Extraction aplha-mangostin from mangosteen pericarp using mixture

solvents

Rawisara Uruekolan
Tanawan Kanno

Advisor
Asst. Prof. Dr. Nuttapol Lerkkasemsan

A Report Submitted in Partial Fulfillment of the Requirements

for the Degree of Bachelor of Engineering (International Program)
Department of Chemical Engineering, School of Engineering, King
Mongkut’s Institute of Technology Ladkrabang
Academic Year 2022
 I

TABLE OF CONTENTS

CHAPTER I INTRODUCTION 1
1.1 Background 1
1.2 Objectives 2
1.3 Scopes of works 2
1.4 Expected benefits 2

CHAPTER II THEORY AND LITERATURE REVIEWS 3

2.1 Mangosteen 3
2.2 Xanthones 4
2.3 Deep Eutectic Solvent 5
2.4 Solid liquid extraction 6
2.4.1 Mechanism of extraction 6
2.4.2 Effect of temperature 8
2.4.3 Effect of extraction time 8
2.4.4 Effect of solvent to solid ratio 9
2.4.5 Effect of particle size 9
2.5 Response surface methodology (RSM) 9
2.6 Box-Behnken design (BBD) 11
2.7 Literature review 12

CHAPTER III RESEARCH METHODOLOGY 14

3.1 Mangosteen powder preparation 14
3.2 Deep Eutectic solvent (DES) preparation and viscosity determination 14
3.3 Shaking method extraction of mangosteen pericarp with deep eutectic solvents to select
the suitable compositions of deep eutectic solvents 15
3.5 Preparation and analysis of alpha-mangostin 17
3.6 The purification method of alpha-mangostin 17
3.7 Planning 18
 II

LIST OF FIGURES
Figure 2.1 Mangosteens 3
Figure 2.2 Xanthone nucleus with IUPAC numbers of carbons and chemical structure of the
most studied xanthones 4
Figure 2.3 Structure of deep eutectic solvent 5
Figure 2.4 Schematic representation of a eutectic on a two-component phase diagram. 6
Figure 2.5 A Box-Behnken design for k=3 11
 III

LIST OF TABLES

Table 2.1 The optimal condition of alpha-mangostin extraction 13

Table 3.1 Solvents used in this study 14
Table 3.2 Experimental design factors and levels of design of experiment 16
Table 3.3 Plan schedule 19
 1

CHAPTER I

INTRODUCTION
1.1 Background

Mangosteen (Garcinia mangostana L.) is an important economic plant grown in Eastern

and Southern Thailand. The fruits are popularly known as the “Queen of fruits”, The fruit pulp
is slightly acidic and sweet. The fruit pericarps are also known for their ethnomedicinal values
and these are a by-product of the fruit processing industry [1]. Generally, one mangosteen
consists of 83% pericarps, 15% pulp, and 2% seeds by weight. Therefore, most of the by-
products and residues are mangosteen pericarps. This creates problems for the mangosteen
processing industry, which may turn into solid waste in large quantities. Therefore, the value-
added creating from by-products and wastes to maximize the utilization of raw materials and
to reduce environmental problems is essential.

Mangosteen has many health benefits originally been used in various folk medicines.
The mangosteen pericarp used to treat diarrhea and it also has properties related to wound
healing it can treat blisters rotting wounds and purulent wounds by grind mangosteen pericarp
mixed with limewater applied to the wound area. Another outstanding property of mangosteen
pericarp is the treatment of skin diseases such as eczema, and rashes as well [2]. From current
research, the mangosteen has been noted to be an abundant source of a class of polyphenols
known as xanthones. The diverse structure and chemical properties of xanthones have been
reported to have a variety of health promoting properties including anti-inflammatory,
antioxidant, anti-proliferative, and anti-cancer activity [3].

Xantones form the core of a variety of natural products, such as mangostin or

lichexanthone. These compounds are sometimes referred to as xanthones or xanthonoids. Over
200 natural xanthones have been identified. Many are phytochemicals found in plants in the
families Bonnetiaceae, Clusiaceae, and Podostemaceae. According to research and studies,
xanthones are mostly found in the pericarp of the mangosteen, which contain alpha-mangostin
as the major xanthones compound followed by gramma-mangostin and minor xanthones
compound [3]. Which these compound have many health benefits such as alpha-mangostin
use as a medicine because of its anti-cancer, antioxidant, and antibacterial
capabilities.Therefore,this study investigate extraction of antioxidant rich xanthones from
mangosteen pericarps to bring the extracted substances for medical use and take the waste that
will be disposed of to be used for maximum benefit.

There are many solvents to extract alpha-mangostin from mangosteen pericarp. Organic
solvents such as methanol, ethanol and chloroform are known as solvents for extracting
bioactive compounds from plant [19]. Most of these organic solvents are toxic and volatile,
resulting in waste that is hazardous to both human health and the environment. Deep eutectic
solvents (DES) which are more environmentally friendly, improving safety and health, as well
as reduce cost have received more attention recently [20]. Therefore, this research study the
 2

optimum conditions for alpha-mangostin extraction from mangosteen pericarps by deep

eutectic solvent with these variables; the molar ratio of DES, the ratio of water content,
extraction time, and temperature of extraction. In addition, a regression model for predicting
the alpha-mangostin content by response surface method (RSM) and three-factor Box-Behnken
design (BBD) will also be studied.

1.2 Objectives

1.2.1 To study the optimum conditions for alpha-mangostin extraction from mangosteen
pericarps using mixture solvent with shaking method extraction.
1.2.2 To construct a regression model for predicting the extracted alpha-mangostin content.

1.3 Scopes of works

1.3.1 To Study factors affecting the extraction method of alpha-mangostin: The molar ratio
and water contents of mixture solvents, temperature, and time.
1.3.1.1 Type of hydrogen bond acceptor (Chorine choline, Betaine anhydrous)
1.3.1.2 Mole ratio of hydrogen bond acceptor and hydrogen bond donor at 1:1,1:2
1:3 and 1:4
1.3.1.3 Water content of deep eutectic solvents 10% w/w, 20% w/w, and 30% w/w
1.3.1.4 Extraction times 120 min,180 min, and 240 min
1.3.1.5 Temperature of extraction 27 °C, 37 °C and 55 °C
1.3.1.6 Liquid to solid ratio 10 ml/g, 20 ml/g, and 30 ml/g

1.4 Expected benefits

1.4.1 Obtain the optimum conditions of alpha-mangostin extraction method form

mangosteen pericarp by using mixture solvents.
1.4.2 Increase value of mangosteen pericarp for pharmaceutical
 3

CHAPTER II

THEORY AND LITERATURE REVIEWS

In the extraction study alpha-mangostin extract from mangosteen pericarp using

mixture solvents. Before the conditions and factors affecting of the extraction had to be studied
to obtain the optimum extraction factor for the most cost alpha-mangostin content for research
purposes. So, we need to study the deep eutectic solvent that used in extraction method. In
addition, xanthones and alpha-mangostin have to be studied which is the compound that we
will extract used in the study according to the purpose.

2.1 Mangosteen

Mangosteen with scientific name Garcinia mangostana L. a tropical tree belonging to

guttifera family that widely grown in southeast Asia also grown in Thailand, is known as
“Queen Fruit”. This tree can reach 6–25 m and it has leathery, glabrous leaves and is slow to
grow. The mangosteen fruit is dark purple or reddish, with white, soft, and juicy edible pulp
with a slightly acid and sweet flavor and a pleasant aroma because it is one of the best tasting
tropical fruits so it widely consumed and strong medical benefits [5].

Mangosteen pericarps is a source of phenolic compounds that have medicinal

properties. It is used as an astringent to cure diarrhea. There are many important phenolic
substances found in mangosteen are xanthone, tannin, and proanthocyanin these chemicals aids
wound healing, reduce inflammation and infection by bacteria that create pus. Xanthones are
most found in mangosteen pericarps, seeds, and pulp respectively which mangosteen consists
of 83% pericarps, 15% pulp and 2% seeds by weight which xanthones from mangosteen is very
useful in pharmaceutical industry [1].

Figure 2.1 Mangosteens

 4

2.2 Xanthones

Xanthone is an organic compound that contains carbon and oxygen as component with
the molecular formula C13H8O2 . It is from the core of a variety of natural products and is
commonly found in higher plants, fungi, and lichens. Xanthones, which are mostly polar
compounds, are the major polyphenolic compounds present in the pericarp. These are soluble
in organic solvent.

More than 50 xanthone derivatives were isolated from the mangosteen pericarp. Among
these is alpha-mangostin, the major xanthone, which has highly functional medicinal
properties. Mangostin is a bright yellow, opaque, needle-shaped crystal derived from various
portions of a mangosteen tree with a melting point of roughly 182–183°C. In addition,
xanthones also contain gramma-mangostin and minor xanthone compounds include gartanin,
8-deoxygartanin, and garcinon E.

Figure 2.2 Xanthone nucleus with IUPAC numbers of carbons and chemical structure of the
most studied xanthones
 5

Alpha-mangostin is a hydrophobic polyphenol compound that accounted for the

superior antioxidant potential. It is formed by 9H-xanthine and replaced by hydroxy groups at
positions 1, 3, and 6, a methoxy group at position 7, an oxo group at position 9, and a phenyl
group at positions 2 and 8.

There are many benefits of alpha-mangostin in mangosteen. Reduce inflammation and

treat various blisters. Naturally, xanthones in mangosteen can inhibit the synthesis of
prostaglandin E2 (PGE2) as well as treat inflammation of various cells in the body, so
mangosteen helps relieve chronic inflammation such as diabetes, cancer, rheumatoid arthritis,
Alzheimer’s disease or memory loss, heart disease and many other serious diseases effectively.
In addition, alpha-mangostin extract will help inhibit the growth of cancer cells in the blood It
can also inhibit the rapid growth of breast cancer, liver cancer, stomach cancer and lung cancer
as well [2].

2.3 Deep Eutectic Solvent

Deep eutectic solvents (DESs) are a class of eutectic mixtures of two or more
compounds. DESs have benign properties such as low volatility, flammability, toxicity, cost,
and tailorable physicochemical properties by altering the type and molar ratio of constituents
[6].
DESs contains two components that are hydrogen bond donor (HBD) and a hydrogen
bond acceptor (HBA) interact via hydrogen bonding. They are usually obtained by the
complexation of a quaternary ammonium salt with a metal salt or hydrogen bond donor (HBD)
as shown in figure 2.3 [7].

Figure 2.3 Structure of deep eutectic solvent

DESs contain large, nonsymmetric ions with low lattice energy and melting points,
which they have a eutectic point or eutectic temperature far below the melting temperatures of
the individual components. Figure 2.4 where the eutectic point is a eutectic mixture containing
compounds A and B, and where the eutectic temperature of the eutectic mixture is lower than
the eutectic temperature of the constituent compounds A and B. When pure compounds are
mixed, there is a greater change in entropy, and when the entropy changes, the melting point
of the deep eutectic solvent will decrease. The relationship between melting and melting point
is that when the melting point decreases, it results in better solubility [8]. The significant
decrease in the melting temperature of the mixture is commonly attributed to strong hydrogen
bonding interactions between the DES constituents. [9].
 6

Figure 2.4 Schematic representation of a eutectic on a two-component phase diagram.

The HBA and HBD have been studied, The hydrogen bond donor is 1,2-propanediol,
and the hydrogen bond acceptors are choline chloride and betaine anhydrous. When the
solvents are mixed together, more hydrogen bonds are formed and there is also charge
delocalization between the hydrogen bonds of HBD and HBA The presence of more hydrogen
bonds stabilizes the mixture and results in a lower melting point temperature of DESs [13].

2.4 Solid liquid extraction

2.4.1 Mechanism of extraction

Extraction methods also include solvent extraction according to the extraction

principle. Solvent extraction is the most widely used method. The extraction of natural
products progresses through the following stages: (1) the solvent penetrates into the
solid matrix; (2) the solute dissolves in the solvents; (3) the solute is diffused out of the
solid matrix; (4) the extracted solutes are collected [10].

The material closest to the surface will dissolve first, leaving a porous structure
in the solid residue if the solute is evenly distributed throughout the solid matrix. To
reach more solute, the solvent must penetrate the outer layer, making the procedure
more complex and decreasing the extraction rate. If the solute represents a significant
portion of the solid, the porous structure may be destroyed, resulting in a fine deposit
of insoluble residue, and more solute may be easily accessible by the solvent [11].

As explained by Fick's law, the internal diffusion of active compounds is driven

by the concentration differential between the plant matrix and the bulk solvent, as the
following equation [12].
𝑑𝐶 (2-1)
𝑁 = −𝐷
𝑑𝑥
 7

Where,
N is the mass flux of the solute (kg/s)
C is the concentration of the solute in the solid particle (kg/m3)
D is the diffusivity or diffusion coefficient for the solute in the solvent (m2/s)
x is the distance in the direction of the transfer (m)

The transfer of the solute onto the surface of the solid particle occurs with
simultaneous molecular and turbulent flow in a batch process where the total volume
of solution (V) is supposed to remain constant. Therefore, the mass transfer rate can be
described as following equations [11].

𝑉" 𝐶! 𝐷!$ (2-2)

𝑁! = = 𝐴# (𝐶 − 𝐶! )
𝑑𝑡 𝐵# !%
Where,
NA is the rate of dissolution of the solute A in solution B (kg/s)
V is the total volume of solvent B (m3)
AT is the area of the solid-liquid interface (m2)
DAB is the diffusivity of solute A dilute in solution B (m2/s)
BT is the thickness of the stagnant layer (m)
CAS is the reference concentration of the solute A in solution B (kg/m3)
CA is the concentration of the solute A at time t (kg/m3)
CA0 is the concentration of the solute A in particles at t = 0 (kg/m3)

By integrating the time takes from the initial concentration of the solution to
rise, which are CA0 to CA, the following is obtained

&!
𝑑𝐶! 𝐴 # 𝐷!$ ' (2-3)
. = . 𝑑𝑡
&!" 𝐶!% − 𝐶! 𝑉𝐵# '()
𝐶!% − 𝐶! !# ,!$ (2-4)
= 𝑒 *+ -$# .'
𝐶!% − 𝐶!)
𝜕𝐶! 𝜕 / 𝐶! (2-5)
= 𝐷!$
𝜕' 𝜕𝑏 /

The mass transfer equation is described by penetration theory following

Equation 2-6 (negligible convection)
From the boundary conditions
 8

𝐶 = 𝐶!) 𝑓𝑜𝑟 𝑡 = 0 (2-6)

𝐶! 2 !
𝐶! = 𝐶!% 𝑎𝑡 𝑏 = 0, 𝑡 > 0

Equations 2-7 can be rearranged as error function as follows:

𝐶!% − 𝐶! 𝑏 (2-7)
= 𝑒𝑟𝑓 ; >
𝐶!% − 𝐶!) 2=𝐷!$ 𝑡

The Stokes-Einstein equation, based on the hydrodynamical theory, has been

developed to estimate the liquid mass diffusivity following Equation

𝜅𝑇 (2-8)
𝐷!$ =
6𝜋𝑟𝜇$

Where,
DAB is the diffusivity of solute A dilute in solution B (cm2/s)
𝜅 is the Boltzmann constant 1.38 x 10-23 (J/K)
T is the absolute temperature (K)
r is the radius of the particles (cm)
𝜇$ is the viscosity (cP)

2.4.2 Effect of temperature

Temperature is known to be an important factor in the extraction of bioactive

components via DESs and NADESs. Increasing temperature cause viscosity to
decrease, resulting in better extraction performance [14]. The solubility and diffusivity
of the material being extracted typically increase with increasing temperature,
increasing the rate of extraction as well. However, this temperature increase should be
reasonable because it can harm thermolabile compounds.

The effect of various temperatures (27-75 °C) on alpha-mangostin extraction

from mangostin pericarp using DESs there are betaine anhydrous with 1,2-propanediol
has shown that with the rise in temperature, DES fraction yield increases. It may be
owing to with the rise in extraction temperatures, the viscosity of DESs solution
reduces, and later on this reduction in the viscosity of DESs for high values of
temperatures has direct impact on the performance of extraction process. [19]

2.4.3 Effect of extraction time

The extraction time is an operation variable critical during the various extraction
processes. Neither too long nor too short extraction time is desirable because high
extraction time may cause oxidative degradation of extracted compounds, making
 9

various process operations uneconomic. While the small-time duration will be

inadequate for finishing the reaction means unreacted quantities of target molecules in
the biomass result in poor extraction efficiency. Therefore, there is a significant need
to obtain the optimal value of time for process viability because this factor governs
process costs and extraction efficiency [14].

2.4.4 Effect of solvent to solid ratio

The solid-to-solvent ratio is critical in keeping the reaction system

homogeneous during the extraction process with deep eutectic solvents (DESs).
Because of the availability of a minimum of DES chemical detected for reducing
reaction efficiency, the reaction system is not found to be homogeneous at lower values
of this ratio. Another plausible explanation could be a poor chemical reaction caused
by insufficient solvent availability to engross the solid biomass sample adequately.

The higher the value of the solid-to-solvent ratio (SLR), the more problems arise
during solvent dispersion in the sample, further reducing extraction efficiency. Its lower
values are also not desirable because it makes the extraction process incompetent on a
large scale because of the large solvent requirements and the insignificant quantity of
sample treated per unit of time [14].

2.4.5 Effect of particle size

The particle size of material is one factor that effect the yield of extraction. The
comminuting or grinding of the raw material is one of the pretreatment steps that must
be considered. Grinding prior to solvent extraction increases the contract area the
solvent and the solid matrix. Reducing the size of material cause the higher the rate of
solute transfer. Because of the shorter diffusional path lengths, the interfacial area
between the solid and the liquid increases, and the intraparticle diffusion resistance
decreases. As a result, extraction efficiency increases as particle size decreases. Smaller
particle sizes, on the other hand, may not be worth the energy [14].

2.5 Response surface methodology (RSM)

The response surface methodology (RSM) is a widely used in mathematical and

statistical method for modeling and analyzing a process in which the response of interest is
affected by various variables and the objective of this method is to optimize the response. The
parameters that affect the process are called independent variables, while the responses are
called dependent variables.
𝑦 = 𝑓(𝑋0 , 𝑋/ , … 𝑋1 ) + 𝜀 (2-9)

In Equation (2-3), 𝜀 represents other sources of variability that were not considered in
f like the error in the determination of the response of y from experimental and the parameter
 10

is X. If determine that 𝐸(𝑦) = 𝑓(𝑋0 , 𝑋/ , … . 𝑋1 ) = 𝜂. Therefore, the equation of the surface or

“Response surface” is

𝜂 = 𝑓(𝑋0 , 𝑋/ , … . 𝑋1 ) (2-10)

Most of the response surface are represented graphically, where η is plotted against the
levels of X1 and X2 in order to aid the viewing of the shape of the response surface which may
be plotted as the contour plot of the response surface. where most problems the relationship
between the response and the independent variable is unknown. First of all, a suitable estimator
must be found to represent the true relationship between y and the set of independent variables.
It may be that the model of the response has a linear relationship with the independent variables.
Functions used as a first derivate power model as shown in the equation (2-11)

𝑦 = 𝛽) + 𝛽0 𝑋0 + 𝛽/ 𝑋/ + ⋯ 𝛽1 𝑋1 + 𝜀 (2-11)

If there is a curvature involved in the system, a higher-powered polynomial function is used

such as a quadratic polynomial as shown in the equation (2-12)

𝑦 = 𝛽) + ∑1'(0 𝛽0 𝑋0 + ∑1'(0 𝛽/ 𝑋/ + ∑ ∑234 +𝜀 (2-12)

In order to determine a critical point (maximum, minimum, or saddle), it is necessary for the
polynomial function to contain quadratic terms according to the equation (2-13)

𝑦 = 𝛽) + ∑1'(0 𝛽2 𝑋2 + ∑1'(0 𝛽22 𝑋2/ + ∑1'(0 𝛽24 𝑋2 𝑋4 + 𝜀 (2-13)

Where,

k is the number of variables

𝛽) is the constant term

𝛽2 is the coefficients of the linear parameters

𝛽22 is the coefficients of the quadratic parameters

𝛽24 is the coefficients of the interaction parameters

Xi is the variables
ε is the residual associated to the experiments
Most of the response surface problems use a first power model or a quadratic model to
determine the response. But neither model could estimate the relationship across the entire
surface of the independent variable. If the surface that we are interested is large. There are
several methods for determining the best value of the response for response surface design, and
one is the fitted model, which focuses on quadratic modeling of the responses [15].
 11

2.6 Box-Behnken design (BBD)

The study of the effects of various variables on the outcomes of a controlled experiment
is the focus of the Design of Experiments (DOE) tool set. A dependent variable or reaction is
typically studied after identifying the independent variables or factors that affect the product
or process. The Box-Behnken method is one technique for choosing the best response value
for a response surface design.
Box-Behnken Design is a three-level design for responsive surface fit. This design was
created by combining a 2k factorial design with an imperfect block design. The design effect
is efficient in terms of quantity of the desired run and this design also has the ability to turning
or almost turning. Due to the Box-Behnken design is a circular design where all points are
placed on a spherical shape of radius 2 and does not include any points which is the vertex of
the cube formed from the upper and lower limits of each variable. This is very useful when
doing avoid the dots on the corners of the cube which is a Factor-level combination that are
impossible to experiment due to the physical limitations of the process [15].

Figure 2.5 A Box-Behnken design for k=3

One way to think about this is that in the central composite design we have a ball in all
of the corner points lie on the surface of the ball. In the Box-Behnken design the ball is now
located inside the box defined by a wire frame that is composed of the edges of the box. If blew
up a balloon inside this wire frame box so that it just barely extends beyond the sides of the
box, it might look like this, in three dimensions. Notice where the balloon first touches the wire
frame this is where the points are selected to create the design.
Therefore, the points are still on the surface of a ball, but the points are never further
out than the low and high in any direction. In addition, there would be multiple center points
as before. In this type of design, not need as many center points because points on the outside
are closer to the middle. The number of center points are again chosen so that the variance of
is about the same in the middle of the design as it is on the outside of the design [16].
 12

2.7 Literature review

Kamarza Mulia et al. [17] studies the extraction of alpha-mangostin from mangosteen
pericarp using natural deep eutectic solvents as a green solvent consisting of choline chloride,
a quarternary ammonium salt, and four hydrogen bond donors: 1,2- propanediol, citric acid,
glycerol, and glucose using shaking method. The result showed that the highest alpha-
mangostin yield obtain from the optimum condition which used choline chloride and 1,2-
propanediol in 1:3 mole ratio and extraction time 4 hours give alpha-mangostin extraction yield
around 2.6 % (w/w).

Farah Fauzia et al. [18] studied alpha-mangostin extraction using deep eutectic solvent
(DES). Deep eutectic solvents (DESs) based on choline chloride (ChCl) with polyalcohols
(ethylene glycol, glycerol, propanediol, and butanediols) as hydrogen bonding donors (HBDs)
were used to extract alpha-mangostin from mangosteen pericarp. After obtaining a suitable
solvent, the following research focused on extraction time for 1-6 hours and mole ratio between
ChCl to polyalcohol. The result showed the suitable condition of ChCl to HBD mole as 1,2-
propanediol, 1,3-propanediol, and 1,2-butanediol ratio of 1:3 afforded the highest extraction
yields 2.40-2.63 % (w/w) of alpha-mangostin.

Y Yoksandi et al. [19] studied extraction of alpha-mangostin from mangosteen pericarp

by deep eutectic solvent. In the experiment, were formed by mixing betaine anhydrous as the
hydrogen bond acceptor with 1,2-propanediol as the hydrogen bond donors in a molar ratio of
3:1. The experiment were conducted in a temperature range of 27-75 °C and extraction time
from 1-4 hours. The result of the experiment concludes that the highest alpha- mangostin
extraction yield of 4.14 % (g/g) was obtained using 1,2-propanediol and betaine anhydrous at
55 °C
Chutima Boonrat et. al [20] studied three extraction techniques (stirring,
ultrasonication, and Soxhlet extractions) and two solvents (methanol and ethanol) for alpha-
mangostin extraction in mangosteen pericarps. Three different extraction techniques were used
to precisely weigh and measure the dried mangosteen pericarp powder. Each extraction method
was carried out using 150 mL of different single extraction solvents and extraction time of 1
hour. According to the research, the difference of solvents and techniques in term of the alpha-
mangostin content, clearly illustrated that the Soxhlet extraction using methanol as solvent
have similar yield compared with stirring extraction.

Werayut Pothitirit et. al [21] have studied to evaluate the content of alpha-mamgostin
in dried pericarp powder using ethanol extract which extraction time of 15 hours by using
soxhlet extraction method which mangosteens are collected from the East and South of
Thailand from 13 locations. From the experiment, show the result that the average content of
total alpha-mangostin (10.39 ± 1.04 % of the dried powder) was higher in samples from the
South which is slightly more than in samples from the East.
 13

Table 2.1 The optimal condition of alpha-mangostin extraction

Yield of
Optimal conditions for Alpha-
Research Method Solvent
extraction mangostin
compounds
1. Solvent to solid ratio
0.2g/2g (1:10)
Chorine Choline:
Kamarza Mulia et al 2. Time:
Shaking 1,2 -Propanediol 2.6 % (w/w)
[17] 240 minutes
(1:3)
3. Temperature:
Room temperature
1.Solvent to solid ratio
0.2g/2g (1:10)
Chorine Choline:
Farah Fauzia et al. 2. Time:
Shaking 1,2- Propanediol 2.63 % (w/w)
[18] 240 minutes
(1:3)
3.Temperature:
Room temperature
1.Solvent to solid ratio
1:10
Betaine
2. Time:
Y Yoksandi et al. anhydrous :1,2-
Shaking 240 minutes 4.14 % (g/g)
[19] propanediol
3.Temperature:
(3:1)
55 °C

1.Solvent to solid ratio

Chutima Boonrat et. al 1:30
Soxhlet Ethanol 3.74 (mg/g)
[20] 2. Time:
1 hours

1.Solvent to solid ratio

10.39 % of the
Werayut Pothitirit et. al 1:30
Soxhlet Methanol dried powder
[21] 2. Time:
15 hours
 14

CHAPTER III

RESEARCH METHODOLOGY

3.1 Mangosteen powder preparation

The mangosteen was received from Chanthaburi Province, Thailand. Mangosteen

pericarp was separated and cleaned from its edible parts and was dried at 60°C to reduce
moisture content until the final moisture was 10%. The dried material was blended using a
coffee blender. After that, the dried mangosteen pericarp was sieved through a 40-mesh sieve
to obtain a uniform size powder. Finally, the mangosteen pericarp powder was collected in
aluminum foil bags and kept at room temperature.

3.1.1 Apparatus
1. Mangosteen
2. Heating Oven
3. Blender
4. Sieve shaker

3.1.2 Procedures
1. Mangosteen pericarp was separated and cleaned from its edible parts.
2. Mangosteen pericarp was dried by heating oven at 60°C.
3. Minimize mangosteen pericarp with the blender.
4. Dried mangosteen pericarp was sieved to obtain fine powder with particle size in mesh
40.
5. Dried mangosteen pericarp powder was collected in aluminum foil bags and kept at
room temperature.

3.2 Deep Eutectic solvent (DES) preparation and viscosity determination

DES used in this study are mixtures of 1,2-propanediol and a HBA having a certain
mole ratio as listed in Table 3.1 These DES were prepared by heating mixtures that consists of
solid-liquid compounds at 50 under constant stirring, respectively. Stirring was continued for
a period from 30-90 min until a clear solution was formed.

Table 3.1 Solvents used in this study

Mixture solvents HBA HBD HBA:HBD

(Hydrogen bond acceptor) (Hydrogen bond donor) Molar ratio

Mixture 1 Choline chloride 1,2-propanediol 1:1

Mixture 2 Choline chloride 1,2-propanediol 1:2

 15

Mixture solvents HBA HBD HBA:HBD

(Hydrogen bond acceptor) (Hydrogen bond donor) Molar ratio

Mixture 3 Choline chloride 1,2-propanediol 1:3

Mixture 4 Choline chloride 1,2-propanediol 1:4

Mixture 5 Betaine 1,2-propanediol 1:1

Mixture 6 Betaine 1,2-propanediol 1:2

Mixture 7 Betaine 1,2-propanediol 1:3

Mixture 8 Betaine 1,2-propanediol 1:4

3.2.1 Apparatus
1. Precision balance
2. Hotplate stirrer
3. Magnetic bar
4. Beaker
5. Kinematic viscosity
3.2.2 Chemicals
1. Choline chloride
2. 1,2-propanediol
3. Betaine anhydrous
4. Deionized water

3.2.3 Procedures
1. Solvents were prepared by mixing choline chloride and betaine anhydrous as the HBA
and 1,2 propanediol as the HBD, in four mole ratios (1:1, 1:2, 1:3, and 1:4).
2. Solvents were prepared by heating mixtures that consist of solid-liquid compounds at
50°C and stirring was continued for a period from 30-90 min until a clear solution was
formed.
3. Measure viscosity of the solvents using kinematic viscosity.

3.3 Shaking method extraction of mangosteen pericarp with deep eutectic solvents to
select the suitable compositions of deep eutectic solvents

3.3.1 Apparatus
1. Precision balance
2. Centrifugal Tube
3. Incubator shaker
4. Centrifuge
 16

5. Membrane filter 0.45 μm

6. Syringes
7. Vial bottles

3.3.2 Chemicals
1. Mangosteen powder
2. Deep eutectic solvents at the suitable molar ratio and water content
3. Deionized water

3.3.3 Procedures
1. Weight 0.2 g of mangosteen powder and mix with 2 ml of each DES in a sealed
extraction tube.
2. The extraction was out at room temperature and 4 hours by shaking method using
incubator shaker.
3. The suspension was then centrifuged for 15 min at 3000 rpm and the residue was
separated using a 0.45 μm membrane filter to obtain the DES and keep the sample in
vial bottles.
4. Analyze the sample with HPLC.

3.4 Shaking method extraction of mangosteen pericarp with deep eutectic solvents to
optimized extraction operating conditions

Box-Behnken design (BBD) is used to design an experiment after find the appropriate
operating condition by varying three variables: water content in DES, Extraction time, and
Temperature which are experimental factors and levels are defined in following table.

Table 2.2 Experimental design factors and levels of design of experiment

Independence variable Symbol Experiment value

Low (-1) Center (0) High (+1)
Water content in DES X1 10 20 30
(% W/W)
Liquid to solid ratio X2 10 20 30
(ml/g)
Extraction time (min) X3 120 180 240
Temperature (°C) X4 25 35 55

3.4.1 Procedures
1. Weight mangosteen powder and mixed with various liquid to solid ratio in a sealed
extraction tube.
2. The extraction was carried out at various condition in Box-Behnken design. by shaking
method using incubator shaker.
 17

3. The suspension was then centrifuged for 15 min at 3000 rpm and the residue was
separated using a 0.45 μm membrane filter to obtain the DES and keep the sample in
vial bottles.
4. Analyze the sample with HPLC.

3.5 Preparation and analysis of alpha-mangostin

HPLC analysis of alpha-mangostin with an injector, a 20 L loop, and a C-18 column

(250 mm long and 4.6 mm diameter). The sample injection volume was 8 μL, and the isocratic
elution was performed at room temperature at a flow rate of 1 mL/min. The mobile phase
contained acetonitrile and 0.1% (v/v) orthophosphoric acid. The wavelengths of the UV-vis
detector were set to 244 and 320 nm, respectively.

3.5.1 Calibration curve preparation

1. Prepare standard alpha-mangostin concentrations of 1000 micrograms of ethanol ml
from the Laboratory of Herbs and Bioactive Substances, Biomass and Bioenergy
Technology Department, Agricultural Product Research and Development Institute,
and agricultural industry Kasetsart University.
2. Dilute the standard solution with ethanol to a concentration of 50, 100, 200, 300, 400,
500, 700, 1000 μg/ml respectively
3. Collect and filter samples for each concentration into a 2 ml black vial using a nylon
syringe filter membrane.
4. Obtain the area under the graph by measuring samples with an HPLC.
5. Plot calibration curve between the area under the graph and concentration of alpha-
mangostin.

3.6 The purification method of alpha-mangostin

DES with the highest alpha-mangostin extraction yield was selected for the purification
method. First, take mangostin-DES extract to elute by ethyl acetate, filtered, and get a
concentrated solution with a rotary evaporator. Then separated by column chromatography and
the results of the sub-fraction were checked for TLC profiles and seen in UV lamps. After that
select fraction for crystallization and recrystallization

3.6.1 Apparatus
1. Column Chromatography
2. Vacuum dryer
3. Vacuum filter
4. TLC plate
5. Cotton wool

3.6.2 Chemicals
1. Ethyl acetate
2. Diethyl ether
 18

3. Silica gel (merck kieselgel 60 GF254 0.2-0.5 mm)

4. Sea sand

3.6.3 Procedures
1. Ethyl acetate 10 mL was added to 20 mL mangostin-DES extract, stirred for 1 hour and
decanted.
2. Evaporated at 70°C with a rotary evaporator until the solvent is gone and obtained a
thick extract.
3. Prepare column chromatography with the silica gel.
4. Take the sample into column chromatography to separate and get their fraction. Then
the fractionation results are analyzed with TLC.
5. Crystallization and recrystallization.
6. The organic phase was dried using a vacuum dryer.
7. Analyze a sample with HPLC to determine purity of alpha-mangostin.

3.7 Planning

The project takes approximately 10 months to complete.

 19

Table 3.3 Plan schedule

Activities Months
Aug. Sep. Oct. Nov. Dec. Jan. Feb. Mar. Apr. May
Topic discusses
Gather information
Plan the experiment
Design experiment
Prepare mangosteen
pericarp
Preliminary
experiments
Extraction experiment
Experiment analysis
Discuss and conclude
the results
 20

REFERENCES
[1] Zarena, A. S., Manohar, B., & Udaya Sankar, K. (2012). Optimization of Supercritical
Carbon Dioxide Extraction of Xanthones from Mangosteen Pericarp by Response
Surface Methodology. Food and Bioprocess Technology, 5(4), 1181ʹ 1188.
https://doi.org/10.1007/s11947-010-0404-7
[2] Abdalrahim F. A. Aisha, “Quantification of α-, β- and γ-mangostin in Garcinia
mangostana fruit rind extracts by a reverse phase high performance liquid
chromatography,” J. Med. Plants Res., vol. 6, no. 29, Aug. 2012, doi:
10.5897/jmpr11.1253.
[3] Shibata, M. A., Iinuma, M., Morimoto, J., Kurose, H., Akamatsu, K., Okuno, Y., et al.
(2011). Alpha-Mangostin extracted from the pericarp of the mangosteen (Garcinia
mangostana Linn) reduces tumor growth and lymph node metastasis in an
immunocompetent xenograft model of metastatic mammary cancer carrying a p53
mutation. BMC Med. 9:69. doi: 10.1186/1741-7015-9-69
[4] Charoenphun, N., Setarnawat, S., & Sai-ut, S. (2018). Chemical Composition and
Trends in Utilization of By - products and Wastes from 4 Types of Tropical Fruit
Processing. Thai Science and Technology Journal, Vol. 28(May), 113–128.
https://doi.org/10.14456/tstj.2020.10
[5] J. Pedraza-Chaverri, N. Cádenas-Rodríguez, M. Orozco-Ibarra, J.M. Pérez-Rojas, 367
Medical properties of mangosteen (Garcinia mangostana), Food and Chemical 368
Toxicology 46 (2008) 3227-3239.
[6] E.L. Smith, A.P. Abbott, K.S. Ryder, Deep Eutectic Solvents (DESs) and Their
Applications, Chem. Rev. 114 (2014) 11060–11082.
https://doi.org/10.1021/cr300162p.
[7] Smith, E. L., Abbott, A. P., & Ryder, K. S. (2014). Deep Eutectic Solvents (DESs)
and Their Applications. Chemical Reviews, 114(21), 11060–11082.
https://doi.org/10.1021/cr300162p
[8] A. Alhadid, L. Mokrushina, M. Minceva, Modeling of Solid – Liquid Equilibria in
Deep Eutectic Solvents : A Parameter Study, (2019) 1–19.
[9] N. Lisa, “Melting point theory.” [Online]. Available:
https://chem.libretexts.org/Bookshelves/Organic_Chemistry/Organic_Chemistr
y_Lab_Techniques_(Nichols)/06%3A_Miscellaneous_Techniques/6.01%3A_
Melting_Point/6.1C%3A__Melting_Point_Theory. [Accessed: 01-Dec-2021].
[10] Zhang, Q. W., Lin, L. G., & Ye, W. C. (2018). Techniques for extraction and isolation
of natural products: A comprehensive review. Chinese Medicine (United Kingdom),
13(1), 1–26. https://doi.org/10.1186/s13020-018-0177-x
 21

[11] S. Chanioti, G. Liadakis, and C. Tzia, “Solid–Liquid Extraction,” 2014, pp. 253–286.
[12] C. H. Chan, R. Yusoff, and G. C. Ngoh, “Modeling and kinetics study of conventional
and assisted batch solvent extraction,” Chem. Eng. Res. Des., vol. 92, no. 6, pp.
1169–1186, 2014, doi: 10.1016/j.cherd.2013.10.001.
[13] A. Kovács, E.C. Neyts, I. Cornet, M. Wijnants, P. Billen, Modeling the
Physicochemical Properties of Natural Deep Eutectic Solvents, ChemSusChem. 13
(2020) 3789–3804. https://doi.org/10.1002/cssc.202000286.
[14] Saini, A., Kumar, A., Panesar, P. S., & Thakur, A. (2022). Potential of deep eutectic
solvents in the extraction of value‐added compounds from agro‐industrial by‐
products. Applied Food Research, 2(2). https://doi.org/10.1016/j.afres.2022.100211
[15] Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008).
Response surface methodology (RSM) as a tool for optimization in analytical
chemistry. In Talanta (Vol. 76, Issue 5). https://doi.org/10.1016/j.talanta.2008.05.019
[16] The Pennsylvania State University, “Box-Behnken Designs,” 2021. [Online].
Available: https://online.stat.psu.edu/stat503/lesson/11/11.2/11.2.2.

[17] Mulia, K., Krisanti, E., Terahadi, F., & Putri, S. (2015). Selected natural deep eutectic
solvents for the extraction of α-Mangostin from mangosteen (Garcinia mangostana L.)
pericarp. International Journal of Technology, 6(7), 1211–1220.
https://doi.org/10.14716/ijtech.v6i7.1984
[18] Mulia, K., Fauzia, F., & Krisanti, E. A. (2019). Polyalcohols as Hydrogen-Bonding
Donors in Choline Chloride-Based Deep Eutectic Solvents for Extraction of
Xanthones from the Pericarp of Garcinia mangostana L. Molecules, 24(3).
https://doi.org/10.3390/molecules24030636
[19] Mulia, K., Yoksandi, Y., Kurniawan, N., Pane, I. F., & Krisanti, E. A. (2019). 1,2-
Propanediol-betaine as green solvent for extracting α-mangostin from the rind of
mangosteen fruit: Solvent recovery and physical characteristics. Journal of Physics:
Conference Series, 1198(6). https://doi.org/10.1088/1742-6596/1198/6/062003
[20] W. Pothitirat and W. Gritsanapan, “QUANTITATIVE ANALYSIS OF TOTAL
MANGOSTINS IN MATERIALS AND METHODS : Chemicals and Reagents,” vol.
22, no. 4, pp. 161–166, 2008.
 22

[21] C. BOONRAT and R. INDRANUPAKORN, “INFLUENCE OF EXTRACTION

TECHNIQUES AND SOLVENTS ON α-MANGOSTIN AMOUNTS FROM
MANGOSTEEN (Garcinia mangostana L.) PERICARP,” Thai Bull Pharm Sci, vol.
10, no. 2, pp. 1–11, 2015.