Study of Some Ginkgo Biloba Contents and Effect of Their Extracts As Antioxidants and Antibacterial

CENTRAL ASIAN JOURNAL OF MEDICAL AND NATURAL SCIENCES
Volume: 04 Issue: 04 | Jul-Aug 2023 ISSN: 2660-4159

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Study of Some Ginkgo Biloba Contents and Effect of their

Extracts as Antioxidants and Antibacterial
1. Aseel Khalil Ibraheem Abstract: Biloba ginkgo leaves are one of the most
2. Mustafa Taha Mohammed popular herbal medicines. The purpose of this study was
to use ginkgo Biloba leaf extracts to find out how many
3. Mohammed Z. Thani free amino acids are in a sample. Phenolic compounds,
particularly flavonoids, were estimated in the two types
of extracts using the high-performance liquid
Received 2nd Jun 2023, chromatography device (HPLC) and many other
Accepted 3rd Jul 2023, important biologically active factors due to their benefits
Online 10th Aug 2023
to human health and antioxidant and antibacterial
effects. Aqueous extracts and ethanol showed nine types
1,2,3 of amino acids, the highest of which was cysteine. In
Department of Chemistry, College of
Science, Al-Mustansiriyah University,
addition to containing good numbers and quantities of
Baghdad-Iraq phenolic compounds, many of which contain multiple
biological properties such as antioxidant and
antibacterial, this research has been carried out. An
antioxidant analysis showed that ethanol extract showed
more antioxidant properties than aqueous extract.
However, compared to vitamin C measurement, both
extract sections were able to inhibit a good ratio of 1, 1-
diphenyl 2-picrylhydrazyl free radical (DPPH) with a
good excellent ratio balance. The maximum activity of
DPPH root rickets for vitamin C (69.4) ppm, ethanol
extract (185.58) ppm, and aqueous extraction (251) ppm
were concentrated. The antibacterial activity showed a
maximum inhibition area of 24 mm for Staphylococcus
aureus as Shigella flexneri at a concentration of 500
ppm.
Key words: Ginkgo biloba, Amino acids, Phenolic
compounds, Antioxidants, Antibacterial.
Introduction:
Ginkgo Biloba is a tree species with a rich, and vibrant history of usage for se medicinal purposes
because of the various benefits to human health it can provide. The name ginkgo is derived from the
Japanese name Yin-Kwo (silver fruit), while the title Biloba refers to the pelvic shape of the leaves.
The species exists without interruption for 270 million years without changes and is the oldest tree in
the world. As a result, it is classified in a separate section: Ginkgo, with the presence of the existing
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CAJMNS Volume: 04 Issue: 04 | Jul-Aug 2023
species G. Biloba. The hallmark of the gene is that it is coniferouside [1]. Researchers are still
studying its mode of action, still discovering new beneficial properties. Active components in Ginkgo
biloba leaves extracts, such as terpenoids and flavonoids, possess antioxidant activity [2]. Studies also
show the ability of these extracts to enhance blood circulation, reduce the risk of clotting, support
capillary walls and their flexibility, and shield neurons from harm, which could be brought about when
deprived of oxygen[3]. Protein, lipids, carbs, vitamins, organic acids, polyphenols, beta carotene,
flavonoids, and terpenoids are abundant in Ginkgo Biloba leaves [4]. Aside from the well-known
flavonol glycosides and terpene lactones, amino acids in Ginkgo Biloba leaves are often found in
foods, beverages, and pharmaceuticals [5]. Amino acids (AAs) are chemical molecules with both
amino and acid groups[6]. Amino acids are essential life-supporting building blocks. Their function in
protein synthesis is widely understood. Still, they also various other intracellular metabolic pathways
that support cell processes organismal functions, such as ATP generation, nucleotide synthesis, and
redox balancing. Immune cells rely on this pathway amended biomass, as well as to alter their
metabolism after activation to sustain their growth, proliferation, and effector activities. Beyond
enhanced protein synthesis essential amino acid metabolism is important for this metabolic rewiring
and supports various immune cell functions[7][8].
Flavonoids are a diverse group of secondary metabolites found in many plants. It has a flavone
structure, which means two benzene rings (A and B rings) are linked by a pyran ring (C ring), which
may contain hydroxyl, methoxy, methyl, isoamyl, and other substituents[9]. Flavonoids give plants
color flavors and pharmacological properties [10]. Fruits and vegetables are the primary sources of
flavonoids [11]. Flavonoids are antioxidants with a lot of power [12], Plants are protected from
adverse environmental conditions [13], in addition, flavonoids have biological properties such as
improved blood circulation, cholesterol reduction, UV protection, angiogenesis inhibition,
antimicrobial, and anti-inflammatory properties[9]. As a result, they have gotten a lot of attention.
They have been tested in of many epidemiological and experimental studies to see if they can help
with a variety of acute and chronic human disorders [14]. Flavonoids have been proven to have anti-
inflammatory and immunomodulatory properties in vitro and animal experiments [15] and potent
anticancer properties [14] [16] [17]. Antioxidants are substances that can counteract the oxidative
effects of free radicals and other oxidants in living systems [18]. Antioxidants are divided into
endogenous and exogenous, based on their presence in the human body[19][20]. They are beneficial to
various human products due to their unique chemical and biological features [21]. The search for new
and natural antioxidants from dietary plants is gaining traction because they can protect the human
body from oxidative damage to biological macromolecules such as lipid, protein, and nucleic acid,
which is primarily caused by secondary metabolism [22].
Fig. (1): Ginkgo Biloba plant leaves [23]
Material and Methods:
1- Collection and preparation of Ginkgo Biloba Leave Extract:
The dried leaves of Ginkgo were collected from the north of Baghdad, Iraq, in September 2021, then
washed with de-ionized water and dried in the shade for several days at the temperature of the room,
and then ground the sample to powder. The conventional Soxhlet extraction method was applied to
samples according to [24] for obtaining aqueous and alcohol extracts.
2- Determination of Amino Acids and Phenols concentrations:
Using an HPLC system (Sykam s3210, Germany) with a C18 column (4.6mm × 150 mm, 5µm), all
the standard materials used were purely 99% of Samarra Pharmaceuticals. For Amino Acids Mobile
phase (acetonitrile: H2O) (5: 95) respectively, Flow Rate (2 mL/min) ,Detection fluorescence system
(340nm-450nm) [25]. For Phenols mobile phase (H2O: acetonitrile) (70:30) respectively, Flow Rate
(1.2 mL/min), Detection fluorescence system (260nm-310nm) [26].
3- Antibacterial Activity studies:
Many microorganisms were used as bacteria, antibacterial activity evaluation of bacteria from selected
microorganisms was performed using a spreading tablet, and antimicrobial activity was done to
Gingko extracts using a well-hired deployment method [27][28]. The study included testing in-kind
water and alcohol extracts from the ginkgo plant in inhibiting the growth of bacteria causing diseases
positive for Kram dye outside the living body; depending on the McFarland method, samples were
loaded on cellulose tablets in diameter (5mm) bacterial insulation was developed, and 0.5 was
transported 1 ml of bacterial and bacterial filaments equivalent to (1.5 × 108) cells/ml to the dish that
contains the center of Müller Hanton's dens, diffuse, leaving the dish for 15-20 minutes at room
temperature until the implant is absorbed Bacterial, tablets loaded with extract and plates are incubated
at 37OC temperature. For 24 hours, the diameters of the tablet areas are measured around the chips for
each type of bacteria used.
4- Determination of free radical scavenging activity:
Ginkgo Biloba extracts' free radical scavenging activity was determined using the DPPH. At varied
concentrations (12.5, 25, 50, 100, and 200 g/mL), the extract was evaluated. To 3 mL of daily prepared
methanol and DPPH solution, a (150L) methanol solution containing various volumes of sample
solution was added. At 517 nm, 20 minutes after the first mixing, optical density changes were
recorded, with a UV spectrophotometer; Vitamin C been used as a benchmark. The sample's
scavenging activity was determined by comparing its absorbance to that of the DPPH reference
solution The following formula was used to calculate the radical scavenging activity percentage of the
DPPH: % Antiradical activity = (𝐀o – 𝐀C) /𝐀o× 𝟏𝟎𝟎
Where Ao is the absorbance of the control and Ac is the absorbance of the sample or standard in the
presence of the control. The results of three independent trials were averaged and expressed as a
percentage of mean radical scavenging activity [29].
3. Results and Discussions:
The results revealed that G. Biloba leaf extracts were high in free amino acids, especially those
considered essential, such as) Arginine, Valine, Isoleucine, Leucine, Phenylalanine, Tyrosine),
addition to (Cysteine, Alanine, Glutamic acid ). The total amount of amino acids in the sample ranged
from (250.197 to 1905.307) ppm in aqueous extract, while it was from (270.346 to 1277.005) ppm in
the alcoholic extract, according to the table (1) and fig. (2).
Table (1) Concentration of amino acids in the aqueous and alcohol extracts of the Ginkgo biloba.
Aqueous extract Alcoholic extract
Retain. Time Amount Amount Retain. Time Amount Amount
Amino acid Mean ± Sd
[min] [ppm] [%] [min] [ppm] [%]
Alanine - - - 9.868 427.075 10.4 10.4 ± 7.4
Arginine 9.132 250.197 5.1 8.976 709.506 17.3 11.2 ± 8.7
Cysteine 11.280 1905.307 39.0 11.212 1277.005 31.2 35.1 ± 5.5
Glutamic acid - - - 7.804 1023.273 25.0 25 ± 17.7
Isoleucine 13.044 274.827 5.6 - - - 5.6 ± 4.0
Leucine 13.256 321.532 6.6 - - - 6.6 ± 4.0
Phenylalanine 14.024 809.927 16.6 13.520 270.346 6.6 11.6 ± 7.1
Tyrosine 12.464 295.353 6.0 12.228 382.716 9.4 7.7 ± 2.4
Valine 11.956 1028.445 21.1 - - - 21.1 ± 15.0
Fig. (2) HPLC analysis for (A) Amino acid in the aqueous extract. (B) Amino acid in ethanol extract.
Fig. (3) Graph shows the concentrations of amino acids in extractors

Amino acids are essential building blocks as a source of energy for cell survival, regeneration, and
growth. Each amino acid has a distinct carbon structure, an amino group, and a carboxylic acid.
Humans use 20 amino acids; most amino of these may be made naturally, but nine are "essential,"
meaning they must be consumed. Amino acids are important energy sources being protein-building
components (ketogenic, glycogenic, or both), are recycled as needed, and serve as building blocks for
Krebs's cycle intermediates and other metabolites [30]. Amino acids (AA) are used of product protein
and a variety of low-molecular-weight molecules with significant physiological significance.
Nutritionally, amino acids are necessary for maximal growth and lactation and optimal health, well-
being, and reproduction [31]. Certain amino acids can be transformed into other amino acids, proteins,
glucose, fatty acids, or ketones in the human body [32]. Chemical messengers in the neurological
system (neurotransmitters) and precursors to nucleic acids, which are components of DNA: glutamate
[33]. Alanine has a direct role in gluconeogenesis; transamination [34]. Arginine has properties of
antioxidant; regulation of hormone secretion; ammonia detoxification; regulation of gene expression
and immune function [35]. Cysteine is a disulfide bond in proteins that allows sulfur to be transported
[36].
Glutamine: The production of arginine connects the urea and Krebs cycles. Isoleucine Synthesis of
glutamine and alanine. Leucine; Regulation of protein turnover and gene expression; activator of
glutamate. Phenylalanine; synthesis of tyrosine; neurological development [37]. Tyrosine is a
precursor of dopamine, epinephrine, norepinephrine, and thyroxin [38]. The HPLC analysis of Ginkgo
Biloba Leave Extracts investigated important antioxidant phenolic compounds in this study. We used
several different standard solutions for phenols. The results showed that G. Biloba leaves interesting
focus extracts because it contains a large number of phenol, which per compound was calculated by
comparing the area of the pick of the standard substance with the size of the picked-choice for the
desired combination and according to the following equation: C (sample) = [A (sample) ×A
(standard)] /C (average)
In the aqueous extract, the concentration of (4-hydroxy benzoic) was the highest and had the best
separation of the peak at retention time (9.720 min) according to the peak of standard substances, and
the presence of all phenolic compounds at range retention time (2-18min), Figure (4) and table (2). In
the alcohol extract, the concentration of (4-hydroxy benzoic) too was the highest and had the best
separation of the peak at retention time (10.020 min) according to the rise of standard substances, and
the presence of all phenolic compounds at the range retention time (2-18min), Figure (4) and table (2).
Table (2) Concentration of phenolic compounds in the aqueous and alcohol extracts of the Ginkgo
biloba.
Result Aqueous Extract Result Alcohol Extract
Retention
Retenti
phenolic conc. -1 Retention Time [Min]
Area[mV.s] -1 on Time Area[mV.s] conc.[µg.mL ]
compounds [µg.mL ] Time [Min] Standard
[Min]
Pyrogallol 3131.303 0.183 2.608 926.427 0.054 2.408 2.782
Gallic acid 3737.326 0.205 3.684 352.120 0.029 3.876 3.607
Rutin 2564.507 *** 4.356 1159.385 *** 4.408 ***
Kaempferol 1234.370 0.229 5.388 1995.025 0.370 5.444 5.448
Cinnamic 1269.683 6.749 8.132 964.322 5.126 8.124 8.0
Catechol 716.155 13.911 8.500 859.589 16.697 8.496 8.300
4-Hydroxy
2555.536 293.470 9.720 2667.033 306.274 10.020 9.953
benzoic
Quercitin 2702.706 10.106 10.000 1357.784 5.077 10.144 10.092
Cinnamaldehyde 2735.988 40.639 10.548 3248.364 48.250 10.488 10.403
Lignin 5735.893 2.722 16.744 3036.491 1.441 16.852 16.740
Chlorogenic 11810.94 261.338 17.160 3123.383 69.110 16.852 17.083
Nigellone - - - 6805.011 12.282 17.036 17.280
Eugenol - - - 2405.560 2.985 13.804 13.890
Fig. (4) HPLC analysis for (A) Phenolic compounds in the aqueous extract. (B) Phenolic compounds
in ethanol extract.
For the research and development of Ginkgo biloba L., it is critical to get a systematic understanding
of flavonoids and their actions. Many studies and research have shown that flavonoids have important
vital role in treating many diseases. An association has been observed between the chemical
composition of flavonoids and their therapeutic properties [39], as an increase in hydroxyl totals
increases anti-tumor activit rise he increase in the number of mitochondrial accommodations increases
anti-cancer someivity [40], Some flavonoids also have anti-inflammatory effects [41], for allergies
[42], for microbes [43], and viruses [44]. Preliminary screening was performed against Staphylococcus
aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella sp., and Candida albicans was
performed. The initial examination was performed against Orius staphylococcus, staphylococcus
epidermis, E. coli, Klebsiella sp., and Candida Albicans. The initial concentrations taken from the
extractors were (500,250, 125, 6 2.5 ppm. The results indicated that there was no effect of aqueous
extract for any type of previous bacteria and that the results of the alcohol extract were as shown in the
table. The efficacy of the aqueous extract was subsequently examined at a concentration of 1500 ppm
and its effect on inhibiting staphylococcus aureus was only at a magnitude of 14 mm table(3).
Table (3) Values the standard deviation and mean of the aqueous and alcohol extracts of the Ginkgo
biloba.
Sample No. S1 S2 S3 S4
Mean - - - -
S.aureus
Sd - - - -
Gram-positive Men - - - -
S.epidermidis
Sd - - - -
Mean - - - -
E .coli
Sd - - - -
Aqueous Gram-Negative Mean - - - -
Extract Klebsiella sp.
Sd - - - -
Mean - - - -
Fungi C.albicans
Sd - - - -
Mean 24 12 10 -
S.aureus
Gram-positive Sd 2.11 0.92 1.24 -
Mean 12 10 - -
S.epidermidis
Alcohol Sd 1.43 1.33 - -
Extract Mean 17 15 14 -
E .coli
Sd 1.56 1.89 1.22 -
Gram-Negative Mean - - - -
Klebsiella sp.
Sd - - - -
Mean 14 11 10 -
Fungi C.albicans
Sd 1.16 0.96 1.11 -
Fig. (5) Inhibition zones caused by Ginkgo Biloba extracts Against Staphylococcus aureus,
Staphylococcus epidermidis, Escherichia coli, Klebsiella sp., and Candida Albicans after 24hrs of the
incubation period.
There are different inhibition mechanisms against microorganisms suchirected as cell membrane
damage, inhibition of protein synthesis, and disruption of cell biological functions and cell membranes
by specific enzymes. The nature of bacterial cell wall is influenced by the presence of compound like
α-Pinene and β-pinene (pinene-type monoterpene hydrocarbons) which is involved in the membrane
cell wall disruption by the lipophilic compounds [45]. In addition, the nature of gram-negative bacteria
cell walls with high lipid content (up to 20 %) compared with 0-2% for gram-positive is responsible
for the resistance of microorganisms such as Escherichia coli, In contrast, positive gram-positive
bacteria show more sensitivity to the antimicrobial compounds found in extracts [40]. It is concluded
that the alcohol extract of the Ginko has antimicrobial activity against gram-positive bacteria higher
than gram-negative bacteria that resist both water extracts and ethanol [46].
Free radical scavenging activity: The DPPH radical scavenging assay is a sensitive antioxidant assay
substrate unaffected by the substrate‟s polarity. The stable free radical DPPH can take an electron or a
hydrogen radical to form a stable diamagnetic molecule. The high phenolic content of the extracts may
explain they are great antioxidant action. Based on these findings, the extracts and the mixed sample
both showed antioxidant activity against DPPH radicals, composite action activity. Therefore, this
exploratory research reveals the intriguing anti-oxidant stress function of Gingko leaves extracts,
suggesting their promising uses as a therapeutic source for the treatment and prevention. It offers uses
benefits therapeutic medicinal illnesses caused by free radicals From Fig(6), data showed a good
percentage of antioxidants that the two extracts contain compared with vitamin C, a strong and
effective antioxidant [47].
Fig. (6): DPPH Free radical scavenging activities of Ginkgo Biloba extracts compared with Vitamin C.
A: Vitamin C (IC50=69.4 ppm), B: Ethanol Extract (IC50=185.58 ppm), C: Water Extract
(IC50=251 ppm).
The results showed the presence of DPPH radical scavenging activity for water extracts and ethanol
from Biloba ginkgo leaves. The highest% scavenging activity was observed in the ethanol extract
according to their different concentration of 10μg/ml to 60 μg/ml. The high DPPH scavenging activity
of Ginkgo biloba leaf extracts aligns with the reports [48]. The extracts were able to donate a hydrogen
atom to DPPH and change the colour. The increasing intensity of the color is directly proportional to
the inhibition of DPPH [49].
Conclusion:
As a result of this research, it was confirmed that Ginkgo Biloba leaf extracts contain abundant
amounts of amino acids, flavonoids, and phenols. This high content of flavonoids and phenols is the
primary driver of their antioxidant activities. Ethanol also showed 99% more extraction of phenolic
content than aqueous extraction, so ethanol extract showed the best antioxidant properties. The
increasing concentration of the extract is increasingly preventing DPPH activity. Ethanol extracts
alsohad a clearance an apparent effect on bacterial inhibition compared to aqueous extracts.
Conclusion:
As a result of this research, it was confirmed that Ginkgo Biloba leaf extracts contain abundant
amounts of amino acids, flavonoids, and phenols. This high content of flavonoids and phenols is the
primary driver of their antioxidant activities. Ethanol also showed 99% more extraction of phenolic
content than aqueous extraction, so ethanol extract showed the best antioxidant properties. The
increasing concentration of the extract is increasingly preventing DPPH activity. Ethanol extracts
alsohad a clearance an apparent effect on bacterial inhibition compared to aqueous extracts.
Acknowledgment:
The authors are grateful to the employees of Mustansiriyah University, Science College, Department
of Chemistry, for their assistance with sample runs and data processing.
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CENTRAL ASIAN JOURNAL OF MEDICAL AND NATURAL SCIENCES

Volume: 04 Issue: 04 | Jul-Aug 2023 ISSN: 2660-4159

Study of Some Ginkgo Biloba Contents and Effect of their

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Fig. (1): Ginkgo Biloba plant leaves [23]

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Fig. (3) Graph shows the concentrations of amino acids in extractors

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