Antiviral Activities of Honey, Royal Jelly, and Acyclovir Against HSV-1
Introduction. Herpes simplex virus type 1 (HSV-1) belongs to the Herpesviridae family and genus simplex virus. This virus is usually acquired during childhood and is transmitted through direct mucocutaneous contact or droplet infection from infected secretions. The aim of the present study was to compare antiviral effects of honey, royal jelly, and acyclovir on herpes simplex virus-1 in an extra-somatic environment. Materials and Methods. Vero cells were cultured in the Dulbecco’s Modified Eagle’s Medium (DMEM) along with 10% fetal bovine serum (FBS) in 12-welled microplates. Various dilutions of honey, royal jelly, and acyclovir (5, 10, 50, 100, 2500, 500, and 800 µg/mL) were added to the Vero cells along with a 100-virus concentration of TCID50. The plaque assay technique was used to evaluate the antiviral activities. Results. The results showed that honey, royal jelly, and acyclovir have the highest inhibitory effects on HSV-1 at concentrations of 500, 250, and 100 µg/mL, respectively. In addition, honey, royal jelly, and acyclovir decreased the viral load from 70 795 to 43.3, 30, and 0 PFU/mL at a concentration of 100 µg/mL, respectively. Conclusion. The results of the present study showed that honey and royal jelly, which are natural products with no reports about their deleterious effect at least in laboratory conditions, can be considered alternatives to acyclovir in the treatment of herpetic lesions. However, it should be pointed out that further studies are necessary to substantiate their efficacy because hard evidence on their effectiveness is not available at present.
Herpes simplex virus infections are ubiquitous around the world. Various physical and medicinal methods have been reported for the treatment of herpetic lesions, including intravenous, oral, and topical preparations of acyclovir, cytarabine, vidarabine, iododeoxyuridine, cryotherapy, and use of chemical agents.1 In many cases, treatment of viral infections with the medications available ends in failure due to drug resistance as a result of viral genetic mutations and presence of some latent and recurrent infections.2 Therefore, there is an ever-increasing need for newly developed antiviral agents. On the other hand, these treatments have problems such as limitations of use with patients who are breastfeeding; limitations of use with patients who are senile due to immune deficiency and the multiple medications these patients are typically taking; emergence of mutant viruses that lack the enzyme thymidine kinase, which is the main target of the medication’s effect; and resistance against these medications due to the toxic effects and the cost of the medications. Even though its use has been limited due to these issues, acyclovir is currently considered an effective drug. In recent years, resistance to acyclovir has also increased due to viral mutations.2,3 This medication only shortens the duration of the disease and cannot prevent transmission of viral infection to other individuals or prevent recurrence of the infection. Therefore, at present there is a need for a new treatment with fewer side effects and less toxicity.4 Honey is a natural, viscous product with highly saturated glucose concentrations; it is produced from the nectar of flowers,4 has few allergic effects, is not toxic, and is readily available.5 It has been demonstrated that honey is an antimicrobial agent with a wide spectrum of activities against different kinds of bacteria, fungi, protozoa, and viruses.5 Honey can contribute to proper and rapid healing of wounds, decrease necrotic tissue debris, eliminate wound malodor, and result in better shrinkage of the wound.6,7 In addition, its high viscosity contributes to the formation of a proper barrier against wound infection. Honey also prevents or decreases scar formation, as well as hypertrophy.8 In addition, the role of honey has been demonstrated in recovery from stomatitis as a result of chemotherapy, healing of gastric ulcers in laboratory rats, and elimination of Helicobacter pylori from the stomach. In addition, its antibacterial activity has also been demonstrated against Pseudomonas aeroginosa, which is resistant to a wide range of antibiotics.9 Royal jelly is a foodstuff specially produced by honeybees for the queen. It has antimicrobial and anti-allergic properties; decreases blood cholesterol level; prevents damage to healthy cells during treatment of cancer and HIV infection; helps wound healing; facilitates growth and development; and increases sexual capabilities.10 Literature review shows no studies to date have compared the effects of honey, royal jelly, and acyclovir on HSV in vitro and some studies have only evaluated and compared the effects of honey and acyclovir on HSV.11-13 In addition, no studies thus far have evaluated the antiviral effects of royal jelly on HSV-1. Therefore, the aim of the present study was to compare the antiviral effects of honey, royal jelly, and acyclovir on HSV-1 in an extrasomatic environment.
Materials and Methods
Manuka honey (UMF 20, Comvita UK Ltd, Maidenhead, Birkshire) and royal jelly supplied by the Faculty of Agriculture, University of Tehran located in Karaj City, Iran, were used. Royal jelly was kept on ice while transferred to the author’s institution. Pure powder of acyclovir was purchased from Sigma-Aldrich (St. Louis, MO) In this study, the dilutions 5, 10, 50, 100, 250, 500, and 800 µg/mL for honey, royal jelly, and acyclovir were considered. Initially, 0.01 g of honey and royal jelly were dissolved in 10 mL of the culture medium, which did not have any serum. Then, 5 µL of the solution was mixed with 995 µL of the culture medium to prepare a dilution of 5 µg /ml. The rest of the dilutions were prepared in the same manner. Regarding acyclovir, 0.01 g of acyclovir was dissolved in 10 mL of 0.1% NaOH. Then 200 µL of each dilution were incorporated into 3 wells in triplicate (containing monolayer cells) plus 2% of fetal bovine serum (FBS). In addition, for each dilution, 1 well was assigned as a control, which contained only the culture medium. Then the plates were incubated at 37ºC and 5% CO2 for 48 hours.14 The Vero cells (African green monkey kidney epithelial cells, Vero 76, ATCC No: CRL 1587) were purchased from the National Cell Bank of Iran (NCBI Code: C101). The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% (v/v) FBS, 2 mM of glutamine and 100 µg/mL of kanamycin in a humidified 5% (v/v) CO2 incubator at 37°C. The standard laboratory strain of HSV-1 KOS (ATCC VR-1493D, ATCC, Manassas, VA) was propagated at 0.01 plaque-forming units (PFU) per cell using standard procedures. Viral titers were determined using a plaque assay.14 The monolayer of Vero cells is shown (Figure 1). Confluent Vero cells (2.3×105/cm2) were infected with 40–50 PFU of HSV for 1 hour. The Vero cells infected with HSV were replaced with overlay medium (RPMI-1640, 0.5% [v/v] FBS and 1% [w/v] carboxymethyl cellulose) and supplemented with test extracts. Infected cultures were incubated in a humidified 5% CO2 incubator for 2 days before staining with 0.8% (w/v) crystal violet in 50% ethanol and then viral plaques were counted under a binocular microscope. The reduction in the number of plaques was calculated as percentage of virus control. The concentration reducing plaque formation by 50% relative to the control was estimated from graphic plots and defined as 50% inhibitory concentration (IC50).14 Duplicate cultures of confluent Vero cells were infected with HSV at a multiplicity of infection as previously indicated. The Vero cells infected with HSV were removed at 1 hour post-infection and maintained with the culture medium containing 0.5% FBS alone, or with various concentrations of extracts, at 37°C for 18 hours. The cultures were frozen and the viral yields were determined by the standard plaque assay method. The IC50 was the concentration of extracts that inhibited viral yields by 50% of control.14 Cytotoxic activity of honey, royal jelly, and acyclovir was evaluated using the sulforhodamine microtiter plate assay method as described by Skehan et al.15 Vero cells (5×104/well) were seeded in 96-well tissue culture plates and incubated at 37°C for 3 days. Each treatment was carried out in triplicate. Fixed cells were stained with sulforhodamine B and optical density was measured at 562 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Bio-Tek, Winooski, VT). The TC50 is the concentration of the extract that inhibits actively replicating cells by 50% of control. The direct inactivation of HSV-1 by selected fractions was tested by direct contact assay. Virus suspensions were mixed with equal volumes of honey, royal jelly, and acyclovir for 1 hour at room temperature. Upon dilution, the surviving infectious virus was measured using the plaque assay method and was compared to the mock pretreated control.14
The threshold toxicities on Vero cells were dilutions of 500 µg/mL, 250 µg/mL, and 100 µg/mL of honey, royal jelly, and acyclovir, respectively. The reading of optical density for each material decreased with an increase in dilutions (ie, the number of viable cells decreased) (Figure 2). There was a significant relationship between various dilutions of honey, royal jelly, and acyclovir, and the effect on Vero cells (P = 0.0001). In other words, with an increase in the dilutions of these materials, more counts of Vero cells were observed in the culture media. Evaluation of the antiviral effects of these materials showed that all the materials were effective on HSV-1. The number of plaques formed decreased—which is a reflection of destruction of Vero cells by the virus—with an increase in the dilutions of each material. Each material had inhibited viral activity on Vero cells and had prevented observation of cytopathic effect (CPE) residues through its antiviral activity. The highest antiviral activities of honey, royal jelly, and acyclovir were observed in 500, 250, and 100 dilutions, respectively (ANOVA test). Honey and acyclovir decreased the number of plaques to zero, but they were completely effective on HSV-1. However, royal jelly decreased the number of plaques, but was not able to reduce the number to 0 (Figure 3). There was no significant relationship between honey, royal jelly, and acyclovir at dilution 5 (P = 0.416). However, there was a significant relationship between the 3 materials at dilution 10 (P = 0.036); post-hoc test revealed a significant difference between honey and acyclovir at this concentration with a higher effect of honey. In addition, there were significant differences between the 3 materials at dilution 50 (P = 0.014); honey and royal jelly were more effective than acyclovir at this dilution. There was a significant difference between acyclovir and honey at dilution 100 (P = 0.012), with a better antiviral activity of acyclovir compared to honey. There was no significant difference between honey and royal jelly at dilution 250 (P = 0.12) (Table 1). The results of viral load determination based on 100-virus TCID50 showed that at dilution 10-3 the viral load or titer was 7 x 104 in each milliliter, with approximately 100 plaques of Vero cells. The results showed that with an increase in dilutions, the viral load decreased, with statistically significant differences between the 3 materials (P = 0.002) (Figure 4). Consistent with the results obtained in the plaque reduction assay, the selectivity index of honey was 10.7 (Table 2). Evaluation of CPE effect under an inverted microscope in the cell control well revealed normal cellular appearance; in the virus control well, the viruses had destroyed the monolayer appearance of Vero cells and the CPE effects of the virus were visible in the form of rounding and separation of Vero cells with granulation of the cytoplasm. In the extract control well, in a manner similar to the cell control well, the cells had a normal appearance. In the experimental wells, a combination of antiviral effect of the extract (the inhibiting effect of extracts on the virus and normal appearance of the cells) and the effect of HSV-1 (in the form of cellular destruction and CPE effects) were observed. There was an increased antiviral effect, of the extract with an increase in the dilution of the extract, and more numerous normal cells and less numerous plaques were observed (Figures 5A, B, and C).
Traditional medicine has a long history of relieving the ills of human beings and is still a source of inspiration for the provision of medicines.16 Various factors have influenced the inclination of human beings toward traditional medicine, including ease of access; interest in self-treatment; and the belief that such treatment modalities are safer, more reliable, and cheaper than other commonly used drugs and techniques.17 The results of the present study showed that all the 3 materials—honey, royal jelly, and acyclovir—have antiviral effects, with more prominent effects seen in acyclovir and honey. Honey has antimicrobial, antifungal, and antioxidative effects, with a high nutritional value. Al-Waili5 published an article in 2001 on the treatment of seborrheic dermatitis with the use of honey. All patients properly responded to the use of honey, and itching, crusting, and flaking of the skin resolved in a period of 1 week. In 2003, Al-Waili18 published results from another study on the treatment of psoriasis and atopic dermatitis with honey. In that study, honey ointment alone or in combination with a steroid was applied; honey ointment alone was effective and resulted in a 75% decrease in symptoms.18 In 2004, Al-Waili19 used honey in the treatment of skin fungal infections. The results showed that 86% of patients with psoriasis, 75% with fungal infections of groin, and 75% with fungal infections were successfully treated. Some studies have evaluated the therapeutic effects of honey and acyclovir on herpetic lesions.19 In a study by Al-Waili19 in 2004, the topical application of honey in the treatment of recurrent labial and genital herpetic infections was compared with that of acyclovir ointment. Eight patients with labial herpetic and 8 patients with genital herpetic infections used honey for 1 herpetic attack and acyclovir ointment for another herpetic attack. In the labial herpetic infection, the means of the duration of attack, pain severity, incidence of crusting and flaking, and healing period were better with 35%, 39%, 28%, and 43% with honey, respectively, in comparison to treatment with acyclovir ointment. In the case of genital herpetic infections, the means of the duration of attack, pain severity, incidence of crusting and flaking, and healing period were better with 53%, 50%, 49%, and 59% with honey, respectively, in comparison to treatment with acyclovir ointment. None of the patients using acyclovir reported complete recovery during the expected period and 3 patients reported local itching with the use of acyclovir.13 Studies have shown that honey can destroy more than 250 bacterial strains, including resistant genera of methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and Helicobacter pylori.13 The results of the present study showed that honey and royal jelly resulted in the formation of less numerous plaques at dilutions 5, 10, and 50, with better effects in comparison to acyclovir. In addition, royal jelly exhibited less numerous plaques at dilutions 50 and 100 in comparison to honey. Several reasons have been suggested for the success of treatment of herpetic ulcers with honey. Since honey is rich in glucose, its absorption through the ulcers inhibits microorganisms. In addition, glucose oxidase is secreted by honeybees into the honey.13 Royal jelly is produced by honeybees. It has a role in the nutrition of larvae and maturation of the queen. Royal jelly is sold as a food supplement for human use. The general composition of royal jelly is 67% water, 12.5% raw proteins, a small amount of various amino acids, 11% monosaccharides, and relatively high concentrations of fatty acids. In addition, it contains minerals, enzymes, antibacterial components, antibiotics, vitamin C, and vitamin B groups.10 Royal jelly has been introduced as an immune agent in the treatment of Graves’ disease;20 as a stimulator of the growth of glial cells21 and brain stem cells;22 as an agent which decreases cholesterol levels;10 and as an anti-inflammatory agent. It also contributes to the healing and treatment of wounds with antibacterial and antibiotic activity against aerobic Streptomyces and gram-positive bacteria;10 as an agent suppressing allergic reactions; and as an agent preventing cellular destruction in cancer and HIV infections.10,23 Studies have shown the 10-HDA in royal jelly can inhibit angiogenesis of tumors. It is believed the antiaging properties of royal jelly and its high content of amino acids have resulted in its widespread use in esthetic products.23 Some studies have evaluated the antibacterial properties of royal jelly. Eshraghi and Seifollahi10 reported that royal jelly has bactericidal effects on E. Coli, S. aureus, and Streptomyces griseus. Boukraâ et al24 showed that royal jelly has strong bacterial activity at concentrations higher than 200 µg/mL. A study by Suemaru et al25 showed that royal jelly ointment has a better healing effect on mucositis in hamsters compared to honey and propolis ointments, and results in the resolution of mucositis in these animals. Yanagita et al26 showed that royal jelly has anti-inflammatory effects on periodontal ligament cells and can be used in the treatment of such problems. The antiviral activity of royal jelly has been attributed to the effect of 10-HDA. This fatty acid stimulates white cells, resulting in the adhesion of white cells to viruses such as HSV and hepatitis viruses, leading to their destruction.27 The only study on the effect of royal jelly on HSV in the genital area has been carried out by Sankum et al.28 The results showed that the viral load decreased from 104.9 to 103.9; therefore, they reported that royal jelly has an antiviral activity.28
The results of the present study showed that royal jelly has a better antiviral activity compared to honey, resulting in a decrease in plaque-forming units from 104.85 to 101.5 and 101.8. The results of the present study showed that honey and royal jelly, which are natural products with no reports about their deleterious effect, at least in laboratory conditions, can be considered as alternatives to acyclovir in the treatment of herpetic lesions. However, it should be pointed out that further studies are necessary to substantiate their efficacy because hard evidence on their effectiveness is not presently available.
Maryam Alsadat Hashemipour Zahra Tavakolineghad Sayed Ali Mohammad Arabzadeh Zahra Iranmanesh Sayed Amir Hossein Gandjalikhan Nassab
acyclovir herpes simplex virus honey royal jelly
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Issue: Volume 26 - Issue 2 - February 2014
Index: WOUNDS. 2014;26(2):47-54.
This study was supported by Kerman University of Medical Sciences, which provided staffing and equipment.
Poster presented at: Thai Society of Virology Conference; April 26-May 1, 2002; Bangkok, Thailand.
Maryam Alsadat Hashemipour, DDS, MSc; and Zahra Tavakolineghad, DDS are from the Kerman Social Determinants on Oral Health Research Center, Kerman University of Medical Sciences, Kerman, Iran. Sayed Ali Mohammad Arabzadeh, PhD; and Zahra Iranmanesh, MS are from the Department of Medical Microbiology, Kerman University of Medical Sciences, Kerman, Iran. Sayed Amir Hossein Gandjalikhan Nassab, MS is from Kerman Oral and Dental Diseases Research Center, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran.
Address correspondence to: Maryam Alsadat Hashemipour Department of Oral Medicine School of Dentistry Kerman University of Medical Sciences Kerman, Iran. email@example.com
Disclosure: The authors disclose the Research Deputy of the Kerman University of Medical Sceinces provided financial support for this study (Thesis No. 653).
Antiviral Activities of Honey, Royal Jelly, and Acyclovir Against HSV-1 | Wounds Research https://www.woundsresearch.com/article/antiviral-activities-honey-royal-jelly-and-acyclovir-against-hsv-1