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Journal of Ethnopharmacology

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Screening for bioactive secondary metabolites in Sri Lankan medicinalplants by microfractionation and targeted isolation of antimicrobialflavonoids from Derris scandensSupun Mohottia,b, Sanjeevan Rajendrana,b, Taj Muhammada, Adam A. Strömstedta,Achyut Adhikaric, Robert Burmana, E.D. de Silvab, Ulf Göranssona, C.M. Hettiarachchib,Sunithi Gunasekeraa,⁎

a Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, SE-751 23, Uppsala, SwedenbDepartment of Chemistry, Faculty of Science, University of Colombo, Thurston Rd, Colombo 03, Sri Lankac Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal

A R T I C L E I N F O

Keywords:Sri LankaMedicinal plantsMicrofractionationAntimicrobial activityCytotoxicityFlavonoids

A B S T R A C T

Ethnopharmacological relevance: Sri Lanka is known to have very diverse flora. Many of these species are used forplant-based remedies, which form the integral part of two Sri Lankan systems of traditional medicine, Ayurvedaand Deshiya Chikitsa. Despite their widespread use, only a limited number of studies have probed into thescientific evidence for bioactivity of these medicinal plants. Such studies rarely progress to the identification ofbioactive natural products.Aim of the study: The primary aim was to develop a bioactivity screening method and apply it to 50 Sri Lankanmedicinal plants where antimicrobial properties could be relevant for its traditional use. The subsequent aim wasthe progression into defining and characterising potent isolates within targeted compound classes from suchplants, i.e. Derris scandens and its antimicrobial flavonoids.Material and methods: The plant collection comprised 24 species of Fabaceae, 15 Rubiaceae, 7 Solanaceae and 4Cucurbitaceae plants. These 50 species were collected based on their ethnopharmacological importance and usein Sri Lankan traditional medicine. Crude extracts from each species were initially subjected to radial discdiffusion and microdilution assays. Subsequently, aqueous extracts of all plants were microfractionated in deepwell plates using reversed-phase HPLC. Fractions were tested for antibacterial and cytotoxic activities andmasses of target bioactive compounds were identified using mass spectrometry. Bioactive compounds with themasses identified through microfractions were isolated from Derris scandens using reversed-phase HPLC. Theisolated pure compounds were characterised using LC-MS and NMR.Results: Crude aqueous extracts from 19 species showed activity against Gram-positive bacteria (Staphylococcusaureus and Bacillus cereus) in the radial disc diffusion assay. Crude aqueous extracts from 34 plant species andorganic extracts from 46 plant species were active against S. aureus (≤4mgmL-1) in the microdilution assay.Microfractionation demonstrated antibacterial activity for 19 plants and cytotoxicity for 6 plants. Furthermore,target bioactive compounds and their molecular ions were identified during microfractionation. Dalpanitin andvicenin-3, two of the flavonoids isolated from Derris scandens gave MICs of 23 μgmL-1 against S. aureus.Dalpanitin also exhibited relevant MICs on Gram-negative bacteria (94 μgmL-1 against Escherichia coli andPseudomonas aeruginosa).Conclusion: The microfractionation protocol developed in this study enabled time-efficient screening of manyplants species, using a small quantity of sample material. In addition, microfractionation served as a guiding toolfor identifying individual antimicrobial compounds. Through this process, flavonoids were isolated from Derrisscandens, out of which dalpanitin and vicenin-3 showed activity in the low micromolar range. The high hit rate

https://doi.org/10.1016/j.jep.2019.112158Received 7 May 2019; Received in revised form 9 August 2019; Accepted 11 August 2019

Abbreviations: HPLC-MS, high-performance liquid chromatography-mass spectrometry; NMR, nuclear magnetic resonance; LCQ-MS, liquid chromatographyquadrupole mass spectrometer; LC-UV-MS, liquid chromatography-ultra violet diode array detector-mass spectrometry; ESI, electrospray ionization; HSQC, het-eronuclear single quantum correlation; HMBC, heteronuclear multiple bond correlation; COSY, correlation spectroscopy; MIC, minimum inhibitory concentration;FMCA, fluorometric microculture cytotoxicity assay; TFA, trifluoroacetic acid; FA, formic acid; DMSO, dimethyl sulphoxide; MHA, mueller hinton agar; ATCC,American Type Culture Collection

⁎ Corresponding author. Pharmacognosy, Department of Medicinal Chemistry, Biomedical Centre, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden.E-mail address: sunithi.gunasekera@ilk.uu.se (S. Gunasekera).

Journal of Ethnopharmacology 246 (2020) 112158

Available online 14 August 20190378-8741/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

T

for in vitro antibacterial properties from this ethnopharmacologically guided sample collection gives credence toSri Lankan traditional herbal medicine as a source for drug discovery.

1. Introduction

Sri Lanka is considered a global biodiversity hotspot due to its di-verse and lush ecosystems (Dharmadasa et al., 2016; Myers et al.,2000). Around 30% of Sri Lanka is covered by forest and the island ishome to 3154 angiosperm species, one quarter of which are endemic(Wijesundara et al., 2012). The usage of plants has penetrated much ofthe Sri Lankan culture, particularly in traditional health care. The tra-ditional medical system in Sri Lanka is mainly composed of four dif-ferent practices: Siddha, Unani, Ayurveda, and Deshiya Chikitsa(Weragoda, 1980). The latter two mainly rely on plant and herbalpreparations, used either as single plants or extracts, or complex mix-tures of several species, for the treatment of diseases. The remedies inAyurvedic medical manuscripts are the fruits of over 3000 years ofempirical experience handed down from generation to generation(Liyanaratne, 1991) and as such represent a valuable pre-screeningsource available in traditional medicine.

Medicinal plants have been a valuable source of therapeutic agents,and still many of today's drugs are plant-derived natural products ortheir derivatives (Chalovich and Eisenberg, 2005; Newman and Cragg,2016). Plants used in traditional medicine for over centuries by humanscan be perceived to have undergone clinical testing for safety and ef-ficacy parameters, albeit without current scientific standards andscrutiny. Thus, compounds derived from these plants presumably havegreater potential to succeed in toxicology screening compared to a denovo synthetic chemical compound. However, among antibacterialagents in conventional clinical use, there is a noteworthy absence ofplant-derived compounds so far, despite the long and widespread tra-ditional use of plant-based remedies.

Although medicinal plants have been used as remedies for humandiseases for centuries, most Sri Lankan medicinal plants have not beenchemically studied (Rathnayake and Weerasinghe, 2018). Given thatthere are dynamic changes in plant metabolite profile based on geo-graphical location and environmental condition (Pavarini et al., 2012;

Sampaio et al., 2016), these plants are an untapped resource of naturalproducts awaiting discovery. Most studies investigating bioactiveproperties of Sri Lankan medicinal plants rarely progress to studies atmolecular level due to limited scientific instrumentation for furtherchemical characterisation and inadequate facilities to conduct bioassayslocally. Typically, scientific efforts are limited to reporting bioactivityof crude plants extracts or decoctions.

One such plant species is Derris scandens, locally called ʽkala walʼ. Itgrows throughout Asia and has been used in Ayurvedic, Thai andChinese herbal medicine to treat a variety of pain-related conditions,including muscle pain, joint pain, arthritis and headaches. The stempart is used as a diuretic, expectorant, anti-tussive and anti-dysenteryagent and for muscle pains (Hussain et al., 2015). It is also used for thetreatment of osteoarthritis and inflammation (Kuljittichanok et al.,2018; Laupattarakasem et al., 2004). Notably, it is used by indigenouspeople in Sri Lanka commonly known as ‘veddas’ for wound healingand in a traditional method of fishing to immobilise the fish to facilitatetheir capture (Chandraratne, 2016). The ethanol extract of the plant hasbeen shown to have anti-metastatic activity in cholangiocarcinoma andhepatoma cell lines (Kuljittichanok et al., 2018).

In the present study, we adapted a microfractionation protocol,coupled with mass spectrometry (MS) and bioactivity screening, toenable rapid screening of a large number of medicinal plants tradi-tionally used in Sri Lanka. This method is a targeted and streamlinedapproach, applicable for the isolation of many natural product classes.As shown in Scheme 1, 50 plant species from four families (Fabaceae,Cucurbitaceae, Rubiaceae and Solanaceae) were selected based ontraditional usage by Ayurvedic practitioners and subjected to the mi-crofractionation protocol followed by antibacterial and cytotoxic ac-tivity screening. By following the targeted masses identified from theinitial screening, four known flavonoids were isolated from Derrisscandens and their structures were then identified by comparison ofnuclear magnetic resonance (NMR) spectroscopic data to literaturevalues.

Scheme 1. Isolation of bioactive compounds from D. scandens.

S. Mohotti, et al. Journal of Ethnopharmacology 246 (2020) 112158

2

Table1

Summaryofbiologicalactivitiesreported

andtraditionalmedicinaluseofselected

plantsfrom

SriLanka.

Plant

Vernacularname

Voucher

specimen

No.

Traditionalusesa

Reported

biologicalactivity

bReference

1Adenanthera

pavonina

L.Madatiya

UOC/NPSD/050

Cholera,boils,diarrhoea,inflam

mations,rheum

atism.

Antimicrobial,cytotoxicity

Rodriguesetal.(2018);Moham

med

etal.(2014)

2Alysicarpusvaginalis(L.)DC.

Aswenna

UOC/NPSD/051

Urinarytractdiseases,fever,swellings,diarrhoea,dysentery.

Antibacterial

Aryaetal.(2016)

3Ba

uhinia

acum

inataL.

Elakoboleela

UOC/NPSD/052

Thyroiddisorders,skindiseases,dysentery,leprosy.

Antimicrobial

Alharbietal.(2018)

4Ba

uhinia

purpureaL.

Rathkoboleela

UOC/NPSD/053

Catarrh,boils,glandular,swellings

Antimicrobial

Negietal.(2012)

5Ba

uhinia

racemosaLam.

Nilkobolela

UOC/NPSD/054

Diarrhoea,epistaxis,abortions.

Antimicrobial,antifungal,

antitum

our

Rashed

andButnariu(2014);G

upta

etal.(2004)

6Bu

teamonosperm

a(Lam

.)Taub.

Gaskela

UOC/NPSD/055

Diarrhoea,dysentery,diabetes,ulcers,sorethroat,w

orninfections.

Antibacterial,anticancer,

antifungal

Sharmaetal.(2019);Kariaetal.

(2018);YadavaandTiwari(2007)

7Ca

ssia

alataL.

Eththora

UOC/NPSD/056

Skindiseases,w

ormdiseases,headache,cough,asthma,anorexia.

Antifungal,antibacterial,

cytotoxicity

Somchitetal.(2003);Olarteetal.

(2013)

8Ca

ssia

auriculataL.

Ranawara

UOC/NPSD/057

Skindiseases,bleeding,dysentery,diabetes.

cytotoxicity,antibacterial

Esakkirajanetal.(2014);

Senthilkum

arandReetha

(2011)

9Ca

ssia

fistulaL.

Ehela

UOC/NPSD/058

Coughs,fevers,constipation,rashes.

Antifungal,cytotoxicity,

antibacterial

Sony

etal.(2018);Zhou

etal.

(2017);Seyyednejad

etal.(2014)

10Ca

ssia

toraL.

Pethithora

UOC/NPSD/059

Eyediseases,liverdisorders,ring

worms,boils,constipation.

CytotoxicityagainstHepG2,

antibacterial

Wuetal.(2001);Sahuetal.,2017

11Co

ccinia

grandis(L.)Voigt

Kowakka

UOC/NPSD/074

Diabetesmellitus,w

ounds,swelling,fever,cough,asthma.

Antibacterial

SakharkarandChauhan(2017)

12Crotalaria

retusaL.

Kaha

andanahiriya

UOC/NPSD/060

Diarrhoea

andskindiseases.

Genotoxicity

Silva-Netoetal.(2010)

13Crotalaria

verrucosaL.

Nilandanahiriya

UOC/NPSD/061

Rashes,diseasesofthemouth,cough,asthm

a.Antibacterial,cytotoxicity

Now

rin(2016)

14Da

tura

metelL.

Ela/Sudu

attana

UOC/NPSR/025

Swellings,rheum

atism,lum

bago,tum

ours,cataract,eyediseases,asthm

a,toothache,

dogbites,hydrophobia,hydrocele,severecolds,tuberculosis,insanity,abscesses,

sciatica,generalpains,rabies.

Cytotoxicity

Guo

etal.(2019)

15DerrisScandens(Roxb.)Benth.

Kalawal

UOC/NPSD/062

Reptile

associated

poisoning,bleeding

ofwounds,stom

achinfections.

Cytotoxicity

Kuljittichanoketal.(2018)

16Desm

odiumheterophyllum

(Willd.)DC.

Udupiyaliya

UOC/NPSD/063

Stom

achaches,sores.

Notreported

17ErythrinavariegataL.

Erabadu

UOC/NPSD/064

Wormdiseases,w

ounds,eardiseases,fever,cough,infertility,swelling.

Antibacterial

Tanaka

etal.(2015)

18Geophila

repens(L.)

I.M.Johnst.

Koturubedde

UOC/NPSR/010

c Cough,treatmentofskinfungi.

Antibacterial,antifungal

Raoetal.(2017);Portilloetal.,2001

19Guetta

rdaspeciosaL.

NilPichcha

UOC/NPSR/011

c Cough,cold,wounds,epilepsy,dysentery,headache,fever,boils.

Notreported

20Haldina

cordifolia

(Roxb.)

Ridsdale

Kolon

UOC/NPSR/012

Sores,dysentery.

Antimicrobial,cytotoxicity

Jayasinghe

etal.(2002);Hasan

etal.

(2015)

21Hedyotis

auriculariaL.

GetaKola

UOC/NPSR/013

c Dysentery,diarrhoea,low

eringofbloodpressure.

Notreported

22Indigofera

tinctoriaL.

Nilawariya

UOC/NPSD/065

Nervous

disorder.

Antibacterial

Selvakum

arandKarunakaran(2010)

23Luffa

acyntangula(L.)Roxb.

Darawatakolu

UOC/NPSD/075

Swellings,fevers,eyedisease,skindisease,urinaryproblem.

Anti-cancer

Vanajothiand

Srinivasan

(2015)

24Kn

oxiazeylanicaL.(endem

ic)

Elaratmal

UOC/NPSR/014

c Inflam

mationduetosnakebites.

Notreported

25MimosapudicaL.

Nidikum

baUOC/NPSD/066

Dysentery,diarrhoea,bronchitis,bladderstones,urinarydiseases

Antimicrobial,cytotoxicity

Akteretal.(2010);Chow

dhuryetal.

(2008)

26Mitragynaparvifolia

(Roxb.)

Korth.

Helam

baUOC/NPSR/015

c Fever,colitis.

Antibacterial

Kaushiketal.(2009)

27Morinda

umbellataL.

Kiriw

elUOC/NPSR/016

Bone

fractures.

Cytotoxic

Chiouetal.(2014)

28Mukia

maderaspatana

(L.)

M.Roem.

Heenkekeiri

UOC/NPSD/076

Skindiseases.

Antimicrobial

Banerjee

andThankamani(2013)

29Myroxylon

balsa

mum

(L.)

Harms

Kattakum

anjal

UOC/NPSD/067

Bone

fractures,rheumatoidarthritis,urinarytractdisorders.

Notreported

(continuedon

next

page)

S. Mohotti, et al. Journal of Ethnopharmacology 246 (2020) 112158

3

Table1(continued)

Plant

Vernacularname

Voucher

specimen

No.

Traditionalusesa

Reported

biologicalactivity

bReference

30Nauclea

orientalis(L.)L.

Bakm

iUOC/NPSR/017

Boils,diarrhoea,toothache,tum

ours.

Cytotoxic,antim

alarial

Liuetal.(2018);Heetal.(2005)

31Oldenlandia

bifloraL.

Pepiliya

UOC/NPSR/018

Fever,gastricirritation.

Antibacterial

Sridharetal.(2012)

32Oldenlandia

herbacea(L.)

Roxb.

Walkoththam

alli

UOC/NPSR/019

Diabetesmellitus,m

enstrualcycledisorders,eardiseases,eye

diseases,vom

iting,

leprosy,naso-pharyngealinfections,stomachdisorders,venerealdiseases,parasitic

infections,liverdiseases.

Notreported

33Ophiorrhiza

mungosL.

Dathketiya

UOC/NPSR/020

Diarrhoea,snake

bites,rabies,dyspepsia,cancers.

Anticarcinogenic

Baskaretal.(2011)

34Paederia

foetidaL.

Apasu

madu

UOC/NPSR/021

Flatulence,Rheum

atism,U

rethritis,Vesiclecalculi,Urine

retention

Antibacterial

Pandaetal.(2016)

35Pavetta

lanceolataEckl.

Pavetta

UOC/NPSR/022

Visceralobstruction,haem

orroidalpains,rheumatism,eye

diseases

Antimycobacterial

Aroetal.(2019)

36Pterocarpusmarsupium

Roxb.

Gam

malu

UOC/NPSD/068

Diabetes,wounds,diarrhoea,cholera.

Antimicrobial

Pantetal.(2017)

37PterocarpussantalinusL.f.

Rathhadun

UOC/NPSD/069

Swellings,vom

iting,inflam

mations,fever,dysentery.

Cytotoxicity

Wuetal.(2011)

38Saraca

asoca(Roxb.)Willd.

Asoka

UOC/NPSD/070

Disuria,diarrhoea,dysentery.

Antimicrobial

Jainetal.(2018)

39Sesbania

grandiflora(L.)Pers.

Kathurumurunga

UOC/NPSD/071

Skininfections,epilepsy,rheumatoidarthritis,fever,w

ounds.

Antibacterial,cytotoxicity

Gandhietal.(2017);Royetal.

(2013)

40Solanummacrocarpon

L.Wam

batu

UOC/NPSR/026

c Throatirritation,stom

achache,earinfections,cardiac

diseases.

Antimicrobial

Ilodibiaetal.(2016)

41Solanummelongena

L.Elabatu

UOC/NPSR/027

Oedem

a,body

achesandpains,heartdiseases,asthm

a,coughs,piles,fevers,facial

paralysis,sciatica.

Anticancer

Fekryetal.(2019)

42Solanumnigrum

L.Kalukanw

eriya

UOC/NPSR/028

c Alterative,aphrodisiac,diuretic,laxative,fevers,diarrhoea,eye

diseases,rabies,

headaches,ringworminfestations,allergic,inflam

mations

Anticancer

Laietal.(2016)

43Solanumtorvum

Sw.

Tibbatu

UOC/NPSR/029

Naso-pharyngealinfections,coughs,pains,oedema,gout,diarrhoea,dysentery,

fevers,kidneystones,urinarydiseases,skindiseases,oraldiseases.

Antimycobacterial,cytotoxicity,

antim

icrobial

Ngutaetal.(2016);Balachandran

etal.(2012)

44SolanumvirginianumL.

Katuwelbatu

UOC/NPSR/030

Cough,asthma,fever,anorexia,chestpains,toothache,dropsy,gonorrhea,catarrh,

worminfestations,pruritus,vaginalcandidiasis,impotency,alopeciaareata,

vomiting,dysurea,renalcalculi,tuberculosis

Antibacterial,cytotoxicity

Prashithetal.(2017);Gnanavel

etal.(2015)

45SpermacocehispidaL.

Hingetakola

UOC/NPSR/023

Dyspepsia,colic,flatulence,generaldebility.

Notreported

46TamarindusindicaL.

Siyambala

UOC/NPSD/072

Eyediseases,swelling,fever,vomiting,oraldiseases.

Antibacterial

Limaetal.(2017)

47Tephrosia

purpurea(L.)Pers.

Kathurupila

UOC/NPSD/073

Anorexia,dyspepsia,cough,liver,kidneyobstructions.

Cytotoxicity

Padm

apriya

etal.(2017)

48TrichosanthescucumerinaL.

Pathola

UOC/NPSD/077

Fever,pneumonia,stomachdisorder,dyspepsia,boils,sores,psoriasis.

Anticancer

Prom

kanetal.(2013)

49WendlandiabicuspidataWight

&Arn.(endemic)

Rawanaidala

UOC/NPSR/024

Dysentery,fever,diarrhoea,ulcers,rheumatism.

Antimicrobial

Jayasinghe

etal.(2002)

50Withania

somnifera(L.)Dunal

Amukkara

UOC/NPSR/031

Rheumatism,ulcers,carbuncles,swellings,H

aemorrhoids,ringw

orminfections,

syphilis,nausea,diarrhoea,consumption,nervoussystem

associated

diseases,

apoplexy,insanity,impotence,skindiseases,eczem

a,hypothyroidism

associated

goiter,psoriasis,worminfestation.

Cytotoxicity

Kimetal.(2019)

aThetraditionalusesoftheplantspecieswereobtained

from

thereference,(InstituteofAyurveda,2017).

bAntimicrobialactivity

broadlyreferstoantibacterial,antifungaland/orantiviralactivities.

cTheinformationon

G.repens,G.speciosa,

H.a

uricularia,K

.zeylanica,M

.parvifolia,S

.macrocarpon

andS.

nigrum

wasobtained

throughdiscussionswith

localtraditionalm

edicinepractitioners.

S. Mohotti, et al. Journal of Ethnopharmacology 246 (2020) 112158

4

2. Materials and methods

2.1. Plant material

Plant materials were collected from Bandaranayake MemorialAyurvedic Research Institute, Mahargama and private lands (inKamburupitiya, Mirigama and Pussellawa). Different plant parts (roots,leaves, stem bark, root bark) of 50 plants were collected based on theirtraditional Ayurvedic usage (Table 1). The plant species were initiallyidentified by a field guide. Later, the identity of the plant specimens wasconfirmed by N.P.T. Gunawardena at the National Herbarium of theRoyal Botanical Garden, Peradeniya. A voucher specimen (No. UOC/NPSD/062) of D. scandens and the other plants studied for antibacterialactivity and cytotoxicity were deposited in the herbarium of Depart-ment of Plant Sciences, University of Colombo, Sri Lanka.

2.2. Small scale extraction

Plant parts (roots, leaves, stem bark, root bark) were separatelywashed with water, air dried for 3 to 5 days at room temperature(30 °C) and separately pulverised using a domestic grinder. A portion(150mg) from each plant part was extracted using dichloromethane(CH2Cl2) and methanol (MeOH) (1:1, v/v, 100mL) using an ultra-sonicbath at room temperature and filtered under reduced pressure. Filtratesof repeated extractions were combined and the organic phase wasconcentrated under reduced pressure at 40 °C using a rotary evaporator.The dried crude extracts obtained were further dried using nitrogen gas(N2) and the dry weights were recorded.

2.3. Microfractionation by HPLC

The dried crude extract (20mg) was re-suspended in 10mL ofMeOH and CH2Cl2 (1:1, v/v) and partitioned using 20mL of distilledwater. The aqueous and organic layers were collected separately andMeOH was removed by rotary evaporation. Freeze-dried aqueous ex-tract (2 mg) was dissolved in 1mL of 5% acetonitrile (CH3CN) andfractionated by Shimadzu LC-10 system equipped with a SPD-M10AVPPhotodiode Array Detector (PDA). A Phenomenex Jupiter C18(250×4.6mm, 5 μm) column with a gradient from H2O/0.05% tri-fluoroacetic acid (TFA) (95%)/CH3CN/0.05% TFA (5%) to H2O/0.05%TFA (5%)/CH3CN/0.05% TFA (95%) over 45min at a flow rate of1mLmin-1 was employed. Each 1mL of 45 fractions were collected intoa 96 deep well plate (VWR, Sweden). 100 μL from each well wastransferred into a 96-well U-bottom plate (Greiner Bio-one, USA) forantibacterial activity screening and a 96-well V-bottom plate (ThermoScientific, Denmark) for lymphoma cell (U-937GTB) toxicity screeningseparately. The plates were dried in a centrifugal evaporator (SavantSpeed Vac plus SC110A) and subjected to the respective assays.

2.4. Identification of target masses for isolation

Active fractions from antimicrobial assays were subjected to liquidchromatography-ultra violet diode array detector-mass spectrometry(LC-UV-MS) analysis to determine the corresponding molecular ionspresent in each well. Each active fraction in the deep well plate wasdiluted to a final concentration of 5% CH3CN and analysed byShimadzu LC-10 system equipped with an PDA detector (Kinetex C18,100×4.60mm 2.6 μm, 100 Å) at a flow rate of 1mLmin-1. A gradientfrom H2O/0.1% formic acid (FA) (95%)/CH3CN/0.1% FA (5%) to H2O/0.1% FA (40%)/CH3CN/0.1% FA (60%) over 12min was employed andUV data were collected between 190 nm and 600 nm. Mass spectro-metric analysis of fractions was performed on a LCQ mass spectrometer(Finnigan LCQ, San Jose, CA, USA), equipped with an electrospray io-nization (ESI) source (capillary temperature of 250 °C, capillary voltageof 5 kV, and sheath gas flow of 80 arbitrary units). Dominant molecularions in the active fractions were identified in both positive and negative

ion modes for 100-2000 m/z range.

2.5. Extraction and isolation

D. scandens leaf powder (500 g) was exhaustively extracted in 2 L ofhexane with sonication for 30 min at room temperature. The extractionwas repeated twice and the organic phase from the combined filtrateswas evaporated to dryness under reduced pressure at 40 °C using arotary evaporator. The crude hexane extract obtained was further driedusing N2 gas and the dry weight was recorded. Remaining plant ma-terial from the hexane extraction was dried and sequentially extractedwith CH2Cl2 (2 L), ethyl acetate (EtOAc) (2 L) and MeOH (2 L) followingthe same above procedure.

D. scandens leaf EtOAc extract was partitioned with water and thefreeze-dried aqueous crude extract (50mg) was dissolved in 10mL of5% CH3CN for fractionation on preparative reversed-phase high per-formance liquid chromatography (RP-HPLC) (Waters 600 Controller). Agradient from H2O/0.05% TFA (95%)/CH3CN/0.05% TFA (5%) toH2O/0.05% TFA (20%)/CH3CN/0.05% TFA (80%) over 75min wasemployed to collect 100 fractions of 8mL each using a fraction collector(Pharmacia LKB FRAC 100). The fractions containing the bioactivecompounds and molecular ions identified from microfractionationprotocol were combined, freeze-dried and further purified by semi-preparative RP-HPLC (Jupiter C18, 250× 10.00mm, 5 μm, 300 Å),using a Waters 600 Controller system at a flow rate of 4mLmin−1. LC-UV-MS was performed on the fractions and selected fractions werefurther purified using LH-20. Freeze-dried fractions were then dissolvedin 20mL of MeOH and loaded to Sephadex® LH-20 column(35 cm×1.5 cm), and eluted with MeOH (500mL) with a flow rate of1.8 mLmin-1 to yield fractions of approximately 10mL.

Analytical RP-HPLC was performed on the isolated compounds todetermine the purity on a Shimadzu LC-10 system equipped with a PDADetector (Kinetex C18, 100×4.60mm 2.6 μm, 100 Å) using a gradientfrom H2O/0.1% FA (95%)/CH3CN/0.1% FA (5%) to H2O/0.1% FA(40%)/CH3CN/0.1% FA (60%) over 18min.

(+)HRESIMS of the pure compounds (0.1 mg mL-1) were analysedon nano Acquity UPLC-QTOF mass spectrometer (Waters, Milford, MA)for the mass of range 100-1000 Da using a nano LC Waters C18 column(150× 0.075mm column, 5 μm) with a 0.30 μLmin-1 flow rate. Massspectrometer was calibrated with standard mass to ensure that the masserror was below five ppm. The mass spectrum was obtained in positiveion mode and data was processed through MassLynx V4.1 software.

NMR was performed to determine the structures of the pure com-pounds; NMR spectra for 1D 1H and 13C, 2D heteronuclear singlequantum correlation (HSQC), 2D heteronuclear multiple bond corre-lation (HMBC) and 2D correlation spectroscopy (COSY) experimentswere recorded at 25 °C on a 600MHz (Bruker) NMR spectrophotometer.

2.6. Biological assays

2.6.1. Agar disc diffusion assay for crude extractsAntibacterial activity of the crude extracts was evaluated against S.

aureus (ATCC 25928), B. cereus (ATCC 11778), P. aeruginosa (ATCC9027) and E. coli (ATCC 35218). The bacterial cell suspensions wereprepared according to the following procedure. The bacterial culturewas streaked on Mueller Hinton (MH) agar medium and incubatedovernight at 30 °C. A small inoculum of overnight grown culture wastaken with a sterile loop and dissolved in sterile saline water (0.9%NaCl, Sigma Aldrich, Korea). The cell suspension was ajdusted to adensity of 1× 108 cfu/mL using 0.5 McFarland standards. Each cellsuspension (200 μL) was distributed evenly on the surface of driedMueller Hinton Agar dishes or plates (Hardy, USA; 38.0 g in 1000mL ofdistilled water). A small portion of the dried crude extract was dissolvedin MeOH and CH2Cl2 (1:1, v/v) to reach a final concentration of20mgmL-1. 20 μL of the sample was delivered onto a sterile blank disc(Whatman grade A filter paper discs of 6mm diameter) to obtain 400

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Table 2Antimicrobial activity of crude plant extracts (aqueous and organic) with the methods of agar disc diffusion assay, two-step microdilution assay (MIC on S. aureus),and fluorometric microculture cytotoxicity assay (FMCA on lymphoma cell line). Active fractions (identified as elution time) obtained from microfractionationprotocol were tested against S. aureus and human lymphoma cells.

Plant Plantparta

Mean diameter of the inhibition zone(mm) ± SE 400 μg/disc (agar discdiffusion assayb)

MIC value against S.aureus (ATCC 29213)mgmL-1

(microdilution assay)

Active fractions from microdilution and cytotoxicity assay

B. cereus S. aureus Crudeaqueousextractc

Crudeorganicextract

Active fractions against < spandata-format="italic"data-type="italic"> S. aureus< /span> ,100% inhibition (elution time in min; massin Da - bracketed)

Active fractions againsthuman lymphoma cells,viability≤ 30% (elutiontime in min/mass in Da)

Adenantherapavonina

Leaves – – > 4 0.06 – –

Alysicarpus vaginalis Leaves – – > 4 2.00 – –Bauhinia acuminata Leaves – – > 4 0.50 – –Bauhinia purpurea Leaves – – > 4 1.00 – –Bauhinia racemosa Leaves 8.6 ± 0.3 8.6 ± 0.3 0.50 0.50 22-23 (633.11)

23-24 (617.22)24-25 (545.01)

–

Bauhinia racemosa Stem bark 9.6 ± 0.3 9.3 ± 0.6 0.50 2.00 9-10 (399.33)37-38 (457.54)38-39 (441.85)39-40 (389.32,409.05)

21-22 (424.21)22-23 (535.36)22-23 (635.23543.25)

Butea monosperma Leaves – – > 4 2.00 – –Cassia alata Leaves – – 0.25 0.50 9-10 (569.30)

10-11 (546.21, 489.32)11-12 (685.12)

29-30 (695.23)30-31 (343.25, 474.38)

Cassia auriculata Leaves 8.6 ± 0.3 8.0 ± 0.0 1.00 4.00 – –Cassia fistula Leaves – – > 4 1.00 – –Cassia fistula Stem bark 10.6 ± 0.3 8.6 ± 0.3 0.50 0.50 20-21(605.32,872.16)

21-22 (563.03)–

Cassia fistulad Root bark 12.6 ± 0.3 8.3 ± 0.3 0.06 0.50 18-19 (841.32)20-21 (825.06)25-26 (857.33)26-27 (809.84, 1065.03)28-29 (793.31, 1049.24)

–

Cassia tora Leaves – – > 4 >4 – –Cassia tora Stem bark – – > 4 >4 – –Cassia tora Root bark 9.3 ± 0.3 9 ± 0.5 0.25 1.00 19-20 (924.32,619.15)

20-21 (633.31)21-22 (481.02,585.23)

–

Coccinia grandis Leaves 7 ± 0.0 9 ± 0.0 0.25 2.00 – –Crotalaria retusae Leaves – – > 4 0.06 – –Crotalaria verrucosa Leaves – – 1.00 1.00 – –Datura metele Leaves 13 ± 0.5 11 ± 0.0 0.13 0.03 19-20 (605.33, 689.09)

22-23 (673.13)24-25 (672.32, 671.26)28-30 (655.24, 691.26, 398.26, 556.24)32-33 (639.25, 398.27)

25-26 (608.16, 284.12,704.13)26-28 (673.33, 691.20,655.30)31-32 (639.27, 242.40,398.32)

Derris Scandensd Leaves 10.3 ± 0.3 12 ± 0.0 0.10 4.00 16-17 (463.08)17-18 (565.14,595.14)18-19 (565.14)19-20 (463,611.15)

–

Desmodiumheterophyllum

Stem bark – – 1.00 0.25 – –

Erythuria variegata Leaves – – > 4 >4 – –Erythrina variegata Stem bark – – 1.00 0.25 – –Geophila repens Leaves,

Stem,Root

9 ± 0.5 11 ± 0.5 1.00 0.25 – –

Guettarda speciosa Leaves – – 2.00 2.00 – –Haldina cordifolia Leaves – – 1.00 0.50 – –Hedyotis auricularia Leaves – – 0.30 0.25 – –Indigofera tinctoria Leaves 10.6 ± 0.3 26.6 ± 0.6 0.50 4.00 – –Knoxia zeylanica Leaves,

Stem– 9 ± 0.0 0.30 1.00 30-31 (625.33) –

Luffa acyntangula Leaves – – > 4 2.00 – –Mimosa pudica Leaves – – 2.00 >4 – –Mimosa pudica Stem bark – – 1.00 0.25 – –Mitragyna parvifolia Leaves – – 0.30 1.00 – –Morinda umbellatad Leaves,

Stem,Root

– – 0.10 0.25 26-27 (710.84)40-41 (398.17, 529.10, 713.28)41-42 (677.20, 713.28)

–

(continued on next page)

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Table 2 (continued)

Plant Plantparta

Mean diameter of the inhibition zone(mm) ± SE 400 μg/disc (agar discdiffusion assayb)

MIC value against S.aureus (ATCC 29213)mgmL-1

(microdilution assay)

Active fractions from microdilution and cytotoxicity assay

B. cereus S. aureus Crudeaqueousextractc

Crudeorganicextract

Active fractions against < spandata-format="italic"data-type="italic"> S. aureus< /span> ,100% inhibition (elution time in min; massin Da - bracketed)

Active fractions againsthuman lymphoma cells,viability≤ 30% (elutiontime in min/mass in Da)

Mukia maderspatana Leaves – – > 4 1.00 – –Myroxylon balsamum Leaves – – 1.00 0.5 – –Nauclea orientalis Leaves – – 0.50 0.50 26-27 (521.09)

28-29 (625.29, 869.37,661.44)–

Oldenlandia biflora Leaves – – 2.00 >4 – –Oldenlandia

herbaceaeLeaves,Stem

– – > 4 0.06 21-22 (557.20, 556.94)24-25 (439.18.995.93)30-31 (581.28)

–

Oldenlandia herbacea Root 9 ± 0.0 8 ± 0.5 1.00 0.25 – –Ophiorrhiza mungos Leaves – – 2.00 0.50 26-27 (996.84, 424.95, 406.13) 23-24 (575.15)

27-28 (521.13, 358.03,425.10)

Paederia foetidae Leaves,Stem

– – 0.30 0.06 27-28 (669.42, 661.41) –

Pavetta lanceolata Leaves – – 1.00 0.25 – 19-20 (447.24, 372.27,318.70, 489.18, 368.23)

Pterocarpusmarsupium

Leaves – – > 4 0.25 – –

Pterocarpussantalinus

Leaves 7.3 ± 0.3 8.3 ± 0.3 2.00 4.00 – –

Saraca asoca Leaves – – 0.12 >4 – –Sesbania grandiflorae Stem bark – – 2.00 0.10 18-19 (467.33)

26-27 (769.31)27-28 (899.24,854.36)

–

Sesbania grandiflora Root bark – – > 4 >4 – –Sesbania grandiflora Leaves – – > 4 >4 – –Solanum

macrocarponLeaves,Stem

– 7 ± 0.0 > 4 1.00 – –

Solanum melongena Leaves – – > 4 1.00 – –Solanum melongenae Root,

Stem– – > 4 0.06 45-46 (580.60, 708.55) –

Solanum nigrum Leaves,Stem

– – > 4 0.12 – –

Solanum torvum Leaves 8 ± 0.5 – > 4 0.50 21-22 (487.00, 470.97)39-40 (714.21)

–

Solanum virginianume Leaves,Stem,Root

9 ± 0.5 8 ± 0.0 > 4 0.06 30-31 (647.26, 632.99, 398.18) –

Spermacoce hispida Leaves,Stem

12 ± 2.0 10 ± 1.5 0.30 0.12 – –

Tamarindus indicae Leaves – – 0.25 0.06 – –Tamarindus indicae Stem bark 8.6 ± 0.3 8.6 ± 0.3 0.50 0.10 22-23 (607.32,547.41)

23-24 (621.52)24-25 (597.35,627.02)

–

Tephrosia purpurea Leaves – – > 4 >4 – –Trichosanthes

cucumerinaLeaves – – > 4 1.00 – –

Wendlandiabicuspidatad

Leaves – 8 ± 0.5 0.06 >4 21-22 (616.99, 599.06, 670.17)22-24 (537.43)23-26 (537.43, 762.76)

–

Withania somniferade Leaves 12 ± 0.5 10 ± 0.0 0.06 0.06 25-26 (805.18, 748.79)31-32 (805.18, 493.23, 963.04)

26-27 (655.18)29-31 (639.22, 493.23)

a Plant species for which no inhibition zones or active fractions were found are indicated by a – mark.b S. aureus (ATCC 25928) and B. cereus (ATCC 11778) were used.c Aqueous extracts of all 50 plants were fractionated.dThese plants displayed MIC ≤0.1mgmL-1 for aqueous layer.

e These plants displayed MIC ≤0.1mgmL−1 for organic layer. Zone of inhibition for the positive control (gentamycin, 20 μg/disc); S. aureus- 23 ± 2mm, B.cereus- 25 ± 2mm.

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μg/disc. A MeOH and CH2Cl2 mixture (1:1, v/v, 20 μL) was used as thenegative control and gentamycin (20 μg/disc) was used as the positivecontrol.

2.6.2. Microdilution assay on bacteria and C. albicansA two-step microdilution assay (Strömstedt et al., 2017) was used in

the screening process to enable the detection of antimicrobial com-pounds sensitive to inhibition by rich growth media. This protocol hasthe added benefit of enhancing solubility of the extract, but shouldotherwise result in similar activity readings as the standard broth mi-crodilution assay. The freeze-dried aqueous and organic fractions fromsmall-scale extractions were subjected to minimum inhibitory con-centration (MIC) determination against S. aureus (ATCC 29213). Thecrude extracts were assayed with a 2-fold serial dilution starting at4mgmL-1, with the organic crude extract being pre-dissolved in 10%DMSOaq (16mgmL-1). The prepared microfractionation microplates,containing fractionated and dried aqueous extract, as well as the pur-ified compounds were tested by the same method. In short, the samplesand microbes were co-incubated for 5 h in Tris buffer, after which 5 μLof concentrated (20%) tryptic soy broth was added (1% final

concentration) followed by a further 9 h of incubation. Pure flavonoidsisolated from D. scandens were subjected to microdilution assay againstS. aureus (ATCC 29213), P. aeruginosa (ATCC 27853), E. coli (ATCC25922) and C. albicans (ATCC 90028). Concentrations that exhibitedtotal inhibition on visual inspection were defined as MICs and pre-sented as medians from triplicate experiments.

2.6.3. Lymphoma cell toxicity assayCell toxicity of fractions from microfractionation were evaluated

using the fluorometric microculture cytotoxicity assay (FMCA)(Lindhagen et al., 2008), which is based on monitoring of fluorescencearising from fluorescein that is produced as a result of fluoresceindiacetate (FDA) hydrolysis by cells with intact cell membranes. Thehistiocytic human lymphoma cell line U-937 GTB (Sundström andNilsson, 1976) suspended in growth medium was dispensed into theextract-containing microtiter plates. Each well was seeded with 200 μLof cell suspension, containing approximately 20,000 cells, to give a totalvolume of 200 μL per well. The plates were then incubated for 72 h at37 °C in 5% CO2 atmosphere. The plates were then centrifuged at1000 rpm for 5min at 37 °C, the medium was removed by aspiration,

Fig. 1. (A) HPLC-UV chromatogram (detected at 254 nm) of the crude aqueous extract of D. scandens with 2mg sample loaded on to the column. Compounds isolatedfrom the peak indicated by dashed line showed 100% of inhibition against S. aureus in the microfractions. (B) HPLC trace at 254 nm and mass profile (M + H)+ ofdalpanitin, rutin, vicenin-2 and vicenin-3.

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and the cells were washed with phosphate buffered saline (PBS)[80.0mg of NaCl (Himedia, USA), 2.0 mg of KCl (Sigma), 14.4mg ofNa2HPO4 (Sigma) and 2.4mg of KH2PO4 in 10.0mL of distilled water].FDA (10mg FDA dissolved in 1mL 100% DMSO, w/v) was added topreheated (37 °C) Q2-buffer (40mL of 125mM NaCl, 10mL of 25mMHepes added up to 400mL with MQ-H2O, pH 7.4). A portion of thissolution (100 μL) was then added to each well and incubated for40min at 37 °C. Fluorescence at 538 nm in each well was measured(Thermoscientific Varioskan Flash) with excitation at 485 nm. Thefluorescence in each well is proportional to the number of living cellsand the cytotoxic activity is thus inversely proportional to fluorescenceintensity. The activity of the microfractionated extracts and the purecompounds isolated from D. scandens is reported in terms of survivalindex (SI). It is defined as fluorescence in the experimental wells, ex-pressed as a percentage of the control wells after the fluorescence of theblank wells is subtracted.

3. Results and discussion

3.1. Plant collection

The plant collection comprised 24 species from the Fabaceae, 4Cucurbitaceae, 15 Rubiaceae and 7 Solanaceae. All species were col-lected based on their ethnopharmacological importance and use in SriLankan traditional medicine and practiced by local people. Informationwas gathered by interviewing traditional Ayurvedic practioners andfrom local Ayurvedic books. A summary of the traditional uses for theseplants (Institute of Ayurveda, 2017) is listed in Table 1.

3.2. Screening of crude extracts for antibacterial activity

3.2.1. Agar disc diffusion assayThe aqueous component derived from the MeOH/CH2Cl2 (1:1 v/v)

crude extracts was first subjected to an agar disc diffusion assay toidentify the presence of potential antibacterial compounds. The in-hibitory activity on bacteria was quantified by the diameter of the in-hibition zone. Nineteen of the 50 species collected showed activityagainst Gram-positive bacteria (S. aureus and B. cereus). None of thecrude extracts showed activity against the Gram-negative bacteria, P.aeruginosa and E. coli (Table 2). Among the tested plants, Withaniasomnifera (common local name: Amukkara), Datura metel (Attana),Spermacoce hispida (Hin Getakola) and Cassia fistula (Ehela) showed aninhibition zone of 12–13mm diameter at 400 μg/disc against B. cereus.Geophila repens (Koturu bedde), S. hispida, D. metel, W. somnifera and D.scandens (Kala wal) showed an inhibition zone of 10-12mm diameter at400 μg/disc against S. aureus.

3.2.2. Microdilution assayThe crude extracts (aqueous and organic extracts) were tested

against S. aureus as this strain was identified as frequently susceptible inthe radial diffusion assay. Aqueous crude extracts from 34 plant species(68% of plants screened) were active against S. aureus (≤4mgmL-1)(Table 2). The highest antibacterial activity was recorded for W. som-nifera, Wendlandia biscuspidata (Ravana idala) and C. fistula(MIC=0.06mgmL-1). In addition, Morinda umbellata (Kiriwel) and D.scandens also showed high antibacterial activity (MIC (≤0.1mgmL-1).Organic extracts from 46 plant species (92% of the total) showed ac-tivity against S. aureus (≤4mgmL-1). Notably, D. metel showed thehighest antibacterial activity (MIC=0.03mgmL-1). Low MIC values(≤0.06mgmL-1) for the organic extracts were also recorded for severalplants. These were Solanum virginianum (Katuwel batu), Solanum mel-ongena (Elabatu), W. somnifera, Paederia foetida (Apasu madu), Old-enlandia herbacea (Wal kothamalli), Adenanthera pavonina (Madatiya),Tamarindus indica (Siyambala) and Crotalaria retusa (Kaha andana-hiriya). Fractions with antibacterial activity showed a pattern of ac-tivity: consecutive sets of wells showed gradual increase in activity to

reach a climax after which a gradual decrease in activity was observed.The width of this pattern was an indication of the amount present in theextract of the active compound and suggested that well contents couldbe further diluted to identify the most potent target masses.

Although only 19 plant species gave activity against S. aureus in theagar disc diffusion assay, the two-step microdilution assay identified 34and 46 plant species that contained antibacterial aqueous and organicfractions, respectively. The two methods present distinct conditions forthe compounds affecting diffusion, scavenging and solubility, all ofwhich may mask activity in the disc diffusion assay (Ge et al., 1999;Strömstedt et al., 2014). The two-step microdilution assay monitorsactivity on both growing and non–growing bacteria and offers moreprecise estimates of potency. It also maximises solubility by avoidingpartial saturation of the medium by broth components and by the op-tion to use DMSO in order to enhance solubility of more hydrophobiccompounds. This makes the microdilution assay more advantageous forthe identification of cationic and less-polar bioactive compounds,compared to the agar disc diffusion method.

3.3. Screening of microfractionated aqueous extracts for antibacterial andcytotoxic activities

The microfractionation protocol was based on RP-HPLC combinedwith PDA and mass detection. The crude extracts from 50 plants weresubjected to solvent-solvent partition separately to yield aqueous andorganic extracts for each plant. Aqueous crude plant extract (2 mg)from each plant was fractionated into 45 fractions (1mL x 45) in a deepwell plate using RP-HPLC. 100 μL from each well was transferred into a96-well plate and the contents in each well were tested by microdilu-tion assay and by FMCA. The results from the microfractionation pro-tocol are shown in Table 2. The wells with antibacterial activity wereanalysed by LC-MS coupled to a PDA detector to identify the molecularions of the bioactive compounds and their purity profile. The RP-HPLCprofile of D. scandens species with the bioactive fractions and masses arehighlighted in Fig. 1. The microfractionation protocol facilitated theidentification of target bioactive fraction/masses, which then served asa guide in the large-scale isolation of compounds. This resulted in time-efficient screening of a large number of plants for bioactivity, using verysmall amounts of crude extracts.

3.3.1. Antibacterial activity of microfractions using microdilution assayA microdilution assay using S. aureus identified 19 of the 50 plant

species (38%) as containing microfractions that exerted complete bac-terial inhibition (Table 2). Although crude aqueous extracts from 34plant species showed activity in the microdilution assay, only 19 ofthese extracts gave activity after microfractionation. For example, In-digofera tinctoria, Saraca asoca, T. indica, Coccinia grandis, Hedyotisauricularia, Mitragyna parvifolia and S. hispida, lost their activity afterfractionation. One possible explanation for this phenomenon is thatseveral compounds are acting additively or in synergy in the crudeextracts to give rise to the observed bioactivity. Once these are sepa-rated, each individual compound activity is below the sensitivitythreshold.

3.3.2. Cytotoxicity of microfractions using FMCAFMCA, using a human lymphoma cell line, was conducted to iden-

tify fractions with cytotoxic activity. Only six plant species (12%)showed any activity on this cytotoxicity assay (Table 2). Of these, onlyPavetta lanceolata showed cytotoxicity but no antibacterial activity.Similar to the microdilution assay, fractions with cytotoxic activityshowed a pattern of activity with gradual increase in activity reaching amaximum, after which a gradual decrease in activity was observed. Forsome species, such as D. metel, O. mungos and W. somnifera, activefractions in this assay were also antibacterial, indicating the presence ofbroadly biocidal agents. In contrast, B. racemosa and C. alata exhibitedboth activities but deriving from separate regions in the chromatogram,

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consequently having different bioactive masses. This indicated moretarget specific compounds within the same plant, mediating either cy-totoxicity or antibacterial activity. Antibacterial and cytotoxicity datafor all species are shown in Table 2.

3.4. Comparison of traditional use of plants with antibacterial and cytotoxicactivities

The 50 plants that were screened in the current study are in mostcases used in traditional medicine for conditions that are associatedwith microbial infections, parasitic diseases or tumours. Thus, anti-bacterial activity and cytotoxicity were likely two of the key biologicalactivities mediated by the active constituents in these plants. Thecomplete list of traditional uses of the plants screened herein, togetherwith previous studies on their biological activities, is given in Table 1.

Crude aqueous extracts of C. fistula, W. bicupidata and W. somniferagave very high antibacterial activity in our bioassays. Of these, thelatter plant species also gave high cytotoxicity. These plants are used intraditional medicine for treatment of conditions including fever, diar-rhoea, cough, worm infestations and skin diseases where the mainbiological activities utilised are most likely antibacterial or cytotoxicactivities.

Cytotoxicity and antibacterial activity at very low concentrationswere confirmed for D. metel in our bioassays. Traditionally, D. metel isused for a variety of conditions including tuberculosis, tumours, eyediseases and worm infestations including filariasis. As given in Table 2,the crude organic extracts of A. pavonina, C. retusa, T. indica, O. her-bacea, P. foetida, S. melongena and S. virginianum showed low MIC valuesagainst tested bacteria but no cytotoxicity. Thus, our findings lendsupport to the traditional use of these plants. For example, urethritis istreated with P. foetida. C. retusa is used in the treatment of diarrhoeaand skin diseases. Markedly, eye diseases and oral diseases are tradi-tionally treated by T. indica. Furthermore, traditional medicinal uses ofS. virginianum include treatment of cough, fever, toothache, gonor-rhoea, vaginal candidiasis, worm infestations, and tuberculosis.

The plants that showed high cytotoxicity in our bioassays, D. meteland O. mungos, are traditionally used against tumours and cancer. Inaddition, skin infections are traditionally treated by plant extracts ofW.somnifera, B. racemosa and C. alata, which also gave high cytotoxicity inFMCA.

D. scandens extract did not exhibit cell line cytotoxic activity in theFMCA despite previous reported activity using other cells and methods(Kuljittichanok et al., 2018). However, it showed substantial anti-bacterial activity in the microdilution assay, which prompted largescale metabolite isolation as described below. Noteworthy is that D.scandens is a plant with extensive traditional use for treatment ofwounds and stomach infections.

3.5. Isolation and structure elucidation of biologically active secondarymetabolites from D. scandens

Microfractionated aliquots were subjected to antibacterial assay andthe fractions with antibacterial activity were further analysed on LC-MS. The target antibacterial compounds, (+)LRESIMS at m/z(M + H)+ for D. scandens were identified at 16min (m/z 463.10),17min (565.07, 595.16), 18min, (565.07) and at 19min (611.14) asmajor masses (Table 2). Interestingly, the aqueous fraction of D. scan-dens showed no cytotoxic activity against the lymphoma cell line.Large-scale sequential extraction with solvents of increasing polarityresulted in four fractions, with the EtOAc fraction showing significantantibacterial activity in agar disc diffusion assay (active radius wasgreater than 10mm). The EtOAc fraction was partitioned with water toyield 2.1 g of crude aqueous extract. MS analysis of the crude aqueousextract partitioned from EtOAc confirmed the presence of targetbioactive compounds at m/z 463.08, 565.14, 595.14 and 611.15(M + H)+. The aqueous extract was subjected to preparative RP-HPLC

and fractions containing the desired masses were pooled and re-purifiedby semi-preparative RP-HPLC. Isolated compounds with>95% UVpurity by analytical RP-HPLC were subjected to further bioassays andstructure elucidation. The four isolated compounds through the mi-crofractionation protocol are shown in Fig. 2.

A compound (3 mg) with a HREIMS at m/z 463.1004 (M + H)+,consistent with the molecular formula of C22H22O11 was confirmed asdalpanitin. 2D NMR data in CD3OD were analysed to elucidate itsstructure. Insufficient NMR data were reported from the previouspublished article due to lack of 13C and 2D NMR data (Adinarayana andRajasekhara Rao, 1972); here, the full spectroscopic data are sum-marised in Table 3. This is the first time that dalpanitin is reported fromD. scandens. Previously, in the early 1970s, dalpanitin was isolated fromanother member of Fabaceae, Dalbergia paniculata (Adinarayana andRajasekhara Rao, 1972). Furthermore, dalpanitin has been reported inrecent times from Dalbergia velutina (Kaennakam et al., 2016).

Rutin (2.5 mg) was identified by (+)HRESIMS analysis and com-parison of 1D NMR spectroscopic data to literature values (Lin et al.,2016; Zhang et al., 2010). Our study is the first report of rutin from D.scandens. Rutin is found in many plants, and recently it was reportedfrom Physalis peruviana (Toro et al., 2014). The antioxidant activity ofrutin is capable of reducing capillary fragility, swelling and bruising,venous insufficiency, and for improving micro-vascular blood flow(Fathiazad et al., 2006).

Vicenin-3 (2 mg) and vicenin-2 (2.5 mg) were obtained from large-scale fractionation. The (+)HREIMS at m/z 565.1442 for [M+H]+ ofvicenin-3 was consistent with a molecular formula of C26H28O14.Vicenin-2 with a molecular formula of C27H30O15 was confirmed by(+)HREIMS at m/z 595.1668 for its [M+H]+. The 13C and 1H NMRdata obtained in the present study for vicenin-2 was compared withalready reported data in the literature (Velozo et al., 2009).

A retention time of 10.9 min for vicenin-2 and 11.0min for vicenin-3 on RP-HPLC were indicative that vicenin-2 was more polar thanvicenin-3. When compared to vicenin-2, the NMR data of vicenin-3showed the loss of an oxymethine at δC 81.5, δH 3.56 (C-5′′) while themethylene resonance at δC 60.6, δH 3.89/3.66 (C-6′′) had shifted to δC81.5, δH 3.54/3.48. This data, combined with the loss of 30 amu, in-dicated that vicenin-3 contained an identical structure to vicenin-2 withthe exception of a D-xylopyranose moiety at C-8.

Fig. 2. Compounds isolated through microfractionation protocol, dalpanitin(1), rutin (2), vicenin-3 (3), and vicenin-2 (4).

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3.5.1. Minimum inhibitory concentration (MIC) determination of isolatedcompounds

The isolated pure compounds were tested against S. aureus, P. aer-uginosa, E. coli and C. albicans in the range of 188 μgmL-1 to 1.46 μgmL-1 using 2-fold serial dilutions. Dalpanitin and vicenin-3 showed thelowest MIC value of 23 μgmL-1 against S. aureus. Furthermore, dalpa-nitin and vicenin-3 also showed moderate activity of 94 μgmL-1 and188 μgmL-1 respectively against E. coli. Notably, only dalpanitinshowed activity against P. aeruginosa at 94 μgmL-1. Interestingly, noneof the tested compounds showed activity against C. albicans.Gentamicin (1.46 μgmL-1) was used as a positive control. MIC valuesare given in Table 4. The MIC of 23 μgmL-1 identified for dalpanitin andvicenin-3 are in the low and intermediary range of the clinical and la-boratory standards institute (CLSI) guidelines for antibiotic potency(4–32 μgmL-1) (CLSI, 2015, 2008).

Although we identified several bioactive plants in addition to D.scandens from our current screen, we could not progress to large-scaleisolation due the limited access to those plant materials. Neverthelesswe offer that our study has highlighted several Sri Lankan plants used intraditional medicine as promising candidates for further antibiotic drugdiscovery studies.

4. Conclusion

The lack of scientific evidence for efficacy is a major concern forfurther development of plant-based traditional medicines. As an at-tempt to investigate the scientific basis of plants used in Sri Lankantraditional medicine, we conducted a bioactivity screen of 50 plant

species, leading to identification of microfractions of 19 plants withantibacterial activity (i.e. fractions which inhibited growth of S. aureus)and 6 plants with cytotoxic activity where cell viability was ≤30%.Notably, a high percentage of plants that were screened in the currentstudy were found to contain antibacterial and cytotoxic compounds. Inmost cases, we were able to correlate the traditional use with theconfirmed antibacterial or cytotoxic activity, providing scientific sup-port for the anecdotal evidence.

Microfractionation of the extracts was valuable to identify thebioactive metabolites in the plant extracts, which then served as aguiding tool to streamline the downstream processes for identificationand large scale isolation of bioactive compounds. Notably, the micro-fractionation protocol is advantageous as it requires only a smallamount of plant material (2 mg). The method allowed rapid screeningof a large number of samples at a time and only required the collectionof plant material in large scale once a target bioactive compound/masswas identified. We exemplified this by large-scale isolation of bioactiveflavonoids from D. scandens, guided by molecular ions associated withbioactive fractions identified during microfractionation. In the presentstudy, vicenin-3 and dalpanitin showed highest activity (23 μgmL-1)against S. aureus but showed no cytotoxicity against lymphoma cells.Follow up studies aimed at identifying the underlying mechanism ofaction will facilitate the design of modified pharmacophores with im-proved antibacterial activity.

To our knowledge, this is the first large-scale bioactivity screeningeffort conducted on Sri Lankan flora. As such it is a first step towardproviding scientific evidence for plant-based medicines used tradition-ally in the region. The high in vitro hit rate for antibacterial and an-ticancer activity we report among the herbal extracts within the SriLankan flora not only enhances the confidence in the traditional SriLankan medicinal system but emphasises the value of the prescreeningapproach for ethno-pharmacologically used plants in search of putativebioactive compounds in plants.

Author contributions

CH, DD, AAS, UG and SG designed the study. SM and SR performedall the experiments. AAS, RB and TM designed and initiated the bio-logical experiments. SM, SR, AAS and SG wrote the manuscript with theinput from other authors. SM and SR equally contributed to this work.

Acknowledgments

This work was supported by Swedish Research Council Linkagegrant (2013-06672), lead by SG. AAS' work on antibiotics from SriLankan traditional medicinal plants was supported by the Lars HiertaMemorial Foundation research grants FO2011-0639 and FO2016-0618.We would like to thank Prof. Björn Hellman for providing facilities toconduct the cytotoxicity assay at Division of Toxicology, Department ofPharmaceutical Biosciences, Uppsala University. We would also like tothank Dr. Cecilia Persson at the Swedish NMR Centre, Gothenburg, forcarrying out the NMR measurements and Dr. Luke Robertson at theDepartment of Medicinal Chemistry, Uppsala University for proof-reading the manuscript.

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Table 3NMR spectroscopic data of dalpanitin (Recorded at 600MHz in CD3OD).

Position δC, type δH (J in Hz) COSY HMBCa

2 155.0, CH 8.20, s 3, 4, 93 124.6, C4 182.7, C5 163.6, C6 100.3, CH 6.30, s 4, 5, 7, 8, 10, 1″7 164.9, C8 106.6, C9 158.2, C10 104.7, C1′ 123.0, C2′ 114.1, CH 7.20, d (2.0) 2, 3, 1′, 3′, 4′3′ 148.2, C4′ 149.0, C5′ 116.4, CH 6.88, d (8.1) 6′ 2′, 4′, 6′6′ 123.8, CH 7.00, dd (8.1, 2.0) 5′ 1′, 2′, 4′, 5′, 37′ 56.6, CH3 3.91, s 2′, 3′1″ 75.5, CH 4.96, d (9.9) 2″ 3″, 4″, 5″, 7, 9, 102″ 71.5, CH 4.13, m 1″, 3″3″ 80.3, CH 3.49, m 2″ 2″, 4″, 5″4″ 73.0, CH 4.04, m 5″ 6″5″ 82.8, CH 3.46, m (6.2, 2.9) 4″ 3″, 4″6″ 62.9, CH2 3.90, dd (2.2)

3.74, dd (12.0, 5.5)5″ 4″, 5″

a HMBC correlations are from proton(s) stated to the indicated carbon.

Table 4Antimicrobial activity of isolated flavonoid compounds. Range of tested con-centration between 1.46 μgmL-1 to 188 μgmL-1.

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E. coli S. aureus P. aeruginosa C. albicans

Dalpanitin 94 23 94 >188Vicenin-3 188 23 >188 >188Vicenin-2 > 188 >188 >188 >188Rutin > 188 >188 >188 >188

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