Antiviral, Antifungal and Antibacterial Activities of the Chinese Medicinal Plants, Houttuynia Cordata, Lobelia Chinensis and Selaginella Uncinata
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SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH ANTIVIRAL, ANTIFUNGAL AND ANTIBACTERIAL ACTIVITIES OF THE CHINESE MEDICINAL PLANTS, HOUTTUYNIA CORDATA, LOBELIA CHINENSIS AND SELAGINELLA UNCINATA Nam Weng Sit1*, Yik Sin Chan1, Bee Ling Chuah1, Ri Jin Cheng1, Wei Min Leong1 and Kong Soo Khoo2 1Department of Biomedical Science, 2Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, Kampar, Perak, Malaysia Abstract. This study was conducted to evaluate the extracts of Houttuynia cordata (Saururaceae), Lobelia chinensis (Campanulaceae) and Selaginella uncinata (Selagi- nellaceae) for their antimicrobial activities against Chikungunya virus, selected yeasts, filamentous fungi, and gram-positive and gram-negative bacteria of medical importance. Fresh preparations of the studied plants were sequentially extracted to obtain six different extracts. The extracts were examined for cytopathic inhibition effect on African monkey kidney epithelial (Vero) cells co-infected with Chikungunya virus. The chloroform extract of L. chinensis had the highest cytopathic inhibition effect with mean (±SD) cell viability of 66.5% (± 2.1)% at 40 µg/ml. The mean (± SD) 50% effective concentration and selectivity index for this extract were 28.4 (± 0.8) µg/ml and 7.5, respectively. A colorimetric broth micro- dilution assay revealed the hexane extract of H. cordata was the only extract with broad spectrum fungistatic activity against all six species of fungi evaluated, with mean minimum inhibitory concentration (MIC) values ranging from 0.08 to 1.25 mg/ml. As for antibacterial activity, the chloroform and ethyl acetate extracts of S. uncinata exhibited broad spectrum bacteriostatic activity against all six species of bacteria, with mean MIC ranges of 0.63-1.25 and 0.63-2.50 mg/ml, respectively. The antimicrobial activities of the extracts were found to be dependent on the species of microorganisms, testing concentration, and extractant used. The leaf extracts of H. cordata and S. uncinata are potential sources of new antifungal or antibacterial agents with broad spectrum activity. The chloroform extract of L. chinensis warrants isolation of the compounds active against Chikungunya virus due to its potency. Keywords: chikungunya virus, cytopathic effect, fungistatic, fungicidal, bacterio- static, bactericidal, Campanulaceae, Saururaceae, Selaginellaceae Correspondence: Dr Nam Weng Sit, Depart- INTRODUCTION ment of Biomedical Science, Faculty of Science, Universiti Tunku Abdul Rahman, Bandar Barat, Medicinal plants have been used in 31900 Kampar, Perak, Malaysia. many cultures as a source of medicine. Tel: 605 4688888 ext 1016; Fax: 605 4661676 They constitute one of the main compo- E-mail: [email protected] nents of traditional medicine which has 616 Vol 48 No. 3 May 2017 CHINESE MEDICINAL PLANTS WITH ANTIMICROBIAL ACTIVITIES long been used for prevention and treat- treat lung (Yang et al, 2014) and colorectal ment of various diseases. In recent years, cancers (Han et al, 2013). medicinal plants have drawn the attention Selaginella uncinata (Desv. ex Poir.) of scientific research due to their acces- Spring, belonging to the family Selaginel- sibility, therapeutic effects and minimum laceae, is a type of fern commonly known side effects (Kong et al, 2008). as blue spikemoss or peacock moss. The Houttuynia cordata Thunb. is a peren- plant is native to China and is used in nial plant native to China, Japan, Korea, traditional Chinese medicine to treat jaun- and Southeast Asia. It belongs to the dice, dysentery, edema and rheumatism Saururaceae family and is commonly (Jiangsu New Medical College, 1986). It is known as Yu-Xing-Cao in Chinese, which also used by traditional Chinese medicine literally means “fishy-smell plant”.Hout - practitioners in Malaysia to treat jaundice, tuynia cordata has a long history of use in acute and chronic nephritis and hemop- traditional Chinese medicine as an anti- tysis (Yang, 2007). Pharmacognostic and septic, febrifuge, diuretic and deobstruant phytochemical studies of S. uncinata have (Zheng et al, 1998). The plant is also used reported the plant to have antiviral (Ma by traditional Chinese medicine practi- et al, 2003), antianoxic (Zheng et al, 2008) tioners in Malaysia as a component in the and antitumor (Li et al, 2014) properties. recipes to treat bronchitis, lung abscesses, Despite the reported pharmacologi- lung cancer, mastitis, pneumonitis and cal properties of these three plants, there urticaria (Lee, 2003; Yang, 2007). Several are few studies of their efficacy against studies have reported a variety of phar- infectious diseases. The lack of specific macological properties: antimutagenic, treatment for Chikungunya virus infec- anti-inflammatory, antiviral, antiobesity, tion, the escalation of invasive fungal anticancer, antiallergic, antidiabetic, an- infections in immunocompromised or tiplatelet aggregation and antioxidant immunosuppressed patients, the develop- activities (Fu et al, 2013; Kumar et al, 2014). ment of resistance against antimicrobial Lobelia chinensis Lour., commonly drugs and the significant side effects of known as ‘Chinese Lobelia’, is a small chemotherapy (Denning and Hope, 2010; flowering perennial plant that belongs Lo Presti et al, 2014; Ciorba et al, 2015) have to the Campanulaceae family. The plant driven a search for newer and safer anti- is distributed widely in East Asian coun- infective agents among natural resources tries, including China, Korea and Japan. such as medicinal plants. This study was It has been used in traditional Chinese thus carried out to evaluate the extracts of medicine as an antidote for snake bite, as H. cordata, L. chinensis and S. uncinata for a diuretic, a hemostat and to treat diar- antimicrobial activities against Chikun- rhea and jaundice (Shibano et al, 2001; gunya virus, selected yeasts, filamentous Yang et al, 2014). It has been used by tra- fungi, and gram-positive and gram- ditional Chinese medicine practitioners negative bacteria of medical importance. in Malaysia to treat gastric, colon and liver cancers (Yang, 2007). The plant has MATERIALS AND METHODS been reported to inhibit the a-glucosidase enzyme (Shibano et al, 2001), to have anti- Collection of plant materials inflammatory activity, to treat herpes sim- The fresh leaves of Houttuynia cordata plex virus infection (Kuo et al, 2011) and to (472.5 g) and Selaginella uncinata (741.0 g) Vol 48 No. 3 May 2017 617 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH were collected from a nursery at Kampar, streptomycin (10 mg/ml) and 3.7 g/l of Perak, and from a forest at Bukit Merta- sodium bicarbonate at pH of 7.4; the cells jam, Penang, Malaysia, respectively. The were incubated at 37ºC in a humidified 5% fresh aerial parts of Lobelia chinensis (81.1 CO2 incubator. g) were harvested from an herb garden Cytotoxicity assay in Endau, Johor, Malaysia. The species A stock solution of each plant extract identity of the plant samples was authen- was prepared in a dimethyl sulfoxide-eth- ticated by Professor Hean Chooi Ong, an anol mixture (60:40, v/v) at a concentration ethnobotanist of the Faculty of Science, of 512 mg/ml, and subsequently diluted University of Malaya, Kuala Lumpur, two-fold serially in maintenance medium Malaysia. Specimen vouchers of the plants (DMEM with 1% FBS) to obtain eight final were prepared, coded (UTAR/FSC/13/004 concentrations ranging from 5 to 640 µg/ml for H. cordata; UTAR/FSC/12/007 for L. chi- for the cytotoxicity assay. The working nensis; UTAR/FSC/12/006 for S. uncinata) solutions (100 µl) were introduced into and deposited at the Faculty of Science, the respective wells in 96-well microplates Universiti Tunku Abdul Rahman, Kam- containing a confluent monolayer of Vero par, Perak, Malaysia. cells and incubated at 37°C in a 5% CO2 Preparation of plant extracts atmosphere for 72 hours. Medium control The respective collected plant materi- and cell control were incorporated in each als were cleaned, cut and blended prior to microplate. The viability of the cells after extraction. The blended plant materials incubation was assessed using a neutral were sequentially extracted using hex- red uptake assay (Repetto et al, 2008). The ane, chloroform, ethyl acetate, ethanol, percent cell viability was expressed as a methanol and sterile distilled water at mean (± standard deviation) for the three room temperature and with agitation at replicates. The 50% cytotoxic concentra- 140 rpm. All the organic solvents used tion (CC50) of the extract was determined were of analytical grade. Three cycles of from the plot of the percent cell viability maceration were performed for each sol- against the concentration of the extract. vent. The filtrate for each organic solvent Antiviral assay was pooled and evaporated in vacuo while The Chikungunya virus used was the the water extract was lyophilized. All the Bagan Panchor strain and belongs to the dried extracts were kept at -20°C prior to Asian genotype. It was obtained from Pro- bioassay. fessor Shamala Devi (Department of Med- Culture of Vero cells ical Microbiology, Faculty of Medicine, African monkey kidney epithe- University of Malaya, Kuala Lumpur, lial (Vero) cell line (ATCC®CCL-81TM), a Malaysia) and propagated in Vero cells. normal mammalian cell line, was used The virus titer was determined using the to propagate the Chikungunya virus, method of Reed and Muench (1938); the and to conduct the cytotoxicity and an- multiplicity of infection of 1 was used in tiviral assays. The cells were grown in the assay.