Published online: 2020-01-22 Original Papers Antiviral Activity of a Arisaema Tortuosum Leaf Extract and Some of its Constituents against Herpes Simplex Virus Type 2 Authors Massimo Rittà1*, Arianna Marengo 2*, Andrea Civra 1, David Lembo 1, Cecilia Cagliero 2, Kamal Kant 3,UmaRanjanLal3, Patrizia Rubiolo 2, Manik Ghosh 3, Manuela Donalisio 1 Affiliations Correspondence 1 Department of Clinical and Biological Sciences, Dr. Manik Ghosh University of Torino, Orbassano, Torino, Italy Department of Pharmaceutical Sciences & Technology, 2 Department of Drug Science and Technology, Birla Institute of Technology University of Torino, Torino, Italy Mesra, Ranchi, Jharkhand 835215, India 3 Department of Pharmaceutical Sciences & Technology, Phone: + 916512276247, Fax: + 916512275401 Birla Institute of Technology, Mesra, Ranchi, India [email protected] Key words Supporting information available online at Arisaema tortuosum ‑ , Araceae, HSV 2, antiviral activity, http://www.thieme-connect.de/products apigenin, luteolin ABSTRACT received July 18, 2019 revised December 19, 2019 Infections caused by HSV-2 are a public health concern world- accepted December 31, 2019 wide, and there is still a great demand for the discovery of novel anti-herpes virus agents effective against strains resis- Bibliography tant to current antiviral agents. In this context, medicinal DOI https://doi.org/10.1055/a-1087-8303 plants represent an alternative source of active compounds published online January 22, 2020 | Planta Med 2020; 86: for developing efficient antiviral therapies. The aim of this – 267 275 © Georg Thieme Verlag KG Stuttgart · New York | study was to evaluate the antiviral activity of Arisaema tortuo- ‑ ISSN 0032 0943 sum, a plant used in the traditional medicine of India. A chloroform soluble fraction of the leaves exhibited anti-HSV- Correspondence 2 activity with a selectivity index of 758. The extract was also Dr. Manuela Donalisio, PhD active against acyclovir-resistant HSV-2 and HSV-1. The Department of Clinical and Biological Sciences, mechanism of action of the extract was investigated evidenc- University of Torino, S. Luigi Gonzaga Hospital ing inhibition of both early and late events of the HSV-2 repli- Regione Gonzole, 10, Orbassano, Torino 10043, Italy cative cycle. A HPLC‑PDA‑MS/MS analysis showed the pres- Phone: + 390116705427, Fax: + 390119038639 ence of flavonoids including apigenin and luteolin in the [email protected] chloroform extract (CE). Apigenin and luteolin showed a high inhibitory activity with EC50 values of 0.05 and 0.41 µg/mL, re- spectively. Both compounds exhibited antiviral activity when This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. added up to 6 h post infection and were able to reduce the vi- ral progeny production. In addition, apigenin interfered with cell-to-cell virus spread. Introduction the disease, herpes can still be spread to sex partners. Clinical manifestations are characterized by recurrent, painful lesions, Herpes simplex virus type 2 (HSV-2) is among the most prevalent named herpes sores, usually appearing on or around the genitals, sexually transmitted viruses. The Centers for Disease Control and rectum, or mouth. Genital herpes may cause painful genital ulcers Prevention (CDC) estimates that, annually, 776,000 people in the that can be severe and persistent in immune suppressed people, United States get new genital herpes infections, and more than 1 out of every 6 people aged 14 to 49 y have genital herpes [1]. Most HSV-2 infections are subclinical, but even without signs of * These authors contributed equally to this work. Rittà M et al. Antiviral Activity of … Planta Med 2020; 86: 267–275 267 Original Papers pulpit” flower and grows to 30cm in height [8]. This plant is wide- ABBREVIATIONS spread in rhododendron forest and alpine meadows in the Hima- ADME‑T absorption, distribution, metabolism, layas, Southern India, Myanmar, and Western China and even at excretion and toxicity elevations of 1500–2200 meters in Nepal [9,10]. The plant tuber AE aqueous extract (dried powder and juice) has been used in traditional medicine of BE n-butanol extract India for its therapeutic properties for piles, ailments related to di- CE chloroform extract gestive tract (constipation, indigestion, abdominal pain, and dys- EaE ethyl acetate extract entery), rheumatism, snake bites, and in treating bone fracture EE ethanolic extract and parasitic infections [11–13]. The tuber extract has shown ESI electrospray ionization antioxidant, anti-inflammatory, and antiproliferative activities; re- HE n-hexane extract cently, n-hexane, chloroform, and ethyl acetate fractions of A. tor- HSV-1 herpes simplex virus type 1 tuosum have been investigated for their antibacterial and antifun- HSV-2 HSV type 2 gal activities [14]. Previous works on A. tortuosum revealed the HSV-2 ACV‑r HSV-2 strain with phenotypic resistance presence of flavonoids and lectins [9, 10]. Among the protein to acyclovir components, lectins purified from A. tortuosum tubers exerted in MOI multiplicity of infection vitro antiproliferative activity against human cancer cell lines and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme- anti-insect activity [9, 10, 15]. Recently, merged molecular dock- thoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium] ing, ADME‑T, and dynamics approaches identified several A. tor- PFU plaque-forming units tuosum phytoconstituents as potential inhibitors of HSV-1 and PIS product ion scan HSV-2 enzymes, namely thymidine kinase and serine protease SI selectivity index [16]. In this context, according to the need to continue searching SIM selective ion monitoring for novel antiviral agents, this study was undertaken to explore SRM selected reaction monitoring whether A. tortuosum leaves and tubers extracts and their identi- fied pure compounds, for which a commercial standard was avail- able, exerted an effective antiviral activity against HSV-2 and ACV‑rHSV-2strainsin vitro. Furthermore, their mode of antiviral such as HIV-infected persons. Recurrent outbreaks of genital her- action was investigated at different steps of the viral infection pes are common during an individualʼs lifespan, since, after the cycle. primary infection, HSV-2 establishes a latency in the neurons of the sensory ganglia and it can be reactivated by several stimuli in- Results and Discussion cluding immunosuppression, stress, and hormonal changes [2]. There is currently no cure or a commercially available vaccine for This study investigated the antiviral activity of A. tortuosum ex- genital herpes. Antiviral medications, like acyclovir, valacyclovir, tracts against HSV-2 infection in vitro on Vero cells. Firstly, etha- and famciclovir, can reduce the severity, shorten outbreaks, and nolic extracts (EE) of both leaves and tubers were prepared, and decrease the rate of transmission to partners [3]. However, these their effect on cellular viability was evaluated. Cell viability assays synthetic nucleoside analogues are unable to suppress the latent were performed on uninfected Vero cells, treated with 3 different infection, and their wide use favors the selection of drug-resistant concentrations (100, 10, 1 µg/mL). Since extracts were dissolved HSV mutants. Genital herpes can be associated with resistance to in DMSO, control wells were prepared by treating cells with equal available drugs especially in immunocompromised patients as re- volumes of DMSO. As shown in ▶ Fig. 1 a, total toxicity was evi- sult of extended viral replication and the reduced elimination of dent at the highest concentration for both samples. On the other resistant strains [4]. Furthermore, acyclovir, the drug of choice hand, partial toxicity was observed at 10 µg/mL for the tuber EE, for treating HSV infections, is characterized by a short half-life while no toxic effect was detected for the leaf EE. Both extracts and incomplete absorption; therefore, it must be taken repeatedly exhibited no toxicity at the lowest tested concentration. Based This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. throughout the day [5]. These drawbacks of available drugs on these results, the EE of the leaves, which exhibit a lower toxic- prompt the discovery and development of novel anti-HSV-2 ity compared to the tubers extract, was further fractionated using agents acting with a different mechanism of action than acyclovir solvents of increasing polarity (n-hexane, chloroform, ethyl ace- and its derivatives, which are inhibitors of viral DNA replication, in tate, n-butanol). The extracts and the remaining aqueous fraction order to improve the efficacy and pharmacokinetics of actual (AE) were submitted to viability assays. As reported in ▶ Fig. 1 b, treatments. It is widely accepted that medicinal plants are rich the n-hexane (HE), ethyl acetate (EaE), and n-butanol (BE) extracts sources of alternative molecules with large structural diversity resulted highly toxic, whereas the chloroform extract (CE) and the and complexity for the treatment of infectious disorders. Natural AE did not have negative effects on cell viability at any tested con- products have been explored as HSV-2 inhibitors, which may be centrations. The nontoxic fractions from the leaves were selected useful for the development of new drugs and act through differ- to investigate their potential inhibitory activity against herpetic ent mechanisms of antiviral action [6, 7]. infections. Arisaema tortuosum (Wall.) Schott,