INTRODUCTION:

The Hepatitis C Virus (HCV) was first noticed a long time ago when an epidemic of jaundice, which was not understood and up till the outbreak of the Second World War. They were particulary noted in armies during times of war then in 1944 British army was troops by three most diseases, hepatitis, malaria, and venereal disease¹. According to the WHO, 58 million people live with chronic hepatitis C infection, which causes approximately 400,000 deaths every year. In 2019 only 21% of 58 million people with chronic hepatitis C had been diagnosed, while only 13% had been treated. However, the target of WHO to eliminate viral Hepatitis is by the year 2030².

The standard therapy of HCV patients is based on the virus genotype, response to treatment, and whether cirrhosis is present or not^3,4,5. The direct-acting antiviral drugs (DAAs) are the standard treatment for HCV that improves the sustained virological response (SVR) in more than 95% of HCV patients. The DAAs are divided into three classes i.e. NS3/4A protease inhibitors, NSSA polymerase inhibitors, and NS5B inhibitors. Even though the DAAs treatments are safe and effective, there are some existing systemic barriers to HCV eradication such as; the cost of DAAs treatment, DAAs accessing HCV treatment, and the resistance-associated substitutions (RASs) which could be important in exhibiting failures to DAAs based therapies^6,7. The DAAs also are known for some side effects (insomnia, nausea, shortness of breath), and limited effectiveness in older patients^8,9. The complementary medicine and or alternatively medicine for HCV is still required due to the HCV characteristic self as the RNA virus. The HCV is a single-stranded RNA virus that lacks of proof-reading activity which makes it easy to mutate. This composition makes it easy for them to mutate and challenging to develop vaccines or antiviral drugs.

Plants have been traditionally used as medicines to treat various diseases, including viral infections. Many compounds derived from plants have demonstrated antiviral properties, and some of them have been incorporated into the development of antiviral drugs^10,11,12,13. One example is the artemisinin compound derived from the plant Artemisia annua, which is used to treat malaria and has shown activity against several viruses, including hepatitis B and C, HIV, and SARS-CoV-2^14,15,16,17. Another example is the compound quercetin, found in many fruits, vegetables, and spices such as apples, onions, and green tea. Quercetin has been shown to possess antiviral activity against several viruses, including influenza, hepatitis B and C, and HIV^18,19.

Artocarpus is a genus of plants belonging to the Moraceae family, which includes species such as jackfruit (A. heterophyllus), breadfruit (A. altilis), and cempedak (A. champeden). The Artocarpus genus is known to contain many flavonoids, phenols, steroids, tannins, saponins, and triterpenoids. Several studies have investigated the antiviral activity of various species of the genus Artocarpus and their compounds^20,21,22. Furthermore, Artocarpus genus has also been reported to be active as an antimicrobial, anti-inflammatory, antioxidant, and anti-analgesic^23,24,25,26. The study aims to determine the anti-HCV activity and toxicity of the 54 extracts of the Artocarpus genus. Furthermore, according to the results which showed the strong activity on the A. sericicarpus, the screening of antiHCV activity of Artosericalcone A-C were performed at a single concentration of 10 µg/mL, and the molecular docking analysis of NS3 HCV protease was determined to predict binding affinity. The NS3 protein of HCV is responsible for most of the cleavages of the viral polyprotein that important on viral replication process. Therefore, the NS3 protein of HCV is considered an ideal target for antiviral therapy.

MATERIALS AND METHODS:

Materials:

Ten species of the Artocarpus genus were collected from the Purwodadi Botanical Garden in Pasuruan and subsequently identified and verified by a botanist from the same institution. The leaves and stem bark were cut, dried, and extracted. The specimens are now stored in the NPMRD laboratory at the Institute of Tropical Disease, Airlangga University, and in the Herbarium for various plant parts.

Methods:

A total of 54 extracts from ten different Artocarpus species were screened for antiviral activity against HCV. The analysis of HCV infection employed Huh7it-1 cells and the HCV JFH1a strain, and the inhibition analysis was determined by 3,3'-diaminobenzidine (DAB) staining in the infected cells.

Extraction of Artocarpus sp:

The powder (25 grams) of various parts of Artocarpus sp was subjected to ultrasonic-assisted extraction using N-hexane as a solvent. The resulting extract was filtered, and the filtrate was then evaporated using a rotary evaporator to yield the N-hexane extract. Subsequently, the residue underwent further extraction using dichloromethane as a solvent to obtain the dichloromethane extract. Finally, the remaining residue underwent extraction with methanol as a solvent to obtain the methanol extract²⁷.

Huh7it-1 Culture and HCV Propagation:

The hepatocellular carcinoma Huh7it-1 was cultured in Dulbecco's Modified Eagle's Medium (GIBCO) supplemented with 10% fetal bovine serum (Biowest, Nuaille, France), 100μg/mL penicillin/streptomycin (GIBCO) and non-essential amino acids (Invitrogen, Carlsbad, CA, USA). Cells with confluent 80% were passaged to maintenance cell growth. The virus propagation was performed in Huh7it-1 cell. In the T75-Flask the cell density 1.8x10⁷was added with 50µL HCV JFH1a (genotype 2a) in 8mL DMEM Medium. After 4 hr incubation, 8 flasks were prepared and filled with 1 mL virus solution and cell and 9 mL DMEM Medium. The supernatants from Huh7it-1 cell cultures infected were collected on days 3, 5, and 7 post-infections continued with concentrated using a 100K Amicon filter, and stored at −80°C^27,28,29.

Anti-HCV Activity Test:

In vitro anti-HCV testing begins by seeding Huh7it-1 cells in 48-well plates (cell density 5.4 x 10⁴) and continuing incubation for 24 hours. After 24 hours, JFH1a virus (titer 4.1x10⁶) with an MOI of 0.1 was added, and 100μL of the sample was added to each well at a concentration of 10μg/mL. However, the concentrations to obtain 50% HCV inhibition were done at 100; 50; 10; 1; 0.1 0.01µg/mL. Subsequently, incubation was carried out for 48hours, and the supernatant was collected to calculate the titer. The supernatant was diluted 40 times and then inoculated on cells in 96-well plates. Incubation was again carried out for 48hours, followed by fixation using 3.7% formalin. The infection was determined by adding HCV patient serum (x200) 100µL followed with secondary antibody (anti Human labelled with HRP) (x750) 100µL. To visualize the infection, staining was performed using DAB staining (Thermo Fisher Scientific).

MTT assay:

The toxicity of the samples was assessed using the MTT [3-(4,5-dimethylthiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (Sigma-Aldrich). The test involved adding the test material at various concentrations of 1000; 500; 250; 125; 62.5; 31.25; 15.625; and 7.8125 µg/mL to the cells and incubating them for 48hours in a 37°C incubator with a CO2 concentration of 5%. After incubation, the old medium was removed, and medium containing 10% MTT was added. The mixture was incubated for an additional 4 hours. DMSO was then added to dissolve the precipitate formed from the MTT reaction. Absorbance measurements of the MTT reaction were conducted at wavelengths of 560nm and 750nm using the GloMax Microplate Multidetection Reader (Promega)^28,30.

Artoserichalcone isolation from Artocarpus sericicarpus.

The Artoserichalcone A–C were isolated as described by Tumewu³¹. In brief, the extraction of A. sericicarpus (800gr dried powder) was performed by ultrasonic-assisted extraction using N-hexane, dichloromethane (DCM), and methanol. The DCM extract (10gr) was subjected to open-column chromatography and resulted in two fractions (AS1 and AS2). AS1 was purified using centrifugal planar chromatography using N-hexane : acetone and acetone, yielding three fractions (AS 1.1-1.3). Fractions AS1.3.2 were then purified using N-hexane : chloroform, resulting in Artoserichalcone A. Otherwise, the purification from AS2 was continued by centrifugal planar chromatography using N-hexane, resulting in 6 fractions AS 2.1-2.6. Fractions AS 2.5 and 2.6 were subjected to Sephadex LH-20 open column chromatography eluted with methanol to obtain each two fractions from AS 2.5 (AS 2.5.1-2) and AS 2.6 (AS 2.6.1-2). Fraction AS 2.5.2.2 was purified using N-hexane : diisoprophyl ether to obtain the compound Artoserichalcone B. Furthermore, fraction AS 2.6.2.6.2 was purified using N-hexane : chloroform to obtain the compound Artoserichalcone C. The molecular structures of the compounds are shown in figure 1.

Figure 1. Isolated compound from A. sericicarpus. (A) Artoserichalcone A, (B) Artoserichalcone B, (C) Artoserichalcone C

Docking Analysis:

Artoserichalcone A, B and C have been integrated into protein such as NS3 (PD-ID: 6NZT). The torsional root and branches of the ligands were selected using Autodock tools 1.5.6, which accommodated all rotatable bonds except the amide bond. Moreover, the Kollman charges for each protein and Gasteiger-Marsili atomic charges for all ligands were assigned using Autodock tools 1.5.6. Next, the chemicals were positioned in the binding site using the Lamarckian genetic process from the AutoDock 4.2.6 software. A grid box with dimensions of NS3 (40 Å × x 40 Å × x 40Å) was used to compute the atom-type maps. The grid boxes were spaced 0.375 A apart and centered in the cocrystallized ligand's binding pocket. We conducted 100runs, evaluating a maximum of 2,500,000 energy values and starting with 150 conformers as beginning populations. The optimal binding mode of each molecule was selected based on the minimum computed free binding energy and the size of the protein cluster. The docking results were analyzed using Autodock tools and Discovery studio 2024, and a validated molecular docking procedure was repeated three times. Consulting with the references³². the binding energy ≤−5.0kJ/mol was used as the criterion to judge the better binding ability of the molecule to the target.

RESULT:

Screening results for anti-HCV activities:

We screened 54 extracts from ten different species of Artocarpus and tested their antiviral activity against HCV. All extracts were administered at a treatment concentration of 10 µg/mL. The samples were then exposed to the respective viruses HCV at an m.o.i. of 0.1.

The viability cells were measured using the MTT assay method at the concentration of 100µg/mL in the Huh7it-1 cells. Based on the data from screening antiviral activity and cytotoxicity assay, we identified 15 extracts that strongly inhibited HCV viruses with low cytotoxicity effect as presented in the table 1.

According to the table 1 N-hexane extracts from the Artocarpus genus both derived from leaves and stem/stembarks didn’t show the HCV inhibition by the 70% HCV grow well in cell culture. Whereas both extracts from methanol and dichloromethane showed various inhibition activity against HCV. Fifteen samples exhibited active as antiHCV at a concentration of 10 µg/mL (more than 70% HCV reduction). The active extracts then continue to analyze the 50% inhibition and toxicity concentrations (IC₅₀ and CC₅₀) to determine the selectivity index (SI) score as presented in the table 2.

Table 1. Activity of Artocarpus genus extracts against HCV at inhibition concentration 10μg/mL and toxicity concentration 100μg/mL.

S. No	Code	Name	Part	Solvent	HCV
S. No	Code	Name	Part	Solvent	% inhibition (mean±SD)	% viability (mean±SD)
1	SC01	Artocarpus altilis	Leaves	DCM	73.38±5.37	19.1±1.55
2				Methanol	0±0	94.53±0.98
3				N-hexane	0±0	86.87±0.99
4	SC04	Artocarpus elestica	Stem/Stem bark	DCM	83.47±4.66	18.63±1.63
5				Methanol	0±0	90.06±1.55
6				N-hexane	7.25±6.79	93.26±0.21
7			Leaves	DCM	60.28±4.75	9.52±0.26
8				Methanol	1.13±1.96	102.64±3.48
9				N-hexane	2.42±4.19	92.14±7.17
10	SC05	Artocarpus fretessi	Stem/Stem bark	DCM	43.32±6.35	81.96±12.06
11				Methanol	69.52±6.58	79.41±7.07
12				N-hexane	0±0	84.97±5.74
13			Leaves	DCM	98.56±0.97	47.94±7.49
14				Methanol	18.63±7.77	86.29±9.64
15				N-hexane	0±0	88.23±4.93
16	SC06	Artocarpus heterophyllus	Leaves	DCM	97.27±1.11	64.33±7.28
17				Methanol	18.85±2.33	63.8±1.12
18				N-hexane	14.77±2.69	96.51±3.11
19	SC07	Artocarpus integer	Stem/Stem bark	DCM	26.79±8,39	32.97±0.88
20				Methanol	11.55±1.97	93.89±0.34
21				N-hexane	7.9±1.29	79.03±3.11
22			Leaves	DCM	98.56±0.97	72.94±3.23
23				Methanol	52.98±5.51	94.64±3.08
24				N-hexane	5.96±5.28	96.3±3.18
25	SC11	Artocarpus rigidus	Stem/Stem bark	DCM	78.75±1.71	12.11±0.14
26				Methanol	26.58±4.65	97.33±2.61
27				N-hexane	6.39±7.55	98.24±3.77
28			Leaves	DCM	76.6±7.07	12.25±1.04
29				Methanol	84.98±4.29	98.89±0.94
30				N-hexane	30.23±8.29	99.44±2.33
31	SC12	Artocarpus sericicarpus	Stem/Stem bark	DCM	30.65±2.91	15.92±0.22
32				Methanol	30.87±2.08	93.46±2.08
33				N-hexane	0±0	91.57±0.57
34			Leaves	DCM	76.6±3.89	64.27±1.09
35				Methanol	88.2±3.05	89.16±1.16
36				N-hexane	7.63±11.97	91.63±2.68
37	SC13	Artocarpus sp	Stem/Stem bark	DCM	43.32±9.75	84.3±2.25
38				Methanol	12.4±6.22	90.02±3.84
39				N-hexane	22.93±11.69	86.78±3.41
40			Leaves	DCM	98.56±0.97	61.96±3.07
41				Methanol	21.21±2.69	95.23±3.89
42				N-hexane	0±0	90.38±0.15
43	SC14	Artocarpus tamaran	Stem/Stem bark	DCM	11.97±4.66	76.75±0.59
44				Methanol	1.56±2.7	90.14±0.88
45				N-hexane	0±0	92.98±1.04
46			Leaves	DCM	86.48±2.33	9.91±0.32
47				Methanol	83.9±4.47	72.18±5.44
48				N-hexane	23.35±6.45	97.18±3
49	SC15	Artocarpus teysmanii	Stem/Stem bark	DCM	96.35±0.75	14.09±1.4
50				Methanol	53.84±6.46	86.8±4.09
51				N-hexane	0.49±0.84	83.72±2.37
52			Leaves	DCM	96.78±1.12	8.53±0.08
53				Methanol	16.48±3.89	88.27±1.25
54				N-hexane	9.4±7.1	97.89±3.16

Table 2. The Selectivity index (CC₅₀/IC₅₀) of active extracts from Artocarpus genus against HCV

No	Sample Number	Name	Part	Solvent	CC₅₀ (mean±SD)	IC₅₀ (mean±SD)	SI
1	1	Artocarpus altilis	Leaves	DCM	43.3±6.03	9.33±2.47	4.64
2	4	Artocarpus elestica	Stem bark	DCM	24.12±1.19	6.19±0.43	3.90
3	13	Artocarpus fratessi	Leaves	DCM	151.78±4.20	4.92±0.31	30.83
4	16	Artocarpus heterophyllus	Leaves	DCM	104.06±6.28	7.82±0.88	13.30
5	22	Artocarpus integer	Leaves	DCM	67.37±5.97	15.25±5.37	4.42
6	25	Artocarpus rigidus	Stem bark	DCM	21.37±0.63	12.78±7.56	1.67
7	28	Artocarpus rigidus	Leaves	DCM	29.79±5.21	4.55±1.40	6.55
8	29	Artocarpus rigidus	Leaves	Methanol	353.44±24.31	10.95±1.08	32.27
9	34	Artocarpus sericicarpus	Leaves	DCM	62.18±1.26	5.4±1.13	11.52
10	35	Artocarpus sericicarpus	Leaves	Methanol	435.54±9.00	5,11±0.12	85.30
11	40	Artocarpus sp	Leaves	DCM	148.16±14.97	5.58±0.01	26.54
12	46	Artocarpus tamaran	Leaves	DCM	16.96±11.12	5.33±1.22	3.18
13	47	Artocarpus tamaran	Leaves	Methanol	185.47±4.02	22.59±0.91	8.21
14	49	Artocarpus teysmanii	Stem bark	DCM	17.73±1.41	5.17±0.07	3.43
15	52	Artocarpus teysmanii	Leaves	DCM	17.11±0.95	16.1±3.82	1.06

Figure 3. Visualization of interaction (3-dimensional and 2-dimensional) among (A) Artoserichalcone A and (B) voxilaprevir. The atom's coloring scheme is as follows: red represents oxygen, grey represents carbon, and blue represents halogen. Green lines represent H-bond interaction, while pink, purple, and orange lines represent phi-bond interaction

DISCUSSION:

Natural medicines, including plants, are recognized as significant sources of novel pharmacologically active compounds. Compounds derived from plants boast a rich history of clinical use, offering better patient tolerance and acceptance. To date, 35,000-70,000 plant species have undergone screening for their medicinal properties. As of the early 21^st century, 11% of the 252
drugs classified as basic and essential by the WHO
originated exclusively from flowering plants³³. Many substances derived from plants are known to have many effects as antiviral in vitro or in silico approach against various virus such as influenza virus, SARS-CoV-2, Rotavirus, HIV, HSV, and HCV. There is no vaccine available in HCV treatment and the complementary drug for treatment still urgently required^{34,35,36,37,38}.

The study results showed the activity of 15 extracts among 54 extracts against HCV. The highest selectivity index score, 85.30, was observed in the methanol extract of Artocarpus sericicarpus, with an inhibition concentration 50% (IC₅₀) value of 5.11±0.12µg/mL and a cytotoxicity concentration 50% (CC₅₀) value of 435.54 ±9.00µg/mL. These results indicate that the Artocarpus genus is a promising source of natural antiviral therapeutic agents against HCV.

Some studies support our findings. A previous publication reported that the subfraction of A. heterophyllus dichloromethane extract was active against HCV with an IC₅₀value of 4.7 ± 1.0 μg/mL²⁷. This result aligns with the findings of our study. According to the results 6 samples were active and not toxic against HCV (selectivity index>10) i.e., A. fratessi leaves DCM (SI 30.83), A. heterophyllus leaves DCM (SI 13.30), A. rigidus leaves methanol (SI 32.27), A. sericicarpus leaves methanol (SI 85.30), A. sp leaves DCM (SI 26.54), and A. sericicarpus leaves DCM (SI 10.52). Those species were known to have been reported as antiHCV, such as A. heterophyllus leaves DCM active against HCV with an IC₅₀ value of 1.5 µg/mL, with inhibition entry process through virucidal activity and targeting host cells. HCV RNA replication and HCV protein expression were slightly reduced by DCM extract treatment at the high concentration²⁷.

According to Suciati, (2024) A. sericicarpus leaves and steam bark showed significant cholinesterase inhibition against AChE and BChE, as well as antioxidant activity. The methanol extract A. sericicarpus was previously reported as antiHCV activity with an IC₅₀ value of 8.04 µg/mL and the DCM extract was active with the an IC₅₀ value of 0.08±0.05 µg/mL^39,40.

The docking modeling was used repeatedly to find the best inhibitor for HCV NS3 protease enzyme. The number of hydrogen bonds in the studied complex ligand is considered to indicate the stability of the ligand when interacting with HCV NS3 protease enzyme. Increasing hydrogen bonds is expected to indicate increasing stability of the docking system. The native inhibitor (voxilaprevir) was used as a benchmark to measure the binding affinity of other recommended drugs. As illustrated in figure 3, the native inhibitor formed 8 hydrogen bond interactions with the residues of NS3 protease enzyme (His1057, Gly1137, Lys1136, Ser1138, Ser1139, Arg1155, Ala1156, Ala1157) and other interactions such as van der Waals, carbon-hydrogen bond, amide π-sulfur, alkyl, π–π stacked, π-alkyl and π-anion with the amino acid residues Lys1136, His1057, Tyr1056, Ala1156, Asp1081, Arg1155, Ala1156 of the target, respectively, through docking modeling (figure 3). The binding affinity of the docking system with the native inhibitor was -13.78±0.01 kcal/mol (table 4). Meanwhile, Artoserichalcone A binding affinity value is not lower than the inhibitor but better than the other two compounds. Artoserichalone A forms 5 H-bond interactions with NS3 protease enzyme residues (His 1057, Leu1135, Ser1139, Arg1155, Ala1157) (figure 3). Substituents that affect the three chalcone compounds in binding to the NS3 protease enzyme are the presence of ketone, aromatic and alkyl groups. H-bonds are the main driving force of interactions between molecules and target receptors, according to this study, and the interaction energy of molecules increases with the increase in the number of hydrogens bonds^41,42.

Artoserichalcone A, Artoserichalcone B, and Artoserichalcone C showed HCV reduction activity of more than 70% at a concentration of 10µg/mL. Another bioactivity report on those three compounds was published, revealing an antimalarial agent with IC₅₀values of 6.63±0.05 µg/mL, 22.19±0.06 µg/mL, and 5.78±0.02 µg/mL, respectively. ²⁶ The anti-HCV activity was predicted through molecular docking, which showed a deep interaction with the NS3 protease. The NS3 protein of HCV is a multifunctional protein that plays pivotal roles in HCV pathogenesis. The molecular weight of NS3 is approximately 70 kDa, NS3 is a cleavage product of the HCV polyprotein. This part of the protein can traverse along RNA or single-stranded DNA (ssDNA) in a 3’ to 5’ direction. These activities are considered essential for the replication of the HCV RNA genome. In addition to these functions, NS3 also plays a role in evading the host’s innate immune response^43,44,45.

CONCLUSION:

The results indicate that the Artocarpus genus is a promising source of natural antiviral therapeutic agents against HCV. The highest selectivity index for antiHCV, 85.30, was observed in the methanol extract of A. sericicarpus leaves followed by A. rigidus (leaves, methanol); A. fratessi (leaves, DCM); A. sp (leaves, DCM); A. heterophyllus (leaves, DCM); and A. sericicarpus (leaves, DCM). The Artoserichalcone A, B, and C which have been isolated from A. sericicarpus inhibited HCV infection more than 70 percent at a concentration of 10 µg/mL. The docking study revealed that Artoserichalcone A-C demonstrated deeply binding interaction with the NS3 protease of HCV.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

The authors would like to thank to the Center of Natural Product Medicine Research and Development, Institute of Tropical Disease for supporting the equipment. The authors are grateful to Professor Hak Hotta and Dr. Chie Aoki Utsubo (Kobe University) for providing HCV-JFH1a and Dr. Yohko Shimizu and Dr Chie Aoki Utsubo for providing Huh7it-1 cells. This study was supported by Fundamental Research with the contract number 1241/UN3.LPPM/PT.01.03/2023 from Kemenristek Dikti, Indonesia.

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Received on 16.09.2024 Revised on 23.01.2025

Accepted on 28.04.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4401-4408.

DOI: 10.52711/0974-360X.2025.00631

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Compound names	% Inhibition (mean ± SD)	% Viability (mean ± SD)
Artoserichalcone A	87.67±3.88	95.65±3.51
Artoserichalcone B	70.55±4.84	90.87±12.16
Artoserichalcone C	81.51±0.97	76.94±5.31

Compound	Binding affinity (Kcal/mol)	H-bond interaction		Hydrophobic interaction
Compound	Binding affinity (Kcal/mol)	Number of interactions	Amino acid residues	Number of interactions	Amino acid residues
Artoserichalcone A	-7.92±0.12	5	His 1057, Leu1135, Ser1139, Arg1155, Ala1157	3	His1057^b, Ala1157
Artoserichalcone B	-7.32±0.16	5	His1057, Gly 1137, Ser1139, Arg1155, Ala1157	3	His1057, Lys1136, Ala1156, Ala1157
Artoserichalcone C	-7.46±0.37	3	Thr1042, Ser1139, Ala1157^a,	3	his1057, Ala1156, Ala1157^a,
Voxilaprevir	-13.78±0.01	8	His1057, Gly1137^a, Lys1136, Ser1138, Ser1139^a, Arg1155, Ala1156, Ala1157^a	7	His1057^a, Tyr1056, Asp1081^a, Arg1155, Ala1156