Gastroprotective and antioxidant effects of Eremurus spectabilis Bieb . methanol extract and its isolated component isoorientin on indomethacin induced gastric ulcers in rats

Purpose: To investigate the gastroprotective effect of methanol extract of E. spectabilis and its major component isoorientin. Methods: Effects of isoorientin and methanol extract of E. spectabilis were investigated in indomethacin-induced gastric damage model on rats. Famotidine was used as the standard antiulcer drug. Numerical density of ulcer areas and oxidative status were determined on stomach tissues of rats. Results: All doses of isoorientin and methanol extract decreased MDA level and increased SOD activity and GSH levels in the stomach tissue of rats. When numerical density of ulcer areas were analized, the 500 mg/kg dose of methanol extract (84%) exhibited a similar effect to 20 mg/kg dose of standart drug famotidine (87%). Conclusions: The gastroprotective effects of E. spectabilis and its major constituent isoorientin in rats for the first time. Detailed analyses suggested that potential antioxidant activity of both plant extract and isoorientin mediates the gastroprotective effect.


■ Introduction
The peptic ulcer is a deterioration in the gastric or duodenal mucosa down to the submucosa.The various causes of gastric ulcer are stress, trauma, some diseases, helicobacter pylori, use of alcohol and cigarettes, and steroidal and nonsteroidal anti-inflammatory drugs (NSAIDs).Peptic ulcer regarded as a important common gastrointestinal disorder which can affect individuals during their lives [1][2][3] .The reactive oxygen species (ROS) play an important role in the gastric ulcer formation induced by stress, ethanol and NSAIDs.The inflammation in the gastric tissue induced by indomethacin causes the formation of ROS 4 .Superoxide dismutase, catalase, glutathione reductase, β-carotene, glutathione peroxidase, β-tocopherol, glutathione, vitamins A and C are good antioxidant agents.In addition, these antioxidants are also effective in prevention stomach damage 5 .
The Eremurus genus is a member of Liliaceae family, including about 50 species.E. spectabilis is grown naturally in the eastern regions of Turkey.This plant, locally known as 'Çiris', and its fresh leaves sold and consumed as an edible vegetable in markets 6 .
According to ethnobotanical studies, E. spectabilis is used for the treatment of numerous ailments like scabies, diabetes and intestinal diseases [7][8][9] .In addition to these, E. spectabilis has also antibacterial and antioxidant activities 10,11 .Carvone, carvacrol, pentane, (E)-caryophyllene, valencene were found to be as main constituents of the essential oil of E. spectabilis 12 .It is reported that inosine, methyl linolenate, chrysophanol, isoorientin, β-sitosterol and sucrose were also isolated from E. spectabilis 13 .In a previous study, we isolated a high amount of isoorientin from E. spectabilis 14 .

Plant material
The aerial parts of E. spectabilis were collected from Iğdasor (Erzurum Province, 1920 m, Turkey) in April 2014.A voucher specimen was stored at the Herbarium of Atatürk University, Faculty of Pharmacy (AUEF1189).It was identified by Esen Sezen Karaoğlan.

Previous extraction and isolation studies
Fresh E. spectabilis leaves were extracted with methanol (MeOH) at 40°C after drying and powdeing.The MeOH extract was filtered and evaporated.The dry MeOH extract was suspended in water and extracted with n-hexane, dichloromethane (CH 2 Cl 2) , ethyl acetate (EtOAc), and n-butanol, respectively.Organic phases were concentrated to dryness under vacuum.Isoorientin was isolated from EtOAc extract via Sephadex LH-20 column eluting with MeOH and then silica gel column eluting with CH 2 Cl 2 : MeOH: H 2 O (90:10:1→50:50:5).The structure of isoorientin was elucidated by 1 H NMR, 13 C NMR, 2D NMR and Q-TOF by Sezen Karaoğlan et al. 14 .

Experimental design of indomethacin-induced ulcer model
Eight experimental groups consisting of six rats were formed: Group

Ulcer model
For ulcer model induction in rats previously described indomethacin-ulcer model was used with no modification 17,18 .NSAID induced ulcer model is one of the the most commonly used gastric ulcer models 19 .In this study; the isoorientin groups received 25, 50, 100 mg/kg doses and the plant extract groups received 250 and 500 mg/kg doses and of drugs as a pretreatment by oral gavage, after which the rats were fed anything for 24 hours.Healthy control and indomethacin control groups received 1 ml of saline as vehicle by oral gavage.Then, famotidine (positive control) 17 , isoorientin and plant extract were administered to the corresponding rat groups in the above-mentioned doses by oral gavage.Five minutes after the famotidine, isoorientin and plant extract treatments, a dose of 25 mg/ kg of indomethacin was suspended in isotonic saline and administered by oral gavage to all groups except healthy group.Six hours after the administration of indomethacin, all rats were euthanized by administering intraperitonal thiopental at a dose of 50 mg/kg.The ulcerated areas of the removed stomachs were examined macroscopically.Ulcer areas were evaluated by stereo-investigator, and numerical densities of ulcerated areas were calculated by software, as described below.

Numerical density (ND)
Numerical density can be defined as the number of particles in the unit volume.For optical dissector, total number of particles (N) counted should be divided to dissector volume calculate Numerical density of that particles.In our study Numerical Density of Ulcerated area has been calculated by dividing total number of ulcer area to total volume of stomach tissue.In our study, stereological analyses were conducted in a stereology workstation using stereology software (Stereo Investigator version 9.0; MBF Bioscience, Colchester, VT).The numerical ulcer hematoma densities were calculated by the following formula: Numerical Density (ND): Total Markers Counted x 1000 (µm 2 )/ Sampling Grid Area (XY) (µm 2 )

Biochemical investigation of stomachs tissues
After the macroscopic analysis, the stomach tissues were stored at -80 C. All stomach tissues were milled in liquid nitrogen using a TissueLyser II grinding jar set (Qiagen, Hilden, Germany).The 50 mg of ground tissues were mixed fully in 1ml PBS buffer in an Eppendorf tube using the TissueLyser II, and then centrifuged.Superoxide dismutase (SOD) activity 20 , malondialdehyde (MDA) 21 and glutathione (GSH) levels 22 from each samples were measured by repeatedly at room temperature, with an ELISA reader 23 .The average absorbances were calculated.A standard curve was drawn and the equation was obtained from the absorbance of the standards.Linear SOD, GSH, and MDA concentrations were calculated considering this equation.The SOD, GSH, MDA levels were explicated as U/mg protein, nmol/mg protein.
The data are offered as mean ± standard deviation (SD) per mg protein.

Protein determination
Protein concentration were carried out by the Lowry method using commercial protein standards (Total protein kit-TP0300-1 KT; Sigma Chemical Co.(Munich, Germany).

Statistical analyzes
Statistical Package for the Social Sciences (SPSS) version 20.0 (SPSS Inc., Chicago, IL) software was used for the statistical analysis.The results are presented as means ± SD.Comparisons between groups were performed using one-way analysis of variance (ANOVA) and the Duncan Multiple Comparison Test which allows inter-comparison of all groups.
Significance was accepted at a level of p<.05.

Biochemical analyses
The all doses of isoorientin and methanol extract significantly (p<0.05)decreased MDA.The most effective decline was observed at the 500 mg/kg dose of methanol extract dose.All doses of isoorientin and methanol extract significantly increased SOD activity (p<0.05) and GSH levels (p<0.05) in the stomach tissue of rats. the 500 mg/kg dose of methanol extract maximally increased the SOD activity and GSH levels.The SOD activity, GSH and MDA levels of isoorientin (25, 50, 100 mg/kg doses), methanol extract (250, 500 mg/kg doses), famotidine (20 mg/kg dose) and control groups (Figures 3 to 5).

■ Discussion
People have been using plants for therapeutic purposes for many years.E. spectabilis is locally known as 'Çiris', and is grown naturally in Erzurum, Erzincan, Sivas, Kars, Yozgat, Bitlis, Usak, Agri, Van, Artvin and Ardahan regions in Turkey.This plant is consumed by people as vegetables.Its fresh leaves sold low-priced in markets and contains a high amount of isoorientin.Any toxic effects are not recorded for this plant.
Peptic ulcer is an important disturbance affecting people's quality of life.NSAIDs is one of the important causes of peptic ulceration process [1][2][3] .It is registered that E. spectabilis is used in gastrointestinal disorders.In a previous study, we isolated a high amount of isoorientin from E. spectabilis.Isoorientin is a substance in the flavonoid structure which has many biological activities.
This study investigated the antiulcer activity and the effect on oxidant and antioxidant parameters of different doses isoorientin and methanol extract of E. spectabilis in rat stomach tissue.It was observed that the gastroprotective efficacy increased both in the groups isoorientin and in the groups methanol extract depending on the dose.When numerical density of ulcer areas were analized, the 500 mg/kg dose of methanol extract (84%) almost exhibited a similar effect to 20 mg/kg dose of standart drug famotidine (87%).
The 500 mg/kg dose of extract showed the highest efficacy, although both extract and isoorientin were observed to be effective when compared to the indomethacin group.In a previous study, ethyl acetate extract of Wilburandia ebracteata showed high anti ulcer effect.This effect was attributed to the substances flavonoid structure, 3',4',5,6,7,8-hexahydroxyflavonol, orientin, luteolin, 6-methoxy-luteolin, vitexin, isoorientin, isovitexin 24 .In our study, we also think that the high efficacy of the plant extract is not due to a single molecule isoorientin, but may be due to the synergistic effect of the different components in it.
We evaluated the effects of isoorientin and methanol extract of E. spectabilis on local oxidative stress parameters in the stomach tissues of rats as a contributing antiulcer mechanism.
Indomethacin dramatically increased stomach MDA content, one of the most widely used parameters as a lipid peroxidation indicator [25][26][27][28][29] , while all doses of isoorientin and methanol extract decreased MDA.In addition, indomethacin caused a significant decrease in stomach antioxidant status, namely decreased SOD activity and GSH levels.Both SOD and GSH are known to be antioxidant agents in inflammatory and ulcerative conditions 30,31 .In previous studies it was reported that the E. spectabilis had significant antioxidant and free radical scavenging activity.In addition, it is known that isoorientin which is a flavonoid, has also highly antioxidant activity 32,33 .In our study, all doses of isoorientin and methanol extract increased SOD activity and GSH levels in the stomach tissue of rats.It has been observed that especially the 500 mg/kg dose of methanol extract increased these parameters at the approximate value of standart drug famotidine.
It is registered that E. spectabilis leaves are used traditionally in gastrointestinal disorders 12 .The results of our work are also supportive of this gastroprotective effect.

■ Conclusions
E. spectabilis is a vegetable that is consumed safely among the population.The fresh leaves of this plant are sold at low prices in the markets.It contains high amount isoorientin, has significant gastroprotective effect.Finding new molecules for the pharmaceutical industry is very important, but these studies are costly and time consuming.For this reason, the importance of effective, low-priced and easily accessible standardized plant extracts is increasing.E. spectabilis extract may be a potential anti-ulcer agent in the future.Scientific research in this area needs to be increased.

Figure 2 -
Figure 2 -Ulcer index areas of the groups underwent indomethacin ulcer model.Columns with the same letter are not significantly different; columns with different letters indicate significant differences between the groups according to the Duncan test (p < 0.05).Results are means ± SD.N ¼ 6 animals per group.(Numerical density can be defined as the number of particles in the unit volume.For optical dissector, total number of particles (N) counted should be divided to dissector volume calculate Numerical density of that particles.In our study Numerical Density of Ulcerated area has been calculated by dividing total number of ulcer area to total volume of stomach tissue).IND: indomethacine; ISO: isoorientin; ES: extract.

Figure 3 -
Figure 3 -Stomach tissue SOD activity in groups underwent indomethacin ulcer model.Columns with the same letter are not significantly different; columns with different letters indicate significant differences between the groups according to the Duncan test (p<0.05).Results are means ± SD.N ¼ 6 animals per group.IND: indomethacine; ISO: isoorientin; ES: extract.

Figure 4 -
Figure 4 -Stomach tissue GSH level in groups underwent indomethacin ulcer model.Columns with the same letter are not significantly different; columns with different letters indicate significant differences between the groups according to the Duncan test (p<0.05).Results are means ± SD.N ¼ 6 animals per group.IND: indomethacine; ISO: isoorientin; ES: extract.

Figure 5 -
Figure 5 -Stomach tissue MDA level in groups underwent indomethacin ulcer model.Columns with the same letter are not significantly different; columns with different letters indicate significant differences between the groups according to the Duncan test (p<0.05).Results are means ± SD.N ¼ 6 animals per group.IND: indomethacine; ISO: isoorientin; ES: extract.