ARIZONA STATE UNIVERSITY
Phenolic Compounds from Methanol Extract Spondias dulcis and Their Antioxidant
Free Radical Scavenging Method
JEFF. M
Background
One of the thriving plants in Indonesian soil is the kedondong or Spondias
dulcis. This plant is referred to as a "living pharmacy" because parts of it can be
processed into effective medicines for treating or preventing diseases. Species
within the Spondias genus contain various secondary metabolites such as
phenolics, tannins, flavonoids, sterols, triterpenes, and saponins. One notable
secondary metabolite in S. dulcis is phenolic compounds, which have a benzene
ring with a hydroxyl group attached. Phenols act as antioxidants by scavenging
reactive oxygen species. The phenolic content is a crucial indicator of the
antioxidant properties of plant extracts. Oxidative compounds or free radicals can
cause cellular damage and even death, as they seek stability by extracting electrons
from biomolecules like proteins, lipids, or DNA. According to the Indonesian FDA
regulation No.38 of 2013 on the maximum limits for antioxidant food additives, the
acceptable daily intake (ADI) for synthetic antioxidants such as Butylated
Hydroxyanisole (BHA) is 0-0.5 mg/kg body weight, while Butylated Hydroxytoluene
(BHT) is 0-0.3 mg/kg body weight. The use of these synthetic antioxidants is
controversial as they may cause adverse effects such as asthma, angioedema,
dermatitis, excessive sweating, joint pain, and gastrointestinal or ocular diseases.
The antioxidant activity of phenolic compounds in methanol extract from S.
dulcis can be assessed using various methods, one of which is the free radical
scavenging technique employing DPPH. In this method, DPPH acts as a source of
free radicals that antioxidants inhibit, resulting in a color change from purple to
yellow. Absorbance is measured using UV-Vis spectrophotometry, and antioxidant
properties are quantified by the Inhibition Concentration (IC50) value. For instance,
the IC50 for methanol extract from S. mombin leaves was found to be 8
μ
g/mL,
close to the standard vitamin C with an IC50 of 9.4
μ
g/mL. Phenolic compounds
detected include galocatechin, kaempferol, quercetin glycosides, and gallic acid.
The antioxidant activity of ethanol extract from S. purpurea bark yielded an IC50 of
6.26
μ
g/mL, nearing the positive control, ascorbic acid, with an IC50 of 9.85
μ
g/mL.
Raw fruit of S. pinnata showed better antioxidant activity compared to ripe fruit in
DPPH radical scavenging tests, with IC50 values of 72.23
μ
g/mL and 97.5
μ
g/mL,
respectively, correlating with higher total phenolic content in raw fruit (384 mg
GAE/100 g) versus ripe fruit (243 mg GAE/100 g). Methanol extract of S. mombin
leaves and fruit demonstrated antioxidant activities with IC50 values of 5.31
μ
g/mL
and 3.92
μ
g/mL, respectively, and the methanol extract had the highest phenolic
content among tested solvents. Ethanol extract of S. dulcis leaves exhibited strong
antioxidant activity with an IC50 of 32.83
μ
g/mL, and methanol was chosen for its
low toxicity compared to chloroform or dichloromethane solvents. The antioxidant
activity of methanol extract from S. purpurea bark had a higher IC50 value of 8.3
μ
g/mL compared to fruit with an IC50 of 280
μ
g/mL. Ethanol extract of S. dulcis
leaves, containing polar phenolic compounds, was evaluated using DPPH with an
IC50 of 13.687
μ
g/mL. The Spondias genus is noted for its pharmacological benefits
beyond antioxidant activity, including cytotoxic, anti-ulcer, diuretic,
hepatoprotective, anti-inflammatory, analgesic, and antihypertensive properties.
Methanol extract of Spondias is noted for containing methyl gallate with potential
for breast cancer treatment, while S. tuberosa showed promising antibacterial
activity. Isolation of compounds from S. dulcis leaves using ethyl acetate resulted in
isorhamnetin with an IC50 of 365.98
μ
g/mL against A549 cancer cells.
Protocatechuic acid isolated by Rudiana et al. exhibited strong antioxidant activity
with an IC50 of 20.97
μ
g/mL, and scopoletin, with an IC50 of 593.14
μ
g/mL, was
tested for antioxidant activity. This study involved isolating and characterizing
phenolic compounds from methanol extract of S. dulcis leaves using various
chromatographic methods, determining chemical structures with UPLC-MS/MS and
NMR, and evaluating antioxidant activity using DPPH free radical scavenging.
Literature Review
Spondias dulcis
The Kedondong plant (Spondias dulcis) is native to South Asia and Southeast
Asia and is widespread in tropical regions. Known in English as ambarella, otaheite
apple, or great hog plum, it is called kedondong in Indonesia and Malaysia, hevi in
the Philippines, gway in Myanmar, and makak farang in Thailand. The plant
typically grows to a height of 12-18 meters, with a smooth bark surface that has
irregular cracks and ranges in color from gray to pale reddish-brown. It exudes a
sticky, transparent sap with a turpentine-like quality. The flowers are white and lack
stalks, while the fruit is yellow, fleshy, and has a hard, serrated seed with a fibrous
surface. The fruit ripens from November to July and can be harvested daily
regardless of weather conditions. There are two distinct types of fruit: large and
mini or dwarf. The fruit is oval-shaped, measuring 5-10 cm in length, with a thin,
hard skin. When ripe, the fruit is green, transitioning to a golden yellow color. A thin
white layer may cover some areas of the skin. The average weight of the fruit ranges
from 150-240 g. Its flesh is juicy, fibrous, and tart. The seeds resemble viruses due to
the various fibers on their surface. The spiky projections from the fibers into the
mesocarp make the flesh difficult to cut.
Bioactivity of spondias genus
The main components found in the kedondong fruit include carbohydrates, which
make up 44-46 kcal/100 g, providing essential energy, and a high water content of
87-90%, which aids in digestion and prevents dehydration. Kedondong leaves
contain flavonoids, saponins, steroids, and tannins, which contribute to their use in
preventing cancer, premature aging, heart disease, diabetes, and cholesterol due to
their antioxidant properties. Additionally, kedondong is employed to enhance
vision and treat eye infections. Extracts from Spondias tuberosa leaves have been
shown to contain phenolic compounds beneficial for anti-inflammatory purposes,
significantly reducing inflammation, leukocyte count in the peritoneal cavity, and
myeloperoxidase activity. Furthermore, two ergosterol triterpenes (SP-40, SP-60)
isolated from the bark of Spondias pinnata were tested for antimicrobial activity
against the resistant strain Pseudomonas aeruginosa MTCC 8158 using an agar disc
diffusion method, resulting in an inhibition zone of 15 mm for streptomycin at a
concentration of 100
μ
g/disc, with SP-40 showing a 20 mm inhibition zone.Ghate et
al. (2013) found that methanol extracts from S. pinnata stems can inhibit cytotoxic
activity in lung (A549) and breast (MCF-7) adenocarcinoma cells. Chauduri et al.
(2015) described that ethyl acetate extracts from S. pinnata produce gallic acid and
methyl gallate compounds, which have potential for inducing effects in
promyelocytic leukemia HL-60RG cells and glioblastoma (U87) cells. Asuquo et al.
(2013) demonstrated that aqueous extracts of S. mombin leaves could enhance
learning and memory in Wistar rats due to observed structural changes in the
cerebral cortex. Ishola et al. (2018) investigated the antioxidant and in vivo
protective effects of hydroethanol extracts from S. mombin leaves against
scopolamine-induced cognitive and memory decline. Das et al. (2013) tested the in
vitro antihypertensive activity of aqueous extracts from S. pinnata fruit, showing
50% inhibition of angiotensin-converting enzyme (ACE) derived from rabbit lungs.
According to Shetty et al. (2016), both ethyl acetate and methanol extracts from S.
pinnata stems exhibit hepatoprotective effects in vivo in rats treated with CCl4,
attributed to flavonoid compounds, indicating potential liver protection. Hazra et al.
(2013) showed that methanol extracts of S. pinnata increase antioxidant enzyme
levels while inhibiting lipid peroxidation, protein oxidation, and liver fibrosis, with
reduced serum enzyme and ferritin levels, suggesting its potential as an iron
chelator for iron overload conditions.
Free radicals
Free radicals come in various forms and can originate from everyday activities;
these radicals are closely associated with antioxidants. To mitigate the negative
effects of free radicals, the human body requires antioxidants. Free radicals are
compounds with unpaired electrons, making them seek electrons from other
cellular components like proteins, fats, or DNA to stabilize themselves. This process
can lead to cancer, premature aging, or heart disease due to diminished bodily
defenses. Free radicals form through three stages: initiation, where homolytic
cleavage generates radicals; propagation, where the number of radicals increases;
and termination, when two radical molecules combine to form a stable molecule.
Excessive production of free radicals can disrupt the body's stable equilibrium,
leading to oxidative stress, which is linked to conditions such as diabetes mellitus,
neurodegenerative disorders (e.g., Alzheimer
’
s disease), cardiovascular diseases
(e.g., hypertension), and respiratory issues (e.g., asthma). Free radicals can be
endogenous, arising from within the body, or exogenous, resulting from external
sources like pollution, microbial infections, or chemicals. Endogenous free radicals
occur through cellular metabolism, such as excessive exercise leading to xanthine
oxidation. On the positive side, free radicals help form white blood cells to combat
viruses or bacteria entering the body through inhalation or digestion. They play
beneficial roles including regulating blood flow through arteries, maintaining focus
in the brain, aiding in immune defense by producing radicals to destroy bacteria
and foreign cells, signaling gene activation or deactivation, and producing radicals
like nitric oxide and superoxide to poison viruses and bacteria. Some free radicals
even target and kill cancer cells, and certain cancer treatments aim to increase free
radical levels in the body.
Antioxidants
Antioxidants are compounds that can slow down or prevent cellular damage
caused by free radicals. They are classified into three main types based on their
function: primary antioxidants, which prevent the formation of new free radicals,
such as transferrin, ferritin, and albumin; secondary antioxidants, which neutralize
free radicals and halt their formation, including Superoxide Dismutase (SOD),
Glutathione Peroxidase (GPx), and catalase; and tertiary antioxidants, which repair
damaged tissues resulting from free radical effects, such as methionine sulfoxide
reductase, DNA repair enzymes, proteases, transferases, and lipases. Antioxidants
also come from different sources: synthetic antioxidants, commonly used in food
products, include BHA, BHT, propyl gallate, and Tertiary-Butyl Hydroquinone
(TBHQ); whereas natural antioxidants are derived from plant parts like wood, bark,
roots, leaves, fruits, flowers, seeds, and pollen, and include vitamins A, C, and E, as
well as phenolic compounds. Phenolic compounds, as secondary metabolites, are
produced in small amounts and do not directly contribute to growth, development,
or reproduction but play a role in responding to foreign substances. They are
essential for plant growth and reproduction, acting as a defense mechanism
against pathogens and stress. Phenolic compounds can also provide specific colors,
flavors, and aromas to plants, such as anthocyanins for red color in apples, eugenol
for banana aroma, and flavanones for bitterness. The antioxidant mechanism of
phenolic compounds involves the phenol group pairing with free radicals by
donating a hydrogen atom through electron transfer, converting the phenol into a
phenoxyl radical.
The free radical scavenging method
The free radical scavenging method describes antioxidant activity based on the
principle that compounds can inhibit oxidation reactions by binding with free
radical molecules, thereby preventing cellular damage, particularly to cell
components such as DNA, brain cells, and skin tissue. Several antioxidant testing
methods are available to measure antioxidant activity, one of which is the 2,2-
diphenyl-1-picrylhydrazyl (DPPH) method. The DPPH radical is an unstable nitrogen
-containing organic compound used to assess antioxidant activity. The DPPH
method quantitatively measures antioxidant activity by capturing DPPH radicals,
which is then evaluated using a UV-Vis spectrophotometer. The extent of inhibition
by antioxidants is expressed as IC50 (Inhibitor Concentration), indicating the
concentration required to capture or reduce 50% of the free radicals.
Isolation of Phenolic Compounds
Isolation of Phenolic Compounds involves extracting secondary metabolites from
plants, which are present in minute quantities, requiring the use of large sample
sizes. To obtain pure compounds, the isolation process must be conducted under
appropriate conditions. Pure compounds are achieved through extraction and
chromatography techniques. Chromatography is a method for separating mixtures
using a mobile phase and a stationary phase. It can be categorized into partition
chromatography, which deals with the distribution of solutes between two
immiscible solvents, and adsorption chromatography, which involves reversible
specific interactions between solute molecules and binding sites on the stationary
phase matrix.
Thin Layer Chromatography (TLC)
Thin Layer Chromatography (TLC) is utilized to separate components based on their
adsorption or partition differences between the stationary and mobile phases. The
TLC plates used for analysis typically have a thickness of 0.1-0.2 mm and measure
20 cm x 20 cm. These plates are usually made of glass or aluminum coated with
silica Kieselgel 60 F254. The retardation factor (Rf) is a parameter used to describe
the migration of compounds in TLC, representing the position of the spot on the
stationary phase after elution. The Rf value is calculated as the ratio of the distance
traveled by the analyte to the distance traveled by the eluent. TLC analysis usually
involves applying very small amounts of the sample onto the plate, which is then
placed vertically in a chamber filled with the mobile phase. The solvent moves up
the plate by capillary action, carrying the sample components along with it and
facilitating separation. To detect natural compounds, UV light is used due to its non
-destructive nature, allowing further analysis of the sample. Detection of spots on
the TLC plate can be done visually using UV light, typically at wavelengths of 254 nm
and 366 nm, as aromatic conjugated compounds and some unsaturated
compounds absorb UV light. These compounds can be analyzed using TLC with
plates impregnated with fluorescence indicators, and detection is carried out under
UV light.
Vacuum Liquid Chromatography (VLC)
Vacuum Liquid Chromatography (VLC) is a technique that employs silica gel as the
adsorbent, with the advantage of time efficiency due to the use of vacuum, and the
capability to separate larger quantities of samples compared to column
chromatography. The choice of silica gel is crucial for achieving effective separation
results; excessively small particle sizes can significantly slow down the elution
process. The selection of solvents in VLC can be determined through literature
review, application of Thin Layer Chromatography (TLC) data to column
separations, and using eluents ranging from non-polar solvents that do not move
the solute to polar solvents that do. Elution can be performed using either a
gradient solvent method or an isocratic system. Gradient elution involves using a
moving phase with varying polarities from the lowest to the highest, allowing for
the separation of compounds with different polarities. The sample can be dissolved
in an appropriate solvent or mixed with the adsorbent (impregnation) and then
placed at the top of the column to be slowly drawn through.
Column chromatography
Column chromatography is a preparative separation technique based on
adsorption. In this method, the sample is placed at the top of the column, and the
eluent or solvent is poured into the column. Due to gravity, the eluent flows
downward through the sample, facilitating the separation process. One drawback
of this technique is the need for a substantial amount of eluent, long elution times,
and the necessity of additional detection methods such as Thin Layer
Chromatography (TLC) for analyzing the separation results. The success of column
chromatography largely depends on the choice of adsorbent, eluent, column
dimensions, and elution rate.
Mass spectrometry
Mass spectrometry, specifically Ultra Performance Liquid Chromatography Tandem
Mass Spectrometry (UPLC-MS/MS), is an analytical instrument that combines liquid
chromatography for separating analyte components within a sample with mass
spectrometry as the detection method. The LC-MS system includes a solvent
reservoir, pump, injector, column, detector, data collection components, and small
-diameter tubing to connect all liquid components before entering the mass
spectrometer. UPLC-MS/MS can produce distinct fragmentation patterns from
parent ions and separate daughter ions for identification processes. Its applications
are broader compared to Gas Chromatography-Mass Spectrometry (GC-MS)
because UPLC-MS/MS is not limited to volatile molecules (typically with molecular
weights under 500 Da) but can also measure highly polar analytes with relatively
simple sample preparation and without the need for derivatization techniques.
Nuclear Magnetic Resonance (NMR)
Nuclear Magnetic Resonance (NMR) spectroscopy is based on the absorption of
radio waves by certain nuclei within organic molecules. Atomic nuclei can be
categorized into two types: those with spin and those without. Both 1H-NMR and
13C-NMR provide information on the types, quantities, and environments of
hydrogen and carbon atoms in a compound. NMR is currently capable of analyzing
natural compounds even in very small amounts, such as a few micrograms. The
sample is dissolved in an appropriate solvent (like CDCl3 or D2O) and placed in a
small glass container or tube between two electromagnetic poles. The sample is
then irradiated with radio waves at a specific electromagnetic frequency (Rf). As the
nuclei interact with the radio waves at this frequency within the applied magnetic
field, they spin due to the energy absorbed from the radio waves. The amount of
energy required for this spinning depends on the type of nucleus and the strength
of the magnetic field. A detector measures the amount of radio wave energy
absorbed during the interaction with the sample, and the detected data is sent to a
recorder.
A UV-Vis spectrophotometer
A UV-Vis spectrophotometer is a device used to measure absorption in a solution
based on the Lambert-Beer Law. Lambert's Law states that absorbance is directly
proportional to the thickness of the sample holder (cuvette), while Beer
’
s Law
states that absorption is directly proportional to the concentration of the substance
(amount of molecules). Combining these principles, the Lambert-Beer Law shows
that absorption is proportional to both the concentration and the path length of the
sample. Therefore, concentration can be determined from the path length and
absorption, as expressed by the equation A = a.b.c, where A is absorbance, a is
absorptivity, b is the cuvette thickness, and c is concentration. A hydrogen or
deuterium lamp is used for UV measurements and a tungsten lamp for visible light
measurements. The wavelength of light is separated by a wavelength separator
such as a prism or monochromator, which then passes through and is absorbed by
the sample. The amount of absorbed light is measured by a detector as absorbance.
UV spectrum characteristics for flavonoids, coumarins, phenolic compounds, and
alkaloids show absorption in the blue region (450-550 nm). For instance, scolopetin
absorbs at wavelengths of 230 nm and 345.5 nm.
Method
Equipment: The equipment used includes glassware and vial bottles, column
chromatography apparatus, a chamber, and a rotary vacuum evaporator
(Heidolph). Instruments employed are a UV-Vis spectrophotometer (Shimadzu
Genesys 10S), an NMR spectroscopy device (Agilent) operating at 500 MHz (1H) and
125 MHz (13C), and an UPLC-MS/MS spectroscopy system (Acquity UPLC® H-Class
System, BEH C18, Xevo G2-S Qtof).
Materials: The materials used consist of methanol extract of Spondias dulcis leaves
from previous research (Rudiana et al., 2023), technical grade organic solvents such
as methanol, ethyl acetate, n-hexane, acetone, and chloroform, DPPH (Himedia),
silica gel TLC plates 60 F254 0.25 mm (Merck), silica gel powder with specifications
G60 (35-70 mesh) and GF254 (200-400 mesh), water, ammonium formate,
acetonitrile, and formic acid.
Work procedure
The procedure for isolating phenolic compounds from the methanol extract of S.
dulcis leaves involves several chromatographic methods: thin layer
chromatography (TLC), vacuum liquid chromatography (VLC), and gravity column
chromatography (GCC). The methanol extract is first diluted slightly with acetone,
spotted on the TLC plate, and developed with a solvent or a mixture of organic
solvents that provides good separation. After the solvent has ascended to the top of
the plate, it is removed from the chamber, dried, and analyzed under UV light to
detect phenolic compounds at
λ
254 nm and
λ
366 nm. In the VLC step, 20 g of the
methanol extract is dissolved in acetone and impregnated with silica G60 at a 1:1
ratio. The sample is then separated using VLC with silica gel GF254 as the stationary
phase, with a sample-to-silica ratio of 1:10, and eluted gradiently with n-hexane and
ethyl acetate. The eluents are collected in 100 mL glass bottles, concentrated using
a rotary evaporator, and analyzed by TLC as described previously. Fractions with
similar TLC patterns are combined and weighed. The GCC method involves packing
a column with cotton at the bottom, followed by silica G60 mixed with n-hexane,
and then loading the acetone-dissolved and G60-impregnated sample. The sample
is eluted gradiently with a suitable solvent. The column eluates are collected in 10
mL vials, evaporated, and analyzed by TLC. Fractions with identical TLC spots are
combined until a phenolic compound isolate is obtained.
The purity test
The purity test of the isolate is conducted to determine whether only a single spot
appears on the TLC plate. Two methods have been employed for this testing. The
first method, Multi-Eluent, involves spotting the sample on a 1x5 cm plate and
performing elution with different eluents. The first eluent used is ethyl
acetate:acetone (9:1), followed by ethyl acetate:methanol (8:2), and the final eluent
is ethyl acetate:acetone:methanol (6:2:2). After elution, the TLC plate is dried and
examined under UV light at
λ
254 nm and 366 nm. If a single spot is observed, it
suggests successful isolation. The second method, 2D TLC, involves spotting the
sample in one corner of a 5x5 cm plate, about 1 cm from the edges. The plate is first
developed for around 15 minutes using ethyl acetate:methanol (8:2) as the mobile
phase. After drying, the plate is rotated 90 degrees and placed in a chamber with
the second mobile phase, ethyl acetate:acetone (7:3). The components separated
during the first development form spots that serve as the origin for the second TLC
separation. After the second elution, the plate is dried and inspected under UV light
at
λ
254 nm and 366 nm. A single spot indicates successful isolation.
Result
Qualitative Testing of Phenolics in Methanol Extract of S. dulcis Leaves
Qualitative phenolic testing was conducted to detect the presence of these
compounds in the methanol extract of S. dulcis leaves. The mobile phase is deemed
suitable if it shows a good separation pattern on the TLC plate. TLC was performed
by varying the composition of the eluent with different volume ratios. A
chromatogram with a good separation pattern indicates that the eluent
composition will be used as the mobile phase in subsequent chromatography
processes. In this study, a mixture of n-hexane and ethyl acetate (3:7) was used as
the mobile phase, as shown in Figure 8, with UV light at different wavelengths
revealing a target compound with an Rf value of 0.60 (marked by a red circle). The
qualitative test results with this eluent showed a non-overlapping pattern,
suggesting effective separation. Effective separation means that the compounds in
the extract are distinctly separated, as evidenced by non-overlapping spots and
adequate spacing between them in the chromatogram, which is important for
successful column chromatography. Figure 8 indicates the presence of a suspected
conjugated aromatic compound (highlighted by a red circle), identified as phenolic
due to its blue fluorescence under UV light.
The methanol extract of S. dulcis leaves
The methanol extract of S. dulcis leaves, obtained through maceration, was
subjected to fractionation to separate phenolic compounds from other substances
present in the leaves. The initial fractionation was performed using liquid column
chromatography due to the large sample size (40 g). The vacuum pump system
allowed for quicker extraction of the sample. The sample was homogenized with
silica G60 and introduced into a column packed with compressed silica GF254. The
purpose of this impregnation was to facilitate the sample's entry and ensure its
even distribution for effective fractionation. Silica GF254 must be free of cracks to
avoid affecting the separation results. The fractionation results were collected and
categorized based on color spots observed through thin-layer chromatography
(TLC). Different colors in the spots indicated the presence of different compounds,
and the collected fractions were then concentrated using a rotary evaporator.
