Study the effect of biosynthesized gold nanoparticles on the enzymatic activity of alpha-Amylase

 

Rusul Y. Hameed1, Israa Nathir2, Waleed K. Abdulsahib3,

Haider Abdulkareem Almashhadani4,5

1Al Hikma College University, Baghdad, Iraq.

2Department of Pharmacy, Al-Rasheed University College, Baghdad.Iraq.

3Pharmacology and Toxicology Department, College of Pharmacy, Al- Farahidi University, Baghdad, Iraq.

4Department of Dentistry, Al-Rasheed University College, Baghdad, Iraq.

5Department of Medical Laboratory Techniques, Dijlah University College, Baghdad, 10021, Iraq.

*Corresponding Author E-mail: H_R200690@yahoo.com

 

ABSTRACT:

In this paper, investigates the biosynthesis of gold nanoparticles (AuNPs) by biochemical method using Myrtus communis leaves extract as reducing agent and Chloroauric acid (HAuCl4) as precursors. X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and FTIR were used in addition to UV-visible spectroscopy (UV) in order to characterize the AuNPs. The biosynthesized AuNPs exhibited inhibitory effects on alpha amylase and alkaline phosphatase in sera of patient with type 2 Diabetes Miletus and the sera of healthy control subjects; the inhibition percentage with alpha amylase was 72 % and 45 % for patient and control group respectively. Oral consent obtained from the most of patients and healthy subjects before them being under study.  Biological activities were investigated against some bacteria species to exploit AuNPs potential. Kinetic studies of alpha amylase exanimated. The goal of this study is to synthesized gold nanoparticles using simple, economical and environmentally green method. This stage is more suited to large-scale manufacturing since it is speedy and removes the complex steps in other bio-based methods.

 

KEYWORDS: Gold nanoparticles, Myrtus communis leaves extract, Biological synthesis, Alpha amylase enzyme, and biological activity.

 

 


INTRODUCTION:

The high surface area that provides distinct features and possible applications compared to their bulk counterparts has sparked a lot of scientific interest in nanoparticle creation and characterization1-3. Gold, gold, copper, and platinum nanoparticles created via physical, chemical, and biological methods. Due to their apparent simplicity, low cost of implementation and environmental friendliness, biological treatments is increasingly becoming a viable alternative to standard methods. Several studies have observed the development of gold nanoparticles with bacteria from biological sources4, fungi 5and plants3,6,7.

 

Due to the extreme high rate of plant extract response and the lack of specific conditions necessary, Plant medium green chemistry has developed as one of modern nano-biotechnology research's active fields.8, 9.

 

Gold nanoparticles have been employed in a wide range of applications, including electronics, biological equipment, and the production of several biological and pharmaceutical products10. This is most likely owing to the nanoparticles' stability, which is ideal for medicinal applications11, 12.

 

Antimicrobial agents such as gold are commonly employed; gold ions can destroy a wide spectrum of microorganisms by modifying the structure and activities of the cell membrane11,13. Microorganisms were destroyed by nano-gold particles at very low concentrations (less than 5 μM), hence they are utilized as a germicide in purified water system13-15. Antibacterial activity against bacteria has been reported to be conflicting.16. gold can be poisonous to mammalian animals at higher doses, freshwater and aquatic organisms17. Apparently, such micromolar gold concentrations have no detrimental impact on humans18, 19.

 

The pancreas and salivary glands create alpha amylase, an enzyme or unique protein. The pancreas is a digestive organ found behind the stomach 20. It produces enzymes that aid in the breakdown of food in your intestines. The pancreas can become injured or inflamed, causing it to generate either too much or too little amylase.  A high level of amylase in the body might indicate a pancreatic problem. Amylase blood tests can identify whether a patient has pancreatic illness by assessing the amount of amylase in the body. If the levels of amylase are either low or too high, it might cause a pancreatic issue.

 

Inhibiting -amylase, enzymes involved in carbohydrate digestion, can dramatically reduce the postprandial rise in blood glucose following a mixed carbohydrate diet, and hence can be an essential technique in the control of postprandial blood glucose levels in type 2 diabetics and borderline individuals 21-23. Currently renewed interest in functional foods and medications based on plants that modulate physiological effects in the prevention and treatment of diabetes and obesity. As a result, appealing targets like as in vitro suppression of -amylase enzymes are now popular 24, 25.

 

This study designed with a simple, cost-effective and ecofriendly synthesis and biosynthesis method of gold nanoparticles (AuNPs) at ambient conditions using Myrtus communis leaves extract as a reducing and stabilizing agent. The AuNPs synthesized in this method has the efficient antimicrobial activity against Staphococcus aurous bacteria (gram positive) and Escherichia coli (gram-negative) bacteria. In addition, it shows an inhibition effect on alpha amylase enzyme in sera of patients with patient with type 2 Diabetes Miletus and control groups.

 

MATERIALS AND METHODS:

Materials:

Chloroauric acid (HAuCl4) that is obtained from Sigma-Aldrich Chemicals. Myrtus communis leaves were obtained fresh from various mulberry farms in Iraq. Myrtus communis leaves were rinsed multiple times in water to remove dust particles before being sun dried to eliminate any remaining moisture. The Myrtus communis leaves extract used for the reduction of gold ions, Au+ to gold nanoparticles, AuNPs was prepared by dissolving 0.296 g from the stock solution in 100 ml of deionized water, boiling 5.0 g of fresh Myrtus communis leaves powder in 100 ml of deionized water. The mixture was stirred and boiled until the color of mixture convert form color less to pale yellow, after that, the extract solution leaves to cooled to room temperature then filtered by using filer paper. To remove the heavy biomaterial from filtered extract solution, centrifuged at 3500 rpm for 5 min. The filtrate extract was kept in the dark at 2oC for future research.

 

Gold nanoparticles synthesis:

1 ml of Myrtus communis leaves extract (5% w/v) added drop by drop to 2 ml of (2 x 10-2 M) Chloroauric acid (HAuCl4) solution at room temperature. After completing the volumes to 20 ml with deionized water, the sample was stirred with heating for one hour at 70o C. the resulting solution change from colorless to maroon indicating the formation of AuNPs. he UV–vis spectrum of the aqueous medium containing gold ion showed peak at 527 nm corresponding to the plasmon absorbance of AuNPs.  The gold nanoparticles were finally dried so order to remove any uncoordinated biological components22.

 

Characterizations Techniques:

Spectrophotometer (with model Shimazu UV-1800) was used to detect absorption spectra of synthesized nano gold. At room temperature, an infrared Fourier transforms infrared (FTIR) spectra obtained by using a Shimadzu FTIR 84005 spectrometer. In order to Preparing an FT-IR analysis sample, The plant extract containing AuNPs was dried for one hour at 60 °C before being combined with an appropriate amount of KBr26.

 

A Shimadzu XRD-6000 diffract meter was used to acquire an X-ray diffraction (XRD) pattern to confirm the biosynthesis of AuNPs. The morphology and contact surface of gold nanoparticles studied by using AA300 Angstrom AFM Atomic Force microscopy. Aliquot of plant extract-filters containing gold nanoparticles evaluated with the SEM S-4160 electron microscope (SEM). Lambert Beer's Law was used to determinate the absorbance of samples.

 

Anti-bacterial Activity:

The technique for well diffusion Staphylococcus aurous and E. coli was used to determinate antibacterial activity of gold nanoparticles. The culture infected with a plate technique. In the case of cultures, brain heart infusion (BHI) broth incubated with 37˚C for 24 hours. Pathogenic bacteria incubation of Mueller-Hinton agar plates. Every plate has been equipped with sterile paper disk, with a diameter of 5mm and plant extract as control and various levels of produced gold nanoparticles. Afterwards, the dishes incubated at 37 °C for 24 hours. Measured and tabulated the inhibitory zones.

 

Effect of AuNPs on the activity of Alpha amylase:

Effect of AuNPs on the activity of Alpha amylase is examined in the present work.  The research was carried out throughout the time period from April 2021 to May 2021 on 50 patients (16-50 years) admitted to Al-Yarmok teaching hospital, where they diagnosed with type 2 Diabetes Miletus. Fifty healthy individual (15-50 years) were participated as control group. Allow the whole blood to coagulate before centrifuging it at 1000 g for 10 minutes to separate the serum, which should then be kept at 4°C.

 

α-amylase catalyzes the hydrolysis of 2-chloro-p-nitrophenyl-α-D-maltotrioside (CNP-G3) substrate at pH 6.0 to generate 2-chloro-p-nitrophenol (CNP) and free glycosides in this direct method. The rate of creation of the colored CNP generated, which is proportional to the activity of the -amylase in the sample, is measured kinetically at 405 nm27. After adding 1 mL of biosynthesized AuNPs (5%) to the serum of each group (patients with type 2 diabetes mellitus and healthy participants), alpha amylase activity was measured again.

 

Kinetic Studies:

The kinetic parameters of alpha amylase are investigated in the presence and absence of AuNPs. The reaction mixture was made and processed in the same manner as described in alpha amylase, alpha amylase, and was determined at various constant reactions of alpha amylase substrate for kinetic investigation. These investigations' data were utilized to construct a linear relationship by Limewater-Burk equation 28.

 

RESULTS AND DISCUSSION:

This work has shown that Myrtus communis leaves extract rapidly created to reduce gold nitrate to gold nanoparticles. UV-visual monitoring of the formation of gold nanoparticles has been performed within 60 minutes with a stirring and heating at 70° C. The reaction was completed. The colorless solution was tinted brown, indicating that gold nanoparticles formed.

 

Nanoparticle’s characterization:

a)    Fourier Transfer Infrared spectroscope (FT-IR):

FTIR technique was used to determination the functional groups of Myrtus communis leaves extract, Figure 1A shows the FTIR spectra of Myrtus communis leaves extract. Myrtus communis leaf extract has a multitude of absorption peaks, indicating its complexity. The stretching of the N–H bond of amino groups resulted in a peak at 3433 cm-1 that is suggestive of bound hydroxyl (-OH) group. The shoulder peak at 1639 cm-1 is ascribed to the carboxylic acid C=O group. The pattern region of CO, C–O, and O–H groups, which exist as functional groups in Myrtus communis leaves extract, is shown by the peak at 1635 cm-1.  The existence of C–O stretching in carboxyl may explain the absorption peaks at 1361 cm-1. The C-N stretching vibrations of aliphatic amines might be assigned to the intense band at 1018 cm-1. According to the results of the FTIR analysis, the carboxyl (-C=O), hydroxyl (-OH), and amine (N-H) groups of Myrtus communis leaves extract are mostly involved in the reduction of Au+ to Au nanoparticles.

 


Figure 1. FT-IR spectra for A) Myrtus communis leaves extract, B)AuNPs in Myrtus communis leaves extract.

 


The FTIR spectrum analysis of the Myrtus communis leaves extract before and after addition of Chloroauric acid and formation of gold nanoparticles figure (1B). We note the occurrence of a displacement of the peaks from 3391 to 3436 cm-1 and from 2823 to 2950 cm-1 referring to the role of phenolic and aliphatic compounds in the stability of gold nanoparticles. For the carbonyl groups, 1703 to 1747 cm-1 became weaker than them before the interaction, which suggests that the carbonyl plays a crucial role in reducing gold ions to gold nanoparticles Several studies have indicated the possibility of using plant extracts for the production of metallic nanoparticles29-31. It contains many secondary metabolites, which is changing the charge and transformation of metal ions and effective nano-sized particles.

 

b)    The atomic force microscope (AFM):

The AFM study used to display surface characteristics and determine topography. The (AFM) provides a three dimensional picture of the surface of a nanoparticles at a microscopic resolution. The averaged nano-scale particle diameter equal to the 41.39 nm as showed in figure (2) that illustrates the two- and three-dimensional images for synthesized AuNPs' by Myrtus communis leaves extract.

 

Figure 2. 2D and 3D AFM images of synthesized gold nanoparticles using Myrtus communis leaves extract.

 

c)     Scanning electron microscopy (SEM)

The size, form, and distribution of produced gold nanoparticles by Myrtus communis leaves extract studied by using a scanning electron microscope (SEM). The particles are spherical, as seen in Figure (3), with a particles size round 39 to 57 nm.

 

Figure 3. SEM image of gold nanoparticles prepared with Myrtus communis leaves extract.

 

d)    X-Ray diffraction (XRD):

To validate the crystalline structure of the particles, an XRD study was done, and the XRD patterns revealed a number of Bragg's reflections that were indexed based on the face centered cubic structure of gold. A comparison of our XRD spectrum to the standard proved that the gold particles generated in our tests were nanocrystals, as demonstrated by the peaks at 2θ of 38.25, 44.25, 64.65, and 77.65 degrees, which correspond to (111), (200), (220), and (311), respectively, Bragg's reflections of gold32-34. The gold nanoparticles generated by the reduction of Au+ ions by the Myrtus communis leaves extract are crystalline in nature, according to the X-ray diffraction data. The average crystallite size of gold nanoparticles produced using the current green process may be calculated using Debye–approximation Scherrer's from the peaks' full width at half maximum (FWHM) (Eq. 1)35.

 

d= kλ/βcosθ                          … (1)

Where, k is the constant, λ=wavelength of CuK equal to 1.542Ao, d=the size of crystallite and θ is angle of diffraction. Figure 4 shows the XRD patterns, the particle average size was calculated by FWHAM, was 50±2.8 nm. The AuNPs synthesized by biochemical method were nanocrystalline in nature figure (4).

 

Figure 4. Gold nanoparticle XRD pattern synthesized using extract leaves of Myrtus communis.

 

e)     UV-visible Spectroscopy:

A UV-visible spectrophotometer with a wavelength range of 200 to 750 nm was used to validate the formation and stability of AuNPs in deionized water.  After mixing Myrtus communis leaves extract in an aqueous solution of gold ion complex for 60 minutes, the reduction of pure Au+ ions to Au0 were tracked by measuring the UV–visible spectra of the reaction medium. The UV–visible spectra indicate that the Surface Plasmon Resonance of gold appears to at 527 nm after 60 minutes. The presence of AuNPs may be attributed to the depletion of gold ions (Au+) in the extract of Myrtus communis leaves.

 

Antibacterial effect of synthesized gold nanoparticles:

Synthesized gold nanoparticles of Myrtus communis leaves showed antimicrobial action against gram-negative E. coli and Gram- positive Staphylococcus aurous and assessed area of inhibition, data obtained tabulated in table 1.

 

The results shown that the varied concentrations of produced gold nanoparticles from the leaves of Myrtus communist show both Gram negative and Gram positive, efficient antibacterial action. Several studies have revealed that gold nanoparticles can kill bacterial spores by damaging membrane integrity 36-38. Other studies suggest that gold nanoparticles may interact with phosphorous and sulphide-containing compounds and can harm the DNA and the proteins resulting to cell death; it is obvious that gold nanoparticles might potentially employed as an efficient bacterial agent against hazardous human infections and can be employed for vital applications in agriculture. AuNPs may readily infiltrate bacteria through the Membrane protein sulfate groups, which cause structural damage of the bacterium. AuNPs can also be transferred to the cytoplasmic fluid that can damage enzyme protein 39-41.

 

Table 1. Zone of inhibition (millimeter) of different concentration AuNPs synthesized using Myrtus communis leaves extract against pathogen.

Name of Organism

Inhibition zone (mm)

AuNPs(1ml)

AuNPs(1.5ml)

AuNPs (2 ml)

Myrtus communis leaves extract

E-coli

6

16

22

0.0

Staphylococcus

9

22

28

0.0

 

Effect of gold nanoparticles on Alpha amylase:

Table (2) shows the influence of gold nanoparticles (AuNPs) on the activity of the enzyme alpha amylase in control and patient groups. The inhibition percent of alpha amylase by AuNPs were (72 %) and (45 %) for patient and control groups respectively, as shown in table (2) this decreasing in alpha amylase activity may due to the interaction between gold nanoparticles (AuNPs) and hydroxyl  group of the starch, which found in enzyme structure 42, 43.

 

Table 2. Effect of gold nanoparticles on Alpha amylase activity of control and patients group

Groups

Alpha amylase concentration without AuNPs(U/L)

Alpha amylase concentration with AuNPs (U/L)

Percent of AuNPs Inhibition

Control (Healthy people)

100

55

(45 %)

Patients

750

210

(72 %)

 

Kinetic studies of alpha amylase:

Analyzing alpha amylase binding data was performed using Lineweaver-Burk equation 28. Table 2, shows the kinetic parameters of AuNPs in control groups as inhibitor was noncompetitive. Where the enzymatic activities of alpha-amylase were reduced, whether of the AuNPs has previously attached to the substrate, it binds as effectively to the enzyme. The Linweaver-Burk equation was used to compute V max, Km, and Keq 44.

 

Table 3. Vmax, Km and Keq in presence and absence of gold nanoparticles in patient group.

Group

Vmax (U/L)

Km (mM)

Keq (mM-1)

Patient

1000

0.01

100

AuNPs-patient

250

0.015

66.7

 

CONCLUSIONS:

The use of green chemistry in the production of nanoparticles offers several benefits, including the simplicity with which the process may be scaled up and its economic feasibility. Devised a rapid, eco-friendly, and easy technique for producing gold nanoparticles with a diameter range of 59.2 nm from Myrtus communis leaves extract. Color change happens as a result of surface plasmon resonance upon contact with plant leaf extract components, resulting in the synthesis of gold nanoparticles, as proved by UV–Visible, XRD, and SEM, with an average mean size of 50 nm. Interestingly, gold nanoparticles showed rather modest concentration of efficient bacterial activity. Based on the current observations, gold nanoparticles employed as a bacterial agent to control various pathogenic agents; however, additional investigation is required to understand the specific process by which gold nanoparticles infiltrate the wall of the bacterial cell.

 

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Received on 06.04.2022           Modified on 20.05.2022

Accepted on 17.06.2022         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(8):3459-3465.

DOI: 10.52711/0974-360X.2022.00579