ARIZONA STATE UNIVERSITY
"Antidiabetic Activity of Malaka Leaf Extract and Fractions Assay"
JEFF M.
Background
Metabolic disorders or conditions where blood glucose levels rise beyond the
normal range are referred to as diabetes mellitus. This condition, also known as
hyperglycemia, is caused by impaired insulin secretion or abnormal insulin function.
Chronic hyperglycemia in diabetes is associated with long-term microvascular
complications, particularly affecting the eyes, kidneys, and nerves, and also
increases the risk of cardiovascular diseases. The number of diabetes cases grows
each year, with global prevalence expected to reach 578 million people by 2030.
Management of blood glucose levels in diabetes patients involves both
pharmacological and non-pharmacological therapies. Physical activity and medical
nutrition therapy are examples of non-pharmacological approaches, while oral or
injectable hypoglycemic drugs are part of pharmacological treatment. Alpha-
glucosidase inhibitors, a type of oral hypoglycemic medication, can be developed
using natural ingredients. Natural ingredients are categorized as traditional
medicine and can serve as an alternative, more efficient treatment for diabetes
mellitus. The use of traditional medicine has been prevalent since ancient times
due to its perceived safety and relatively lower side effects compared to modern
drugs. The malacca plant (Phyllanthus emblica), from the Euphorbiaceae family
and native to tropical Southeast Asia, is widespread in many subtropical and
tropical countries, including Indonesia, India, China, and Malaysia. The malacca
plant holds potential as a traditional medicine for antidiabetic activity. Malacca
leaves are beneficial for treating asthma, leucorrhea, bronchitis, fever, and vomiting,
as well as aiding conditions related to poor digestion and chronic dysentery. In
India, it has even been used to treat cancer, diabetes, liver issues, heart problems,
and anemia. The utilization of malacca leaves is due to their secondary metabolite
content. Both ethanol extracts and simplicia contain compounds such as flavonoids,
phenolics, tannins, quinones, saponins, monoterpenes, and sesquiterpenes.
Research by Nath and Dhivya in 2019, using acetone as a solvent, revealed the
presence of secondary metabolites, including flavonoids, saponins, sterols,
terpenoids, and tannins. In contrast, using petroleum ether as a solvent showed
positive results for alkaloids, carbohydrates, glycosides, steroids, flavonoids,
saponins, tannins, and proteins. Another study by Gupta et al., in 2014,
demonstrated that the isolation of compounds from malacca leaves resulted in
quercetin in the ethanol extract and
β
-sitosterol in the ethyl acetate extract, both
exhibiting antioxidant and antidiabetic effects. According to Srinivasan et al., in
2018, quercetin is a potent antidiabetic and antihyperglycemic agent, mediated by
changes in glucose, cholesterol, and triglyceride levels, as demonstrated in both in
silico and in vivo studies. The research by Fauzi et al., in 2018, showed that the
aqueous fraction of malacca leaves could inhibit
α
-glucosidase activity, with an
IC50 value of 0.87% w/v, compared to 0.78% w/v for acarbose. The hydro-methanol
extract of malacca leaves, as studied by Nain et al., in 2012, effectively normalized
disrupted antioxidant status in streptozotocin-induced diabetic rats. Oral
administration of 400 mg/kg extract significantly improved impaired glucose
tolerance in diabetic rats with altered plasma insulin levels. Therefore, malacca
leaves have potential as an antidiabetic agent, warranting further exploration of
their antidiabetic activity using methanol extracts. Each extract and fraction should
be tested for phytochemicals, total phenolics, and total flavonoids. The extract or
fraction with the best antidiabetic activity will likely have its compounds identified
using Ultra Performance Liquid Chromatography Tandem Mass Spectrometry
(UPLC-MS/MS).
Literature Reviev
Malacca Plants
The Malacca plant, known as kimalaka in Indonesia, is also referred to by various
names across different regions, such as "bak rem" in Aceh, balakka in North
Sumatra, metengo in Ternate, and kemloko in Java. In English, it is commonly
called Indian gooseberry, while in Malaysia it is known as popok melaka, in
Germany as amla, in Thailand as ma-khampom, and it is also known by names like
amla and aonla in India. Morphologically, the Malacca plant is a small to medium-
sized tree with greenish-yellow flowers and greenish-gray bark. The bark turns
brown and flakes off in irregular pieces. Its branches can extend up to 40 cm, with
over 100 small leaves arranged along them. The leaves are hairy, oblong-linear,
with rounded bases and blunt or pointed tips, measuring 1.25 to 2 cm in length and
only 3 mm in width, with fine venation. The fruit is soft, green, and fleshy, with a
sour and astringent taste, round in shape, and shiny, with a diameter ranging from
1.8 to 2.5 cm, turning pale yellow or brick red when ripe. The Malacca plant is
renowned for its nutritional value, containing various chemical compounds such as
tannins, mucic acid, amino acids, alkaloids, flavone glycosides, phenolic glycosides,
flavonol glycosides, phenolic acids, sesquiterpenoids, norsesquiterpenoids, and
carbohydrates. The fruit, roots, and leaves are rich in polyphenols, with the roots
containing flavonoids, and the leaves containing both flavonoids and saponins.
Malacca Leaf Bioactivity
Ethanol extract of malacca leaves at doses of 250 mg/kg BW, 500 mg/kg BW, and
750 mg/kg BW demonstrated a reduction in total blood cholesterol levels, with a
14.0% decrease observed at the 750 mg/kg BW dose, making it highly effective in
anti-obesity activity in male white rats, with Orlistat used as a positive control. This
reduction in total blood cholesterol is attributed to the flavonoid compounds
present in malacca leaves. Ethanol extract at a concentration of 1000 mg/kg
contains a total flavonoid content of 35.838 mg/g quercetin and has been shown to
lower blood glucose levels in alloxan-induced rats. Additionally, malacca leaf
extract in an alcohol solvent exhibited significant activity against *Staphylococcus
aureus* at 20 mg/ml with an inhibition zone diameter of 22 mm. FTIR analysis
based on detected wavelengths identified functional groups C-OH, C=O, C-H, and O-
H, indicating that malacca leaf tea contains flavonoid compounds, which are
organic substances with antioxidant properties that combat free radicals. Duncan's
test showed that the antioxidant activity of malacca leaf tea using the indirect
sunlight method was 79.05%, significantly different from the drying method, which
yielded 68.99%, with the highest average total phenol content obtained from the
drying method being 71.08 mg gallic acid/50 mL, significantly different from the
indirect sunlight method, which was 50.31 mg gallic acid/50 mL.
Extraction Method
Extraction is defined as the process of separating a soluble substance from a
material by using a solvent, based on the physical properties of the material to be
extracted, whether it is liquid or solid. Solid particles are dispersed in the solvent,
leading to relative movement between the solid particles and the solvent, as well as
between the solid particles themselves. The extraction process can be either liquid-
liquid or solid-liquid. Extracts are created by separating soluble substances from
plant tissues using appropriate solvents such as alcohol, water, or ether. The
resulting liquid is concentrated through evaporation to obtain a liquid extract or
further concentrated to near dryness to obtain a solid extract. Maceration is a
simple extraction method that is both cost-effective and popular for producing
bioactive compounds and essential oils. This method involves placing plant
material in a closed container filled with an extraction solvent at room temperature
for a specified period, ranging from a few hours to several weeks, depending on the
properties of the material and solvent, as well as the experimental goals.
Maceration is suitable for thermolabile compounds. Methanol is commonly used in
the isolation of natural organic compounds because it is a polar solvent known as a
universal solvent, capable of extracting both polar and nonpolar compounds such
as fats and waxes. Methanol can extract flavonoids, saponins, alkaloids, and
steroids from plants.
Fractination
A single separation technique is unlikely to yield a pure compound from a crude
extract. Therefore, it is often necessary to fractionate the crude extract into various
fractions containing groups of compounds with similar polarity or molecular size.
The separation process based on differences in polarity can also be referred to as
fractionation, which involves purifying active substances or plant macerates to
optimize their potential and broaden their spectrum of activity. Fractionation is
conducted through liquid-liquid extraction, where molecules are transferred from
one liquid to another that is immiscible, resulting in two phases after the addition
of a solvent. In this process, the added solvent must dissolve the solute but not mix
with the solution
’
s solvent, allowing the transfer of the solute from one solvent to
another at the liquid-liquid interface. Crude extracts are typically mixtures
containing a variety of polar, semi-polar, and non-polar compounds, and based on
varying polarities, the compounds are usually pre-purified by solvent partitioning
using solvents of increasing polarity. A typical process for partitioning natural
product extracts involves initially extracting the crude extract with n-hexane or
petroleum ether to yield a fraction of non-polar components like lipids, terpenoids,
and others.
Phytochemicals
Phytochemicals are simply chemicals derived from plants. The term "phyto"
originates from the Greek word for plant and refers to secondary metabolites
produced by plants. As previously mentioned, these metabolites are typically
synthesized as a defense mechanism against pathogens, ultraviolet exposure,
environmental hazards, and other threats. Phytochemicals are distinct from
essential nutrients (primary metabolites) such as carbohydrates, proteins, fats,
minerals, and vitamins. The term phytochemicals is used to refer to functional
foods with properties like antioxidants, nutraceuticals, phytonutrients,
antinutrients, phytotoxins, and so forth.
Alkaloids
Alkaloids represent the largest group of secondary metabolites. They are essentially
nitrogenous bases in the form of ammonia compounds, where various radicals
replace one or more hydrogen atoms. Most alkaloids, such as atropine, exist in solid
form and contain oxygen. However, some alkaloids like lobeline or nicotine are
found in liquid form and consist of carbon, hydrogen, and nitrogen. Alkaloids are
characterized by their solubility in organic solvents, being easily soluble in alcohol
but only slightly soluble in water. The names of alkaloids typically end with the
suffix "-ine." Common examples of official alkaloid salts in various pharmacopeias
include codeine, atropine, morphine, ergotamine, and ephedrine.
Flavonoids
Flavonoids are the largest group of phenolic compounds found in nature,
characterized by a C6-C3-C6 carbon skeleton consisting of 15 carbon atoms. They
often contain glucose or hydroxyl groups, making them polar and soluble in polar
solvents. Flavonoids are known for their ability to inhibit various oxidation
reactions, both enzymatic and non-enzymatic, and function effectively as reducers.
Less polar flavonoid aglycones, such as flavanones, isoflavones, flavones, and
methoxylated flavanols, are more soluble in solvents like chloroform and ether.
Polar solvents are typically used for extracting flavonoids, including aglycones and
flavonoid glycosides, while less polar solvents like chloroform, ethyl acetate, and
ether are preferred for extracting less polar flavonoids. Flavonoid presence can be
tested using methods such as the Bate-Smith test, 10% NaOH test, and Wilstatter
test. Additionally, adding HCl and magnesium to a sample extract can produce an
orange or red flavilium salt, indicating the presence of flavonoids.
Tannins
A polyphenolic compound with a high molecular weight that consists of carboxyl
and hydroxyl groups can be referred to as a tannin. Tannins are classified into two
types: hydrolyzable tannins and condensed tannins. Hydrolyzable tannins are
formed through ester bonds between hydroxyl groups on glucose and carboxyl
groups from phenolic acids, while condensed tannins result from condensation
reactions that create dimeric compounds and then higher oligomers. Tannins,
which are polyphenolic or phenolic compounds, have astringent and bitter tastes
and can react with and coagulate proteins or other organic substances containing
alkaloids and amino acids. Tannin solutions exhibit a range of colors from light to
dark brown or red, with distinct colors depending on their source. Tannins are
water-soluble and can impart color to aqueous solutions.
Terpenoids
Terpenoids represent the largest group of metabolites. In nature, most terpenoids
are components of essential oils. These compounds contribute to the taste, smell,
and color of plants. Terpenoids are commonly found in the leaves and fruits of
higher plants, such as pine and citrus trees. They include various types of
compounds, such as sesquiterpenes and monoterpenes (C15 and C10), which are
volatile and present in essential oils, diterpenes (C20), which are less volatile, and
triterpenoids and sterols (C30), which are non-volatile, as well as carotenoid
pigments (C40). Terpenoids are essentially oxygenated and dehydrogenated
derivatives of terpenes, also known as isoprenoids, due to their carbon framework
resembling that of isoprene (C5H8). Chemically, terpenoids are composed of
isoprene units, which can be cyclic or linear, and may contain hydroxyl groups,
double bonds, carbonyl groups, or other functional groups. Non-hydrolyzed organic
sterol or steroid compounds are derived from the degradation of terpenes or
squalene. Steroids are a class of triterpenoids containing a cyclopentane
perhydrophenanthrene nucleus, consisting of one cyclopentane ring and three
cyclohexane rings. Cholesterol, estrogen, and ergosterol are steroid derivatives, as
are squalene, retinol, and carotene. Tests for triterpenoids and sterols in samples
can be conducted using the Salkowski test, with a positive result indicated by a
golden-yellow color for triterpenoids and a red color in the lower layer for sterols,
and the Liebermann-Burchard test, with a positive result indicated by a red ring for
triterpenoids and a reddish-brown color for sterols.
Saponins
Saponins are a group of steroid aglycones or triterpenoid glycosides, consisting of
one or more sugar groups attached to a sapogenin or aglycone. They feature
various glycosyl groups linked to the C3 position, although some saponin
compounds also have two glucose chains attached to the C3 and C17 positions. The
term saponin is derived from the Latin word "sapo," meaning soap, as saponins can
cause foaming on water surfaces when shaken due to their ability to lower water
surface tension. Steroid saponins with carbohydrate molecules consist of a steroid
core (C27) and yield a non-sugar compound known as sapogenin upon hydrolysis.
Triterpenoid saponins with carbohydrate compounds have a triterpenoid core and
produce a non-sugar compound known as sapogenin when hydrolyzed. Saponins
are yellow, can form amorphous crystals, have a taste ranging from very sweet to
extremely bitter, and possess a sharp odor. They are known to be non-volatile
compounds that offer high solubility in both cold and hot water and alcohol, but
they can produce colloidal foam in water.
UV-Vis Spectrophotometer
The UV-Vis spectrophotometer is an instrument used to measure the absorption of
a sample by analyzing the interaction between electromagnetic radiation and an
atom or molecule within the wavelength range of 200-400 nm (UV region) and 400-
800 nm (visible region). Bonding and non-bonding electrons (free electrons) within
a molecule react quickly to both UV and visible light. This light represents energy
that, when interacting with electrons, causes them to move from their ground state
to a higher energy level. The resulting excited electrons are recorded as a spectrum,
indicating absorbance and wavelength, based on the type of electrons in the
analyzed molecule. Spectrophotometers generally come in two types: single-beam
and double-beam. Single-beam instruments measure absorbance at a single
wavelength, while double-beam instruments use two beams created by a V-shaped
mirror or beam splitter, allowing simultaneous measurement of sample and blank
cuvettes in the same chamber, thus minimizing the impact of electrical voltage
changes. Deuterium lamps provide UV light, and tungsten lamps are used for visible
light. Quartz or glass cuvettes of varying widths hold the sample, while optical
filters and prism lenses act as monochromators. Photodiode or thermal detectors
capture transmitted light and convert it into an electrical current. The UV-Vis
spectrophotometer operates on the principle that when light passes through a
monochromator, it becomes monochromatic and then travels through a sample
cell. Some of the light is absorbed by the sample, and the rest reaches a
photoelectric cell that converts light energy into electrical energy. The amount of
absorbed light generates an electrical signal detected as absorbance. The amount
of light passing through is proportional to the concentration of the solution in the
cuvette. Absorbance refers to the amount of light or energy absorbed by particles in
a solution, while transmittance is the amount of light that passes through the
sample. Absorbance and transmittance are inversely related, as described by the
Lambert-Beer Law, where absorbance is zero if no light is absorbed and infinite if all
light is absorbed. Absorbance (A) measures absorbed light, whereas transmittance
(T) measures scattered light.
Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-
MS/MS)
Ultra Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-
MS/MS) involves separating components within a mixture, a process known as
chromatography, which is a key method for both qualitative and quantitative
analysis. Components are distributed between two phases: a stationary phase that
remains fixed and a mobile phase that moves in a specific direction. The process
relies on the equilibrium of the targeted component's concentration between these
non-mixing phases, chosen so that each component has different affinities or
solubilities. LC-MS has been widely applied across various fields including
pharmaceuticals, food, and environmental samples, combining the effective
separation capabilities of chromatography with the high selectivity and sensitivity
of mass spectrometry. UPLC-MS/MS has seen exponential growth in the last decade
due to its unmatched sensitivity, exceptional selectivity, and rapid analysis speed.
Mass spectrometry operates by producing ions from the analyte, which are then
separated based on their mass-to-charge ratio (m/z). Most quantitative
bioanalytical applications use tandem mass spectrometry (MS/MS), which employs
two mass analyzers: one for precursor ions and another for product ions. MS-MS
detectors are superior due to their high sensitivity, ability to detect very small
quantities, high selectivity, time resolution, and longevity. Q-TOF-MS integrates the
advantages of quadrupole and time-of-flight (TOF) mass analyzers, leveraging the
high fragmentation efficiency of quadrupole technology with the rapid analysis and
high mass resolution of TOF. The Q-TOF-MS instrument is similar to a triple
quadrupole mass spectrometer, but the third quadrupole is replaced by a TOF tube.
The first quadrupole (Q1) acts as a mass filter for specific ions based on their m/z
ratio, or operates in radio frequency (RF) mode where all ions pass through. The
second quadrupole (Q2) serves as a collision cell where ions interact with neutral
gas molecules, resulting in ion fragmentation through collision-induced
dissociation (CID). Q2 can also operate in RF mode without further ion
fragmentation. Post-Q2, ions are accelerated back to the TOF ion modulator, where
they are driven by an electric field. Ions with the same kinetic energy enter the TOF
tube, a field-free region where mass separation occurs; lighter ions have shorter
flight times, while heavier ions take longer to reach the detector.
Diabetes
Diabetes, or diabetes mellitus (DM), is a chronic condition that can disrupt the
metabolism of carbohydrates, proteins, and fats and is associated with prolonged
hyperglycemia. Symptoms include increased thirst and hunger, as well as frequent
urination. If untreated, diabetes can lead to serious complications such as
ketoacidosis, stroke, heart disease, kidney failure, eye damage, foot ulcers,
impotence, and death. The onset of diabetes mellitus is due to metabolic
disturbances in carbohydrates, proteins, and lipids, often caused by impaired
insulin secretion from the
β
-cells in the pancreas or reduced cellular response to
insulin. Normal fasting blood glucose levels range from 70 to 105 mg/dl, while levels
reaching 126 mg/dl are classified as hyperglycemia. The two most common types of
diabetes are type 1 and type 2. Type 1 diabetes may result from an autoimmune
reaction attacking insulin-producing
β
-cells in the pancreas, leading to minimal or
no insulin production. This destructive process may be triggered by a combination
of genetic susceptibility and environmental factors, such as viral infections. Type 2
diabetes is often genetically predisposed and involves insulin resistance and beta-
cell dysfunction. Insulin resistance means that insulin does not effectively act on
muscle, liver, and fat cells, causing the pancreas to produce more insulin. Chronic
hyperglycemia occurs if the pancreas cannot compensate for this resistance.
Gestational diabetes is a metabolic disorder characterized by elevated blood
glucose levels during pregnancy, typically appearing around 24 weeks of gestation
and normalizing post-delivery. Other types of diabetes may be related to genetic
dysfunction of beta cells, insulin action, pancreatic exocrine diseases, endocrine
disorders, infections, medications or chemicals, rare immunological conditions,
and other genetic syndromes associated with diabetes. Diabetes management can
involve pharmacological therapy with oral medications, but due to their side effects,
herbal alternatives are sought as they generally have fewer adverse effects
compared to modern drugs.
α
-glucosidase inhibition method
The
α
-glucosidase inhibition method involves the use of
α
-glucosidase, an enzyme
crucial for the hydrolysis of carbohydrates into glucose. Inhibition of this enzyme
delays the absorption of sugars.
α
-glucosidase inhibitors prevent the absorption of
carbohydrates from the small intestine by competitively inhibiting the enzyme that
converts complex carbohydrates, which are not absorbable, into simple
carbohydrates that can be absorbed. By delaying carbohydrate absorption, these
inhibitors reduce postprandial glucose spikes and contribute to better glycemic
control. The inhibition power is measured in vitro based on the absorbance of p-
nitrophenol, which results from the hydrolysis of p-nitrophenyl
α
-D-
glucopyranoside into p-nitrophenol and D-glucose by
α
-glucosidase. The
absorbance value is influenced by the yellow intensity of p-nitrophenol, with a
higher inhibition activity resulting in less p-nitrophenol and a decreased yellow
intensity. The
α
-glucosidase inhibitory activity is tested using UV-Vis
spectrophotometry at a wavelength of 410 nm. The types of solvents known to
inhibit
α
-glucosidase in vitro include polar and semi-polar solvents such as water,
methanol, ethanol, ethyl acetate, and acetone. The use of polar solvents in several
studies has also resulted in high
α
-glucosidase inhibitory activity with low IC50
values. Antidiabetic activity through
α
-glucosidase inhibition can be determined by
measuring blood sugar levels after administering plant extracts and by assessing
the percent inhibition or IC50 value. The IC50 value indicates the concentration of
an extract or sample needed to inhibit 50% of
α
-glucosidase activity, with a lower
IC50 value reflecting higher inhibition activity, and vice versa. IC50 values are
categorized as very active for antidiabetic properties if < 11 ppm, active if 11-100
ppm, and inactive if > 100 ppm. Additionally, IC50 values < 50
μ
g/mL are considered
very strong, 50-100
μ
g/mL strong, 100-150
μ
g/mL moderate, 150-200
μ
g/mL weak,
and > 200
μ
g/mL very weak.
Method
The equipment used includes a set of laboratory glassware, a digital balance, a
rotary vacuum evaporator, a water bath, a UV-Vis single beam spectrophotometer
(Agilent Technologies), and Ultra Performance Liquid Chromatography Tandem
Mass Spectrometry (UPLC-MS) Xevo G2-XS QToF Waters (Waters MS Technologies).
The materials employed are Malacca leaves obtained from the Advanced Chemical
Research Center, National Research and Innovation Agency (BRIN) Serpong,
solvents such as methanol, n-hexane, and ethyl acetate, aquades, DMSO (Merck),
phosphate buffer pH 7, p-nitrophenyl
α
-D-glucopyranoside (pNPG) (Wako Pure
Chemical Industry),
α
-glucosidase enzyme from Saccharomyces cerevisiae (Wako
Pure Chemical Industry), quercetin, chloroform, acetic anhydride, sulfuric acid,
Dragendorff
’
s reagent, zinc powder, hydrochloric acid, ethanol, acetic anhydride,
ferric chloride, gallic acid, Folin-Ciocalteu reagent, sodium carbonate, sodium
nitrite, aluminum chloride, sodium hydroxide, formic acid, and acetonitrile.
Work procedure
1. Extraction of Malaka Leaves
The malaka leaves are cleaned, dried, and ground into a powder. A container is
filled with 100 g of the malaka powder, which is then soaked in methanol at a 1:10
ratio for 72 hours at room temperature. The sample is filtered using filter paper, and
the filtrate is collected. The residue obtained from filtration is re-extracted with
methanol twice, following the same method. The filtrate is evaporated using a
rotary evaporator at 40°C to obtain a concentrated extract. The yield of the extract
is calculated using the formula: % Yield = (Weight of Extract Obtained (g) / Weight of
Simplicia Powder Extracted (g)) × 100%.
2. Fractionation
Fractionation is performed using liquid-liquid partitioning. Twenty grams of the
crude extract are added to a separating funnel and dissolved in 100 mL of water.
The solution is then partitioned with 100 mL of n-hexane, gently shaken for 5
minutes, and allowed to separate into n-hexane and aqueous layers. The n-hexane
fraction is separated from the aqueous layer and partitioned again until a clear
solution is obtained. The aqueous fraction is then partitioned with ethyl acetate
using the same procedure as for n-hexane. The liquid fractions of water, n-hexane,
and ethyl acetate are evaporated with a rotary evaporator to yield thick extracts.
3.
α
-Glucosidase Inhibition Assay
The enzyme inhibition activity for
α
-glucosidase is assessed by preparing a reaction
mixture containing 250
μ
L of 5 mM p-nitrophenyl
α
-D-glucopyranoside (pNPG), 495
μ
L of 100 mM phosphate buffer (pH 7.0), and 5
μ
L of malaka leaf methanol extract, n
-hexane, ethyl acetate, and aqueous fractions dissolved in DMSO at various
concentrations (100, 50, 25, 10, and 5
μ
g/mL) in an Erlenmeyer flask. The reaction
mixture is pre-incubated for 5 minutes at 37ºC, and the reaction is started by adding
250
μ
L of
α
-glucosidase (0.065 Units/mL). The incubation continues for 15 minutes,
and the reaction is stopped by adding 1 mL of 0.2 M Na2CO3.
α
-Glucosidase activity
is determined by measuring the p-nitrophenol produced at a wavelength of 400 nm,
with quercetin used as a standard. The percentage of
α
-glucosidase inhibition is
calculated using the formula: % Inhibition = (C - S) / C × 100, where S is the
absorbance of the sample (difference between absorbance with and without
enzyme) and C is the absorbance of the blank (DMSO). The inhibition activity is
represented by IC50 (ppm), which indicates the extract concentration needed to
inhibit enzyme activity by 50%. IC50 is determined using a linear regression
equation where x is the sample concentration and % inhibition is the y-axis. Based
on the equation: y = ax + b, IC50 is calculated as: IC50 = (50 - b) / a.
Phytochemical Testing
1. Alkaloid Test: To 500 mg of the best extract or fraction from the Malaka plant, 1
mL of 2 N HCl and 9 mL of water are added, then heated in a water bath for 2
minutes, cooled, and filtered to obtain the filtrate. This solution is then tested with
Dragendorff's reagent. If 1 mL of the filtrate combined with 2 drops of Dragendorff's
reagent
—
prepared by dissolving 8 g of Bi(NO3)3 in 20 mL of concentrated HNO3
and adding 27.2 g of KI in 50 mL of water
—
produces an orange-brown precipitate, it
indicates the presence of alkaloids.
2. Flavonoid Test: A few milligrams of the sample are mixed with 4 mL of ethanol
until the extract dissolves. To 2 mL of this solution, 0.5 grams of magnesium and 2
mL of 2 N HCl are added, allowed to stand for 1 minute, and then 10 drops of
concentrated HCl are added. The appearance of an intense red color within 2-5
minutes indicates the presence of flavonoids.
3. Tannin Test: A few milligrams of the sample are mixed with 15 mL of hot water,
then boiled for 5 minutes. After filtering, a few drops of 1% FeCl3 are added. The
development of a green-violet to black color indicates the presence of tannins.
4. Terpenoid Test: A few milligrams of the sample are dissolved in 2 mL of
chloroform in a test tube, then 10 drops of acetic anhydride and 3 drops of
concentrated H2SO4 are added. The initial formation of a red solution, which
subsequently changes to blue and green, indicates the presence of terpenoids.
5. Saponin Test: 500 mg of the sample is placed in a test tube with 10 mL of hot
water, cooled, and shaken vigorously for 10 seconds. If the foam formed is stable at
1-10 cm for at least 10 minutes and remains unchanged after the addition of 1 drop
of 2 N HCl, the sample is considered positive for saponins.
Total Phenol Test
In the Total Phenol Test, 1 mg of gallic acid was weighed and dissolved in 1 mL of
methanol to make a stock solution with a concentration of 1000
μ
g/mL. This stock
solution was then diluted serially to achieve final concentrations of 5, 10, 20, 30,
and 40
μ
g/mL. For the phenolic content measurement, 4 mg of extract or fraction
was dissolved in 4 mL of methanol to obtain a sample concentration of 1000
μ
g/mL.
From this, 500
μ
L of the sample solution and standard gallic acid solutions (25, 50,
100, 150, and 200
μ
L) were pipetted into test tubes, which were then filled to 4 mL
with distilled water and mixed with 250
μ
L of Folin-Ciocalteau reagent. After 8
minutes of incubation, 750
μ
L of 20% Na2CO3 was added, and the mixture was
shaken thoroughly. The solution was left to stand for 2 hours at room temperature,
and absorbance was measured at 765 nm using a UV-Vis spectrophotometer.
Calibration curves were constructed using the regression equation y = bx + a, and
measurements were repeated twice. The phenolic content was expressed as gallic
acid equivalents (mg/g extract).
Total flavonoid
To measure total flavonoid content, 4 mg of quercetin is dissolved in 4 ml of
methanol to create a stock solution of 1000
μ
g/ml. Standard solutions of 10, 20, 30,
40, and 50
μ
g/ml are prepared by pipetting 50, 100, 150, 200, and 250
μ
l of the stock
solution into test tubes, respectively, and adding 2 ml of distilled water. To each
tube, 150
μ
l of 5% NaNO2 is added, followed by 150
μ
l of 10% AlCl3 after 5 minutes.
Six minutes later, 2 ml of 1M NaOH is added, and the volume is adjusted to 5 ml with
distilled water. The solution is then homogenized, and absorbance is measured at
λ
510 nm using a UV-Vis spectrophotometer. The flavonoid content is reported as
quercetin equivalent (mg/g extract). For total flavonoid quantification, sample
solutions of 250 and 500
μ
L are prepared similarly to the standards. A calibration
curve is generated by plotting absorbance values against standard solution
concentrations. Mass spectrometry is performed using a Xevo G2-XS QTof (Waters
MS Technologies) with ESI+ ionization, scanning from 100 to 1200 m/z. The capillary
and cone voltages are set at 0.8 kV and 30 kV, respectively, with positive
electrospray mode. Desolvation gas is set to 1000 L/hour at 500°C, and cone gas to
50 L/hour, with the source temperature at 120°C. UPLC analysis is carried out with a
Waters Acquity Ultra Performance LC system using an ACQUITY UPLC HSS T3 C18
column (100 mm x 2.1 mm, 1.7
μ
m) at 40°C. The mobile phase consists of solvent A
(0.1% formic acid in water, v/v) and solvent B (0.1% formic acid in acetonitrile), with
a gradient polarity from 95:0.5 (A:B) to 0.5:95 (A:B). The flow rate is set to 0.3
mL/min, and the column and auto-sampler are maintained at 40°C and 20°C,
respectively. The injection volume is 1
μ
L, and data acquisition and processing are
performed using UNIFI. Retention times are in the range of 1-15 minutes.
Results and Discussion
Extract and Fractionation of Malaka Leaves
The extraction method employed is maceration, chosen for its simplicity and use of
cold extraction, which is suitable for compounds that are either heat-sensitive or
heat-stable, thus preserving the active compounds in the plant material. Methanol
was selected as the solvent because it is a "magic solvent" that extracts both polar
and non-polar compounds from the plant. Fractionation was carried out using
solvents of increasing polarity, starting with non-polar n-hexane followed by semi-
polar ethyl acetate. This approach aimed to separate the chemical compounds in
the Malaka leaves based on their polarity, resulting in distinct fractions from each
solvent. The partitioning process began with the addition of water to dissolve the
crude extract. Partitioning with 100 mL of n-hexane was performed four times until
a clear n-hexane layer was obtained, indicating that the layer no longer contained
extractable compounds. The upper layer was the organic phase (n-hexane) and the
lower layer was the aqueous phase. Partitioning with 100 mL of ethyl acetate was
also done four times until a clear ethyl acetate layer was obtained, indicating no
further extractable compounds. The upper layer was ethyl acetate, and the lower
layer was water. According to Table 4, the ethyl acetate fraction had the highest
mass and yield at 5.1 grams with a 25.5% yield, followed by the aqueous fraction
with 3.8 grams and a 19% yield, and the concentrated n-hexane fraction with 1.6
grams and an 8% yield. This suggests that the compounds in Malaka leaves are
predominantly semi-polar and are more attracted to the semi-polar solvent, such
as flavonols, aglycone flavonoids, isoflavones, flavanones, and methylated flavones.
The inhibition activity of
α
-glucosidase by the methanol extract
Ethyl acetate fraction, n-hexane fraction, and water fraction of Malaka leaves was
assessed using the
α
-glucosidase inhibition method. The aim was to determine the
potential of these extracts or fractions as antidiabetic agents based on their IC50
values.
α
-Glucosidase inhibitors, such as acarbose, work by delaying glucose
absorption in the intestine, thereby preventing postprandial blood sugar spikes.
Consequently,
α
-glucosidase is a key target enzyme for type II diabetes treatment.
The test was conducted using various extract concentrations (100, 50, 25, 10, and 5
μ
g/mL) to evaluate the impact of concentration variation on
α
-glucosidase
inhibition. Samples were analyzed using UV-Vis spectrophotometry at 400 nm
wavelength. The inhibition percentage was calculated by comparing the
absorbance of samples with a blank, and the IC50 values were determined
accordingly. The IC50 values obtained for each extract and fraction were compared
with a standard solution, quercetin, known for its strong
α
-glucosidase inhibitory
activity compared to gallic acid, rutin, and acarbose. Quercetin was chosen because
it is a more sensitive inhibitor for
α
-glucosidase derived from Saccharomyces
cerevisiae, whereas acarbose is less effective against enzyme sources from bacteria
and yeast. The p-nitrophenyl-
α
-D-glucopyranoside substrate was used to represent
carbohydrates, as the enzyme breaks down the substrate into glucose and p-
nitrophenol. The enzyme's activity is influenced by factors like secondary
metabolite content in the extracts or fractions. The antidiabetic test results
indicated that the ethyl acetate fraction of Malaka leaves had a higher
α
-
glucosidase inhibition activity compared to other extracts, with an IC50 value of
5.93±0.17
μ
g/mL, meaning it inhibited 50% of
α
-glucosidase activity at this
concentration. This suggests that the ethyl acetate fraction contains more active
antidiabetic compounds compared to other extracts. Although quercetin, with an
IC50 of 2.17±0.02
μ
g/mL, showed better inhibition, the ethyl acetate fraction's IC50
value indicates its significant antidiabetic potential, though still lower than the pure
quercetin.
Phytochemical screening of the extract and fractions from Malaka leaves
Involves a qualitative examination of the chemical content to identify the types of
compounds present in the test extract. In this case, the extracts and fractions of
Malaka leaves are examined. Secondary metabolites play a crucial role in
α
-
glucosidase inhibition assays, and thus, the results of the phytochemical test can
indicate which chemical groups may contribute to inhibiting
α
-glucosidase activity.
These secondary metabolites, which are not directly involved in plant growth but
are produced in response to certain conditions, include antibiotics, enzyme
inhibitors, pesticides, and antitumor agents. Alkaloids, saponins, and flavonoids
have been identified for their potential antidiabetic properties through
α
-
glucosidase inhibition mechanisms. Tannins, as antioxidants, may prevent glucose
oxidation in the blood and show antidiabetic effects. Additionally, steroids, as part
of the saponin aglycone structure, can stimulate insulin release. The phytochemical
tests performed include evaluations for alkaloids, flavonoids, tannins, saponins,
and terpenoids. The phytochemical analysis of the Malaka leaf extracts and
fractions revealed the presence of secondary metabolites, including tannins,
saponins, flavonoids, terpenoids or steroids, and alkaloids. Tannin compounds in
the extracts and fractions produced a greenish-black solution when tested with
FeCl3, indicating the formation of a complex between tannins and Fe3+ ions. This
complex forms due to covalent coordination bonds between the central metal
atom and the non-metal donor atoms. The central atom, located at the center of
the bond, connects with the donor atoms, which are either ions or neutral atoms
capable of forming bonds with other atoms. Thus, it can be concluded that all
extracts and fractions contain tannins. In the test for saponin compounds in the
extract and fractions of Malaka leaves, a positive result was indicated by the
formation of stable foam lasting about 10 minutes, which was observed only in the
methanol extract, ethyl acetate fraction, and water. It has been explained that
compounds with both polar and nonpolar groups exhibit surface-active properties,
so when saponin is shaken with water, it forms micelles. In micelles, the polar
groups face outward while the nonpolar groups face inward, creating a foam-like
appearance. When the sample is shaken, the hydrophilic groups in saponin bind
with water, while the hydrophobic groups bind with air, leading to foam formation.
The stable foam formation is also attributed to the fact that glycosides can generate
foam in water and then undergo hydrolysis into glucose and other compounds.
