The Activity of Sterculia quadrifida R.br Stembark against Hepatitis

C Virus

Maria Ayu Wandira Moi Sola

, Adita Ayu Permanasari

, Myrna Adianti

2,4

, Lidya Tumewu

, Aty

Widyawaruyanti

2,3

, Achmad Fuad Hafid

2,3

Graduate Master Student of Pharmaceutical Science Master Course Program, Faculty of Pharmacy, Universitas

Airlangga, Surabaya 60286, Indonesia;

Institute of Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia;

Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Airlangga, Surabaya 60286,

Indonesia;

Department of Health, Traditional Medicine Study Program, Faculty of Vocational, UniversitasAirlangga,

Surabaya 60286, Indonesia.

Keywords: Sterculiaquadrifida R.br, stembark, anti-HCV, JFH1a, cytotoxicity

Abstract: Sterculia quadrifida R.br is commonly known in East Nusa Tenggara, Indonesia as “Faloak”. Itsstembark

has been traditionally used to cure liver disease. One of the active agentscausing liver disease is theHepatitis

C Virus (HCV). Meanwhile, there is no information on the activity of this plant for anti-HCV. This study

was aimed to investigate the anti-HCV activity and cytotoxicity of extracts and fractions from S. quadrifida

R.br stembark. The stembark of S. quadrifida R.br was extracted using several different solvents. The

stembark was gradually extracted using n-hexane, dichloromethane, and methanol. It was also extracted

using 70% ethanol by ultrasonic-assisted extraction method as well. In addition, it was extracted using water

by decocta method. All samples were further analyzed for their anti-HCV activity using Huh7it cell and

HCV JFH1a virus, while the cytotoxicity was determined by MTT assay. The most active extract was

further separated by column chromatography and the fractions were tested for their anti-HCV activity and

cytotoxicity. The anti-HCV assay results showed that water, 70% ethanol, and methanol were active against

HCV with an IC

value of 6.06 µg/ml, 9.44 µg/ml, and 10.39 µg/ml, respectively. Meanwhile, the hexane

and dichloromethane extracts were less active against HCV with IC

values of 51.93 µg/ml and 179.31

µg/ml, respectively. The fractionation of water extract as the most active extract resulted in seven fractions.

Fractions 5 and 6 showed highest activity with IC

values of 7.60 µg/ml and 8.87 µg/ml, respectively.

Furthermore, the cytotoxicity of these two active fractions exhibited no toxicity with CC

value of >2,000

µg/ml. Methanol extract, 70% ethanol extract, water extract, fraction 5, and fraction 6 of water extract from

S. quadrifida R.br stem bark had potential activity as anti-HCV.

1. INTRODUCTION

Hepatitis is one of the dangerous diseases caused by

Viruses. HCV is a virus include flaviviridae family,

a family with dengue virus and yellow fever, and is

an RNA virus. HCV, a virus with high envelope

heterogeneity and has variations of seven genotypes.

Hepatitis C virus spreads through direct contact with

blood or blood products infected with HCV [1,2]. In

the world, there are more than 170 million people

suffering from chronic hepatitis C infection with

about 3 million new infections each year and 1-3%

global prevalence [3].

Medicinal plants are a promising source of drug

candidates for HCV infection. The previous study

about natural product that ethanol extract M. latifolia

as antiviral activity with IC

value of 3.5±1.4µg/ml

against HCV JFH1a with CC

>100 µg/ml [4]. In

plants that contain various types of active chemical

components such as flavonoids, terpenoids, lignin,

sulfites, polyphenols, coumarins, saponins,

alkaloids, proteins and peptides, tend to inhibit the

replication cycle of various types of DNA or RNA

viruses [7] . Based on these there is a need to

develop safe, cheap, and is well tolerated for HCV

infection [13].

Sterculia quadrifida R.br, commonly known by

the name Faloak, has been used by the Timorese in

East Nusa Tenggara society to cure various diseases.

Faloak stembark can cure jaundice, typhoid, ulcers,

and hepatitis. The stembark of faloak is commonly

used for healing various diseases. In East Nusa

Moi Sola, M., Permanasari, A., Adianti, M., Tumewu, L., Widyawaruyanti, A. and Haﬁd, A.

The Activity of Sterculia quadriﬁda R.br Stembark against Hepatitis C Virus.

DOI: 10.5220/0009842800002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

Tenggara (NTT), people consume Faloak by cutting

the stembark into small pieces and then boiled it

with water for as much as 3 cups and then take them

regularly after eating. The use of faloak stembark as

a traditional medicine was commonly found in

Timor NTT.

Previous studies reported isolated naptokuinon

derivate compound from faloak stembark, which

was identified as 2,3-dihydro-6-hydroxy-2

methylenenaphtho [1,2-b] furan-4,5-dione active as

an anticancer with IC50 value in breast cancer cells

of 9.88 μg/mL and with an index selectivity value of

30.23 [6]. The family sterculiceae contains chemical

compounds of alkaloids, phenyl propanoids,

flavonoids, terpenoids and other types of

compounds, including hydrocarbons, sugars,

quinones, phenolic acids, lactones, lignans, amines

and amides. Test results by the Institute of Integrated

Research and Testing (LPPT) UGM showed

secondary metabolite compounds in faloak

containing phenolic acids, flavonoids, alkaloids, and

terpenoids. However, further studies to identify the

active extract and fraction which are responsible for

anti HCV activities have not been conducted yet.

Therefore, this study was conducted to identify

active compound from Sterculia quadrifida R.Br and

analyzed it for anti-HCV.

2. EXPERIMENTAL

2.1 Plant Material

S.quadrifida stembark was collected from Penfui

Kupang East Nusa Tenggara, Indonesia.

Authentication and determination of plants were

carried out at Purwodadi Botanical Garden-

Indonesia Institute of Science, East Java.

2.2 Extraction and Fractionation of

S,quadrifida stembark were dried at room

temperature and grinded for as much as 800 grams.

The stembark of S. quadrifida R.br was extracted

using several different solvents. The stembark was

gradually extracted using n-hexane (sqsh),

dichloromethane (sqsd), and methanol (sqsm). It was

also extracted using 70% ethanol (sqse) by

ultrasonic assisted extraction method as well. In

addition, it was extracted using water (sqsw) by

decocta method. All samples were further analyzed

for their anti-HCV activity using Huh7it cell and

HCV JFH1a virus, while the cytotoxicity was

determined by MTT assay. The most active extract

was further separated by column chromatography,

and the fractions were tested for their anti-HCV

activity and cytotoxicity.

2.3 Cells and Viruses

Huh7it hepatocyte cells cultured in the medium

Dulbecco's modified Eagle (Invitrogen, Carlsbad,

CA, USA) with an additional 10% Fetal bovine

serum (Biowest, Nuaille, France), kanamycin

(SigmaAldrich, St. Louis, MO, USA) and non-

essential amino acids(Invitrogen). Every cell growth

in the petridish reaches >80% passage cell. Viral

culture is done with collecting supernatants from

Huh7it cell cultures infected by HCV JFH1.

Supernatants collected on the 3th to 5th days after

infection are then concentrated using Amicon filters

and stored at -80°C.

2.4 Cytotoxicity Test

Toxicity testing of anti-HCV assay material was

performed on Huh7it hepatocyte cells with the

addition of the sample without inoculation HCV

JFH1a being visible from the ingredients for

hepatocyte cells without the presence of HCV

infection. The toxicity test results were obtained by

measurement absorbance at wavelengths 560 nm

and 750 nm. The cytotoxicity test was performed on

extract and fraction.

2.5 Analysis of Anti-HCV Activities

S.quadrifida extract and fraction were dissolved in

dimethyl sulfoxide (DMSO) to obtain stock solution

at a concentration of 100 mg/ml. The stock solution

was stored at-20°C until it was used. Huh7it cells

were seeded in 48-well plates (5x10

cells/well). A

fixed amount of JFH1a, with multiplication infection

(MOI) of 0.1 was infected on Huh7it cell then

treated with the presence of extract and fraction of

S.quadrifida.The virus titer was counted after being

stained with DAB thermo staining.

3. RESULTS

The anti-HCV assay results showed that water

(sqsw), 70% ethanol (sqse) and methanol (sqsm)

were active against HCV with IC

values of

6.06±0.09 µg/ml, 9.43±0.12 µg/ml, and 10.37±0.96

µg/ml, respectively. Meanwhile, the hexane (sqsh)

and dichloromethane (sqsd) extracts were less active

against HCV with IC

values of 51.94±0.62 µg/ml

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

and 179.63±1.88 µg/ml, respectively. The results

were then treated with further separation. The

fractionation of water (sqsw) extract as the most

active extract resulted in seven fractions. Fractions 5

and 6 showed highest activity with IC

values of

7.62±0.04 µg/ml and 8.6±0.21 µg/ml, respectively.

Tests were conducted on Water Extract,

Methanol, Ethanol 70%, Dichloromethane, Hexane,

and Water Faction 1-7. Furthermore, the water

extracts and two active fractions exhibited no

toxicity with CC

value of >2,000 µg/ml.

Table 1. The anti-HCV activity (IC

), toxicity (CC

), and selectivity index (SI) of extracts and fractions

Sample

(µg/ml)

SI (CC

/IC

)

Sqsh

51.94±0.62

291,4

5,611

Sqsd

179.63±1.88

1967,9

10,97

Sqsm

10.37±0.96

2108

202,88

Sqse

9.43±0.12

>2000

211,86

Sqsw

6.06±0.09

>2000

330,03

Sqsw fraction 1

64,67±0.53

>2000

30,92

Sqsw fraction 2

100.04±1.37

2028,6

20,27

Sqsw fraction 3

105.08±2.44

>2000

19,03

Sqsw fraction 4

95.05±4.13

>2000

21,04

Sqsw fraction 5

7.62±0.04

1957,2

256,82

Sqsw fraction 6

8.6±0.21

>2000

232,58

Sqsw fraction 7

36.38±3.83

>2000

54,97

4. DISCUSSION

The development of natural materials as drug

candidates, extraction, fractionation, and activity

testing and toxicity should be performed

simultaneously, which is known as bioassay guided

500

1000

1500

2000

2500

IC50

CC50

The Activity of Sterculia quadriﬁda R.br Stembark against Hepatitis C Virus

isolation. This means that the subsequent separation

is only performed on the selected extract or active

fraction as anti-HCV. The anti-HCV antiviral

activity screening was conducted on a concentration

of 30 µg/ml, which found that the water extract,

70% Ethanol, and Methanol had 100% inhibition

value, while the Hexane and DCM extract did not

have anti-HCV activity. The results of inhibition

percentage were showed, then IC

value was

calculated using probit log and the result showed

that Methanol had resistance to Hepatitis C antivirus

with an IC

value of 10.39 μg/ml. Ethanol 70% had

activity against Hepatitis C antivirus with an IC

value of 9.44 μg /ml and Water had activity against

Hepatitis C with an IC

value of 6,06μg/ml. The

selected water extract was included in the next

separation process.

The profile of TLC S. quadrifida with stationary

phase RP 18 and mobile phase Methanol : Water

TFA 0.03% (1:2) showed the presence of blue and

green on uv 366, possibly containing phenolic group

compounds. Plants containing a wide variety of

active chemical components, such as flavonoids,

terpenoids, lignins, sulfites, polyphenols, coumarin,

saponins, alkaloids, proteins, and peptides, tend to

inhibit replication cycles of different types of DNA

or RNA [7]. Previous studies onS. Quadrifide

showed that it contains phenolic acid compounds

that can inhibit the growth of C. albicans bacteria,

with 3-hydroxyoctadecanoic compound is the main

compound in the extractive substance of S.

quadrifida to inhibit the growth of C.albicans fungi

[8]. Previous study to analyze the antibacterial and

antioxidant of ethanol extract of S. quadrifida bark.

Fraction 3 showed the highest antibacterial activity

(IC50) against B. subtilis bacteria (90.51 μg/mL), E.

coli (80.12 μg/mL), S.aureus (77.87 μg/mL), and S.

thypi (61.23 μg/mL). The antioxidant activity test

showed that fraction 2 had the highest phenol

content (34.16±0.76 mg ) and antioxidant activity

[9].The main content of phenolic acids and

flavonoids in the stembark of S. quadrifidahad

various effects on various organisms, including to

cure lumbago, kidney, rheumatic, liver, and other

internal diseases [10,11].

The toxicity test was conducted on Water Extract

(sqsw), Methanol (sqsm), Ethanol 70% (sqse),

Dichloromethane (sqsd), Hexane (sqsh), and Water

Faction 1-7. Toxicity testing was performed using an

MTT reagent, which was then converted to crystals

of purple formazan by succinic dehydrogenase in the

mitochondria in living cells. The higher intensity of

purple color produced meant more number of living

cells [7,12]. From the toxicity test results, it is

known the extraction and fraction of water had a

good safety value and were safe to use at the

separation stage to obtain the active substances

contained in faloak.

CONCLUSION

Methanol extract (sqsm), 70% ethanol extract (sqse),

water extract (sqsw), fraction 5, and fraction 6 of

water extract from S. quadrifida R.br stembarkhad

potential activity as anti-HCV.

ACKNOWLEDGEMENTS

The authors are grateful to NPMRD (Natural

Product Medicine Research and Development),

Institute of Tropical Disease, Universitas Airlangga.

REFERENCES

Moradpour D, Penin F, Rice CM: Replication of hepatitis

C virus. Nat Rev Microbiol. 2007, 5: 453-463.

Gottwein JM, Scheel TK, Jensen TB, Lademan JB,

Prentoe JC, Knudsen Mlet al. Development and

characterization of hepatitis C virus genotype 1-7 cell

culture system: role of CD81 and scavenger receptor

class B type and effect of antiviral drugs. Hepatology.

2009. 49:364-377.

WHO, 2012. Preventation and Control of viral Hepatitis

Infection: Framework for Global Action. WHO

Geneva.

Wahyuni TS, Tumewu L, Permanasari AA, Apryani E,

Adianti M, Rahman A, et al. 2013. Antiviral Activities

of Indonesian Medicinal Plants in The East Java

Region Against Hepatitis C Virus. Virology Journal

10:259.

Hafid, A.F., Aoki, C., Permanasari, A.A., Adianti, M.,

Tumewu, L.,Widyawaruyanti, A., Wahyuningsih,

S.P.A., Wahyuni, T.S.,Soetjipto, Lusida, M.I., Hotta,

H., 2017. Antiviral activity of the dichloromethane

extracts from Artocarpusheterophyllus leaves against

hepatitis C virus. Asian Pac J Trop Biomed, Vol 7(7),

Page 633–639.

Rollando R, Prilianti KR. 2017. Isolasi Senyawa Turunan

Naptokuinon Dari Kulit BatangFaloak (Sterculia

quadrifida R.BR) Dan Uji Aktivitas Antikanker Pada

Sel Kanker Payudara Jenis T47D.Indonesian E-

Journal of Applied Chemistry Volume 5, 13-17.

Javed, T.A., Usman, A., Riaz,. S., Rehman, S., and

Riazuddin, S., 2011. In-vitro antiviral activity of

Solanumnigrum against hepatitis C virus.Virology

Journal, Volume 8 Nomor. 23, Page 1-2.

Ranta, Fabianus, 2012. Aktivitas Anticendawan Zat

Ekstraktif Faloak (Sterculia comosa Wallich Jurnal

Kehutanan IPB, 67, 6-11.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Rollando R, Siswadi S. 2016. Penelusuran Potensi

Aktivitas Sitotoksik Fraksi Kulit Batang Tumbuhan

Faloak (Sterculia quadrifida R. Br). E-Publikasi

Ilmiah Fakultas Farmasi Unwahas Semarang,

13(1):27–32.

Robinson, T. 1991. Kandungan Organik Tumbuhan

Tinggi (Edisi Keenam) Padmawinata K.,

Penerjemah: Sutomo T., Penyunting. Bandung:

ITB. Terjemahan dariThe organic constituents of

higher plants, 6thedition.

Balai Penelitian Kehutanan Kupang, 2010. Faloak si

Jangkung yang Berkhasiat. Jurnal penelitian

kehutanan vol.1

Ravikumar, Y.S., Ray, U., Nandhitha, M., Perween, A.,

Raja Naika, H.,Khanna, N., Das, S., 2011. Inhibition

of hepatitis C virus replication by herbal extract:

Phyllanthusamarus as potent natural source. Virus Res.

158 (1-2):89-97.

Manns,M.P.,Foster,G.R.,Rockstroh,J.K.,Zeuzem,S.,Zouli

m,F.,Houghton,M.,2007.The way forward in VHC

treatment finding the rightpath.Nat Rev Drug

Discov;6, 991-1000.

The Activity of Sterculia quadriﬁda R.br Stembark against Hepatitis C Virus