Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
640
INVESTIGATE THE ANTIBACTERIAL ACTIVITY OF AMYGDALIN
AGAINST SOME TYPES OF ANTIBIOTIC RESISTANT PATHOGENIC
BACTERIA
Ahmed A. Mhawesh , Dhafar N. Al-Ugaili ,Yasameen Mad olaiwy
College of Biotechnology, Al-Nahrain University,Baghdad,Iraq
Keywords; (HPLC), Pseudomonas aeruginosa, Inhibition zone.
Corresponding Author: [email protected]
ABSTRACT
Extraction of Amygdalin from apple seeds by alcoholic extraction procedure were done
and applied a high-performance liquid chromatographic (HPLC) procedure for quantification of
it to investigate extraction efficiency and (16) bacterial isolates ( 4 isolates of Staphylococcus
aureus, 4 isolates of Escherichia coli , 4 isolates of Pseudomonas aeruginosa and 4 isolates of
Streptococcus pyogenes from skin infections samples are choose by accomplished of drug
sensitivity tests as a multidrug resistant, the antimicrobial activity of methanolic extract of
Amygdalin from crushed apple seeds was tested against each group of four pathogenic bacteria
species of gram positive (Staphylococcus aureus and Streptococcus pyogenes ) and gram
negative (Escherichia coli and Pseudomonas aeruginosa) by well diffusion method, the results
shown that plant extract gave antibacterial activity through the inhibition zone , but the
concentration of the raw material was more inhibited than the dilute substance concentration,
inhibition of the substance was obtained on all types of bacteria were in Staphylococcus aureus,
inhibition zone of substances concentrations (100 mg/ml, 50 mg/ml) were (40, 28 mm)
respectively , in Pseudomonas aeruginosa inhibition zone of substances concentrations (100
mg/ml, 50 mg/ml) were (50, 35 mm) respectively, in Escherichia coli, inhibition zone of
substances concentrations (100 mg/ml, 50 mg/ml) were (40, 25 mm) respectively, also this
results showed that three types of bacteria above were inhibited in synergistic effect, while
inhibition zone of Streptococcus pyogenes in concentration 100 mg/ml was 30 mm and in
concentration 50 mg/ml was 20 mm.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
641
The present study is aimed to evaluate the antimicrobial effects of Amygdalin from crushed
apple seeds plant extract against some pathogenic bacterial species (Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa and Streptococcus pyogenes), and determination the
synergistic effect of alcoholic extract of it.
INTRODUCTION
Amygdalin is classified as a cyanogenic glycoside because each amygdalin molecule
includes a nitrile group, which can be released as the toxic cyanide anion by the action of a betaglycosidase:
eating amygdalin will cause it to release cyanide in the human body and may lead to
cyanide poisoning. Cyanogenic glycosides are plant natural toxicant that has the ability to
produce toxic hydrogen cyanide. Concentrations of cyanogenic glycosides vary widely in plants
as a result of genetic and environmental factors, location, season and soil types. (1)
Cyanogenic glycoside is a large group of secondary metabolites that are widely,
distributed in the plant kingdom, including many plants that are commonly consumed by
humans. The diverse chemical nature of cyanogenic glycoside means that extraction and analysis
of individual compounds can be difficult. In addition, degradation can be rapid under appropriate
condition. (2 ).
Amygdalin is a cyanogenic glycoside derived from the aromatic amino acid
phenylalanine. Amygdalin and prunasin are common among plants of the family Rosacea,
particularly the genus Prunes, Phocaea (grasses), Fabaceae (legumes), and in other food plants,
including flaxseed and manioc. Within these plants, amygdalin and the enzymes necessary to
hydrolyze them are stored in separate locations so that they will mix in response to tissue
damage. This provides a natural defense system (3).
Although apple contains compounds which may confer significant health benefits to
humans (4)., apple seeds contain amygdalin. Amygdalin (from Ancient Greek: ἀμυγδαλή
amygdale "almond") is a naturally occurring chemical compound, famous for falsely being
promoted as a cancer cure. It is found in many plants, but most notably in the seeds (kernels) of
apricot, bitter almonds, apple, peach, and plum. (5) .
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
642
MATERIALS AND METHODS
Reagents and standards
Amygdalin, ethanol, diethyl ether, and HPLC-grade methanol were all achieved from
Sigma-Aldrich commercially. Water was prepared using a Millipore Milli-Q purification system.
All other reagents were of analytical grade.
Microorganisms Isolates collection:
The pathogenic microorganisms (Staphylococcus aureus, Escherichia coli, Pseudomonas
aeruginosa and Streptococcus pyogenes) which choose in this study were form different samples
of skin infections from different hospitals in Iraq/ Baghdad and tested by bacteriological and
biochemical tests in Microbiology laboratory / College of biotechnology / AL-Nahrain
University.
Fruit Sample Collection:
Firstly, the red apples purchase from local market were cutting to separate the seeds from
it and collect a suitable quantity of seeds and wash them to get rid of the dust particles or any
other triad, then leave it to dry and grind it by using the blender to become small molecules, then
prepare this powder for their where been extraction process.
Alcoholic Extraction Method:
Weigh (2g) of dry powder (crushed apple seeds) and place it in flask (500ml) and add
100ml of methanol alcohol with a concentration of 80% and mixing it , then close the flask
tightly to avoid evaporation and left it to heating on the hot plate in 50 C 0 for 100 min, after
completing the boil cool the mixture ,then filter it by Filter paper for the disposal of impurities
and residues non-solvent and after finishing the extract put in the rotary evaporator for 15 min to
separate the alcohol from the powder residue ,and remain for (24-48) hour in room temperature
to dry and become a powder and then take the dry extract and put it in 20 ml of Diethyl ether
and mixing for 1 min for the purpose of dissolving the extract and then purify the extract by
Millipore filter to avoid any pollution during the extraction process, by Millipore filter unite was
used to sterilize crushed apple seeds extract. Size of Millipore filter was 0,4 mm. (6).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
643
Preparation of dilution of materials:
The dilution of the extracted substance is 100% and 50% concentration. The dilution of
100% is the substance extracted only, the dilution of 50% is taking 1ml of the extract and adding
it to 1ml of double distilled water thought to has been 50%.
Microorganisms Preservation:
The isolates have been used in this work were maintained by weekly activation in
nutrient broth and were incubated at 37 0C for 24 hours .(7).
Minimum Inhibitory Concentration and Minimum Bactericidal Concentration Estimation:
Culture: Overnight Mueller Hinton broth cultures of Staphylococcus aureus and Streptococcus
pyogenes, Escherichia coli and Pseudomonas aeruginosa at 370C were prepared. The culture
was adjusted to obtain turbidity comparable to that of the turbidity of MacFarland 0.5 standard3.
and then further diluted 1: 200 in Mueller Hinton broth. The inoculums thus prepared expected to
obtain 105 to 106 C.F.U/ml. This procedure done according to Hossain MD., 2000 method. (8).
Testing the effect of alcoholic apple seeds extracts on bacterial isolates:
To detect the susceptibility of the bacteria and their effect on the plant extract, Muller
Hinton agar medium, prepared, then inoculated by using cotton swab with a culture of four
bacteria species after comparing it with standard suspension containing 1.5x1012 g (McFarland
Turbidity Standard ), then two well work on agar and fill one well with appropriate amount
from plant extract only and fill other well with appropriate amount from diluted extract , after
complete all this, should take these petri dishes and put them in the incubator at 37C0 for 24
hours, After this period , taken this dishes and determine the inhibitory activity (mm) of the
bacteria by measuring diameter of the inhibition zone around the bacteria .measure the
inhibitory effect of the substance and the effect the bacteria on it by using the rulers. then two
well used on agar, one well filled with suitable amount from plant extract handiest and other
filled properly with suitable quantity from diluted extract , after entire all this, have to take those
petri dishes and positioned them within the incubator at 37C0 for 24 hours, After this period ,
taken this dishes and determine the inhibitory interest (mm) of the bacteria by measuring
diameter of the inhibition area across the microorganism .degree the inhibitory impact of the
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
644
substance and the effect the bacteria on it by using the rulers.
Preparation of extracts for HPLC analysis:
Aliquots of the extract was dispensed into Eppendorf tubes (1.5 ml), centrifuged (10 min,
22 oC, 14000 rpm, using a micro centrifuge) and filtered with 0.45 μm PTFE filters.
HPLC determination of amygdalin
Amygdalin contents of desiccated apple seeds was determined by RP-HPLC, using a
Shimadzu HPLC consisting of a 20ADXR pump, SIL-20ACXR autosampler and degasser. (4.60
mm, 3 μm) placed in a column oven set at 40 o C. The mobile phase was an isocratic elution that
consisted of methanol and water (25:75, v:v) and the flow rate was 1 ml/min. The mobile phase
was sonicated (20 min, 22o ± 2 oC) to remove gas bubbles before use. The sample injection
volume was 5 μl. Amygdalin was detected using a photodiode array detector at 214 nm. Results
were expressed as the amount of amygdalin in mg per gram or mg per milliliter of extracted
samples (9).
Statistical analysis
The data obtained in this study were assessed statistically by using of The IBM SPSS
statistics version 20 software was used for the analysis.
RESULTS AND DISCUSSION
HPLC analysis of amygdalin
Amygdalin detection was achieved by UV detection in an isocratic elution with an
excellent peak area and the concentration of amygdalin. The amygdalin peak was completely
separated from other materials without any pre-treatment. The recovery of amygdalin was
greater than 98%.
Amygdalin content of apple seeds
The amygdalin contents of seeds from different varieties of apple are given ranged from 0.5-1
mg g-1. Which was not accepted with Haque and Bradbury (10). who reported the amygdalin
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
645
contents of Fuji apple seeds to be 5.4 mg g-1. This value is lower than the amygdalin content of
Fuji apple seeds (1.89 mg g-1) reported in this study. The variation in the amygdalin content of
apple seeds could be due to cultivation practices (e.g. different levels of fertilization, irrigation
and use of pesticides) or environmental factors such as drought or infection by pathogens during
fruit formation. Application of fertilizer to a field before planting has been reported to decrease
cyanogenic glycoside levels in cassava tubers (11).
MIC and MBC Of Amygdalin Evaluation
The results of MIC were 12.5 mg/mL for all strains used in this study and MBC were 200
mg/mL for all strains used in this study.
Antibacterial Activity:
After preparation stock solution from the dried powder of crushed apple seeds extract
(alcohol) ,and diluted it to one concentration (50%) by take 1ml from it and add to 1ml from
distilled water ,for testing antibacterial activity on gram positive bacteria of (Staphylococcus
aureus and Streptococcus pyogenes) and gram negative bacteria of (Escherichia coli and
Pseudomonas aeruginosa) both, diluted extract and raw extract (without dilution) were positive
,but the concentration of the raw material was more inhibited than the dilute substance
concentration, inhibition of the substance was obtained on all types of bacteria, both well are
filled as control positive (trioxane antibiotic) and control negative (sterile water)
Table (1): Antibacterial activity of Amygdalin from alcoholic apple seeds extractions against
Staphylococcus aureus.
Type of
extraction
Concentration
mg/ml
Type of
microorganism
Inhibition
zone
Alcoholic
extraction
100mg/ml Staphylococcus
aureus
40
50mg/ml 28
This table shows that inhibition zone of Staphylococcus aureus in concentration 100 mg/ml
was 70 mm and in concentration 50 mg/ml was 70 mm, this type was inhibited in synergistic
effect with other concentrations were tested as shown in Figure (1).
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
646
Figure (1): Inhibition zone of Amygdalin from the apple seeds extract on Staphylococcus aureus
culture.
Table (2): Antibacterial activity of Amygdalin from alcoholic apple seeds extractions against
Streptococcus pyogenes.
Type of extraction Concentration
mg/ml
Type of
microorganism
Inhibition zone
(mm)
Alcoholic
extraction
100mg/ml Streptococcus
pyogenes
30
50mg/ml 20
This table shows that inhibition zone of Streptococcus pyogenes at concentration 100
mg/ml was 30 mm and at concentration 50 mg/ml was 20 mm.
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
647
Figure (2): Inhibition zone of the Amygdalin from apple seeds extract on Streptococcus
pyogenes culture.
Table (3): Antibacterial activity of Amygdalin from alcoholic apple seeds extractions against
Pseudomonas aeruginosa.
Type of
extraction
Concentration
mg/ml
Type of
microorganism
Inhibition
zone
Alcoholic
extraction
100mg/ml Pseudomonas
aeruginosa
50
50mg/ml 35
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
648
This table shows that inhibition zone of Pseudomonas in concentration 100 mg/ml was 40
mm and in concentration 50mg/ml was 35 mm ,this type was inhibited in synergistic effect as
shown in Figure (3).
Figure (3): Inhibition zone of the Amygdalin from apple seeds extract on Pseudomonas
aeruginosa culture.
Table (4): Antibacterial activity of Amygdalin from alcoholic apple seeds extractions against
Escherichia coli.
Type of
extraction
Concentration
mg /ml
Type of
microorganism
Inhibition
zone (mm)
Alcoholic
extraction
100mg/ml Escherichia coli 40
50mg/ml 25
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
649
This table shows that inhibition zone of Escherichia coli in concentration 100 mg/ml
was 40 mm and in concentration 50 mg/ml was 25 mm, this type was inhibited in synergistic
effect with other concentrations were tested as shown in figure (4).
Figure (4): Inhibition zone of the Amygdalin from apple seeds extract on Escherichia coli
culture.
In these test all types of bacteria affected with effective substance and the result was
positive due to high concentration of substance and according to the size of each zone measured
the inhibitory effect of the substance and its effect, through the measure of inhibition zones of
activity of bacteria proved that three types of bacteria were inhibited in synergistic effect with
other concentrations were tested due to increasing of the size of inhibition zone that could be
attributed to presence of active compounds of apple seed alcoholic extract against these types of
isolates.
It seems that amygdalin has antibacterial activity. The antibacterial activity may brought
by amygdalin decomposition by bacterial enzymes to give hydrogen cyanide and benzaldeyde,
Basrah Journal of Veterinary Research,Vol.17, No.3,2018
Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
650
the toxic compounds which killed the bacteria. The results of this report are in agreement with
the study of (12). They reported the antibacterial activity of bitter apricot extract against several
bacterial strains. They supposed that the antibacterial activities might be brought by amygdalin,
alkaloids, flavanoids, tannins and phenolic compounds.
CONCLUSION
Through this study the active substance of apples seeds plant alcoholic extract prove the
ability to inhibit some types of pathogenic bacteria growth , were methanolic extract of crushed
apple seeds has antibacterial activity against (Staphylococcus aureus ,Escherichia coli
,Pseudomonas aeruginosa and Streptococcus pyogenes ), the activity inhibition zone increased
when the raw extracted substance (without dilution ) increase in concentration. Finally, it is
necessary to determinetoxicity and their side effects.
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Proceeding of 6th International Scientific Conference,College of Veterinary Medicine
University of Basrah,Iraq
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