Antiherpetic Effect of Topical Formulations Containing Sulfated Polysaccharide from Adenanthera pavonina

Abstract

Adenanthera pavonina is a native tree of Africa and Asia, introduced in Brazil for reforestation and wood industry. Several pharmacological activities have described scientifically, including antiviral activity. This study evaluated the antiviral effect of sulfated polysaccharide of Adenanthera pavonina (SPAp) against acyclovir (ACV)—resistant (AR-29) and sensitive (KOS) herpes simplex virus strains. The 50% cytotoxic concentration (CC₅₀) was determined by MTT method and the 50% inhibitory concentration (IC₅₀) was evaluated by plaque reduction assay. The in vivo SPAp antiviral activity was performed in Balb/c mice infected by skin scarification and treated with topical 0.5% (w/w) SPAp formulations. SPAp showed a CC₅₀ of 47.81 μg/mL and the IC₅₀ were 0.49 μg/mL (SI = 97.5) and 0.54 μg/mL (SI = 88.5) for the strains KOS and AR-29, respectively. Our results demonstrated that mice treated with SPAp presented a delay in the development and progression of skin lesions compared with the control group.

Introduction

Herpes simplex virus (HSV) is an agent of remarkable diseases in the public health, with high prevalence in human. Acyclovir (ACV) is the drug of choice for the treatment, nevertheless, for several reasons, drug-resistant HSV strains have emerged. Therefore, is urgent the search for new sources of antiherpetic drugs with different mechanisms of action and, consequently, less prone to select resistant strains [1]. Polysaccharides from several origin present conspicuous biological activities, as antiviral activity, and have a low toxicity. These activities can be enhanced by chemical modifications, such as sulfation [2]. This process provides a higher charge density and interferes with electrostatic interactions of viral glycoprotein and the cell surface receptor, preventing the viral adsorption and penetration into the host cells. However, the accurate antiviral effect of polysaccharides in vivo is still unknown [3]. Adenanthera pavonina (Ap) L. (family Fabaceae, subfamily Mimosoideae), commonly known as pigeon eyes, dragon eyes, carolina and red-bead tree, is a native tree of Africa and Asia, introduced in Brazil for reforestation and wood industry [4]. In traditional medicine Ap has been used for diarrhea, fever, gout, rheumatism, ulcers and asthma [5–7]. Triterpenoids, flavonoids, steroids and saponins are the major secondary metabolites [8, 9]. Pharmacological properties have been reported, such as, anti-inflammatory, antioxidant, antidiabetic, antihypertensive, anticancer and antimicrobial [10–15]. Herein, we aimed to investigate the activity of sulfated polysaccharide of Ap against the HSV-1 infection, in vitro and in vivo.

Materials and Methods

Compound

Ap seeds were harvested at the Pici Campus, UFC, Fortaleza, Brazil, in the months of November and December 2010. The polysaccharide (PAp) was obtained by submitting Ap seeds to a treatment of boiling water for 20 min and maintained 12 h at 4 °C until swelling. Seeds coats were removed and the endosperm submitted to aqueous extractions with hot water (85 °C) [16]. The polysaccharide solution was filtered in 5-μm polyester membrane and lyophilized. The sulfation of Ap polysaccharide (SPAp) was obtained from 300 mg of the lyophile with pyridine: N,N-dimethylformamide (v/v) in the proportion 50:10. The resulting solution was neutralized with NaHCO₃, dialyzed and lyophilized as previously [17] and its chemical characterization was performed according to Godoi et al. [18].

Cells and Virus

Vero cells (ATCC CCL-81) cultivated in DMEM (Dulbecco’s modified Eagle’s medium) (Invitrogen—Gibco, USA) were used throughout. The cultures were supplemented with fetal bovine serum (10%, Invitrogen—Gibco, USA), glutamine (2 mM, Sigma Chem. Co., USA) and treated with antimicrobials streptomycin (100 μg/mL, Gibco BRL, USA), penicillin (100 IU/mL, Novafarma Ind. Farm., BR) and amphotericin B (2.5 μg/mL, Meizler Biopharma S/A, BR). The ACV-sensitive (KOS) and -resistant (AR-29) HSV-1 strains, supplied by the LITDFC/DBS/UEM, BR, were stored at − 20 °C with glycerol (10%) and titrated by plaque assay.

Cytotoxicity Assay

Cultured Vero cells, in 96-well microplates (Nunc A/S, Denmark), were exposed to various concentrations of the compound (0.95–2000 μg/mL). After 72 h, the SPAp cytotoxicity was evaluated by MTT assay (Sigma Chem. Co., USA). The concentration capable of reducing by 50% the cellular viability (CC₅₀) was determined by linear regression analysis [19].

The In Vitro Antiviral Activity

Cultured Vero cells, in 24-well microplates (Nunc A/S, Denmark) were treated with different concentrations of SPAp (0.18–3 μg/mL) and simultaneously infected (time 0 h) with KOS or AR-29 strain (10⁵ PFU/mL). After 72 h incubation cells were fixed, stained and the viral inhibition was calculated by: % VI = [1 − (PFU in treated cells/PFU in virus control)] × 100 [19]. The concentration of the compound capable to reduce PFU in 50% (IC₅₀) was calculated by linear regression and the selectivity index (SI) by ratio of CC₅₀/IC₅₀. The ACV (Zynvir, Novafarma Ind. Farm., BR) was used as positive control.

Formulations

O/W emulsion was prepared using 7% (w/w) cetearyl alcohol, 3% (w/w) ceteareth-20, 2% (w/w) glyceryl stearate, 10% (w/w) mineral oil and 2% (w/w) caprylic/capric triglycerides, dispersed in the oil phase and 2% (w/w) glycerin and 0.2% (w/w) methylparaben, dispersed in distilled water until 100 mL. The oil and aqueous phases were heated separately to 75 ± 2 °C. The oil phase was slowly added to the aqueous phase with stirring until 40 °C, forming the base cream and 0.5% (w/w) SPAp dispersed in 20% (w/w) propylene glycol was added to the emulsion at 25 °C [20].

The gel was prepared using 5% (w/w) glycerin and 0.2% (w/w) methylparaben in distilled water until 100 mL and heating at 75 °C. Under stirring, 2% (w/w) hydroxyethylcellulose was sprayed until the formation of gel. When the temperature reached 40 °C, 1.5% (w/w) cyclomethicone was added, forming the base gel. Finally, 0.5% (w/w) SPAp dispersed in 20% (w/w) propylene glycol was added to the formulation.

Commercially available ACV cream (50 mg/g—Medley Pharm. Ltd, BR) was used as positive control.

The In Vivo Antiviral Activity

The Male Balb/c mice (5 weeks old; weight 22–27 g; n = 6 per group) were used and the experiment was approved by the UEL Animal Ethics Committee (CEUA nº 24414.2015.49). The right mid flank was depilated, under anesthesia, and 1 day later, the skin was scarified with sterile needle and 50 μL of HSV-1 (KOS or AR-29 strain—at 10⁵ PFU/mL) were applied. The mice were randomly separated into nine groups: (I) animals scarified, not infected and not treated; (II) animals not scarified, not infected and treated with base gel; (III) animals not scarified, not infected and treated with base cream; (IV) control group—mice scarified, infected and not treated; (V) animals scarified, infected and treated with base gel; (VI) animals scarified, infected and treated with base cream; (VII) animals scarified, infected and treated with 0.5% (w/w) SPAp gel; (VIII) animals scarified, infected and treated with 0.5% (w/w) SPAp cream; (IX) animals scarified, infected and treated with ACV. Formulations containing SPAp or ACV were administered topically 4 h after infection, 4 times a day for 8 successive days, except at night. The skin lesions were daily scored, as follows: 0—no lesion; 1—vesicles in local region; 2—erosion and/or ulceration locally; 3—mild zosteriform lesion; 4—moderate zosteriform lesion; 5—severe zosteriform lesion and 6—death [21].

Statistical Analysis

GraphPad Prism 5.0 software (GraphPad Software Inc., CA, USA) was used to perform statistical analysis. The Tukey’s test (Anova) were applied to analyze the difference among experiments with SPAp and control group. Values of p < 0.05 were considered significant.

Results

SPAp showed a CC₅₀ of 47.81 μg/mL and the in vitro SPAp antiviral activity for KOS and AR-29 strains is shown in Fig. 1. In the treatment simultaneously with infection (time 0 h), with SPAp at 3 μg/mL, 1.5 μg/mL and 0.75 μg/mL it has shown the percentages of viral inhibition (%VI) of 100%, 88.7% and 70% respectively, for the KOS strain. Similar values were found for AR-29 strain, at the same concentrations, with %VI of 97.8%, 85.7% and 50.7%, respectively. The SPAp IC₅₀ were 0.49 μg/mL (SI = 97.5) and 0.54 μg/mL (SI = 88.5) for the strains KOS and AR-29, respectively. While the ACV showed a CC₅₀ > 2000 μg/mL with IC₅₀ of 0.13 μg/mL and 5.22 μg/mL for the strains KOS and AR-29, respectively.

Fig. 1.

The effect of the SPAp added concomitantly (time 0 h) to HSV-1 infection (KOS and AR-29 strains), in Vero cells by plaque reduction assay. The experiments were conducted independently in triplicate and the viral inhibition (%) was expressed as the mean ± SD of experiments

Regarding the in vivo SPAp activity, mice infected with the KOS strain treated with 0.5% (w/w) SPAp gel or cream followed an evolution similar to that of the control group until day 3 post-infection (pi). However, on day 4 pi, in the control group all animals showed ulcerated skin and some developed a mild zosteriform lesion. While most of the animals treated with the cream had only vesicles and for those treated with gel half had only vesicles and half had skin ulcerated, but without zosteriform lesions. On day 8 pi, there was a predominance of moderate to severe zosteriform lesions in the control group. The groups treated with 0.5% (w/w) SPAp gel or cream showed a milder evolution of the lesions. Comparatively, mice treated with ACV had a milder evolution of the lesions, because on day 7 pi all animals showed retrieved skin (Fig. 2).

Fig. 2.

The effect of 0.5% (w/w) SPAp gel and cream on skin lesions caused by HSV-1 KOS strain in Balb/c mice. The animals were infected by skin scarification and topically treated, 4 h after infection, 4 times a day, for 8 days. Each point represents the mean score values of 6 animals per group (± SD)

Concerning mice infected with the strain AR-29, controls and groups treated with 0.5% (w/w) SPAp gel or cream presented only vesicles on day 2 pi. On day 3 pi, all mice treated with the gel had ulcers, as well as, those of the control group, however, some animals treated with the cream still had vesicles. On day 8 pi, most of the mice in the control group had moderate to severe zosteriform lesions, with one death. While for the group treated with the gel, most of the lesions were mild zosteriform and most of the mice treated with the cream still had only focal ulceration (Fig. 3).

Fig. 3.

The effect of 0.5% (w/w) of SPAp gel and cream on skin lesions caused by HSV-1 AR-29 strain in Balb/c mice. The animals were infected by skin scarification and topically treated, 4 h after infection, 4 times a day, for 8 days. Each point represents the mean value of 6 animals per group (± SD)

Discussion

Sulfated polysaccharides have been reported as potent inhibitors of virus. They present a high density of negative charge that mimics the cell receptor heparan sulfate (HS) interacting with the positively charged viral glycoproteins and blocking the attachment of virus to cell [2]. Fucoidans derived from marine brown alga Padina tetrastromatica exhibited potent anti-herpetic activity, inhibiting the virus adsorption to the cell [22]. Other fucoidan isolated from brown algae Kjellmaniella crassifolia, showed in vitro activity against influenza A virus [23]. Moreover, the intranasal therapy with fucoidan in infected mice decreased significantly the viral replication and increased animals’ survival. The fucoidan cream improved the healing time and decreased the discomfort in two patients with recurrent oral herpes labialis [24].

However, the biological properties of sulfated polysaccharides are also associated to structural characteristics, such as sulfation degree, molecular weight and specific positioning of the sulfate groups [2]. The compounds (sulfated xylogalactofucan and alginic acid) from Laminaria angustata, a seaweed, significantly inhibited the infectivity of HSV while the content of sulfate groups enhances the inhibition [25]. An oral preparation enriched with sulfated polyanions, such as galactofucan of Undaria pinnatifida, inhibited the HSV reactivation and improved injuries in patients with active infections. [26]. The polysaccharides from green algae Enteromorpha compressa presented anti-herpetic activity and demonstrated an improved antiviral activity after chemical derivatization of ulvan [27]. Corroborating with these results, the sulfated depolymerized carageenans were more effective than native carrageenan against influenza virus A, suggesting that the sulfate content and molecular weight potentiate the antiviral activity of carrageenan [28].

The polysaccharide obtained from Agaricus brasiliensis and their chemically modified derivatives were tested against human and bovine herpesvirus type 1 replication and only the sulfated form demonstrated stronger inhibition [29]. Similar findings were described for glucomannan polysaccharide, isolated from the mycelium of same basidiomycete and chemically sulfated, demonstrating an improve of anti-HSV-1 activity, with SI > 439 and 208, for KOS and AR-29 strains, respectively [30]. Posteriorly, the glucomannan demonstrated antivirus potential in vivo for cutaneous herpes by oral treatment. However, its topical application on skin lesions was not effective [31]. In our study, we found for SPAp, lower selectivity indexes, but less discrepant between the two strains.

The sulfated galactomannans from Adenanthera pavonina and other plants of Caatinga biome inhibited the early stages of dengue infection as, for example, inhibiting the entry of virus into cells [32]. Previous studies also investigated the antiviral activity of A. pavonina against poliovirus and HSV-1 and showed antiviral effect almost 50 times higher than that native [18, 19]. Godoi et al. [19] also found a CC₅₀ for SPAp of 500 μg/mL in HEp-2 cells, differing of the value found in our study of 47.81 μg/mL, since we used another cell line. Regarding the IC₅₀, they found a value of 15 μg/mL, but the virus used was isolated from a clinical sample, already in our work we used standard strains sensitive or resistant to ACV. Based on these studies and knowing the in vitro mechanism of action of SPAp [19, 33], a galactomannan extracted from A. pavonina, semisolid pharmaceutical formulations were developed for tests in vivo.

The mice infected with the strain AR-29 and treated with 0.5% (w/w) SPAp gel or cream presented a mean lesion score lower than the ACV-treated group only in the first days of infection. Then, it is suggested that this occurred due: the HSV-1 inhibition by SPAp involves the blockage of early stages of replication, which precede the step of DNA synthesis; the concentration of SPAp used in the cream or gel be much lower compared to the concentration of ACV. However, in summary, our results demonstrated that mice treated with SPAp presented a delay in the development and progression of skin lesions compared with the control group.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.