitus, the nonenzymatic glycation of proteins occurs in a wide variety of proteins, and this phenomenon could enhance diabetic complications[25]. Therefore, in medicine, the inhibition of protein glycation has been proposed as an important strategy for the restriction of diabetic complication development[26]. The use of some medicinal plant extracts to reduce of diabetic complications has been widely investigated[17,27].

In folk medicine, Boraginaceae species have been used for the treatment and prevention of many ailments and diseases such as hypertension, diarrhea and diabetes mellitus and have been a tremendous resource for the development of new drugs[28-31]. Many species of Boraginaceae family are grown in Iran and some of them are used in ethnomedicine[4]. However, there were no studies about their antiglycation properties and therefore, in the present study, we investigated the in vitro effects of aqueous and hydroethanol extracts of E. italicum, A. arvensis and T. incanum on albumin, hemoglobin, and crystalline glycation reactions. The results from this study revealed that the all prepared extracts possessed antiprotein glycation. These findings correlated with those reported by other investigators about antiglycation efficacy of some natural plants, such as Allium cepa L.[32], Curcuma longa L.[33], Rosmarinus officinalis L.[34], Cinnamomum verum[35] and Origanum officinalis[36] under in vitro conditions. Under identical conditions, the antiglycation potential of extracts on albumin was higher than hemoglobin, and crystalline glycation, respectively. According to the previous studies, this difference may contribute to the protein structure[37]. The hydroethanol extracts of each tested plant have more inhibitory effects on protein glycation reaction, compared to aqueous extracts. These results may be related to effects of extracting solvents on the isolation of phytochemical constituents[38].

Under identical conditions, the extracts of E. italicum have more potent antiglycation activity compared to A. arvensis and T. incanum extracts. Moreover, our data revealed that the antiglycation potential of E. italicum was higher than A. sativum, Zingiber officinale, A. cepa and Thymus vulgaris that their antiglycation activities were reported previously[33,34]. Thus, the ability of E. italicum to modulate glycation might be providing its use as a significant source of the natural product in preventing the diabetic complications.

In summary, the present study was carried out to evaluate the antiglycation potential of E. italicum, A. arvensis and T. incanum extracts on albumin, hemoglobin, and crystalline glycation reaction. Results indicated that the hydroethanol extracts of these plants might be promising antiglycation agents for the prevention of diabetic complications through inhibition of protein glycation. Therefore, the prepared extracts could be considered as a promising strategy for developing the antidiabetic drugs.

Acknowledgments

The authors would like to acknowledge the staff of Islamic Azad University.

Financial support and sponsorship

This study was supported by Islamic Azad University, Borujerd Branch, Iran.

Conflicts of interest

There are no conflicts of interest.

References