Anticholinergic, antidiabetic and antioxidant activities of Anatolian pennyroyal (Mentha pulegium)-analysis of its polyphenol contents by LC-MS/MS

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Highlights

  • Polyphenol contents of Mentha pulegium were analyzed by LC-MS/MS.

  • Kaempferol-3-O-rutinoside was found the most abundan polyphnol in Mentha pulegium extracts.

  • Anticholinergic and antidiabetic of Mentha pulegium was determined.

  • Antioxidant activity of Mentha pulegium was performed using several bioassay.

Abstract

Methanol and water extracts of pennyroyal (Mentha pulegium) were evaluated for their antioxidant profiles by eight distinguished bioanalytical methods and for their inhibitory effects against enzymes linked to different diseases, namely acetylcholinesterase, butyrylcholinesterase, α-glycosidase and α-amylase. Additionally, the antioxidant properties of both extracts were determined and their polyphenolic compositions were evaluated by LC-MS/MS. For estimation of the antioxidant capacities of methanolic extract of pennyroyal (MEP) and water extract of pennyroyal (WEP); 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid radical (ABTS•+), 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH), and N,N-dimethyl-p-phenylenediamine (DMPD•+) scavenging activities, Fe3+-2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), Fe3+, and Cu2+ reducing assays were studied. The IC50 values of the MEP and WEP indicated that they were potent effective DPPH· (16.92 and 18.52 μg/mL), ABTS•+ (7.92 and 9.37 μg/mL) and DMPD•+ (36.02 and 38.94 μg/mL) scavengers, as well as acetylcholinesterase (AChE) (40.76 and 60 μg/mL), butyrylcholinesterase (BChE) (49.51 and 63.03 μg/mL), α-glycosidase (20.38 and 21.65 μg/mL) and α-amylase (23.11 and 36.47 μg/mL) inhibitors. Plant materials are potential sources for novel products in food and pharmaceutical applications. Also, biologically active compounds from plants have proven to be desirable in cosmetics, foods, and pharmaceuticals. This study clearly showed that both MEP and WEP had a broad screening of active compounds and phytochemicals. As a result of this abundant active ingredient, both extracts possessed antioxidant, anticholinergic and anti-diabetes properties.

Introduction

Plants are potential sources for the improvement of novel products in cosmetics, foods, and pharmaceuticals (Elmastas et al., 2018; Rodrigues et al., 2018). Recently, biologically active compounds of plants have proven to be desirable post natural agents. Medicinal plants have a crucial role in basic healthcare and the cosmetic industry in many developing and developed countries. It was recently reported that approximately 400.000 plant taxa have been identified to date, and approximately 30.000 have been documented and used in traditional medicine (Dalar et al., 2018). It was estimated that Turkey flora includes approximately 11.000 taxa, and close to 1300 plants were used in traditional Anatolian folk medicine (Coskun and Gençler-Özkan, 2005). Turkey possesses rich plant biodiversity for use in foods, cosmetics, and the pharmaceutical industry (Mahomoodall et al., 2018). They contain significant amounts of bioactive compounds and have organoleptic, as well as biochemical properties. They have different usage in the food, pharmaceutical and cosmetic industries (Villaverde et al., 2016). Additionally, secondary metabolites of plants have had extensive use in traditional medicine due to their well-established potential in terms of pharmaceutical and biological properties. Secondary metabolites have preventive roles in plants and demonstrate different biological and pharmaceutical properties including anti-inflammatory, neuroprotective, antioxidant, and antidiabetic activities (Zengin et al., 2018a). Biologically active secondary metabolites have been isolated and used for the treatment of some health disorders. Recently, there have been growing demands and many studies for the validation, production and utilization of herbal medicines to treat many diseases in Anatolia (Mahomoodall et al., 2018).

The Labiatae family is commonly used for different aims worldwide (Ishtiaq et al., 2014). Mentha is a genus of the Labiatae family, which contains approximately 220 genera and 3300 species. This genus includes more than twenty species all over the world. This plant family had a large spectrum of polyphenolic compounds, which have well-known different biological properties (Erhan et al., 2012). They are commonly used in traditional and alternative medicines. Additionally, these plants are generally used as powders, decoctions and infusions. They also may have anti-inflammatory, antiemetic, carminative, diaphoretic, analgesic, antispasmodic, antitussive and stimulant effects (Kamkar et al., 2010). Also, they have curative effects against some gastrointestinal diseases such as dyspepsia, vomiting, stomachache, nausea, infections, diarrhea and bloating (Khonche et al., 2017). These health-healing properties are particularly due to their rich secondary metabolite contents including phenolics, essential oils and flavonoids (Kasrati et al., 2015; Kumar et al., 2018; Mahboubi and Haghi, 2008).

Pennyroyal (Mentha pulegium) is a perennial, aromatic, popular and herbaceous plant, which can reach up to half a meter in height. It was extensively used as a preservative ingredient in the food industry and as a natural flavor for folk medicine (Rodrigues et al., 2013). It has been used as an infusion, herbal tea or powder for the treatment of some diseases such as bronchitis, whooping cough, the common cold, sore throat and digestive disorders (Ahmed et al., 2018).

Reactive oxygen species (ROS) are described as short-lived molecules, ions or radicals. Their half-lives differ from nanoseconds to hours. They occur in many chemical reactions and during some biological processes such as the electron transport chain. They can be countable as hydrogen peroxide (H2O2), hydroxyl radicals (·OH), peroxynitrite (ONOO), hypochlorite ions (ClO), superoxide anion radicals (O2·−), singlet oxygen (1O2), and hydroperoxyl radicals ((HOO·) (Gulcin, 2006a, 2006b, 2012). Increased ROS levels can provoke oxidative stress and lead to hazardous cellular and molecular damage. As a result of this, various types of diseases such as neurological disorders, lung diseases, cardiovascular diseases, cancer and inflammation can occur in living systems (Gulcin et al., 2005; 2006a). Recently, oxidative stress and ROS have been accepted as important environmental risks for different types of chronic disorders such as cancer, immunodeficiency syndrome, age-related pathologies, cardiovascular diseases, arteriosclerosis, diabetes, and obesity (Ak and Gulcin, 2008; Gulcin, 2009). ROS occur in living organisms during normal cellular metabolism and can be harmful to decisive biomolecules including proteins, nucleic acids, lipids and carbohydrates (Elmastas et al., 2006; Gulcin et al., 2006b; Taslimi and Gulcin, 2018). Additionally, ROS, which have been implicated in many diseases, are produced in all living organisms as a primary immune defense (Gulcin, 2010). The antioxidant defense system includes antioxidant components and antioxidant enzymes. They can repair or remove damaged biomolecules including lipids, carbohydrates, nucleic acids and proteins in living organisms. Antioxidants delay, prevent and inhibit the oxidation of these biomolecules. They include phenols and polyphenols, which are very effective agents that reduce or neutralize the undesired and hazardous effects of ROS (Gulcin et al., 2009). In terms of pharmaceutical products, they can easily scavenge ROS and reduce the lipid autoxidation of foods and pharmaceuticals during production and storage processes (Bursal and Gulcin, 2011; Gulcin and Beydemir 2013). In terms of food, antioxidants are described as molecules that prevent food products from oxidation in low quantities or concentrations. Additionally, they easily delay or inhibit the oxidation of biological substrates (Halliwell et al., 1995; Koksal and Gulcin, 2008). Therefore, attention has recently turned to research for effective antioxidants including phenolic compounds from natural and accessible resources (Gülçin et al., 2012; Koksal et al., 2009). Plants include many biological active phytochemicals such as phenols and polyphenols that possess structural features that have antioxidant activities (Cakmakçı et al., 2015; Lu et al., 2019). According to nutritionists and toxicologists, the undesired side effects of synthetic antioxidants including BHT and BHA have also been documented and are well known. However, the usage of synthetic antioxidants has been limited due to their carcinogenic and toxic effects in living organisms (Gulcin et al., 2010; Koksal and Gulcin, 2008). Hence, plants have been intensely searched for their possible antioxidant abilities. For this reason, many works have been performed for pure molecules and crude plant extracts (Wang et al., 2015). Thus, there are growing demands for safer natural antioxidants from plant origins for pharmaceutical and food applications (Gülçin et al., 2010b). Phenolics are active secondary metabolites that scavenge ROS and eliminate oxidative damage (Gulcin et al., 2012). Their biological effects make them crucial products for treatment of some degenerative disorders including diabetes, cancer, arteriosclerosis, hypercholesterolemia and cardiovascular diseases (Sehitoglu et al., 2015).

Alzheimer's disease (AD) generally affects the memory and behavior of elderly people worldwide. This neurological disease clinically includes the growing degeneration of brain tissue, which is effected by an acetylcholine (ACh) deficiency (Yiğit et al., 2018, 2019). The acetylcholinesterase enzyme (AChE), as a component in the nervous systems, converts ACh to choline (Ch) and acetate (Biçer et al., 2019; Zengin et al., 2018b). It was reported that the reduced levels of Ach in the hippocampus and cortex had great biochemical changes in patients with AD (Huseynova et al., 2018). Natural substances such as AChE inhibitors (AChEIs) were commonly used in clinical trials, especially for AD treatment. Phenolics have also been identified as AChEIs and provide pioneering molecules for AD treatment (Rezai et al., 2018; Wilkinson, 2007).

Diabetes mellitus (DM) is a metabolic disease that is hallmarked by abnormal levels of glucose in the blood, as well as some disorders including neuropathy, retinopathy, cardiovascular diseases, atherosclerosis, and neural damage (Rendra et al., 2019). It was reported that oxidative stress and ROS are major mediators of DM. In the case of hyperglycemia, the excessive glucose loading triggers ROS generation in mitochondria. In this case, the mitochondrial functions are impaired (Giacco, 2011; Pitocco et al., 2013). Digestive enzymes hydrolyze polysaccharides into monosaccharide units. Thus, digestive enzyme inhibition is an important therapeutic route for the treatment of DM (Gülçin et al., 2018). α-Amylase and α-glycosidase enzymes were released from small intestine cells. Both enzymes hydrolyze oligosaccharides and polysaccharides to monosaccharide units such as glucose (Koçyiğit et al., 2018). In humans, digestive enzyme inhibitors (DEIs) are very important for controlling DM and hyperglycemia (Gulcin et al., 2018). DEIs can decrease the absorption of carbohydrates and suppress postprandial glucose levels, hyperglycemia and Type-2 diabetes mellitus (T2DM). Therefore, DEIs compete with the oligosaccharides to bind to the active site of digestive enzymes. Thus, they efficiently reduce the postprandial glucose in T2DM (Demir et al., 2018; Taslimi et al., 2018).

Many studies have been conducted with evaluating antioxidant activity of various samples of research interest using by different methods in food and human health. Methods based on inhibited autoxidation are the most suited for termination-enhancing antioxidants and, for chain-breaking antioxidants while different specific studies are needed for preventive antioxidants (Aras et al., 2019; Turkan et al., 2019). For this purpose, the most commonly methods used in vitro determination of antioxidant capacity of food constituents are reviewed for both pennyroyal (Mentha pulegium) extracts.

In this study, the cupric (Cu2+) and ferric (Fe3+) ion reducing abilities, 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid radical (ABTS•+), 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH), and N,N-dimethyl-p-phenylenediamine (DMPD•+) scavenging activities of the MEP and WEP were investigated. These biochemical methods are used for the determination of the antioxidant profiles of foods, and tested materials. This study also aimed at identifying the polyphenols in both extracts by LC-MS/MS. Furthermore, another significant aim of this study is to demonstrate the inhibitory abilities of the MEP and WEP against the BChE, AChE, α-amylase and α-glycosidase enzymes, which are linked to global and common health diseases.

Section snippets

Chemicals

Neocuproine, DMPD, ABTS, BHA, DPPH·, BHT, α-tocopherol, curcumin and trolox were purchased from Sigma-Aldrich (Germany). The other compounds used were of analytical grade and were obtained from either Merck or Sigma-Aldrich.

Plant material

Mentha pulegium was collected in the vicinity of the Doşemealtı-Ayanlar (Antalya) district in open forestry and macchie areas at 800–1100 m altitude in June 2017 by Ömer Kılıç. The taxonomic description of Mentha pulegium was made by taxonomist Dr. Ömer Kılıç from Adiyaman

Results and discussion

Most medicinal and aromatic plants contain chemicals with antioxidant properties. Among them, phenols are one of the major chemical groups and act as antioxidants, metal chelators and radical scavengers (Benabdallah et al., 2016). Pennyroyal demonstrated a large spectrum of biological activities including antibacterial and antifungal (Amalich et al., 2016), insecticidal (Lamiri et al., 2001), anti-inflammatory and antioxidant (Kogiannou et al., 2013), anti-genotoxic (Romero-Jiménez et al., 2005

Conclusion

As a conclusion, the evaluation of the bioactivity and phytochemical screening of pennyroyal is important. The MEP and WEP, as natural sources of phenolic compounds, were examined for their biological activities including antioxidant activities and some metabolic inhibitory properties. The MEP and WEP were found as having potent antioxidant properties in several bioanalytical assays including Fe3+, Cu2+ and FRAP reducing abilities, as well as DPPH·, DMPD•+ and ABTS•+ radical scavenging

Author statement

İlhami Gülçin: Data curation, Writing-Original draft preparation, Ahmet C. Gören: Conceptualization, Methodology, Visualization, Parham Taslimi: Visualization, Investigation. Saleh H. Alwasel: Writing - Review & Editing, Formal analysis. Omer Kılıc: Resources, Investigation. Ercan Bursal: Data Curation, Methodology.

Declaration of competing interest

The authors report no conflicts of interest.

Acknowledgements

S.H.A would like to extend his sincere appreciation to the Researchers Supporting Project (RSP-2019/59), King Saud University, Saudi Arabia.

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