Pistagremic acid, a glucosidase inhibitor from Pistacia integerrima

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FITOTE-02527; No of Pages 5 Fitoterapia xxx (2012) xxx–xxx

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Pistagremic acid, a glucosidase inhibitor from Pistacia integerrima Ghias Uddin a,⁎, Abdur Rauf a, Abdulaziz M. Al-Othman b, Simona Collina c, Muhammad Arfan a, Gowhar Ali d, Inamullah Khan d,⁎⁎ a b c d

Institute of Chemical Sciences, Centre for Phytomedicine & Medicinal Organic Chemistry, University of Peshawar, Peshawar 25120, Pakistan Department of Community Health Sciences, College of Applied Medical Science, King Saud University, Riyadh 11433, Saudi Arabia Department of Drug Sciences, University of Pavia, Viale Taramelli 12, Pavia 27100, Italy Department of Pharmacy, University of Peshawar, Peshawar 25120, Pakistan

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Article history: Received 30 July 2011 Accepted in revised form 16 September 2012 Available online xxxx Keywords: Pistagremic acid Docking α-Glucosidase Pistacia integerrima Anti-diabetic

a b s t r a c t Pistacia integerrima Stewart in traditionally used as folk remedy for various pathological conditions including diabetes. In order to identify the bioactive compound responsible for its folk use in diabetes, a phytochemical and biological study was conducted. Pistagremic acid (PA) was isolated from the dried galls extract of P. integerrima. Strong α-glucosidase inhibitory potential of PA was predicted using its molecular docking simulations against yeast α-glucosidase as a therapeutic target. Significant experimental α-glucosidase inhibitory activity of PA confirmed the computational predictions. PA showed potent enzyme inhibitory activity both against yeast (IC50: 89.12 ± 0.12 μM) and rat intestinal (IC50: 62.47 ± 0.09 μM) α-glucosidases. Interestingly, acarbose was found to be more than 12 times more potent an inhibitor against mammalian (rat intestinal) enzyme (having IC50 value 62.47 ± 0.09 μM), as compared to the microbial (yeast) enzyme (with IC50 value 780.21 μM). Molecular binding mode was explored via molecular docking simulations, which revealed hydrogen bonding interactions between PA and important amino acid residues (Asp60, Arg69 and Asp 70 (3.11 Å)), surrounding the catalytic site of the α-glucosidase. These interactions could be mainly responsible for their role in potent inhibitory activity of PA. PA has a strong potential to be further investigated as a new lead compound for better management of diabetes. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Pistacia chinensis var. integerrima J. L. Stewart ex Brandis belongs to family anacardiacea. P. integerrima is commonly known as kakar singhi. It is found in eastern Himalayan range from Indus to Kumaon [1] at a height of 12,000 to 8000 ft. P. integerrima is a medium sized deciduous tree that can attain a height of forty feet. P. integerrima is an important medicinal plant and is used as an anti-inflammatory, antidiabetic agent, blood purifier, a remedy for gastrointestinal disorders and as an

⁎ Corresponding author. ⁎⁎ Correspondence to: I. Khan, Department of Pharmacy, University of Peshawar 25120, Pakistan. Tel.: +92 91 9216750. E-mail addresses: [email protected], [email protected] (I. Khan).

expectorant. P. integerrima is also an important traditional herbal drug, which is used in oxidative stress and has potential to counter hyperuricemia [5]. In India it is used as a herbal remedy for diseases such as cough, asthma, fever, vomiting and diarrhea [2]. P. integerrima is reported to possess CNS depressant activity [7]. Galls of P. integerrima are used as a herbal drug for diarrhea in northern India [3] and for treatment of hepatitis and other liver disorders in Pakistan [4]. Ground galls are aromatic, astringent and expectorant and used in Indian traditional medicine for the treatment of asthma, phthisis and other disorders of the respiratory tract, dysentery, chronic bronchitis, hi-cough, vomiting of children, skin diseases, psoriasis, fever, and as an appetizer [6]. Furthermore its galls in combination with other herbal drugs are also used for the treatment of snake bites and scorpion stings [1]. Taking into account that the

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Please cite this article as: Uddin G, et al, Pistagremic acid, a glucosidase inhibitor from Pistacia integerrima, Fitoterapia (2012), http://dx.doi.org/10.1016/j.fitote.2012.09.017

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discovery of new bioactive natural products based on ethnopharmacological investigations is a well established methodology [8], in the current study, we have made an effort to identify the potential bioactive compound(s) responsible for the folk use of P. integerrima galls in diabetes. To the best of our knowledge, no scientific paper on P. integerrima galls as a source of new antidiabetic compounds is published. 2. Experimental 2.1. Plant material P. chinensis var. integerrima galls were collected from Khall, Toormang, Razagram area of district Dir, Khyber Pukhtun Khawa province of Pakistan in the month of February, 2010. The plant material was identified by plant taxonomist, Department of Botany, University of Peshawar, Pakistan. A voucher specimen no (RF-895) was deposited in the herbarium of the said department. 2.2. Extraction and isolation Dried and crushed galls of P. chinensis var. integerrima (5 kg) were subjected to cold extraction with MeOH. The MeOH extract (400 g) was suspended in water and successively partitioned with hexane, CHCl3, EtOAc and BuOH. The CHCl3 fraction (10 g) was subjected to Column chromatography on silica gel (Merck Silica gel 60 (0.063–0.200 mm), 5 × 60 cm). The column was first eluted with hexane-EtOAc (100:0→ 0:100) as a solvent system. A total of 13 fractions, RF-1 to RF-13 were obtained based on TLC profiles. Fraction RF-3 afforded a triterpene named Pistagremic acid, previously isolated and identified as reported earlier [9]. 2.2.1. Pharmacological screening α-Glucosidase inhibitory activity. α-Glucosidase (E.C.3.2.1.20) from Saccharomyces species was purchased from Sigma Aldrich. In the case of mammalian enzyme, rat-intestinal acetone powder (Sigma Aldrich) was used. Rat intestinal α-glucosidase was processed according to the method reported in the literature [10]. The acetone powder (100 mg) of rat intestine was dissolved in 2 ml of 50 mM cold PBS and was subjected to sonication for 20 min at 4 °C. After thorough mixing for 25 min, the resulting suspension was further subjected to centrifugation (12,000 g, 4 °C, 25 min) and then the final supernatant (FS) was used for enzyme inhibition assay. α-Glucosidase inhibitory activities of enzyme from yeast and rat intestine were measured spectrophotometrically through continuous monitoring of the nitrophenyl produced by the hydrolysis of the substrate p-nitrophenyl α-D-glucopyranoside (PNP-G) (0.7 mM) and 500 milliunits/ml of the enzymes used. The enzymatic reaction was performed at 37 °C for 30 min. The increment in absorption at 400 nm, due to the hydrolysis of PNP-G by α-glycosidase, was monitored continuously on a microplate spectrophotometer (Spectra Max, Molecular Devices, U.S.A.). A phosphate saline buffer (pH 6.9), containing 50 mM sodium phosphate and 100 mM NaCl was used. Acarbose (0.78 mM) was used as positive controls.

2.3. Cytotoxicity assay The in-vitro cytotoxicity assay was conducted using LCMK-2 monkey kidney epithelial cells and mice hepatocytes [11]. The test samples were incubated for 24 h, and finally the cell viability was determined employing the MTT assay. For the MTT assay, cells were maintained in RPMI 1640 medium (Gibco BRL) containing 10% FBS (Gibco BRL), 110 μg/ml penicillin sodium salt, 2 mg/ml sodium bicarbonate solution, and 100 μg/ml streptomycin sulfate. Initial seeding of the 7.1 × 10 3 LCMK-2 cells and 8.6 × 10 3 mice hepatocytes, was conducted in 96 well plates. The cells were treated with test samples at various concentrations as well as with vehicle (0.2% DMSO) and then incubated for 48 h followed by performing an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay (Sigma Chemical Co., St. Louis, MO, USA). 2.4. Molecular docking simulation FRED 2.1 [12] was used in this study to dock the OMEGA pre-generated multi-conformer library mentioned above. FRED 2.1 strategy is to exhaustively dock/score all possible positions of each ligand in the binding site. The exhaustive search is based on rigid rotations and translations of each conformer within the binding site defined by a box. FRED filtered the poses ensemble by rejecting the ones that clash with the protein (α-glucosidase) or that does not have enough contacts with the protein. The final poses can then be scored or re-scored using one or more scoring functions. In this study, the smooth shape-based Gaussian scoring function (shapegauss) was selected to evaluate the shape complementarily between each ligand and the binding pocket. Default FRED protocol was used except for the size of the box defining the binding sites. In an attempt to optimize the docking-scoring performance we performed exhaustive docking with shapegauss applying the “Optimization” mode. The “Optimization” mode involves a systematic solid body optimization of the top ranked poses from the exhaustive docking. 3 different boxes were explored for α-glucosidase. Three different simulations were carried out with an added value of 8 Å around the reference ligand. 3. Results and discussion In the current ethnopharmacological study, the major aim was to identify the potential bioactive compound that could be responsible for the folk medicinal use of P. integerrima in diabetes. α-Glucosidase inhibition is one of the important targets to control abnormal plasma levels of glucose in diabetic patients. α-Glucosidase hampers the release of free glucose as catalyzed by this enzyme inside the brush border of the intestinal lumen thus blocking the postprandial rise in blood glucose level. α-Glucosidase enzymes from both mammalian and non-mammalian sources are available for discovery of new α-glucosidase inhibitors. Potencies of the well established α-glucosidase inhibitors (including acarbose, which is clinically used in diabetes) vary from one source to another. After isolation and structure elucidation of Pistagremic acid, its molecular docking study was conducted in comparison to acarbose. In molecular docking, binding affinity is

Please cite this article as: Uddin G, et al, Pistagremic acid, a glucosidase inhibitor from Pistacia integerrima, Fitoterapia (2012), http://dx.doi.org/10.1016/j.fitote.2012.09.017

G. Uddin et al. / Fitoterapia xxx (2012) xxx–xxx

Fig. 1. Chemical structure of Pistagremic acid.

calculated and predicted based on binding energy (in kcal/mol) resulting from the favorable molecular interactions between the protein (yeast α-glucosidase) and ligands (PA or acarbose). Interestingly, results of molecular docking simulations revealed better binding energy for PA (−78.51 kcal/mol) as compared to the standard drug acarbose (−32.29 kcal/mol). In order to validate the computational predications based on the calculated binding affinity (in terms of binding energy), in-vitro enzyme inhibitory activity was performed. PA showed significant microbial (yeast) α-glucosidase (yeast) inhibitory activity (IC50: 89.12 ± 0.12 μM), as compared to activity shown by the standard drug acarbose (IC50: 780.21 μM). Interestingly, PA also showed significant mammalian (rat intestinal) α-glucosidase inhibitory activity (IC50: 62.47 ± 0.09 μM) in comparison to the standard drug acarbose (IC50: 38.92.47 ± 0.14). Experimental results supported the prediction of better binding affinity of PA.

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The difference between enzyme inhibitory activities of PA against microbial (yeast) and mammalian (rat) enzymes was comparatively lesser in contrast to the standard drug acarbose. According to the current results, acarbose showed an approximately 11 times lesser potency than PA against the microbial enzyme. Furthermore, results of the MTT assay showed no considerable cytotoxicity using LCMK-2 monkey kidney epithelial cells and mice hepatocytes. The preliminary safety profile at the cellular level indicated its potential for further studies regarding its antidiabetic potential. To explore the molecular basis of the inhibitory activity of the PA, the binding mode was analyzed by molecular docking simulations. At the molecular level, α-glucosidase is composed of amino acids arranged in a single polypeptide chain and chloride and calcium ions, which are bound to it as cofactors (Fig. 1). There are two important catalytic domains in the tertiary structure of α-glucosidase, which are Domains A and B. Domain C is loosely associated with the other two domains but its function is unknown. Domain A forms the β-barrel in which Asp197, Glu233, and Asp300 along with the chloride binding site, play important catalytic roles. Domain B is lying adjacent to domain A, which comprises the calcium binding site. A narrow pathway to both the chloride binding and calcium binding sites is surrounded by various amino acid residues like Asp70, Arg69 and Asp62. So for normal catalysis, the substrate must pass through this tunnel. Therefore, any compound that is capable of making favorable and strong binding interactions with the aforesaid amino acid residues can play the role of an α-glucosidase inhibitor with a potential antibiotic profile. In the microbial (yeast) enzyme, the binding cavity seems to be slightly narrow as compared to the binding pocket of the mammalian enzyme. Keeping this background in view, the difference in size of the binding cavities of both enzymes affected the inhibitory activity of the

Fig. 2. Binding mode of Pistagremic acid inside the active site of α-glucosidase. Hydrogen atoms (except polar ones) were omitted for clarity.

Please cite this article as: Uddin G, et al, Pistagremic acid, a glucosidase inhibitor from Pistacia integerrima, Fitoterapia (2012), http://dx.doi.org/10.1016/j.fitote.2012.09.017

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Fig. 3. Surface contacts of Pistagremic acid inside the catalytic site of α-glucosidase. Hydrophobic regions are represented as yellow colored regions while hydrophilic regions are represented as blue colored regions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

standard drug acarbose. Acarbose showed approximately more than 12 times lesser potency against the yeast enzyme in comparison to its potency against mammalian enzyme. On the other hand, the molecular shape and size of PA made it capable of easily penetrating the binding pockets of both enzymes as evidenced by slightly comparable inhibitory activities. The elongated skeleton of PA, along with the polar functional group rendered it an active compound. Interestingly, Pistagremic acid showed considerable molecular interactions with the amino acid residues surrounding the narrow tunnel leading to the catalytic sites of α-glucosidase (Figs. 2 and 3). Both hydrophobic and hydrophilic interactions were identified between the ligand and α-glucosidase. On one side the terminal hydroxyl group revealed hydrogen boding (at a distance of 2.98 Å) with Asp 62 lining the inner rim of the border surrounding the long and narrow catalytic pocket of α-glucosidase. On the other side the carboxylic group tightly held Pistagremic acid in contact with amino acid residues Arg 69 (3.04 Å) and Asp 70 (3.11 Å) in the terminal pocket via hydrogen bonding. In between these two terminal interactions the central tetracyclic frame matches

Fig. 4. Main skeleton of pentacyclic triterpenes (PTs).

the shape of the elongated and curved pocket through hydrophobic interactions with comparatively less polar amino acids (Fig. 3). This combination of both hydrophobic and hydrogen bonding interactions could be the main reason behind the α-glucosidase inhibitory profile of Pistagremic acid. Slightly similar experimental results of PA in the case of both enzymes clearly indicate the involvement of structurally similar amino acid residues surrounding the binding pocket of the mammalian enzyme. According to chemical structure, PA is a typical triterpene acid. Various pentacyclic triterpenes (PTs) (i.e. betulinic acid, bartogenic acid, oleanolic acid, dehydrotrametenolic acid, corosolic acid, ursolic acid etc.) are reported for their significant α-glucosidase inhibitory activity. Furthermore, the in-vivo antidiabetic activities of these compounds have been reported at molecular and cellular levels [13–17]. According to structure activity relationships (SAR) of PTs, various positions of the main triterpene core or nucleus (Fig. 4) are important. Hydroxy group at positions R1 and R2 (R1, R2_OH) results in optimum inhibitory effect of PTs [18], This effect might be due to the involvement of hydrogen bonding and electrostatic interactions between hydroxyl moieties and the binding pocket of α-glucosidase. Replacement of the hydroxyl group by the carbonyl group (R1, R2_C_O) leads to a decrease in potency [15]. Interestingly, the presence of the β-O-coumaryl group at position R2 (R2_β-O-coumaryl) slightly increases the potency of PTs [18]. Furthermore, the inhibitory effect of PTs decreases in the case of glycosides attached at positions R1 and R2 (R1, R2_-O-glycoside) [14,15,18]. The carboxylic acid group at position R3 (R3_COOH) plays a critical role in the SAR of PTs and is partly responsible for the optimum activity of PTs as α-glucosidase inhibitors. Replacing the hydrogen of the carboxylic acid (COOH) group with glycoside at the R3 position (R3_COO-Glycoside) decreases the inhibitory effects of PTs [15,18]. Based on the current experimental results, PA will attract researchers to explore its therapeutic potential against diabetes mellitus. Synthetic modifications guided by experimental and computational (including molecular docking, pharmacophore modeling and 3D QSAR) studies will provide a firm base for PA on the way to becoming the best lead

Please cite this article as: Uddin G, et al, Pistagremic acid, a glucosidase inhibitor from Pistacia integerrima, Fitoterapia (2012), http://dx.doi.org/10.1016/j.fitote.2012.09.017

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