Universiti Teknologi MARA Cawangan Perak Kampus Tapah

Faculty of Applied Sciences

Diploma in Science

BIO301

Mini Project Written Report

Journal article review

Title:
Extraction Method and Pharmacological Properties of Ginkgo Biloba

Supervisor:
Siti Nur Azmu’i Abdullah

Class:
A4AS1205_13

No. Name of Students Student No.

1. Nurul Ain Binti Azni 2020847984
2. Nurul Ashikin Adila Binti Nor Katini 2020817628
3. Syaura Nurmizan Qistina Binti Sumadee 2020826604
4. Amirah Sofiya Binti Anis 2020815282
5. Syahidatul Badriah Binti Bachtiar 2020460672
6. Aina Najwa Binti Mohd Syaifuddin 2020610716
7. Nur Ilya Natasya Binti Mohd Sabri 2020479414
ABSTRACT

The world has been astonished by the enormous amount of non-vitamin and non-mineral
nutritional and bioactive components found in Ginkgo biloba (G.biloba), a living fossil. G.biloba
is a unique plant with a variety of advantages because of its eccentric properties that it gave to
humans. This plant consists of two primary active ingredients at different concentrations, terpene
lactones, and Ginkgo flavone glycosides. Besides that, the presence of ginkgolides, Ginkgo
flavonoids, and ginkgolic acid in G.biloba, contributes to its wider use in the field of medications
to prevent central nervous system regulated by cytokinin, antioxidants enzymes, kinases, and
receptors thus putting it as a wonder tree with multifarious uses. These bioactive compounds also
provide anti-inflammation, antioxidant, anticancer, antitumor, antibacterial, and anti-radiation.
Thus, in this study, we want to analyze the extraction method that could be used to separate the
active compounds in this plant and examine its pharmacological properties. This study covers
some parts of G.biloba that can be extracted: leaves and seeds. There are many methods to extract
and separate these active compounds. The extraction methods consist of supercritical carbon
dioxide fluid extraction (SFE-CO2), reflux, and extraction using ethanol, methanol, and acetone.
The review mainly summarizes the extraction procedures using this different solvent and its
pharmacological properties include anti-inflammation, antioxidant, anticancer, antitumor,
antibacterial, and anti-radiation. Thus, this study aims to provide a detailed and collective scientific
evaluation of the extraction procedure and its pharmacological properties for possible future use.

KEYWORDS

G.biloba plant extract, extraction method, pharmacological properties.

1.0 INTRODUCTION

Ginkgo biloba (G. Biloba), also known as yinhsing (Chinese), ginkyo (Japanese), maidenhair tree,
and silver apricot (Fang et al., 2020). It is an ancient species belonging to the Ginkgo genus of
Ginkgoaceaea family that was used as a traditional Chinese herb for many years (Liu et al., 2020).
It is the oldest gymnosperm left by the Quaternary glacial movement hundreds of millions of years
ago and the only surviving species of the Ginkgo genus in the Ginkgo family.

G. biloba is considered a ‘living fossil’ and is about 250 million years old as it contains a
number of biologically active compounds that act as defense measures against insects, bacteria,
and fungi, making it one of the oldest medicinal trees in China, Japan, and South Korea and
economically important plant that is now cultivated in China, Japan, Korea, France, Germany and
in some parts of India, especially in Uttarakhand state for its aesthetic and the medicinal value (Ma
et al., 2021; Gong et al., 2021; Ražná et al., 2020; Sati et al., 2019). The Ginkgo tree can hold under
reduced light and nutrient conditions and is resistant to bacteria, fungi, viruses, and air pollution
(Tian et al., 2017).

G. biloba nuts (Baiguo) are mature dry seeds of G.biloba, which are rich in flavonoids,
starch, lipids, ginkgo polysaccharide, and vitamin C. They are usually applied for treating lung
disorders in ancient China and have been proven to have anti-cancer, anti-inflammatory, and
antioxidant bioactivities in modern research (Liu et al., 2020). G.biloba is a valuable tree with high
medical, ecological, ornamental, and scientific values, which is widely introduced and cultivated
all over the world (Guo et al., 2022).

G. biloba medical use was first mentioned in the 16th century in “Ben Cao Gang Mu” by Li
Shizhen of the Ming Dynasty (Abouheif et al., 2022). The medicinal and antimicrobial properties
of G. biloba can be attributed to two important chemical constituents, ginkgolides and bilobalide,
and flavonoid glycosides (Sati et al., 2019). G. biloba, is one of the most extensively used botanical
medicines and dietary supplements in the world.

Nowadays, G. biloba ranks first prescribed herbal medicine in Germany and is one of the most
commonly used herbal supplements in the USA and Japan. Total worldwide sales of G. biloba
products were 1.26 billion U.S. dollars in 2012 based on IMS Health MIDAS data, among which
China (46% sales) and Germany (12% sales) occupied the most sales (Xiaoyan et al., 2018). The
species is largely used in the treatment of central nervous system (CNS) disorders, such as
Alzheimer’s disease and cognitive deficits (Belwal et al., 2018). The seeds of G. biloba have been
consumed as a food and versatile medicine in Chinese, Japanese, and Indonesian traditional
medicine owing to their reliable activities to treat asthma, cough, and pyogenic skin infections
(Fang et al., 2020).

The bacteriostatic and bactericidal effects of ginkgolic acid and phenol have been used to treat
respiratory infectious diseases. The leaves of G. biloba contain shikimic acid, while their fruit
contains isoflavones, and sterols, which are used to treat hypertension, cerebral vasospasm,
hypercholesterolemia, and cardiovascular and cerebrovascular diseases. Therefore, G. biloba
feedstock is desirable (Ma et al., 2021).

This review paper discusses the studies that have been undertaken on the pharmacological
properties and the detailed and collective scientific evaluation of the extraction procedure of G.
biloba. This study was carried out for future use that involves G. biloba.
2.0 EXTRACTION

Extraction methods such as hot water extraction, ultrasonic-assisted extraction, and enzymatic
extraction, as well as purification methods such as ion-exchange chromatography and gel filtration,
have all been used. Extraction conditions such as extraction solvent, pH, the raw material to solvent
ratio, temperature, and time all have a significant impact on extraction rates. Jiang et al. used an
orthogonal experiment to improve the extraction from Ginkgo biloba leaves. We mainly focus on
solvents such as ethanol, acetone, and methanol (Fang et al., 2020).

2.1 Ethanol

In this research, ethanol is the most commonly used to extract Ginkgo biloba (G.biloba). The dried
G.biloba extracts were refluxed three times with 86% ethanol for one hour each time (Chen et al.,
2013; Wang et al., 2022). The extract was concentrated using a rotary evaporator, and the sample
was obtained. The detection of total ion chromatogram (TIC) and multiple reaction monitoring
(MRM) in Ginkgo biloba extracts was done in both positive and negative ion modes, and the
ingredients were classified qualitatively and quantitatively using secondary spectral information
and triple quadrupole mass spectrometry using ultra-performance liquid chromatography-Q-trap-
mass spectrometer (UPLC/Q-TRAP-MS/MS) system.

After the pretreated samples were placed into the extraction kettle of the supercritical
extraction device, each sample was extracted with ethanol at a specific extraction time, pressure,
temperature, and cosolvent concentration (ethanol/water solution). Furthermore, the amount of
cosolvent used is 15% of the mass of the raw material. After cooling to ambient temperature, the
extract was centrifuged, precipitated with four volumes of ethanol, and incubated for twelve hours.
The resulting bold material was labeled Ginkgo leaves (FGPs), and the FGPs extraction yield was
determined. In this investigation, utilizing the supercritical fluids extraction with carbon dioxide
(SFE-CO2) to extract FGPs considerably increased FGPs yield while avoiding FGPs degradation
throughout the extraction process (Fang et al., 2020).

Four extraction procedures (maceration, reflux, shaker, and soxhlet) were used to acquire
their respective extracts. Extraction was carried out for two and a half hours using 3 mL of
concentrated hydrogen chloride (HCl) and 5 mL of water (H2O) in the reflux method. The
extractions used the same solid-to-solvent ratio of 5 g powdered leaf sample to 50 mL ethanol
(99.7%). These findings are consistent with the earlier findings of (Kaur et al., 2012), who found
that reflux with 60% aqueous ethanol was more efficient than maceration, ultrasound/orbital
shaker, and microwave techniques for extracting flavonoid glycosides from G.biloba. This can
also be linked to a successful extraction under reflux conditions, which results in a larger release
of some bound phenolic.

To extract crude polysaccharides from G. biloba leaves, the concentrate was maintained
overnight after being precipitated with four liters of absolute ethanol (95%) (Su & Li, 2020). The
leaf powder was soaked in 200 ml of 60% ethanol solutions with 0, 100, and 200 mg/g
concentrations of Trichoderma vride cellulase each for twenty-four hours at room temperature.
The filtered extracts were gathered for the detection of total flavonoid content. Deionized water
was used to leach enzyme-treated G.biloba leaf residue (GBLR) for two hours at room temperature
(Guo et al., 2022). After filtering, oven drying, and storage in airtight glass jars pending additional
examination, all samples were collected. With an increase in enzyme concentration, the yield of
total flavonoids extracted from G.biloba leaves a rose (Pinelo et al., 2006).

Reflux extraction of G.biloba leaves with 50% ethanol was followed by ethanol
precipitation, water precipitation, macroporous resin enrichment and purification, and ethyl acetate
extraction. The result was an aqueous phase rich in flavonoid glycosides and an ethyl acetate phase
rich in terpene lactones. Acid hydrolysis, separation of macroporous resin, crystallization, and
deacidification with an n-hexane/ethanol system were used to obtain total flavonoid aglycones
(TFA) from the aqueous phase. Total terpene lactones (TTL) were isolated from the ethyl acetate
phase via deacidification twice with activated carbon and crystallization in ethanol/water systems.
(Liang et al., 2022)

In research from Xiaoyan et al. (2018), to obtain homogeneous powders with an 80 mesh
size, dried G. biloba leaves were pulverized and sieved. The powder was then accurately weighed
and placed in a 1000 mL round bottom flask. A specific concentration of ethanol solution was
added to soak the leave powders. A heating jacket was then used to heat the flask (TC-15, Haining
Huaxing Instrument Co. Ltd, and China). After two rounds of reflux extraction, the two extracts
were combined, centrifuged at 10,000 rpm/min for ten minutes, and filtered through a 0.22 m filter.
The supernatant that resulted was collected. Total flavonoids, ginkgolide A (GA), ginkgolide B
(GB), ginkgolide C (GC), and bilobalide (BB) concentrations in the supernatant were determined.

2.2 Acetone

Second, the most commonly used solvent to extract from G.biloba leaves is acetone. G.biloba
aqueous acetone extract (G24) was then submitted to solid or liquid extraction using aqueous
acetone as an extraction solvent. The extracted fractions are then concentrated under low pressure
to remove the extraction solvent before being purified using liquid/liquid extractions. In both cases,
the organic solvent was then replaced with water, and the resulting aqueous phase was
concentrated and dried to yield the powdered extract (Stefano et. al., 2019).

Patented standardized extraction and purification processes were used to obtain extracts
with consistent compositions in pharmacologically active compounds. From these processes, some
compounds such as flavonoids, terpenes, ginkgolides, and bilobalide in G.biloba leaves with G24
extract were determined (Figure 1). To determine flavonoids in the acetone extract of G.biloba
leaves, 200 mg of the extract was dissolved in 20 mL of methanol, with the addition of the previous
solution to dilute the methanol at 100° C water bath for twenty-five minutes (Stefano et. al., 2019).
Quercetin dihydrate was used as a reference compound whereas terpene was determined by
dissolving 120 mg of extract in 10 mL with pH 5.8 of phosphate buffer solution by stirring.

Figure 1: EP current edition; Flavonoids in acetone (G24) extract (Stefano et. al, 2019)
 According to Gargouri et al. (2018), a special G. biloba leaf extract, designated as EGb
761 is one of the most commonly used natural medicinal plants based on its remarkable biological
activities. To obtain this kind of extraction from a special G. biloba leaf, different solvents were
used. EGb 761 is an investigational product that is a dry extract from G. biloba leaves (a drug-to-
extract ratio 35-67:1) with an extraction solvent of acetone 60% (w/w). The extract is adjusted to
22.0-27.0% ginkgo flavonoids calculated as ginkgo flavone glycosides, which include ((3-0-(2′′-
0-(6""-O-(p-hydroxy-trans-cinnamoyl)-ẞ-D-glucosyl)-α-L-rhamnosyl)-quercetin (FLA 269) and
3-0-(2′′-0-(6′′-0-(p-hydroxy-trans-cinnamoyl)-ẞ-D-glu- cosyl)-a-L-rhamnosyl)- kaempferol (FLA
270) and 5.0-7.0% terpene lactones, consisting of 2.8-3.4% ginkgolides A, B, C and 2.6-3.2% BB,
and contains less than 5 ppm ginkgolic acids.
2.3 Methanol

Another solvent would be methanol used in the extraction of G. biloba leaves. Extraction with
ionic liquid has been used to extract flavonoids and terpene tri lactones from G. biloba leaves
(Feng et. al., 2019). The combination of ionic liquid and methanol reflux extraction at 80◦C, which
then provided an extraction yield of 0.14%, this extraction yield was 1.4 times higher than
bilobalide extraction that used only methanol as their extraction solvent (Liu, 2020). Methanol
extraction was then obtained analytes from a qualitative analysis of twenty-seven compounds, a
quantitative analysis of six compounds which are BB, ginkgolide J (GJ), ginkgolide B (GB),
quercetin (QCT), kaempferol (KMF), and isorhamnetin (ISR) by using the ultra-high performance
liquid chromatography-tandem mass spectrometer (UHPLC-MS/MS), the ultra-high performance
liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) and an
assay of platelet aggregation (Zheng et al., 2019).

According to Wang et al. (2022), 100 mg from each 1-year-old G.biloba leaf (Y1), 4-year-
old ginkgo leaves (Y4), and 7-year-old leaves (Y7) have been obtained to dissolve in 0.6 mL of
70 % methanol and stored in the refrigerator overnight at 4◦C. The content of flavonoids in
G.biloba leaves according to various years was measured. TFA content decreased with increasing
age and the total flavonoid content in Y1-Y4 is higher than in Y5-Y7 G. biloba leaves (Figure 2.1)
Furthermore, the TFA content was significantly higher in July than in May and September. The
TFA content of 1-year-old G.biloba leaves harvested in July was 32.4 ±1.96 mg/g, which was
nearly twice that of seven-year-old G.biloba leaves (17.89 ± 1.29 mg/g) as shown in Figure 2.1.
 Figure 2.1: Total flavonoids content in different months (Wang et. al., 2022)

In July, G.biloba leaves were chosen for additional research on TFAs. The content of QCT
decreased significantly with age. The QCT content of 4 and 7-year-old G. biloba leaves were lower
by 15.1% and 38.7%, respectively, when compared to 1-year-old G. biloba leaves. The KMF
content followed a similar pattern. The content of ISR in 4-year-old ginkgo leaves was higher, at
0.71 ± 0.04 mg/g. The TFA content of 7-year-old G. biloba leaves was 6.06 ± 0.91 mg/g, which
was significantly lower than the content of 1-year-old (9.88 0.27 mg/g) and 4-year-old (8.39 ±
0.37 mg/g) G. biloba leaves as shown in Figure 2.2.

Fig. 2.2: Total flavonol aglycones (TFAs) contents of leaves of 1-, 4-, and 7-year-old G.biloba
trees. (Wang et. al., 2022)
3.0 PHARMACOLOGICAL PROPERTIES

Ginkgo biloba (G. biloba) is a traditional Chinese medicine that has been used in many different
disorders for many centuries (Zuo et al., 2017). There are some specific validations in order to
verify the effectiveness of the ailment treatments.

Liu et al. (2022) have cited that the primary chemical components of G. biloba are
flavonoids, terpene tri-lactones (TTL), and phenolic acids. Flavonoids include quercetin (QCT),
rutin, kaempferol (KMF), and isorhamnetin (ISR), while TTL includes ginkgolides and bilobalide
(BB). There are still certain crucial nutritional components left over, such as polysaccharides,
proteins, fatty acids, carbohydrates, and nucleoside compounds, among others.

Liu et al. (2022) also stated that in G. biloba leaves, the content of flavonoids is the highest
(45.60%) compared to G. biloba exocarp (2.21%) and G. biloba seeds (0.063%). However, the
exocarp of G. biloba contains the largest percentage of polysaccharides (23%) compared to the
leaves (5%) and seeds (7.68%). Additionally, the amount of ginkgoic acid (12.88%) is larger in
G.biloba exocarp than in G. biloba seeds (0.21%) and leaves (0.113%).

These substances typically have pharmacological properties that are anti-oxidant, anti-
inflammatory, neuroprotective, anti-tumor, and anti-bacterial activities.

3.1 Antioxidant effect

Antioxidants are natural substances that are capable to delay and inhibit the cell damage or
oxidation of other molecules. G. biloba extracts are rich in polysaccharides and flavonoids which
could be used as a natural antioxidant. From crude Ginkgo leaves polysaccharides (GBPSs),
GBPS-2 and GBPS-3 are two common acidic heteropolysaccharides. GBPS-2 and GBPS-3 had
good 2, 2-diphenylpicrylhydrazyl (DPPH) radical scavenging activity effects on superoxide
radicals and 2, 2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals which led to
a good antioxidant capacity (Liu et al., 2022; Joanna et al., 2020). Wang et al. (2021) also cited
that the polysaccharide from Dendrobium officinal passed an in vitro antioxidant test,
 demonstrating good DPPH radical scavenging ability as well as high hydroxyl radical scavenging
ability and metal chelating activity.

Other than that, Li et al. (2021) have stated that polysaccharides and flavonoids in G.biloba
extracts include the inhibition of lipid peroxidation and prevent oxidative stress. It also scavenges
free radicals, superoxide anions, and nitric oxide.

Table 3.1: Antioxidant activities of G. biloba

Plant part - Extract Extract Result References

Method
IC50 ABTS TPC
(TEAC)

Leaves- ethanol DPPH 0.69 mg/mL 4.0 mg/mL NA (Gong et al.,

2021)

Leaves- ethanol DPPH 64.2 μg/mL 1.545 mg 2.804 (mg (Ražná et al.,
(TEAC)/g GAE/g 2020)
FM FM)
Seeds- hot water DPPH 100.7 μg/mL, NA NA (Boateng et
al., 2021)

Leaves- ethanol DPPH 0.325 mg/mL NA NA (Sati et al.,

2018)

Leaves- ethanol DPPH 83.91 g/mL 8.0 mmol/L NA (Cao et al.,

2018)

Leaves- methanol DPPH 0.103 mg/mL 0.052 NA (Wang et al,

mg/mL 2019)
3.2 Anti-Inflammatory effect

Liu et al. (2020) found that the most specific and competitive plateplatelet-activatingor receptor
antagonist with anti-inflammatory properties was ginkgolide B (GB).

Li et al. (2021) and Boateng et al. (2021) stated that some biginkgosides have anti-
inflammatory properties. Ginkgolide A (GA) could lessen the inflammatory response brought on
by lipopolysaccharide both in vivo and in vitro and discovered that G. biloba extracts may control
inflammatory cytokines and mediators both in vivo and in vitro, thereby inhibiting inflammation.
Nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), Interleukin 6 (IL-6), prostaglandin E 2

(PGE ), inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX-2) are all
2

downregulated by ethyl acetate and chloroform sections of the ginkgo male flower and G. biloba
seed extract. (Li et al., 2021; Boateng et al., 2021). In lipopolysaccharide-induced monocyte
macrophage-like cells (RAW264.7), the primary active component flavonoid glycosides may
prevent the release of inflammatory cytokines (Li et al., 2021). Gargouri et al. (2017) found that
flavonoids in G. biloba leaf extract lower eicosanoid formation via inhibiting phospholipase
activity, protein kinases, histamine release, and transcription of genes.

3.3 Anti-cancer effect

Research indicates that G. biloba extracts have several components with Ginkgo leaf extracts,
including flavonoids and terpenoids. Additionally, it has been demonstrated that Ginkgo leaves,
nuts, and their extracts or active components have anti-lung cancer activity. G.biloba extracts may
therefore potentially be able to treat lung cancer (Liu et al., 2022).

Li et al. (2021) showed that the bioflavonoids in the male flowers of G. biloba have anti-
cancer properties. Bilobetin and isoginkgetin, two of them, had the ability to stop the cell cycle in
the G2 phase, second growth period of the cell cycle following Deoxyribonucleic acid (DNA)
replication and preceding prophase and M phase, cell division occurs during which duplicated
chromosomes are split evenly between two parent cells, specifically inhibiting the growth of HeLa
cells by activating the apoptosis-associated protein and investigation obtained the anti-cancer
action of G. biloba extracts in vitro on human gastric cancer cells SGC-7901 and MGC-803
discovered that G. biloba extracts might prevent gastric cancer metastasis in a dose-dependent
way. According to Li et al. (2021) findings, ginkgolic acid from Ginkgo exopleura can reduce the
expression of vital adipogenesis-related enzymes such as acetyl CoA carboxylase and fatty acid
synthase, hence slowing the growth of pancreatic tumor cells.

Polysaccharides derived from G. biloba leaves inhibit the multiplication of breast cancer
cells and human endometrial cancer cells. G. biloba exocarp polysaccharides (GBEP) capsules can
be used to treat gastric cancer by boosting apoptosis and inducing cancer cells and differentiation
(Zuo et. al., 2017).

The leaves of G. biloba are enriched in selenium-containing polysaccharide (Se-GBLP),

which has an antitumor impact, particularly against human bladder cancer cells, due to a change
in B-cell lymphoma 2 (Bcl-2) (Zahraa et. al., 2021)
 Table 3.2: Anti-cancer activities of G. biloba

Plant part - Bioactive Cancer cell lines Result References

Extract Compound

Leaves - Methanol Flavonoids Caspase-3 cell, Induce apoptosis of every (Boateng et.
Human prostate cell lines. al., 2021)
(P53) cell
Nuts - NA Flavonoids Caspase-3 cell Induce cell apoptosis in (Zuo et. al.,
melanoma cells. 2017)

Leaves - NA Flavonoids Caspase-3 cell Inhibit cancer cell growth (Belwal et. al.,
and when used with 2018)
anticancer medicine,
synergistic effect with
enhanced apoptic cell
concentrations was seen.
Seed - Water Polysaccharides Sarcoma 180 cancer Varying dosages of GBE (Wang et. al.,
cell can diminish drug 2019)
resistance in ascites tumour
cells, suppress tumour
development, and extend
mouse lifetime.
Leaves - Hot water Flavonoids, Human gastric Limit the growth of human (Liu et. al.,
terpene lactones cancer cell (SGC- melanoma cell lines. 2022)
7901)

Leaves - Methanol Ethyl acetate Human esophageal Induce HeLa cell death and (He et. al.,
cancer (EC-109) decrease HeLa cell growth 2020)
cell and migration.
3.4 Antimicrobial effect

Antimicrobial activity is the substances that destroy bacteria or stop them from growing or their
ability to reproduce and cause disease. G. biloba seeds extract (GBSE) and G. biloba leaves extract
(GBLE) have obvious inhibitory effects on fungi and bacteria. GBSE and GBLE inhibit a variety
of microorganisms, and the inhibitory properties of extracts made with various solvents on various
microbe kinds also vary (Liu et al., 2022; Ražná et al., 2020).

Wang et al. (2022) stated that G. biloba exocarp polysaccharides (GBEP) and ginkgolic
acids in G. biloba exocarp extracts (GBEE) have strong antibacterial effects. Ginkgolic acids could
enter the interior of bacteria for a short time and inhibit their growth by inhibiting the biosynthesis
of nucleotides and proteins. It was found that GBEE was able to alter the expression of genes
related to bacterial biofilm formation and inhibit biofilm formation, and thus exhibited
antibacterial effects. A number of plant fungi that causes disease were also inhibited by GBEE and
ginkgolic acids. The most notable effect on fungi Alternaria brassicae was seen when ginkgolic
acids were used to suppress the growth of pathogenic fungi in five different types of vegetables.

Boateng et al. (2021) said that antibiotic activity is present in a number of G. biloba seeds
(GBS) constituents, including polysaccharides, phenolic acids, ginkgolic acid, and proteins. Gram-
negative bacteria were less sensitive to the extract, typically because of the permeability barrier in
their outer membrane, which makes them more resistant to antimicrobial treatment when ginkgolic
acid is present in GBS.
 Table 3.3: Antimicrobial activities of G. biloba

Plant part- Microbe/bacteria Antimicrobial Result References

Extract Assays
Root, stem, leaf, Endophytic fungi Potato dextrose Endophytic fungi (He et al., 2020)
seed, bark agar medium strains J-1, J-2 and
tissue- ethyl J-3 producing
acetate podophyllotoxin
compound
Leaves- ethanol E. coli, K. Disc Diffusion G. biloba leaf (Ražná et al.,
pneumoniae, S. extracts did not 2020)
enteritidis, S. sonei, effectively stop E.
and Y. coli from growing.
enterocolitica
Leaves- alcohol E. coli, C. albicans, Mueller-Hinton E. coli> S. aureus> (Zhang et al.,
S. aureus and S. agar S. pneumoniae >C. 2020)
pneumoniae albicans
Extract- ethanol S. aureus and C. Agar diffusion Growth of (Hu et al., 2019)
albicans microorganisms
inhibited.
Leaves B. subtilis, M. Disc Diffusion Maximum (Priyanka et al.,
roseus, P. putida, S. antibacterial 2018)
marcescens, F. activity against the
oxysporum and T. Gram +ve bacteria
hirsuta (B. subtilis), then
Gram -ve bacteria
(P. putida) and
fungi.
Exocarp- S. aureus Anthrone-sulfuric Alter the expression (Wang et al.,
ethanol acid method of genes involved in 2022)
bacterial biofilm
formation

3.5 Neuroprotective effect

Neuroprotective refers to the malfunction of a cell's function caused by the slow loss or degradation
of its neurons and myelin sheaths over time (Liu et al., 2022).

Belwal et al. (2018) said that at a dose of 240 mg/kg/day, the leaf extract has been shown
to be effective against Alzheimer's disease by reducing the cell death brought on by Aβ.
Furthermore, G. biloba leaf extract was also found to be helpful in lowering the behavioral deficit
when tested against 6-hydroxydopamine-induced neurotoxicity in rats.

Table 3.4: Neuroprotective activities of G. biloba

Plant part- Extract Active Compound Result References

Leaves Ginkgolide B Lowering the level of (Li et al., 2021)

nitric oxide
Leaves Ginkgolide J Reducing the cell death (Zahraa et al., 2021)
brought on by Aβ

Leaves- ginkgolic Bilobalides Prevents the brain's (Lu et al., 2018)

acid phospholipase A2 from
activating and causing
cell death
3.6 Cardioprotective effect

Belwal et al. (2018) findings, through the production of nitric oxide (NO) and prostaglandin (PG),
G. biloba extracts were reported to enhance blood flow, prevent hypoxia and platelet aggregation,
improve blood rheology, and reduce capillary permeability. The cardioprotective efficacy of G.
biloba leaves extract (GBLE), terpenoids (ginkgolide A and B), and a terpene-free extract on
isolated ischemia and reperfused rat hearts was investigated and the result obtained was G. biloba
extracts and isolated terpenoids both delayed the onset of contracture during ischemia and
postischemia and enhanced functional recovery (Belwal et. al., 2018).

G. biloba seeds (GBS) have a hypocholesterolemia impact by blocking lipid metabolism

(Boateng et al., 2021). According to Boetang et al. (2021) findings, the GBS might regulate blood
cholesterol levels by affecting Apolipoprotein B production and low-density lipoprotein receptors
in the liver. In the mouse feeding research, GBS supplementation lowered hepatic cholesterol
levels while raising serum cholesterol levels when compared to the control group. The lipid-
soluble fraction had reduced hepatic cholesterol, while the water-soluble fraction had greater
serum cholesterol. As a result, the lipid-soluble part of GBS could be used to prevent
cardiovascular diseases (Boateng et al., 2021).

3.7 Anti-radiation effect

Li et al. (2022) cited that by using X-ray radiation, G. biloba extract was able to minimize the
micronucleus rate of bone marrow polychromatic erythrocytes and the chromosomal aberration
rate of bone marrow cells. Li et al. (2021) found the anti-radiation effects of Ginkgo leaves and
Ginkgo blossoms extract on C57BL/6J mice irradiated with 60 Co rays. Both leaves and Ginkgo
flower extract could increase the average survival time of irradiated mice, but Ginkgo flowers
performed better and greatly enhanced the recovery of irradiated mice's peripheral blood cell level.
3.8 Anti-obesity effect

G. biloba leaves polysaccharide (GBLP) reduces weight gain and insulin resistance by lowering
blood glucose and insulin levels (Fang et al., 2020).

3.9 Anti-depressive effect

Fang et al. (2020) conclude that depression is a frequent and long-lasting mental disorder that
affects mood and emotions, causing global economic losses. Gut microbiota, particularly
probiotics, have been shown to influence depression onset and therapy, and polysaccharides have
anti-depressive effects by modulating the microbiota-gut-brain axis. G. biloba seeds (GBS)
decreased immobility periods and anxiety-like behavior in the tail suspension test and open field
test in unpredictable chronic moderate stress treatment mice, which was equivalent to the
antidepressant paroxetine (Fang et al., 2020).
4.0 CONCLUSION

In conclusion, the review has shown various methods of extraction from different locations of
G.biloba such as leaves and seeds, and the pharmacological properties of G.biloba. G.biloba
extract was rich in bioactive compounds that can be used as biomedicine to treat various
treatments. Mainly the leaves and seeds of G.biloba exhibit many active compounds such as
flavonoids, ginkgolide, and bilobalide that can benefit in therapeutic effect on various cancers.
Among all those extraction methods, reflux was the most efficient extraction method to extract
leaves and seeds of G.biloba and also for obtaining its pharmacological properties. The reflux
method using ethanol, acetone, and methanol yields a higher percentage of bioactive compounds
that have pharmacological properties such as antioxidant, anti-inflammatory and anti-tumor.
5.0 RECOMMENDATION FOR FUTURE DIRECTION RESEARCH

Ginkgo Biloba (G.biloba) known worldwide for ages for its medical properties that provide
humans with a variety of benefits. Much research has been done through various methods to prove
this fact but there are still shortcomings. Some questions have emerged through this review, and it
will enlighten us for the following research on G. biloba. Future research will need to offer
additional evidence for the medical properties of G. biloba so that it can be used. This paper
systematically reviewed the extraction and pharmacological properties of G. biloba.

From this research, we found that ethanol is the most common solution and useful for the
extraction of G. biloba. Ethanol was used in the reflux extraction method. Reflux is one of the
suitable methods for future research since it results in a high percentage of bioactive compounds.
The limitation of our research is that we only cover some parts of G. biloba, which are the leaves
and the seeds, as we lack time and resources. Future research may cover more parts of G. biloba,
such as nuts and exocarp. G. biloba nuts are said to be rich in flavonoids, starch, lipids, ginkgo
polysaccharide, and Vitamin C and have proven to have anti-cancer, anti-inflammatory, and
antioxidant bioactivities in modern research (Liu et al., 2020) but what kind of extraction method
can be used. Additionally, more solutions and extraction procedures should be discovered other
than the four extraction procedures that were used (maceration, reflux, shaker, and soxhlet) to get
various kinds of accurate results. More chromatographic methods for extraction can be used such
as high-performance thin-layer chromatography (HPTLC). HPTLC is one of the most useful
methods for analysis in pharmaceutical industries as it is the only method in chromatography that
results in an image form. It is also less costly.

Research indicates that G. biloba is rich in pharmacological properties such as antioxidant,

anti-inflammatory, anti-cancer, and antimicrobial effects. However, there may be undiscovered
pharmacological properties. Future research may focus on this since pharmacological properties
have many benefits and are important for medicine. More research on anti-obesity and anti-
depressive effects can be discovered as only a few research have been done on these two properties.
 The various phytochemical categories are essentially the same across G. biloba. In these
three components, the same components have distinct contents, though. The pharmacological
effects of G. biloba leaves, seeds, and exocarp are very diverse from one another, which may be
caused by the various contents. Studying monomeric substances like flavonoids, terpene lactones,
and ginkgolic acids have allowed many studies to try and determine the basis of the active
ingredient. It is much more unlikely to explain the various pharmacological properties of the
leaves, seeds, and exocarp of G. biloba because these chemicals are virtually exclusively found in
these three plant components. Since the Ming Dynasty of China, the leaves and seeds of G. biloba
have been mentioned as medicinal plants in the ancient books Diannan Bencao’ and ‘Compendium
of Materia Medica’. As a result, a lot of research has concentrated on these two components of G.
biloba.

According to historical usage records and current scientific studies, these two G. biloba
plant components are currently mostly used to make healthful foods and beverages. However, due
to its poisonous nature and bad smell, the exocarp of G. biloba is typically regarded as industrial
waste and has no known traditional usage. Statistics show that China discards at least 75,000 tons
of fresh exocarp annually, which results in significant resource loss as well as soil and water
contamination. It is important to find a solution for how to utilize the exocarp resource of G. biloba
to its full potential. Because of its toxicity, the high ginkgolic acid content of G. biloba exocarp is
the main factor limiting its usefulness as a medicine. According to studies, n-hexane extraction
can remove the majority of ginkgolic acids compared to ethanol, methanol, ethyl acetate, and
petroleum ether. The low effectiveness and use of harmful chemicals are just two of the many
drawbacks of this technology. Thus, a sustainable, effective, and secure technique to eliminate
ginkgolic acids is required. Additionally, the exocarp of G. biloba is a source of pharmacologically
potent substances including ginkgolides and biflavonoids. An effective technique for enhancing
macroporous adsorption resins with high-purity biflavonoids on a wide scale and targeted basis
from the exocarp of G. biloba. This suggests that it has enormous potential as a source of the active
components in G. biloba.
 The lethal G. biloba endotoxins 4’-methoxy pyridoxine (MPN) and 4’-O-methyl
pyridoxine (MPNG) are frequently found in the leaves, seeds, and exocarp of G. biloba. As a
common nut, they have highly restricted G. biloba seeds. Heat treatment is a common, low-cost
processing approach for removing poisonous MPN from G. biloba seeds. However, it is ineffective
in removing MPNG. MPN and MPNG can be removed from G. biloba seeds more efficiently with
enzymatic hydrolysis when paired with resin adsorption but the production cost will go up and
there may be some unidentified security risks. Therefore, creating straightforward and effective
detoxification techniques will greatly aid in utilizing G. biloba's resources to their utmost potential.
(Liu et al., 2022)
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