Antiviral Activity of an Extract from Leaves of the Tropical Plant Cynometra cauliflora

Medicinal plants of the Malaysian forest were reportedly rich in biological activities. Several interesting natural products were isolated from local medicinal plants such as styrylpyrone derivatives isolated from G. umbrosus have shown a potent antiviral activity against HSV-1 and dengue virus type 2 (DENV-2).1-3 Other in vitro studies also showed SPD is active against several cancer cell lines namely; HL-60 (leukemia), HepG2 (liver), PANC-1 and Hela cells,4-5 geraniin extracted from the rind of Nephelium lappaceum that were reported has been shown to exhibit antiviral properties against several types of viruses, as well as crude methanolic extract of Psidium guajava leaves extract that were reported to have antibacterial activities against foodborne pathogens.6 This plant extract also was reported to have antiviral activity against DENV-2.7 Previous studies have shown antioxidant, antiinflammatory, antitumor, antimicrobial and antidiabetic activity of C. cauliflora.8-10 C. cauliflora L. or commonly known as ‘Nam-Nam’ among native Malaysian is a tropical plant under the Fabaceae family. It is also commonly known by local as NamNam or Buah Katak Puru in Malaysia.11 The fruit which are kidney-shape pod, greenish yellow to brown, with a sandy and wrinkled surface. It can be consumed as fruit salad (ulam).12


INTRODUCTION
Medicinal plants of the Malaysian forest were reportedly rich in biological activities. Several interesting natural products were isolated from local medicinal plants such as styrylpyrone derivatives isolated from G. umbrosus have shown a potent antiviral activity against HSV-1 and dengue virus type 2 (DENV-2). [1][2][3] Other in vitro studies also showed SPD is active against several cancer cell lines namely; HL-60 (leukemia), HepG2 (liver), PANC-1 and Hela cells, [4][5] geraniin extracted from the rind of Nephelium lappaceum that were reported has been shown to exhibit antiviral properties against several types of viruses, as well as crude methanolic extract of Psidium guajava leaves extract that were reported to have antibacterial activities against foodborne pathogens. 6 This plant extract also was reported to have antiviral activity against DENV-2. 7 Previous studies have shown antioxidant, antiinflammatory, antitumor, antimicrobial and antidiabetic activity of C. cauliflora. [8][9][10] C. cauliflora L. or commonly known as 'Nam-Nam' among native Malaysian is a tropical plant under the Fabaceae family. It is also commonly known by local as NamNam or Buah Katak Puru in Malaysia. 11 The fruit which are kidney-shape pod, greenish yellow to brown, with a sandy and wrinkled surface. It can be consumed as fruit salad (ulam). 12 Herpes Simplex Virus type-1 (HSV-1) is a common pathogen which causes cold sores or common cold and orolabial infection. Normal sites of infection are mucosal epithelium, hence keratitis labial herpes, gingivostomatitis, and genital herpes. Infection can disseminate from mucosal epithelium to other tissues with slow healing and more detrimental outcome in immunocompromised individual. 13 This includes in newborn babies, transplant patient or HIV patient who are readily struggling with immature immunity, immune suppressive drugs regiment and prolonged toxicity and prophylaxis, respectively. 14 Generally, HSV-1 infections can be treated successfully with acyclovir. However, drug resistant variants emerged as a result of long-term treatment of immunocompromised patients with acyclovir. This subsequently led to treatment failure. 15 Thus, a new target is required to ensure alternative possible treatments for HSV-1 resistant strains. In order to combat this resistant HSV-1 strain, new antiviral agents with different mode of actions are indeed important. Therefore, the aim of this study was to investigate the potential of crude methanolic extract of C. cauliflora leaves as an antiviral agent against HSV-1 infection.

Plant material
The fresh leaves parts were collected from the state of Terengganu, Malaysia. The leaves were cleaned with tap water to remove dirt and oven-dried at 60°C. Dried leaves powder of C. cauliflora was extracted with methanol. C. cauliflora leaves (100 g) was macerated with methanol (300 mL) to produce crude methanol extract. The extracts were filtered and solvent was evaporated under reduced pressure using rotary vacuum evaporator.

Cells and virus
Vero cell from American Type Culture Collection (ATCC) CCL-81 was used for both cytotoxicity and antiviral test. Dulbecco's Modified Eagle's Medium (DMEM) (SigmaAldrich, USA) supplemented with 5% fetal bovine serum (FBS) (Sigma-Aldrich, USA) was used for cell maintenance throughout the experiment. Clinical strain of HSV-1 used was obtained from the stock culture of Faculty Science and Technology, Universiti Kebangsaan Malaysia.

Cytotoxicity test
Briefly, Vero cells (2.5×10 5 cells/mL) were seeded into 96-well plates and incubated overnight at 37°C. Upon 80% confluence, the cells were treated with several concentrations of extract, ranging from 3.13 mg/mL to 100 mg/mL. After incubation of about 72h, the growth medium was discarded and replaced with 100 μL of MTT solution and incubated for 3h. After that, the MTT solution was discarded, and formazan crystal was dissolved using 100 μL of dimethyl sulphoxide (DMSO) to lyse the cells. Colour development was detected using a microplate reader (TECAN Infinite 200 PRO, Austria) at 540 nm. Optical density (OD) of individual well was quantified using spectrophotometer at 540nm. 16 Cells viability was calculated using formula below: Cell viability (%) = ODtest -ODblank / ODcell -ODblank x 100 where ODtest = optical absorbance of cells treated with SPD, ODblank = optical absorbance for well filled with DMSO and OD-cells = optical absorbance for cells without treatment with SPD. Nonlinear regression was done to obtain the CC50 value (cytotoxic concentration which killed 50% of cells).

Antiviral assay
Antiviral activity was also evaluated by the plaque assay method. Screening for antiviral activity was performed using 3 different treatments. 17

RESULTS
Cytotoxicity evaluation of C. cauliflora extract MTT assay was conducted to determine the cytotoxicity of C. cauliflora extract towards Vero cells. The cytotoxicity assay result, as presented in Figure 1, shows the percentage of cell viability versus C. cauliflora extract concentration. The estimated CC 50 value towards the Vero cells was 36.0 mg/mL.

Anti-HSV-1 activity of C. cauliflora
Plaque reduction assays were done to screen for anti-HSV-1 activity using C. cauliflora extract with different concentrations. Figure 2A, 2B and 2C shows the percentage of plaque reduction in post-treatment, pre-treatment and virucidal assays, respectively. The results from posttreatment assay showed that 100% plaque reduction was achieved at the concentration of 18 mg/mL. In pre-treatment assay, more than 50% plaque reduction was observed at 9 mg/mL. Meanwhile, C. cauliflora extract at any concentrations had no virucidal effect on HSV-1.
Effectiveness of certain compounds or extracts can be evaluated by using selective index (SI). In post-treatment assay, C. cauliflora extract exhibited potent antiviral activity against HSV-1 with EC 50 = 2.14 mg/ mL and with SI value of 16.8 (Table 1). Pre-treatment of Vero cells with C. cauliflora extract exhibited the prophylactic activity of extract against HSV-1 infection with EC 50 = 8.5 mg/mL and with SI value of 4.23 (Table 1). C. cauliflora extract when added simultaneously with the virus not showed any anti-adsorption activity against HSV-1 (Table 1). Result revealed that C. cauliflora extract had greater SI value in posttreatment. Any antimicrobial compound that has SI values higher than 10 (SI>10) ensures the potential to be developed as an agent of antiviral drug. 18 Selectivity index of C. cauliflora extract against HSV-1 was more than 10 indicating potential as antiviral agent.

DISCUSSION
Based on phytochemical analyses the findings in previous study, C. cauliflora leave extract has been reported to be rich in secondary metabolites such as tannin, flavonoid, saponins, cardiac glycosides and terpenoids. 19 Lyu and collaborators 20 reported the elucidation of the mechanism of the antiherpetic (HSV-1) activity in vitro via plaque reduction assay of flavonoid. Similarly, Sieniawska 21 demonstrated that tannins and related compounds, exhibit antiherpes activity in vitro. In addition, Perez 22 reported that saponins inhibit the replication of HSV-1 and poliovirus type 2 as shown by inhibition of cytopathic effect and reduction of virus production. Thus, the richness of secondary metabolites in C. cauliflora plant may contribute to anti-HSV-1 properties. In this study, we investigated whether C. cauliflora methanolic extracts could confer protection to cells before or after the initiation of HSV-1 infection. The ability of the extract to act directly against HSV-1 virion particle was observed in virucidal assay. This antiviral analysis was performed on Vero cells as a model of infection in mammalian cells.
Screening for antiviral activity involves post-, pre-and virucidal treatment to determine the best mode for antiviral administration. In this part of the study, C. cauliflora extract treatment was found to not interfere directly to infectious particle and confer mild protection when given as prophylaxis. Instead, evidence showed that extract-HSV-1 treatment most effective when administered as post-treatment. C. cauliflora extract anti-HSV-1 activity was observed to be concentration dependent. The ability of C. cauliflora to confer protection to the cells before HSV-1 infection was tested by pretreating the cells with C. cauliflora methanol extracts for 24 h prior to viral infection. Protection could be conferred through extracellular mechanisms. The C. cauliflora extracts might interrupt the interaction of several envelope glycoproteins with cell surface receptors requires for fusion of the virion envelope with a cell plasma membrane, resulting in ineffective viral infection. 23 Pre-treatment was done to study the effect of the extract as prophylactic agent in protecting the cell from HSV-1 adsorption and penetration. C. cauliflora extracts presented low to mild prophylactic effects, perhaps due to the presence of various plant alkaloids in the crude extract of C. cauliflora, which may act synergistically to decrease the effective interaction of the active compounds. Additionally, the results are presented as some of the antiviral compounds in these extracts may be present at low levels in a non-cytotoxic dilution of the extract. 24 Therefore, extract can act as partial prophylactic agent to protect Vero cells against HSV-1 infection. Virucidal agents are chemical substances that attack and inactivate the extracellular viral particles by damaging the protein coat or penetrating the virion or by destroying the viral genome resulting in decreased infectivity of the virus. 25 The possibility of this occurring was demonstrated using a virucidal assay. C. cauliflora extract treatment was found to not interfere directly to infectious particle because no inhibition was observed.

CONCLUSION
As a conclusion, our findings suggest that crude extract prepared from C. cauliflora contains antiviral active compounds and could be potential antiviral agent.