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, Indonesi

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

(Moradpour et al., 2007; Gottwein et al., 2009). 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 (WHO, 2012).

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 (Wahyuni

106

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: 10.5220/0008358201060110

In Proceedings of BROMO Conference (BROMO 2018), pages 106-110

ISBN: 978-989-758-347-6

et al., 2013). 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 (Javed et al., 2011) . 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

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 (Rollando & Prilianti, 2017). 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 METHODS

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

Sterculia quadrifida R.Br

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.

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

107

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 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

value of >2,000 µg/ml.

Table 1: The anti-HCV activity (IC

), toxicity (CC

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

Sample IC

(µg/ml) CC

(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

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

108

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

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 (Javed et al., 2011). 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

(Ranta, 2012). 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 (Rollando et al., 2016). 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 (Robinson, 1991; Balai

Penelitian Kehutanan Kupang, 2010).

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

500

1000

1500

2000

2500

IC50

CC50

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

109

mitochondria in living cells. The higher intensity of

purple color produced meant more number of living

cells (Javed et al., 2011; Ravikumar et al., 2011).

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.

5 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

Balai Penelitian Kehutanan Kupang, 2010. Faloak si

Jangkung yang Berkhasiat. Jurnal penelitian kehutanan

vol.1

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.

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.

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.

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

Zoulim, F., Houghton, M. 2007. The way forward in

VHC treatment finding the righpath.Nat. Rev. Drug

Discov; 6, 991-1000.

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

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

Ranta, Fabianus, 2012. Aktivitas Anticendawan Zat

Ekstraktif Faloak (Sterculia comosa Wallich Jurnal

Kehutanan IPB, 67, 6-11.

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.

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.

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.

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.

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.

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

110