Article

Cytotoxic Activity of Polymethoxyflavone Compounds Ethyl Acetate Extract of

Sungkai Leaves (Peronema canescens Jack) as Anticancer Candidates

Zairaini Adriliana¹, Nindita Clourisa Amaris Susanto², Muhammad Fikriansyah³, Indra Lasmana

Tarigan³, Sutrisno^3*

¹Department of Food Sciences and Technology, Faculty of Agricultural Technology, IPB University, Indonesia

²Department of Pharmacy, Vocational School, Universitas Sebelas Maret, Surakarta, Indonesia

³Department of Chemistry, Faculty of Science and Technology, Universitas Jambi, Indonesia

Abstract

This study presents the findings of a phytochemical screening of the ethyl acetate fraction of Sungkai leaves,

revealing the presence of various secondary metabolites, including flavonoids, steroids, phenolics, and tannins.

Liquid vacuum chromatography resulted in the separation of four distinct fractions: Fraction 1 (F1), Fraction 2 (F2),

Fraction 3 (F3), and Fraction 4 (F4). An isolate obtained from Fraction 2 was designated as F2.1. Characterization of

this isolate was conducted using UV-Vis and FT-IR spectrophotometry. The UV-Vis analysis revealed two absorption

peaks at 235.29 nm (band 1) and 286.87 nm (band 2), indicating the presence of a conjugated diene system and an

aromatic system with specific substituents, respectively. FT-IR spectroscopy of the F2.1 isolate from the ethyl acetate

fraction of Sungkai leaves exhibited absorption bands at 2851.89 cm⁻¹, suggesting the presence of methoxy (-OCH₃)

functional groups, and at 1261.13 cm⁻¹, corresponding to C-O-C stretching vibrations. Additional absorption peaks

at 1467.27 cm⁻¹ and 840.65 cm⁻¹ indicated the presence of aromatic C=C and C-H bonds, respectively, while the

absorption at 1729.25 cm⁻¹ was characteristic of carbonyl functional groups. These spectral characteristics suggest

that the isolate belongs to the flavonoid class, specifically the flavone subclass, with structural similarities to

polymethoxyflavones such as sinensetin. The cytotoxic evaluation, performed using the Brine Shrimp Lethality Test

(BSLT) and analyzed via the probit method in SPSS version 25 (SPSS Inc., Chicago, IL, USA), determined the LC₅₀value

of the ethyl acetate extract to be 408.32 ppm. The LC₅₀values for the VLC fractions (F1, F2, F3, and F4) were 168.66

ppm, 242.66 ppm, 1936.42 ppm, and 857.03 ppm, respectively. Meanwhile, the isolate exhibited an LC₅₀value of

191.43 ppm, indicating potential anticancer properties.

Keywords: Biactive compound, cytotoxic; Peronema canescens.

Graphical Abstract

*

Corresponding author

Email addresses: herasutrisno@unja.ac.id

DOI: https://doi.org/10.22437/chp.v6i4.41660

(https://creativecommons.org/licenses/by/4.0 )

140

Chempublish Journal, 6(4) 2022, 140-148

Introduction

Cancer is

alternative treatment materials [6]. Traditional

medicine has been widely used and developed in

several countries [8].

a

disease characterized by the

uncontrolled growth and spread of abnormal

cells [1]. Four main factors cause cancer such as

environment, food, biological and psychological.

Cancer is a disease with no known exact cause

but is influenced by many factors, such as

Traditional medicine is a treatment derived from

herbal plants and has been known since ancient

times. Traditional medicine has been relied on

for generations because it is inexpensive and

often has fewer side effects than modern

medicine. Additional research on plants used as

traditional medicines is still needed through

preclinical trials and clinical trials to be used

safely in healthcare facilities. Many herbal plants

have no known degree of cytotoxicity. One of the

natural ingredients that is often used in

traditional medicine is sungkai leaves [9].

smoking/exposure

to

cigarette

smoke,

consuming alcohol, excessive exposure to

ultraviolet light on the skin, obesity, an unhealthy

diet, lack of physical activity, and infections

related to cancer. Cancer can be prevented by

reducing the risk factors for cancer. In

developments in the health sector, anticancer

drugs have been discovered, and chemotherapy

has been carried out, but the high-cost factor is

an obstacle [2]. Cancer is a deadly disease for

humans. The World Health Organization (WHO)

states that cancer is the main cause of morbidity

and mortality worldwide, with approximately 14

million new cases and 8.2 million deaths from

cancer in 2012. In Indonesia, the prevalence of

cancer is 1.4 out of 1000 population or around

330 thousand Persons. The number of cancer

patients in Indonesia is very high. This was

published based on data on people affected by

cancer from the government and cancer

institutions [3]. Deaths caused by cancer

continue to grow. From the World Health

Organization (WHO), 9.6 million people died from

cancer in 2018 [4].

The sungkai leaf plant is one of the plants used

as a source of traditional medicine for the

community and is unique (endemic) to Indonesia.

There are bioactive compounds from Sungkai

leaf extract, including flavonoids, alkaloids,

steroids, phenolics, tannins, and saponins [10–

12]. Several studies reported that Sungkai leaves

have

bioactivities

such

as

antimalarial,

antibacterial,

analgesic,

immunomodulatory,

antiplasmodial, antidiabetic, anthyperusemia,

and immunostimulant [13].

Secondary metabolite compounds (such as

alkaloid compounds, terpenoids, flavonoids,

phenolics, and organic acids) indicate that plants

have secondary metabolite compounds that

inhibit the growth of cancer cells. Potential

anticancer activity of chloroform subfraction

from Sungkai leaves against HT-29 cancer cells in

vitro for secondary metabolite compounds from

chloroform extract, namely alkaloids, terpenoids,

steroids, flavonoids, and phenolics. The cytotoxic

activity (IC₅₀) value against HT-29 colon cancer

cells ranged from 14.807 to 34.448 µg/ml. Tests

showed that chloroform subfraction 3 showed

potential cytotoxic activity against human HT-29

cancer cells with an IC₅₀value of 14.807 µg/ml

[14].

Most patients with cancer receive chemotherapy,

which involves using anticancer drugs and

supporting drugs to reduce the side effects of

using anticancer drugs. One example of an

anticancer drug is doxorubicin, but continuous

use of doxorubicin can be toxic to organs [5].

Chemotherapy drugs kill not only cancer cells but

also normal cells [6]. Early prevention against the

risk of cancer is to be able to consume fruits and

vegetables that contain phytochemicals that are

beneficial to the body [7]. Thus, it is necessary to

develop therapies using natural medicines as

141

Chempublish Journal, 6(4) 2022, 140-148

Cytotoxic activity of Peronema canescens Jack

sungkai leaves in human cells: HT-29 and primary

colon cancer adenocarcinoma methanol extract.

Cytotoxicity value (IC₅₀) to AdenoCa cells 1.897

μg/ml. The inhibitory activity of the synthesis and

mitotic phases in the cell cycle showed that

different concentrations of SF3 had inhibitory

activity on HT-29 (29,614 µg/ml) of 26.79% and

0.16%, AdenoCa cells (14,807 µg/ml) of 10.27%

and 19.29% respectively. The apoptotic activity

induced in HT-29 cells (29,614 µg/ml) and

AdenoCa cells (14,807 µg/ml) were 26.58% and

11.50% [15].

L n-hexane solvent for 2 x 24 hours with two

repetitions. The simplicia was macerated again

using ethyl acetate solvent in the same way. Both

maserates were evaporated at 60 °C. The yield

obtained was calculated as a weight percentage

(w/w). Sungkai leaf phytochemical screening,

including tests for alkaloids, saponins, phenolics,

tannins, steroids, and flavonoids [10].

Separation

and

Purification.

Thin

Layer

Chromatography (TLC) was prepared 1 x 5 cm

with a lower and upper limit of 1 cm and 0.5 cm,

respectively, so the eluent travelled 3.5 cm.

Eluents are prepared by comparing organic

solvents based on their polarity. The extract was

spotted using a capillary tube at the bottom of

the plate, and elution was carried out using the

mobile phase. After the mobile phase reaches

the upper limit on the plate, the elution process

is stopped. Then the stain was examined directly

under a UV lamp with a wavelength of 254 or 395

nm. All fractions were subjected to the TLC test,

and the fractions with the same stain spots were

put together and analyzed by TLC.

Material and Methods

Materials and Instrumentations

The sample used in this study was sungkai leaves

(Peronema

canescens Jack). Samples were

obtained from Kademangan Village, Jaluko

District, Muaro Jambi Regency, Jambi Province,

Indonesia. The chemicals were used ethyl

acetate, n-hexane, 2N sulfuric acid, Dragendorff

reagent, Meyer reagent, Lieberman-Burchard

reagent, concentrated HCl, Mg powder, 2N HCl,

FeCl3, acetic acid. Anhydrous (Sigma Aldrich), fine

silica gel (packing) 0.040 - 0.063 mm and coarse

silica gel (imprent) 0.063 -0.200 mm (Merck),

Artemia salina larvae eggs, NaCl.

Furthermore, Isolation of compounds was

carried out using vacuum liquid chromatography

(VLC). VLC was performed using silica gel as the

stationary phase with a sample:silka gel ratio

(1:20). The sample extract was impregnated

using silica gel and added to the column

containing the stationary phase. At the same

time, the mobile phase used is a mobile phase

with gradient polarity. 10-15 grams of sample

was impregnated with 15 grams of coarse silica

gel (0.063 – 0,200 mm silica imprent). VLC was

carried out using a column with a diameter of 5

cm with the stationary phase in the form of fine

silica gel (silica packing 0.040-0.063mm) and the

mobile phase in the form of a solvent with

gradient polarity. Gradient elution increases the

resolution of complex mixtures, especially when

the sample has a broad polarity range. VLC starts

from 100% n-hexane, n-hexane: ethyl acetate,

ethyl acetate: methanol, to 100% methanol. The

resulting fraction is then accommodated in a vial.

The eluate is accommodated based on each band

The equipments and instrumentations used in

this study were maceration bottles, filter paper,

rotary

evaporator

components,

funnels,

measuring cups, Erlenmeyer cups, VLC and GCC

columns, vacuum pumps, capillary tubes, TLC

plates (Merck), drip plates, test tubes, test tube

rack, dropping pipette, 1 ml micropipette, 10 ml

micropipette, KBR pellet, volumetric flask, vial,

stir bar, watch glass, label paper, aerator, Artemia

salina larvae breeding vessel, incandescent lamp,

loop

(magnifying

glass),

UV-Vis

spectrophotometer (Shimadzu, Japan), FTIR

spectrophotometer (Bruker, USA)

obtained

and

then

evaporated.

Column

Methods

chromatography results were carried out by TLC

again. Rf values that are close to and have

identical spots are combined into one fraction.

Extraction and Screening. Sungkai leaves as much

as 1.5 kg were macerated in stages starting in 1.5

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Chempublish Journal, 6(4) 2022, 140-148

BSLT Method Cytotoxic Test. The cytotoxicity test

treatment was carried out on four repetitions.

Artemia salina larvae were prepared by

incubating the eggs 48 hours before testing and

Secondary Metabolites

Phytochemical screening was carried out

qualitatively using specific reagent solvents for

each secondary metabolite, with the colour test

method to determine the content of chemical

compounds (Table 2 dan Table 3). There is a

difference in the phytochemical screening using

hexane and ethyl acetate solvents. Ethyl acetate

solvents contain more diverse secondary

metabolite compounds than hexane solvents

because hexane solvents tend to be non-polar,

which can extract non-polar compounds such as

steroid compounds. The existence of various

metabolite compounds in phenolic ethyl acetate

solvents, tannins, steroids and flavonoids are a

class of compounds that have cytotoxic activity.

prepared

mother

liquor

and

serial

concentrations of 1000 ppm, 100 ppm, and 10

ppm solvent used according to the fraction. Sea

water was used as a negative control. Added 5 ml

of engineered seawater and 1 ml of each test

solution which had been evaporated for 24 hours

into a test tube and then homogenized, then ten

larvae were added. Observations were made (24

hours) on the death of shrimp larvae with the

help of a magnifying glass

Characterization and Data analysis. Isolate was

characterized using a UV-Vis spectrophotometer

and FTIR spectrophotometer. Cytotoxicity data

analysis was carried out by knowing the mortality

of Artemia salina larvae, then looking for the

probit number using the probit analysis program

SPSS version 25 (SPSS Inc., Chicago, IL, 250 USA).

Table 2. Phytochemical screening of extracts

Fractions

Secondary

Metabolites

Hexane

EtAc

Results and Discussions

Alkaloids

-

- Dragendorf

- Meyer

A dry sample of 1500 g was macerated in stages.

The initial solvent used for maceration was n-

hexane, as much as 1.5 L. From the maceration

-

Saponins

-

results,

a

yellow

extract

was

obtained.

Phenolics/Tanins

Steroids

-

+

Maceration with hexane was repeated two times

until the yellow extract faded. Then, maceration

was continued with 1.5 litres of ethyl acetate and

two repetitions. The EtOAc extract fraction

obtained was dark green. The extracts from the

two solvents were then concentrated using a

rotary evaporator. The dry hexane extract

fraction from Sungkai leaves has a sticky

+

-

Flavonoids

There is a difference in the phytochemical

screening using hexane and ethyl acetate

solvents. Ethyl acetate solvents contain more

diverse secondary metabolite compounds than

hexane solvents because hexane solvents tend

to be non-polar, which can extract non-polar

compounds such as steroid compounds. The

existence of various metabolite compounds in

phenolic ethyl acetate solvents, tannins, steroids

and flavonoids are a class of compounds that

have cytotoxic activity.

character

and

a

yellowish-green

colour.

Meanwhile, the dry EtOAc extract fraction also

has a sticky character and is green-black. The

mass and yield of the extract are presented in

Table 1.

Table 1. Mass and yield of dried crude extracts

Fraction extracts

Hexane

Mass (g)

14.2

Yield (%)

0.94

Ethyl acetate

18.3

1.22

143

Chempublish Journal, 6(4) 2022, 140-148

Table 3. Phytochemical screening of VLC EtOAc fraction

Secondary

Metabolites

F1

-

F2

-

F3

F4

-

Alkaloids

Saponins

-

Phenolics/tanins

Steroids

+

-

+

-

+

Flavonoids

+

In the separation process using VLC, 21 vials were

obtained and then evaporated at room

temperature to approximately ½ vial. After that,

TLC was performed on each eluate in the vials to

identify the fractions in the 21 vials. The eluate in

the vial, which has the same stain pattern as the

TLC, is combined into 1 fraction. The results of

this VLC are 4 fractions, namely F1, F2, F3, and F4.

The four fractions were then subjected to a

phytochemical screening test. The results of the

phytochemical screening showed that all the VLC

fractions of the EtOAC extract of Sungkai leaves

were still positive for several classes of secondary

metabolite compounds in Table 3. This

phytochemistry aims to classify secondary

metabolite compounds that can describe isolate

from the isolate as belonging to a specific class of

secondary metabolite.

Characterization of the Isolate

The UV-Vis spectrum showed that the isolate

gave two absorption peaks: band 1 λ = 235.29 nm

and band 2 λ = 286.87 nm (Figure 1). The

absorption at 235 nm indicates the presence of a

conjugated diene/double system in the structure.

Meanwhile, the absorption at 286 indicates the

presence of an aromatic system with specific

substituents.

1,0

0,8

0,6

0,4

0,2

0,0

200 250 300 350 400 450 500 550 600 650 700 750 800

In the F2 fraction, there is a precipitate, and then

the precipitate is separated from the solution to

be recrystallized. Recrystallization is carried out

for the purification stage of impurities that come

down during the elution process. The isolate is

recrystallized with n-hexane solvent then ethyl

acetate is called F2.1. After recrystallization was

complete, TLC and phytochemical screening

were carried out. In isolate, the phytochemical

screening usually shows 1 positive test on the

results of the phytochemical screening.

Figure 1. UV-Vis spectrum of the isolate

Based on the TLC test for isolate F2.1, ethyl

acetate: methanol (3:7) was used with an Rf value

of 0.51 and acetone: methanol (6:4) with an Rf

value of 0.77. In this study, the isolate was

positive for secondary metabolite compounds,

namely flavonoids, on the results of the

phytochemical screening

Figure 2. FTIR spectrum of the isolate

Figure 3. Chemical structure of Sinensetin [16]

144

Absorbance

Wavelenght (nm)

235,29

286,87

-0,1

0,0

0,1

0,2

0,3

0,4

0,5

0,6

4000 3500 3000 2500 2000 1500 1000 500

Transmitance (%)

Wavenumber (cm^-1

)

3340,54

2920,88

2851,89

1729,25

1605,24

1467,27

1261,13

1164,23

1019,69

716,64

840,65

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Chempublish Journal, 6(4) 2022, 140-148

Table 4. IR spectrum comparison and interpretation

Adsorption (cm^-1)

Interpretation

Isolate

PMF

Sinensetin [16]

2851.89 cm^-1

2854 cm^-1

2838 cm^-1

-OCH₃

1261.13 cm^-1

1467.27 cm^-1

840.65 cm^-1

1729.25 cm^-1

1266 cm^-1

1463 cm^-1

-

1264 cm^-1

1486 cm^-1

-

-C-O-C streching

-C=C stretching aromatic

-C-H bending aromatic

-C=O streching

1733 cm^-1

1639 cm^-1

IR spectrum on isolate F2.1 ethyl acetate fraction

of Sungkai leaves showed absorption in the area

of 2851.89 cm^-1, indicating the presence of –OCH₃

from methoxy, stretching vibration of C-O-C in

the absorption area of 1261.13 cm^-1, stretching

vibration in the absorption area of 1467 .27 cm^-1

which indicates an aromatic C=C characteristic,

bending vibrations in the absorption area of

840.65 cm^-1indicate the presence of aromatic C-

H, and stretching vibrations in the absorption

area of 1729.25 cm^-1which indicates the

characteristics of carbonyl compounds (Figure 2).

This IR absorption is significant in indicating the

characteristics of the secondary metabolite class

of flavonoids from sub-flavonoids, namely

flavones. Based on the comparison of the IR

spectra in Table 4, shows that these compounds,

second instar stage. In this second stage, the

mouth and digestive system are complete [¹⁸].

Artemia salina larvae are known to have skin

sensitive to the environment, for example,

foreign

compounds

such

as

bioactive

compounds that can diffuse into cells. If the

bioactive compound has cytotoxic activity,

Artemia salina larvae will die [19].

The testing time for Artemia Salina larvae

significantly affects the mortality rate of the

larvae. In previous studies, the 12-hour test time

was classified as an acute time for larvae, for 24

hr was a chronic time.

From Table 5, the results of the cytotoxic test

show successively increased toxicity of F4, EtOAc

Extract Fraction, F2, F2.1 and F1 with LC₅₀values

of 857.03 ppm, 408.32 ppm, 242.66 ppm, 191.43

ppm and 168 .66 ppm is classified as toxic, for F3

it is classified as non-toxic with an LC₅₀value of

1936.42 ppm. While the hexane fraction, the LC₅₀

value of 1066.66 ppm, is classified as non-toxic,

this is the basis for isolating the ethyl acetate

fraction. LC₅₀values have a toxic range, LC₅₀

values < 30 ppm are very toxic, LC₅₀values are 30-

1000 ppm toxic and LC₅₀values > 1000 ppm are

not toxic [²⁰]. When compared to the LC₅₀values

of the EtOAc, F2, and F2.1 (Isolate) extract

fractions indicates the presence of bioactive

compounds, with a decrease in the LC₅₀value on

the mortality of Artemia salina larvae.

including

polymethoxyflavone

(PMF),

are

particularly close to the IR spectrum of sinensetin

(5,6,7,3',4'-pentamethoxyflavone) (Figure 3).

Cytotixic Activity

The cytotoxicity test of a compound can be

carried out using the Brine Shrimp Lethality Test

(BSLT) method. The BSLT method was carried out

to see the effect of cytotoxicity on cells and is

often used in preliminary tests for screening or

screening

of

pharmacological

activity

in

medicinal plants and is also widely used for

screening new anticancer compounds derived

from plants. Toxicity test results with this method

have been shown to correlate positively with the

cytotoxicity of anticancer compounds [17].

The LC₅₀value of F1 is more toxic than F2.1. This

is possible because of the synergistic effect of

other compounds in Sungkai leaves, which can

increase cytotoxicity, whereas F3, classified as

The use of Artemia salina larvae eggs is used after

48 hr. It was the first instar phase when Artemia

salina was 24 hr old. The larvae did not have a

complete mouth and digestive system in this

phase. While at the age of 36-48 hr after breeding

the larvae, the larvae will metamorphose into the

non-toxic,

can

be

influenced

by

other

compounds in Sungkai leaves with antagonistic

effects.

antagonistic

The

synergistic

(inhibiting)

(supporting)

effects affect the

and

146

Chempublish Journal, 6(4) 2022, 140-148

bioactivity of the compounds. Based on the

results of the characterization of the isolated

compounds, it is possible that they belong to the

flavonoid class, which has a methoxy functional

group. In previous studies, flavonoid compounds

that have a methoxy functional group have the

potential to inhibit leukaemic cancer cells HL60

and prostate cancer cells (PC-3 and DU145)

[21,22].

Table 5. BSLT method cytotoxic test result

Samples

Conc (ppm)

Total deaths

Probit values

LC₅₀(ppm)

n-hexane extract

fraction

10

8

3.028

1066.6

100

1000

10

100

1000

10

100

1000

10

100

1000

10

100

1000

10

100

1000

10

11

23

7

14

27

6

7

37

3

6

35

3

5

18

4

EtOAc extract fraction

2.611

2.227

2.385

3.287

2.933

2.282

408.32

168.66

242.66

1936.42

857.03

191.43

F1

F2

F3

F4

10

21

4

F2.1 (Isolate)

100

1000

8

36

Conclusion

S; Formal Analysis, ZA and MF.; Investigation, MF

and ZA.; Resources, ILT and S.; Data Curation, S

and MF; Writing – Original Draft Preparation, ZA,

MF, ; Writing – Review & Editing, S and ILT;

Visualization: ZA and MF.; Supervision, S and ILT;

Project Administration, S.

The profile of bioactive compounds from the

ethyl acetate fraction of Sungkai leaves refers to

the flavonoid group, especially flavone, namely

polymethoxyflavone. Based on the results of the

cytotoxic test, the bioactive compound from the

ethyl acetate fraction had an LC50 value of

191.43 ppm against Artemia salina.

Conflic of Interest

The authors declare no conflict of interest

References

Acknowledgement

This research is supported by LPPM Universitas

Jambi through Basic Research Scheme 2022.

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