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Anti-HSV and Cytotoxicity Properties of Three Different Nanoparticles Derived from an Indian Medicinal Plants

K. VASANTHI, G. REENA, G. SATHYANARAYANAN, and ELANCHEZHIYAN MANICKAN

Department of Microbiology, Dr. ALM PG1BMS, University of Madras, Taramani, Chennai, Tamil Nadu, India

ABSTRACT

Currently, the medical field kept their step in nanotechnology which employs the nanoparticles for treating the diseases. The study highlighted the use of nanotechnology in the virus aqueous extracts from the medicinal plants and their effect against herpes virus. Aqueous extract of Ptmica granatum (P. granatum) (Peels, Juice), Camellia sinensis (C. sinensis), Nilavembu Kudineer Chooranam (NKC), and Acalypha indica (A. indica). Three different nanoparticles (NP) such as silver, gold, and bimetallic were synthesized from the above aqueous extracts and characterized. Anti-HSV (both 1 and 2) activities of these nanoparticles were done by CPE assay. Toxicity of the extracts was determined by MTT assay. Among the tested nanoparticles P granatum peels silver NP (PgPSNP) exhibiting a potent anti-HSV activity followed by P granatum juice silver NP > C. sinensis Silver NP > NKC Silver NP > A. indica silver NP. Neither bimetallic nor gold NP exhibited significant anti-HSV activity. Except P. granatum other extracts showed more toxicity. This study indicated that P granatum peels silver NP (PgPSNP) showed the maximum anti-HSV activity besides its minimal toxicity observed. Thus, PgPSNP is a novel anti-HSV drug which is worth pursuing.

INTRODUCTION

Herpes simplex viruses (both types, HSV-1 and -2) are pathogenic to humans with a worldwide morbidity. HSV has a capacity to hide into our neurons and can reactivate, causing frequent recurrent infections in some patients, while most people experience few recurrences. Infections with HSV-1 and HSV-2 are highly prevalent. HSVs infected more than 3.7 billion people under the age of 50-60. The WHO estimated that over 500 million people are infected with HSV-2 worldwide with approximately 20 million cases annually [3]. Also, the global rates of either HSV-1 or HSV-2 are between 60% and 5% in adults. In India, the seropositivity has been reported to be 33.3% for HSV-1 and 16.6% for HSV-2 [3].

At present, there is no complete cure for this virus. Treatment focuses on getting rid of sores and limiting outbreaks. The medication includes acyclovir (ACV), famciclovir, valacyclovir, and these can help infected individuals reduce the risk of spreading the virus to others. Chronic use of anti-herpes virus drugs results in severe side effects and drug-resistant viruses. Use of ACV is unsuitable for pregnant women and neonates because of incorporation of drug into the host DNA and yields adverse effects. Also, effective vaccines against HSV infection are not yet identified. Furthermore, the available therapeutic vaccines does not protect the patients from recurrences because failure to stimulate the antibody-specific responses against HSV virus. Therefore, there is an urgent requirement of alternative agents to cure and prevent the HSV infection also these agents should be cheap, readily available, and less toxic.

Natural therapy is an alternative to allopathic medication which exploits the least side effects. Currently, there was an unlimited plants and herbs resources, therefore their phyto-chemicals were useful in concerned. Wide range of global population also prefers the use of natural products in treating and preventing the medical problems. But there is a few number of studies have used known purified plant chemicals, very few screening programs have been initiated on crude plant materials. Also, the drug delivery into the human body is very poor while using the plants as choice of drug [5].

Nanotechnology is an emerging area in pharmaceutical and medical field. Physicochemical and biological properties of materials would be vaiying fundamentally from their bulk part at their nanometric scale; this is due to the size-dependent quantum effect. The nanoparticles such as gold and carbon are surface-functionalized, have unique dimensions, and controlled drug release, thus can be used in the drug delivery [20-22]. On this basis the present study was highlighted the use of nanotechnology in the synthesis of drug from the medicinal plants and their effect against herpes virus.

MATERIALS AND METHODS

13.2.1 PLANT MATERIALS

The plants used for this study namely: Ptmica granatum (P. granatum), Camellia sinensis (C. sinensis), Nilavembu Kudineer Chooranaum (NKC), and Acalypha indica (A. indica) were collected from Chennai and some were purchased commercially from the stores (Figure 13.1). All these plants were taxonomically identified by the Department of Botany, University of Madras. Peels of P granatum and leaves of the A. indica were shade dried, ground into a uniform powder using a blender, and stored at 4°C. Fruits of the P granatum were grinded and the juice was lyophilized. Leaves of Camellia sinensis were purchased from commercial source available at Chennai. Dried leaves were ground into a uniform powder using a blender and stored at 4°C in refrigerator. NKC was purchased from the TAMPCOL and the dried leaves were ground into a uniform powder using a blender and stored at 4°C in refrigerator.

13.2.2 PREPARATION OF AQUEOUS EXTRACT

About 10 grams of the dried C. sinensis, NKC, and Peels of P granatum, and A. indica powder was soaked in 100 ml of distilled water overnight at room temperature. It was then cotton filtered to remove the coarse particles and then filtered through Whatman No. 1 filter paper. Then the extracts was passed through 0.45 and 0.2 micron membrane filter and the water content of extract is removed by lyophilization and stored at 4°C until use. Juice of the P granatum were filter by Whatman filter followed by 0.45 pm and 0.2 pm, lyophilized, and stored at 4°C until use.

13.2.3 SYNTHESIS OF NANOPARTICLES FROM THE EXTRACTS

Synthesis of gold nanoparticles was done as described previously by Tiwari (2011). Briefly, lyophilized powder of Plant extracts were reconstituted with 1 ml of sterile distilled water and mixed with 0.002 M of chlororauric acid (SRL Cat. No) (HAuC14) in dark conditions with a preincubation at 90°C. After incubation the color of the solution were turned its color to ruby pink (Figure 13.2) indicates the gold nanoparticle formation. According to Klaus (2001) synthesis of silver Nanoparticles was done. Briefly, lyophilized powder of plant extracts were reconstituted with 1 ml of sterile distilled water and mixed with 20 ml of 1СГ3 M AgN03 (SRL Cat. No: Cat. No for (HAuC14)- 12023, Cat. No for (AgN03) - 94118) (99.99%) aqueous solution and kept at room temperature. After 1 hour the color of the solution were changed from colorless to honey brown (Figure 13.2) indicating the formation of silver nanoparticles and this is confirmed by UV-visible spectroscopy and other methods. Synthesis of bimetallic nanoparticles (Silver-Gold) were done according to the Pal et al. Briefly, lyophilized powder of plant extracts were reconstituted with 1 ml of sterile distilled water and mixed with equal amount of КГ3 M AgN03 and 0.002 M of chlororauric acid and incubated at room temperature. After incubation the color of the solution were turned its color in the combination of ruby pink and honey comb color (Figure 13.2).

Plants used in this study and methodology

FIGURE 13.1 Plants used in this study and methodology.

Synthesis of three different nanoparticles from medicinal plants, nanoparticles of (a) NKC, (b) P. granatum peels, (с) C. sinensis, (d) P. granatum juice, (e) A. indica

FIGURE 13.2 Synthesis of three different nanoparticles from medicinal plants, nanoparticles of (a) NKC, (b) P. granatum peels, (с) C. sinensis, (d) P. granatum juice, (e) A. indica.

CHARACTERIZATION OF NANOPARTICLES

The newly synthesized nanomaterials was characterized using ultra violet visible (UV-Vis) spectrophotometer, high resolution scanning electron microscopy (HR-SEM), high resolution transmission electron microscopy (HR-ТЕМ), FTIR spectra. X-ray diffraction (XRD) patterns (Figure 13.2).

13.3.1 IN VITRO STABILITY TEST

In vitro stability studies were performed with aqueous solutions of nanoparticles (0.5 ml) and 0.5 ml of 10% NaCl, 0.2 M cysteine, 0.2 M histidine, 0.5% HSA and 0.5% BSA solutions. The stability of nanoparticles will also investigated in phosphate buffer at pH 4, 5, 6, 8, 9, and 10 respectively. The stability of the nanoparticles will be measured by using a UV-Vis spectrum after 24 hrs and 360 hrs.

  • 13.3.2 IN VITRO ANTI-HSV TESTING
  • 1. Cell Culture and Virus: African green monkey kidney (Vero) cells were grown in DMEM (Sigma) supplemented with 10% heat- inactivated Fetal bovine serum (Gibco BRL Co., Germany) and 1% antibiotics (Penicillin [100 IU/ml] and Streptomycin, [100 pi/ ml] Himedia) at 37°C in a humidified atmosphere of 5% COv Wild- type HSV-1 (753166) and HSV-2 (753167) strains obtained from Dr. S. Rajarajan (Department of Microbiology and Biotechnology, Presidency College, Chennai). Vims stocks were propagated in Vero cells and titer was calculated from plaque numbers (15 x lOVrnl) for HSV-1 and PFU (20 x lOVml) for HSV-2 according to the method of Reed and Muench (1938).
  • 2. Standard Antivirals: ACV (Sigma) was used as standard antiviral drug (1 mg/ml) dissolved in PBS and was serially diluted with respect to test compounds at a concentration of 500 pg/ml (1:2) to
  • 3.9 pg/ml (1:256).
  • 13.3.3 CYTOPATIC EFFECT ASSAY (CPE ASSAY)

Vero cells were plated on 96-well plate, at a density of 10,000 cells/well for 70-80% confluency. Cells was infected with HSV-1 and HSV-2 and incubated for 90 min at 37°C. Varying concentrations of the different nanoparticles were added to the virus infected 96 well plates and incubate for 7 days at 37°C in 5% CO, environment. Cytopathic effects (CPE) and their reduction were observed for 7 days (Figure 13.3). Cell control and Virus control, Drug control were put along with the assay.

13.3.4 MTT ASSAYS TO MEASURE THE CYTOTOXICITY OF EXTRACT

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was used for the screening of extracts toxicity using standard protocol. Briefly, verocells were incubated at 37°C on 96-well plates at a density of 10,000 cells/ well, with 5% CO, in a humidified atmosphere with 10% Dulbecco’s Modified Essentials Medium. After 24 hours, nanoparticles were added on monolayer (70-80% confluency) to final concentrations of 50 pg to 10 mg. The plate was incubated for further 5 day under the same conditions mentioned above. 200 pi MTT (Sigma-Aldrich, Catalogue No. M2003) solutions (5 mg/ml in phosphate buffer) was added to each well and incubated at 37°C for 4 hours. The MTT solution was decanted off, and Formosan was extracted from the cells with 250 pi of DMSO in each well. Color was measured with a 12-well ELISA plate reader at 550 mn (Figure 13.4). Toxicity Control used as 1% Triton X-100 (Qualigens, catalog No. 10655). All MTT assays were repeated three times. Cell viability was calculated using the following formula:

Characterization of nanoparticles,

FIGURE 13.3 Characterization of nanoparticles, (a) UV-Vis reading of P granatum,(b) ТЕМ image of P. grcmatum peels of silver Np’s,(c) SEM image of P. granatum peels silver Np’s,(d) XRJD of P. granatum peels silver Np’s, (e) FTIR spectra of P granatum peel synthesized silver nanoparticles and aqueous extract of P. granatum peel.

Antiviral assay, (a) CPE ofVero cell line, (b) reduction in CPE by P. granatum peels silver Np’s, (c) cell control, and (d) virus control

FIGURE 13.4 Antiviral assay, (a) CPE ofVero cell line, (b) reduction in CPE by P. granatum peels silver Np’s, (c) cell control, and (d) virus control.

 
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