Exploring of phytochemical constituents of Achillea arabica Kotschy. ethanolic flower extract by LC-MS/MS and its possible antioxidant and antidiabetic effects in diabetic rats

doi:10.21203/rs.3.rs-1207243/v2

The aim of this study was to reveal the antidiabetic and antioxidant effects of ethanolic lyophilized extract of Achillea arabica flower extract against streptozotosine (STZ)-induced in diabetic rats and to determine its phytochemical content by liquid chromatography with tandem mass spectrometry (LC-MS/MS). After toxicity test, 35 female rats were divided into 5 groups. Control, Diabetes mellitus (DM), A. arabica (400 mg/kg) extract, DM + A. arabica (400 mg/kg) extract and DM + Glibenclamide (2 mg/kg). It was determined that while diabetic rats treated A. arabica plant extract significantly decreased blood glucose level, serum glucose, HbA1c, liver and kidney damage biomarker levels, and malondialdehyde (MDA) content compared to the DM group, it caused fluctuations in antioxidant enzyme levels. According to LC–MS/MS results of A. arabica flower extract, quinic acid (2439.9 μg/g), cyranoside (858.4 μg/g), chlorogenic acid (698.7 μg/g) and cosmosiin (347.8 μg/g) were determined as major compounds, respectively. In addition, two new compounds were determined in this extract according to NMR and Mass analyses and these compounds were named edremitine and achillosine, respectively. Thus, A. arabica flower extract has possible therapeutic effects to prevent high blood glucose level and oxidative stress casued by DM in liver and kidney via its high phenolic content.

A. arabica

diabetes mellitus

LC-MS/MS

NMR

oxidative stress

rat

Achillea genus, which belongs to the Asteraceae family, consists of more than 100 species, The Achillea species are used for treatment for many diseases such as gynecological and hepatobiliary disorders, rheumatic pain, spasmodic gastrointestinal, pneumonia, hemorrhages, inflammation and wound healing in folk medicine (Wichtl 2002; Burk et al. 2010). Members of this genus contain volatile oil and other secondary metabolites, including terpenes (especially sesquiterpene lactones), flavonoids, polyphenols and others (Salehi 2020).

The genus Achillea L. (Compositae) commonly used as medicinal plant have various pharmacological effects because of presence of active compounds since a long time. These plants contain bioactive compounds like terpenes, flavonoids, alkaloids, tannins and lignins (Aburjai and Hudaib 2006). Achillea arabica Kotschy. (Synonym: Achillea biebersteinii Afanasiev), which is known as Hanzabel in Turkey. In folk medicine, A. arabica is used for the treatment of stomachache and abdominal pain, and antinociceptive action (Jaffal and Abbas 2019), inflammatory and spasmodic gastrointestinal disorders, hepatobiliary and cardiovascular ailments (Akkol et al. 2011), DNA damage (Varasteh-Kojourian et al. 2017), antiulcer and hypoglycemic (Abd-Alla et al. 2016). A. arabica flowers contains various chemical components such as sabinene, p-cymene, α-terpenine, 1.8-cineol, linalool, trans-sabinene, terpinen-4-ol, nerol, ascaridole, cis-piperitone epoxide, trans-piperitone epoxide, trans-sesquisabinene hydrate, thymol, carvacrol, iso-ascaridole, eugenol, lavandulyl-2-methylbutanoate, hexadecene, γ-eudesmol, octadecene, hexadecenoic acid methyl ester, hexadecenoic acid, tricosane (N-) and pentacosane (N-) (Jaffal and Abbas 2019). In addition, ethanolic extracts of A. arabica have been reported to contain flavonoids, quercetin, routine and myrisetin (Hammad et al. 2013). Nowadays, the interest of people onto medicinal plants is increasing as therapeutic agents against some diseases such as neurodegenerative, cardiovascular, skin disorders and chronic disturbance like diabetes mellitus (DM) (Zengin et al. 2014). DM is the most common disorder of the endocrine system and the levels of fasting glucose (≥ 126 mg/dL) and/or glycolyzed hemoglobin % (HbA1c %) ≥ 6.5 are used for hyperglycaemic diagnosis (Agirman et al. 2020). There are two common types of diabetes, Type 1 and Type 2. Type 2 make up for approximately 90-95 % of current patients, and here lifestyle, nutrition and stress play an important role. In addition, DM prevalence is increasing in the world, it was approximately 415 million diabetic adult in 2015 and the number will be increased to more than 800 million in 2040 (Ogurtsova 2017). Complications of DM are including such as nephropathy, neuropathy, foot amputation and chronic diseases cause both quality of life and serious economic losses for countries globally 673 billion US dollars in 2015 (Ogurtsova 2017). People with diabetes are inetrested in the use of natural products or crude extracts obtained from traditionally medicinal plants to avoid possible negative side effects of drugs and its complications or to make their daily life more comfortable (Dewanjee et al. 2020).

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 (Yilmaz et al. 2018). The main aim of this study was to determine the role of A. arabica lyophilized flower extract on hyperglycemia, hyperlipidemia, lipid peroxidation content, oxidative biochemical markers and antioxidant enzyme alterations in serum and in liver and kidney tissues in STZ (50 mg/kg) induced diabetic rats. Additionally, we aimed to identify the phytochemical content in the flower extract by LC-MS/MS and to reveal new compounds by 1H-, 13C-NMR spectrometric analyses.

2.1. Plant material

The A. arabica plant collected by Hanife Ceren HANALP and Abdulahad DOGAN (PhD) in the Edremit district (GPS coordinates: 38° 25' 09 " N; 43° 15' 54" E), Van, Turkey, during May 2018. The samples were authenticated by Süleyman Mesut Pinar (PhD, Department of Botany, Van Yuzuncu Yil University, Turkey). The voucher specimen was placed into the Van Yuzuncu Yil University, Faculty of Science, Department of Biology (VANF) herbarium (Herbarium Code:164098).

2.2. Preparation of lyophilized extract

The plant ethanolic lyophilized extract was prepared according to the method of Dogan et al. (2018), with some modifications. The flower samples were dried in the shade and crushed into a fine powder. The 50 g of flower powder was mixed into 1L of 80 % of ethanol and 20 % of distilled water (dH₂O) for 2 hours using a magnetic stirrer to prepare extract. After that, it was filtrated through strainer and cheesecloth in order, liquid part was centrifuged at 10 000 rpm for 20 min. Supernatant was filetered through a 0.45 μm hydrophilic filter with the help of an injector. The combined supernatants were evaporated with a rotary evaporator at 37°C (Rotavapor R-205; Buchi, Switzerland). After the liquid extracts were evaporated, they were frozen and stored at -80 °C for 48 hours. The samples were freeze dried under a vacuum in a lyophilizer device at -51 °C to obtain fine powder of flower extract.

2.3. Animals

A total of 40 Wistar albino female rats (5 rats for acute toxicity test and 35 rats for diabetes model), which were 2–3 weeks old and weighed 140–180 g, were supplied from the Experimental Animal Research Center of Van Yuzuncu Yil University. All of the experimental procedures on animals during the study were followed according to Animal Experiments Ethics Committee of Van Yuzuncu Yil University for ethical concerns (YUHADYEK-2019/06). During the experiments, the ethical regulations that were followed in line with the institutional and national guidelines for animal welfare protection. The rats were housed in cages made of plastic at a steady room temperature of 25 ± 2 °C and a 12-h light/dark photoperiod. They were supplied ad libitum with drinking water and a diet that was wheat and soybean meal-based.

2.4. Acute toxicity test

For evaluation of the acute toxicity of the lyophilized ethanolic A. arabica extract on the rats, increasing dosages of 25, 50, 100, 250, 500, 1000, and 2000 mg/kg of body weight (bw) were given via oral gavage for 5 rats. Signs or symptoms of toxicity and mortality were not determined for the first 72 h. Thereafter, the favorable dosage of the extract used in the experimental study was ascertained to be 400 mg/kg bw.

2.5. Induction of diabetes and experimental design

Cold and fresh citrate buffer (0.1 M, pH 4.5) containing STZ was prepared for the rats that were fasted for 12 hours. After that, a single dose of 50 mg/kg was injected intraperitoneally (i.p.) to each rat. 72 hours after STZ administration, fasting blood glucose levels were measured with Accu-Chek Go (Roche) glucometer device strips from the tails of the rats. At the end of the determined period, it was observed that all rats given STZ had blood glucose levels above 200 mg/dL and they were diagnosed with diabetes.

The rats were randomly divided into 5 groups, each containing 7 rats.

Control group: The rats in this group were fed only with feed and water ad libitum during the 21-day experiment period.

Diabetes mellitus (DM) group: The rats in this group with diabetes mellitus with STZ (50 mg/kg bw) were fed only with feed and water ad libitum during the 21-day experiment period.

A. arabica (400 mg/kg) extract group: During the 21-day experiment period the rats fed with feed and water ad libitum, and A. arabica (400 mg/kg) extract was given as gavage once a day.

DM+A. arabica (400 mg/kg) extract group: During the 21-day experiment, diabetic rats were fed with feed and water ad libitum, and A. Arabica (400 mg/kg) extract was given as gavage once a day.

DM+Glibenclamide (2 mg/kg) group: During the 21-day experiment, diabetic rats were fed with food and water ad libitum, and glibenclamide (2 mg/kg) was administered orally once a day.

Additionally, the first and the last live body weight (LBW) and blood glucose level of the rats were examined during the experiment.

2.6. The obtained blood and preparation of tissues supernatant

At the end of the experiment, the rats were anesthetized by ketamine (10 mg/kg, ip), and blood was drawn from rat’s heart with injectors. Blood put into hemogram tubes were used for HbA1c % measurement. The blood taken into biochemistry tubes was centrifuged at 3000 g for 15 minutes at 4 °C and biochemical parameters were measured in the serum samples obtained.

Tissue samples taken from rats were placed in tubes after washing with physiological water and stored in a ultra freezer at at –78 °C until analysis. The tissues were homogenized for 3 min in 50 mM of ice-cold KH₂PO₄ solution (1:5 w/v) using a stainless steel probe homogenizer (SONOPULS HD 2200, Bandelin, Berlin, Germany), and then subsequently centrifuged at 9000 rpm for 30 min. All of the processes were carried out at 4 °C. Then, supernatants were used to determine malondialdehyde contents and antioxidant defence systems constituents as described previously method (Dogan and Celik 2012).

2.7. Measurement of HbA1c and biochemical parameters

The glycosylated haemoglobin % (HbA1c %) were determined using an auto analyser device (Cobas 6000 c501, Germany) with kits Roche Cobas kits. Alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatinine (CREA), uric acid (UA), urea, glucose (GLU), triglyceride (TRIG), cholesterol (CHOL), high density lipoprotein cholesterol (HDL_c) and low density lipoprotein cholesterol (LDL_c) were measured using a auto analyzer (ARCHITECT 16200, Abbott park, IL 60064, USA) and Abbott biochemistry kits.

2.8. Lipid peroxidation and antioxidant enzyme analyses

Total protein content of the tissues was determined using the Bradford method (Bradford 1976). The level of reduced glutathione (GSH) in tissues was measured using the method described by Beutler et al (1963). Tissue MDA content based on TBA activity was determined according to the method of Buege and Aust (1978). Glutathione peroxidase (GPx) activity was measured using a method of the Paglia and Valentine (1976). Superoxide dismutase (SOD) activity was measured at 505 nm by calculating the inhibition percentage of formazan dye formation (McCord and Fridovich 1969). Glutathione S-transferase (GST) was assayed by following the conjugation of glutathione with CDNB at 340 nm, as described by Mannervik and Guthenberg (1981). Catalase (CAT) activity was determined using the method described by Aebi (1974).

2.9. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis

The LC-MS/MS phytochemical content analysis of the A. arabica flower extract was performed based on 53 phytochemical compounds previously optimized and validated by Yilmaz (2020).

2.10. Compounds isolation from A. arabica extract

The crude ethanol extract (2.16 g) was subjected to column chromatography over silica gel eluted with chloroform and gradually increasing the polarity, by addition of methanol (MeOH) (up to 100%). Totally, 12 fractions were collected. Similar fractions were combined to obtain in total 10 fractions. A flavone glycoside was obtained from fraction 4 and a sesquiterpene lactone was obtained from fraction 5 and purifed on preparative silica gel thin layer chromatography (TLC) plates using from a solvent system (chloroform/methanol) (9.5:0.5).

2.11. Nuclear magnetic resonance (NMR) and Mass analyses

The NMR spectra were measured on a Bruker Avance NEO NMR Specrometer at 500 and 125 MHz (¹H at 500 MHz, ¹³C at 125 MHz) instrument at 24 ⁰C. The tetramethylsilane resonance was used as internal standard. Chemicals shifts were given in parts per million (ppm) and coupling constants (J) in Hertz. The liquid chromatography–high resolution mass spectrometry (LC–HRMS) spectral analyses were obtained on Thermo Orbitrap Q-exactive was used for mass analyses of the isolated compounds.

2.12. Statistical analysis

The statistical analyses were performed using the Minitab 13 for Windows package program. All data were expressed as the mean ± standard deviation (SD). The one-way analysis of variance (ANOVA) statistical test was used to determine differences between the means of the experimental groups with statistical significance accepted at P < 0.05. The student-t test was used for determination of the differences in body weight changes.

3.1. Acute toxicity test and body weight

Animals showed good tolerance to testing seven doses (25, 50, 100, 250, 500, 1000 and 2000 mg/kg) 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 3^rd day (Data not shown).

The body weight of each rat was recorded at the 1^st day and 21^st day. As seen in Fig. 1 (A), in all groups (except the DM + A. arabica group), there was a significant increase in body weights in the last week compared to the first week, while a decrease was found in the DM group.

3.2. Effect of A. arabica extract on glucose, HbA1c and biochemical parameters

Glucose level measured from tail by glucometer at the 1^st day and 21^st day was presented in Fig. 1 (B). According to results, while glucose level in DM group was not significantly different between measurements, it was significantly decreased in the DM + A. arabica group and DM + Glibenclamide group between measurements.

As shown in Table 1, HbA1c and glucose levels were significantly increased in DM group compared to all other groups while DM-treatment (extract or 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 or 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 1).

3.3. Effect of A. arabica extract on MDA content, GSH level and antioxidant enzyme activities

As shown in Table 2, 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 (Table 2). The differences in kidney tissue GSH levels were not statistically significant between the groups.

As shown in Table 2, 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 2).

3.4. Phytochemical content analysis of A. arabica plant extract by LC-MS/MS

Phenolic compounds of A. arabica species were determined by LC-MS/MS and detailed information of this phenolics were given (Fig. 2 and Table 3). Quinic acid, fumaric aid, aconitic acid, gallic acid, protocatechuic acid, gentisic acid, chlorogenic acid, protocatechuic aldehyde, 4-OH benzoic acid, caffeic acid, vanillin, p-coumaric acid, ferulic acid, salicylic acid, cynaroside, miquelianin, rutin, isoquercitrin, hesperidin, genistein, cosmosiin, astragalin, quercetin, naringenin, luteolin, kaempferol, apigenin, amentoflavone, chrysin and acacetin were detected in the ethanolic flower extract of A. arabica (Table 3). The major compounds of the extract were determined as Quinic acid, cynaroside, protocatechuic acid and cosmosiin (2439.9, 858.4, 698.7 and 347.8 µg/g extract, respectively).

3.5. NMR analysis findings of A. arabica flower extract

In the present study, ethanol extract of Achillea arabica afforded a new sesquiterpene lactone (1) and a new phenolic glycoside (2) (Fig. 3). Compound 1 was a sesquiterpene which has a lactone ring displayed by ¹H NMR spectra. 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 was 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 compound 1 was found for the first time and named as edremitine (Fig. 3).

The new compound 2 was a glycoside which has phenolic skeleton (Fig. 3). 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. The compound 2 name was 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 (Fig. 3). 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”).

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 (Ahmadi et al. 2017; Jaffa and Abbas 2019). 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 (Mottaghi et al. 2016; Kaffash et al. 2020). Similarly, in different studies, it has been reported that Achillea species are quite rich in terms of phenolic and sesquiterpene lactone compounds (Salehi et al. 2020) such as 𝛼-terpinene (41%), p-cymene (13%) (Dehghan and Elmi 2015), 1,8-cineole (9–37%), camphor (16–30%) (Morteza-Semnani et al. 2005), piperitone (35%) and eucalyptol (13%) (Sökmen et al. 2004).

Live animal body weight changes between first and last measurements are as in Fig. 1 (A). The the final body weight of the DM group was significantly decreased compared to the first day; however, final avarege body weight of the other groups except DM + A. arabica group was significantly increased compared to the first measurement. 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. Our findings are consistent with changes in body weight caused by previous STZ-induced diabetes studies (Dogan et al. 2015; Demirbolat et al. 2019).

Circulating glucose cannot be taken into the cells and the glucose level continues to be present in a high rate of circulation due to the low or absolute absence of insulin released from the β-cells of the pancreas in diabet disease. 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 (Bhatti et al. 2017).

As shown in Fig 1 (B), 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 1, glucose, HbA1c, AST, ALT, creatinine, urea, TRIG and LDL levels 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 (Abd-Alla et al. 2016; Rezaei et al. 2020). In another study, it has 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 (Ahmadi et al. 2017). STZ application caused histopathological changes in islets, significantly decreased islet diameter/area, β-cell index and blood insulin and C-peptide levels; in addition, 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 (Hanalp et al. 2021). In liver and kidney damage indicator parameters such as AST, ALT, ALP, LDH, urea and CREA are releasedfrom cells into the circulation and increase their levels in diabetes (Dogan et al. 2015). Similarly, diabetes-induced oxidative stress causes an increase in lipid profile parameters LDL, CHOL, TRIG and a decrease in HDL levels (Daisy et al. 2008). Achillea arabica essential oil (0.2 ml/kg/day) caused 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 (Al-Said et al. 2016). 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 (Rezaei et al. 2020).

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 (West 2000). 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 (Dogan et al. 2015). These enzymes protect cells against ROS, including CAT, H₂O₂breaks 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 (Dogan and Anuk 2019).

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 (Table 2). Also, our results found that Achillea arabica extract or glibenclamide led to a significant increase in GSH level (Table 2) and antioxidant enzyme activities in liver and kidney tissues (Table 2). Abd-Alla et al. (2016) reported that the aerial parts of the A. arabica ethyl acetate extract (200 mg/kg/day) decreased the amount of lesion and MDA level against ethanolic gastric ulcer and increased antioxidant enzyme activities. Morever, they demonstrated that this extract has the ability to reduce starch-induced postprandial glycemic deviations through its potent intestinal α-amylase inhibitory activity. 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 (Varasteh-Kojourian et al. 2017). 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) (Mohammed et al. 2016). 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 (Al-Said et al. 2016).

In this study, 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 (2020). 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 human 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 (Pero et al. 2009). Chlorogenic acid, an ester of caffeic acid and quinic acid, has been reported to have potent antioxidant and multiple antiviral activities against HIV (McDougall et al. 1998; Tamura et al. 2006). 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 (Patocka and Navratilova 2019). In addition, it has been reported that cyranoside and cosmosiin 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 (Ceylan et al. 2021).

In the present study, ethanol extract of A. arabica afforded a new sesquiterpene lactone (1) and a new phenolic glycoside (2) (Fig 3). Compound (1) is a sesquiterpene which has a lactone ring. According to literature research, Compound (1) 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. In fact, the literature search showed that similarity between structure of compound (2) and quercetin 3-O-vicianoside which was previously isolated (Lee et al. 2019). 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. Achillea L. genus is a rich source for volatile oil and other secondary metabolites, including terpenes (especially sesquiterpene lactones), flavonoids, polyphenols and phenolics (Salehi 2020). Obtained a new chlorine-containing sesquiterpene lactone together with two new epimeric secoguaianolides of tanaphallin and the known vomifoliol from aerial parts of Achillea cretica L. has been reported (Hichri et al. 2015). It has been reported that a new sesquiterpene compound, namely as santolinoidol, has been found by HPLC analysis of the Achillea santolinoides subsp wilhelmsii (Fahed et al. 2016). In another study, three new sesquiterpene lactones and eight known compounds were reported from the aerial parts of Achillea clavennae. One of the new compounds has been reported to exhibit higher cytotoxicity than cisplatin on partially apoptotic death in human U251 and rat C6 glioma cell lines (Trifunović et al. 2014). Furthermore, clinical studies have demonstrated that some Achillea species have protective or treatment potential against irritable, multiple sclerosis, bowel syndrome ulcerative colitis, episiotomy wound, primary dysmenorrhea and oral mucositis (Salehi 2020). It was shown that these activities are due to the sesquiterpene lactones and phenolics compounds in the structure of the plant (Loggia et al. 1992).

When the data are evaluated as a whole, the antioxidant and antidiabetic potential of A. arabica flower extract can be attributed to the presence of major compounds determined in the plant. Ethanolic flower extract of A. arabica were found to be rich in phenolic compounds. Among phenolics, mainly quinic acid, cyranoside, chlorogenic acid and cosmosiin were found to be high amounts in the extract. Additionally, a new sesquiterpene lactone namely ((3ab, 9ba)- 6-(hydroxymethyl)- 2,7-dioxo-4a- (2-butenoyloxy)- 2,3,3a,4,5,7,9a,9b-octahydroazuleno [4,5-b] furan) (1) and a new glycoside which has phenolic skeleton as namely 3,6,4'-trihydroxy-5,3',5'-trimethoxyflavone-3-O-α-L-rhamnopyranoside (2) were isolated and characterized by NMR. Thus, A. arabica should be considered as a rich source for these compounds with their antioxidant and antihyperglycemic properties. Therefore, we propose that A. arabica extract may be useful in curing patients suffering from diabetes and its complications. However, since in vivo studies are negligible, further studies are required to determine the optimal doses of A. arabica plant.

Acknowledgements

Authors are thankful to Süleyman Mesut PINAR for botanical identification of the plant materials. Authors are also grateful to Dicle University Science and Technology Researchand Application Center (DUBTAM) for providing laboratory facilities.

CRediT authorship contribution statement

A. Dogan: Study design, writing-review & editing. H. C. Hanalp: Performing in vivo analyzes. T. Kusman Saygı: In vitro analyzes to be performed. F. Donmez: Contributed to manuscript review and biochemical analysis. A. Battal: Contributed in manuscript writing and review.

Funding

This study was supported by Van Yuzuncu Yil University Scientific Research Project Commission (Grant number TYL-2019-7865).

Data availability

The data and material used and analyzed during the current study are available from the corresponding author on reasonable request.

Declaration of Competing Interest

The authors declare that they have no competing interest

Ethical approval

The procedures conducted herein were in line with those of the National and Institutional Regulations for the Protection of Animal Welfare. The animal used, and all of the experimental procedures during the study, were approved of by the Animal Experiments Ethics Committee of Van Yuzuncu Yil University for ethical concerns (YUHADYEK-2019/06).

ORCIDs:

Hanife Ceren Hanalp https://orcid.org/0000-0001-5235-4167

Abdulahad Dogan http://orcid.org/0000-0002-5438-8560

Tuba Kuşman Saygı http://orcid.org/0000-0002-1771-1453

Fatih Donmez http://orcid.org/0000-0003-3958-1028

Abdulhamit Battal http://orcid.org/0000-0001-6098-3908

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Table 1. Quantitative screening of phytochemicals in ethanol extract of Achillea arabica by LC–MS/MS.

No	Compounds	Retention time	Amount (μg analyte/ g extracts)
1	Quinic acid	3.0	2439.9
2	Fumaric aid	3.9	44.1
3	Aconitic acid	4.0	55.2
4	Gallic acid	4.4	2.6
5	Protocatechuic acid	6.8	103.1
6	Gentisic acid	8.3	9.4
7	Chlorogenic acid	8.4	698.7
8	Protocatechuic aldehyde	8.5	8.6
9	4-OH Benzoic acid	10.5	13.6
10	Caffeic acid	12.1	43.3
11	Vanillin	13.9	14.8
12	p-Coumaric acid	17.8	14.2
13	Ferulic acid	18.8	7.7
14	Salicylic acid	21.8	43.3
15	Cynaroside	23.7	858.4
16	Miquelianin	24.1	79.5
17	Rutin	25.6	1.4
18	isoquercitrin	25.6	326.8
19	Hesperidin	25.8	1.9
20	Genistein	26.3	0.3
21	Cosmosiin	28.2	347.8
22	Astragalin	30.4	37.3
23	Quercetin	35.7	165.2
24	Naringenin	35.9	23.1
25	Luteolin	36.7	264.0
26	Kaempferol	37.9	8.4
27	Apigenin	38.2	60.0
28	Amentoflavone	39.7	0.1
29	Chrysin	40.5	0.4
30	Acacetin	40.7	0.8

Table 2. Effects of A.arabica extracts on rat blood glucose levels during the application period

Groups	Day	Glucose level ( mg/dL)
Control	1	89.33 ± 6.62^Aa
	7	92.67 ± 4.32^Aa
	14	83.83 ± 11.43^Aa
	21	85.33 ± 6.38^Aa
DM	1	462.67 ± 63.11^ABbc
	7	441.50 ± 68.49^ABbc
	14	423.83 ± 37.40^Bbc
	21	508.50 ± 57.27^Abc
A. arabica (400 mg/kg) extract	1	93.83 ± 6.79^Aa
	7	91.33 ± 17.61^ABa
	14	82.00 ± 10.24^Ba
	21	84.17 ± 7.07^Ba
DM + A. arabica (400 mg/kg) extract	1	480.60 ± 46.83^Abc
	7	354.75 ± 25.08^Bb
	14	358.00 ± 80.61^Bbc
	21	244.00 ± 83.48^Cb
DM + Glibenclamide (2 mg/kg)	1	281.40 ± 32.85^Ab
	7	127.50 ± 28.68^Bb
	14	167.67 ± 41.52^Bb
	21	141.83 ± 33.15^Bb

Two way ANOVA was used to determine whether the means were significantly different or not between the days and hours (p < 0.05). Following analysis of variance, the Tukey test was used to determine whether the means were significantly different or not between the days and groups (p < 0.05).

A, B, C: Capital letters represent the difference between days in the same group (p < 0.05).

a, b, c: Lowercase letters represent the same day difference between different groups (p < 0.05).

Table 3. Effects of A.arabica extract on biochemical parameters during the application period

Parameters	Groups
	Control	DM	A. arabica (400 mg/kg) extract	DM + A. arabica (400 mg/kg) extract	DM + Glibenclamide (2 mg/kg)
AST (U/L)	167.33 ± 17.92	1093.80 ± 255.28^a	101.17 ± 15.17^ab	205.67 ± 70.69^bc	100.00 ± 22.99^abd
ALT (U/L)	67.17 ± 11.29	887.00 ± 83.93^a	44.50 ± 8.62^ab	77.17 ± 18.80^bc	61.20 ± 13.03^bc
LDH (U/L)	912.50 ± 202.96	917.00 ± 126.13	851.17 ± 163.29	1111.17 ± 157.08^bc	928.60 ± 133.22
ALP (U/L)	313.67 ± 65.29	1222.20 ± 230.74^a	221.67 ± 51.68^ab	775.60 ± 153.34^abc	577.00 ± 139.68^abc
HbA1c (%)	3.79 ± 0.16	7.61 ± 0.48^a	3.51 ± 0.11^ab	5.38 ± 1.03^abc	5.16 ± 1.44^bc
CREA (mg/dL)	0.32 ± 0.03	0.44 ± 0.03^a	0.33 ± 0.03^b	0.37 ± 0.09	0.37 ± 0.05^ab
Urea (mg/dL)	50.00 ± 4.34	81.80 ± 4.15^a	42.83 ± 6.37^ab	52.00 ± 10.26^b	43.80 ± 7.66^b
GLU (mg/dL)	123.00 ± 20.83	755.20 ± 39.12^a	163.83 ± 22.34^ab	531.17 ± 82.22^abc	446.40 ± 110.79^abc
TRIG (mg/dL)	111.50 ± 18.06	120.80 ± 9.23	91.50 ± 11.50^ab	72.33 ± 17.50^abc	74.40 ± 17.87^ab
CHOL (mg/dL)	67.00 ± 11.24	75.80 ± 15.35	67.50 ± 15.40	66.67 ± 12.32	61.00 ± 8.75
HDL (mg/dL)	31.92 ± 6.67	33.44 ± 4.30	38.70 ± 5.86	42.57 ± 6.12^ab	35.10 ± 3.49^d
LDL (mg/dL)	40.25 ± 7.75	47.44 ± 6.45	30.37 ± 2.93^ab	25.90 ± 4.69^ab	29.10 ± 5.63^ab

Data are expressed as the mean ± SD. One way ANOVA was followed by the Tukey test, when appropriate (n=7). Differences between the groups were considered significant (p < 0.05).

^aDifference between the Control group and the other groups was significant (p < 0.05).

^bDifference between the DM group ant the other groups wassignificant (p < 0.05).

^cDifference between the A.arabica group and the other groups was significant (p < 0.05).

^dDifference between the DM + A. arabica group and the DM + Glibenclamide group was significant (p < 0.05).

AST: aspartate aminotransferase; ALT: alanine aminotransferase; LDH: lactate dehydrogenase; ALP: alkalane phosphatase; HbA1c:glycosylated haemoglobin; CREA: creatinine; GLU: glucose; TRIG: Triglyceride; CHOL: cholesterol; HDL: high density lipoprotein; LDL: low density lipoprotein.

Table 4: Effects of A. arabica extracts on antioxidan defence system parameters during the application period

		Groups
Tissue	Enzym	Control	DM	A. arabica (400 mg/kg) extract	DM + A. arabica (400 mg/kg) extract	DM + Glibenclamide (2 mg/kg)
Liver	SOD U/g Prt	56.92 ± 11.84	43.47 ± 12.47	60.38 ± 14.76	55.59 ± 13.50	46.14 ± 12.58
	GPx U/g Prt	18.34 ± 1.35	15.80 ± 4.43	22.53 ± 4.03^b	21.68 ± 4.40	17.00 ± 3.91
	CAT nmol/g Prt	8.15 ± 2.33	10.48 ± 2.24	17.02 ± 5.21^ab	10.63 ± 4.70^c	14.20 ± 2.06^ab
	GST nmol/g Prt	18.29 ± 3.99	18.05 ± 2.72	22.85 ± 6.57	17.28 ± 2.38	16.34 ± 1.76^c
Kidney	SOD U/g Prt	92.89 ± 15.57	83.98 ± 9.89	87.84 ± 10.53	80.82 ± 9.08	76.68 ± 16.59
	GPx U/g Prt	32.33 ± 8.68	27.33 ± 6.92	34.40 ± 6.51	26.98 ± 5.46	28.18 ± 3.10
	CAT nmol/g Prt	13.29 ± 3.32	14.27 ± 3.70	15.07 ± 3.91	12.13 ± 3.27	16.24 ± 2.88^d
	GST nmol/g Prt	1.70 ± 0.66	3.31 ± 0.96^a	1.85 ± 0.61^b	1.96 ± 0.56^b	2.29 ± 0.46^b

Data are expressed as the mean ± SD. One way ANOVA was followed by the Tukey test, when appropriate (n=7). Differences between the groups were considered significant (p < 0.05).

^aDifference between the Control group and the other groups was significant (p < 0.05).

^bDifference between the DM group ant the other groups wassignificant (p < 0.05).

^cDifference between the A.arabica group and the other groups was significant (p < 0.05).

^dDifference between the DM + A. arabica group and the DM + Glibenclamide group was significant (p < 0.05).

SOD: superoxide dismutase; GPx: glutathione peroxidase; CAT: catalase. GST: glutathione S-transferase; Prt: protein.

Exploring of phytochemical constituents of Achillea arabica Kotschy. ethanolic flower extract by LC-MS/MS and its possible antioxidant and antidiabetic effects in diabetic rats

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Version 2

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusion

Declarations

References

Tables

Supplementary Files

Status:

Version 2