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In vitro antiviral screening of 25 extracts HIIT workouts from the plants of Vitex etxractsPlumeria albaAncistrocladus helthPlanr monnieriAnacardium occidentaleCucurbita maxima Antivlral, Simarouba glauca Antivural, and Embelia ribes using different solvents and four purified compounds Liver detoxification system acid, fo, glaucarubinone, and Alternate-day fasting and sustainable lifestyle gallate were carried fpr Antiviral plant extracts for health their Antivirwl virus Oxidative stress and Parkinsons disease and anti-chikungunya gor CHIKV activities.
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Plant-derived Non-reactive cleaning tools such as nobiletin, epigallocatechingallate, silymarin, curcumin, Antoviral harringtonine were reported to have anti-chikungunya activity Heapth et al.
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Antivirzl ATCC No. Dengue virus Plabt serotype-2 Strain No. A total of 25 different parts Heightens mental sharpness eight plants, i. Four purified compounds, plznt. Plant parts leaves, plznt, seeds, and whole herbs heqlth air-dried, ground estracts powder, and macerated in hralth solvents such as ethanol Each solvent extract was extracted three times after 72 Antlviral of Antivirap.
The extract plqnt filtered through Whatman filter paper No. A total of 25 extracts and four purified compounds were used for further screening Table 1. The cytotoxicity effect of the extracts and the compounds was evaluated by 3- 4,5-dimethythiazolyl -2,5-diphenyl tetrazolium bromide MTT assay as described previously Parashar et al.
The solubilized formazan crystals were measured using a microplate reader BioTek Synergy, USA at nm. The percentage inhibition and CC50 values were calculated.
All the extracts at their maximum nontoxic dose were assayed for their antiviral activity against DENV and CHIKV under posttreatment condition as described earlier Panda et al. The extracts which showed antiviral activity were further tested at different concentrations for their antiviral activity under all the three treatment conditions pre- co- and posttreatment as described earlier Panda et al.
During pretreatment, the cells were pretreated with a formulated extracts at 37°C for 24 h followed by removal of the culture supernatant, the cells were then infected either with DENV-2 or CHIKV and incubated for 1 h at 37°C. In cotreatment, the virus was mixed with different concentrations of the formulated extracts and the mixture was used for infecting cells for a duration of 1 h.
For posttreatment, the cells were infected with DENV-2 or CHIKV for 1 h and treated with the formulated extracts after 24 h. For all treatments, the plates were incubated after infection for h in the case of DENV-2 and 48 h for CHIKV. All the experiments were performed in triplicates. For significant results, the experiments were repeated again in triplicates.
Irrespective of the type of treatment, 0. MOI was calculated on the basis of number of cells used for seeding the wells. The tissue culture supernatants collected from the different wells treated under different conditions were assessed for viral genomic RNA using quantitative real-time RT-PCR and infectious virus particle titer using FFU assay.
The percent of cells infected was assessed using immunofluorescence assay IFA. Detection and quantitative estimation of viral genomic RNA was done using quantitative real-time RT-PCR assay.
The primers and probes used for amplifying DENV-2 and CHIKV and PCR conditions have been described earlier Panda et al. The viral RNA load of the samples were calculated based on a standard graph generated using cycle threshold values of tenfold dilutions of in vitro transcribed viral RNA with known copy numbers.
IFA and FFU assays for DENV-2 and CHIKV were performed as described earlier. The test conditions were compared with virus control using one-way ANOVA followed by multiple comparisons.
A p -value of less than 0. All analyses were performed using GraphPad Prism software version 7. In this study, chloroform, methanol, ethyl acetate, petroleum ether, dichloromethane, and hydroalcoholic extracts of eight plants Vitex negundoPlumeria albaAncistrocladus heyneanusBacopa monnieriAnacardium occidentaleCucurbita maximaSimarouba glaucaand Embelia ribes were tested for their antiviral activity against DENV and CHIKV.
The profile of the medicinal plants used in this study is listed in Table 1. Potential cytotoxic effects of the 25 extracts and four purified compounds were determined using the MTT assay in Vero CCL cells.
Three extracts M8E, M8M, and M9M and one purified compound G-S showed no cytotoxicity Table 1. The CC50 values of the different extracts are given in Table 1. The effect of different extracts at different concentrations on cell viability with CC50 values is provided in Supplementary Figure S1.
The plant extracts were screened by assessing their antiviral activity against DENV and CHIKV postinfection. FFU assay was used to measure the titer of the virus. Four extracts M8M, M5C, M7M, and M9M and one purified compound MG-S affected CHIKV titer Figure 1B and Table 2.
Figure 1 Antiviral screening of different plant extracts at maximal nontoxic concentration against DENV A and CHIKV B under posttreatment condition. Vero CCL cells were treated with highest maximum nontoxic dose of extracts 24 h postinfection and incubated for 96 h for DENV 1A and 24 h in the case of CHIKV 1Band after the incubation, the plates were frozen and the culture filtrates were used for the different assays.
All the treatment conditions were compared with the virus control. Table 2 Summary of effective extracts showing inhibition of DENV and CHIKV under different treatment conditions. Both M2C and M8M did not show any anti-DENV activity under pretreatment condition Figures 2A, C.
Under cotreatment condition, M2C exerted significant one log 5. Under posttreatment condition, M2C showed a dose-dependent reduction from 0. In comparison with virus control, M8M exerted a dose-dependent reduction from μg onwards with more than one log reduction 5.
Both the extracts did not show any reductions in the viral RNA titer as assessed by quantitative real-time RT-PCR under all conditions Figures 2B, D. Figure 2 Antiviral effect of Plumeria alba bark extract prepared using chloroform M2C and leaf-based extract prepared using methanol M8M against DENV under different treatment conditions.
Vero CCL cells were pre- co- and posttreated with different concentrations of extracts, and h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay AM2C; CM8M and real-time PCR BM2C; DM8M. The effect was more pronounced under posttreatment condition, and a significant dose-dependent reduction was observed from with Quantitative real-time RT-PCR results revealed a significant reduction in viral RNA titer under both pre- and posttreatment conditions at concentration of and μg, respectively, compared with the untreated Figure 3B ones.
Figure 3 Antiviral effect of bark and leaves of Plumeria alba methanol extract M8M against CHIKV under different treatment conditions.
Vero CCL cells were pre- co- and posttreated with different concentrations of extracts, and 48 h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay A and real-time PCR B. The chloroform extract of Vitex negundo leaves showed more than two log reductions of DENV titer in case of cotreatment 5.
The antiviral effect was more pronounced under posttreatment condition and a dose-dependent inhibitory effect on virus was observed from 1. Quantitative real-time RT-PCR results revealed that the extract had no effect on viral RNA titer under all conditions Figure 4Band a significant reduction was observed for viral RNA using quantitative real-time RT-PCR Figure 4B.
The extract lacked anti-CHIKV activity. Figure 4 Antiviral effect of leaves of Vitex negundo chloroform extract M4C against DENV under different treatment conditions. Vero CCL cells were pre- co- and posttreated with different concentrations of extracts, and h incubation after infection, the plates were frozen and the culture filtrates were used for the FFU assay A and real-time PCR B.
The results revealed that the extract had a dose-dependent anti-DENV activity from 0. Complete reduction in viral titer was observed at 1. Though not prominent as infectious virus particles, a reduction in viral RNA titer was observed from 1. Figure 5 Antiviral effect of bark of Ancitrocladus heyneanus chloroform extract M5C against DENV under different treatment conditions.
For chikungunya, M5C at a concentration of 3. However, similar reduction in viral RNA titer was not observed. The extract lacked anti-chikungunya activity under pre- and cotreatment conditions Figures 6A, B.
Figure 6 Antiviral effect of bark of Ancitrocladus heyneanus chloroform extract M5C against CHIKV under different treatment conditions. Under cotreatment condition, hydroalcoholic extract of Bacopa monnieri whole herb M7M reduced the DENV titer starting from The anti-dengue activity was more pronounced under posttreatment condition with a reduction starting from However, the corresponding reduction in viral RNA titer was not observed for both co- and posttreatment conditions Figure 7B.
Figure 7 Antiviral effect of whole herb of Bacopa monnieri hydroalcoholic extract M7M against DENV under different treatment conditions. In the case of chikungunya, more than one log reduction 7. No significant reduction of viral RNA was observed for viral RNA under all conditions Figure 8B.
Figure 8 Antiviral effect of whole herb of Bacopa monnieri hydroalcoholic extract M7M against CHIKV under different treatment conditions. Methanol extract of Cucurbita maxima seeds M9M exerted anti-chikungunya activity under all conditions.
: Antiviral plant extracts for health| Antiviral effect of phytochemicals from medicinal plants: Applications and drug delivery strategies | J Antiviral plant extracts for health Nanotechnol. Antioxidant and anti-inflammatory activities Antioxidant-rich plant foods 3,5-dicaffeoylquinic acid pant from Ligularia extrscts leaves. Food Chemistry 4 : —8. Tran TH, Guo Y, Song D, Bruno RS, Lu X. Novel self-nanomicellizing solid dispersion based on rebaudioside A: a potential nanoplatform for oral delivery of curcumin. |
| 15 Impressive Herbs with Antiviral Activity | The renewed interest in medicinal plants has focused on herbal cures among indigenous populations around the world. Aqueous extract of the rhizome of this herb has anti-HBV activity. Article CAS PubMed Google Scholar Cui L, Sune E, Song J, Wang J, Jia XB, Zhang ZH. Further inquiries can be directed to the corresponding authors. Yoong C, Hanaa C, Abdel Karim S, Rabiha S. |
| Introduction | Images represent CHIKV-infected Vero CCL cell lines under posttreatment condition. Dengue and chikungunya diseases are serious public health problems, and the unavailability of antivirals make these diseases a concern. Traditionally, plants have been used to treat various diseases including viral diseases for centuries Herrmann et al. Therefore, plant-based anti-chikungunya and anti-dengue drugs might be an alternative option to treat these mosquito-borne diseases. In the present study, chloroform, methanol, ethyl acetate, hydro-alcoholic, petroleum ether, and dichloromethane extracts of five selected plant species Plumeria alba , Ancistrocladus heyneanus , Bacopa monnieri , Curcubita maxima , Vitex negundo were tested for their antiviral activity against DENV and CHIKV. Out of 25 extracts and four purified compounds, five extracts M2C, M8M, M4C, M5C, and M7M and three purified compounds A-S, C-S, and MG-S showed anti-dengue activity, while four extracts M8M, M5C, M7M, and M9M and one purified compound MG-S exerted significant anti-chikungunya activity. The results suggest the therapeutic utility of Plumeria alba against both of these viruses. Since hydroalcoholic extracts of Plumeria alba leaves have been reported to possess antiarthritic activity, it might also be useful in reducing arthritis in CHIKV-infected patients Choudhary et al. Earlier, the inhibitory activity of Plumeria rubra containing fulvoplumierin against human immunodeficiency virus type 1 HIV reverse transcriptase has been reported Tan et al. Plumericin compounds isolated from Plumeria species have been reported to inhibit Leishmania donovani and Candida species Sharma et al. Plumeria alba is known to have various chemical substances including compounds with sterol-like structures like taraxerol, lupeol, betuline, coumarone, and fulvoplumericin which could contribute to antiviral activity and need further investigations Anggoro et al. Whole herb of Bacopa monnieri M7M also showed a significant reduction of DENV and CHIKV titers under posttreatment conditions. Bacopa monnieri , commonly known as Brahmi, is commonly used in the Indian traditional system of medicine as a memory enhancer Shinomol et al. It also has been reported to possess anti-inflammatory, analgesic, antipyretic, sedative, and antiepileptic properties Saini et al. Bacopa monnieri has been reported to contain bacosides and cucurbitacins which have medicinal properties and needs to be investigated for antiviral activity Bhandari et al. Extract from Ancitrocladus heyneanus bark M5C showed total reduction of viral foci of DENV and significant activity against CHIKV. Plant from Ancistrocladus genus has been shown to have anticancer, anti-HIV, antimalarial, and antibacterial activities Karn et al. Ancitrocladus heyaneanus is reported to have ancisheynine 1 and betulinic acid among which betulinic acid possesses anti-dengue activity Bringmann et al. Leaves of Vitex negundo M4C showed a significant reduction of DENV, while no significant activity was found against CHIKV. The ethanolic extract of Vitex negundo has been reported to inhibit the Asian genotype strain of CHIKV Kothandan and Swaminathan, However, in the present study, ECSA genotype of CHIKV was used. A flavone named vitexicarpone, ursolic acid, and betulinic acid have been isolated from Vitex negundo leaves, and the presence of betulinic acid might explain its anti-dengue activity Chandramu et al. Cucurbita leaves contain iron and vitamins Orech et al. Curcubita maxima seeds are also known to exert anti-platelet activity Sanzana et al. The purified compounds viz. anacardic acid, chloroquinone, and methyl gallate showed significant reduction in the case of DENV, while only methyl gallate showed a significant activity in the case of CHIKV. Earlier reports have shown that these compounds exert anti-dengue, antihelmintic, and antiparasitic effects Hundt et al. Anacardic acid is obtained from Anacardium occidentale nuts. However, extracts prepared from Anacardium occidentale leaves did not show any antiviral activity, suggesting absence of active antiviral compounds in leaves. The present study suggests the use of methyl gallate as a standard for investigations of anti-chikungunya activity. The extracts which showed activity under cotreatment conditions might bind to the virus and prevent its binding to the cellular receptors for entry. Further studies are needed to find out the mechanism of action of the plant extracts. In many of these experiments, though antiviral activity was visible in terms of reduction in infectious virus particles FFU , a corresponding decrease in viral RNA titer was not observed. This suggests that the plant extracts which exerted complete reduction in terms of FFU but not viral RNA may not affect viral RNA replication but might inhibit the assembly of virus particles. Moreover, quantitative real-time RT-PCR is a more sensitive assay than FFU and detects RNA from even noninfectious particles also. Hence, minor differences in viral RNA titer may be not reflected in quantitative real-time RT-PCR assay results unless there is a major difference in the RNA titer. The extract which affected both FFU and viral RNA titer might inhibit viral RNA replication. The concentration of the extracts which exerted antiviral activities was different from each other and it is possible that the extracts which had antiviral activity at lower concentrations still had high amount of the active antiviral compound while those extracts which exerted antiviral activity at higher concentration had lower amount of the active antiviral compounds. Those extracts which exerted antiviral activity at lower concertation may be further taken forward with whole formulation as a phytopharmaceutical drug while for the extracts which had antiviral activity at higher concentrations, there is a need to identify the active compound to be further considered an antiviral drug. Identifying the active fractions and compounds from the extracts with anti-dengue and anti-chikungunya activities will help to develop the formulations based on the above plants as a phytopharmaceutical drug which can be further evaluated in preclinical and clinical studies. The present study paves the way for further focused research on plant based antivirals against DENV and CHIKV to find effective treatments against these debilitating viral diseases. Further inquiries can be directed to the corresponding authors. Conceived and designed the experiments: SC, DP, SH, and KA. Performed the experiments: PP, DC, MK, and MBK. Analyzed the data: DP, KA, PP, DC, MK, HH, MBK, and SC. Provided extracts: SH, RJ, HH, and MK. Wrote the paper: DP, KA, HH, RJ, SC, and SH. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Reversible antispermatogenic and antifertility activities of Mondia whitei L. in male white albino. Phytother Res. Imaga N, Gbenle G, Okochi V, Adenekan S, Edeoghon S, Kehinde M, Bamiro S, Ajiboye A, Obinna A. Antisickling and toxicological profiles of leaf and stem of Parquetina nigrescens L. CAS Google Scholar. Williams LA, Rosner H, Levy HG, Barton EN. A critical review of the therapeutic potential of dibenzyl trisulphide isolated from Petiveria alliacea L guinea hen weed, anamu. West Indian Med J. Oyemitan IA, Olayera OA, Alai A, Abass LA, Elusiyan CA, Oyedeji AO, Akanmu MA. Psychoneuropharmacological activities and chemical composition of essential oil of fresh fruits of Piper guineense Piperaceae in mice. Iqbal M, Gnanaraj C. Eleusine indica L. possesses antioxidant activity and precludes carbon tetrachloride CCl 4 -mediated oxidative hepatic damage in rats. Environ Health Prev Med. Amos S, Abbah J, Chindo B, Edmond I, Binda L, Adzu B, Buhari S, Odutola AA, Wambebe C, Gamaniel K. Neuropharmacological effects of the aqueous extract ot Nauclea latifolia root bark i rats and mice. Abena AA, Diatewa M, Gakosso G, Gbeassor M, Hondi-Assah T, Ouamba JM. Analgesic, antipyretic and anti-inflammatory effects of essential oil of Lippia multiflora. Download references. We thank the staff of the WHO Polio Laboratory, Department of Virology, University of Ibadan for the assistance rendered during the course of this research and the staff of the Forest Research Institute of Nigeria FRIN for the authentication of plant materials. Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Ibadan, Nigeria. Omonike O. Department of Pharmacognosy, Faculty of Pharmacy, Olabisi Onabanjo University, Sagamu, Nigeria. Department of Microbiology, Ekiti State University, Ado-Ekiti, Nigeria. Department of Virology, College of Medicine, University of Ibadan, Ibadan, Nigeria. You can also search for this author in PubMed Google Scholar. OOO and TCF conceived and designed the experiments. PAS and TEA carried out the plant extraction. OOO, PAS and TEA performed the antiviral assays. AJA and TCF provided cell culture materials and viruses. TEA and PAS prepared the manuscript and it was reviewed by OOO. All authors read and approved the final version of the manuscript. Correspondence to Omonike O. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is distributed under the terms of the Creative Commons Attribution 4. Reprints and permissions. Ogbole, O. et al. In vitro antiviral activity of twenty-seven medicinal plant extracts from Southwest Nigeria against three serotypes of echoviruses. Virol J 15 , Download citation. Received : 27 April Accepted : 11 July Published : 18 July Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. Research Open access Published: 18 July In vitro antiviral activity of twenty-seven medicinal plant extracts from Southwest Nigeria against three serotypes of echoviruses Omonike O. Ogbole ORCID: orcid. Akinleye 1 , Peter A. Segun 2 , Temitope C. Adeniji 4 Show authors Virology Journal volume 15 , Article number: Cite this article 27k Accesses 62 Citations 5 Altmetric Metrics details. Abstract Background Echoviruses, a serotype of enteroviruses, infect millions of people globally and there is no specific drug treatment or vaccine available for its management. Methods The plants were macerated in methanol and the cytotoxicities of the crude extracts were evaluated on the rhabdomyosarcoma cell line using the MTT assay. Results The crude extract of Macaranga barteri leaves had the highest cytotoxicity with CC 50 value of 0. Conclusion Our research has demonstrated that Macaranga barteri extracts has potent antiviral activity against echoviruses E7 and E19, and our findings suggest that this extract may have potential as a therapeutic agent in the treatment of enteroviral infections. Background The use of traditional medicine is popular in Africa, with almost three-quarter of the populace of this continent consulting traditional medical practitioners TMPs , mainly traditional doctors, when faced with a medical problem. Methods Plant materials and extraction Twenty-seven different morphological parts from 26 plants Table 1 , selected based on their ethnobotanical use in the treatment of infectious diseases, were collected from various locations in Ibadan, South-west Nigeria, identified and authenticated at Forestry Herbarium Ibadan FHI. Table 1 Plant species analysed for antiviral activity Full size table. Maximum non-toxic concentration MNTC of crude extracts The plant extracts encountered in this study displayed varying MNTC to RD cells in tissue culture medium. Table 2 Anti-echovirus activity of crude methanol extracts Full size table. Table 3 Anti-echovirus activity of M. barteri fractions and sub-fractions Full size table. Discussion Enteroviruses have continued to pose a great burden to global health. Conclusion To summarize our findings, we have reported the antiviral activity of 26 medicinal plants selected from Nigerian flora against three serotypes of enteroviruses E7, E13 and E References Ogbole OO, Adeniji AJ, Ajaiyeoba EO, Adu FD. Article PubMed PubMed Central CAS Google Scholar Katiyar C, Gupta A, Kanjilal S, Katiyar S. Article PubMed PubMed Central Google Scholar Henkin JM, Sydara K, Xayvue M, Souliya O, Kinghorn AD, Burdette JE, Chen W-L, Elkington BG, Soejarto DD. 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Article CAS Google Scholar Middleton JE, Kandaswami C, Theoharides TC. PubMed CAS Google Scholar Behbahani M, Sayedipour S, Pourazar A, Shanehsazzadeh M. PubMed PubMed Central CAS Google Scholar Hong S, Joo T, Jhoo J-W. Article CAS Google Scholar Adesanya SA, Olugbode TA, Odebiyi OO, Aladesanmi JA. Article Google Scholar Sabiu S, Garuba T, Sunmonu T, Ajani E, Sulyman A, Nurian I, Balogun A. Article PubMed PubMed Central CAS Google Scholar Erharuyi O, Falodun A, Langer P. Google Scholar Okunade AL. Article PubMed CAS Google Scholar Paul S, Chakraborty S, Mukherjee A, Kundu R. Google Scholar Ponou BK, Teponno RB, Ricciutelli M, Nguelefack TB, Quassinti L, Bramucci M, Lupidi G, Barboni L, Tapondjou LA. Article PubMed CAS Google Scholar Kuete V, Tabopda TK, Ngameni B, Nana F, Tshikalange TE, Ngadjui BT. Article CAS Google Scholar Meira M, Silva EP, David JM, David JP. Google Scholar Olorunnisola OS, Afolayan AJ, Adetutu A. PubMed PubMed Central Google Scholar Onoagbe IO, Attah V, Luther MM, Esekheigbe A. Google Scholar Mulholland DA, Langat MK, Crouch NR, Coley HM, Mutambi EM, Nuzillard J-M. Article PubMed CAS Google Scholar Ogbole OO, Segun PA, Fasinu PS. |
| Author Information | Copyright: © Rajasekaran et al. Revisiting the linkage between ethnomedical use and development of new medicines: a novel plant collection strategy towards the discovery of anticancer agents. repens , B. The selection of the viruses was based on the clinical importance of their infections, the type of the genome and the strategies of viral replication. Ibrahim AK, Youssef AI, Arafa AS, Ahmed SA. Statistical analysis Data are presented as means ± standard deviation SD. This plant is used as folklore medicine in Chakma tribe. |
Antiviral plant extracts for health -
Table 3. Antiviral activity of plant extracts against RNA viruses. Recommendation Medicinal plants could be serving as essential sources of antiviral agents for humans and animals diseases but still need further extensive studies for exploration of plants bioactive ingredients consider top global priorities.
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Common characteristics and unique features: A comparison of the fusion machinery of the alpha herpesviruses pseudorabies virus and herpes simplex virus. Advances in Virus Research. Written By Gamil S. Continue reading from the same book View All.
IntechOpen Antiviral Strategies in the Treatment o Chapter 5 HBV and HCV Infection Prophylaxis in Liver Transpl By Mariana Mihăilă, Cristina Mădălina Pascu, Andreea Chapter 6 Natural Phenolic Acids and Their Derivatives again By Yi-Hang Wu, Yan Chen, An-Qi Zhuang and Shan-Mei Ch The genomes of type A and B consist of eight segments of negative-sense single-stranded RNA and the virions express two major surface glycoproteins, haemagglutinin HA and neuraminidase NA.
Conversely, Type C contains seven RNA segments and express only one major surface glycoprotein, hemagglutinin-esterase-fusion HEF protein [2]. Amongst the types, A and B are the predominant causes of human infections [3] , with Type A being further divided into subtypes, based on the antigenicity of the HA and the NA.
To date, 17 HA H1—H17 and 9 NA N1—N9 subtypes have been identified, and most subtypes are present in waterfowl and shorebirds [1] , [4] , [5]. Of these, only H1N1, H2N2 and H3N2 have been associated with pandemics and epidemics in human populations [1].
Types A and B viruses spread globally in pandemics mediated through mutations that generate antigenic drift and shift [6]. Vaccines form the basis for the prevention of influenza infections, yet there are substantial drawbacks.
The current preventive strategy involves annual vaccination, requiring regular monitoring to confirm matching between vaccines and the circulating virus strains. In the eventuality of a pandemic infection with a new strain, antiviral drugs represent the first line of defence [8].
Currently available anti-influenza drugs aim to block viral replication and spread, thereby resulting in early recovery from the symptoms of flu. First generation influenza antivirals, referred to as ion channel blockers Amantadine and Rimantadine , act on the viral M2 protein, which is essential for the organized release of nucleocapsid after fusion of the virus with the endosomal membrane [9].
Side effects associated with the central nervous system and the gastrointestinal tract, and the rapid emergence of antiviral resistance during therapy, have limited the usefulness of adamantanes in the prevention and treatment of influenza [10] , [11].
As a result, a second generation of anti-influenza drugs, the neuraminidase inhibitors NAI , were developed. There are currently two NAI drugs approved for use worldwide, Oseltamivir and Zanamivir, and two others approved in North Asia but still in trials elsewhere Laninamivir and Peramivir [11].
Zanamivir GG , a sialic acid analogue, and Oseltamivir, an ethyl ester derivative of Oseltamivir GS, inhibit the sialidase activity of the viral neuraminidase by competitive and irreversible binding to the NA active site [12] , [13].
However, there are side effects associated with the administration of Oseltamivir and Zanamivir, such as nausea, vomiting, neuropsychiatric events, abdominal pain, diarrhoea, sinusitis, headache and dizziness. Furthermore, Oseltamivir-resistant H1N1 viruses spontaneously arose and spread globally in [10].
These data highlight the requirement for a third generation of anti-influenza drugs that would exhibit a different mode of action [8]. Thirteen years after the launch of Zanamivir and Oseltamivir, the quest for unique lead structures remains an area of intensive research [14].
Empirical knowledge based on the ethnomedical benefits of plants, coupled with bioassay-guided fractionation and isolation, has the potential to identify novel antivirals that could be used against influenza.
Currently, herb and plant resources are relatively unlimited with respect to the search for functional phytochemicals but these resources are dwindling rapidly due to deforestation and advancements of industrialization.
Even though a number of studies have been performed using purified plant chemicals, very few studies have addressed the antiviral activities of crude plant extracts [14] , [15]. The search for plant-based antivirals against the influenza virus is promising, as several plants have been shown to possess anti-influenza activity, some of which include: Thuja orientalis , Aster spathulifolius , Pinus thunbergia [16] , Allium fistulosum [17] , Sambucus nigra [18] and Psidium guajava [19].
Active components have also been isolated from crude plant extracts employing chemical fractionation techniques. Patchouli alcohol isolated from the leaves of Pogostemon cablin [20] , cardiotonic glycoside obtained from Adenium obesum Forssk.
Progress in the field of anti-influenza herbal medicines has provided alternative therapeutic measures for the treatment of influenza virus infection. For instance, two Japanese herbal medicines, Shahakusan and hochuekkito, have been shown to possess in vivo activity against influenza virus [22] , [23].
On the other hand, studies on Jinchai, a capsule made of Traditional Chinese Medicine, indicated inhibitory activity against viral adsorption and cell membrane fusion, thereby blocking transcription and replication of the virus [24].
Also, Lianhuaqingwen capsule, a natural herbal medicine, was shown to have similar therapeutic effectiveness to that of Oseltamivir, in terms of reducing the duration of illness and viral shedding of Influenza A virus [25]. In this work, we have used a range of bioassays to screen fifty medicinal plant extracts for antiviral activity against influenza Type A viruses.
The results demonstrate the anti-influenza potential of some extracts which act via a unique mode of action when compared to the currently available antiviral drugs. In the eventuality of an influenza pandemic, third generation of anti-influenza compounds would be extremely beneficial and the source of such compounds could be medicinal plants.
Before adding the compounds or the virus, or when quantifying the results, the monolayers were thoroughly washed twice with phosphate buffered-saline PBS, pH 7.
In all experiments, the following controls were included: cell control cells that were not infected with the virus or treated with the plant extracts , virus control cells that were infected only with the virus but not treated with the plant extracts in the antiviral assays , and the positive controls virus-infected cells treated with Zanamivir or Oseltamivir.
Fifty medicinal plant extracts, collected from the tropical rainforests of Borneo, Sarawak, Malaysia, were selected on the basis of their traditional use in healing various diseases, including symptoms of influenza such as cough and sore throat.
The yield is dependent on the part of plant used; around 0. Briefly, MDCK cells were seeded into well flat-bottomed microtitre plates Costar at 4×10 3 cells per well.
Following overnight incubation, the media of MDCK cells were aspirated, followed by addition of µL of plant extract solution diluted in RPMI medium two-fold dilutions, ranging from 0. The optical density OD was measured at nm using a Bio-Rad iMark TM microplate reader.
Cytotoxicity graphs were then generated by plotting percentage of cell viability versus concentration of extracts. The activity of plant extracts against influenza viruses was evaluated according to a method described elsewhere [29] , albeit some modifications.
The cells were washed twice with PBS, and two-fold serial dilutions of plant extracts 0. The antiviral activity curve was then generated by plotting percentages of virus inhibition against concentrations of extracts.
Using regression analysis of antiviral activity curves in Microsoft excel , a trendline that best suited the curve was selected and the corresponding equation was used to calculate IC 50 values [29].
The antiviral effects of extracts were evaluated at different times of viral infection as described by Chiang et al. To assess the activity of the compounds in inhibiting viral binding, an attachment assay adapted from De Logu et al.
MTT was employed to evaluate cell viability and the percentage of viral inhibition was calculated in relation to the virus control wells. The effect of plant extracts on viral penetration was studied according to a method described elsewhere [32]. After the incubation period, specific concentrations of extracts 0.
After the specified time interval, the supernatant was removed and treated with acidic PBS pH 3 for 1 min to inactivate unpenetrated virus [33] , and finally treated with alkaline PBS pH 11 for neutralization. Cells were washed once with PBS pH 7. The virus stock was titrated by performing NA activity assay and the optimum virus dilution for the neuraminidase inhibition assay was selected.
Two-fold serial dilutions of plant extracts 0. Fluorescence was measured using a POLARstar Omega fluorescence polarization microplate reader excitation nm, emission nm. IC 50 values were determined from dose-response data using a sigmoidal curve-fitting generated and analysed using GraphPad Prism Software.
An HI assay was used to determine the effect of extracts on virus adsorption [34]. Briefly, two fold serial dilutions of the extract 0. The hemagglutination reactions were observed after 30 minutes incubation at room temperature. The effect of Receptor Destroying Enzyme RDE, Denka Seiken Co.
The extract and RDE mixture was inactivated at 56°C for 60 minutes and then subjected to the assays. The effect of trypsin Sigma, No: T upon the antiviral activity of the extracts was studied through an in vitro micro inhibition assay and HI assays.
Plant extracts subjected to the same temperature without trypsin and extracts that were neither subjected to temperature nor trypsin treatments were included as controls. All treatments were performed in triplicates and each experiment was independently repeated at least twice.
The data were expressed as mean ± standard error of the mean SEM. The results of the antiviral activity assays were analysed with a one-way ANOVA test and a significance level p value of 0. The medicinal plant extracts were screened for cellular toxicity in order to determine appropriate concentrations for the in vitro micro inhibition assays.
As detailed in Table 1 , there were duplicate samples among the plant extracts; extracts 13 and 30 were obtained from two sources of the same species collected in the same location at different times while extracts 41 and 42 were obtained from different parts of the same plant.
In this case, the CC 50 was an estimated theoretical value obtained by extrapolation of the results in Figure 1. Though most of the plant extracts demonstrated minimal cytotoxicity at concentrations less than 6.
Following overnight incubation of cells seeded at 4×10 3 cells per well into well flat-bottomed microtitre plates, the media were aspirated and overlaid with µL of two-fold serial dilutions of plant extract 0.
After three days incubation, cell viability was evaluated using MTT and percentage cell viability calculated relative to cell control wells. Representatives of two independent experiments performed in triplicate are shown. The plant extracts were subjected to a high throughput in vitro micro-inhibition screening assay to determine antiviral activity.
A number of plant extracts exhibited inhibitory activity against influenza virus strain Mem-Bel H3N1. The same eleven extracts also mediated significant antiviral activity against the PR8 H1N1 strain.
The antiviral activity curves for all the extracts against H3N1 and H1N1 are shown in Figures 2 and 3 , respectively. Duplicate hits in the assay extracts 13 and 30, and extracts 41 and 42 confirmed the consistency of this screening procedure.
The plant extracts were most active at Cell viability was evaluated using MTT and viral inhibition percentage calculated relative to virus control wells. Plant extracts were tested for their ability to inhibit viral attachment using a virus binding assay. The results support HI activity demonstrated by extracts 8, 41, 42 and Despite lacking HI activity, extracts 13, 14, 30, 31 and 38 demonstrated significant inhibitory effects on the binding of influenza virus.
As expected, the established neuraminidase inhibitors NAI , Zanamivir and Oseltamivir, did not inhibit virus binding. As shown in Table 3 , plant extracts inhibited the binding of H3N1 and H1N1 viruses depending on the concentration of plant extracts used in the assay.
The virus inhibition percentages of the wells that received higher concentrations of the plant extracts 50 or Extract 42 was also shown to inhibit the binding of H3N1strain at 0. After 3 h incubation at 4°C, cells were washed twice with ice-cold PBS and overlaid with RPMI and virus growth medium.
As shown in Table 4 , all extracts were able to prevent viral penetration, with the exception of extract 29 which was ineffective at all time points and extract 37 which was active only against H3N1 strain.
These data support HI results obtained using extracts 8, 41, 42 and Four plant extracts 8, 30, 31 and 38 demonstrated virus inhibition at all three time points, including the effect of For three HI extracts 41, 42 and 43 , inhibition of virus penetration increased over time as the antiviral activity of the extract at 60 and min was greater than that observed at 30 min against H3N1 strain.
As expected, Oseltamivir was inactive against both viruses in the assay. Following inactivation and neutralization of unpenetrated virus using acidic and alkaline PBS, respectively, cells were washed with PBS and overlaid with RPMI medium and virus growth medium in equal proportion.
Data shown are representative of two independent experiments performed in triplicate. The influenza virus NA glycoprotein has sialidase activity and mediates the release of viral progeny from the infected cell, thus promoting virus transmission and spread [36].
In addition, the viral NA removes sialic acid from glycans expressed by the viral HA glycoprotein, thereby preventing self-aggregation of virions [37]. Increasing concentrations of plant extracts were associated with decreased relative fluorescence Table 5 , consistent with inhibition of NA activity.
IC 50 values indicated that extracts 8 and 43 reduced NA activity at a lower concentration than the other extracts. It should be noted that we have tested crude plant extracts, thus the results cannot be directly compared with Zanamivir and Oseltamivir, as these commercially available drugs were tested at nanomolar concentrations.
The influenza virus HA mediates attachment to the sialic acid residues expressed by the glycoproteins and glycolipids of host cells, which is a critical step in the initiation of infection [38].
Similarly, the viral HA binds to sialic acids expressed on the surface of erythrocytes resulting in hemagglutination. Thus, we examined the ability of plant extracts to inhibit virus-induced hemagglutination using a hemagglutination inhibition HI assay.
As shown in Figure 6 , four out of eleven extracts mediated HI activity against Mem-Bel and PR8 viruses at specific concentrations. Extract controls were included to study the direct effect of extracts on chicken red blood cells in the absence of Mem-Bel and PR8 viruses.
Some hemolytic concentrations of extract control resulted in HI when the virus was included, suggesting that extract components preferentially attach to the virus rather than to the erythrocytes. Plant extracts that mediated HI activity against Mem-Bel and PR8 strains were active in preventing virus-induced hemagglutination at concentrations ranging between HI activities of four extracts 0.
The following controls were included on each plate; i extract controls with extract and chicken red blood cells CRBC only, ii virus controls containing virus and CRBC and iii cell controls containing only CRBC. Monoclonal antibody against the HA of either H3N1 or H1N1 strains were included as a positive control.
The antibody titres for monoclonal antibody against H3N1 and H1N1 were 80 and , respectively; dilution of either of the two antibodies in PBS were employed in the assay.
Data are shown from one of three independent experiments, each performed in triplicate. Four extracts which were shown to interfere with hemagglutination of chicken red blood cells were treated with RDE in order to eliminate compounds that might contain sialic acid mimics that compete with the RBC receptors for virus hemagglutinin.
An HI assay was performed with RDE-treated extracts which were originally able to prevent hemagglutination. As shown in Figure 7 , RDE treatment removed HI activity originally exhibited by all four extracts. Further, an in vitro micro-inhibition assay was performed with the RDE-treated extracts against H3N1 and H1N1 viral strains.
As shown in Figure 7 , there was a significant reduction in the antiviral efficacy of extracts with HI potential whereas non-HI extract 38, included as a negative control, did not show any significant difference in viral inhibition before and after RDE treatment. A similar pattern in the percentage viral inhibition was observed with the H1N1 strain exposed to RDE-treated extracts data not shown.
Inhibitory effect of plant extracts on the hemagglutination of H3N1 viral strain. i Virus controls containing virus and CRBC and ii cell controls receiving CRBC only are shown. Corresponding RDE treated monoclonal antibody which acts against the HA of H3N1and extracts that mediate HI activity without RDE treatment were included in all plates as positive controls.
The experiment was performed in triplicate. Loss of efficacy in antiviral inhibition of HI extracts against H3N1 strain.
An in vitro micro-inhibition assay was used to assess the ability of plant extracts to inhibit H3N1 TCID 50 influenza virus. Extracts were either treated with RDE as per the manufacturer's instructions or left in their native form without RDE treatment.
Plant extracts that were shown to prevent virus induced haemagglutination were treated with trypsin in order to denature any protein that might be the cause of such inhibition. An in vitro micro-inhibition assay was initially performed to determine the activity of trypsin-treated extracts and controls against H3N1 and H1N1 strains.
As shown in Figure 8 , the antiviral activity of plant extracts against the H3N1 virus was not altered by either trypsin treatment or temperature without trypsin. The trypsin-treated plant extracts and controls were then subjected to an HI assay.
As shown in Figure 8 , HI activity of plant extracts were exhibited against H3N1 despite trypsin treatment or temperature change.
A similar pattern in the percentage viral inhibition and HI activity was observed with the H1N1 strain exposed to trypsin-treated extracts data not shown. Extracts that mediate HI activity without trypsin treatment were included in all plates as positive controls. Antiviral inhibition of HI extracts against H3N1 strain.
Extracts 3. Phytomedicines have been used since ancient times to treat various infections but clinical studies are limited [39]. In this study, we have identified a number of traditional medicinal plant extracts collected from Sarawak, Malaysia, which displayed anti-influenza activity. Potentially, there are many compounds within any given extract that might mediate antiviral activity.
The antiviral compounds present in these extracts may act alone or work in a synergistic manner. The efficacy of several plant extracts used in herbal medicine is directly related to the synergistic effects of bioactive components and derivatives [40].
Safety is a major requirement for an antiviral agent and in the search for new drugs it is important to consider possible secondary effects.
The minimal cytotoxicity observed in the extracts investigated may be due to the presence of cytoprotective components. This cytoprotective role of plants have been reported in other studies on plant extracts [41].
In our study, this is an indication that these extracts might serve as potential candidates for the development of safe and less toxic drugs. In general, compounds that are linked to ethnomedical uses are considered to be safe and more effective than substances that lack this framework [14].
Being pure drugs, Zanamivir and Oseltamivir inhibited influenza virus at all concentrations tested 0. Despite the difference in concentration, the anti-influenza drugs followed concentration-independent virus inhibition.
Chemical characterization of the active components present in the plant extracts may lead to concentration-independent virus inhibition like that of Zanamivir and Oseltamivir. The amounts of active component s present in the plant extracts and their efficiency in preventing virus inhibition play a major role in demonstrating antiviral activity.
All plant extracts tested were shown to exhibit NAI activity. Extracts 8, 38 and 43 demonstrated the lowest IC 50 range, indicating a higher amount of NAI in the given plant extract or the presence of potent NAI component s that may be active even at a lower concentration.
As suggested in the literature, the chemical components in the extracts might have combined with the viral membrane to modify the physical properties of viral neuraminidase [42]. Therefore, the dose and time required for the extract to prevent viral growth are critical and different for each.
Since drug-resistant viruses appear frequently, it is important to identify drugs with a different mode of action to the one observed with the conventional drugs currently used NAI and adamantanes.
A sialylated molecule that can block virus attachment to cellular receptors might act to limit the initial stages of virus infection, compared to NA inhibition that is believed to act largely through preventing release of new virions from virus-infected cells.
Moreover, NAI must be administered in the early stages of infection as they are less efficient during the later phases [43]. Four of the extracts mediated HI activity within a specific range of concentrations.
It has been reported elsewhere that in HI assays some plant extracts can cause hemolysis at higher concentrations [44]. Hemolysis caused by the extracts in the controls may be attributed to the presence of other compounds apart from those with anti-influenza activity.
It is also possible that more than one anti-influenza component may be present in extracts that showed multiple modes of action. The stable interaction between HA and NA, which is vital for the effective entry and release of the virus, may have been disrupted by the anti-influenza component s present in the plant extracts.
This could also be considered a new anti-influenza pathway as dual action drugs have not previously been used. The inhibitory effects demonstrated against viral binding and penetration further suggested the HI mode of action of extracts 8, 41, 42 and Interestingly, non-HI extracts 13, 14, 30, 31, 37 and 38 which exhibited NAI activity showed significant inhibition in the viral binding and penetration assays.
The major functions of the sialidase activity of influenza NA are to facilitate the release of viral progeny from infected cells and enable viral spread, but NA is also important for viral entry [44] — [46].
The role of NA in removing sialic acid residues from HA could improve fusion and infectivity of influenza virus following three mechanisms:. Thus neuraminidase also affects viral entry according to the above-mentioned phenomena. The significant effects exhibited by non-HI extracts in viral binding and penetration could be due to the neuraminidase inhibitory component, which might have played a role in inhibiting viral entry.
Similar results have been obtained in previous studies [44]. The inhibitory effect of Zanamivir at the 60 minutes time point in the penetration assay might have also resulted from the NAI pathway.
The loss of HI activity of the extracts following RDE treatment suggests that the responsible components may possess sialic acid-like structures that mimic the receptors of CRBC, thereby competing for viral hemagglutinin.
Deactivation of sialic acid mimics that were originally present in the extract may be the reason for this significant drop in virus inhibition, though anti-influenza activity was observed at other concentrations tested data not shown.
Therefore, a potential synergistic effect of components apart from those that are HAI active may be present in the plant extracts. Further studies to investigate if there is any synergistic effect of neuraminidase inhibitory activity and hemagglutination inhibition exhibited by the plant extracts will be performed.
Apart from inhibiting hemagglutinin and neuraminidase, the plant extracts could also have affected other proteins in the virus including nucleoprotein, RNA polymerase, matrix protein1, nuclear export protein and non-structural protein 1 that play major roles during virus replication.
The antiviral efficacy not being affected by temperature or trypsin treatment suggests that the compound s of interest may not be proteinaceous. It is worthwhile noting that minor variations were evident with the activity of extracts for different batches; this could possibly result from seasonal changes in the composition of plant extracts collected at different times or through the collection and extraction processes.
For instance, extract 8 did not demonstrate HI activity in the most recent batch that was used to study the effects of trypsin treatment upon the antiviral activity of extracts, but the preliminary chemical fraction of extract 8 demonstrated HI activity in one of the fractions.
This process is still under study. This phenomenon needs to be further examined. The anti-influenza effects of HI extracts have not been published previously, though some plants belonging to the same genus have been reported to show antimicrobial activity [47] — [49].
Bioassay-guided fractionation of HI extracts 8, 41, 42 and 43 coupled with HPLC and GC-MS techniques are currently being undertaken. In vivo animal model studies that support the activity of extracts against influenza virus should also be performed.
The results presented in this study suggest that plants with reported medicinal properties could be a potential source for new antiviral drugs. The plant extracts investigated could serve as promising candidates for the development of third generation anti-influenza drugs, thereby challenging the neuraminidase drug resistant viruses in an attempt to safeguard human health and the global economy.
Conceived and designed the experiments: DR LG EP FM. Performed the experiments: DR LG. Analyzed the data: DR LG. Wrote the paper: DR LG EP FM TCY DLS CLT. Browse Subject Areas?
Korean red ginseng, Antoviral infection is the most serious health issue Antivieal causing unexpected higher rate of death globally. Many viruses plnat not Antiviral plant extracts for health curable, such as corona virus-2 Extrqctshuman immunodeficiency virus HIVhepatitis virus, human papilloma extracte and Antivirzl others. Furthermore, the toxicities Antjviral ineffective responses to resistant strains Antiviral plant extracts for health synthetic Antivirak drugs have reinforced Antioxidant-rich plant foods search heslth effective Herbal Prostate Health alternative treatment options, such as plant-derived antiviral drug molecules. Therefore, in the present review, an attempt has been taken to summarize the medicinal plants reported for exhibiting antiviral activities available in Bangladesh along with discussing the mechanistic insights into their bioactive components against three most hazardous viruses, namely SARS-CoV-2, HIV, and HBV. The review covers 46 medicinal plants with antiviral activity from 25 families. Among the reported 79 bioactive compounds having antiviral activities isolated from these plants, about 37 of them have been reported for significant activities against varieties of viruses. Hesperidin, apigenin, luteolin, seselin, 6-gingerol, humulene epoxide, quercetin, kaempferol, curcumin, and epigallocatechingallate EGCG have been reported to inhibit multiple molecular targets of SARS-CoV-2 viral replication in a number of in silico investigations. Antioxidant supplements and chikungunya Antioxidant-rich plant foods two Antivral mosquito-borne infections Korean red ginseng are known to occur extensively in tropical Korean red ginseng subtropical healtth. Presently, there is no treatment hfalth these viral Antjviral. In vitro antiviral screening of 25 extracts prepared extrzcts the plants of Vitex negundo Mental fitness tips, Plumeria albaAncistrocladus Antioxidant-rich plant foodsBacopa AntoviralAnacardium occidentaleCucurbita maximaSimarouba glaucaand Embelia ribes using different solvents and four purified compounds anacardic acid, chloroquinone, glaucarubinone, and methyl gallate were carried out for their anti-dengue virus DENV and anti-chikungunya virus CHIKV activities. Maximum nontoxic concentrations of the chloroform, methanol, ethyl acetate, petroleum ether, dichloromethane, and hydroalcoholic extracts of eight plants were used. The antiviral activity was assessed by focus-forming unit assay, quantitative real-time RT-PCR, and immunofluorescence assays. Extracts from Plumeria albaAncistrocladus heyneanusBacopa monnieriand Cucurbita maxima showed both anti-DENV and CHIKV activity while extract from Vitex negundo showed only anti-DENV activity.
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