VIETNAM NATIONAL UNIVERSITY – HOCHIMINH CITY

INTERNATIONAL UNIVERSITY

INVESTIGATION OF ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF

VARIOUS EXTRACTS FROM HOUTTUYNIA CORDATA THUNB.

A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Applied Biochemistry

Student name: NGUYEN TRAN THANH HUYEN – ID: BTBCIU15043

Supervisor: MSc. LE TRAN HONG NGOC

March, 2020
ACKNOWLEDGEMENT

I would like to express my special thanks of gratitude to my mentor MSc. Le Tran Hong Ngoc
for her passionate guidance during several months. She saved my life in the difficult stage of
my life. Her instruction, suggestions, advices throughout the field of lab work made me believe
that this research is possible. Her unending support will be one of the most unforgettable
memory in my life.

I would like to extend my gratitude to Dr. Hoang Le son for providing me a lot of health
knowledge. By following his advises, I can stay strong physically for a long period.
Concurrently, my sincere thanks also goes to lab technicians Ms. Vy Ha and Mr. Giang for
providing me with all facility in my thesis project.

Besides that, I would like to thank my thesis friends Anh Thu, Nhu Hao, Minh Truc for their
encouragement and for all the fun we have had in the last semester together.

Last but not least, I would like to thank my family and my special friend for always being by
my sides and supporting me spiritually.
 TABLE OF CONTENT
1. ABSTRACT: ..................................................................................................... 1
2. INTRODUCTION: ............................................................................................ 1
2.1. Overview of Houttuynia cordata Thunb.: ........................................................ 1
2.2. Pressurized solvent extraction: ..................................................................... 3
3. MATERIAL AND METHOD: ............................................................................... 6
3.1. Sample preparation: ................................................................................... 6
3.2. Conventional solvent extraction: ................................................................... 6
3.3. Pressurized solvent extraction: ..................................................................... 7
3.3.1. Sample loading..................................................................................... 7
3.3.2. Extraction: ........................................................................................... 7
3.4. Temperature optimization for ethanol extraction: ............................................ 7
3.5. Phytochemical analysis: ............................................................................... 7
3.5.1. Total extraction yield: ........................................................................... 7
3.5.2. Total Phenolic content (TPC) quantification: ............................................. 8
3.5.3. Total Flavonoid content (TFC) quantification:............................................ 8
3.5.4. Antioxidant activity – DPPH assay: .......................................................... 8
3.5.5. Antimicrobial evaluation: ....................................................................... 9
3.5.6. Statistical analysis: ............................................................................. 10
4. RESULTS: ..................................................................................................... 11
4.1. Conventional method versus Pressurized solvent extraction method: ............... 11
4.1.1. Methanol extraction: ........................................................................... 11
4.1.2. Ethanol extraction: ............................................................................. 12
4.1.3. Hexane extraction: ............................................................................. 13
4.1.4. Ethyl acetate extraction:...................................................................... 14
4.2. Temperature optimization: ......................................................................... 15
4.3. Antimicrobial activities: ............................................................................. 15
5. DISCUSSION: ............................................................................................... 16
5.1. Comparison of PSE with percolation: ........................................................... 16
5.1.1. The effectiveness of pressure in higher yield: ......................................... 16
5.2. The effect of different solvents on extraction yield, total phenolic and flavonoid
contents: ............................................................................................................. 16
5.3. Temperature optimum in ethanol extraction of SpeedExtractor: ...................... 17
5.4. Antioxidant activities – DPPH assay:............................................................ 17
5.5. Total phenolic content and total flavonoid content:........................................ 18
5.6. Antimicrobial evaluation: ........................................................................... 19
6. CONCLUSION: .............................................................................................. 21
LIST OF ABBREVIATIONS:

H. cordata: Houttuynia cordata Thunb.

PSE: Pressurized solvent extraction

TPC: total phenolic content

TFC: total flavonoid content

DPPH: 2,2-diphenyl-1-picrylhydrazyl

MIC: minimal inhibitory concentration

IC50: inhibitory concentration at 50% of a maximum effect

QE: Quercetin equivalent

GAE: Gallic acid equivalent

DS: dried sample

Na2CO3: sodium carbonate

AlCl3: aluminum chloride

CH3COOK: potassium acetate

S. aureus: Staphylococcus aureus

P. aeruginosa: Pseudomonas aeruginosa

E. coli: Escherichia coli

LB: Luria-Bertani broth

ND: Not detected

LIST OF FIGURES:

Page

Figure 1. Houttuynia cordata Thunb 2

Figure 2. The decanoyl acetaldehyde can be oxidized or decarboxylated into

2-undecanone 3

Figure 3. Schematic diagram of an accelerated solvent extractor 4

Figure 4. Schematic diagram of SpeedExtractor E-916 5

Figure 5. The process of H. cordata extraction by PSE and conventional methods 6

Figure 6. Extraction cell of SpeedExtractor E-914/E-916 7

Figure 7. The percentage yield from H. cordata by PSE and conventional

method at 30℃ 11

Figure 8. The total phenolic content from H. cordata by PSE and conventional

method at 30℃ 11

Figure 9. The total flavonoid content from H. cordata by PSE and conventional

method at 30℃ 11

Figure 10. The IC50 values from H. cordata by PSE and conventional

method at 30℃ 11

Figure 11. The percentage yield from H. cordata by PSE and conventional

method at 30℃ 12

Figure 12. The total phenolic content from H. cordata by PSE and conventional

method at 30℃ 12

Figure 13. The total flavonoid content from H. cordata by PSE and conventional

method at 30℃ 12

Figure 14. The IC50 values from H. cordata by PSE and conventional

method at 30℃ 12
Figure 15. The percentage yield from H. cordata by PSE and conventional

method at 30℃ 13

Figure 16. The total phenolic content from H. cordata by PSE and conventional

method at 30℃ 13

Figure 17. The total flavonoid content from H. cordata by PSE and conventional

method at 30℃ 13

Figure 18. The IC50 values from H. cordata by PSE and conventional

method at 30℃ 13

Figure 19. The percentage yield from H. cordata by PSE and conventional

method at 30℃ 14

Figure 20. The total phenolic content from H. cordata by PSE and conventional

method at 30℃ 14

Figure 21. The total flavonoid content from H. cordata by PSE and conventional

method at 30℃ 14

Figure 22. The IC50 values from H. cordata by PSE and conventional

method at 30℃ 14

Figure 23. DPPH structure before and after accepting an electron or hydrogen 18

Figure 24. Polyphenol classification 19

LIST OF TABLES:

Page

Table 1. A briefly comparison of traditional extraction and pressurized liquid extraction 4

Table 2. Percentage yield, TPC, TFC and antioxidant activity of H. cordata extract

obtained from PSE 15

Table 3. MIC value of H. cordata extracts and positive control on 3 strains of bacteria 15

Table 4. Inhibition zone of H. cordata extracts and positive control on 3 strains of

bacteria 16
INVESTIGATION OF ANTIOXIDANT AND ANTIMICROBIAL PROPERTIES OF VARIOUS
EXTRACTS FROM HOUTTUYNIA CORDATA THUNB.

NGUYEN TRAN THANH HUYEN a, LE TRAN HONG NGOC a

a
Department of Biochemistry, International University – Vietnam National University HCM

1. ABSTRACT:

The extraction of Houttuynia cordata Thunb. (H. cordata) is carried out by two methods:
pressurized solvent extraction (PSE) and percolation with different solvents such as methanol,
ethanol, hexane and ethyl acetate. This study mainly focused on percentage yield, total
phenolic content, total flavonoid content, antioxidant capacity and antimicrobial activity of
various extracts from the two methods. Overall, the conventional method had lower yield than
PSE, but the antioxidant capacity of extracts between two methods were not significantly
different across corresponding solvents. Meanwhile, methanol was the most efficient solvent
showing the highest percentage yield (8.66±2.38), total phenolic and flavonoid contents
(3.34±0.69 mg GAE/g DS and 2.68±0.11 mg QE/g DS), and the lowest IC50 value – the
concentration needed to decrease 50% concentration of free radical in fixed DPPH solution
(0.87±0.053 mg/mL). In terms of scavenging capacity, methanol extract was 111 times
weaker than ascorbic acid followed by ethyl acetate extract with 143 times, ethanol extract
with 161 times and hexane extract with 248 times. Because ethanol was always applied in
pharmaceutical industry as it is relatively safe for consumption, ethanol was used for
subsequent optimization in SpeedExtractor – PSE machine. At optimum temperature at 70℃
in this machine, ethanol extract reached 12.44±1.67 in percentage yield, 75.25±7.72 mg
GAE/g DS in total phenolic content, 17.00±1.10 mg QE/g DS in total flavonoid content and
0.486±0.037 mg/mL in IC50 value. At this point, talking to antioxidant capacity, ethanol
extract was 62 times weaker than ascorbic acid, proven to be strong scavenger. Finally, all
extracts did not show any antimicrobial activities on three strains bacteria including
Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa.

Keywords: Houttuyia cordata Thunb., pressurized solvent extraction, conventional extraction,

methanol, ethanol, hexane, ethyl acetate, temperature optimization, antioxidant activity,
antimicrobial activity.

2. INTRODUCTION:
2.1. Overview of Houttuynia cordata Thunb.:

1
Houttuynia cordata Thunb. (H. cordata) is an aromatic medicinal herb belonging to family
Saururaceae (Kumar, Prasad, & Hemalatha, 2014). It was described the first time by Carl
Peter Thunberg, who is a Swedish naturalist in 1784. It is distributed in a wide native range
from Nepal to the mountains of Java (Indonesia). It is a creeping herb and the height of its
trees ranges from 30 to 60 cm, with thin and spreading rhizomes. Its stem has green or
purplish-red and either smooth or pubescent on the nodes. Its leaf shape looks like a heart
and its height ranges from 4 to 10 cm long, with 2.5 to 6 cm wide. Its flowers are small, which
is grown into short spike around 2 cm long. In Vietnam, H. cordata grows wildly in moist to
wet locations.

Figure 1. Houttuynia cordata Thunb.

Previous studies have shown that there are many active compounds contained in Houttuynia
cordata Thunb. such as volatile oils, organic acids, flavonoids and water soluble
polysaccharides (Yang & Jiang, 2009). This sheds light on usage of H. cordata as a medicinal
herb. Firstly, volatile oils found in H. cordata include decanoyl acetaldehyde, myrcene, lauric
aldehyde, α-pinene, d-limonene and methyl nonyl ketone. These volatile oils which are
isolated by steam distillation show a wide range of pharmaceutical activities. This is because
decanoyl acetaldehyde plays significant role in these activities. However, it is easily oxidized
to 2-undecanone during the distillation process and storage (Figure 2)(Zeng, Shi, Zeng, &

2
Lai, 2003). These volatile oils are proposed to be able to inhibit the growth of Staphylococcus
aureus, Escherichia coli, Bacillus subtilis, Penicilium, Aspergillus niger and budding fungus
(Xiong, Xi, & Deng, 2002). Secondly, flavonoids found in H. cordata by traditional extraction
method have a wide range of pharmaceutical activities such as anti-hepatotoxic, anti-
inflammatory and anti-tumor activities. The previous study has shown that the yield of
flavonoids in H. cordata is 0.586% including rutin, hypersoide, quercetin and quercitrin(Guo
& Xu, 2007). The flavonoids can be characterized by Thin Layer Chromatography (TLC) and
Ultraviolet-visible detection (UV-Vis).

Figure 2. The decanoyl acetaldehyde can be oxidized or decarboxylated into 2-undecanone

(Zeng et al., 2003).

2.2. Pressurized solvent extraction:

Pressurized solvent extraction (PSE) is one of the most promising techniques for bioactive
compound extraction, which combines extraction process with high temperature and
pressure. High temperature makes solutes in the solvent become more soluble and increase
diffusion rates. Meanwhile, the high pressure enhances the penetration of the solvent into the
sample. The sample is placed in a stainless steel extraction cell. The solvent is filled into the
cell, which is heated to the desired temperature and pressurized concurrently. In the next
step, the extract is removed and the cell is flushed with fresh solvent. The cycle can be
repeated. When the extraction is complete, the compressed nitrogen releases all of solvent
from the cell.

3
 Figure 3. Schematic diagram of an accelerated solvent extractor (Mandal, Mandal, & Das,
2015).

Solvent extraction is the most common method. In this method, the solvent penetrates into
the solid matrix of the sample to dissolve the solutes. Then, the solutes are removed out of
solid matrix. Finally, the extracted solutes are collected.
Table 1.
A briefly comparison of traditional extraction and pressurized liquid extraction (Q. W. Zhang,
Lin, & Ye, 2018).
Method Solvent Temperature Pressure Time Volume of Polarity
organic of natural
solvent products
consumed extracted
Percolation Water, Room Atmospheric Long Large Dependent
aqueous temperature, on
and non- occasionally extracting
aqueous under heat solvent
solvents
Pressurized Water, Under heat High Short Small Dependent
liquid aqueous on
extraction and non- extracting
aqueous solvent
solvents

4
The SpeedExtractor E-916/ E-914 is an automated instrument, which takes advantage of
parallel extraction. In comparison with conventional method, the solvent in this machine is
used at elevated temperature and high pressure is applied to maintain solvent in liquid state.
Moreover, multiple extraction cycles are used to achieve higher percentage yield. When first
extraction cycle finishes, the next cycle is run automatically. In maxima, this machine can
perform 3 cycles. After extraction step, the extracts could be evaporated in parallel by using
Multivapor P-6. All the extracts would be evaporated concurrently to achieve high productivity.
In this study, the SpeedExtractor E-914 and Multivapor P-6 are applied to create process
workflow in parallel up to 4 samples.

Figure 4. Schematic Diagram of SpeedExtractor E-916 (E-914/E-916 Operation manual)

In this study, flavonoids from H. cordata will be extracted by Pressurized Solvent Extraction
(PSE) and traditional solvent extraction method with various solvent. By doing this, the
differences between modern and traditional method would be identified. The total flavonoids
and phenolic contents are concurrently estimated, which are expressed by Quercetin and
Gallic acid equivalent, respectively. Moreover, free radical scavenging activity of H. cordata is
determined by using DPPH due to the elimination of DPPH radicals. The flowchart in Fig. 5
indicates the extraction process of H. cordata by the two methods. After that, temperature
optimization in SpeedExtractor is performed to determine a temperature point, in which
percentage yield, total phenolic and flavonoid are the highest. In comparison with Spe-ed SFE
system (Applied Separations, Allentown, PA, USA), the optimum temperature is expected at
70℃ (Y. Zhang, Li, & Wu, 2008). Spe-ed SFE system is also a pressurized liquid extraction.

5
 Figure 5. The process of H. cordata extraction by PSE and conventional methods.

3. MATERIAL AND METHOD:

3.1. Sample preparation:

Freshly harvested H. cordata was purchased at markets in District 12, Ho Chi Minh city,
Vietnam. The plant including stem, leaf and flower was cleaned with tap water and cut into
slice approximately 10cm. Then, they were placed in oven machine to dry at 55℃. Samples
were ground into fine powder before putting in sealed plastic bags and storing at room
temperature (25℃).
3.2. Conventional solvent extraction:
5g of H. cordata powder was soaked in 50mL of various solvents including methanol, ethanol,
hexane and ethyl acetate in 3 days, then the mixture were filtered by Whatman no.1 filter
paper and vacuum Buchner funnel to collect the liquid. The obtained solid continued to soak
in 50mL solvent in the next 3 days. After that, approximately 100mL collected extracts were
evaporated by MultivaporTM P-6 of Buchi Switzerland to obtain the total extract. The
extraction for each solvent was triplicated.

6
 3.3. Pressurized solvent extraction:
SpeedExtractor E-914/E-916 machine of Buchi Switzerland was used for this extraction
3.3.1. Sample loading
A cellulose filter paper was placed at the bottom of the sample cell. A metal
frit was placed onto the filter and a cell was closed by plug screw. A sample
cell was inserted with H. cordata powder and quartz sand at specific portion
and ratio. Firstly, one third of a sample cell was filled with quartz sand.
Then, a mixture containing 5g of H. cordata powder and 5g of quartz sand
was placed at the middle layer. Finally, a sample cell was completed by
quartz sand layer on the top to reach about 1cm below from the upper end
of the cell. Any dust or sand remaining in the upper space was wiped off
with a small brush to prevent physical damages to sample cell due to high
pressure extraction. The cell was sealed with a cellulose filter paper that
was pressed tightly by using a plunger.
Figure 6. Extraction cell of SpeedExtractor E-914/E-916 (E-914/E-916 Operation manual)

3.3.2. Extraction:
Every extraction process had to go through a quick tightness test to ensure the presence of
nitrogen gas and all cells are in place on activated positions. After that the cells were heated
up to increase the temperature to the desired level while the pressure increased gradually to
reach the set number. The extraction process took place with three cycles. Each extraction
cycle included three steps – heat up, hold and discharge, which were set for 1, 5 and 2
minutes, respectively. The extracted solutions were released into 250mL collection vials. After
the final discharge of the last cycle, the extraction cells were flushed with fresh solvent and
nitrogen gas.
5g of H. cordata powder was loaded in the extraction cells. The extraction was carried out by
the SpeedExtractor E-914/E-916 machine by Buchi with various solvents including methanol,
ethanol, hexane and ethyl acetate. Then, the extracted solutions were evaporated by
MultivaporTM P-6, Buchi Switzerland to obtain the total extract.
3.4. Temperature optimization for ethanol extraction:
Regarding temperature, other factors were kept constant, including pressure at 100Bar and
three extracting cycles. The extraction was conducted at different temperature of 30℃, 40℃,
50℃, 60℃, 70℃ and 80℃. Each temperature was conducted in triplicate.
3.5. Phytochemical analysis:
3.5.1. Total extraction yield:

7
Total extracts from various solvents including methanol, ethanol, hexane and ethyl acetate
by conventional extraction and PSE methods were obtained after solvent evaporation. All the
extractions were done in triplicates. The percentage yields of all extracts were calculated by
using the following equation:
𝒘 𝒏𝒆𝒕 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒆𝒙𝒕𝒓𝒂𝒄𝒕𝒔 (𝒈𝒓𝒂𝒎𝒔)
𝑷𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 𝒚𝒊𝒆𝒍𝒅 (% ) = × 𝟏𝟎𝟎%
𝒘 𝒕𝒐𝒕𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒅𝒓𝒊𝒆𝒅 𝒔𝒂𝒎𝒑𝒍𝒆 (𝒈𝒓𝒂𝒎𝒔)

3.5.2. Total Phenolic content (TPC) quantification:

Phenolic compounds which were considered as secondary metabolites in plant were
responsible for antioxidant activity (Huyut, Beydemir, & Gulcin, 2017). The TPC was
investigated by using Folin-Ciocalteau reagent (Škerget et al., 2005). The results were derived
from calibration curve (y = 0.0045x – 0.0071, R2 = 0.99) of Gallic acid (0-250µg/mL) and
expressed as milligram of equivalent gallic acid per gram of dried sample (mg GAE/g DS).
The reaction solution contained 100µL of diluted extracts, 500µL of Folin-Ciocateau reagent
(Sigma Aldrich, diluted 10 times with distilled water) and 400µL of 7.5% Na2CO3. The samples
were incubated at room temperature for 1 hour. After that the absorbance of samples was
read at 760nm by using a Biotek Synergy HT 96 well plate (Biotek, USA). All analyses was
done in triplicate.
3.5.3. Total Flavonoid content (TFC) quantification:
Flanonoids were a critical class of natural products, which also were considered as secondary
metabolites in plant having a polyphenolic structure (Panche, Diwan, & Chandra, 2016). The
TFC was measured by using the aluminum chloride colorimetry method (Do et al., 2014).
Quercetin was used as a standard prepared in methanol. The reaction solution contained
100µL of diluted extract or standard, 300µL of methanol, 20µL of 10% aluminum chloride,
20µL of 1M potassium acetate and 560µL of distilled water. The samples were kept at ambient
temperature for 30mins. After that the absorbance of samples was read at 415nm by using a
Biotek Synergy HT 96 well plate (Biotek, USA). The results were derived from calibration
curve (y = 0.0041x + 0.0111, R2 = 0.99) of Quercetin (0-220µg/mL) and expressed as
milligram of equivalent quercetin per gram of dried sample (mg QE/ g DS). All analyses were
done in triplicate.
3.5.4. Antioxidant activity – DPPH assay:
The antioxidant test was performed by using DPPH scavenging method with slight modification
(Zahratunnisa, Elya, & Noviani, 2017). The initial DPPH solution was prepared by dissolving
1mg of DPPH in 10mL of methanol. Then, this solution was diluted until the absorbance at
517nm fell in the range of 0.98 ± 0.02. L-ascorbic acid was used as a standard at 1.0-
15µg/mL. The reaction solution consisted of 250µL of extract or standard at different

8
concentrations and 750µL of DPPH solution, which was incubated for 30mins in the dark at
the room temperature. The absorbance of reaction solutions was read at 517nm by using a
Biotek Synergy HT 96 well plate (Biotek, USA).
The scavenging activity was measured following the equation:
(Acontrol − Asample )
%𝐃𝐏𝐏𝐇 𝐪𝐮𝐞𝐧𝐜𝐡𝐞𝐝 = × 100%
Acontrol
Where Asample was the absorbance at 517nm of 250µL of extract or standard with 750µL of
DPPH solution after 30mins; Acontrol was the absorbance at 517nm of 250µL of methanol with
750µL of DPPH solution after 30mins.
IC50 value was defined as the concentration of extract or standard was required to quench
50% of DPPH free radical absorbance (Al-Reza, Rahman, Sattar, Rahman, & Fida, 2010). IC50
could be derived from the calibration curve. The scavenging activity of the extract was
compared to that of the standard solution.
3.5.5. Antimicrobial evaluation:
3.5.5.1. Disc – diffusion test:
Disc diffusion test was performed to determine antimicrobial resistance with slight
modification (Bauer, Kirby, Sherris, & Turck, 1966). A wire loop was used to transfer a few
colonies of the desired organism from the original culture plate to a test tube containing 10mL
of LB broth (Luria Bertani Broth, Miller, HiMedia, India). Then, this tube was incubated for 4
hours to produce bacterial suspension with moderate cloudiness. This meant that the bacterial
suspension of the tested organism should equivalent to 0.5 McFarland standard by measuring
absorbance at 620nm (0.08 – 0.13) using a Biochrom WPA Biowave II UV/Vis
spectrophotometer. If necessary, the bacterial suspension was diluted by LB sterile broth.
100µL of the suspension was transferred to agar plates by 100µL micropipette with a matched
tip, which was streaked onto the surface of the agar plate by sterile cotton swabs. The extracts
were dissolved by Tween 80 and distilled water (v/v = 2:8). Each sterile filter paper disc
(6mm in diameter by Whatman no. 1 filter paper) was load with 20µL of a known
concentration of extracts. Plates were incubated at 37℃ for 24 hours. The standard antibiotic
disc was Gentamycin for E. coli, S. aureus and P. aeruginosa. A mixture of Tween 80 and
distilled water was considered as negative control. After overnight incubation, the zone
diameters (including 6mm in disc) were measured by a ruler as inhibition zone.
3.5.5.2. Determination of minimum inhibitory concentration (MIC):
MIC value was defined as the lowest concentration of substance needed to result in inhibiting
the growth of specific bacteria strain (Zaidan et al., 2005). The MIC values were identified by
using 96-well micro-plates with serial dilution technique (Wiegand, Hilpert, & Hancock, 2008).
Two bacterial suspensions were prepared and incubated for 4 hours at 37℃. If necessary, the

9
suspensions were adjusted with sterile LB broth to 1x108 CFU/mL. The extracts were dissolved
in Tween 80 and distilled water (v/v=2:8) to different concentrations. For each well in a sterile
96-well micro-plate, 100µL of different concentrations of extract or standard were mixed with
100µL of working bacterial suspension. Gentamycin was used as standard antibiotic in the
range of 0.0025mg/mL – 0.1 mg/mL. A mixture of Tween 80 and distilled water was
considered as negative control. Finally, the plates were covered by a lid and incubated at 37℃
for 24 hours.
3.5.6. Statistical analysis:
All the analyses were conducted in triplicate and the experimental data were analyzed by
SPSS. The results were expressed as mean ± standard deviation (S.D.). One-way analysis
(ANOVA) was applied to determine the significant differences between means at p <0.05.

10
 4. RESULTS:
4.1. Conventional method versus Pressurized solvent extraction method:
4.1.1. Methanol extraction:
The percentage yield, TPC, TFC and IC50 value of methanol extraction from PSE were 8.66 ±
2.38, 3.34 ± 0.69 (mg GAE/g DS), 2.68 ± 0.11 (mg QE/g DS) and 0.87 ± 0.053 (mg/mL),
respectively. Similarly, conventional method followed the same order with 4.02 ± 0.02, 1.31
± 0.08 (mg GAE/g DS), 1.57 ± 0.19 (mg QE/g DS) and 0.82 ± 0.23 (mg/mL). In the exception
of IC50 values, the percentage yield, TPC and TFC from two methods were significant
difference (p<0.05). Values were presented as means ± standard deviation. Error bars
represented standard deviation.

11
 4.1.2. Ethanol extraction:
The percentage yield of ethanol extraction increased from 1.62 ± 0.00079 to 3.99 ± 0.24
when changing from conventional method to PSE. Concurrently, the TPC and TFC rose from
0.78 ± 0.06 to 1.97 ± 0.04 (mg GAE/g DS) and from 0.52 ± 0.11 to 1.15 ± 0.14 (mg QE/g
DS). Values between PSE and conventional method were significantly different (p<0.05). In
contrast, IC50 of two methods was the same with 1.26 ± 0.14 (mg/mL) for PSE and 1.32 ±
0.12 (mg/mL) for conventional method. Values were presented as means ± standard
deviation. Error bars represented standard deviation.

12
 4.1.3. Hexane extraction:
The percentage yield, TPC, TFC and IC50 value of hexane extraction from PSE were 2.22 ±
0.21, 0.19 ± 0.01 (mg GAE/g DS), 0.68 ± 0.05 (mg QE/g DS) and 1.94 ± 0.45 (mg/mL),
respectively. Similarly, conventional method follows the same order with 0.83 ± 0.003, 0.058
± 0.00051 (mg GAE/g DS), 0.23 ± 0.06 (mg QE/g DS) and 3.29 ± 0.85 (mg/mL). In the
exception of IC50 values, the percentage yield, TPC and TFC from two methods were
significant difference (p<0.05). Values were presented as means ± standard deviation. Error
bars represented standard deviation.

13
 4.1.4. Ethyl acetate extraction:
The percentage yield of ethyl acetate extraction increased from 1.25 ± 0.08 to 3.15 ± 0.03
when changing from conventional method to PSE. Concurrently, the TPC and TFC rose from
0.54 ± 0.11 to 0.91 ± 0.07 (mg GAE/g DS) and from 0.43 ± 0.07 to 0.57 ± 0.01 (mg QE/g
DS). Values between PSE and conventional method were significantly different (p<0.05). In
contrast, IC50 of two methods was the same with 1.12 ± 0.078 (mg/mL) for PSE and 0.97 ±
0.13 (mg/mL) for conventional method. Values were presented as means ± standard
deviation. Error bars represented standard deviation.

14
 4.2. Temperature optimization:
Table 2.
Percentage yield, TPC, TFC and antioxidant activity of H. cordata extract obtained from PSE
Extraction Percentage Total phenolic Total flavonoid Antioxidant
temperature yield (%) content (mg content (mg activity – IC50
GAE/g DS) QE/g DS) (mg/mL)
Ethanol, 30℃ 3.95 ± 0.27 1.97 ± 0.05 1.10 ± 0.23 1.26 ± 0.136

Ethanol, 40℃ 3.98 ± 0.24 2.33 ± 0.20 0.433 ± 0.095 1.08 ± 0.099

Ethanol, 50℃ 6.77 ± 0.52 5.98 ± 0.64 1.48 ± 0.15 0.486 ± 0.033

Ethanol, 60℃ 9.14 ± 1.05 8.72 ± 2.36 2.84 ± 0.84 0.481 ± 0.136

Ethanol, 70℃ 12.44 ± 1.67 75.25 ± 7.72 17.00 ± 1.10 0.486 ± 0.037

Ethanol, 80℃ 15.37 ± 1.28 54.85 ± 18.61 2.64 ± 1.24 0.645 ± 0.195

4.3. Antimicrobial activities:

A different concentration of extracts from 10mg/mL to 80mg/mL was tested on three strains
of bacteria on Petri dishes. Among three bacteria strains, E. coli was the strongest ones,
requiring the highest concentration of Gentamycin, following by P. aeruginosa and S. aureus.
As a result, all extracts do not have ability to inhibit the growth of these bacteria.
Table 3.
MIC value of H. cordata extracts and positive control on 3 strains of bacteria.
Bacteria MIC value (mg/mL)
Extracts Gentamycin
Staphylococcus aureus ND 0.005
Escherichia coli ND 0.01
Pseudomonas aeruginosa ND 0.008
ND – Not detected

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Table 4.
Inhibition zone of H. cordata extracts and positive control on 3 strains of bacteria.
Bacteria Inhibition zone (mm)
Extracts Gentamycin
Staphylococcus aureus ND 0.49 ± 0.0074
Escherichia coli ND 0.72 ± 0.0125
Pseudomonas aeruginosa ND 0.57 ± 0.0085
ND – Not detected

5. DISCUSSION:
5.1. Comparison of PSE with percolation:
Pressurized solvent extraction and percolation with corresponding solvents were performed
as what referred in Section 3. The results are exhibited in Fig. 7-22. These results demonstrate
the obviously higher percentage yield, higher phenolic and flavonoid content of PSE in various
solvents. In terms of scavenging activity, two methods show no difference by IC50 value.
5.1.1. The effectiveness of pressure in higher yield:
High pressure is one of the factors affecting extraction yield. High pressure can increase the
rate of mass transfer and disrupt the cellular walls. By doing this, the ability of solvent
penetration into cells can be improved, which results in high permeability (Prasad, Yang, Yi,
Zhao, & Jiang, 2009). As shown in Fig. 7-9, Fig. 11-13, Fig. 15-17 and Fig. 19-21, total
percentage yield, phenolic and flavonoid content was ultimately influenced by pressure. Take
methanol extraction as an evidence, percentage yield in conventional method was 4.02 ±
0.02, which increased to 8.66 ± 2.38 when 100Bar was applied in extraction process. Also,
the phenolic content increased from 1.31 ± 0.08 to 3.34 ± 0.69 (mg GAE/g DS) and the
flavonoid content went up to 2.68 ± 0.11 compared with 1.57 ± 0.19 (mg QE/g DS) without
pressure. These results confirm the hypothesis that pressure would affect the percentage
yield during extraction process.
5.2. The effect of different solvents on extraction yield, total phenolic and
flavonoid contents:
As a result, methanol and ethanol extractions show the maximum percentage yield and TPC
followed by ethyl acetate and hexane. This result indicates that the percentage yield and TPC
increases with increasing polarity of solvent used in extraction. Based on the polarity and
“like dissolves like” principle, hexane is applied to extract non-polar compounds such as
carotenoid and ethyl acetate extraction are used to extract polar limonoids and flavonoid

16
aglycones and glucosides. Meanwhile, methanol and ethanol extractions are performed to
release medium polar and polar compounds such as aglycones and glucosides of flavonoids,
limonoids, ascorbic acid and sugars (Jayaprakasha, Girennavar, & Patil, 2008). Methanol can
be considered as the most efficient solvent for extraction (the highest percentage yield, TPC,
TFC and lowest IC50 value). Other compounds such as proteins and carbohydrates are more
soluble in methanol than ethanol, which contribute to higher yield in methanol extraction (Do
et al., 2014). Although ethanol extraction is second level in percentage yield, TPC and TFC,
ethanol is safe for human consumption. From this reason, ethanol extraction is chosen for
subsequent optimization.
5.3. Temperature optimum in ethanol extraction of SpeedExtractor:
As shown in Table 2, the percentage yield increases when temperature increases. However,
TPC and TFC peaked at 70℃ with 75.25 ± 7.72 (mg GAE/g DS) and 17.00 ± 1.10 (mg QE/g
DS), respectively. Meanwhile, the lowest IC50 value emerged at 60℃ with 0.481 ± 0.136
(mg/mL). This result indicates the effectiveness of temperature on extraction yield, TPC and
TFC contents. Theoretically, under the heat treatment, plant tissue becomes softer and the
interactions of phenol-protein and phenol-polysaccharide become weaker. As a result, more
phenolic compounds could be easily released into the solvent (Shi et al., 2003). However,
high temperature can cause the degradation of phenolic compounds (Brglez Mojzer, Knez
Hrncic, Skerget, Knez, & Bren, 2016). In comparison with IC50 value at 60℃, the IC50 value
at 70℃ is relatively lower (0.486 ± 0.037 mg/mL). Therefore, 70℃ is believed to be the
optimum temperature.
5.4. Antioxidant activities – DPPH assay:
DPPH assay is considered as reliable method since DPPH appears a deep violet color, having
strong absorption band at 517nm (Akar, Kucuk, & Dogan, 2017). The antioxidant can convert
DPPH• radical into its non-radical form by donating an electron or hydrogen to odd electron
of nitrogen atom in DPPH• (Fig. 23). By doing this, the mixture of DPPH• radical and
antioxidant would lose violet color. DPPH assay is applied to measure the ability of all extracts
to act as free radical scavengers or hydrogen donors. The results are expressed as IC50 value,
which mean the concentration of substance needed to neutralize 50% concentration of fixed
DPPH solution. The lower IC50 values are, the better antioxidant capacities of the extracts
are proven.

17
 Figure 23. DPPH structure before and after accepting an electron or hydrogen (Kedare &
Singh, 2011).

As shown in the Fig. 10, Fig. 14, Fig. 18, Fig. 22, IC50 values of various extracts follow the
order methanol, ethyl acetate, ethanol and hexane extracts, meaning that the more polarity
solvents are, the more antioxidant capacity extracts are observed, which are compared to
IC50 value of ascorbic acid as standard with 7.83x10 -3 mg/mL. In comparison with antioxidant
activity to DPPH of ascorbic acid, methanol extract is weaker than 111 times followed by ethyl
acetate extract with 143 times, ethanol extract with 161 times and hexane extract with 248
times. However, previous studies have shown that IC50 values of methanol, ethanol and
hexane extracts were 0.05 ± 0.00, 0.10 ± 0.01 and 0.00 mg/mL (Tuyen, Anh, Trang, & Xuan,
2018), respectively, which were higher than IC50 values across corresponding solvents in this
study. This might be because of the difference in pedology, time of gathering a crop and
climate in the two different areas. Therefore, further investigation are needed to conduct to
H. cordata species information in different areas.
On the other hand, IC50 value in temperature optimization of ethanol extraction peaked at
60℃ with 0.481 ± 0.136 mg/mL. In comparison with ascorbic acid, ethanol extract was
weaker than 61 times. At the optimum temperature 70℃, IC50 value was 0.486 ± 0.037
mg/mL being insignificantly different with this value at 60℃, which was weaker than ascorbic
acid 62 times. Therefore, ethanol extracts at 70℃ are apparently observed to be the highest
scavenger, which is preliminary screening for further experiments to optimize other factors in
SpeedExtractor – PSE machine.
5.5. Total phenolic content and total flavonoid content:
Generally, polyphenols are divided into two main classes including flavonoids which are
separated to flavones, flavononse, flavonols, flavanols and isoflavones, and phenolic acids
consisting of hydroxybenzoic and hydroxycinnamic acids (Abbas et al., 2016) (Fig.24). In

18
plants, there are three types of phenolic acids including free, soluble conjugated and
insoluble-bound forms (Jung, Jeon, & Bock, 2002; Madhujith & Shahidi, 2009; Robbins,
2003). Free forms phenolic are easily released by extraction, while conjugated and bound
forms need to go through hydrolysis to be detected. Gallic acid belongs to conjugated forms.
In this study, Gallic acid is used as standard for equivalent total phenolic content. Hence, only
conjugated gallic acid is detected. However, other conjugated compounds, free-bound and
bound phenolic compounds still contribute to scavenging capacity. Similarly, Quercetin which
is considered as a conjugated soluble and bound flavonoid (Dong, Hu, Hu, & Xie, 2016) is
used as standard for flavonoid equivalent. Therefore, only conjugated and bound quercetin is
detected by spectrophotometer. Other types of flavonoid is ignorable. Because all types of
phenolic and flavonoid are not exactly measured, this causes in some cases TPC to be lower
than TFC from the same extracts. It might be the case in hexane extracts from the
conventional and PSE methods. The TFC (0.65±0.05 mg QE/g DS) was seen as higher figure
than the TPC (0.19±0.01 mg GAE/g DS). More experiments should be performed to invest
more in depth the TPC and TFC measurement. Moreover, the phenolic and flavonoid contents
of extract from PSE and conventional method need to be identified.

Figure 24. Polyphenol classification (Hardman, 2014).

5.6. Antimicrobial evaluation:

According to literature review, H. cordata extracts have shown the great ability to inhibit
the growth of some bacterial strains, especially S. aureus and E. coli, which oppose to the

19
findings in this study that all extracts show no significant antimicrobial activities on these
bacteria. This might be caused by two strains of bacteria. Due to financial constraint, the
ATCC strains could not be investigated, these trains of bacteria were collected from drug
resistance bacteria research lab by transferring cultivation. These types of bacteria may
have already developed antibiotics resistance, in turn affecting the overall results in this
study. Besides that, the pedology, climate and harvesting conditions have taken into
account. There are many variable for the antimicrobial evaluation. More studies should be
designed to assess and establish reliable conclusion.

20
6. CONCLUSION:
In comparison between PSE and conventional method, PSE has been proven to have
always resulted in higher percentage yield, TPC and TFC. The effect of temperature on the
SpeedExtractor E-914/E-916 for ethanol extraction of H. cordata was optimized. Under
optimal temperature (70℃), percentage yield, TPC and TFC achieved 12.44±1.67,
75.25±7.72 (mg GAE/g DS) and 17.00±1.10 (mg QE/ g DS). Most noticeably, ascorbic
acid is only stronger than this extract 62 times, which prove that this extract has strong
antioxidant ability. In the future, other factors in the SpeedExtractor machine should be
optimized to form a whole process, which allows to produce high-qualified and high-
quantified H. cordata extract. In addition, further investigation is highly recommended to
confirm the antimicrobial property of H. cordata extracts.

21
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APPENDICES:

APPENDIX A. Gallic acid standard curve

APPENDIX B. Quercetin standard curve

24
 Ascorbic acid
100 y = 6.002x + 3.0322
90 R² = 0.9989
80
% DPPH quenched 70
60
50
40
30
20
10
0
0 2 4 6 8 10 12 14 16
Concentration (ug/mL)

APPENDIX C. Scavenging capacity of ascorbic acid against DPPH

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APPENDIX D. Differences between PSE and conventional methods of the methanol extracts
in terms of percentage yield, TPC, TFC and IC50 values by T-test.

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APPENDIX E. Differences between PSE and conventional methods of the ethanol extracts in
terms of percentage yield, TPC, TFC and IC50 values by T-test.

27
28
APPENDIX F. Differences between PSE and conventional methods of the hexane extracts in
terms of percentage yield, TPC, TFC and IC50 values by T-test.

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APPENDIX G. Differences between PSE and conventional methods of the hexane extracts in
terms of percentage yield, TPC, TFC and IC50 values by T-test.

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31
32
33
APPENDIX H. Differences in temperature of ethanol extracts in PSE method in terms of yield,
TPC, TFC and IC50 by using One-way ANOVA.

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SUPERVISOR’S APPROVAL

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