Editor in Chief
Prof. Dr. Cornelia M. Keck
Philipps-Universität Marburg
Marburg, Germany

Bibliography
Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).
Journal Highlights
Abbreviation: J App Pharm
doi: http://dx.doi.org/10.21065/19204159
Frequency: Annual 
Current Volume: 9 (2017)
Next scheduled volume: December, 2018 (Volume 10)
Back volumes: 1-9
Starting year: 2009
Nature: Online 
Submission: Online  
Language: English

EVALUATION OF ANTIOXIDANT, CHOLINESTERASE INHIBITORY PROPERTIES, AND ANTIBACTERIAL POTENTIALS OF GLYCOMIS PENTAPHYLLA LEAF EXTRACT RELEVANT TO THE TREATMENT OF ALZHEIMER’S DISEASE
Md. Rezanur Rahman, Tania Islam, Hossain Md Faruquee*, Sharmin Akhter, and Md Anwarul Haque
1 Department of Biotechnology and Genetic Engineering, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh. 2 Department of Applied Nutrition and Food Technology, Faculty of Applied Science and Technology, Islamic University, Kushtia-7003, Bangladesh.
Keywords: Alzheimer’s disease; Glycomis pentaphylla; anticholinesterase; anti-oxidant; antibacterial; radical scavenging; lipid peroxidation.
Abstract

There is a tremendous unmet need to discover more potent and safe drugs for the treatment of Alzheimer’s disease (AD). Reduced cholinergic activity and oxidative stress have been recognized as a major contributing factor in the pathogenesis of AD. Therefore, inhibition of cholinesterase and oxidation are the two promising strategies in the development of a drug for AD. This study determined the anti-acetylcholinesterase (AChE) activity, anti-butyrylcholinesterase (BChE) activity, DPPH free radical scavenging and antioxidant properties of Glycomis pentaphylla (Rutaceae). The objective of this study is to measure G. pentaphylla anti-AChE, anti-BChE, DPPH free radical scavenging, lipid peroxidation inhibition, antibacterial potentials to find out the MIC (minimum inhibitory concentration) against different pathogenic bacteria. G. pentaphylla leaf extract (GPEx) is exploited in the presented research to estimate its anticholinesterase, antioxidant potentials, and antibacterial properties. The cholinesterase inhibitory properties was quantified by modified Ellman method, and antioxidant potentials were evaluated by the assay of radical scavenging, and inhibition of lipid peroxidation. The antibacterial activity and minimum inhibitory concentration (MIC) were determined using agar well diffusion method. The methanolic extract exhibited significant dual acetylcholinesterase (AChE) and butyryl cholinesterase (BChE) effect. The IC50 values of AChE and BChE were 325.1±0.91, and 42.14±3.31 µg/ml. Furthermore, the extract showed radical scavenging ability, and lipid peroxidation inhibitory effect. The IC50 values of the extract for DPPH and hydroxyl free radical scavenging, and lipid peroxidation inhibition assay were 95.6±0.68, 198.0±1.39, and 288.7±0.91 µg/ml, respectively. Phytochemical screening of the extract revealed the presence of significant total phenolics and flavonoids contents. Additionally, the extract showed good effect with the zone of inhibition ranging 12–16 mm in diameter against Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus. The tested sample reflects potential antioxidative and anticholinesterase inhibitory potentiality which may warrant its effectiveness in the treatment of AD along with good antibacterial properties.

Article Information

Identifiers and Pagination:
Year:2017
Volume:9
First Page:17
Last Page:30
Publisher Id:JAppPharm (2017 ). 9. 17-30
Article History:
Received:October 9, 2017
Accepted:December 3, 2017
Collection year:2017
First Published:December 17, 2017

1. INTRODUCTION

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by gradual memory loss, cognitive deficit and behavioural aberration, and the most predominant cause of dementia in the elderly having age more than 65 years[1–3]. It has been estimated that around 35 million people are now afflicted by the AD, and currently it is the fourth leading cause of death in the elderly person [3,4] and it is inferred that it may be reached to 65.7 million by 2030 [2].

Many factors are hypothesized to be involved in the pathophysiology of the AD such as oxidative stress, deposition of senile plaques, the formation of neurofibrillary tangles of the microtubule-associated protein tau, and profound deficits in cholinergic transmission is to be the hallmarks of neuropathophysiology of AD [2,5–8]. Understanding the etiology and pathogenesis of AD has been progressed, effective drugs remain limited yet. Reduced cholinergic activity and oxidative stress have been recognized as a major contributing factor in the pathogenesis of AD [2,9]. The deficit of cholinergic neurons and the associated decrease in levels of acetylcholine (ACh) has been found to correlate well with the cognitive impairment seen in AD patients [10].

The cognitive impairment observed in the AD is correlated with the loss of cholinergic neurons and its associated acetylcholine reduction [2,10]. The undue breakdown of acetylcholine by the increased activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) is implicated in the progression of the AD. Thus, inhibition of these two enzymes are the main strategy followed for AD and currently majority of drugs such as tacrine, donepezil, rivastigmine and galantamine are available for AD treatment are cholinesterase inhibitors (ChEI) despite it is only used for mild to moderate AD and could not reverse the disease progression simply they improve the symptoms [7,11,12]. In addition, these available drugs have adverse effects such as hepatotoxicity and gastrointestinal disturbances [13]. Therefore, in recent years much attention has been drawing to develop safe, cost-effective and active anti-cholinesterase from natural sources.

It has been reported that the reactive oxygen species (ROS) and other free radicals generated from activated neutrophils and macrophages during oxidative stress play an important role in the pathogenesis of AD [14]. A number of studies indicated that oxidative stress and Aß protein are linked each other because Aß induces oxidative stress in vivo and in vitro [15,16] and oxidative stress increases the production of Aß [17,18]. Therefore, antioxidant therapy has been suggested to be successful in improving cognitive function and behavioural deficits in patients with mild to moderate AD [19]. Natural products are the important source of antioxidants which can be beneficial for AD therapy.

The emergence of multi-drug resistance pathogenic bacterial strains is a major medical concern globally and these antibiotics have an adverse reaction such as hypersensitivity, immune-suppression and allergic reactions lead to a search for new and active antibacterial compounds to combat infections against these pathogenic bacteria [19–21]. Therefore, plants or natural products would be an enormous resource to discover new compounds or drug-like molecule which will be helpful for antibiotic discovery and development.

The G. pentaphylla (Orange berry) is an evergreen shrub and it grows usually 4m tall and widely distributed throughout the world including various parts of Bangladesh [22]. It is widely distributed in Bangladesh, India Malaysia and Southern China to the Philippine Islands but this plant is native to eastern, southern, and southeastern Asia and north-eastern Australia [23]. Traditionally, this plant is used to reduce fever, liver complications, various intestinal parasites [23], and toothache and bleeding [22], all forms of cancers, hepatic disorder, rheumatic fever [24]. This traditional use should be supported by the scientific evaluations. In addition, it is reported that G. pentaphylla possessed anti-hepatocellular carcinoma activity, hepatoprotective activity, and antibacterial activity [23].

Therefore, the present study was carried out with an aim to explore the cholinesterase (AChE and BChE) inhibitory activity and antioxidant properties of G. pentaphylla as well as antibacterial activity with particular emphasis in order to treat AD.

2. MATERIALS AND METHODS

2.1 Plant material

The plant leaves of G. pentaphylla were collected from the Islamic University premises, Kushtia, Bangladesh in February 2017 by the authors and collected plant sample was identified by an expert taxonomist. A voucher specimen has been deposited at the Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh. The freshly collected plants were cleaned to remove dust.

2.2 Extraction of plant material

The leaves of G. pentaphylla plant were air dried and ground. The powdered plant (500 g) was subjected to extraction with methanol at room temperature for 7 days with sporadic shaking. The extractive solution was filtered and concentrated under vacuum in a rotary evaporator at 45 °C to yield 17 g of the crude extract (GPEx).

2.3 Enzyme sources

Adult Long-Evans rat (125-150g) brain homogenate was used as the source of acetylcholinesterase enzyme and human blood was used as the source of butyrylcholinesterase enzyme. The rats were purchased from the animal research branch of the International Centre for Diarrhoeal Disease and Research, Bangladesh (ICDDR, B). The approval for this study was taken from the Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia, Bangladesh.

2.4 Chemicals

The chemicals used in this study were analytical grade. DPPH (2, 2'-diphenyl-1-picrylhydrazyl), aluminum chloride, potassium ferricyanide, ascorbic acid, Folinciocalteu reagent, TrisHCl and triton X-100 were obtained from Sigma-Aldrich (India). Gallic acid was obtained from Wako Pure Chemical Company Ltd., Japan. 5,5'-dithio-bis-(2-nitro) benzoic acid (DTNB), acetylthiocholine iodide (ATCI), S-butyrylthiocholine, galantamine, and donepezil were obtained from Sigma-Aldrich, Japan. Unless otherwise specified, all other chemicals were of analytical grade.

2.5 Determination of total phenolic content

The concentration of total phenolics of GPEx was determined by the method Folin-Ciocalteu [25]. Briefly, a 0.5 ml of plant extract was mixed to 2.5 ml of Folin-Ciocalteu reagent which was diluted 10 times with water and 2.5 ml of sodium carbonate (7.5%) solution and the reaction mixture was allowed to incubate for 20 min at room temperature (25°C) to complete the reaction thereafter the absorbance of the reaction mixture was measured at 760 nm by spectrophotometer. To quantify the total phenolics, the Gallic acid standard curve was exploited and the results were represented as mg of Gallic acid equivalent (GAE)/g of dried extract.

2.6 Determination of total flavonoid content

The aluminium chloride colourimetric method with slight modification was employed to determine the total flavonoid content of GPEx [26]. In short, 1.0 ml of each concentration of crude extract of GPEx was added with 3.0 ml of methanol, 0.2 ml of 10% AlCl3, 0.2 ml of 1 M potassium acetate and 5.6 ml of distilled water was added to the resulting mixture which was then incubated at room temperature for 30 min to complete the reaction. At 420 nm, the absorbance was recorded of the reaction mixture by spectrophotometer. Gallic acid was used as standard and the results were expressed as milligrams of gallic acid equivalents per gram of dried fraction [2].

2.7 Determination of DPPH radical scavenging activity

DPPH assay was used to assess the free radical scavenging activity of the extract according to the method described by Choi et al. with slight modifications [27]. Briefly, 3 ml of a methanol solution of DPPH and 2 ml of a methanol solution of plant extract or reference standard catechin at various concentrations was mixed into the test tube and allowed to incubate at room temperature (250C) for 30 min in dark place to complete the reaction. The absorbance of the solution was measured out by spectrophotometer at 517 nm. DPPH free radical scavenging ability (%) was calculated by using the formula:

 [(An absorbance of control - An absorbance of sample) /An absorbance of control] × 100

 

2.8 Determination of hydroxyl radical scavenging activity

The hydroxyl free radical scavenging activity of the methanol crude extract of GPEx was determined by the method as described by Elizabeth and Rao with a slight modification [28].  At various concentrations the plant extract or reference compound was mixed with a reaction mixture contained, in a final volume of 1 ml: 2-deoxy-2-ribose (2.8 mM); KH2PO4- KOH buffer (20 mM, pH 7.4); FeCl3 (100 µM); EDTA (100 µM); H2O2 (1.0 mM); and catechin (100 µM). The mixture was then incubated for 1 h at 37 °C and 0.5 ml of the reaction mixture was heated at 90 °C for 15 min after addition of 1 ml of 2.8 % TCA and 1 ml of 1 % aqueous TBA to develop the colour. After cooling, the absorbance was measured at 532 nm against an appropriate blank solution [2]. Hydroxyl radical scavenging ability (%) was calculated by using the formula:

 [(An absorbance of control - An absorbance of sample) /An absorbance of control] × 100

2.9 Determination of cholinesterase (ChE) inhibitory activities

The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory assay were performed according to the colourimetric Ellman method with slight modification [2,29]. For the AChE enzyme source, rat brain was homogenized in a homogenizer with 5 volumes of a homogenization buffer [10 mM TrisHCl (pH 7.2), which contained 1 M NaCl, 50 mM MgCl2 and 1 % Triton X-100], and centrifuged at 10,000 g for 30 min. The resulting supernatant was used as an enzyme source. For the BChE enzyme source, human blood from anonymous healthy men subject was provided by the Islamic University Medical Center, Kushtia, Bangladesh and collected in EDTA treated (1 mg/ml) glass tubes. The tubes were centrifuged at 2000 g for 10 min to eliminate the red blood cells. The resulting plasma (supernatant) was then recuperated, diluted (1/200) with 50mM phosphate buffer (pH 7.4) and was used immediately for studying butyrylcholinesterase activity. The rates of hydrolysis by acetylcholinesterase were monitored spectrophotometrically. Each extract or standard (500 µl) was mixed with an enzyme solution (200 µl) and incubated at 37 °C for 15 min. Absorbance at 405 nm was read immediately after adding an Ellmans reaction mixture [3.5 ml; 0.5 mM acetylthiocholine iodide (ATCI), 1 mM DTNB in a 50 mM sodium phosphate buffer (pH 8.0)] with the incubated reaction mixture. The hydrolysis of ATCI was monitored by the formation of yellow 5-thio-2-nitrobenzoate anion as a result of the reaction of DTNB with thiocholine. Reading was repeated for 10 min at 2 min intervals to verify that the reaction occurred linearly. The blank reaction was measured by substituting saline for the enzyme. Percentage of inhibition of AChE/BChE was determined by comparison of rates of reaction of samples relative to blank sample using the formula (E-S)/E× 100, where E is the activity of enzyme without test sample and S is the activity of the enzyme with the test sample. The experiments were done in triplicates. Donepezil (anticholinesterase drug) was used as a standard. Assessment of BChE inhibition was performed as described above except that the enzyme solution was 50 µl and acetylthiocholine iodide was replaced by butyrylthiocholine iodide. Galantamine was used as positive control. The percentage inhibition of butyrylcholinesterase activity was calculated using the same formula as mentioned above for acetylcholinesterase activity.

2.9 Antibacterial Susceptibility Test and Minimum Inhibitory Concentration (MIC) Determination Test

To assess the antibacterial potentials of the crude methanol extract of GPEx, in vitro antibacterial susceptibility test was performed by agar well diffusion technique [30]. Human pathogenic bacterial culture (0.1%) was swabbed uniformly over the surface of solid nutrient agar media using sterile cotton swab under asceptic condition. After complete drying of the media, 4 mm small bore is filled with the crude extracts at the different concentration (0.5-10 mg/ml) with appropriate control, the inoculated plates were allowed for 24 h incubation. Finally, inhibition zones formed around the well were measured using a calibrated scale within millimeter. All the tests for antibacterial activity were carried out in triplicate.

The methanol crude GPEx extracts were subjected to serial dilution technique (0.5-10mg/ml) to prepare various concentrations for the determination of MIC. The various diluted extracts were applied to wells posed in the nutrient agar media containing inoculums and incubated for 24 h. After overnight incubation, the growth of the test organisms was observed to determine the MIC.

2.10 Statistical analysis

The data were analyzed by one-way ANOVA followed by Tukeys test to estimate significant differences between the test and control groups, and IC50 was determined with GraphPad Prism Data Editor for Windows, Version 5.0 (GraphPad Software Inc., San Diego, CA). Values were expressed as a mean ± Standard error of the mean (± SEM). *p<0.05 were considered as statistically significant.

3. RESULTS

3.1 Antioxidant activity

3.1.1 Total phenolics and flavonoid contents of G. pentaphylla

This research aims to evaluate the antioxidative and anticholinergic effects of the GPEx. For assessing the antioxidative effects, total phenolic and flavonoid contents of GPEx, DPPH radical scavenging capacity, hydroxyl radical scavenging effect and lipid peroxidation inhibitory effects were screened. Total phenolic and flavonoid contents were calculated using the standard curve for Gallic acid. Phenolic compounds have been reported to show anti-Aß (amyloid ß protein) aggregation effects in AD indicating the pivotal role of phenolics in preventing the Alzheimer’s progression [2,10,31]. Flavonoids are known to serve as potential antioxidants for their radical scavenging, an ion chelating and lipid peroxidation inhibiting properties [10,32] preventing or slowing the progression of the AD by interfering the generation of amyloid-ß peptides and its polymerization into neurotoxic oligomeric aggregates thereby reducing aggregation of tau proteins [31]. Higher content of total phenolic (28.23 mg of GAE/g of dried extract) and flavonoid (181.76 mg of GAE/g of dried extract) in TGEx, therefore, may carry an immense importance to evaluate its role in controlling AD.

3.1.2 DPPH radical scavenging activity of G. pentaphylla

Radical scavenging activity is very important to prevent the deleterious role of free radicals in AD  [2,33]. DPPH is a stable free radical contains an odd electron and DPPH antioxidant assay is based on the ability of DPPH to decolourize in the presence of antioxidants. Decolorization of DPPH after accepting an electron donated by an antioxidant compound is quantitatively measured by the change in absorbance and % of scavenging activity. DPPH radical scavenging assay for G. pentaphylla is shown in Figure 1(A). The results of this study showed very significant DPPH scavenging activity with an IC50 value of 95.6±0.68 µg/ml and found to be comparable with that of the standard antioxidative agent ascorbic acid showing IC50 value 1.97±0.27µg/ml.

Figure 1: Radical scavenging activity of G. pentaphylla extract compared with the standard. (A) Percent of DPPH radical scavenging by different concentrations of the extract and reference standard ascorbic acid as assessed by spectrophotometric method using DPPH free radicals. (B) Percent of hydroxyl radical scavenging by different concentrations of the extract and reference standard (+)-catechin as assessed by the Fenton-reaction initiated deoxyribose degradation method.Notes: Results represent mean ± SEM (n = 3). *p < 0.05 significantly different as compared with control.

Figure 2: Percentage of lipid peroxidation inhibition at different concentrations of G. pentaphylla extract and the reference standard (+)-catechin.

Notes: Results represent mean ± SEM (n = 3). *p < 0.05 significantly different as compared with control.

Figure 3: Percentage of inhibition of acetylcholinesterase (A) and  butyrylcholinesterase (B) activity at different concentrations of G. pentaphylla extract and the reference standard donepezil and galantamine, respectively.

Notes: Results represent mean ± SEM (n = 3). *p < 0.05 significantly different as compared with control.


Table1. IC50 value of the test extract and standards obtained in the radical scavenging and enzyme inhibitory activity assays

Sample

IC50 (µg/ml)

DPPH radical

scavenging

Hydroxyl radical

scavenging

Inhibition

of lipid

peroxidation

AChE

BChE

Ascorbic Acid

1.97±0.27

-

-

-

-

(+)-Catechin

-

25.44±0.63

19.50±0.56

-

-

Donepezil

-

-

-

85.13±0.57

-

Galantamine

-

-

-

-

11.37±0.56

Extract

95.6±0.68

198.0±1.39

288.7±0.91

325.1±0.91

42.14±3.31

         Note: Values are expressed as a mean ± SEM (n = 3).

Table2. Minimum Inhibitory Concentration (MIC) (mg/mL) of methanolic extract of G. pentaphylla L. leaves.

Conc.

(mg/ml)

Amount in µg

St (DIZ)

Pa (DIZ)

Sa (DIZ)

Ec (DIZ)

Ec14 (DIZ)

.5

50

NG

SG

NG

NG

NG

1

100

NG

SG

NG

NG

NG

2

200

NG

12.01±0.06

NG

NG

NG

3

300

12.1±0.15

12.06±0.06

SG

NG

NG

4

400

12.1±0.20

16.19±0.36

12.1±0.05

NG

NG

5

500

12±0.16

16.20±0.41

12.18±0.09

NG

NG

6

600

14.10±0.06

12±0.17

14.03±0.09

NG

NG

7

700

14.1±0.15

14.05±0.10

12.09±0.04

NG

NG

8

800

11.08±0.04

SG

12.10±0.05

NG

NG

9

900

12.04±0.04

12.12±0.06

14.04±0.04

NG

NG

10

1000

14.1±0.05

12.01±0.08

15.13±0.06

NG

NG

Control

-

NG

NG

NG

NG

NG

Note: St:Salmonella typhi; Pa: Pseudomonas aeruginosa; Sa: Staphylococcus aureus; Ec: Escherichia coli; Ec14: Escherichia coli 14; SG: Slight growth; NG: No growth. Conc.: Concentration; DIZ: Diameter of Zone of Inhibition in mm. Note: Values are expressed as a mean ± SEM (n = 3).

3.1.3 Hydroxyl radical scavenging activity

Hydroxyl radicals are the major reactive oxygen species that severely damage the neurons in AD [34]. In the hydroxyl radical scavenging activity, the ability of GPEx to remove the formed hydroxyl radical in solution was evaluated quantitatively by Fenton-reaction initiated deoxyribose degradation assay and the result has been shown in Figure 1(B).

The test sample significantly scavenged the hydroxyl radicals generated in the reaction in a dose-dependent manner with an IC50 value of    198.0±1.39 µg/ml which was significant compared to that (25.44±0.63 µg/ml) of the reference standard (+)-catechin. Removal of hydroxyl radicals from the reaction mixture decolourized the pink chromogen which was quantitatively measured by the change in absorbance at 532 nm. The results demonstrate very promising hydroxyl radical scavenging potentials for therapeutic uses. because the cut-off value for antioxidative compounds is 1000 µg/ml