Antioxidant potential, biochemical activity and hypoglycemic effects of R. obtusifolius L. seed extracts used in Armenian traditional medicine

The diversity of plants in Armenia is due to the singularity of natural environment. However, biochemical activity of these plants has not been studied well. The goal was to investigate biological activities as well as antihyperglycemic properties of Rumex obtusifolius L. in rabbits with hyperglycemia. According to obtained data, R. obtusifolius extracts possessed high antioxidant activity in chemical-based tests. GS/MS analysis of its methanol extracts allowed identification of several compounds, which could have high contribution on biological activities. The sub-cytotoxic concentration of investigated extract was 10 mgmLon Murine microglial BV-2 WT cell lines. Oral administration of ethanol extract showed significant effect on hyperglycemia, reducing fasting glucose levels (57.3%, p<0.05) and improving glucose tolerance. These findings suggest that R. obtusifolius extracts could be source of new biologically active compounds and recommended for treatment of diabetes mellitus.


Introduction
Armenia is positioned at the junction of several biogeographical zones and appears to be rich in health promoting edible or medicinal plants. These zones are closely linked, resulting in relatively few endemic species of Armenian flora. Overall, 123 endemic plant species are described. These plants are commonly employed in traditional medicine for the prevention and treatment of various diseases from the 15th century and even earlier time. The privilege is the less cytotoxicity of plants to the humans. Armenian flora is rich with with herb species which have been widely used in traditional medicine since ancient times [1]. However, this biodiversity was not studied properly for their biological activities, biochemical properties, including antioxidant properties [2], and hypoglycemic activities [3]. Therefore, there could be hidden a great potential for their antioxidant properties and antihyperglycemic effects which can has huge value for therapeutic uses, as well as food industry [4].
The aim of this research was to evaluate antioxidant potential of crude extracts of R. obtusifolius, identify biologically active compounds using GC-MS technique and to investigate biochemical properties and antihyperglycemic properties of R. obtusifolius alcohol extract in rabbits with hyperglycemia.

Plant materials
Collection of plant material and preparation of plant crude extracts R. obtusifolius L. (seed) samples (voucher specimen number ERCB 13208) were collected from Tavush region (1300-1600 m above sea level). The collection, identification and preparation of plant material was done according to already established protocol [1]. R. obtusifolius L. samples were deposited to the Herbarium of Yerevan State University. Plant crude extracts were prepared by maceration technique using methanol (98%), ethanol (96%) and acetone (99.8%), according to method described previously [1]. Dry crude extracts stored in freezer (-18-20 0 C). For determination of hypoglycemic properties, the dried seeds were extracted with 40% ethanol for 20 min at 60 ∘ C. Briefly, 270-280 mg of dry matter are in 6 ml of ethanol. The extract was filtered with Watman filter paper (N1, Unichem, China) and after cooling at room temperature was orally administrated.

Hydrogen peroxide reducing activity
Hydrogen peroxide (H2O2) reducing ability of R. obtusifolius was evaluated by the method described by Ruch et al. [6]. Percent reduction of hydrogen peroxide calculated by following formula: H2O2 reduction =Ac-At/Ac⸱100% where Ac -absorption of control solution, At absorption of test solution. Ascorbic acid was used as positive control at 10 μg mL -1 concentration. *

Metal chelating activity
Metal chelating activity of R. obtusifolius extracts was assessed by color change due to the formation of ferrozine-Fe 2+ complexes [7]. Percent metal chelating activity of plant extracts was calculated according the following formula: where Acabsorption of control solution, Atabsorption of test solution. EDTA was used as a positive control at 22 μg⸱mL -1 concentration.

Determination of extent of lipid peroxidation
TBARs assay was used for evaluation of R. obtusifolius extracts on extent of lipid peroxidation [8]. Mice brain tissue homogenate (in 20 mM phosphate buffer (pH 7.4)) used during the test. Test solution contains 0.3 mL of plant extract (3 mg/mL concentration), 0.7 mL 250 mM HCl, 1 mL tissue homogenate and 2 mL TBA solution (0.375%). The absorbance of mixtures measured at 532 nm. Malondialdehyde concentration calculated using an extinction coefficient of 1.56⸱10 5 M -1 cm -1 .

MTT assay
The cell proliferation and/or mitochondrial activity were measured using 3-(4,5-dimethyltrazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. Cells, plated in 96-wells plates, were treated for 24-72 h with different concentrations of extracts (50 -5⸱10 -4 µL⸱mL -1 in dimethyl sulfoxide. Cells were incubated for 2 h with MTT dye followed by the absorbance (Abs) measurement at the 570 nm with a microplate reader and the subcytotoxic concentration was selected for further investigations [9]. The MTT test was carried out in order to clarify the influence of R. obtusifolius crude methanol extract cell viability. * Corresponding author: mikayel.ginovyan@ysu.am

Identification of volatile compounds by GC-MS technique
For identification of volatile compounds contained in R. obtusifolius crude methanol extract, GC-MS technique was applied using a Hewlett-Packard 5890 Series II gas chromatograph, fitted with a fused silica HP -5MS capillary column (30 m × 0.25 mm, in thickness 0.25 μm) [10], The identification of peaks was tentatively carried out based on library search using National Institute of Standards and Technology (NIST)-2013.

Induction of hyperglycemia in experimental rabbits and blood sampling
The domestic rabbits (Oryctolagus cuniculus domesticus) (1800-1900g) were divided into three groups ( = 9) as follows: group 1: normoglycemic, group 2: hyperglycemic control, putting immobilization, and group 3: hyperglycemic experimental, in common with immobilization received R. obtusifolius seed extract (150mg kg -1 body weight.). Hyperglycemia was induced by immobilization stress in the rabbits during 21 days (5 h daily) [11]. The animals were authorized by the "International Recommendation on Carrying out of Biomedical Researches with use of Animals," and the study plan has been approved by the National Center of Bioethics (Armenia).

Biochemical Analysis
Blood samples were taken from the aural vein and collected in serum separation tubes (Clot Activator & Gel, Turkey). Blood clot was removed by centrifugation at 3000 g for 10 min in a centrifuge at 4°C. The resulting supernatant was designated as a serum. The biochemical analysis was performed to measure the serum level of glucose, total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglycerides (TG). All parameters were assayed using enzymatic kit. Serum glucose level (mmol/L) was determined using glucose test kit based on the glucose oxidase method [12]. TC and TG were estimated by the method, as described [12]HDL and LDL were measured using the method, as described [13]. Analytical tests were conducted using automatic biochemical analyzers VITROS-5.1/FS (Germany) and MINDRAY B1-120 (China).

Oral Glucose Tolerance Test
On the 10 th day of treatment an oral glucose tolerance test (OGTT) was carried out. Animals were fasted overnight before commencing of experiments. 20% glucose solution (2g kg -1 b.w.) was administrated a signal oral dose to all groups of rabbits. The blood glucose level was measured by portable glucometer (Contour TS, Bayer, Switzerland). Blood samples were collected from aural vein at 0, 30, 60, 90, and 120 min after glucose loading.

Data processing
All experiments were independently repeated at least 3 times. Obtained data were processed; mean values and standard deviations were calculated using GraphPad Prism 8.0.1 (GraphPad Software, Inc.; USA) software. The results obtained in the study are reliable (p <0.05), unless another value is followed.

Results and Discussion
R. obtusifolius or broad-leaved dock is well known herb which have been widely used in traditional medicine from ancient times for treatment of infectious diseases, skin rash, mucosal inflammations, etc. [14]. In previous research works of our group, promising antimicrobial, antibiotic modulatory, and antiviral activity of its crude extracts was shown [1,15]. TLC-bioautographic analysis allowed separating active antimicrobial fractions from R. obtusifolius extracts against Staphylococcus aureus MDC 5233 and describing the chemical nature of those compounds [14]. It was hypothesized, that polar compounds could have considerable contribution to the antibacterial activity of acetone and methanol extracts of R. obtusifolius [14].
In current research based on chemical tests it was shown that crude methanol and acetone extracts of seeds of R. obtusifolius possessed promising antioxidant activity. Particularly high DPPH, hydrogen peroxide reducing and metal chelating activity were shown (Table  I). It was revealed that methanol extract of R. obtusifolius had 25.29 µg mL-1 IC50 value in the presence of DPPH 0.05 mg mL-1 concentration. It was shown that at the presence of 100 μg mL-1 concentration R. obtusifolius acetone extracts have 99.30% hydrogen peroxide reducing activity when the concentration of H2O2 in the mixture was 36 mM. Its methanol extract exhibited moderate activity leading to 40.78% hydrogen peroxide reduction. On the other hand, R. obtusifolius methanol extracts exhibited expressed metal chelating activity as well at the concentration of 125 μg mL-1 by reducing the number of Fe2+-ferrozine complexes by 73.02% at the presence of 0.125 mM FeCl2. Its acetone extract had low metal chelating ability at tested concentrations. TBARs assay revealed that R. obtusifolius methanol and ethanol extracts have not possessed anti-peroxidative activity till 225 µg mL-1 concentration.

GC-MS Analysis of R. obtusifolius methanol extract.
In order to identify biologically active volatile and semivolatile constituents R. obtusifolius methanol extract GC-MS technique was used. Based on obtained data various biologically active compounds were identified, which could play important role in their antioxidant effect. In general, 21 compounds were identified in methanol extract of R. obtusifolius. The identified compounds and their characteristics are shown in Table II. Some of the identified compounds are considered to possess biological activities. These were linoleic acid (41.32%), cis-vaccenic acid (24.43%), palmitic acid (12.25%), oleic acid (3.65%) 1,2,4-benzenetriol(0.46%), N-[4-bromo-n-butyl]-2piperidinone (0.64%) which present in the extracts with relatively high concentrations and have different biological activities based on literature data [16,17]. These compounds could have a contribution to the antioxidant and antimicrobial activities of this plant extracts. Other bioactive compounds also identified in the extract but only at low concentrations ( Table 2).

The effect of R. obtusifolius methanol extract on BV2 cell viability (MTT assay)
According to this test the sub-cytotoxic concentration of investigated extract was 10 mgmL -1 (Fig. 1) (p < 0.05).

Effect of R. obtusifolius on Fasting Glucose Levels.
Antihyperglycemic activity of R. obtusifolius seeds ethanol extract was evaluated on hyperglycemic rabbit model. Fasting blood glucose levels in the hyperglycemic control (56.2%) and hyperglycemic + R. obtusifolius extract (48.0%) groups during the first day of immobilization were significantly increased, compared to the normoglycemic group, p<0.05 (Fig. 2).
Therefore, it may be noted that disposable strong stressful pressure provokes of hyperglycemia. Treatment with R. obtusifolius (150mg/kg BW) single dose for 21 days showed a significant reduction in fasting glucose to hyperglycemic rabbits (57.3%, p<0.05) in comparison with 1 th day value.

Effect of R. obtusifolius on OGTT
The results of effect on OGTT were observed that the seeds extract showed a significant effect on hyperglycemia compared to the hyperglycemic group (Fig.3).
Blood glucose in all groups were increased at 30 min time point after glucose load, and then gradually decreased. following hours. At 120 min blood glucose levels were significantly reduced in treated group of rabbits (25.3%) and hyperglycemic control group (14.7%) compared to the values at 30min. Therefore, glucose tolerance was significantly improved in the R. obtusifolius treated animal groups, when compared to the hyperglycemic control group (p<0.05).

Effect of Ethanol Extract of R. obtusifolius on Serum Lipid Profiles
The data of effect of R. obtusifolius ethanol extract on serum lipid profiles showed that the TC and LDLcholesterol levels in the hyperglycemic control group were significantly increased (68.8% and 61.9% respectively), TG level was increased (17.5%) compared to the normoglycemic group (Table III). After 21 days of oral treatment, physiological levels of blood lipids parameters demonstrated significantly decreased TC and LDL-cholesterol levels (53.3% and 38.4%, respectively), and reduced TG level (17.5%) compared to the hyperglycemic control group. The HDL levels of the extract treated hyperglycemic group did not differ significantly from the hyperglycemic animals group.
Although the hyperglycemic control group of animals demonstrated a tendency towards increased LDLcholesterol level compared to the other groups.

Conclusion
Thus, high antioxidant activity of R. obtusifolius seed extracts was revealed. GS/MS analysis of R. obtusifolius seed methanol extracts allowed identification of several compounds, which could have high contribution on biological activities. On the other hand, it was shown that the sub-cytotoxic concentration of investigated extract was quite low (10 mgmL -1 ) on Murine microglial BV-2 WT cells. The ethanol extract of R. obtusifolius seeds revealed hypoglycemic activity, improved lipid profile in hyperglycemia induced by immobilization stress in rabbits. Further investigations should be performed to clarify the mechanisms of action. These findings suggest that R. obtusifolius extracts could be source of new biologically active compounds and might be recommended for treatment of diabetes mellitus.