key: cord-0887280-eyan7n7x authors: Wahab, Noor Zarina Abd; Azizul, Aziah; Ibrahim, Nazlina title: Phytochemistry, cytotoxicity and antiviral activity of Catharanthus roseus date: 2020-10-03 journal: Iran J Microbiol DOI: 10.18502/ijm.v12i5.4608 sha: d33a7683a58fef922a72c3288b5db8ae0a676c47 doc_id: 887280 cord_uid: eyan7n7x BACKGROUND AND OBJECTIVES: Catharanthus roseus is generally used to treat many diseases in folklore remedies. The present study is aimed at determining phytochemical constituents, cytotoxicity and antiviral activities for crude extract of the plant. MATERIALS AND METHODS: The whole plant of C. roseus was extracted using methanol extraction method. Phytochemical qualitative screening was carried out for C. roseus extract according to standard procedures used to test for the presence of alkaloid, saponin, terpenoid and steroid. Cytotoxicity was assessed using 3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) assay. Plaque reduction assays were carried out to evaluate the antiviral activity of C. roseus extract against herpes simplex virus type 1 (HSV-1). These include post-treatment, pre-treatment and virucidal assays. RESULTS: C. roseus extract contain secondary metabolites such as alkaloid, saponin and terpenoid but does not contain steroid. Cytotoxicity screening against Vero cells using MTT assay showed that the CC(50) values for crude extract of C. roseus was 0.5 mg/mL. The extract prepared from C. roseus possesses phytochemical compound that was non-cytotoxic to the cell with potential antiviral activity. Plaque reduction assays against herpes simplex virus type 1 (HSV-1) showed that the selective indices (SI = CC(50) / EC(50)) of C. roseus extract in post-treatment, pre-treatment and virucidal assays were 36, 20 and 4.7 respectively. The results revealed that the extract prepared from C. roseus possesses phytochemical compound that was non-cytotoxic to the cell with potential antiviral activity. CONCLUSION: This study showed that C. roseus extract has promising potential to be explored as anti-HSV-1 agent regardless of the mode of treatment. Catharanthus roseus is a perennial tropical medicinal plant belongs to the Family Apocynaceae in which the leaf extracted from C. roseus showed significantly higher activity, suggesting that its bioactive compounds can be potentially exploited as antibacterial agents (1) . Out of 36 alkaloids of C. roseus evaluated, nine showed a degree of antiviral has been reported (2) . Pericalline, perivine, periformyline, leurosivine, leurocristine, vincaleukoblastine, perividine, vindolinine and carosine all showed antiviral activity against the vaccinia and polio type III viruses. Extract of C. roseus has been reported to exhibit antivirus activities against dengue virus type 2 (3). C. roseus also exhibited broad-spectrum antibacterial activity against Salmonella Typhi and Shigella boydii (4) . In addition, C. roseus extract also showed antifungal properties against Rigidoporus microporus, Ganoderma philippii and Phellinus noxius (5) . Herpes Simplex Virus type-1 (HSV-1) is a common pathogen that causes cold sores or common cold and orolabial infection. Normal mucosal epithelium including keratitis labial herpes, amino acid. The cell culture was maintained in an ingingivostomatitis and genital herpes (6) . However, cubator at 37°C and humidified 5% CO atmosphere. these common symptoms can develop into serious illness when infection disseminates from mucosal epithelium to other tissues with slow healing. The effect has more detrimental outcomes in immunocompromised individuals (7) which includes newborn babies, transplant patients or HIV patients who are readily struggling with immature immunity, immune-suppressive drugs regiment and prolonged toxicity and prophylaxis, respectively (8) . HSV infection can be successfully treated with Acyclovir and related nucleoside analogue but the emergence of drug resistance to acyclovir has created a barrier for the treatment especially in immunocompromised patients (9) . Acyclovir resistance has been increasingly described and is caused by mutations in either the thymidine kinase or the DNA polymerase genes (10) . Therefore, alternative therapies for patients with documented resistance are required to reduce the clinical impact of drug-resistant herpes viruses (11) . So, in order to combat this resistant HSV-1 strain, new antiviral agents with a different mode of actions are indeed important. until cytopathic effects developed. The titer of the virus was estimated and stored at -80°C until used. Phytochemical screening. Phytochemical qualitative screening was carried out for C. roseus extract according to standard procedures used to test for the presence of alkaloids, saponins, terpenoid and steroids (12) . Determination of alkaloid. 10 mg/mL of extract was diluted in 2 mL of 25% ammonia for several minutes. After that, 5 mL of chloroform was added and shaken gently to extract the alkaloidal base. Mayer's reagent was added. The formation of a cream with Mayer's reagent was observed indicating the presence of alkaloids. Determination of saponin. 2 mL of distilled water was added to extract suspended in ethanol and was shaken vigorously. The formation of copious foam layer indicates the presence of saponins. Determination of terpenoid. 2 mL of chloroform was added to 10 mg of the extract. After that, 3 mL of concentrated sulphuric acid (H SO ) was carefully 2 4 Plant Material. The fresh plant of C. roseus was collected from the state of Terengganu, Malaysia. The plants authentication was performed by competent botanist from Faculty of Bioresources and Food added to form a layer. A reddish brown colouration of the interface indicates the presence of terpenoids. Determination of steroid. 2 mL of chloroform and Industry, Universiti Sultan Zainal Abidin. The plants 2 mL of concentrated H SO was added to 10 mg/mL were cleaned with tap water to remove dirt and oven-dried at 60°C. The dried whole plants consisting of leaf, stem and root were cut into smaller pieces and ground into powder using a grinder. The powder (200 g) was socked in 500 mL methanol for three days. The extracts were filtered and solvent was evaporated under reduced pressure using rotary vacuum evaporator (Eyela). Vero cells used in this study were obtained from stocks available at the Virology Laboratory, Faculty of Science and Technology, Universiti Kebangsaan Malaysia. Vero cells were maintained in Dulbecco's Modified Essential Medium (DMEM) supplement-of extract and shaken well. Chloroform layer appeared red. This confirm the presence of sterols steroid. Cytotoxicity test. Cytotoxicity test was performed to determine the maximum non toxic dose of the plant extracts. Confluent Vero cells (2.0 × 10 4 cell/ well) grown in 96-well microtitre plate were treated with different concentrations of extract. After 48 h of incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added into each well and was further incubated for 3 h. Excess MTT was removed and 100 μL of dimethylsulfoxide (DMSO) was added. The absorbance was measured at 540 nm by using microplate reader (Tecan Infinite 200 Pro). The percentage of cell viability was calculated and ed with 5% Fetal Bovine Serum (Nacalai Tesque), the CC value was obtained directly from graph of penicillin/streptomycin (100 U/L) and non-essential cell population versus extract concentration. IRAN Antiviral assay. Antiviral assays were deter-C. roseus methanol extracts at concentrations rangmined by plaque assay. Antiviral assays composed ing from 0.039 to 1.25 mg/mL. The related CC was of post-treatment, pre-treatment and virucidal assays. then calculated using Graph Pad Prism for Windows, version 5 (Fig. 1) . In this assay, the CC value of C. Post-treatment assay. Vero cells (1.0 × 10 5 cell/ well) were seeded into 24 well plate and incubated overnight. Upon confluency, cells were inoculated with 200 μL of virus at ~50 PFU (plaque forming unit). Cells were incubated for 2 h to allow virus adsorption. After adsorption period, the cells were washed twice with PBS to remove any residual unbound viruses. This was followed by the addition of serial dilutions of plant extracts in triplicate mixed with DMEM+methyl cellulose. Cells were then incubated for 48 h. After incubation, cells were stained using crystal violet and plaques were counted. Pre-treatment assay. Vero cells (1.0 × 10 5 cell/ well) were seeded in 24 well plate and incubated overnight. Cells were pre-treated with six concentrations of extracts for 24 h before being removed and infected with 200 μL of virus at ~50 PFU. After adsorption period, DMEM+methylcellulose was added and incubated for 48 h. After incubation, cells were stained using crystal violet and plaques were counted. roseus extract was determined at 0.5 mg/mL. Anti-HSV-1 activity of C. roseus. Plaque reduction assays was used to evaluate the in vitro anti-HSV-1 activities of C. roseus extract. C. roseus extract was added at the different phases of viral infection: i) pre-treatment for 24 hours prior to infection for its prophylactic activity, ii) treatment for 48 hours post-adsorption and iii) directly to cell free virus suspension to examine its direct virucidal effect. Fig. 2 shows the percentage of plaque reduction in post-treatment, pre-treatment and virucidal assays, respectively. The results from post-treatment assay showed more than 75% plaque reduction was observed at 0.250 mg/mL. In pre-treatment assay, 100% plaque reduction was achieved at a minimum concentration of 0.125 mg/mL. For virucidal assay, more than 50% plaque reduction was observed at a minimum concentration of 0.016 mg/mL. The effectiveness of the C. roseus extract as an Table 1 . Phytochemical analysis of C. roseus methanolic extract Virucidal assay. Direct virucidal effect of the extract was investigated by incubating virus with extract for 1 hour before it was inoculated on the cells. After adsorption period, DMEM+methylcellulose was added and incubated for 48 h. After incubation, cells were stained using crystal violet and plaques were counted. Table 1 indicates the methanolic extract of C. roseus contained a few secondary metabolites such as alkaloid, saponin and terpenoid. Phytochemical analysis also indicates the C. roseus extract does not contain steroid. Alkaloid Saponin Terpenoid Steroid +: Present; -: Absent + + + -Cytotoxicity of C. roseus extracts. Cytotoxicity of C. roseus extracts on Vero cells were evaluated using the MTT assay. To determine the nontoxic dose, Vero cells were exposed to two-fold serially diluted antiviral compound expressed as selectivity index (SI). In post adsorption assay, C. roseus extract exhibited potent antiviral activity against HSV-1 with = 0.014 mg/mL and with SI value of 36 (Table 2) . Pre-treatment of Vero cells with C. roseus extract exhibited the prophylactic activity of extract against ent secondary metabolites and part of them has been already used for the treatment of various chronic diseases in human (14) . Interestingly, different secondary plant metabolites such as flavonoids, saponins, lignans, tannins, alkaloids and thiophenes, phenolic acids were found to have significant antiviral activity HSV-1 infection with EC = 0.025 mg/mL and with against variety of viruses (15, 16) . Based on phyto-SI value of 20 (Table 2) . C. roseus extract when added simultaneously with the virus showed anti-adsorp-chemical analyses, C. roseus extract has been proven to be rich in secondary metabolites such as alkation activity against HSV-1 with EC = 0.106 mg/ loids, saponin and terpenoid. According to Moradi mL and with SI value of 4.7 (Table 2) . Results from direct virucidal activity assessment of C. roseus extract showed that the extract exhibited a weak extracellular anti-HSV-1 activity. Result revealed that C. roseus extract had greater SI value in post-treatment followed by pre-treatment and virucidal assays. Any antimicrobial compound that has SI values higher than 10 (SI>10) ensures the potential to be developed as an agent of antiviral drug (13) . Selectivity index of C. roseus extract against HSV-1 was more than 10 indicating potential as antiviral agent. (19) demonstrated that some triterpenoidal saponins exhibit anti-HSV-1 activity in vitro. Wen et al. (20) reported the elucidation of the mechanism of the anti-severe acute respiratory syndrome associated coronavirus (SARS-CoV) activity of plant terpenoids. In this study, we investigated whether C. roseus extract could confer antiviral activities against HSV-1 infection. This antiviral analysis was performed on Vero cells as a model of infection in mammalian cells. The cytotoxicity evaluation assay was performed to determine the cytotoxic concentration that kills 50% cell population which is known as CC value. The CC value of C. roseus extract was determined at 0.5 mg/mL. The results of the greater SI value was identified with C. roseus extract might be due to the presence of the unique phytochemical constituents. The anti-HSV-1 activity can be related to the presence of active compounds in C. roseus methanol extract such as alkaloids (21) , saponins (22) and terpenes (23) . The calculated selective index (SI) value of more than 10 for C. roseus extract in post-treatment and pre-treatment which its worth to be further studied as antiviral agent. C. roseus extract can affect virus infection to pre-treated and post-treated cells. The pre-treatment assay was conducted to investigate the effect of the extract on the cells be- or block the HSV-1 from binding to the cell surface (24) . Meanwhile, the post-treatment assay was conducted to investigate whether HSV-1 viral replication cycle and assembly in infected cells, could be affected (25) . Extracellular anti-HSV-1 activity of C. roseus extract was investigated by virucidal assay. Results from direct virucidal activity assessment of C. roseus extract showed that the extract exhibited a weak extracellular anti-HSV-1 activity. According to calculated SI value, C. roseus extract showed the ability to decrease viral replication more in the post-treated cell compared to the pre-treated cell. This result indicated that C. roseus extract was capable in controlling viral infection after 2 h which is the initial phase in virus replication cycle involving virus attachment process (26) . This result proposed that C. roseus extract was effective in inhibiting early viral entry including attachment, penetration phases and viral replication but incapable inactivate free virus particles. In conclusion, C. roseus extract exhibited potent anti-dengue activity in vitro and contains antiviral active compounds and could be potential antiviral agent. Results from this study can help further in vivo anti-viral studies as part of the developmental process for development of C. roseus extract as potential anti-HSV-1 therapeutic. Indole alkaloids from Catharanthus roseus: Bioproduction and their effect on human health Antiviral activity of selected Catharanthus alkaloids Efficacy of Catharanthus roseus extract against dengue virus type 2 infection in vitro Antibacterial activity of different extracts from the Catharanthus roseus Antifungal compound isolated from Catharanthus roseus L. 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