Phytochemical content analysis of A. arabica plant extract by LC-MS/MS
Achillea arabica is a perennial aromatic herb widely used in folk medicine for the treatment of the stomachache and abdominal pain, wound healing, gout, cancer, diabetes and such [9, 28]. In studies on the A. arabica plant, it has been reported that the plant contains antioxidant compounds and major essential fatty acids such as p-cymene, piperitone, camphor and 1,8-cineole [29,30]. In addition, Achillea species are very rich in phenolic and sesquiterpene lactone compounds .
Therefore, in this study, phenolic compounds of A. arabica species were determined by LC-MS/MS and detailed information of this phenolics were given (Fig.1 and Table 1). A. arabica were investigated by LC–MS/MS and the major compounds were detected to be quinic acid (2439.9 μg/g), cyranoside (858.4 μg/g), chlorogenic acid (698.7 μg/g) and cosmosiin (347.8 μg/g), respectively. The analytical method validation parameters in this study were made according to the LC-MS/MS method developed by Yilmaz .
Quinic acid and its derivatives, which are compounds rich in polyphenols, are an important compound abundantly found in various plant products. Quinic acid is a normal component of our diet and can be converted to tryptophan and nicotinamide via the gastrointestinal tract microflora, thus providing humans with an in situ physiological source of these essential metabolic components . Chlorogenic acid, an ester of caffeic acid and quinic acid, has been reported to have potent antioxidant and multiple antiviral activities against HIV [32,33]. Plants of the genus Achillea, which are rich in protocatechuic, vanilic, chlorogenic, ferulic and quinic acid contents, have been reported to have antiinflammatory, analgesic, antimicrobial, antitrypanosomal, antidiabetic and antitumor effects .
It has been stated that Achillea species may have the potential to develop new functional and nutraceutical products due to their strong antioxidant and anticytotoxic effects . In addition, It has been reported that cyranoside and cosmosilin compounds determined by LC-MS/MS in various plant extracts may have antidiabetic effects due to their antioxidant, antimicrobial and in vitro diabetic enzyme inhibition effects .
NMR analysis findings of A. arabica flower extract
In the present study, ethanol extract of Achillea arabica afforded a new sesquiterpene lactone and a new phenolic glycoside. Compound 1 is a sesquiterpene which has a lactone ring.
The 1H NMR spectra of compound 1 displayed. Looking at the 1H NMR spectrum δ 5.06 and 5.36 doublet signals (J= 8.2 Hz) indicate the presence of an isolated double bond in the structure.
The 13C NMR spectra of compound 1 showed 3 carbonyl carbon atoms and two methylene carbons one of them is isolated methylene between C-15 and C-16 (δ 126.0 and 117.0, respectively) and the other one pentacyclic methylene with C-3 and C-4 (δ 136.8 and 136.3). HRMS spectrum of compound 1 confirmed its molecular structure (C17H18O6) molecular ion peak at m/z 318. HRMS; Exact Mass: 318.11034, Calculated m/z [M+H]+: 319.11816, Experimental m/z [M+H]+: 319.11496. According to literature research, it was determined that this substance was found for the first time and named as edremitine (1). The other new compound 2 is a glycoside which has phenolic skeleton were determined (Fig. 2). The 1H NMR spectra of compound 2 displayed 2 aromatic singlet protons (this protons appeared at δ 7.68 and 7.69 at H-2’ and H-6’ respectively) and 2 aromatic doublet protons signals (δ 7.69 and 7.05 at protons 7 and 8 with J=8.3 Hz). The 13C-NMR spectra of compound 2 showed 3 methoxyl carbons, 1 carbonyl carbon and 10 quaternary carbons. The methoxyl carbons appeared at δ 56.11, 60.17 and 60.93. HRMS spectrum of compound 2 approved its molecular structure (C29H34O16) molecular ion peak at m/z 638; [M-glc]: 360. HRMS; Exact Mass: 638.18469, Calculated m/z [M-H]+: 637.17686, Experimental m/z [M-H]+: 637.30078.
In fact, the literature search showed that similarity between structure of compound 2 and quercetin 3-O-vicianoside which was previously isolated . The compound 2 name is 6, 4’ two hydroxy, 5, 3’, 5’ three methoxy 3-O-vicianoside. Herewith compound 2 was isolated from nature for the first time and named as achillosine. Structures of these compounds were elucidated based on the 1H-, 13C-NMR spectrometric analyses.
(1): 1H-NMR (600 MHz, CD3OD): 5.98 (1H, d, H-3), 5.62 (1H, d, H-4), 3.42 (1H, m, H-5), 3.87 (1 H, d, J = 12.0, 10.1 Hz, H-6), 3.79 (1H, m, H-7), 4.07 (1H, m, H-8), 2.19 (2H, dd, J = 11.6; 8.4 Hz, H-11), 4.83 (1H, s, H-13), 5.06 (1H, d, J= 8.2 Hz, H-15), 5.36 (1H, d, J= 8.2 Hz, H-16), 2.95 (3H, m, H-17). 13C-NMR (150 MHz, CD3OD): 126.2 (C-1), 190.0 (C-2), 136.8 (C-3), 136.3 (C-4), 49.0 (C-5), 78.0 (C-6), 66.4 (C-7), 73.6 (C-8), 42.0 (C-9), 134.4 (C-10), 41.0 (C-11), 180.0 (C-12), 24.4 (C-13), 144.0 (C-14), 126.0 (C-15), 117.0 (C-16), 22.5 (C-17).
(2): 1H-NMR (600 MHz, CDCl3): 7.69 (1H, d, J= 8.3Hz, H-7), 7.05 (1H, d, J= 8.3Hz, H-8), 7.68 (1H, s, H-2’), 7.69 (1H, s, H-6’), 3.98 (3H, s, 5-O-CH3), 3.85 (3H, s, 3’-O-CH3), 3.64 (3H, s, 5’-O-CH3), 5.24 (1H, d, H-1”), 3.57-5.15 (4H, m, H-2”-6”and 2”’-5”’). 13C-NMR (150 MHz, CD3OD): 156.0 (C-2), 151.79 (C-3), 179.0 (C-4), 169.80 (C-5), 169.90 (C-6), 122.76 (C-7), 127.90 (C-8), 156.00 (C-9), 106.19 (C-10), 110.83 (C-1’), 122.44 (C-2’), 146.36 (C-3’), 148.38 (C-4’), 114.61 (C-5’), 130.00 (C-6’), 56.11 (5-O-CH3), 60.17 (3’-O-CH3), 60.93 (5’-O-CH3), 93.08 (C-1”).
Acute toxicity test and body weight
Animal showed good tolerance to testing seven (25, 50, 100, 250, 500, 1000 and 2000 mg/kg) doses of the ethanolic lyophilized A.arabica flower extracts. No any noticeable signs of toxicity and mortality were observed after daily administration of the extract orally at the end of the 3rd day (Data not shown).
The body weight (bw) of each rat was recorded every week during the 21-day experimental period. DM and treatment with extract/drug groups bw were shown in Fig. 3. The the final week bw of the DM group was significantly decreased compared to the first week; however, final week bws of the other groups were a significantly increased compared to the first week. In patients with DM, circulating blood glucose cannot/or little be taken into the cell in the absence or insufficiency of insulin, and when this happens, the body starts burning fat and muscle for energy, causing a decrease in total body weight. According to the findings obtained in our study, DM body weight caused a significant decrease compared to other groups. On the other hand, 400 mg/kg dose of A. arabica extract and glibenclamide (2 mg/kg) prevented STZ-induced diabetes as demonstrated by significant reduction of bw levels in DM-treatments (A. arabica extract and glibenclamide) groups. Our findings are consistent with changes in body weight caused by previous STZ-induced diabetes studies [38,39].
Effect of A. arabica extract on glucose, HbA1c and biochemical parameters
During the 21-day treatment, weekly the differences in blood glucose levels within the group and between the groups are given in Table 2. Blood glucose levels in DM group were significantly increased compared to all other groups (except the 1st day of DM+A.arabica 400 mg / kg). In addition, the 21th day blood glucose level of the DM group was increased according to the first three measurements. On the other hand, blood glucose levels of DM+A.arabica (400 mg/kg) and DM+Glibenclamide (2 mg/kg) groups were significantly decreased as weeks progressed, and these glucose levels were down by about half when compared to the first measurement.
As shown in Table 3. HbA1c and Glucose levels were significantly increased in DM group compared to all other groups while DM-treatment (extract and drug) groups showed a significant decrease compared to DM group. AST, ALT, ALP, CREA and urea biochemical parameters were significantly increased in the DM group compared to the control and A. arabica groups. On the other hand, AST, ALT, ALP, CREA (only, DM+Glibenclamide group) and urea levels were significantly decreased in DM-treatment (extract + drug) groups compared to DM group. Among the lipid profile parameters, TRIG and LDL levels were significantly decreased in A.arabica and DM-treatment (extract and drug) groups compared to control and DM groups. HDL level was significantly increased in DM+A.arabica (400 mg / kg) group compared to control, DM and DM + Glibenclamide (2 mg / kg) groups (Table 3).
Due to the low or absolute absence of insulin released from the β-cells of the pancreas, circulating glucose cannot be taken into the cell and the glucose level continues to be present in a high rate of circulation. There are many factors that cause pancreatic β-cells damage and insulin release disorder, such as oxidative stress, sedentary lifestyle, malnutrition, genetic predisposition, ATP synthesis disorder in the electron transport system, etc. If the ATP/ADP ratio is not sufficient in mitochondria, the K+ channels cannot depolarize and closed, so the Ca2+ channels cannot open and the Ca2+ cannot enter cell, and if the Ca2+ level is not at a sufficient in the cell, the insulin release will not circulate or little release into the circulation . In another study, STZ application caused histopathological changes in islets, significantly decreased islet diameter/area, β-cell index and blood insulin and C-peptide levels; however, they reported that Achillea arabica extract application caused improvement in islet histology, diameter/area, β-cell index values, blood insulin and C-peptide levels, compared to diabetic rats, similar to rats treated with glibenclamide .
As shown in Table 2, our results identified that there was a significant decrease in weekly blood glucose level in the treatment extract or drug groups compared to the DM group. Similarly, as shown in Table 3, glucose and HbA1c, liver and kidney damage biomarker parameters, and TRIG and LDL levels from lipid profile parameters showed significant decrease in treatment groups (A. arabica extract and glibenclamide) compared to DM group. Decrease in the treatment groups of blood glucose, HbA1c, liver and kidney damage biomarkers indicate that the plant extract has an antidiabetic and antioxidant role. In previous studies reported that similar results from Achillea species on blood glucose and biochemical parameters levels in rats [12,42]. In another study, it have been reported that Achillea arabica (synonymous: Achillea biebersteinii) ethanolic extract may have α-amylase enzyme inhibition, hypoglycemia and therapeutic effects on pancreatic damage in diabetes mellitus . In liver and kidney damage, AST, ALT, ALP, LDH, urea and CREA parameters leak into the circulation and increase their levels. Similarly, diabetes-induced oxidative stress causes an increase in lipid profile parameters LDL, CHOL, TRIG and a decrease in HDL levels . Achillea arabica essential oil (0.2 ml/kg/day) has been reported to cause a decrease in AST, ALT, GGT, ALP, bilirubin, cholesterol, triglycerides LDL and VLDL levels and an increase in HDL level against CCl4-induced toxicity in rats over 21 days . In another study, it was reported that 25 mg/kg/day or 100 mg/kg/day Achillea millefolium extract had a significant reduction in blood sugar, serum liver enzymes, triglycerides, and total CHOL and LDL cholesterol levels compared to the diabetic groups .
Effect of A. arabica extract on MDA content, GSH level and antioxidant enzyme activities
As shown in Fig. 4A. Liver and kidney (except A.arabica group) MDA contents were significantly increased in DM group compared to all other groups. The decrease in liver GSH level in DM group was significant determined compared to all other groups (Fig. 4B). The differences in kidney tissue GSH levels were not statistically significant between the groups.
As shown in Table 4. Liver GPx enzyme activity was significantly decreased in DM group compared to A.arabica group. The liver CAT enzyme activity of DM and control groups were significant decreased compared to A.arabica and DM+Glibenclamide groups. Also, liver CAT enzyme activity was significantly increased in the A.arabica group compared to the DM+A.arabica (400 mg/kg) group. Similarly, liver GST enzyme activity was significantly increased in the A.arabica group compared to the DM+Glibenclamide (2 mg/kg) group. Kidney CAT enzyme activity was significantly increased in DM+Glibenclamide (2 mg/kg) group compared to DM+A.arabica (400 mg/kg) group. In addition, kidney GST enzyme activity was significantly increased in DM group compared to all other groups (Table 4).
Free radicals such as hydroxyl (HO.), hydroperoxyl (HOO.), superoxide (O2.-), lipid (L.), lipid peroxyl (LOO.), peroxy (ROO.), lipid alkoxyl (LO.), nitric oxide (NO.), nitrogen dioxide (NO2.), and protein (P.) play an important role in the formation of diabetes . Increasing free radicals cause an increase in MDA content, liver and kidney damage biomarkers, and a decrease in antioxidant defense system enzymes such as SOD, GPx, CAT and GR . These enzymes protect cells against ROS, including CAT, H2O2 breaks down into H2O and O2; SOD, which removes O2.−; GST, which can remove xenobiotics by forming thiol groups; GR, which converts oxidized glutathione (GSSG) into reduced GSH . According to our results, diabetes caused a considerable increase in liver and kidney levels of MDA contents while treatment with Achillea arabica extract or glibenclamide drug significantly mitigated these elevations (Fig. 4A). Also, our results found that Achillea arabica extract or glibenclamide led to a significant increase in GSH level (Fig. 4B) and antioxidant enzyme activities in liver and kidney tissues (Table 4). The treatment of gastric ulcer by the A. arabica (200 mg/kg) extract showed significant decrease in oxidative stress markers by 84.61, 54.43 and 64.80 % for GSH, MDA and SOD, respectively, while ranitidine drug exhibited significant decrease in the OS markers by 79.80, 33.54, and 52.51 % . In another study, it was reported that the ethanolic lyophilized extract of A.arabica leaf showed significant antioxidant activity and DNA protective effect, as well as, the plant extract was not cytotoxic even at the highest dose studied (512 µg/ml) and inhibited H2O2-induced cell toxicity . Isolated all compounds from A.arabica plant were examined for their anti-inflammatory activity to inhibit lipopolysaccharide-induced NO production in RAW264.7 macrophage cells, and compounds 3 and 4 produced a promising anti-inflammatory effect (76% and 80% inhibition, respectively) . It was reported that the AST, ALT, GGT, ALP enzymes and bilirubin concentrations as well as the level of MDA, nonprotein sulfhydryl and total protein contents in liver tissues were significantly reinstated towards normalization by the A. arabica essential oil (0.2 mL/kg) against CCl4-induced hepatotoxicity in rats . It is known that A. arabica plant extract have major compounds such as 𝛼-terpinene (41%), p-cymene (13%) , 1,8-cineole (9–37%), camphor (16–30%) , piperitone (35%) and eucalyptol (13%) .