Anti-infective activities of 11 plants species used in traditional medicine in Malaysia

Accepted Manuscript Anti-infective activities of 11 plants species used in traditional medicine in Malaysia Nadiah Syafiqah Nor Azman, Mohd Shahadat Hossan, Veeranoot Nissapatorn, Chairat Uthaipibull, Parichat Prommana, Khoo Teng Jin, Rahmatullah Mohammed, Tooba Mahboob, Chandramathi Samudi Raju, Hassan Mahmood Jindal, Banasri Hazra, Mohd Ridzuan Mohd Abd Razak, Vijay Kumar Prajapati, Rajan Kumar Pandey, Norhaniza Aminudin, Khozirah Shaari, Nor Hadiani Ismail, Mark S. Butler, Vladimir V. Zarubaev, Christophe Wiart C

PII:

S0014-4894(18)30117-6

DOI:

10.1016/j.exppara.2018.09.020

Reference:

YEXPR 7618

To appear in:

Experimental Parasitology

Received Date: 1 March 2018 Revised Date:

2 July 2018

Accepted Date: 23 September 2018

Please cite this article as: Nor Azman, N.S., Hossan, M.S., Nissapatorn, V., Uthaipibull, C., Prommana, P., Jin, K.T., Mohammed, R., Mahboob, T., Raju, C.S., Jindal, H.M., Hazra, B., Mohd Abd Razak, M.R., Prajapati, V.K., Pandey, R.K., Aminudin, N., Shaari, K., Ismail, N.H., Butler, M.S., Zarubaev, V.V., Wiart C, C., Anti-infective activities of 11 plants species used in traditional medicine in Malaysia, Experimental Parasitology (2018), doi: https://doi.org/10.1016/j.exppara.2018.09.020. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Anti-infective activities of 11 plants species used in traditional

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medicine in Malaysia

Nadiah Syafiqah Nor Azman1, Mohd Shahadat Hossan1, Veeranoot Nissapatorn2,***, Chairat Uthaipibull3, Parichat Prommana3, Khoo Teng Jin1, Rahmatullah Mohammed4,**, Tooba Mahboob5, Chandramathi Samudi Raju6, Hassan Mahmood Jindal6 , Banasri Hazra7, Mohd Ridzuan Mohd Abd Razak8, Vijay Kumar Prajapati9, Rajan Kumar

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Christophe Wiart C1,*

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Pandey9, Norhaniza Aminudin10, Khozirah Shaari11, Nor Hadiani Ismail12, Mark S Butler13, Vladimir V. Zarubaev14,

1

School of Pharmacy, Faculty of Science, University of Nottingham Malaysia Campus, 43500 Semenyih, Malaysia.

2

School of Allied Health Sciences and 3Research Excellence Center for Innovation and Health Products (RECIHP), Walailak University, 80161 Nakhon Si Thammarat, Thailand.

3

National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Khlong Luang, 12120 Pathum Thani, Thailand.

Department of Pharmacy, Faculty of Life Science, University of Development Alternative, 1207 Dhaka, Bangladesh.

5

Department of Parasitology and 6Department of Medical Microbiology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.

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4

Department of Pharmaceutical Technology, Jadavpur University, 70032 Kolkata, India.

8

Herbal Medicine Research Center, Institute for Medical Research, 50588 Kuala Lumpur, Malaysia.

9

Department of Biochemistry, School of Life Sciences, Central University of Rajasthan, 305817 Rajasthan, India.

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7

Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.

11

Laboratory of Natural Products, Institute of Bioscience, University Putra Malaysia, 43400 Serdang, Malaysia.

12

Atta-ur-Rahman Institute for Natural Products Discovery, Universiti Teknologi MARA Puncak Alam, 42300 Kuala Selangor, Malaysia.

13

Institute for Molecular Bioscience, University of Queensland, QLD 4072 St Lucia, Australia.

14

Pasteur Institute of Epidemiology and Microbiology, 14 Mira str., 197101 St. Petersburg, Russia.

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* Corresponding author. E-mail address: [email protected] (C. Wiart). ** Corresponding author. E-mail address: [email protected] (M. Rahmatullah). *** Corresponding author. E-mail address: [email protected] (V. Nissapatorn).

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Abstract

Treatment of drug resistant protozoa, bacteria, and viruses requires new drugs with alternative

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chemotypes. Such compounds could be found from Southeast Asian medicinal plants. The present study examines the cytotoxic, antileishmanial, and antiplasmodial effects of 11 ethnopharmacologically important plant species in Malaysia. Chloroform extracts were tested for

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their toxicity against MRC-5 cells and Leishmania donovani by MTT, and chloroquine-resistant Plasmodium falciparum K1 strain by Histidine-Rich Protein II ELISA assays. None of the

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extract tested was cytotoxic to MRC-5 cells. Extracts of Uvaria grandiflora, Chilocarpus costatus, Tabernaemontana peduncularis, and Leuconotis eugenifolius had good activities against L. donovani with IC50 < 50 µg/mL. Extracts of U. grandiflora, C. costatus, T. peduncularis, L. eugenifolius, A. subulatum, and C. aeruginosa had good activities against P.

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falciparum K1 with IC50< 10 µg/mL. Pinoresinol isolated from C. costatus was inactive against L. donovani and P. falciparum. C. costatus extract and pinoresinol increased the sensitivity of Staphylococcus epidermidis to cefotaxime. Pinoresinol demonstrated moderate activity against

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influenza virus (IC50 = 30.4 ± 11 µg/mL) and was active against Coxsackie virus B3 (IC50 = 7.1 ± 3.0 µg/mL). β-Amyrin from L. eugenifolius inhibited L. donovani with IC50 value of 15.4 ± 0.01

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µM. Furanodienone from C. aeruginosa inhibited L. donovani and P. falciparum K1 with IC50 value of 39.5 ± 0.2 and 17.0 ± 0.05 µM, respectively. Furanodienone also inhibited the replication of influenza and Coxsackie virus B3 with IC50 value of 4.0 ± 0.5 and 7.2 ± 1.4 µg/mL (Ribavirin: IC50: 15.6 ± 2.0 µg/mL), respectively. Our study provides evidence that medicinal plants in Malaysia have potentials as a source of chemotypes for the development of antiinfective leads.

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Keywords: Antimalarial; Antileishmanial; Antiviral; Antibacterial; Medicinal plants; Natural

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products; Malaysia

1. Introduction

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Natural products from medicinal plants in India, Thailand, China, and Indonesia have substantial potentials as a source of anti-infective chemotypes (Pras et al., 1991; Kayser et al.,

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2003; Misha et al., 2009; Hazra et al., 2012; Somsak et al., 2015; Chokchaisiri et al., 2015; Yenjai et al., 2004). Prior to current intense palm oil-induced deforestation, Peninsular Malaysia was endowed with a one of the richest medicinal flora on earth (Gimlet, 1930; Andaya & Andaya, 2016). The antiprotozoal potentials of medicinal plants used in traditional Malay

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medicine remain unknown despite the use of several hundred plants for the treatment of infectious diseases (Burkill, 1966).

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Leishmania donovani (L. donovani), the protozoa responsible for visceral leishmaniasis, has developed resistance to antileishmanial drugs such as pentavalent antimonials (Croft & Olliaro,

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2011). In India, resistance of L. donovani to pentavalent antimonials resulted in the banning of this drug in the nineties (Prajapati, personal communication). Miltefosine is also used for the treatment of visceral leishmaniasis, but it is teratogenic and requires a long oral treatment (Croft & Olliaro, 2011). As per WHO recommendation there is an urgent need to develop safe and effective antileishmanial medicines to tackle this epidemiological situation (WHO, 2017).

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Plasmodium falciparum (P. falciparum) infection is a major health concern (Snow et al., 2005). According to the latest World Malaria Report, there were 216 million cases of malaria reported in 2016, up from 211 million cases reported in 2015 (WHO, 2017a). Most malaria cases

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in 2016 were in the WHO African Region (90%), followed by the WHO Southeast Asia Region (3%), and the WHO Eastern Mediterranean Region (2%) (WHO, 2017b). Treating P. falciparum infection is difficult because of resistance to quinine, chloroquine, artemisinin (Farnert et al., and

even

artemisinin-based

combination

therapy

(Wongsrichanalai,

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2012),

2013;

Wongsrichanalai & Sibley, 2013). In brief, enlarging the chemotherapeutic antileishmanial and

necessity (Walochnik & Duchêne, 2016).

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antiplasmodial arsenal with safe, affordable, and effective compounds is an urgent therapeutic

In an attempt to control bacterial resistance, WHO recommends to developing new

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antibacterial agents (WHO 2014). However, approvals for new antibacterial compounds by the FDA have been decreasing (Blakovitch et al., 2017). Traditional antibiotic structures have been almost exhausted to the point that antibacterial research is literally crying for new chemotypes

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that could be found by using fresh and different research approaches. The resistance of Grampositive Staphylococcus epidermidis to cefotaxime has been problematic in hospital settings for

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decades (Raad et al., 1998; Tacconelli et al., 2007). Medicinal plants in Malaysia have the ability to synthesize a fascinating array of low molecular weight compounds with structures completely unrelated to antibiotics. The pandemic influenza A/H1N1 (Spanish flu) in 1918-19, the Asian flu virus A (H2N2) in 1957, and the Hong Kong influenza (H3N2) in 1968 claimed the life of millions of patients (Swartz & Luby, 2007). Vaccination can only provide a limited control of the spread of infection due to continuous antigenic drift (Cox, 2005). For this reason, the search

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for inhibitors of influenza A virus among natural products from medicinal plants is a promising route in the selection of lead compounds for preclinical studies (Arakawa et al., 2009). Despite the large variety of illnesses caused by Coxsackie virus, very few specific antiviral drugs are

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available. Coxsackie virus B3 is developing resistance to ribavirin (Beaucourt & Vignuzzi, 2014).

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In this context, we examined the effects of ethnopharmacologically selected medicinal plants used by Malays in two preserved villages of the State of Perak against L. donovani and P.

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falciparum. The aims of our study were: (i) to examine the properties of 11 ethnopharmacologically important medicinal plants in Malaysia against L. donovani and P. falciparum, (ii) to identify at least 1 plant with good or antileishmanial and/or antiplasmodial activity (iii), and to identify at least 1 compound with anti-infective activity from active plant, to

products.

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2. Material and methods

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contribute to the development of safe, effective, and inexpensive anti-infectious plant-based

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2.1. Chemicals and reagents

Chloroform and analytical grade acetonitrile were purchased from RCI Labscan (Bangkok, Thailand). 3-(4,5-dimethylthiazolyl-2) 2,5-diphenyltetrazolium bromide was purchased from ICN Biochemicals Inc. (Ohio, USA). DMSO was purchased from Sigma Aldrich (St Louis, USA). Chloroquine and dihydroartemisinin (>98% purity) were purchased from Sigma Aldrich

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Sigma Aldrich (St Louis, USA). Vincristine and β-amyrin (>98% purity) were purchased from ChemFaces (Wuhan, China). Normal human lung cells (MRC-5 cells), MDCK, and Vero cells were purchased from the American Tissue Culture Collection (Rockville, USA). RPMI-1640,

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minimal essential medium and fetal bovine serum were purchased from Life Technologies, Grand Island (New York, USA). Glutamine-Penicillin-Streptomycin was purchased from GIBCO. SYBR Green I was purchased from Takara BioMedicals (Tokyo, Japan). 96-well plates

were

purchased

from

Microlon,

Greiner

(Frickenhausen,

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ELISA

Germany).

Immunoglobulin M (IgM) capture antibody was purchased from ICL Inc. (Newberg, USA). 5,5-

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Tetramethylbenzidine chromogen was purchased from Zymed Lab. Inc. (San Francisco, USA). Miltefosine and L. donovani (BHU-1251 strain) were kind gifts from Professor Shyam Sundar, Banaras Hindu University, India. P. falciparum 3D7, K1, and V1/S strains were obtained from the Malaria Research and Reference Reagent Resource Center (MR4) (Virginia, USA).

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Oseltamivir carboxylate was obtained from Hoffmann LaRoche (Basel, Switzerland) and

rimantadine from Sigma-Aldrich (Missouri, USA).

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2.2. Collection of medicinal plants

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Survey for evaluation and documentation of the use of medicinal plants by Malays was performed from 1th to the 25th of August 2015 according to ethnopharmacological criteria (Cotton 1996). The survey was done around and in Manong and Kuala Kangsar, State of Perak, Malaysia (4°77’ North, 100°9’East) (Figure 1). Information gathered allowed the selective collection of 11 medicinal plants, which were identified by botanists at the National Herbarium of the Forest Research Institute of Malaysia (FRIM). Voucher herbarium specimens with

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vernacular names, collecting localities, and dates of collections were deposited at the Department of Pharmacy, Faculty of Science, University of Nottingham Malaysia Campus. The collected leaves, stems, fruits, or rhizomes were separated and air-dried at room temperature for two

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weeks. The dried materials were then finely pulverized by grinding using aluminum collection blender (Philips, China) and the powders obtained were weighted with top loading balance

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(Sartorius AG, Germany).

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2.3. Preparation of extracts

Dried plant powders (200 g) were soaked at room temperature with chloroform for selective extraction of mid-polar compounds (Harborne 1998). Each extraction was performed in triplicate by maceration of plant powder-to-solvent ratio of 1:5 (w/v) for 3 days at room temperature. The

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respective liquid extracts were subsequently filtered through qualitative filter papers No 1, Whatman International Ltd. (Maidstone, UK) using aspirator pump (EW-35031-00, 18 L/min, 9.5l Bath, 115 VAC), and the filtrates were concentrated to dryness under reduced pressure at

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40°C using rotary evaporator, Buchi Labortechnik AG (Flawil, Switzerland). The dry extracts obtained were weighed and stored in tightly closed glass scintillation vials (Kimble, USA) at -

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20°C until further use. The yields of chloroform extracts were calculated using the following formula:

Mass of dry extract % yield =

______________________________________________

×

100

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Mass of dried plant powder

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2.4. Cytotoxic assay

The microtetrazolium test (MTT) of Mosmann (1983) was used to study the cytotoxicity of

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extracts or compounds in vitro. Briefly, the extracts or compounds were serially diluted 2-fold in minimal essential medium from 0 to 100 µg/mL and from 0 to 10 µg/mL, respectively. Cells

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were incubated for 48 h at 37°C in 5% CO2 in the presence of the dissolved substances. Cells were washed twice with saline, and a solution of 3-(4,5-dimethylthiazolyl-2) 2,5diphenyltetrazolium bromide (0.5 mg/mL) in saline was added to the wells. After 1 h incubation, the wells were washed and the formazan residue dissolved in DMSO (0.1 mL per well). The

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optical density of cells was then measured on a Victor2 1440 multifunctional reader, Perkin Elmer (Turku, Finland) at a wavelength of 535 nm and plotted against the concentration of the extracts or compounds. The optical density was plotted against the concentration. Vincristine

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was used as positive cytotoxic control drug. All experiments were performed in triplicate and results were expressed as the concentration reducing the number of living MRC-5, MDCK, or

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Vero cells by 50% (CC50). Selectivity Index (SI) was determined as the ration of CC50 (cells) to the IC50 (Protozoa).

2.5 Antileismanial assay

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The method of Prajapati et al. (2013) was used to determine the in vitro toxicity of extracts or compounds against L. donovani (BHU-1251 strain). Parasites were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum and 1% L-Glutamine-Penicillin-Streptomycin-

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Solution at 26°C temperature in BOD incubator. Stationary stage of L. donovani promastigotes was used for the antileishmanial assay. Parasites (1×105 promastigotes) were added to each well. Together, media and parasites were used as negative control. Miltefosine was used as the

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positive antileishmanial control drug. Following 72 h of incubation at 26°C, MTT assay was performed and the absorbance was measured at 540 nm, to check the viability of promastigotes.

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All experiments were performed in triplicate and results were expressed as the concentration reducing the number of living promastigotes by 50% (IC50).

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2.6. Antiplasmodial assay

Toxicity of extracts against chloroquine-resistant P. falciparum K1 strain was evaluated using the Histidine-Rich Protein II ELISA technique following Noedl et al. (2002). Parasites were

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grown in a continuous culture as described by Trager and Jensen (1977). Synchonization of malaria culture to one stage was done according to Lambros and Vanderberg (1979).

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Chloroquine and dihydroartemisinin were used as the positive antiplasmodial control drugs. All experiments were performed in triplicate. Results were expressed as the concentration reducing the number of living parasites by 50% (IC50).

The toxicity of pinoresinol against P. falciparum 3D7 (chloroquine-sensitive), K1, and V1/S (chloroquine-resistant) strains was determined using malaria SYBR Green I-based fluorescence

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(MSF) assay (Smilkstein et al., 2004). Synchonized ring stage parasites at 1% parasitemia, and 2% hematocrit were put into the individual well of 96-well plates, while non-parasitized erythrocytes at 2% hematocrit served as reference controls. Pinoresinol was prepared serially

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diluted in complete medium and dispensed into the test wells. The plates were incubated at 37°C 48 h. At the end of the incubation period, the SYBR Green I solution was added to each well and mixed by a microplate mixer. After 1 h incubation in the dark at room temperature, the

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fluorescence signal was measured with a Spectramax M5 Multi-Mode Microplate Reader (Molecular Devices, USA) with excitation and emission wavelength bands centered at 485 and

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535 nm, respectively. Chloroquine and dihydroartemisinin were used as the positive antiplasmodial control drugs. All experiments were performed in triplicate. Results were expressed as the concentration reducing the number of living parasites by 50% (IC50).

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2.7. Isolation and identification of compounds

Pinoresinol (1): Chloroform extract of stems of Chilocarpus costatus Miq. (Apocynaceae) (5

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g) was dissolved in acetonitrile-water (5 L; 90:10) to make a solution with a concentration of 1 mg/mL. A total volume of 60 mL was separated using high-pressure liquid chromatography

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(HPLC) (Dionex Ultimate 3000, Thermo Fisher (Massachusetts, USA), which was equipped with a binary pump, diode array detector (DAD), autosampler, and automatic fraction collector (AFC). Acetonitrile/ ultrapure water (90: 10 ratio) was used as mobile phase with flow rate of 1 mL/min, detected by DAD at 210 nm UV wavelength, injection of 100 µL/min, at 37ºC, using a reverse phase Hypersil™ ODS C18 column, 250 × 4.6 mm, particle size 5 µm, Thermo Fisher (Massachusetts, USA). A linear gradient system was applied to the separation: 20-80%

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acetonitrile in 14 min, 80% acetonitrile in 3 min, 80-20% acetonitrile in 1 min, and 20% in 7 min. Collection of one major peak at retention time of 9 min allowed the isolation of compound

literature (Guz & Stermitz, 2000; Diep et al., 2007).

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1 (3 mg) identified as pinoresinol by comparison of its 1H-NMR and EIMS data with that from

Zeylenol (2) and ferrudiol (3): Chloroform extract of leaves of Uvaria grandiflora Roxb. ex

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Hornem (Annonaceae) (1.5 g) was dissolved in acetonitrile-water (90:10; 1.5 L) to make a solution with a concentration of 1 mg/mL. A total volume of 60 mL from this solution was

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passed into HPLC, Agilent 1200 (Santa Clara, USA) equipped with a binary pump, diode array detector (DAD), autosampler, and automatic fraction collector (AFC). Acetonitrile/ ultrapure water (90:10 ratio) was used as mobile phase with flow rate of 1 mL/min, detected by DAD at 210 nm UV wavelength, injection of 60 µL/min, at 37oC, using reverse phase C18 column of

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Biphenyl 100A, size 150 x 4.6 mm, particle size 5 µm, Kinetex® Phenomenax (California, USA). A linear gradient system was employed for the separation: 10% acetonitrile in 2 min, 1075% acetonitrile in 12 min, 75% acetonitrile in 10 min, 75-90% acetonitrile in 4 min, 90%

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acetonitrile in 4 min, 90-10% acetonitrile in 2 min, and 100% acetonitrile in 3 min. Two peaks were collected at a retention time of 11.01 min and 16.25 min allowing the isolation of

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compound 2 (2 mg) and 3 (1.5 mg), respectively, which were identified as zeylenol and ferrudiol by comparison of their 1H-NMR and EIMS data with that from literature (Jolad et al., 1981; Kijjoa et al., 2002; Wirasathien et al., 2006).

Leuconolam (4) and epigallocatechin (5): The chloroform extract of leaves of L. eugenifolius (10.01 g) was subjected to silica gel 60 (0.063-0.2nm, 70-230 mesh ASTM), Merck

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(Darmstadt, Germany) open column chromatography eluted with ethylacetate- hexaneisopropanol (50:10:40), and chloroform-methanol (100:0 to 10:90) to afford 185 fractions Each collected fraction was monitored by analytical thin layer chromatography (TLC) on silica gel 60

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F aluminium sheet (0.2 mm thickness, Merck (Darmstadt, Germany) using chloroform-methanol (90:10 to 95:5) as eluent. The spots of TLC were visualized under ultraviolet light at 254 nm and 365 nm and were next sprayed with Dragendorff’s reagent to detect the presence of alkaloids.

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Fractions with similar TLC profiles were combined to give 8 major fractions (LC-1 – LC8). The fraction LC-7 was passed on open column using silica gel 60 (0.063-0.200 mm, 70-230 mesh

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ASTM), Merck (Darmstadt, Germany) eluted with ethyl acetate-diethylamine-toluene (80:10:10) to provide 10 fractions (LC-7.1-LC-7-10). Combination of LC-7-1 and LC-7-2 afforded after recrystallization compound (4) 1.1 mg. Compound (4) was identified as leuconolam by comparison of its 1H-NMR and EIMS data with that from literature (Abe & Yamauchi, 1993).

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The fraction LC-8 yielded after recrystallization compound (5) 0.8 mg, identified as epigallocatechin by comparison of its 1H-NMR and EIMS data with that from literature (Abe &

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Yamauchi, 1993).

Furanodienone (6): Chloroform extract (0.5 g) of rhizomes of Curcuma aeruginosa Roxb.

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presented white solid material that was separated from the oil by recrystallization with cold ice hexane into a solid crystalline material Compound (6) (10.2 mg). Compound (6) was identified as furanodienone by comparison of its 1H-NMR and EIMS data with that from literature (Makabe et al., 2006).

2.8 Antibacterial assay

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Stock cultures of bacteria used for this study were kindly provided by the Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Malaysia. The following

25923),

methicillin-resistant

Staphylococcus

aureus

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human pathogenic bacteria were used as tested organisms: Staphylococcus aureus (ATCC (clinical

isolate),

Staphylococcus

epidermidis (ATCC 12228), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC

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25922), Pseudomonas aeruginosa (ATCC 15442), and Klebsiella pneumoniae (clinical isolate). Determination of minimum inhibitory concentration (MIC) was performed by broth

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microdilution assay according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2012). Briefly, bacterial strains were grown for 18-24 h at 37°C. Direct suspension of the colonies was made in cationically adjusted Müeller-Hinton broth (CAMHB) and adjusted to OD625 0.08-0.1 which corresponds to 1 ~ 2×108 CFU/mL followed by serial 10-fold dilutions to

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give 1×106 CFU/mL. Bacterial suspension (50 µL) was added to 96-well round bottom microtiter plates containing an equal volume of extracts or compounds at different concentrations and the 96-well plates were incubated for 24 h at 37°C. The minimum inhibitory concentration (MIC) is

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defined as the lowest concentration of material tested that completely inhibits the growth of

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bacteria. Vancomycin and rifampicin were used as the positive antibiotics control drugs.

2.9 Synergistic interaction assay

The ability of C. costatus extract and pinoresinol to increase the sensitivity of Staphylococcus epidermidis towards cefotaxime or vancomycin was measured by fractional inhibitory concentration (FIC) indices (FICI) (Giacometti et al. 1999). FICI is the sum of the FIC of extract

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or compound and FIC of antibiotic calculated according to the following formula (Berenbaum 1978):

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MIC (extract or compound in the presence of antibiotic) FIC (extract or compound) = _______________________________________________

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MIC (extract or compound alone)

MIC (antibiotic in the presence of extract or compound) ______________________________________________

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FIC (antibiotic) =

MIC (antibiotic alone)

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FICI = FIC (extract or compound) + FIC (antibiotic)

The FICI results are interpreted as such: ≤ 0.5 synergistic, >0.5 to 1 additive, 1 to 4 indifferent; ≥

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4 antagonistic (Schelz et al., 2006).

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2.10 Anti-influenza virus assay

Influenza virus A/Puerto Rico/8/34 (H1N1) was obtained from the collection of viruses of Influenza Research Institute, St Petersburg, Russia. Prior to the experiment, the virus was propagated in the allantoic cavity of 10-12 days-old chicken embryos for 48 h at 36ºC. Infectious titer of the virus was determined in MDCK cells (ATCC # CCL-34) in 96-well plates in alphaMEM medium, Biolot (St. Petersburg, Russia). Pinoresinol and furanodienone at increasing concentrations (4-300 µg/mL) were incubated with MDCK cells at 36°C for 1 h. The cell culture 14

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was then infected with corresponding viruses at a multiplicity of infection (moi) 0.01 and incubated for 1 h at 36°C in the presence of 5% CO2. The culture medium was then removed and replaced with fresh one containing the same concentrations of material to be tested. Plates were

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incubated at 36°C in the presence of 5% CO2 for 24 h followed by virus titer determination by TCID50 for 48 h. Pinoresinol and furanodienone were tested in triplicate. The 50% inhibiting

used as the positive control antiviral drugs.

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2.11 Anti-Coxsackie virus B3 assay

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concentration (IC50) and SI were calculated from the data obtained. Oseltamivir carboxylate was

Coxsackie virus B3, strain Nancy, provided by the D.I. Ivanovsky Institute of Virology, Moscow, Russia, was propagated in Vero cells and stored at -70°C until use. Vero cells were grown in minimum essential medium (MEM) with 7% cattle serum, 100 U/mL of amphotericin

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B and 100 U/mL of ceftriaxone per mL at 36.5°C in an atmosphere of 5 % CO2. Furanodienone (3-300 µg/mL) was incubated with Vero cells at 36°C for 1 h. The cell culture was then infected with CVB3 (m.o.i. 0.01) and incubated for 1 h at 36°C in the presence of 5% CO2. The culture

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medium was then removed and replaced with fresh one containing the same concentrations of the compound. Plates were incubated at 36°C in the presence of 5% CO2 for 24 h following by virus

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titer determination by TCID50 for 48 h. All concentrations of the compounds were tested in triplicate. The 50% inhibiting concentration (IC50) and the selectivity index (SI, CC50 to IC50 ratio) were calculated from the data obtained.

2.12. Statistical analysis

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Results were expressed as the mean ± standard deviation for three independent experiments. Student’s unpaired t-test was used to evaluate the difference between the test sample and the

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negative (solvent) control. A p-value below 0.05 was considered statistically significant.

3. Results

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3.1. The collection of medicinal plants

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Survey for evaluation and documentation of the use of medicinal plants used in day-to-day practice by Malays in two preserved villages (Kuala Kangsar and Manong) in the State of Perak, Peninsular Malaysia (Figure 1) afforded the collection of 11 plants from eight different families in the Class Magnoliopsida (Dicotyledons): Annonaceae, Apiaceae, Apocynaceae, Clusiaceae,

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Lamiaceae, Myrsinaceae, Piperaceae, and Zingiberaceae (Table 1) (Takhtajan 2009). Six plants were used to treat infections. Chilocarpus costatus Roxb., Leuconotis eugenifolius A.DC and

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Tabernaemontana peduncularis Wall. belong to the Apocynaceae Juss. (1789).

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3.2. The preparation of extracts

Leaves, stems, fruits, or rhizomes powders were extracted with chloroform. The average yield values ranged from 0.01 (T. peduncularis) to 10% (Garcinia atroviridis Griff. ex T. Anderson, Clusiaceae) (Table 2).

3.3. The cytotoxic activity of extracts against MRC-5 cells

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Prior to antileishmanial and antiplasmodial testing, it was necessary to evaluate the potential toxicity of chloroform extracts against mammalian cells to calculate SI Extracts were tested

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against normal human lung fibroblast cells (MRC-5) (Table 3). The lowest cytotoxic concentration 50% (CC50) was demonstrated for Piper longum L. (Piperaceae) with a value of 20.0 ± 1.5 µg/mL. C. costatus and Curcuma aeruginosa Roxb. (Zingiberaceae) exhibited CC50

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values above 100 µg/mL. The remaining extracts had CC50 above 20 µg/mL.

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3.4. The antileishmanial activity of extracts against L. donovani

We investigated the susceptibility of L. donovani to chloroform extracts in vitro (Table 3). Nine extracts out of 11 tested had 50% inhibitory concentrations (IC50) below 100 µg/mL.

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Chloroform extract of stems of C. costatus inhibited L. donovani with IC50 of 17.3 ± 0.01 µg/mL and SI above 5.7. L. eugenifolius, T. peduncularis, and U. grandiflora demonstrated IC50 of 21.7 ± 3.4, 27.3 ± 0.03, and 40.5 ± 0.05 µg/mL, respectively. Eryngium foetidum L. (Apiaceae), G.

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atroviridis, Ocimum americanum L. (Lamiaceae), Embelia ribes Burm.f. (Myrsinaceae), P. longum, and Amomum subulatum Roxb. (Zingiberaceae), and C. aeruginosa had IC50 above 90

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µg/mL.

3.5. The antiplasmodial activity of extracts toward P. falciparum K1

We further examined the toxicity of chloroform extracts against chloroquine-resistant P. falciparum K1. Six extracts out of 11 demonstrated IC50 values below 5 µg/mL (Table 3).

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Extract of T. peduncularis had the lowest IC50 (1.1 ± 1.0 µg/mL) and SI of 86.8. C. costatus had an IC50 value of 1.3 ± 1.5 µg/mL and SI > 76.9. Extract of C. aeruginosa yielded an IC50 value of 4.0 ± 0.05 µg/mL (SI> 25). L. eugenifolius, U. grandiflora, and A. subulatum demonstrated IC50

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values of 3.0 ± 1.3, 3.7 ± 1.0, and 4.8 ± 2.5 µg/mL, respectively. E. foetidum, G. atroviridis, O.

3.6 The isolation and identification of compounds

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americanum L, E. ribes, and P. longum demonstrated IC50 above 100 µg/mL.

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HPLC analysis of the chloroform extract of stems of C. costatus extract yielded a major compound (1) which was identified as pinoresinol (Figure 2 and 4). HPLC analysis of the chloroform extract of leaves of U. grandiflora yielded compound (2) and (3), identified as zeylenol and ferrudiol, respectively (Figure 3 and 4). Column chromatography of the chloroform

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extract of L. eugenifolius yielded leuconolam (4) and epigallocatechin (5) (Figure 4). The extract of C. aeruginosa yielded by recrystallization compound (6) identified as furanodienone (Figure

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4).

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3.7 The cytotoxic, antileishmanial, and antiplasmodial activity of compounds

The toxicity of pinoresinol, ferrudiol, and zeylenol, β-amyrin, a known constituent of L. eugenifolius (Sim et al., 1971), and furanodienone towards MRC-5 cells were examined (Table 4). All 5 compounds tested demonstrated CC50 value above 20 µM. β-Amyrin had the highest activity against L. donovani with IC50 and SI: 15.4 ± 0.01 µM and 13, respectively, followed by furanodienone (39.5 ± 0.2 µM; SI> 1.0). Comparatively, L donovani was less susceptible to

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pinoresinol, ferrudiol, and zeylenol with of IC50 of 213.4 ± 0.2, 101.1 ± 0.3, and 110.0 ± 0.1 µM, respectively. Pinoresinol demonstrated IC50 of 203.1 ± 5.9, 180.8 ± 5.7, and 202.5 ± 12.7 µM for chloroquine-sensitive P. falciparum 3D7, chloroquine-resistant P. falciparum K1, and V1/S

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strains, respectively.

3.8. The antibacterial and synergistic activity of the chloroform extract of C. costatus and

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pinoresinol

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We next examined the antibacterial activity of the chloroform extract of C. costatus and its major constituent pinoresinol against 4 Gram-positive bacteria: Staphylococcus aureus, methicillin-resistant S. aureus, S. epidermidis, and Enterococcus faecalis and 3 Gram-negative bacteria: Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae by broth

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microdilution technique. The extract and pinoresinol inhibited S. epidermidis with MIC value of 187.5 ± 0.2 and 750 ± 0.5 µg/mL, respectively and demonstrated MIC values above 1500 µg/mL against the remaining 6 bacteria tested. (Table 5). We examined the synergistic effects of the

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chloroform extract of C. costatus and pinoresinol with vancomycin or cefotaxime towards S. epidermis (Table 6). Both extract and pinoresinol increased the sensitivity of S. epidermidis to

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cefotaxime. No synergistic effects were observed with vancomycin.

3.9 Antiviral activity of pinoresinol and furanodienone

Pinoresinol demonstrated moderate activity against influenza virus A/Puerto Rico/8/34 (H1N1) with IC50 of 30.4 ± 11 µM (SI: 6) and was active against Coxsackie virus B3 with IC50 =

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7.1 ± 3 µM (SI: 18) (Ribavirin IC50: 15.6 ± 2.0 µg/mL). Furanodienone inhibited the replication of H1N1 in MDCK with an IC50 value of 4.0 ± 0.5 µg/mL and SI of 5.9. Furanodienone inhibited the replication of Coxsackie virus B3 in Vero cells with an IC50 value of 7.2 ± 1.4 µg/mL and SI

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of 8 (Ribavirin IC50: 15.6 ± 2.0 µg/mL).

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4. Discussion

There is an urgent necessity to identify new leads for the treatment of infectious diseases.

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Medicinal plants could be of value as a source of anti-infective drugs (Kayser et al., 2003). In this context, we examined the antiprotozoal properties of 11 ethnopharmacologically selected medicinal plants used by Malays in preserved villages of the state of Perak, Peninsular Malaysia. The choice of collecting medicinal plants from this geographical area was based on the fact that

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(i) it escaped deforestation and (ii) no antiprotozoal reports are available on these plants. Plants were selected when fruits or flowers were present to allow absolute botanical identification by FRIM botanists. Plants were extracted with chloroform to extract mid-polar materials (Harborne,

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1998) which could penetrate protozoal cells (Surade & Blundell, 2012).

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We first assessed the in vitro toxicity of extracts to normal human diploid embryonic lung cells (MRC-5) at a serially diluted range of assay concentrations (0-100 µg/mL). The American National Cancer Institute defines extracts as toxic to human cells when CC50 values are below 20 µg/mL (Hashim et al., 2012). According to this classification, none of the extracts were toxic to MRC-5 cells. The least cytotoxic extracts were the extracts of C. costatus and C. aeruginosa with CC50 values above 100 µg/mL.

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We next examined the in vitro toxicity of extracts against L. donovani. Osorio et al. (2007) classifies the antileishmanial activity of extract as high: EC50 < 10 µg/mL, good: IC50:10 > and <

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50 µg/mL; moderate: EC50 > 50 and < 100 µg/mL, and inactive for extract with EC50 > 100 µg/mL. Accordingly, U. grandiflora, C. costatus, L. eugenifolius, and T. peduncularis, had good antileishmanial activity. SI for C. costatus was above 5, which according to Zirihi et al. (2005)

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indicates selective toxicity for protozoa. To the best of our knowledge, the selective antileishmanial activity of C. costatus is being reported for the first time. Apocynaceae are

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known for their antileishmanial properties (Munoz et al., 1994; Roberts, 2013). The good activity of U. grandiflora against L. donovani which corroborate previous study (Ankisetty et al., 2006).

We next examined the in vitro toxicity of extracts against chloroquine-resistant P. falciparum

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K1 strain. Rasoanaivo et al. (2011), and Bagavan et al. (2011) both define good activities for extract for IC50 below 10 µg/mL. Accordingly, U. grandiflora, C. costatus, T. peduncularis, L. eugenifolius, A. subulatum, and C. aeruginosa had good activities for P. falciparum K1

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(Rahman, 2012). Members of the genus Leuconotis Jack (1823) and Tabernaemontana L. (1753) are known for antiplasmodial activity (Nugroho et al., 2012; Marinho et al., 2016). To the best of

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our knowledge, good activities from these 6 medicinal plants are being reported for the first time. We found that E. foetidum and E. ribes were both inactive confirming previous studies (Roumy et al., 2007; Chenniappan & Kadarkarai, 2010). SI were all above 5, which according to Zirihi et al. (2005) indicates a selective toxicity for protozoa. T. peduncularis was the most active plant against P falciparum K1 with SI of 86.8. We were unable to isolate any antimalarial compounds

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from this plant due to an insufficient amount of extract and further studies are warranted. The next most active plant was C. costatus with SI above 76.9.

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HPLC analysis of the chloroform extracts of stems of C. costatus yielded a major constituent identified as pinoresinol. To the best of our knowledge, it is the first report on the occurrence of pinoresinol in the genus Chilocarpus Blume (1823). We tested pinoresinol for toxicity against

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MRC-5 and found and it was not toxic according to the American National Cancer Institute, which defines the threshold for cytotoxicity of pure at 10 µM (Efferth & Kuete, 2010). Mbongo

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et al. (1997) define antileishmanial activity for compounds with IC50 below 50 µM. Liu et al. (2003) classify antileishmanial activity of compounds as very good : EC50 value < 5 µM, good: 5 – 10 µM, moderate: EC50 10 - 20 µM, and weak: EC50 > 20 µM. Chollet et al. (2008) define as moderately active compounds with IC50 value of 50 µM. Accordingly, pinoresinol had no

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activity against L. donovani. In a previous study, pinoresinol was inactive against L. major (Glaser et al., 2015). Mahmoudi et al. (2006) classifies the antiplasmodial activity of pure compounds as follow: IC50 < 0.06 µM: very active, IC50 between 0.06 and 5 µM: active, and IC50

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> 5 µM: inactive. Accordingly, pinoresinol was inactive for chloroquine-sensitive P falciparum 3D7, chloroquine-resistant P. falciparum K1, and V1/S strains. To the best of our knowledge, the

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inactivity of pinoresinol is being reported for the first time against P. falciparum 3D7, and V1/S strains. Our results contradict Apisantiyakom et al. (2013) who reported that pinoresinol inhibited P. falciparum K1 with IC50 of 3.4 µg/mL. With pinoresinol being inactive, it is reasonable to infer that some antiplasmodial minor constituents are present in the extract. The HPLC profile demonstrated minor peaks, which could not be analyzed by spectroscopy and further work is required to characterize these minor constituents.

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We tested the extract of C. costatus and pinoresinol against a panel of 7 human pathogenic bacteria. Fabry et al. (1998) define active extracts as having MIC values below 8000 µg/mL.

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Kuete (2010) and Cos et al. (2006) used stricter endpoint criteria, in which crude extracts with MIC values less than 100 µg/mL are active. Further, Kuete (2010) classifies as weakly active extracts with MIC above 625 µg/mL. Accordingly, the extract of C. costatus had a moderate

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activity against S. epidermidis. According to Kuete (2010) the antibacterial activity of pure compounds are classified in 3 categories: MIC < 10 µg/mL: high; MIC between 10 and 100

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µg/mL: medium and weak for MIC above 100 µg/mL. Accordingly, pinoresinol had a weak activity against S. epidermidis. Minor constituents in the chloroform extract may have interesting antibacterial effects and further studies are needed. We cannot explain the selectivity of the extract and pinoresinol against S. epidermidis. Wright (2000) proposed to develop agents

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“resisting” resistance as a strategy to antibiotic resistance. We looked into the synergic effects of C. costatus and pinoresinol with cefotaxime or vancomycin against S. epidermidis and found the extract and pinoresinol to be synergistic with cefotaxime. To the best of our knowledge, the

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synergistic effects of C .costatus and pinoresinol against S. epidermidis are reported for the first time. We examined the antiviral properties of pinoresinol and found that pinoresinol

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demonstrated moderate activity against influenza virus A/Puerto Rico/8/34 (H1N1) and activity against Coxsackie virus B3. To the best of our knowledge the antiviral activity of pinoresinol against Coxsackie virus B3 is reported for the first time.

Column chromatography of the chloroform extract of leaves of L. eugenifolius yielded the indole alkaloid leuconolam and epigallocatechin, which could not be tested for cytotoxicity,

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antileishmanial, and antiplasmodial activities due to an insufficient amount available. It should be noted that indole alkaloids elaborated by members of the family Apocynaceae are known to have antileishmanial activity (Ferreira et al., 2004). Nugroho et al. (2012) isolated series of

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indole alkaloids from the leaves of L. griffithii, which demonstrated antiplasmodial effect against P. falciparum 3D7. Epigallocatechin itself is not known for antiplasmodial activity. Therefore more work needs to be done with this plant. β-Amyrin is known constituent from L. eugenifolius

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(Sim et al., 1971). Although we did not isolate β-Amyrin from L. eugenifolius, we decided to test β-amyrin commercial standard. Testing commercial natural product standard is a norm in natural

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product pharmacology (Lee et al., 2013; Menon et al., 1999; Mahboob et al., 2017). This pentacyclic triterpene was not toxic for MRC-5 cells according to National Cancer Institute guidelines (Abu-Dahab & Afifi, 2007) and was moderately but selectively (SI> 5) active against L. donovani. Passos et al. (2017) reported that hexane fraction of Brosimum glaziovii Taub.

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(Moraceae) containing both α and β-amyrin was active against L. amazonensis. In a previous study, lup-20(29)-en-ol was active against L. amazonensis and β-amyrone against L. donovani (Fournet et al., 1992). Also, hexane extracts of seeds and leaves from Artemisia annua L.

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(Asteraceae) were active against L. donovani, a property ascribed to their content of β-amyrin acetate and β -amyrin (Islamuddin et al., 2012). We could not test the activity of β-amyrin

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against P. falciparum because of limited amount available. To the best of our knowledge, it is the first report on the moderate but selective antileishmanial properties of β-amyrin against L. donovani. To date, there are no antileishmanial drugs based on triterpene structure, and it would be of interest to further assess the antileishmanial activities of β-amyrin synthetic derivative.

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Ankisetty et al. (2006) isolated grandiuvarone A from U. grandiflora which inhibited L. donovani with IC50 of 0.7 µg/mL (positive control pentamidine IC50 of 1.7 µg/mL). We therefore examined constituents in the chloroform extract of leaves of U. grandiflora by HPLC and

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isolated zeylenol and ferrudiol. We were unable to characterize the other peaks because of impurity and then insufficient amount and further work need to be done with this plant. Zeylenol is a known constituent of U. grandiflora (Seangphakdee et al., 2013) whereas ferrudiol is

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reported for the first time from this plant. We tested zeylenol and ferrudiol for cytotoxicity against MRC-5, which were both non-toxic. Zeylenol and ferrudiol were both inactive against L.

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donovani. Our result diverge from Roumy et al. (2009) reporting that mixture of 4,6,20 trihydroxy-6-[10’ (Z)-heptadecenyl]-1-cyclohexen-2-one and 1,4,6-trihydroxy-1,20 -epoxy-6[10’ (Z)-heptadecenyl]-2-cyclohexene from Tapiria guianense Aubl. (Anacardiaceae) were active against both P. falciparum and L. amazonensis with IC50 values below 5 µM. A reason for

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that difference could be that our study strain is different from the strain mentioned in the above studies. It is well known that strains present in the Indian subcontinent have developed tolerance against antileishmanial drugs because of drug pressure. We hypothesize that because of this there

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is a differences between our study and Roumy et al. (2009). Zeylenol and ferrudiol could not be tested for antiplasmodial activity due to a limited amount available. It is reasonable to infer that

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other constituents participate in the activity of U. grandiflora and more work needs to be done with this plant.

The Zingiberaceae A. subulatum and C. aeruginosa had both good and selective activities against P. falciparum K1. Heilmann et al. (2000) reported antiplasmodial activity from A. aculeatum. Our results with C. aeruginosa are in agreement with Murnigsih et al. (2005). We

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isolated furanodienone from C. aeruginosa and found that it is weakly active against L. donovani and inactive against P. falciparum K1. Other constituents in the chloroform extract of C. aeruginosa may account for antiplasmodial activity and further work need to be done with this

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plant. We examined the antiviral activity of furanodienone and found that it inhibited the replication of H1N1 and Coxsackie virus B3. Furanodienone was 2.1 times more active than ribavirin against Coxsackie virus B3. To the best of our knowledge, the strong antiviral activity

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of furanodienone against H1N1 and Coxsackie virus B3 is reported for the first time.

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In conclusion, we collected 11 ethnopharmacologically important medicinal plants used by the Malays of Perak in Peninsular Malaysia for traditional medicine. Out of these plants, C. costatus, L. eugenifolius, T. peduncularis, U. grandiflora, A. subulatum, and, C. aeruginosa demonstrated good antiprotozoal effects in vitro. More phytochemical work needs to be done

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with these plants. β-Amyrin from L. eugenifolius, pinoresinol from C. costatus, and furanodienone from C. aeruginosa have to potential be used for the development of antiinfective leads. We demonstrate that the medicinal plants of Malaysia have significant potential

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as a source of anti-infective drugs and further investigation in that field is warranted on that

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incredibly rich medicinal flora before its complete extinction owed to palm oil deforestation.

Acknowledgments

This work was financed by the Ministry of Higher Education Malaysia (Fundamental Research Grant Scheme FRGS/1/2014/SG01/UNIM/02/1), the University of Malaya Research Grants UMRG 544/14HTM and UMRG 362-15AFR, the Thailand National Science and

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Technology Development Agency (P1450883), and the National Center for Genetic Engineering and Biotechnology and Thailand Research Fund (RSA5880064). We acknowledge the support. We would like to express our gratitude to Professor Shyam Sundar for providing L. donovani and

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miltefosine. We are also very grateful to Mr. Hassan Saberi bin Ibrahim and Mr. Muhammad Kamarul Ikhwan bin Idris for their kind assistance during field plant collection.

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Table 1. Traditional therapeutic properties of 10 medicinal plants from Malaysia

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________________________________________________________________________________________________________________________________________________ Family, genus species and authority

Voucher No

Datea

Locality

Malay name

Part

Medicinal uses

Annonaceae: 048

15/6/2016

Eryngium foetidum L.

031

22/6/2016

Apiaceae:

Apocynaceae: 035

23/6/2015

036

18/6/2015

Tabernaemontana peduncularis Wall

063

19/6/2015

Stems

Wounds Yaws

Manong

Deranjang

Stems

Syphilis

19/6/2015

Manong

Asam gelugor

Fruits

Unspecified

27/6/2015

Kuala Kangsar

Kemangi

Leaves

Unspecfied

26/6/2015

Kuala Kangsar

Akar sulur kerang

Fruits

Intestinal worms

20/6/2015

Manong

Ladah pnjang

Fruits

Post-partum infection

065

19/6/2015

Manong

Kenamgi

Fruits

Unspecified

066

19/6/2015

Manong

Temu hitam

Rhizomes Medicinal food

036

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Curcuma aeruginosa Roxb.

044

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Piperaceae:

Amomum subulatum Roxb.

Stomach aches

Leaves

Myrsinaceae:

Piper longum L.

Leaves

Getah gerip puteh

Lamiaceae:

Zingiberaceae:

Ketumbar Jawa

Wounds

Akar getah garah

Garcinia atroviridis Griff. ex T. Anderson 077

Embelia ribes Burm.f

Kuala Kangsar

Leaves

Kuala Kangsar

Clusiaceae:

Ocimum americanum L.

Pisang pisang

Manong

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Chilocarpus costatus Miq. Leuconotis eugenifolius A.DC

Manong

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Uvaria grandiflora Roxb. ex Hornem

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________________________________________________________________________________________________________________________________________________ a

Date of collection: day/month/year

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Table 2. Percentage yields (w/w) of selected 10 medicinal plants from Malaysia __________________________________________________________________________ Part extracteda

Family, genus species

% Yield

Leaves

Fruits

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Stems Leaves Stems

0.1

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Leaves

0.5

0.2 0.9 0.01

10.0

1.0

Stems Fruits

2.3 2.0

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Leaves

Fruits Rhizome

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Annonaceae: Uvaria grandiflora Apiaceae: Eryngium foetidum Apocynaceae: Chilocarpus costatus Leuconotis eugenifolius Tabernaemontana peduncularis Clusiaceae: Garcinia atroviridis Lamiaceae: Ocimum americanum Myrsinaceae: Embelia ribes Piper longum Zingiberaceae: Amomum subulatum Curcuma aeruginosa

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0.6 0.2

__________________________________________________________________________ a

Maceration of plant powder-to-solvent ratio of 1:5 (w/v) at room temperature for 3 days.

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Table 3. Cytotoxic, antileishmanial, and antiplasmodial activities of extracts (µg/mL) ____________________________________________________________________________________________________________ MRC-5 [CC50 ]

L. donovani [IC50 ]

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Family, genus species

P. falciparum K1 [IC50 ]

____________________________________________________________________________________________________________ 40.5 ± 0.05 (SI: 0.6)

3.7 ± 1.0 (SI: 6.9)

34.6 ± 0.05

93.2 ± 1.2

>100

>100 57.6 ± 1.5 95.5 ± 1.3

17.3 ± 0.01 (SI> 5.7) 21.7 ± 3.4 (SI: 2.6) 27.3 ± 0.03 (SI: 3.4)

1.3 ± 1.5 (SI> 76.9) 3.0 ± 1.3 (SI: 19.2) 1.1 ± 1.0 (SI: 86.8)

>100

>100

92.4 ± 0.01

>100

87.5 ± 0.6

96.0 ± 0.2

>100

20.0 ± 1.5

>100

>100

35.9 ± 0.8 >100

98.5 ± 2.5 91.0 ± 1.6

4.8 ± 2.5 (SI: 7.4) 4.0 ± 0.05 (SI> 25)

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25.5 ± 0.4

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40.8 ± 0.08

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25.6 ± 2.3

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Annonaceae: Uvaria grandiflora Apiaceae: Eryngium foetidum Apocynaceae: Chilocarpus costatus Leuconotis eugenifolius Tabernaemontana peduncularis Clusiaceae: Garcinia atroviridis Lamiaceae: Ocimum americanum Myrsinaceae Embelia ribes Piperaceae: Piper longum Zingiberaceae: Amomum subulatum Curcuma aeruginosa

Positive control drugs: Vincristine Miltefolsine Chloroquine Dihydroartemisinin

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____________________________________________________________________________________________________________ 2.4 ± 1.5 NT NT NT

NT 1.8 ± 2.5 NT NT

NT NT >31.9 0.001 ± 0.6

____________________________________________________________________________________________________________ Each value represents mean ± standard deviation of 3 independent experiments. SI: Selectivity index NT: Not tested

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Table 4. Cytotoxic, antileishmanial, and antiplasmodial activities of compounds (µM)

MRC-5 [CC50]

L.d [IC50 ]

P.f 3D7 [IC50]

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__________________________________________________________________________________________________________ P.f. K1 [IC50 ]

P.f. V1/S [IC50]

__________________________________________________________________________________________________________ 201.0 ± 0.05 > 27.9 > 20.4 > 26.0 > 43.4

15.4 ± 0.01 (SI: 13.0) 213.4 ± 0.2 101.1± 0.3 110.0 ± 0.1 39.5 ± 0.2 (SI> 1.0)

NT 203.1± 5.9 NT NT NT

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β-Amyrin Pinoresinol Ferrudiol Zeylenol Furanodienone

NT 180.8± 5.7 NT NT 17.0 ± 0.05 (SI> 2.5)

NT 202.5± 12.7 NT NT NT

____________________________________________________________________________________________________________ 0.3 ± 1.5 NT NT NT

NT 4.6 ± 2.5 NT NT

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Positive control drugs: Vincristine Miltefolsine Chloroquine Dihydroartemisinin

NT NT 0.01 ± 2.8 0.004 ± 0.4

NT NT 0.1 ± 14.6 0.005 ± 0.6

NT NT 0.15 ± 14.7 0.002 ± 0.3

_________________________________________________________________________________________________________

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Each value represents mean ± standard deviation of 3 independent experiments L.d: L. donovani; P.f 3D7: P. falciparum 3D7; P.f. K1: P. falciparum K1; P.f. V1/S: P. falciparum V1/S SI: Selectivity Index NT: Not tested

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Table 5. Minimum inhibiting concentration of C. costatus extract and pinoresinol against bacteria (µg/mL) ____________________________________________________________________________________________________________ Mean MIC

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_____________________________________________________________________________________

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Gram-positive ______________________________________________________ S.a MRSA S.e E.f

Gram-negative __________________________________________ E.c P.a K.p

___________________________________________________________________________________________________________ Extract Pinoresinol

>625 >1500

>625 >1500

187.5 ± 0.2 750 ± 0.5

>625 >1500

>625 >1500

>625 >1500

>625 >1500

__________________________________________________________________________________________________________ 0.7 NT

1500 ± 0.03 NT

1.5 NT

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Antibiotic standards: Vancomycin Rifampicin

11.7± 0.1 NT

NT 11.7± 0.5

NT 11.7± 0.1

NT 11.7± 0.01

____________________________________________________________________________________________________________

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Each value represents mean ± standard deviation of 3 independent experiments b S.a: Staphylococcus aureus (ATCC 25923); MRSA: Methicillin-resistant Staphylococcus aureus (clinical isolate); S.e: Staphylococcus epidermidis (ATCC 12228); E.f: Enterococcus faecalis (ATCC 29212); E.c: Escherichia coli (ATCC 25922); P.a: Pseudomonas aeruginosa (ATCC 15442); K.p: Klebsiella pneumoniae (clinical isolate) NT: Not tested

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Table 6. Fractional inhibitory concentration index (FICI) of different combination of C. costatus extract and pinoresinol and antibiotics against Staphylococcus epidermidis

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FICI Effect ________________________________________________________________________________________________________ Cefotaxime Vancomycin

0.5 1.1

Synergistic Indifferent

Pinoresinol

Cefotaxime Vancomycin

0.5 2.2

Synergistic Indifferent

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Extract

________________________________________________________________________________________________________

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Values are given as mean of triplicate. FICI: ≤ 0.5 synergistic, >0.5 to 1 additive, 1 to 4 indifferent; ≥ 4 antagonistic (Schelz et al. 2006).

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Table 7. Antiviral activity of pinoresinol and furanodienone against influenza virus A/Puertorico/8/34 (H1N1) (MDCK cells) and

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Coxsackie virus B3 (Vero cells) (µg/mL )

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_____________________________________________________________________________________________________________________ H1N1 Coxsackievirus B3 ____________________________ ____________________________ CC50 IC50 SI CC50 IC50 SI _____________________________________________________________________________________________________________________

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Pinoresinol 182.4 ± 2.5 30.4 ± 11 6 127.8 ± 1.5 7.1 ± 3.0 18 Furanodienone 23.9 ± 1.9 4.0 ± 0.5 5.9 58.8 ± 4.7 7.2 ± 1.4 8 _____________________________________________________________________________________________________________________ Antiviral positive standard: Oseltamivir carboxylate 50.6 ± 4.3 0.051 ± 0.006 992 NT NT NT Ribavirin NT NT NT 203.3 ± 16.4 15.6 ± 2.0 13 _____________________________________________________________________________________________________________________

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Each value represents mean ± standard deviation of 3 independent experiments SI: Selectivity index NT: Not tested

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Figure 1. Map of Perak State in Peninsular Malaysia and survey areas (circled). 34

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Figure 2: High-performance liquid chromatography (HPLC) profile of the chloroform extract of stems of C. costatus (0-25 min). The peak of compound (1) is indicated.

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Figure 3: High-performance liquid chromatography (HPLC) profile of the chloroform extract of leaves of U. grandifolia (0-35 min). The peak of compounds (2) and (3) are indicated. 36

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Figure 4: The structure of pinoresinol (1), zeylenol (2), ferrudiol (3), leuconolam (4), epigallocatechin (5), and furanodienone (6)

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Treatment of drug resistant protozoa, bacteria, and viruses requires new drugs with alternative chemotypes.

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The present study examines the cytotoxic, antileishmanial, and antiplasmodial effects of 11 ethnopharmacologically

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important plant species in Malaysia.

Chloroform extracts were tested for their toxicity against MRC-5 cells and Leishmania donovani by MTT, and chloroquine-resistant Plasmodium falciparum K1 strain by Histidine-Rich Protein II ELISA assays.

None of the extract tested was cytotoxic.

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Uvaria grandiflora, Chilocarpus costatus, Tabernaemontana peduncularis, and Leuconotis eugenifolius had good

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activities against L. donovani with IC50 < 50 µg/mL.

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Extracts of U. grandiflora, C. costatus, T. peduncularis, L. eugenifolius, A. subulatum, and C. aeruginosa had good activities against P. falciparum K1 with IC50< 10 µg/mL.

Pinoresinol isolated from C. costatus was inactive against L. donovani and P. falciparum. C. costatus, increased the

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sensitivity of Staphylococcus epidermidis to cefotaxime, and was active against Coxsackie virus B3 (IC50 = 7.1 ± 3.0 µg/mL).

β-Amyrin from L. eugenifolius inhibited L. donovani with IC50 value of 15.4 ± 0.01 µM.

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Furanodienone from C. aeruginosa inhibited L. donovani and P. falciparum K1 with IC50 value of 39.5 ± 0.2 and 17.0

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± 0.05µM, respectively. Furanodienone also inhibited the replication of influenza and Coxsackie virus B3 with IC50

value of 4.0 ± 0.5 and 7.2 ± 1.4 µg/mL (Ribavirin: IC50: 15.6±2.0 µg/mL), respectively.

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Our study provides evidence that medicinal plants in Malaysia have potentials as a source of chemotypes for the

development of anti-infective leads.