minerva medica copyright

REVIEWS MINERVA BIOTEC 2012;24:117-21

In vitro evaluation of the biological activity of Lebanese medicinal plants extracts against herpes simplex virus type 1

IN C ER O V P A Y R M IG E H DI T C ® A

A. M. SAAB 1, I. LAMPRONTI 2, A. FINOTTI 2, M. BORGATTI 2, R. GAMBARI F. ESSEILY 3, S. SAFI 4, M. DIAB-ASSAF 1, H. RABENAU 5, J. CINATL 5, H. W. DOERR

Medicinal plants extracts are interesting novel drugs for use as antimicrobial and antiviral agents. In this study we investigate the in vitro antiviral activity of eight ethanol medicinal plant extracts against Herpes simplex virus (HSV-1) infection on monkey kidney cells. Acyclovir, an antiviral agent currently applied for treatment of herpes virus type 1 infection, was used to compare the plant extracts therapeutic activity. The inhibitory concentrations (IC50) were determined for eight medicinal plants extracts obtained from the following plants: Calamintha origanifolia, Satureja thymbra, Prangos aspurela, Sidiritis Perfoliata, Aspurela glomerata, Erythreae Centaurium, Hyssopus officinalis and Salvia accetabulosa. Cytotoxicity was evaluated by MTT assay in Vero cells. The selective index (SI) of these medicinal plant extracts was used to prove the therapeutic activity. We found that C. origanifolia and S.thymbra extracts have the highest selective index (SI) in our data and are therefore potentially be used for treatment of HSV-1 disease. Key words: Herpes simplex - Plants, medicinal - Antiviral agents - Acyclovir.

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ebanon has an area of 10425 sq km, despite its relative small size; it still harbors a wide variety of ecosystems and landscapes and is regarded as a floristically rich country with an estimated 2600 plant species.1 Medicinal plants are sold in small herbalist stores under different names like “Dabbous”, “Aashab” or “others”. Herbalists, according to their experience concerning the biological activity of each plant, usually prescribes the herb needed for specific Corresponding author: Dr. A. M. Saab, Department of Chemistry, Faculty of Sciences II, Lebanese University, Lebanon. E-mail: [email protected]

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1Department

of Chemistry, Faculty of Sciences II Lebanese University, Lebanon 2Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy 3Faculty of Public Health II, Lebanese University, Lebanon 4Department of Biology, Faculty of Sciences II Lebanese University, Lebanon 5Institute for Medical Virology University Hospital of the Johann Wolfgang GoetheUniversity, Frankfurt, Germany

purposes.2 More than a hundred of plants are endemic and reported to have biological activities. Some of them have been used and are still used by people as folk medicine. Discovery of biological activities in medicinal plants, found in different countries in the world, helps the agriculture and pharmaceutical institutes to progress towards achieving better economic and social benefits.3 Calamintha origanifolia Vis is grown on the slope of Sanin Mountain, at the altitude of 2000 m above the sea in Lebanon, and belongs to Laminaceae family. It is rich in essential oils and its extract reduces blood pressure and also, possesses anti-fungal, diaphoritic and expectorant activity.4-8 Saturea thymbra L is grown in several regions in Lebanon. It belongs to Laminaceae family. Known to be rich in essential oils and extract, it reduces blood pressure, possesses antimicrobial anti inflammatory and antinociceptive activities.5-9 Prangos aspurela boiss is quite an interesting medicinal plant, for agriculture and pharmaceutical institutes, due to its higher oils yield production and the valuable use of traditional medicine. It grows in several slopes of “Mont-Liban”, and belongs to the Apiaceae

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herpes infection. Acyclovir (ACV) and other nucleoside derivatives, penciclovir, cidofovir, famciclovir, valaciclovir and granciclovir have been approved for treatment of HSV-1 infections worldwide.17 However, ACV-Resistant strains of HSV-1 and drug toxicity have been recently reported.5-8, 18, 19 The aim of the present study was to investigate the in vitro inhibition of the herpes virus HSV-1 infection by eight ethanol plants extracts derived from areal parts of flowers and leaves. Plant material


family. It reduces blood pressure, cures skin disease, digestive disorder, hemorrhoids and possesses anti microbial activity.5-8, 10 Sideritis perfoliata L. is grown in several sites in Lebanon, especially in Mont-Liban near Kfrakaab town. It belongs to the Laminaceae family. It is rich in essential oils and is known to reduce blood pressure, and joint pain. In addition, it possesses anti oxidant, anti-inflammatory and anti microbial activities, decongestant of the respiratory tract, astringent property.5-8, 11 Asperula glomerata (M. Bieb) Griseb belongs to the Rubiaceae family and is grown on the highest slopes of ‘Sanin’ mountain at the altitude of 2000 m above the sea. It is also recognized for its reduction in blood pressure, inflammation and edema.5-8 Hyssopus officinalis L is grown in several sites of Lebanon and belongs to the Labiateae family. It is rich in essential oils and extract. Known to reduce blood sugar, blood pressure, spasm in the smooth muscle of the gastrointestinal tract and possess anti microbial and anti oxydant activities.5-8, 12 Centaurium erythreae L is quite an interesting plant for inflammatory therapeutic use in folk medicine. Found near Kfarkaab town in Mont-Liban, it belongs to the Gentianaceae family. It reduces spasm in the gastrointestinal tract, fever, inflammation and edema. Also cures digestive and stomach disorders.5-8, 13 Salvia acetabulosa L, is an aromatic plant, grown in several sites in Lebanon and especially in “Dair Al Ahmar” village. It belongs to Laminaceae family. It reduces blood sugar and blood pressure. Possess anti oxidant, antimicrobial, eupeptic, antihydrotic effects and also known to reduce pain in joints, anxiety and depression.5-8, 14 In this paper, we performed anti-HSV-1 extracts from these herbal medicines native from Lebanon. Herpes simplex viruses (HSV-1) are important pathogens for humans, especially in the case of highly susceptible adults. Therefore, the discovery of novel anti-HSV-1 drugs deserves great efforts. Herpes simplex virus (HSV-1) is a single large double-standard DNA virus that is widespread in humans. It is responsible for a broad range of diseases causes several clinical conditions such as keratitis, cutaneous herpes, genital herpes and encephalitis.15 Severe forms of disseminated HSV-1 infection are often seen in patients with reduced cell mediated immunity as in bone marrow transplant recipients or patients with immunodeficiency syndrome AIDS.16 After the primary infection, HSV-1 tends to persist in the neuron of the ganglia. Reactivation of latent HSV, which is very common during the deficiency of immunity, causes recurrent

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IN VITRO EVALUATION LEBANESE MEDICINAL PLANTS EXTRACTS IN HSV 1

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Calamintha origanifolia Vis, Satureja thymbra L, Prangos aspurela Boiss, Sideritis perfoliata L, Aspurela glomerata, Hyssopus officinalis L, Centaurium Erythreae L and Salvia acetabulosa L were collected from several site locations in Lebanon during July and August 2007. These selected medicinal plants were recommended by several herbalists in Lebanon and authenticated by Prof. S.Safi from the biology department, Faculty of Sciences II and also by Prof. P. Malichef, a well known herbalist and pharmacist-ecologist in Lebanon. A voucher specimen of each species was deposited in the herbarium, Faculty of Sciences II, Lebanese University. The dried plant materials were ground and stored in brown glass bottle until extraction at 25 °C. Preparation of plant extracts

The dried and ground sample (1 g) was extracted with 10 ml of ethanol by maceration using vortexing for 30s and sonication for 1 min. After centrifugation at 3000 rpm for 20 minutes, the material was transferred into 50 ml round bottom flask and then the plant extracts were obtained with rotary vacuum evaporator. This extraction was performed three times.20 The percent yields (w/w) ranged from 10.4% to 15.7% for ethanol extracts plants (Table I). Cell culture

Monkey kidney cell line (Vero) (ATCC:CCL 81, Manassas, VA) were culture at 37 oC in 5% CO2 atmosphere in Eagle’s Minimum Essential Medium (MEM), Supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 100 U/mL peniciline, and


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Table I.—List of Lebanese extract plants with their sites locations and the percent yield (w/w). Latin name

Family name

Locations

Laminaceae Laminacea Apiaceae Laminaceae Rubiacea Lamiaceae Gentianaceae Laminaceae

Sanin Sanin Baskinta Kfarkaab Sanin Kfarkaab Kfarkaab Dair-ElAhmar

Material Studied

% yield w/w

Voucher SpecimenNo.

Leaves and flowers Leaves and flowers Leaves and seeds Leaves and flowers Leaves Leaves and flowers Leaves and flowers Leaves and flowers

12.4±0.7 14.6±0.9 15.7±1.2 10.4±0.5 13.6±0.6 12.4±0.7 14.5±0.8 11.5±0.4

1215 1217 1225 1230 1245 1250 1265 1275

Altitude

2000 2000 1700 1200 2000 1200 1200 1000

m m m m m m m m


Calamintha origanifolia vis Satureja thymbra L Prangos aspurela L Sideritis perfoliata L Asperula glomerata Hyssopus officinalis L Centurium erythreae L Salvia acetabulosa L

100 mg/mL streptomycin. Cells were used for experiments after 5 days at 100% confluence. Viruses

HSV-1 laboratory strain Maclntyre (F Strain ATCCVR733) was used for the in vitro experiments. The virus identities were confirmed by indirect immunofluorescence staining (Monoclonal primary and secondary antibodies purchased from Syva, Palo Alto, CA). The viruses were propagated in Vero cells as described previously,21 and virus stocks were quantified as 50% tissue culture infective doses (TCID50) by endpoint dilution, with the infectious titer determined by method of Reed and Muench. Cytotoxicity assay

Cytotoxicity of extracts towards Vero cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction. Briefly, cells in exponential growth phase were seeded in 96-well plates. After cell reached confluence, 50 mL medium per well containing various concentrations of the test compounds, or control medium without drug, were added and incubated (37 °C, 5% CO2). The fourth day of treatment, a 2% MTT solution was added to each well for 4 hours. After incubation with MTT solution cells became lysed using sodium dodecyl sulfate. The absorbance at 570 nm was determined with a multi-well ELISA reader. The results were expressed as the percentage of metabolizing (viable) cells relative to control cultures incubated without tested samples (Tissue Culture 50, TC50) value represent the sample concentrations that reduced the cell growth by 50%.

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Antiviral assay

Antiviral action against HSV replication of the plants extracts and ACV- used as positive control was tested as follows. Tested extracts were solved in DMSO. Working solutions were prepared freshly before their use. Vero cells were seeded in 96-well plates. For virus adsorption, confluent Vero cells were infected with HSV-1 at multiplicity of infection (MOI) of 0.01 Virus and different concentrations of the tested compounds or ACV were added at the same time in MEM supplemented with 2% FBS. For each dilution step, 8 wells were used in parallel. Virus infection was assessed by visually scoring the virus induced CEP 72 hours post infection. The effective concentration inhibiting 50% of virus growth (IC50) was determined as concentration of compounds required to inhibit the CPE effect to 50% of the control value. The Selective Index SI was calculated as quotient of TC50/IC50. Results and conclusion

Medicinal plants have been used for treatment of many infectious diseases including bacterial and viral infection. The utilization of medicinal plant extracts as drugs depends on their biological activities. During recent years, scientists have tried to increase the number of anti-viral drugs. Most of these substances belong to the class of nucleoside example acyclovir and ribavirin. In our present investigation we report the antiviral activity against HSV-1 of eight medicinal plants extracts which were obtained with ethanol solvent by maceration. Our results proved that medicinal plant extract of Calamintha origanifolia vis exhibited (IC50) value of 0.233mg/mL and selective index (SI) superior of 4,29. On the other


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Table II.—Anti viral activity drugs testing (mg/mL) of Lebanese plant extracts against herpes simplex virus type 1 infection in vitro. Plant extracts

TC50 (mg/mL)

C.origanifolia S.thymbra P. aspurela S. Perforiata A.glomerata H. officinalis E.centaurium S. acetabulosa Acyclovir

IC50 (mg/mL)

>1 1 >1 1 >1 >1 >1 1 >100µM

SI

0.233 0.22 0.66 0.41 0.69 0.373 0.29 0.27 3.77µM

4.29 4.55 >1.5 2.44 >1.45 2.68 3.45 3.7 26.5


IC5: concentration required to inhibit 50% of virus growth; TC50: drug concentration that reduces the cell growth 50% (cellular toxicity); SI: selective index (TC50/IC50)

hand, Satureja thymbra L exhibited (IC50) of 0.25 mg/mL and (SI) of 4.55. Moreover Salvia accetabulosa L exhibited (IC50) of 0.27mg/mL with (SI) superior of 3.7. In other side, Centaurium erythreae L exhibited (IC50) of 0.29 mg/mL with (SI) superior of 3.5. Hyssopus officinalis L exhibit (IC50) of 0.373mg/ mL with (SI) superior of 2.68. Sidiritis perforiata L exhibit (IC50) of 0.41 mg/mL with (SI) of 2.44. Asperula glomerata exhibit (IC50) of 0.69 mg/mL with (SI) of 1.45. Finally, Prangos aspurela Boiss exhibit (IC50) of 0.66 mg/mL and (SI) superior of 1.5 (Table II). In our previously antiviral work, Laurus nobilis seeds proved good inhibitory activity against HSV1, while Cedrus libani Leaves, Juniperus oxycedrus seeds, Thuya oriontalis seeds, Salvia officinalis leaves, Pistacia palestinea seeds, Cupressus simpervirens pyramidalis leaves presented anti viral activities against Herpes Simplex Type 1(HSV) similar to our present investigation.5-8 In other side relating to anti viral activities against HSV-1, Calamintha origanifolia Vis possesses anti-proliferative activity and induction of erythroid differentiation in vitro on human erythroleukemic K562 cells 22, 23 and proved a good inhibitory activity against digestive enzymes (α-Amylase and α-glycosidase) related to diabetes.5-8 Moreover, Satureja thymbra L proved activities against C32 and ACHN cells lines, used as in vitro Human Tumor Models 24 beside antiproliferative activity and induction of erythroid differentiation on human erythroleukemic K562 cells.22 Salvia accetabulosa exhibited antiproliferative activities against human tumor cell in vitro models which are COR-L23, Caco-2, A549, A375, C32 and ACHN.25 Beside inhibitory activities against angeotensin

conversting enzyme (ACE) and digestive enzymes (α-Amylase and α-glycosidase) related to diabetes.5-8 Prangos aspurela proved anti proliferative activity in human renal adenocarcinoma and amelanotic melanoma cells.5-8 Sidiritis perforiata L possesses activities against C32 and ACHN cell lines.24 Conclusions

In conclusion, the eight ethanol plants extracts possess antiviral activity against Herpes Simplex Type 1 (HSV) which their selective index (SI) range between 1, 5 and 4, 5 beside their previously anti cancer activities. We utilized the ethanol extract, because this solvent is highly polar and can contain extract terpenes, alkaloids, fatty acids and other compounds. In future this study merits testing the activity of the other two different extracts (butanol and ethylacetate) and other investigation on antiviral activity against HSV by isolation and purification of pure compounds from these medicinal plants extracts which contain antiviral drugs.

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Received on February 15, 2012. Accepted for publication on June 15, 2012.

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