RESULTS

Table of Contents:

HSVs are unique viruses that target sensory neurons and after the first primary infection of HSV, which heals without a trace. However, the virus finds its way to dorsal root ganglion where they remain latent until the next recurrence episode. HSV type 1 (HSV-1) stays latent in the trigeminal ganglia and HSV type 2 (HSV-2) remain latent in the lumbo-sacral ganglia. During latency, the virus remains dormant and the only transcription found is latency associated transcripts (LAT). At the time of recurrence, the virus multiplies and exit through motor neurons and cause painful recurrent lesions at the neurodermatome where the nerves supply. The mechanism of latency and recurrence is poorly understood though the stress, radiation, etc., are attributed for those mechanisms. This kind of latent infections and periodic recurrences are the hallmarks of HSV infections. Nanoparticles have an extraordinary property to deliver a drug without losing its ability.

In this study, we evaluated the anti-HSV activity of three different nanoparticles (gold, silver, and bimetallic) which was derived from the four different plants. Figure 13.3a shows the UV-spectrophotometric reading of P granatum silver nanoparticle showing 420 nm. Figure 13.3b shows the ТЕМ image of P granatum silver Np showing 20 nm of each particle. Figure 13.2c SEM showing surface morphology of P. granatum peels silver nanoparticles, showing well-separated spherical nanoparticle. Figure 13.2d shows the XRD analysis, structural information about crystalline metallic nanoparticle. XRD analysis showed intense peaks corresponding to (111), (200), (220), and (311) Bragg’s reflection based on the face-centered cubic structure of silver nanoparticles. The broadening of Bragg’s peaks indicates the formation of nanoparticle and its crystalline nature. There is no other peak in the XRD pattern, which confirmed the stability and high purity of silver nanoparticles. XRD pattern of newly synthesized silver nanoparticles showed intense peaks of (111), (200), (220), and (311) which confirmed the monophasic nature of pure Ag with face-centered cubic symmetry. Figure 13.3e shows FTIR Spectra of P granatum peel synthesized silver nanoparticles and aqueous extract of P granatum peel. The spectrum of P granatum extract displayed important peaks at 3408, 2939 (O-H stretch hydroxyl of phenols or alcohols), 1641 (C=0 stretch of amides), 1384, 1326 (N=0 bend of Nitro groups), 1104 (C-0 stretch of Carboxylic acid) and 830 cm'1 (C-H stretch of Aromatics). On the other hand. FTIR spectra of aqueous extract of P granatum synthesized silver nanoparticles exhibited peaks at 3351, 1634, 1360, 1320, 1069, and 800 cnr1. This confirms the involvement of these functional groups during formation of silver nanoparticles. The hydroxyl and carboxylic acid from the polyphenols and amino acid residues of proteins have ability to produce silver nanoparticles.

Table 13.1 shows a dose-response analysis of gold, silver, and bimetallic nanoparticles of aqueous extracts of P granatum (Peels, Juice), C. sinensis, Nilavembu Kudineer Chooranam, and A. indica for both HSV-1 and HSV-2. Experiments showed that strong anti-HSV activity (both 1 and 2) were observed for silver nanoparticles of P granatum peels extracts (1 pg/ml for HSV-1 and 3.9 pg/ml for HSV-2) followed by Silver nanoparticles of Juice (7.8 pg/ml for HSV-1 and 15.9 pg/ml for HSV-2) revealed by the complete reduction of CPE on vero cells (Table 13.1). However, the anti-HSV activity of nanoparticles derived from other aqueous plant extract was much milder (Table 13.1). Positive control (ACV) does not show CPE. MTT assay was done with vero cell line for all the nanoparticles (Figure 13.6). Except P. granatum nanoparticles, other nanoparticles had cytotoxicity at the various concentrations tested on vero cell line and their percentages were calculated using the formula. Selectivity of the nanoparticles was founded against HSV-1 and 2. Based on the results, it was found that the PgPSNP showed highest selectivity index (SI) (155 for HSV-1 and 150.1 for HSV-2), therefore the promising antiviral activity against HSV-1 and HSV-2, is followed by PgJSNP (33.21 for HSV-1 and 29.1 for HSV-2) whereas nanoparticles showed moderate Sis (Figures 13.4-13.10 and Table 13.1).

MTT assay after 48 hours incubation

FIGURE 13.5 MTT assay after 48 hours incubation.

Percentage of cell viability for P. granatum juice nanoparticles using MTT assay at 48 hrs

FIGURE 13.6 Percentage of cell viability for P. granatum juice nanoparticles using MTT assay at 48 hrs.

Percentage of cell viability for P. granatum peels nanoparticles using MTT assay at 48 hrs

FIGURE 13.7 Percentage of cell viability for P. granatum peels nanoparticles using MTT assay at 48 hrs.

Percentage of cell viability for C. sinensis nanoparticles using MTT assay at 48 hrs

FIGURE 13.8 Percentage of cell viability for C. sinensis nanoparticles using MTT assay at 48 hrs.

Percentage of cell viability for NKC nanoparticles using MTT assay at 48 lirs

FIGURE 13.9 Percentage of cell viability for NKC nanoparticles using MTT assay at 48 lirs.

Percentage of cell viability for A. mdica nanoparticles using MTT assay at 48 lirs

FIGURE 13.10 Percentage of cell viability for A. mdica nanoparticles using MTT assay at 48 lirs.

TABLE 13.1 Antiviral Activity and Cytotoxicity Effect of Nanoparticles

SI.

No.

Nanoparticles

Cytotoxicity

CC.0(pg/inl)

Antiviral Activity

IC_0 (pg/ml) Selectivity Index

HSV-1

HSV-2

HSV-1

HSV-2

1.

P. grcmatum peel silver nanoparticle (PgPSNP)

453

2.93

3.01

155

150.1

2.

P. grcmatum peel gold nanoparticle (PgPGNP)

416.08

42.09

45.70

9.89

9.10

3.

P. granatum peel bimetallic nanoparticle (PgPBNP)

418.04

34.39

39.78

12.16

10.51

4.

P. granatum juice silver nanoparticle (PgJSNP)

353

10.63

12.09

33.21

29.1

5.

P. granatum juice gold nanoparticle(PgJGNP)

456

47.80

47.98

9.54

9.50

6.

P. granatum

juice bimetalllic

nanoparticle(PgJBNP)

398

50.67

56.80

7.85

7

1.

C. sinensis silver nanoparticle (CsSNP)

415

89.12

96.34

4.66

4.31

8.

C. sinensis gold nanoparticle (CsGNP)

402

100.65

120.87

4

3.3

9.

C. sinensis bimetallic nanoparticle (CsBNP)

437

95.69

107.04

4.6

4.1

10.

Nilavembu Kudineer Chooranaum Silver nanoparticle (NKCSNP)

429

226.10

224

1.9

2

11.

Nilavembu Kudineer Chooranaum Gold nanoparticle (NKCGNP)

312

250

223.41

1.2

1.4

12.

Nilavembu Kudineer Chooranaum Bimetallic nanoparticle (NKCBNP)

395

212

206.93

1.9

2

13.

A. indica silver nanoparticle (AiSNP)

453

160.04

178

2.8

2.5

14.

A. indica gold nanoparticle (AiGNP)

378

198.23

178.02

1.9

2.1

15.

A. indica bimetallic nanoparticle (AiBNP)

409

145

178.96

2.8

2.3

16.

Acyclovir (ACV)

250

1.5

2

167

125

DISCUSSION

HSVs infection is common in worldwide. After the primary infection the virus, remain latent in the host body till the end. There is no exact treatment for this infection. Currently using drugs not completely eliminating the vims from the human body, it is giving timely cure from the infection. Extensive and long term clinical use of anti-heipes virus agents like ACV, and its derivatives results in severe side effects and drug-resistant viruses. Further, ACV is reported to incorporate into the cellular DNA, yielding adverse drug reactions and thus, unsuitable for pregnant women and neonates. Moreover, the major determinants of effective immunity against HSV infection are not yet identified. Furthermore, the therapeutic vaccines failed to induce antibody- specific responses to protect recipients from recurrences. Therefore, there is an urgent need for cheap, readily available, less toxic alternate agents to control and prevent HSV infection and its transmission [11].

Nanotechnology is an emerging area in the drug development. It has a huge capacity and thus used drug delivery system. Since the host cells were actively absorbs the nanoparticles than the other larger micro molecules, the drug delivery system is effectively achieved by the nanoparticles [12]. Limited number of studies revealed the approach of metal-based nanoparticles such as tin, silver, gold, and zinc oxide nanoparticles in heipes infection treatments. Reduction of the progeny viruses with weak cytotoxicity was due to the interaction between silver nanoparticles and HSV-2 by in vitro [16]. Formation of the bond by the nanoparticles between the glycoprotein membrane of HSV-2 and the receptor of the host inhibits the entry of virus in the cell [ 16]. Modified tannic acid with silver nanoparticles has been reported in reduction of HSV-2 infection in both in vitro and in vivo [17]. The antiviral activities of the nanoparticles were greatly influenced by the particle size and the dose of the formulation. Because the smaller-sized nanoparticles were characterized by the production of cytokines and chemokines which was greatly useful for anti-viral response [17].

Discovery of water-soluble gold nanoparticles were useful in preventing the herpes virus infection by interacting the viral attachment and penetration [19]. Baram-Pinto et al. developed a non-toxic fonnulation such as gold- based mercapto ethane sulfonated nanoparticles. These nanoparticles can be widely used for topical application, due to its non-toxic formulation it can blindly use as therapeutic and prophylactic application. The mechanism behind this discovery was blocking of viral attachment to the cell and thus preventing the cell-to-cell spread of virus [20]. Another nanoparticle such as zinc oxide nanoparticles also developed to inhibit the entry of HSV-2

virus and preventing the cell to cell spreading in the vaginal lining by its negatively charged surface [18].

Our study underlines the capacity of the nanoparticles which was derived from the plants extract against the HSV. Thus, our nanoparticles such as PgPSNP and PgJSNP revealed anti-HSV activity due to the presence of more active compounds punicalagin. Our also study supports the previous reporting, punicalagin (active compound) of the extract and juice of P. grcmatum were responsible for the antiviral activity. Thus, the fractionation of these bioactive compounds against the viral activity should be explored further [14, 15]. The active compounds in the remaining extracts might be less in quantities to inhibit the virus particles. Elucidation of those active compounds from those plants might be providing the new and effective antiviral agents. This study clearly indicated that P grcmatum 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.

KEYWORDS

  • cytopathic effects
  • cytotoxicity
  • herpes simplex-1
  • herpes simplex-2
  • nanoparticles
  • plant extracts

REFERENCES

  • 1. Steiner, I., & Benninger, F., (2013). Update on herpes virus infections of the nervous system. Cun: Neurol. Neurosci. Rep., 13,414.
  • 2. Aggarwal, A., & Kaur, R., (2004). Seroprevalence of herpes simplex virus 1 and 2 Antibodies in STD clinic patients. Indian J. Med. Microbiol., 22, 2446.
  • 3. Bogaerts, J., Ahmed, J., Akhter, N., Begum, N., Rahman, M., Nahar, S., et al., (2001). Sexually transmitted infections among married women in Dhaka, Bangladesh: Unexpected high prevalence of herpes simplex type 2 infection. Sex Transm. Infect., 77, 1149.
  • 4. Mathiesen, T., Linde, A., Olding-Stenkvist, E., & Waliren, B., (1988). Specific IgG Subclass Reactivity in Herpes Simplex Encephalitis.
  • 5. Jassim, S. A. A., & Naji, M. A., (2003). Novel antiviral agents: A medicinal plant perspective. Journal of Applied Microbiology’, 95,412-427.
  • 6. Bradley, H., Markowitz, L. E., Gibson, X, et al., (2014). Seroprevalence of herpes simplex virus types 1 and 2-United States, 1999-2010. J. Infect. Dis., 209, 325-333.
  • 7. Luker, G., Bardill, J., Prior, J., Pica, C., Piwnica-Worms, D., & Leib, D., (2002). Primary infection with HSV-1 and reactivation from latency. J. Virol, 76, 12149-12161.
  • 8. Corey, L., Adams, H. G., Brown, Z. A., & Holmes, К. K., (1983). Genital herpes simplex virus infections: Clinical manifestations, course, and complications. Ann. Intern. Med., 98, 958-972.
  • 9. Forsgren, M., Skoog, E., Jeansson, S., Olofsson, S., & Giesecke, J., (1994). Prevalence of antibodies to herpes simplex virus in pregnant women in Stockholm in 1969, 1983, and 1989: Implications for STD epidemiology. Int. J. Sex. Transtn. Dis. AIDS., 5, 113-116.
  • 10. Cowan, F. M., Johnson, A. M., Ashley, R., Corey, L., & Mindel, A., (1996). Relationship between antibodies to herpes simplex virus (HSV) and symptoms of HSV infection. J. Infect. Dis., 174, 470-545.
  • 11. Bag, P., Chattopadhyay, D., Mukherjee, H., Ojha, D., Mandal, N., Sarkar, M. C., et al., (2012). Therapeutic vaccines failed to induce antibody-specific responses. Journal of Virology, 9, 98.
  • 12. Yokoyama, M., (2005). Drug targeting with nano-sized carrier systems. J. Artif Organs., 8, 77-84. doi: 10.1007/S10047-005-0285-0.
  • 13. Khan, M. X, Ather, A., Thompson, K. D., & Gambari, R., (2005). Extracts and molecules from medicinal plants against herpes simplex viruses. Antiviral Res., 67,107-119.
  • 14. Lin. L. X, Chen. X Y„ Chung, C. Y„ Noyce, R. S„ Grindley, X B„ McCormick, C„ et al., (2011). Hydrolyzable tannins(chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread. J. Virol, 85, 4386-4398.
  • 15. Lu, J., Wei, Y., & Yuan, Q., (2007). Preparative separation of punicalagin from pomegranate husk by high-speed countercurrent chromatography. J. Chromatogr В Analyt. Technol. Biomed Life Sci., 857, 175-179.
  • 16. Hu. R. L„ Li. S. R„ Kong, F. J.. Hou, R. J., Guan, X. L„ & Guo, F„ (2014). Inhibition effect of silver nanoparticles on herpes simplex virus 2. Genet. Mol. Res., IS, 7022-7028.
  • 17. Orlowski, P., Tomaszewska, E., Gniadek, M., Baska, P, Nowakowska, J., Sokolowska, J., Nowak, Z., Donten, M., Celichowski, G., Grobelny, J., et al., (2014). Tannic acid modified silver nanoparticles show antiviral activity in herpes simplex virus type 2 infection. PLoS One, 9, el04113.
  • 18. Antoine, X E., Hadigal, S. R., Yakoub, A. M., Mishra, Y. K., Bhattacharya, P., Haddad, C., Valyi-Nagy, X, et al., (2016). Intravaginal zinc oxide tetrapod nanoparticles as novel immuno protective agents against genital herpes. J. Immunol, 196,4566-4575.
  • 19. Sarid, R., Gedanken, A., & Baram-Pinto, D., (2012). Pharmaceutical Compositions Comprising Water-Soluble Sulfonate-Protected Nanoparticles and Uses Thereof. U.S. Patent 20120027809 Al.
  • 20. Baram-Pinto, D., Shukla, S., Gedanken, A., & Sarid, R., (2010). Inhibition of HSV-1 attachment, entry, and cell-to-cell spread by functionalized multivalent gold nanoparticles. Small, 6, 1044-1050.
  • 21. Angshuman, P., Sunil, S., & Surekha, D., (2007). Preparation of silver, gold, and silver- gold bimetallic nanoparticles in w/o micro emulsion containing TritonX-100. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 302(1-3), 483-487.
  • 22. Piotr, O., Andrzej, K., Emilia, T., Ranoszek-Soliwoda, K., Agnieszka, W., Jakub, G., Grzegorz, C., et al., (2018). Antiviral activity of tannic acid modified silver nanoparticles: Potential to activate immune response in herpes genitalis. Viruses, 10(10), 524.
  • 23. Tiwari, P. M., Vig, K., Dennis, V. A., & Singh, S. R., (2011). Functionalized Gold Nanoparticles and Their Biomedical Applications. Nanomaterials, 7(1), 31-63.
  • 24. Klaus. X, Joerger, R., Olsson, E., & Granqvist, C. Gr., (1999). Silver-based crystalline nanoparticles, microbially fabricated. Proc Natl Acad Sci USA, 96,13611-13614
  • 25. Reed, L. J., & Muench, H., (1938). A Simple method of estimating fifty per cent endpoints. American Journal of Epidemiology, 27(3),493-497.
 
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