ANTI-VIRAL RESEARCH - Anti-Viral Activity of Pimpinella anisum Extract In Vitro Study

INTRODUCTION

In the tropics and subtropics, dengue fever is one of the ut­most dreaded vector-borne flavivirus illnesses due to its rising prevalence. 55% of the world’s population, or 3.6 billion people, are at a higher risk of contracting dengue virus (DENV) infection, according to worldwide estimates. Dengue fever is estimated to affect 390 million people each year, with 96 million cases involv­ing dengue hemorrhagic fever or dengue shock syndrome (DSS) and 300 million cases involving moderate or asymptomatic illnesses.¹ Recent WHO categorization categorizes the disease as dengue without warning symptoms (DWOS), dengue with warn­ing symptoms (DWWS), and severe dengue.² A DWOS DENV in­fection may be asymptomatic or manifest as a “flu-like illness,” but (DWWS) is distinguished by a rapid onset of fever accompa­nied by non-specific signs and symptoms, such as back pain, headache, flushed facial skin, and stiffness.³ Leaking plasma and a low number of platelets can kill people with severe dengue in­fections, especially after hypovolemic shock.

For viral infections, herbal therapies, including traditional Chinese medicine, have also been proposed as alternatives.⁴ Due to their multivalent properties, they are typically safer than chem­ical medications and are less likely to cause resistant infections. Moreover, a number of herbal remedies may target both the virus and the signs of DENV infection, some of which are caused by the viral-induced overproduction of inflammatory mediators, such as cytokines.^5-8 P. anisum is an Apiaceae flowering plant endemic to India and southwest Asia.⁹ A 1-m annual herbaceous plant, the lower leaves are simple, 2-5 cm long, and shallowly lobed, whereas the upper leaves are feathery pinnate and formed of many leaflets. The 3-mm white flowers are in dense umbels. The fruit is a 3-5-mm long, oblong, dry schizocarp.¹⁰ P. anisum has been utilized as a medicinal plant for its stimulating impact on digestion, antiparasitic, antifungal, and antipyretic properties.^11,12 In addition, it has demonstrated anticonvulsant properties and has been used to treat constipation and possesses anti-ulcer properties.^13-15 Recent reports indicate its oil possesses antioxidant and antibacterial properties.¹⁶ There are limited reports of P. anisum’s antiviral activity. Therefore, this study was done to evaluate the antiviral effectiveness of this plant against dengue virus.

MATERIALS & METHODS

Plant Specimens

Ethno Resources Sdn Bhd in Selangor, Malaysia provided the seeds for P. anisum. By contrasting them with the voucher speci­men kept in the Herbarium of Rimba Ilmu at the Institute of Sci­ence and Biology of the University of Malaya in Kuala Lumpur, they were verified.

Ethanol Extraction

The crude-dried extract was made by extracting 100 grams of plant material for 48 hours in 900 mL of 95% ethanol, then filtering and evaporating the ethanol extract under low pressure with a Buchi-type rotary evaporator. It was determined the yield of ethanol extracts was 7.2% (w/w).

In Vitro Antioxidant Screening

DPPH Radical Scavenging Activity Assay: The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used in the modified technique to measure the antioxidant ac­tivity of plant extract based on an electron transfer reaction between the DPPH reagent and the plant extract.¹⁷ Five sep­arate concentrations were obtained by di­luting a stock solution (1 mg/1 mL) of the plant extract and the antioxidant standard (gallic acid) to five different concentra­tions. DPPH was mixed with five mL of plant extract and standard (195 mL). After that, the combination was incubated at 37°C for 30 minutes. The absorbance value was measured at 517 nm using a UV-1601 spectrophotometer (Shimadzu, Japan).

Ferric Reducing Antioxidant Power Assay (FRAP): FRAP was completed using Erel’s previous method.¹⁸ The FRAP reagent was made using a 1:1:10 ratio of 300-mmol/L acetate buffer (pH 3.6), 10-mmol/L 2,4,6-tri [2-pyridyl]-s-triazine (TPTZ) in 40 mmol/L HCl, and 20-mmol/L FeCl3 at 37°C. TPTZ working reagent served as the blank solution, while ferrous sulphate heptahydrate (FeSO4*7H2O) served as the standard. The standards (300 mL) and the sample solution (10 mL, 1 mg/mL of plant extract) were combined with FRAP reagents. The mixture was incu­bated at 37°C for 0 to 4 minutes before the absorbance at 593 nm was spec­trophotometrically determined. The data were then shown as the moles of gallic acid contained in 1 mg of extract.

Phytochemical Screening

Total Phenolic Content (TPC) & Total Flavonoids Content (TFC): The total phe­nolic components of ethanol plant extract were measured using a modified version of the prior approach, and the total flavonoids content (TFC) was assessed using Chang et al aluminum’s chloride colorimetric method.^19,20

Cytotoxicity

For this test, the American Type Cul­ture Collection provided the Hs888Lu cell line, which is a human normal lung fi­broblast cell line (ATCC, The Global Biore­source Centre, Manassas, VA, USA). Using the Promega Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation (MTS) assay, the cytotoxic potential of the P. anisum extract was evaluated.²¹ First, Hs888Lu cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma, USA), which has a high glucose content, 1% non-essential amino acids (PAA Labo­ratories GmbH, Austria), 2% L-glutamine (200 mM), 1% penicillin/streptomycin (100 times), and 1% sodium pyruvate (FBS, PAA Laboratory GmbH, Austria). The Hs888Lu was incubated at 37°C in an in­cubator (Contherm Scientific Ltd., New Zealand) with 5% CO₂ and 95% humidity. The cell lines were seeded (1 x 105 cells/mL) on a 96-well plate and cultured for 24 hours at 37°C in a moistened envi­ronment before being added to the plant extract. The diluted extract solution, rang­ing from 100 to 500 g, was applied in triplicate to the culture plate, which was then cultivated for 24 hours under the same circumstances. After the treatment, each of the 96 wells received 20 mL of 37°C-prewarmed MTS reagent, and the plate was incubated for 3 hours at 37°C. The Glomax multi-detection system was used to measure the absorbance at 492 nm (Promega, USA).

Anti-Viral Activity of P. anisum

Dengue Virus: The Dengue virus type-2 was propagated in confluent C6/36 cells. Briefly, the medium was discarded, and the virus stock was added slowly and gen­tly. It was then incubated at 37°C for 1-1.5 hours. Post-infection, L-15 medium sup­plemented with 2% FBS was added post-infection and incubated at 30°C. Cytopathic effect (CPE) was examined daily, and the virus was harvested when most of the cells showed CPE. The content of the flask was spun down, and the su­pernatant, which contains the virus, was aliquoted and stored at -80°C.

Sample Preparation: Vero cells were plated at a density of 5.0 104 per well in a 24-well plate and incubated overnight. Three different types of treatment were used, namely pre-, simultaneous-, and post-treatment. For pre-treatment, the ex­tracts were first added 24 hours prior to the infection. For simultaneous treatment, both the virus and the extracts were added simultaneously, while the extracts were added 24 hours after the infection was es­tablished. After that, the plates were kept in an incubator for three days at 37°C. The supernatant and the attached cells were harvested and subjected to RNA ex­traction.

RNA Extraction: RNA was extracted from the samples using the Bioneer Viral RNA extraction kit. Briefly, 200 mL of sample and 400 mL of binding buffer (VB) were added and vortexed for 10 seconds. Fol­lowing a 10-minute incubation at room temperature, 100 mL of isopropanol was added to the mixture. Vortex and quick spin for 10 seconds before transferring the content into the spin column provided. The 500 mL of washing buffer 1 (W1) was added and rotated at 8,000 rpm for 1 minute, followed by the addition of 500 mL of Washing Buffer 2 (W2) and spinning at 8,000 rpm for 1 minute. The column was further spun at 13,000 rpm for 1 minute before the column was transferred into a new tube. After adding 30 mL of elution buffer and letting it sit for 5 minutes at room temperature, the tube was spun at 8,000 rpm for 1 minute.

Real Time-PCR: The antiviral properties of the extracts were determined using real-time PCR. For Dengue virus, the primer se­quence and procedure described in the reference were utilized.²² The thermal cy­cling parameters were reverse transcrip­tion for 30 minutes, Taq activation for 15 minutes, 35 cycles of denaturation for 30 seconds at 94°C, annealing for 40 sec­onds at 60°C, elongation for 50 seconds at 72°C, and a final elongation for 10 minutes at 72°C, followed by a hold for 4°C.

Statistical Analysis

The t-test was used to examine all in vitro test results, which are reported as mean SEM. All in vivo test results are shown as mean standard error of the mean, and they were analyzed using one-way ANOVA and Tukey’s post-hoc test in the Statistical Program Sciences version 20 (SPSS Inc, USA). The statistical significance of the differences between the means is determined by whether or not the p value is less than 0.05.

RESULTS

Evaluation of Antioxidant Activities & Phytochemical Screening

The FRAP value of P. anisum was 1134.42 ± 0.1 mmol/g, which is lower than the standards for gallic acid, 1503.44 ± 0.3 mmol/g (Table 1). The positive control and plant extract DPPH free radical scavenging findings, on the other hand, are presented as a percentage of inhibition. The DPPH of the P. anisum was 71.39 ± 0.71 inhibition, while the standard gallic acid was 77.31 ± 0.83 in­hibition, respectively. The TPC of the P. anisum was 495.56 ± 0.090 mg/g, while the TFC value was 0.435 ± 0.0298 mg/g. This suggested that P. anisum contained sufficient antioxidant efficacy.

Cytotoxicity Screening

Figure 1 summarizes the findings of P. anisum cytotoxic activity, which was rep­resented as a percentage of the value seen with no plant treatment (control). Normal lung cells were not killed by any of the plant extract concentrations, as can be seen in Hs888Lu.

 Evaluation of Antiviral Activity of P. anisum Against Dengue

P. anisum was evaluated for antiviral activity against dengue virus and is sum­marized in Figure 2. P. anisum exhibits anti-viral activity against dengue at concentrations of 200 g/ml to 500 g/ml, which could inhibit the virus’ reproduction.

DISCUSSION

A FRAP test was performed to exam­ine the antioxidant components of P. anisum free radical scavenging activity. This technique was created by Huan et al.²³ During the procedure, an antioxidant, such as polyphenol (ArOH), may provide a single electron. The blue Fe (II)-TPTZ complex is decreased from the Fe (III)-TPTZ complex, which is then detected at 700 nm. Antioxidant chemicals, such as polyphenol, which function as reducing agents, exert their impact by donating hy­drogen atoms to the ferric complex, there­fore interrupting the Fe (III)-TPZ radical chain reaction. Depending on the reduc­tion potency of each antioxidant sample, test solutions changed color from bright yellow to green or dark blue after the re­action. In vivo, the FRAP assay has been criticized for its low pH, which may inhibit electron transmission. The antioxidant ac­tivity of ferric (Fe3+) is too reliant on its ca­pacity to decrease iron, which is a downside of its usage.²⁴ The Fenton Reac­tion describes how P. anisum may protect against oxidative damage by eliminating ferrous ions (Fe2+) that can participate in hydroxyl radicals. By suppressing the gen­eration of reactive oxygen species and lipid peroxidation, reducing ions (Fe2+) protect against oxidative damage. Further­more, the significance of the DPPH test in determining the free radical scavenging ability of several antioxidants has been highlighted by Ozcelik.²⁵ The presence of phytochemicals is a common indicator of antioxidant action. A higher content of al­kaloids, phenolics, flavonoids, terpenoids, and other phytochemicals was associated with higher antioxidant activity.²⁶ The pres­ence of TFC and TPC in P. anisum phyto­chemical screening explains its scavenging activity. Similarly, it was discovered that P. anisum seed is a powerful antiperoxidative agent and has a wide range of applica­tions and uses in the food and pharma­ceutical industries.²⁷ Therefore, the findings of the DPPH experiment demon­strate the antioxidant activity of P. anisum is attributable to the presence of phyto­chemicals in its content. Interestingly, it has been demonstrated that the antioxidant and antibacterial effects of P. anisum ex­tracts can be related to their phenolic con­tent because several phytochemical investigations have revealed that P. anisum contains considerable levels of phenolic chemicals.^28,29 We may deduce from the findings of these two antioxidant assays that no one antioxidant test delivers the best results as various assays may yield dif­ferent kinds of antioxidant capabilities. This is due to the fact that in theory, neither a specific combination of phytochemicals nor a specific individual component can be attributed to having overall antioxidant activity. Consider the antioxidant capacity when all antioxidants contribute to the an­tioxidant activities by acting synergistically or additively. Much of the antioxidant scav­enging action is determined by the quan­tity and type of antioxidant chemicals as well as the polarity of the solvents. Yu et al have found that the polarity of a solvent affects the antioxidant properties of a spe­cific group of antioxidant compounds and changes how well the solvent works with molecules from that group.³⁰

To analyze the toxic effect of a plant on human health, it is necessary to exam­ine the toxicity of a plant extract. The data showed no cytotoxicity against the Hs888Lu cell line. This result was consis­tent with a previous study that showed MTT and LDH experiments demonstrated ethanolic extract had cytotoxic action against the human prostate cancer cell line at quantities deemed safe for normal rat skeletal muscle cells.³¹ As previously mentioned, traditional medicine is used by a large number of people in developing countries for their primary healthcare needs. Numerous illnesses, including ul­cers, wound healing, and hyperlipidemia, are treated with medicinal herbs.³² The flavonoid content of the plant extract sug­gested that P. anisum had substantial an­tiviral activity against DENV in vitro. Indicators of selectivity for P. anisum when infected cells or uninfected cells were treated with 200, 400, or 500 g/ml, re­spectively. The observed discrepancies in P. anisum concentration levels may be the result of intracellular P. anisum accumula­tion during therapy. At a low concentra­tion, however, a minor influence on the activity of P. anisum was also found. These results imply the predominant anti-dengue activity of P. anisum is probably related to its action alongside the various phases of intracellular reproduction of DENV, as op­posed to early stages of the virus’s repli­cation cycle, such as viral attachment or entrance. P. anisum suppresses DENV replication, and the considerable decrease in DENV-specific RNA proposes that P. anisum may also target the viral replication machinery, specifically by inhibiting RNA polymerase. According to reports con­ducted by Ibrahim, waste residues of P. anisum are prospective new sources of phenolic antibacterial chemicals, offering the pharmaceutical sector new economic prospects.³³ They propose that combining P. anisum waste extracts with some antibi­otics yields a novel option for treating infectious disorders. Also, Schnitzler dis­covered the antiviral action of anise oil on enveloped viruses.³⁴ P. anisum, on the other hand, was stated to be efficient against herpes viruses; it is more specific for HSV-1.³⁵ However, the effects of various plant extracts on cellular RNA polymerases and the formation of complexes with RNA have been described, indicating they may similarly alter the replication enzymes.³⁶ Lastly, three antiviral and immune-stimu­lating compounds (LC1, LC2, and LC3) were identified from a hot water extract of P. anisum seeds. LCs demonstrated antivi­ral activity against HSV-1 and 2, HCMV, and measles virus.

CONCLUSION

The present study demonstrates P. anisum showed significant antioxidant activity by DPPH and FRAP, which may be due to the contents of TPC and TFC, which are considered potent antioxidants. The cytotoxic activity and acute toxicity of P. anisum did not show any signs of toxicity in vitro. Antiviral results showed that P. anisum has antiviral activity in a dose-dependent manner.

ACKNOWLEDGMENTS

The authors express gratitude to the staff of the Faculty of Dentistry (UiTM) and the Faculty of Medicine (UM). This study was financially supported by University Technology MARA, Grant no. 600-RMI/FRGS 5/3 (62/2012).

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Dr. Fouad Hussain M.H. AL-Bayaty is currently Professor Faculty of Dentistry, Universiti Teknologi MARA, Malaysia. He earned his Master’s and PhD in Clinical Periodontology in France in 1984, and also earned his Master’s and PhD in General Immunity (Distinction Degree) in France. He was promoted to Professor in 1994. He earned his BDS in1973, Baghdad University. He is Chief Editor of the Journal of Advanced Medical Research (JAMR), and supervisor and member of the implant center. Teaching periodontology, he supervises 32 PhD and MSc students, he is an invited speaker and external examiner, has published more than 180 research papers and 6 books. He earned the Prize of Distinguished Professor for 3 years and excellence in service in faculty of dentistry, UiTM. He also earned the prize and Diploma of Honor from the higher committee of Lyon university association as distinguished post-graduate student in 1982, earned 92 medals and 4 patents. He is currently a member of several international and national societies.

Dr. Mazen M. Jamil Al-Obaidi is an Assistant Professor at the University of Technology and Applied Sciences in the Sultanate of Oman. He has spent over a decade (Master’s, PhD, and post-PhD) growing as a scientist. His doctoral dissertation at MARA University of Technology (UiTM/Dental Faculty) focused on the effects of Ellagic Acid on tooth socket repair in diabetic and nicotinic rats. After earning his PhD, he did a 1-year post-doctoral fellowship in the Medical Microbiology Department of the Faculty of Medicine at the University of Malaya. Additionally, he worked as a post-doctoral researcher at the University Putra Malaysia’s Department of Medical Microbiology (Faculty of Medicine and Health Sciences). During his journey, he has published a number of ISI-indexed articles and presented a number of projects at national and international conferences. In addition, he finished writing two book chapters about his field. He has been a reviewer for the Molecular Medicine journal for more than 4 years.

Dr. Omar Emad Ibrahim is an academic pathologist/histopathologist earning his PhD in Pathology from the University Putra Malaysia with 18 years of experience in academic, teaching, histopathology slide consultation, and research in the field of medical, oral, experimental, and comparative pathology. He worked as a lecturer in the Pathology Department, Faculty of Medicine and Health Sciences, Thamar University, Yemen. He is also a lecturer in the Pathology Department, International Medical School, Management and Science University, Malaysia, a senior lecturer in Pathology Department, Faculty of Medicine, Lincoln University College, Malaysia, and senior lecturer in Pathology Unit, Centre of Preclinical Science Studies, Faculty of Dentistry, Universiti Teknologi MARA (UiTM). He is currently Associate Professor in Medical Pathology in the Department of Pathology, International Medical School, Management and Science University, Malaysia. He had a good experience in Medical Pathology to Medical, Dental, and Allied health sciences students. He has authored or co-authored more than 40 peer-reviewed abstracts and manuscripts in the areas of pathology. He has been a reviewer to the BMJ for more than 6 years.

Maryam Haki Ismail is currently an IGCSE lecturer at Cambridge International schools. She earned her Bachelor’s in Biomedical Science in Taylor’s University, Malaysia. In addition, she had completed her internship at Advanx Health, worked at the Science Department, and conducted a number of research related to genetics. Her university degree in Biomedical Science equipped her with an excellent combination of skills in both dry and wet labs. Due to her interest in human health, she has published several research papers about human health and diseases.