Abstract
Context: Thirty-one medicinal plant species from Hawaii, Morocco, and the Sonoran Desert, USA have been shown in past studies to be highly inhibitory to pathogenic bacteria, fungi, and certain cancer cell lines. However, none were tested for antiviral activity.
Objective: Acetone and methanol extracts from these species were bio-assayed for antiviral activity against herpes simplex virus types 1 and 2, and for cytotoxicity to the Vero C1008 cell line.
Materials and methods: Extracts from these species were tested in vitro for antiviral activity using an immunoperoxidase mini-plaque reduction assay to detect viral structural protein synthesis. A 50% inhibitory concentration (IC50) was computed. Sulforhodamine B and neutral red assays were used to qualitatively and quantitatively assess the cytotoxicity of extracts to C1008 cells, and to compute a 50% cytotoxic concentration (CC50) using a dose response curve.
Results: Eight of the 31 plant species assayed showed significant antiviral activity against HSV 1 and HSV 2 viruses. The acetone extract of Kalanchoe pinnata Pers. (Crassulaceae) produced an IC50 of 0.025 mg/mL and a CC50 of 1.25 mg/mL yielding a therapeutic index of 50. Additionally, this extract reduced plaque numbers to zero or near zero at a concentration of 0.1 mg/mL when added 30 min before or 30 min after virus infection.
Discussion and conclusion: The mechanism of inhibition against HSV 1 and HSV 2 viruses is now being investigated, along with fractionation of the acetone extract in search of the active compound or compounds.
Introduction
Natural products play an important role in the discovery of leads for the development of drugs to treat human diseases (CitationNewman & Cragg, 2007). Over 2,000 compounds extracted from higher plants are used in a medical setting throughout the world, and at least 46% of these have not been used in the USA (CitationFabricant & Farnsworth, 2001). Knowledge about the medicinal use of these species may be held individually, tribally, or documented in well established tomes (CitationNewman & Cragg, 2007). But often scientific information is lacking as to whether these plants contain anti-carcinogenic, antibiotic, antifungal, and antiviral properties (CitationCordell et al., 1991).
Researchers in the Natural Products Laboratory, Brigham Young University have collected and tested 157 medicinal plant species for activity against pathogenic microbial diseases, cancer cell lines, and for cytotoxicity (CitationDonaldson et al., 2004; CitationDonaldson & Cates, 2005). From these, 31 plant species (one Hawaiian plant, 15 from Morocco, 15 from North America) were selected that had inhibition levels at 70% or greater against one or more of the above organisms or cancer cell lines. However, none of these species have been tested for activity against human herpes simplex virus types 1 or 2 (HSV 1, HSV 2).
HSV 1 and HSV 2 are common human pathogens which cause several symptoms such as vesiculo-ulcerative lesions at mucocutaneous junctions, gingivostomatitis, keratitis, and encephalitis (CitationArduino & Porter, 2007). In immunocompetent hosts these clinical symptoms are often benign, but in immunocompromised patients they become severe, may be progressive, and require more time for healing (CitationWhitley, 2002). Life threatening herpes infections may be transmitted to neonates during delivery where a wide spectrum of clinical manifestations can occur including encephalitis. HSV may account for 10 to 20% of all viral encephalitis infections in the USA (CitationTang et al., 1999). Many medicinal plants may have a variety of chemical constituents known to inhibit the replication cycle of various types of DNA or RNA viruses (CitationSchang, 2006), including herpes simplex. As a consequence, attention has been given to the development of antiviral agents from traditional plant medicines (CitationJassim & Naji, 2003).
Our objective was to determine whether the selected 31 plant species would demonstrate significant activity against HSV 1 and HSV 2 viruses in vitro. Acetone and methanol extracts from these species were bio-assayed for activity against these viruses using the immunoperoxidase mini-plaque assay. Cytotoxicity was determined using a neutral red assay. A therapeutic index (TI), defined as the ratio of the cytotoxic concentration in 50% of the cell monolayers (CC50) to the inhibiting concentration of the extract that showed 50% antiviral effects on the cell monolayers (IC50) (CitationBurns, 1999), was computed for the four most inhibitory species.
Materials and methods
Plant tissue collection
Medicinal plant species were collected in Hawaii, Morocco, and in the Sonoran Desert, USA, between 2000 and 2004 (CitationDonaldson et al., 2005; CitationDonaldson & Cates, 2004). Samples from the USA were labeled and placed in plastic bags, frozen in coolers containing dry ice, and then shipped to the Natural Products Laboratory, Brigham Young University. These were stored in −80°C ultralow until analyzed. Samples from Morocco were air dried, ground to a fine powder using a Wiley Mill, and stored in labeled sample bags in boxes. Species analyzed in this study along with their collection number and traditional medicinal plant use are found in .
Table 1. Plant species*, family, collection number, and their medicinal use by indigenous peoples.
Tissue extraction and drug preparation
Air dried leaves, stems, or roots, 3 g, or 5 g of frozen tissue, were ground using a mortar and pestle in liquid nitrogen in 15 mL hexane and then filtered through cheese cloth and VWR grade 415 filter paper. The filtrate was collected in vials. This process was repeated twice using 10 mL and 5 mL, respectively. These were combined into one pre-weighed vial and taken to dryness using nitrogen gas. The remaining plant material was extracted in acetone using the same protocol as above. The 15, 10, and 5 mL aliquots were combined into a pre-weighed vial and dried using nitrogen gas. The remaining plant material was extracted with 70% methanol following the same protocol as above, combined into a pre-weighed vial, and dried using nitrogen gas.
Distilled water was added to the dried acetone or methanol extract vial to make an 8 mg/mL concentration. Each vial was vortexed and sonicated to homogenize the extract, filtered through 0.2 µm Minisart filter (Sartoirus Stedim Biotech, Aubagne, France), placed into 1.5 mL Ependorf tubes, and stored at −80°C until assayed.
Cell culture and viruses
African green monkey kidney cells (ATCC CRL-1586, sub-line C1008) were maintained at 37°C and 5% CO2 in Dulbecco modified eagle medium (DMEM, Sigma, St. Louis, MO) supplemented with 5% cosmic calf serum (HyClone, Logan, Utah), 10 µM HEPES buffer, and 50 µg/mL gentamicin (Sigma-Aldrich). Stocks of HSV 1 (McIntyre strain) and HSV 2 (strain 333) (CitationJensen & Johnson, 1994) which were stored at −80°C were used to determine the activity of the plant extracts.
Mini-plaque reduction assay
C1008 cell monolayers were used to determine if any of the plant extracts inhibited HSV 1 or HSV 2 infection in vitro (). Cells were seeded into 24-well plates, and when cell monolayers were 90% confluent the extract was introduced to each well. Three concentrations were used, and each concentration was replicated three times. The old media was removed and to the appropriate well was added 1 mL DMEM containing 6.25, 12.5, or 25 µL of plant extract resulting in concentrations of 0.05, 0.1, and 0.2 mg/mL, respectively. Control wells contained C1008 cells growing in l mL of medium without extracts and were randomly located. After 30 min 0.1 mL virus suspension was added to each well for each concentration at a multiplicity of infection of one (MOI = 1) and incubated at 30°C and 5% CO2 for 30 h (CitationTaniguchi & Yoshino, 1964).
Table 2. Percent inhibition by methanol and acetone plant extracts of plaque formation of HSV 1 and HSV 2 as measured by the immunoperoxidase assay.
A concern was that the plant extracts might prevent entry or destroy the virus particles. Consequently, C1008 cells were seeded into 24-well plates, incubated to 95% confluency, and then HSV 1 or HSV 2 were introduced to the monolayers 30 min before the extract was added. Viruses were added by removing the old media and adding 0.1 mL of virus suspension at MOI = 1 to test wells; 0.1 mL media was added to the control wells. Plates were incubated at 37°C and 5% CO2 for 30 min. Then 1.1 mL of media with extract in one of three concentrations (0.05, 0.1, and 0.2 mg/mL) were added and plates were incubated for 30 h.
Microscopic observations and plaque counts to measure extract inhibition
In vitro HSV 1 and HSV 2 infections and plant extracts that induced cytotoxicity might cause observable changes in C1008 cell monolayers. Consequently, microscopic observations were recorded to determine if HSV infections and/or plant extracts might adversely affect cell growth, and therefore indicate cytotoxicity. Plaques due to virus infection appeared as holes in the monolayer due to cell death and syncytia formation. In the case of K. pinnata, only foci (individual stained cells indicating the presence of virus structural proteins) were observed, and these were counted to determine percentage inhibition for this species. Observable cellular changes due to adding plant extracts were minimal, but rounding up of cells and changes in cell membrane shape was noted for extracts (especially at 0.2 mg/mL) (). However, none of the changes influenced quantification of the effects of viruses on cell monolayers.
Immunoperoxidase staining to determine plaque number in cell monolayers
Following CitationLucker et al. (1991), all wells were stained using an immunoperoxidase staining protocol to detect plaque and syncytia numbers or presence of foci. Each well was separated into quadrates and plaques were counted manually using an inverted phase contrast microscope (Nikon Eclipse TS100). Overlapping plaques were deemed individual when lobes were apparent (CitationZielinska et al., 2005). Plaque number was calculated as a mean of three replicates, and growth inhibition of infected cells was given as a percentage of the control (). If the established cell monolayer appeared disrupted, detached, or absent from the well after the extract was added and just before staining, the extract was assumed to be toxic to the C1008 cells. This was noted as “tx” in . The IC50 (50% inhibitory concentration of viral effect) was determined from the dose response curve (). Positive controls used in the IC50 determination were infected cell monolayers without extract.
Neutral red assay for cytotoxicity of active extracts
The four extracts confirmed to be inhibitory to HSV 1 and 2 by the mini-plaque assay were tested for cytotoxicity to C1008 cells using a neutral red (NR) assay (Cytotox NR Kit, Xenometrix, Allschwil, Switzerland). The assay is based on the ability of viable cells to incorporate and bind neutral red within lysosomes (CitationMotohashi et al., 2003). C1008 cells were treated with serial dilutions of each plant extract (0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/mL), and the concentrations were replicated three times. After 72 h cell viability was measured using a spectrometer (Fusion α-HT Universal Microplate Analyzer, Packard Instruments, Meriden, CT) with a 540 nm filter and a 690 nm reference filter. Results were determined from the dose-response curves and a CC50 (50% cytotoxic concentration) was reported () (CitationRajbhandari et al., 2009).
Table 3. Cytotoxic concentration (CC50), inhibition concentrations (IC50), and therapeutic index (TI) values for HSV 1 and HSV 2 infected C1008 cells monolayers in the presence of four plant extracts.
Virucidal assay
Antiviral screening assays must be viewed cautiously when apparent antiviral activity is observed until a determination is made that the extract is not destroying or disrupting the virus particle. To determine if the acetone extract of K. pinnata interfered with virus particle structure, the extract was allowed to incubate with the virus prior to exposure to the cell monolayer. The acetone extract of K. pinnata (0.2 mg/mL) was added to the thawed HSV 2 virus stock of 8 × 107 plaque forming units per mL (pfu/mL) and incubated at 37°C for 30 min. Six 10-fold serial dilutions of virus stock (10−1 to 10−6) with and without extract were added to cell monolayers in 24-well plates and incubated at 37°C and 5% CO2 for 36 h. Staining followed that of the mini-plaque assay described above. Plaque numbers were counted as described for the mini-plaque assay. The concentration of virus, in plaque forming units (pfu as a measure of the number of particles capable of forming plaques per unit of volume), was calculated for infected wells with and without extract exposure.
Data analysis
Cytotoxicity and mini-plaque assays were performed in triplicate in two independent experiments. The CC50 and IC50 values were calculated and a therapeutic index (TI, selective index; defined as CC50/IC50), was reported for the four most promising extracts (). Statistical comparisons were made using a one-sample t statistic (significant differences at P <0.05).
Results
Inhibition of extracts against HSV 1 and HSV 2 using the mini-plaque reduction assay
Eight of the 31 plant species assayed showed significant (P <0.05) antiviral activity against one or both of these viruses at one or more concentrations when compared to the control (). Also, percentage inhibition was not significantly different when cells were infected with viruses before or after the addition of the extracts. Consequently, the protocol of adding the virus before the extract was added was chosen for subsequent experiments because this modeled the natural order of infection.
Species were selected for further tests based on an extract being at least 60% inhibitory to HSV 1 or HSV 2 infection and showing no obvious cytotoxicity at any concentration based on microscopic observations (). This group included the acetone extracts of Atractylis macrophylla Desf. (Asteraceae) and Hymenoclea salsola Torr. & A. Gray (Asteraceae), and the methanol and acetone extracts of K. pinnata (). The other extracts showing significant inhibition (Clematis cirrhosa L. (Ranunculaceae), Gnaphalium chilense Spreng. (Asteraceae), Lithospermum officinale L. (Boraginaceae), Psilostrophe cooperi Greene (Asteraceae), and Tetraclinis articulata Mast. (Cupressaceae) were not pursued due to cytotoxicity (indicated by “tx” in ).
Cytotoxicity of extracts on C1008 cells
Upon initial contact with any of the four extracts at 0.2 mg/mL, changes in the shape of some cell membranes were observed. Since these observations may indicate cytotoxic effects, cytotoxicity of these four extracts was determined using the neutral red assay. Based on CC50 values, K. pinnata was two to five times less toxic than the extracts of A. macrophylla and H. salsola (). The CC50 for the acetone and methanol extracts of K. pinnata were 1.25 ± 0.123 mg/mL (mean ± SE) and 0.95 ± 0.15 mg/mL, respectively. This indicated that the acetone-derived extract was less toxic to the C1008 cells than the methanol extract. Additionally, K. pinnata extracts had a 50% cytotoxic concentration up to 50 times higher than the concentration necessary for 50% virus inhibition as indicated by the TI value ().
Virucidal activity of the acetone extract of K. pinnata
The virucidal assay for the acetone extract of K. pinnata showed that titers of surviving virus were 8.2 × 107 pfu/mL for the control culture and 8.3 × 107 pfu/mL for the extract-exposed cultures. These data indicate that the acetone extract was not directly interfering with the virus particles before their attachment and entry into the C1008 cells.
Discussion
For all eight of the 31 species that showed antiviral activity, the acetone fractions showed greater inhibition than the methanol extracts (). If the acetone extract was toxic to the cell monolayer, the methanol extract from the same species displayed similar toxicity regardless of whether the virus was added before or after the extract. In addition, virucidal assays indicated that extracts were not inhibiting entry of the virus or destroying virus particles. Extracts of the remaining 23 species did not protect C1008 cells from HSV induced cytopathic effects or they were toxic to the cells. Of the eight species that originally showed activity for at least one of the extracts, five (C. cirrhosa, G. chilense, L. officinale, P. cooperi and T. articulata) showed high cytotoxicity at extract concentrations less than 0.1 mg/mL. These observations, and previous research identifying specific organic compounds responsible for the cytotoxicity of some of the species, lowered their priority for further testing (CitationHerz et al., 1970).
Previous studies identified specific compounds in H. salsola and their adverse effects on cellular activities (CitationTorrance et al., 2006). The neutral red assay in this study confirmed the low CC50 (0.28 mg/mL) for H. salsola indicating that a low concentration of extract was cytotoxic to the cell monolayer. Similarly, the acetone extract of A. macrophylla was found to be cytotoxic to C1008 cells at a low concentration (0.25 mg/mL). Alternatively, the acetone extract (CC50 of 1.25 mg/mL) and the methanol extract (CC50 of 0.95 mg/mL) of K. pinnata showed a higher CC50 indicating a higher concentration of extract was needed to bring about a similar level of cytotoxicity. Furthermore, K. pinnata TI values of 50 (acetone extract for both HSV 1 and HSV 2) and 19 (methanol extract for HSV 1) indicated significant antiviral activity at lower cytotoxic concentrations.
Conclusion
Of the 31 species tested, only the acetone and methanol extracts of K. pinnata were not cytotoxic to host cells. The acetone extract from the roots of K. pinnata revealed plaque numbers near zero at concentrations less than 0.1 mg/mL. Due to significant antiviral activity against HSV 1 and HSV 2 and low cytotoxicity to C1008 cells in vitro, the acetone extract was selected for additional studies. Studies underway include fractionation of the extract in search of an active compound or compounds, and investigation of the molecular mechanisms involved in the antiviral properties of the acetone extract of K. pinnata.
Acknowledgements
We thank Jack Donaldson, Brandon Odum, Brad Prestwich and Analiesa Leonhardt for help with this project.
Declaration of Interest
The authors are grateful to the Wesley T. Johnson Memorial Virus Research Fund, the Professional Development Fund in the Department of Biology at Brigham Young University and the Public Education Job Enhancement Committee, in association with the Governor’s Office of the State of Utah, for financial support of this work.
References
- Arduino PG, Porter SR (2007): Herpes simplex virus type 1 infection: Overview on relevant clinico-pathological features. J Oral Pathol Med 37: 107–121.
- Bean LJ, Saubel KSS (1972): Temalpaki [From the earth]: Cahuilla Indian Knowledge and Usuage of Plants. Banning, CA, Malki Museum Press.
- Burns M (1999): Management of narrow therapeutic index drugs. J Thrombosis Thrombolysis 7:137–143.
- Chopra RN, Nayar SL, Chopra IC (1980): Glossary of Indian Medicinal Plants. New Delhi, Council of Scientific and Industrial Research.
- Cordell GA, Beecher CWW, Pezzuto JM (1991): Can ethnopharmacology contribute to the development of new anticancer drugs? J Ethnopharmacol 32: 117–133.
- Donaldson JR, Cates RG (2004): Screening for anticancer agents from Sonoran Desert plants: A chemical ecology approach. Pharm Biol 42: 478–487.
- Donaldson JR, Warner SL, Cates RG, Young DG (2005): Assessment of antimicrobial activity of fourteen essential oils when using dilution and diffusion methods. Pharm Biol 43: 687–695.
- Epple AO (1995): A Field Guide to the Plants of Arizona. Guilford, CT, Globe Pequot Press.
- Fabricant DS, Farnsworth NR (2001): The value of plants used in traditional medicine for drug discovery. Environ Health Perspec 109: 69–75.
- Ghanzanfar SA. (1994): Handbook of Arabian Medicinal Plants. Boca Raton, FL, CRC Press.
- Herz W, Aota K, Holub M, Samek Z (1970): Sesquiterpene lactones and lactone glycosides from Hymenoxys species. J Org Chem 35: 2611–2624.
- Hocking GM (1956): Some plant materials used medicinally and otherwise by Navaho Indians in the Chaco Canyon, New Mexico. Palacio 56: 146–165.
- Hoffman J, Timmermann B, McLaughlin S, Punnapayak H (1993): Potential antimicrobial activity of plants from the southwestern United States. Int J Pharmacog 31: 101–115.
- Jassim SAA, Naji MA (2003): Novel antiviral agents: A medicinal plant perspective. J Appl Microbiol 95: 412–427.
- Jensen C, Johnson FB (1994): Comparison of various transport media for viability maintenance of herpes simplex virus, respiratory syncytial virus and adenovirus. Diag Microbiol Infect Disease 19: 137–142.
- Johnson T (1999). CRC Ethnobotany Desk Reference. Boca Raton, FL, CRC Press.
- Lucker G, Chow C, Richards DF, Johnson FB (1991): Suitability of infection of cells in suspension for detection of herpes simplex virus. J Clin Microbiol 29: 1554–1557.
- Moerman DE (1989). Native American Ethnobotany. Portland, OR, Timber Press.
- Moore M (1995): Herbal/Medical Contraindications. Bisbee, AZ, Southwest School of Medicine.
- Moore M (1989) Medicinal Plants of the Desert and Canyon West: A Guide to Indentifying, Preparing, and Using Traditional Medicinal Plants Found in the Deserts and Canyons of the West and Southwest. Santa Fe, NM, Museum of New Mexico Press.
- Motohashi N, Wakabayashi H, Kurihara T (2003): Cytotoxic and multidrug resistance reversal activity of a vegetable, “Anastasia Red”, a variety of sweet pepper. Phytother Res 17: 348–352.
- Muzitano MF, Tinoco LW, Guetter C, Kaiser CR, Rossi-Bergmann B, Costa SS (2006): The antileishmanial activity assessment of unusual flavonoids from Kalanchoe pinnata. Phytochemistry 67: 2071–2077.
- Newman DJ, Cragg GM (2007): Natural products as sources of new drugs over the last 25 years. J Nat Prod 70: 461–477.
- Palmese MT, Manganelli REU, Tomei PE (2001): An ethno-pharmacobotanical survey in the Sarrabus district (south-east Sardinia). Fitoterapia 72: 619–643.
- Rajbhandari M, Mentel R, Jha PK, Chaudhary RP, Bhattarai S, Gewali MB, Karmacharya N, Hipper M, Lindequist U (2009): Antiviral activity of some plants used in Nepalese traditional medicine. Evid Based Complement Alternat Med 6: 517–522.
- Schang LM (2006): Herpes simplex viruses in antiviral drug discovery. Current Pharm Des 12: 1357–1137.
- Tang YW, Mitchell PS, Espy MJ (1999): Molecular diagnosis of herpes simplex virus infections in the central nervous system. J Clin Microbiol 37: 2127–2136.
- Taniguchi S, Yoshino K (1964): An analysis of the plaque assay of herpes simplex virus in chick embryo monolayers. Arch Virol 14: 537–552.
- Torrance SJ, Wiedhopf RM, Cole JR (2006): Ambrosin, tumor inhibitory agent from Hymenoclea salsola (Asteraceae). J Pharm Sci 64: 887–888.
- Whitley RJ (2002). Herpes simplex viruses, in: Fields BN, Knipe DM, Howley PM, eds, Fields Virology. Philadelphia, PA, Lippincott-Williams & Wilkins, pp. 2461-2498.
- Zielinska E, Liu D, Wu H, Quiroz J, Rappaport R, Yang D (2005): Development of an improved microneutralization assay for respiratory syncytial virus by automated plaque counting using imaging analysis. Virol J (online). Available at: http://www.pubmedcentral.nih.gov/doi/10.1186/1743-422X-2-84, (Accessed on 20 January 2006).