Antiviral, Antifungal and Antibacterial Activities of the Chinese Medicinal Plants, Houttuynia Cordata, Lobelia Chinensis and Selaginella Uncinata

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 microdilution 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, bacteriostatic, 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 (Konget 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. Each plant extract was evaluat- Dulbecco’s modified Eagle’s medium ed at six concentrations: two-fold serially (DMEM) (Sigma-Aldrich, St Louis, MO) diluted in maintenance medium (DMEM supplemented with 5% fetal bovine serum with 1% FBS) starting from the highest (FBS), 1% penicillin (10,000 units/ml), 1% concentration (with cell viability ≥ 90%)

618 Vol 48 No. 3 May 2017 Chinese Medicinal Plants with Antimicrobial Activities based on the results obtained from the used with some modifications. A stock cytotoxicity assay. The Vero cells (4x104 solution of each plant extract was pre- cells/well) were incubated with the plant pared at 10 mg/ml in a methanol-water extract and Chikungunya virus at 37ºC mixture (2:1, v/v). The stock solution was in 5% CO2 for 72 hours. Controls in each two-fold serially diluted with RPMI-1640 microplate included medium (DMEM medium in 96-well microplates to achieve only), virus (cells with virus only) and cell final concentrations ranging from 0.02 to (cells with medium only). Chloroquine 2.50 mg/ml. Amphotericin B (Bio Basic, diphosphate (MP Biomedicals, Illkirch, Ontario, Canada; purity >75%) with a France; purity > 99.9%) with a concentra- final concentration range of 0.06 to 8 µg/ tion range of 0.39 to 12.4 µM was used as ml was used as the positive control. Fifty the positive control. Following incubation, microliters of the prepared fungal inocu- cell viability was assessed using a neutral lum was added into the wells, making the red uptake assay (Repetto et al, 2008). The total volume of each well 100 µl. Sterility percent cell viability was expressed as (medium only), growth (fungus only) the mean (± standard deviation) for three and negative (extract only) controls were replicates. The 50% effective concentra- included in each microplate. The micro- tion (EC50) of an extract was determined plates were incubated at 35°C for 48 hours from the plot of the percent cell viability for the Candida spp; 35°C for 72 hours for against the concentration of the extract. C. neoformans and A. fumigatus, and 28°C The selectivity index was calculated as for 7 days for T. mentagrophytes. After µ the ratio of CC50 to EC50. incubation, 20 l of the growth indicator, Fungal cultures p-iodonitrotetrazolium chloride (INT, 0.4 Four species of yeast (Candida albi- mg/ml; Sigma-Aldrich, St Louis, MO; pu- cans ATCC®90028tm, Candida parapsilosis rity ≥98%) was added to each well. Obser- ATCC®22019tm, Candida krusei ATCC®6258™ vation of the color change of the indicator (teleomorph) and Cryptococcus neofor- was made and a minimum inhibitory con- mans ATCC®90112tm) and two species of centration (MIC) was recorded. Twenty filamentous fungi (Aspergillus fumigatus microliters of the content from wells with ATCC®204305tm and Trichophyton men- inhibitory activity (the indicator remained tagrophytes ATCC®9533tm) were used in colorless) was inoculated onto SDA/PDA the study. Prior to inoculum preparation, and incubated accordingly. Formation of the yeast species were subcultured on the fungal colony on the agar plate was Sabouraud dextrose agar (SDA) (Merck, observed and the minimum fungicidal Darmstadt, Germany) and the filamentous concentration (MFC) was determined. fungi were subcultured on potato dextrose The MIC and MFC values were reported agar (PDA) (Merck, Darmstadt, Germany). as the mean of three replicates. The fungal inocula were prepared for an- Bacterial cultures tifungal assay according to the guidelines Two species of gram-positive bacteria published by Clinical Laboratory Stan- (Bacillus cereus ATCC®11778tm and Staphy- dards Institute (CLSI, 2002a, b). lococcus aureus ATCC®6538tm) and four Antifungal assay species of gram-negative bacteria (Acineto- A colorimetric broth microdilution bacter baumannii ATCC®19606tm, Escherich- method described by Eloff (1998) was ia coli ATCC®35218tm, Klebsiella pneumoniae

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Table 1

Fifty percent cytotoxic concentrations (CC50) of various extracts of Chinese medicinal plants against African monkey kidney epithelial (Vero) cells.

Extracts 50% Cytotoxic concentration (µg/ml)

Houttuynia cordata Lobelia chinensis Selaginella uncinata

Hexane 198.5 ± 4.8 256.3 ± 6.8 365.1 ± 50.7 Chloroform 107.2 ± 4.3 213.8 ± 13.3 135.6 ± 10.8 Ethyl acetate 506.9 ± 9.0 132.8 ± 12.4 39.7 ± 3.3 Ethanol 126.1 ± 2.7 - 255.9 ± 17.9 Methanol 227.2 ± 6.4 - 440.5 ± 22.8 Water - - -

“-” no significant toxicity observed at the highest concentration (640 µg/ml).

ATCC®13883tm and Pseudomonas aerugi- of 100 µl. Sterility (medium only), growth nosa ATCC®27853tm) were evaluated in the (bacteria only) and negative (extract only) study. All the bacteria were subcultured controls were included in each test. The on Mueller-Hinton agar (MHA) (HiMe- microplates were incubated at 37°C for dia Laboratories, Mumbai) at 37°C for 24 24 hours. Following incubation, 20 µl of hours prior to inoculum preparation. The INT at 0.4 mg/ml was added to each well. bacterial inocula were prepared according The color of each well was observed and to Clinical Laboratory Standards Institute the MIC was determined and recorded. guidelines (CLSI, 2005). Twenty microliters of the content from the Antibacterial assay wells with inhibitory activity (the indicator remained colorless) was inoculated We used a colorimetric broth microdi- onto MHA and incubated at 37°C for 24 lution method to determine the minimum hours. Formation of the bacterial colony inhibitory concentration (MIC) of the on the agar plate was observed and the tested plants (Eloff, 1998). A stock solution minimum bactericidal concentration of each plant extract (10 mg/ml) was pre- (MBC) was determined. The MIC and pared in a methanol-water mixture (2:1, MBC values were reported as the mean v/v). The stock solution was two-fold se- of three replicates. rially diluted with Mueller-Hinton broth (MHB) (HiMedia Laboratories, Mumbai) Data analysis in 96-well microplates to achieve final The susceptibility index for a microor- concentrations ranging from 0.02 to 2.50 ganism is calculated as 100 x number of ex- mg/ml. Chloramphenicol (Nacalai Tesque, tracts effective against the microorganism Kyoto, Japan; purity ≥98%) or tetracycline strain divided by number of total extracts (Sigma-Aldrich, St Louis, MO; purity (Bonjar, 2004). The percentages of cell vi- ≥95%) with a final concentration range of ability at the different concentrations of 1 to 128 µg/ml was used as the positive plant extract to evaluate for cytotoxicity control. Fifty microliters of the prepared and antiviral assays were analyzed with bacterial inoculum was added into the one-way analysis of variance using SPSS®, wells, making a total volume in each well version 20 (IBM, Armonk, NY). Signifi-

620 Vol 48 No. 3 May 2017 Chinese Medicinal Plants with Antimicrobial Activities

------NA NA NA 1.25 0.63 2.50 0.16 1.25 0.63 1.25 MFC

Trichophyton 2.5 NA NA NA NA NA NA NA 1.25 0.31 1.25 0.31 1.25 0.04 0.04 0.08 0.63 MIC 0.002 0.002 0.002

- - - - NA ------NA NA NA MFC - - - -

fumigatus mentagrophytes Filamentous fungi Aspergillus NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA 2.50 1.25 1.25 MIC 0.001 - 0.001 - 0.004

- - - 2.5 NA NA NA NA 0.31 0.63 0.63 1.25 0.16 0.16 0.16 0.16 0.16 0.31 0.31 0.63 MFC - neoformans

Cryptococcus NA 0.31 0.31 0.08 0.16 0.31 0.16 MIC 0.0001 0.0005 0.0001 0.16 ------2.50 MFC Table 2 Table krusei Candida

NA NA NA NA NA NA 0.31 0.63 0.16 0.31 0.63 1.25 0.63 0.31 0.08 0.08 0.04 0.16 0.31 0.08 0.31 0.63 0.16 1.25 NA 0.16 0.63 0.31 0.16 0.16 0.31 0.63 MIC 0.002 0.002 0.002 -

against medically-important fungi. medically-important against ------NA NA NA NA NA NA NA NA 0.31 0.63 1.25 1.25 MFC

Candida Yeasts parapsilosis NA NA 0.63 1.25 1.25 0.63 1.25 1.25 0.16 0.31 0.63 0.63 0.63 0.63 MIC 0.0005

- - - NA - NA - NA - NA - - 0.001 - - - 0.001 2.5 NA NA NA MFC albicans a Candida

NA NA NA NA NA NA NA NA NA 1.25 0.31 1.25 0.31 0.63 1.25 0.63 2.50 1.25 0.63 1.25 0.001 - 0.001 0.001 MIC

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of Chinese medicinal plants plants medicinal Chinese of (MFC) concentration fungicidal minimum and (MIC) concentration inhibitory Minimum Extracts Houttuynia cordata Hex Chl EA EtOH MeOH Water AMB Lobelia chinensis Hex Chl EA EtOH MeOH Water AMB Selaginella uncinata Hex Chl EA EtOH MeOH Water AMB The MIC and MFC values are expressed as a mean of three replicates in mg/ml. Hex, hexane extract; Chl, chloroform extract; EA, ethyl acetate in mg/ml. Hex, hexane extract; Chl, chloroform replicates as a mean of three expressed The MIC and MFC values are a , no activity; “-” unavailable. NA Amphotericin B (positive control); AMB, extract; EtOH, ethanol MeOH, methanol

Vol 48 No. 3 May 2017 621 Southeast Asian J Trop Med Public Health

80 80 80 Hexane Chloroform Ethyl acetate 60 60 60 Ethanol Methanol Water 40 40 40 Viability of Vero cells (%) of Vero Viability cells (%) of Vero Viability cells (%) of Vero Viability 20 20 20

0 0 0 0.1 1 10 100 1,000 0.1 1 10 100 1,000 0.1 1 10 100 1,000 Concentration of extract (µg/ml) Concentration of extract (µg/ml) Concentration of extract (µg/ml) Fig 1–Viability of African monkey kidney epithelial (Vero) cells co-incubated with Chikungunya

virus and plant extracts. The cells were incubated at 37ºC with 5% CO2 for 72 hours. Cell viability was assessed by a neutral red uptake assay. (A) Houttuynia cordata, (B) Lobelia chinensis, (C) Selaginella uncinata. The x-axis is in a log scale.

cance was set at p < 0.05. A post hoc test, (compared to untreated Vero cells which either using the Tukey’s (equal variance is the cell control of the assay) were used assumed) or Dunnett’s (equal variance in the antiviral assay. Of the 18 extracts not assumed) test was conducted to de- evaluated in this study, only the chloro- termine which concentration of extract form, ethanol and methanol extracts of produced a significant result. H. cordata, the chloroform, ethyl acetate, ethanol and methanol extracts of L. chi- RESULTS nensis, and the chloroform extract of S. uncinata exhibited a significant (p < 0.05) Antiviral assay protective effect for the Vero cells against The cytotoxicity of each plant extract cytopathic effects caused by the Chikun- against Vero cells was determined prior gunya virus. The highest percentage of to the antiviral assay. The water extracts cell viability was seen with the chloroform of the three studied plants were not sig- extract of L. chinensis (66.5% ± 2.1% at 40 nificantly toxic (p > 0.05) to the Vero cells, µg/ml), followed by the methanol extract even at the highest concentration of 640 of H. cordata (38.8% ± 2.4% at 80 µg/ml) mg/ml. The ethanol and methanol extracts and the chloroform extract of S. uncinata of L. chinensis were not significantly toxic (33.8% ± 1.8% at 80 µg/ml). In the virus to the Vero cells (p>0.05). The mean 50% control wells, the Vero cells were com-

cytotoxic concentrations (CC50) for those pletely lyzed (cell viability = 0%) after 72 extracts which were significantly toxic to hours of co-incubation with the virus titer the Vero cells are shown in Table 1. The at the multiplicity of infection of 1. The ethyl acetate extract of S. uncinata was chloroform extract of L. chinensis was the the most toxic extract having the lowest only extract that provided greater than µ mean CC50 value of 39.7 g/ml. Concentra- 50% protection for the Vero cells against tions of an extract which were associated virus infection; the mean 50% effective

with greater than 90% Vero cell viability concentration (EC50) for this extract was

622 Vol 48 No. 3 May 2017 Chinese Medicinal Plants with Antimicrobial Activities

------NA NA NA 2.50 1.25 0.63 1.25 1.25 2.50 1.25 whereas whereas MBC

aeruginosa Pseudomonas NA NA 1.25 0.63 0.008 0.004 0.008 K. pneumoniae 0.63 0.63 1.25 - 0.63 - 0.63 and 2.50 0.31 NA 1.25 NA 1.25 NA - - NA - - NA - NA

Klebsiella S. aureus S. aureus pneumoniae , 2.5 NA NA NA NA NA NA NA 0.63 NA 0.63 1.25 1.25 NA 2.50 0.63 1.25 NA 0.31 1.25 1.25 1.25 0.31 2.50 1.25 MIC MBC 0.004 - 0.002 - 0.004 - B. cereus

------NA NA MBC

Gram-negative bacteria

NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA NA Escherichia coli 2.50 1.25 MIC 0.002 0.002 0.004

------NA NA NA NA

Table 3 Table ------

NA, no activity; “-” unavailable. baumannii Acinetobacter NA NA NA 1.25 NA - 1.25 - 1.25 - NA - NA - NA - NA - NA - 0.001 - 0.001 - 0.002 - - - NA NA NA 2.50 NA 0.63 NA 2.50 NA 2.50 NA 0.31 2.50 2.50 1.25 A. baumannii; MBC MIC against medically important bacteria. important medically against and aureus aureus

Staphylococcus NA NA NA NA NA NA NA NA NA NA 0.63 0.31 1.25 1.25 2.50 0.16 0.63 1.25 MIC 0.004 0.004 0.004

P. aeruginosa P. a

, ------NA NA NA NA 2.50 0.16 0.31 0.63 0.16 0.63 1.25 Gram-positive bacteria E. coli MBC a

Bacillus cereus

NA NA NA NA NA NA NA 1.25 1.25 2.50 0.16 0.31 0.63 0.63 1.25 0.16 0.63 0.63 0.004 0.004 0.004

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Chinese medicinal plants plants medicinal Chinese of (MBC) concentration bactericidal minimum and (MIC) concentration inhibitory Minimum Extracts MIC Houttuynia cordata Hex Chl EA EtOH MeOH Water Antibio Lobelia chinensis Hex Chl EA EtOH MeOH Water Antibio Selaginella uncinata Hex Chl EA EtOH MeOH Water Antibio The MIC and MBC values are expressed as a mean of three replicates in mg/ml. Hex, hexane extract; Chl, chloroform extract; EA, ethyl acetate EA, extract; Hex, hexane extract; Chl, chloroform in mg/ml. replicates of three a mean as expressed are values MBC MIC and The a Antibio, chloramphenicol was used for extract; EtOH, ethanol MeOH, methanol tetracycline was used for

Vol 48 No. 3 May 2017 623 Southeast Asian J Trop Med Public Health

28.4 (± 0.8) µg/ml. The selectivity index ethyl acetate extracts of S. uncinata had for this extract was 7.5. broad spectrum antibacterial activity Antifungal assay against both the gram-positive and all four of the gram-negative tested bacte- The MIC and minimum fungicidal ria. Their MIC ranges were 0.63-1.25 and concentration (MFC) for each tested plant 0.63-2.50 mg/ml, respectively. These two extract are shown in Table 2. Considering extracts were also the only extracts active one extract against one fungus as one against E. coli in this study. Escherichia bioassay, 58.3% (63/108) of the bioassays coli was the least susceptible bacterium showed fungistatic activity and 38.0% towards the plant extracts tested with a (41/108) of the bioassays exhibited fun- susceptibility index of 11.1%. None of the gicidal activity. The lowest MIC value extracts of L. chinensis were active against recorded from this study was 0.04 mg/ A. baumannii or P. aeruginosa. ml, from the hexane extract of L. chinensis against C. neoformans, and the hexane and chloroform extracts of S. uncinata against DISCUSSION T. mentagrophytes. The lowest MFC value The Vero cell line was used in this (0.08 mg/ml) from this study was the study because it is one of the most com- hexane extract of L. chinensis against C. mon and well established normal mam- krusei. The hexane extract of H. cordata malian cell lines used to assess the effects had broad spectrum antifungal activity of chemicals, toxins and other substances, against all the fungi evaluated in this such as plant extracts, on cells at the study, with MIC values ranging from molecular level (Ammerman et al, 2008). 0.08 to 1.25 mg/ml. The hexane extract of This cell line is known to be susceptible to H. cordata was the only H. cordata extract many viruses, such as Chikungunya virus active against the three species of Candida (Sourisseau et al, 2007), and has visible tested. This extract and the chloroform cytopathic effects found upon infection. and ethyl acetate extracts of H. cordata This study reported for the first time had fungistatic activity against Aspergillus the antiviral activity of H. cordata, L. fumigatus. Houttuynia cordata is the only chinensis and S. uncinata against Chikun- plant extract active against this filamen- gunya virus. A literature search revealed tous fungus. Aspergillus fumigatus gave the H. cordata has potential antiviral activity lowest susceptibility index (16.7%) among against human (Hayashi et al, 1995; Lau et the fungi tested in this study. al, 2008; Lin et al, 2009) and animal (Choi Antibacterial assay et al, 2009) viruses. Houttuynia cordata has Extracts of the three medicinal plants been reported to have activity against generally had less antibacterial activity human immunodeficiency virus type 1, than antifungal activity. Forty-three point herpes simplex virus type 1, influenza five percent of the bioassays showed bac- virus (Hayashi et al, 1995), herpes simplex teriostatic activity and 25.9% of the bioas- virus type 2 (Chiang et al, 2003), severe says showed bactericidal activity (Table acute respiratory syndrome coronavirus 3). The hexane extracts of L. chinensis and (Lau et al, 2008), enterovirus-71 (Lin et al, S. uncinata had the lowest MIC and MBC 2009), porcine epidemic diarrhea virus values in this study, both with 0.16 mg/ (Choi et al, 2009), dengue virus type 2 ml against B. cereus. The chloroform and and mouse hepatitis virus (Chiow et al,

624 Vol 48 No. 3 May 2017 Chinese Medicinal Plants with Antimicrobial Activities

2016). The methanol extract of L. chinen- and S. aureus) and K. pneumoniae were sis has been found to have in vitro and susceptible to the non-polar or less polar in vivo inhibitory activity against herpes extracts (hexane, chloroform or ethyl ac- simplex virus type 1 (Kuo et al, 2008). Two etate) while A. baumannii was only suscep- compounds, uncinoside A and B, isolated tible to the ethanol and methanol extracts, from S. uncinata have been reported to which are polar in nature. Escherichia coli have antiviral activity against respiratory was resistant to all extracts examined, syncytial virus and parainfluenza type 3 but P. aeruginosa was susceptible to all virus (Ma et al, 2003). The ethyl acetate the extracts of H. cordata. This plant has extract of S. uncinata has been found to also been reported to have antibacterial have antiviral activity against herpes sim- activity against acne-inducing bacteria plex virus type 1 and coxsackie B3 virus (Propionibacterium acnes and Staphylococ- (Jiang et al, 2008). In our study, we found cus epidermidis) (Chomnawang et al, 2005), L. chinensis had stronger antiviral activity typhoid fever-causing bacteria (Salmo- against Chikungunya virus than H. cordata nella typhimurium) (Kim et al, 2008) and and S. uncinata. methicillin-resistant Staphylococcus aureus This study also reported the antifun- (Sekita et al, 2016). These results support gal activity of H. cordata and S. uncinata the traditional use of H. cordata as an an- for the first time. The hexane extract of tiseptic (Zheng, 1998). The antibacterial H. cordata had broad spectrum antifungal activity of L. chinensis was also reported activity against all six fungi tested in the by Zhang et al (2013) using the ethanol study. Extracts of S. uncinata also had extract of L. chinensis. antifungal activity against all the tested We found resistance to the extracts of fungi except A. fumigatus. All the extracts the studied plants by some microorgan- of L. chinensis had antifungal activity isms, such as the filamentous fungus A. against at least one species of fungus. In a fumigatus and E. coli bacterium. Aspergil- study of 58 traditional Chinese medicinal lus fumigatus and E. coli are regarded as plants, Zhang et al (2013) found the etha- nosocomial pathogens among humans. nol extract of L. chinensis had no activity Resistance to antimicrobials has made against C. albicans or A. fumigatus, even treatment difficult or even impossible at the highest concentration of 1.0 mg/ml. (von Baum and Marre, 2005; Arendrup, Their findings agree with our findings: the 2014). The search for medicinal plants ethanol extract of L. chinensis had a MIC against these microorganisms needs to value of 1.25 mg/ml against C. albicans continue. but had no inhibitory activity against A. The antimicrobial activities of the fumigatus even at a dose of 2.50 mg/ml. extracts of H. cordata, L. chinensis and S. The broad spectrum antibacterial uncinata were found to be dependent activities of S. uncinata were reported for on the species of microorganism, tested the first time in this study. These activi- concentration and type of solvent used ties were only found in extracts derived for extraction. The chloroform extract of from less polar solvents, such as hexane L. chinensis was the most potent extract (except against E. coli), chloroform and against Chikungunya virus; this warrants ethyl acetate. The different extracts of H. further investigation to identify the active cordata had different antibacterial activi- compounds and mechanisms. The plant ties. Both gram-positive bacteria (B. cereus extracts of H. cordata and S. uncinata could

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