Kasetsart J. (Nat. Sci.) 44 : 816 - 823 (2010) Antimutagenicity and Anti-HSV-2 Activity of Mulberry Tea (Morus rotunbiloba Koidz) Thipamon Patharakorn1, Sulak Talawat2, Amornrat Promboon2, Nuanchawee Wetprasit3 and Sunanta Ratanapo2* ABSTRACT Hot water extract from mulberry leaves, Morus rotunbiloba Koidz was extracted with diethyl ether, and its components were analyzed using high-pressure liquid chromatography (HPLC). Polyphenolic compounds constituted the major component (79.8%), consisting of mainly tannic acid (37.9%), epigallocatechin-3-gallate (21.1%) and caffeic acid (11.2%). The genotoxicity of the extract was evaluated by the Ames mutagenicity test, using Salmonella typhimurium strain TA 98 induced by a mutagen Trp-P-1. It was found that the number of revertant colonies was significantly decreased with an IC50 value of 4.5 mg/mL. The extract of Morus rotunbiloba Koidz also exhibited marked antiviral activity against herpes simplex virus type 2 (HSV-2) with an IC50 of 0.52 µg/mL. The results suggested the benefit of consumption of mulberry tea for prevention of cancer and HSV-2 infection. Keywords: mulberry tea, Morus rotunbiloba, antimutagenicity, anti-HSV-2 INTRODUCTION providing clinical merit (Khan and Mukhtar, 2007). M. rotunbiloba and the other Morus Leaves of the mulberry have been species are widely cultivated in many Asian reported to be a rich source of flavonoids and other countries. In Thailand, besides being used mainly polyphenolic compounds (Doi et al., 2001). Nine for feeding silkworms (Bombyx mori L.), the dried flavonoids isolated from Morus alba L. leaves leaves of M. rotunbiloba have been consumed as were identified and some contained free radical a mulberry tea beverage and in food supplements. scavenging properties (Asano et al., 2001). The Among all herbal health teas consumed in the antioxidant and antihyperglycemic roles of world, tea derived from the dried leaves of mulberry leaves have been reported in black Camellia sinensis is the best for chemoprevention mulberry, Morus indica L. (Andallu and of degenerative diseases, such as cardiovascular Varadacharyulu, 2003). Ingestion of mulberry diseases, arthritis, and diabetes (Sharangi, 2009). leaves was reported to reduce blood glucose It has been suggested that the polyphenolic concentration and total serum lipids in patients content, found in high levels, is an active ingredient with type 2 diabetes (Andallu et al., 2001). 1 Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. 2 Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. 3 Department of Biotechnology, Faculty of Science, Ramkhamhaeng University, Bangkok 10900, Thailand. * Corresponding author, e-mail: [email protected] Received date : 20/01/10 Accepted date : 26/05/10 Kasetsart J. (Nat. Sci.) 44(5) 817 Recently, hot-water extract from mulberry (M. Preparation of mulberry tea-leaf extract rotunbiloba Koidz) was shown to exhibit potent Tea leaves of the mulberry were ground, scavenging effects on the ABTS+ (2,2’-azino- then 150g were brewed with hot water (1.5 L) for bis(3-ethylbenzthiazoline-6-sulfonic acid) free 20 min and the resulting tea extract was decanted. radical, as well as having antibacterial activities After the mixture was centrifuged at 8,000 × g for against some human-pathogenic bacteria 15 min, pool supernatant was recovered, freeze- (Patharakorn et al., 2006). Prenylated flavonoids dried to reduce the volume to 120 mL and stored obtained from the root bark of Morus mongolica in airtight containers at 4°C until use. Diethyl ether and M. alba L. were found to exhibit strong (3 mL) was added into the tea-leaf extract. After antibacterial and antifungal activities (Shon et al., mixing and standing for 2 h, the upper diethyl ether 2004). Two prenylated flavonoids (leachinone G soluble fraction was separated, concentrated to and mulberroside C), from the root bark of M. alba dryness in vacuo, redissolved in absolute L., were shown to inhibit the activities of the methanol, and filtered, prior to furnishing a stock herpes simplex virus type 1 (Du et al., 2003). solution for HPLC analysis. Recently, tea polyphenols, particularly catechin derivatives, have been shown to have antiherpetic Chemical analysis by high-pressure liquid activity (Savi et al., 2006). chromatography Herpes simplex virus type 2 (HSV-2) Stock solution (2 µL) was analyzed using causes neonatal infections and fatal infections in an analytical HPLC unit (Perkin Elmer system, humans, with symptoms that are similar to those comprising auto-sampler and quaternary pump of meningitis and cervical cancer types (Corey and coupled to a diode array) and a reverse phase Spear, 1986). Nowadays, acyclovir and other Micro-Bonapak C18 silica column (300 × 3.9 antiviral drugs are used for the treatment of genital mm) from Waters, which was pre-equilibrated with herpes infection. However, HSV-2 has been acetonitrile. The solvent system used a gradient reported to acquire resistance to these drugs of acetonitrile and 0.1% trifluoroacetic acid, (Wagstaff et al., 1994). Therefore, research on increasing from 0-100% acetonitrile over 30 min seeking new anti-HSV-2 drugs from medicinal at 35°C, with a solvent flow rate of 1 mL/min. plants that provide effective treatment with the The chromatogram was recorded at 255 nm. lowest cytotoxicity and low cost is of high interest. Quantification was achieved by the absorbance The objectives of this study were to recorded in chromatograms relative to the identify the chemical composition of the diethyl reference compounds, epigallocatechin-3-gallate ether extract of tea derived from leaves of a (EGCG), tannic acid, caffeic acid, chlorogenic mulberry, M. rotunbiloba Koidz (Mon-Noi) and acid, quercetin, quercitrin and rutin. to investigate its antimutagenicity and antiviral activity against HSV-2. Antimutagenicity assay Antimutagenicity of the mulberry tea MATERIALS AND METHODS extract against a mutagen, Trp-P-1, was assessed using the standard plate incorporation assay, as Plant materials described by Maron and Ames (1983). Salmonella Tea leaves of the mulberry, M. typhimurium tester strain (TA 98) and a quantity rotunbiloba Koidz (Mon-Noi) were provided by of S9 mixture were obtained from the Institute of the Udon Thani Sericultural Research Center, Food Research and Product Development (IFRD), Thailand. Kasetsart University. For the plate incorporation 818 Kasetsart J. (Nat. Sci.) 44(5) inhibition assay, 0.05 mL of Trp-P-1 (1µg/mL in thawing at -70°C and 37°C, respectively, in order DMSO), 0.05 mL of the mulberry tea extract of to break the infected cells, followed by various concentrations, 0.1 mL of the S9 mixture centrifugation at 1,200 × g for 10 min. The and 0.1 mL of a bacterial culture (grown overnight supernatant was collected as stock seed and kept in Oxoid nutrient broth No. 2 at 37°C, with at -70°C until use. shaking) were mixed carefully with 2 mL molten For the determination of virus titers, the top agar, containing 0.05 mM biotin-histidine and BHK-21 cells in 6-well tissue culture dishes were dispersed onto minimal glucose agar plates. The incubated with serially diluted infected cell culture mutagenicity of Trp-P-1 was monitored against TA supernatants for 1 h at 37°C and, after removal of 98 in the presence of the S9 mixture. A series of the inoculum, the dishes were overlaid with culture control plates containing only the tea extracts and medium containing 0.5% agar for visualization of the bacteria in the absence and presence of the S9 induced plaque formation. Plaques were counted mixture were also included to screen the different by neutral red assay and titers were calculated as tea preparations for mutagenic effect. Control plaque forming unit (PFU) mL-1. plates containing only DMSO, which was used as The antiviral activity of the mulberry tea the solvent vehicle, were also included to obtain extracts was studied using a plaque reduction assay the background or spontaneous revertant count. (Wetprasit et al., 2000), under two conditions. All plates were incubated at 37°C for 48 h. (1) Inactivation assay: Mulberry leaf hot- Thereafter, the histidine revertants were counted. water extracts and the diethyl ether soluble- There were three replicates of each treatment. The fractions were diluted to 1:300 in MEM medium percentage of mutagenicity was calculated and passed through a 0.25-µm pore diameter filter. according to the formula in Equation 1: Each extract was twofold serially diluted in MEM percentage of antimutagenicity and then each was mixed with 100 PFU of virus. = [1- (T / M)] × 100 (1) After incubation at 37°C for 30 min, 0.2 mL was where: T = the number of revertants per plate in inoculated into BHK-21 monolayer cells. After 1 the presence of the mutagen and test sample and h adsorption, the unadsorbed virus was discarded, M = the number of revertants per plate and the cells were washed with the MEM medium. in positive controls. The cells were further cultured in MEM medium The number of spontaneous revertants was and inspected periodically until 80% virus-induced subtracted from the numerator and denominator. cytopathic effects (CPE) were observed in the control cell cultures containing no mulberry Anti-HSV-2 assays extracts. Absence of CPE indicated complete Herpes simplex virus type 2 (HSV-2) inactivation of the virus. The infected cells were (LB-strain) was propagated in BHK-21 (baby stained and the number of plaques was counted. hamster kidney) cells. The cells were cultured in The 50% inhibition concentration (IC50) was Eagle’s minimal essential medium (MEM) (Nissui determined from the curve constructed using Pharmaceutical Co., Tokyo) containing 1.2 g/L of different sets of values of plaque number and the NaHCO3 and supplemented with 100 U/mL of concentration of the extract. penicillin, 50 µg/mL of streptomycin, and 10% of (2) Post-treatment assay: BHK-21 fetal bovine serum (FBS) (Biowhittaker, monolayer cells were added to 0.2 mL virus Walkersville, Maryland, USA), and incubated in corresponding to 100 PFU.