NaturalProducts: Bioactive Alkaloids from the Bhutanese Medicinal Plants

PhurpaWangchukJ, SamtenJ and John B. Bremner2

IPharmaceuticaland Research Unit, Institute of Traditional Medicine Services

Ministryof Health, Thimphu, Bhutan

2Departmentof Chemistry, Faculty of Science, University of Wollongong

NSW2500, Australia

ABSTRACT

Naturalproducts had been indispensably used in traditional medicines for thousands of years. These

traditionalmedicines cater to about 85% ofthe world population for their primary health care needs.

Besides,the natural products have been an important chemotherapeutic pool providing indispensable

bioactivepharmacophores.1n fact, they are the skeletal frameworks of about 60% ofthe modem drugs

thatareavailable today.

Fromtheseperspectives,Bhutanis fortunatetobe richinboththebiodiversityandthetraditionalmedical

knowledge.However, not much of these resources have been studied before. Therefore, this study was

perfonnedto describe and evaluate Bhutanese medicinal plants with an aim to discovering novel new

antimalarialand antibacterial drugs as well as provide scientific validation to gSo-ba rig-pa. About four

alkaloidpositive medicinal plants: Aconitum orochryseum Stapf, Corydalis gerdae Fedde, Rununculus

brotherusiFreyn and TribulusterrestrisLinn, with antimicrobial claims were studied fortheir phytochemical

constituentsand bioactivities. Aconitum orochryseum furnished three new hetisane-type diterpenoid

alkaloidsnamed Orochrine, 2-0-acetylorochrine, and Lingshinaline, together with two known alkaloids

AtisinilllTIchloride and Vlfescenine and six other unidentified alkaloids. Corydalis gerdae, gave fourknown

protopinetype and protoberberine type isoquinoline alkaloids: Protopine, Scoulerine, Cheilanthifoline,

andStylopinealong with one unidentified alkaloid. The crude methanol extracts, crude alkaloids and the

purealkaloidsof thesetwoplantsgaveverysignificantantimalarialactivityagainstPlasmodiumfalciparum,TM4and Kl strains when tested in vitro.

Theseresults proved for the first time at the molecular level that Bhutanese traditional medicines have

clinicalefficacy.Thus, Bhutanese traditional medicines have potential to be an effective treatment for rp.any

diseasesespeciallythose formulations that use the above medicinal plants for treating malaria. This medicine

alsohaslotsof scope forbioprospectingthat would contributeto discoveringnew drugs aswell as new

mechanismof drug action.

Keywords: Natural products, Bhutanese traditional medicine, medicinal plants, alkaloids and anti-malarialactivities

122

1. Introduction

Withthe advancementin scienceand technology,

remarkableprogresshas been made in the fieldof

medicine including diagnosis, treatments and

pharmaceutics.Recentdrugdiscoverytechniques

based on Structure-Activity Relationships,

ComputerModelling, CombinatorialChemistry,

HighThroughputScreeningandSpectroscopyhave

triggeredandspearheadedthe discoveriesofmany

naturalandsyntheticdrugs.In 1999,worldsalesof

pharmaceuticals,excludingveterinarymedicines,were valued at ca US$ 325billion1.

Despite these developments, of the known 30,000

human diseases or disorders, only one-third can be

treated symptomatically with drugs and that too at

a great economic and social cost. This is because

of the fact that the medicines including traditional

medicines available today are stillnot very effective

particularly with respect to the fight against drug

resistant pathogens and newly emerging infections.

The infectiousdiseaseswill continue tobe the leading

causes of premature death in human beings of both

developed and developing nations as their resistance

to many conventional drugs is increasing. Therefore,

microbial resistance especially by bacteria and

protozoa to drugs is of special concern tocommunities worldwide and to researchers and there

is an urgent need to find concrete solutions for

combating such epidemics.

Prevention of famines, droughts,poverty,floods,

wars, political upheaval, economic failures,

environmental depletion andpollution would be

good solutions to help reduce infections andmicrobial resistance. However, such natural

calamities are unpredictable and uncontrollable.

Anothermeasuretopreventmicrobialreinfectionis

to use the currently available antimicrobialdrugs

morejudiciouslyandoptimally.Choosingthecorrect

drug, dose, dose interval and duration oftherapy

maymoreefficientlyprovideclinicalbenefitswhile

contributinglesstoresistancethanchoosingthedrug

withthe narrowestspectrum2.

Above all, the most important strategyto control,

cure and toprevent reinfection is to developmore

appropriatetreatmentregimensincludingthesuperdrugs and the traditional medicines. Super drugs

couldbe developedsynthetically,but as experience

hastaughtus,naturalproductsarerichinstructurally

diversebioactivemolecules.Naturalproductshad

beenindispensablyusedintraditionalmedicinesfor

thousandsof years.Recently,the natural products

werealsoexploredextensivelyformedicinalagentsthatquiteoftenbecomepotentialcandidatesforthe

development of new drugs. The natural products

usedintraditionalmedicinehavegivennewhorizon

and the dimension to the new drug discovery

programs.

1.1Natural Productsin TraditionalMedicines

Because Homo sapiens are herbivores to some

degree, they first encountered bioactive age~tsinvegetalfooditems3.Having livedharmoniouslyin

closeassociationwiththeenvironment,theylearned

toutilisethetoxicandmedicinalpropertiesofplants

and other natural products. Some of those toxic

plants were used as poisons forcausing death andas arrow poisons for hunting food, warfare,

depredatingwild animalsand forgainingmasteryoverahostileenvironmenrt,5.Themostremarkable

of all the ancientinventionswas the artofutilising

these toxic and medicinal natural products fortreating various ailments but the ethno-medical

knowledge was restricted to a few elites such as

priests, medicine men, shamans, magicians and

witch doctors3. Although some cultures used

individual herbs or plants as medicines, many

traditionspropoundedpowerfulcombinations.with

123

differentingredientsknownaspoultices,tincturesandmixtures.

Itwas the Mesopotamians who first used the herbs

likeoils of cypress, cedar, liquorice and poppy juice

fortreatingdifferent ailments in 2600 B.C6followed

by a Buddhist system of medicine known as gSo-

baRig-pa that dates to 2500 BC7.The gSo-ba Rig-

pa is practiced in Bhutan and Tibet and its

phannacopoeia records the use of the use of as

many as 2200 traditional prescription drugs8 and

eachofthese drugs are multi-ingredient products

compounded using medicinal plants, animal parts

andminerals as the main ingredients

In 1500 BC, Egyptian's developed the Ebers

Papyrus that documented some ofthe 700 drugs

includingformulas such as gargles, snuffs,poultices,

infusions,pills and ointments6.The Chinese materia

medicaWu-Shi Er-Bing Fang which contains

52prescriptions date back to 1100BC6.9and the

IndianAyurvedicMedicine that dates to 1000BC(Susrutaand Charaka) documents the medicinal

,useof plants like Datura, Aconitum, Cannabisand Sarcostemma3.

romthese ancient cultures, some of the knowledge

reachedMediterranean countries through traders

Fd migrations and it was in Hippocrates's time in~O-377BCthatphannacognosyreachedasummit

tnGreece3.In 300 to 322 BC, Theophrastus-who

~as a philosopher and naturalist, was the first to

dealwith the history of plants, which later on helped

rn the classification of plants including herbs.

h 78AD,PedaniusDioscorides,aGreekphysician!roducedDe materia medica, which described

norethan 500 medicinal plants and their usesildetail.

ralen(ca 129-199AD) founded "Galenics" and

\UghtphannacyandmedicineinRomelD.Avicenna124

,

(980-1037AD), a Persian phannacist, physi~ian,philosopher and apoet described 1400drugs andmedicinalplants which greatlycontributedto theformationof acodifiedGreco-RomanMedicinein

the 5th century3,6. Paracelsus (1493-1541)

administered dosage formulations separating

"Arkanum" fromnon-activeingredientsof drugs.

Westernmedicine andpharmacy originatedfromthismedicalsystem.

In the USA, homeopathy, that includes

hydrotherapy,nutritional therapy,herbal therapy,

manual manipulationandmidwifery,whichwere

foundedbyGermanphysicianHahnemann(1755-18-43),became popular in 1830s.

The discovery of antibiotics and'Vaccines in th~ 20th

century dramatically changed medical practice

worldwideandas a result a separatefieldof ethno-

medicine emerged as an academic specialization

focusingon traditionalhealing systems1I. Theinvestigation of the principles of drug action of

JapanesetraditionalSino-medicineat themolecular

level have resulted in obtaining many novel

compoundsandtheuncoveringofnewmechanisms

of drugaction12. ClinicaltrialsofgSo-baRig-pamedicine, the PADMA Products, also provedsuccessful in the treatment of irritable bowel

syndrome!3andfibrinolysiswithstableintermittent

claudication!4.ls.Thisproductsalsofurnishednewanti-oxidativemechanismsatthemolecularlevep6.

Thus, traditional medicines were found to be

effectivelyaddressingthehealthneedsof millions

ofpeopleincludingdevelopednationsbycompletely

differentstrategiesandwelldefinedapproaches,andgenerally with minimal side effects1!. In fact,

traditionalmedicinesprovide primaryhealth care

needs to almost65-85%ofthe world's population

including developed nationsI7.18.In terms of

economic value, traditional therapies contribute

to US $ 60 billion a year and the USA alone

spendsUS $ 2.7billionperyearfollowedbyChinawith US $ 1.8 billion and Australia.with Aus

$ 1billion ayear19.

1.2 Natural Products in Modern DrugDiscoveries

Bioactive natural products are mainly secondarymetaboliteswhichareusedby thehost asdefensive

andprotectivemechanismsagainsttheirenemiesand

predators2°.Generally,screeningofthesesecondarymetabolitesandthe developmentof drugsis avery

hard task requiring much effort starting frombotanical identification, collection, extraction,

isolation,purificationandcompoundidentification

to pharmacologicalandclinical testing.However,theenormouschemicaldiversityandhighlyunusual

structuresprovided bynatural products is greater

thanthatprovidedbymost availablecombinatorial

approachesbasedonheterocycliccompounds21,22.When screening fornatural products, three main

types of search strategies, namely biorational,chemo-rational and random approaches23 are

followed.Thebio-rationalapproachincludesethno-

medicallydirectedscreening(asdiscussedabove)

and random screening.Random high throughput

screeningisbasedonfieldobservationsuchaspest-

plantanalysisandplantcharacteristics.Drugssuchas artemisinin,morphine,quinine, and ephedrine

were discovered using the ethno-directed bio-

biorational approach24. Conocurvone25, an anti-HIV

agent was discovered as a result of random high

throughputscreening.Thechemo-rationalapproachisbasedonchemo-taxonomicalconsiderationssuch

as alkaloid surveys, and investigation of specificfamilieswhichareknownto containpotentialdrug

leads26. Employing these three search strategies as

many as 88,000natural product compoundshavebeenisolatedfromdifferentsourcessuchasplants,

animals, marine organisms, insects and micro-

orgarusms.

It is estimatedthat ca.250,0006to 500,000species

of plants grow on earth17,18and 70% of all the

world'sbiodiversityofvertebrates,higherplantsand

butterfliesareharbouredby 12countries;Mexico,Colombia, Ecuador, Peru, Brazil, Zaire,

Madagascar,China,mdia,Malaysia,mdonesiaand

Australia27.It is reported that only 10-15%of this

terrestrialbiodiversityespeciallyhigherplantswere

explored phytochemically for medicinal

applications17,and the main categories of plant-

derived drugs are terpenes (34%), glycosides

(32%), alkaloids (16%) and others (18%)l8.Around85% to 90 % ofterrestrial resources are still intact

and await exploration.

Amongst the animal kingdom, lower groups of

animalsespeciallythosesecretingvenomsandtoxins

such as insects, amphibians, reptiles, wasps, and

spiders, produce interesting and novel bioactive

compounds.Thereare30millionspeciesof insectsand very few have been studied for bioactive

secondarymetabolites23.Outof ca 100,000species

of spiders, only ca 30 have been studied until

date29.Themarineworldrepresents70%of earth's

surface,but only5%of themarine organismshave

been explored chemically and a huge diversity ofmarine fauna and flora are yet to be investigated2O.

Marine secondarymetabolites are often observed

to haveanticanceractivity.

Micro-organisms have been a frequent source of

antibiotics,eversincethediscoveryofpenicillinfrom

the filamentous fungus Penicillium notatum by

Alexander Fleming in 1928. Cyclosporins and

rapamycin (an immunosuppressive agent),

ivermectin(ananthelminticsandantiparasiticdrugs),

streptomycin,chloramphenicol,tetracyclinesand

125

fromtheplantsused intraditionalmedicines6.

However, in using this ethno-directed search

strategy,it is crucialto haveintimateunderstanding

of the disease concepts of the culture whose

pharmacopeiais underexamination.Theproducts

used as medicines by local people are usuallynot

those that aretested in the laboratory.Most ofthe

effectivebrews or formulariesaremulti-ingredientcompounds.Chemicalreactionsoccurwithinthese

mixturesorpoulticesandaremost oftenassociated

withsynergismmakingthemmoreeffectivethanthe

singleisolatedleadcompound.Whenthemedicinal

plants are subjected to phytochemical screening,

researchers often target only one compound, or a

fewlimitedcompounds,whichquiteoftenturnouttobebiologicallyinactiveowingtothelossof other

active components duringthis screeningprocess.

Therefore, the ethno-medical indication maynot

necessarilybeproductivewhenscreeningisdirected

towardsonlyspecificphytochemicalisolations.

1.5 Rationale ofthe Study

Sincethe infectiousdiseasesincludingmalariais aleadingcauseof prematuredeathworldwide,there

is an urgent need to find effective treatment

regimens,be it traditionalmedicinesor new drugstocombatthem.Besides,Bhutanhasrichtraditional

medicalknowledgeprovisionedwith rich natural

medicinal resources which could provide new

mechanismofdrug actionandalsonew drugleads.

Above all, in order to provide the patients withclinicallysafeandeffectivetraditionalmedicine,there

is an urgent need to scientifically validate the

traditionalclaims.For doingthis, the best strategy

is to first focus the study on single individual

ingredientsratherthanonmulti-ingredientproductswhich isdifficult to analyseby any means. Therefore,

thispaper sets to describe the studyperformed on

individualalkaloid-containingmedicinalplantsusedintheBhutanesetraditionalmedicine.

2. Objectives

a) To isolate and identify the alkaloids from the

selectedmedicinalplantsofBhutan

b) Todetermineandelucidatethe structuresof thenew alkaloids isolated from the selected

medicinalplants

c) To assess the antibacterial and antimalarial

propertiesofthe isolatedalkaloids

d) Toprovide validationand clinicalevidenceto

gSo-baRig-paclaims.

3. Study Design, Materials, Methodology,Results and Discussions

The study was purely experimental laboratoryanalysis involving little bit of field work while

collectingthemedicinalplantsinBhutan.Otherwise,

almost all the laboratory work was done in the

UniversityofWollon.gonginAustralia.Ratherthan

studying the compounded finished products, this

studywas'targetedto individual six high altitude

medicinalplants,whichwere purposivelyselectedbased on certain selection criteria. The

phytochemicalandpharmacologicalanalyses:werethenperformedonthose selectedmedicinalplants.The methanol extracts, crude alkaloids and the

selected pure alkaloids were then tested for their

antimalarialandantibacterialpropertiesasindicatedintraditionaluses.

3.1 Material Selection Method

At first, a list of medicinal plants was generated

consistingof 156plantspecies.Themedicinalplantslist was then analysed and only those medicinal

plants, which were of interest, were selected for

the studybased on the followingselectioncriteriagive in Scheme 1.

127

- ~

Free listing of medicinal plants (160 species)

.0.

Medicinal plants used for treating malaria and

infectious diseases (55 species)

.0.

Medicinalplants thatare endemicto theHimalavasandBhutan(26 species)

.0.

Alkaloid-containing medicinal plants (15 species)

.0.

Final selection (5 species)Scheme1.Flowchart for the selectionofthe medicinal

plantsbasedon the four selectioncriteria.

Although,many plants were found being used intreatingfeverarisingITomdifferentdisorders,onlyCorydalisgerdae was specifically used againstmalaria.Aconitum orochryseum was used for

treatinginfections,snakebite, andbilious feveror

feverarisingITombile disorders.Therewere also

manymedicinalplantsused fortreatinginfectionsincluding blood infections, skin infections,andinfections of internal organs.About fifty five

medicinal plants were selected based on thefirstcriterion.

Here,it should be noted that the medicinal plantsusedinBhutanesetraditionalmedicinewerenever

usedas single plant components, but rather as amixture of two or more active ingredients. The

theorybehindusingmulti-ingredientcompoundsisthatthose mixtures of ingredients are believed to

actsynergistically by neutralising the toxicity ofindividualingredientandenhancingthetherapeuticvalues of the mixture. So, for this reason the

therapeuticindicationof anindividualingredientis

quiteoften different ITomthat of fonnulated finished

products.For example, Tribulusterrestrisis usedfor formulating four different multi-ingredient

products such as Go-yu-28, Seng-lden-25, Ba-

sam-fha-rlung and Bdud-rtsi-nga-fum incombinationwith other ingredients.Although,the

~ =-~

therapeutic properties of Tribulus terrestris are

retained while fonnulated, the individualtherapeutic

propertiesof otheringredientsused inthe fonnulation

of four other products were lost.

Similarly, Aconitum orochryseum is used for

fonnulating eighteen different multi-ingredient

products such as Tig-ta-8, Tig-ta-16, Gser-mdog-

11, Hong-Ien-9, Thang-chen-25, Gyu-rgyal, Bol-

sman-7, Rta-ze-dmar-po, Spang-rtsi-12, Gtso-bo-

8, Dbang-po-kuen-sel, Gur-gum-13, Man-sil, Ko-

la-19, Dvag-sman-15, Brag-zhun-9, Mkhris-phyi-

7 and Ded-pon-lO. Only three of these products

(Product 8, 9 and 10) have similar therapeutic

indications, as those of the individual plants and the

remainingproducts have entirelydifferenttherapeutic

indications to that of individual therapeutic

properties. Corydalis gerdae and Codonopsis

bhutanica are used for the fonnulation of aproduct

called kLu-bdd-8 which is used for treating

dennatitis and leprosy.

The second criterion was to select only those

endemic species (endemic to Himalayas and

Bhutan) growingat high altitudes of 4000metersabove sea level. These species were the least studied

phytochemically and could contain new molecules

that would potentially be new drug leads. About 26

medicinal plants were short-listed based o~ thiscriterion.

Only those medicinal plants that contained alkaloids

were then selected from these 26 species. This is

because alkaloids have interesting structural types

with awide range of biological activities and have

quite often become potential drug leads. An effort

was alsomade to selectmedicinalplants representing

different families since different families usually

contain different classes of alkaloids. So, the

preliminary tests for the presence of alkaloids were

conducted on the selected medicinal plants ofBhutan at the Pharmaceutical and Research Unit in

Bhutan using the Culvenor and Fitzgerald39 test

procedure. Only fifteen plants were found to contain

128

alkaloids (Table 1).The result was reported only

as alkaloid positive or alkaloid negative and thedegreeof alkaloidcontentwas not estimated.

Finally, only five alkaloid-containing medicinal

plants that were not studied before were selected

for furtherphyto-chemical andpharmacologicalanalysis(Table 2).

3.2 Collection of medicinal plants

The collection was done ITomJuly to September

2002 by the medicinalplant collectionteam ITomthePhannaceuticalandResearchUnitoftheInstitute

forTraditionalMedicineServices.Corydalisgerdae

Table 1. Medicinal plants investigated for alkaloids at ITMS using the Culvenor andFitzgerald procedure39.

gSo-ba Rig-pa name

Btsan-dug

Bong-dkarDam-bu-kara

Spang-ram

gYa-kyi-ma

Klu-bdud-dOljiRe-skon

Tong-ri-zil-pa

sTong-zil

Bya-rgod-sug-pa

Bya-rgod-spos

Dkar-po-chig-thub

Dngul-tigRtsad

Ra-mne

Che-rtsa

Da-li

Se-rgod

Sgno-sprin

Bre-ga

Botanical name

Aconitum lacinatum

Aconitum orochryseum

Aletris pauciflora

Bistorta macrophylla

Chrysospleniumforrestii

Codonopsis bhutanica

Corydalisflabellata

Corydalisgerdae

Corydalis megacalyx

Corydalis stracheyi

Delphinium brunonianum

Fritillaria devalayiPamassia ovata

Pleurospermum amabile

Polygonatum verticillatumRanunculus brotherusi

Rhododendron anthopogon

Rosa macrophylla

Thalictrum reniforme

Thlapsi arvense

....

Feddeis endemicto BhutanandtheChumbivalleyin Tibet4°andwascollectedITomtheupperMochu

(Yha-Ie-La near Lingshi), Chomolhari (4400-

4900m) bases and ITomnearby mountain scree.Codonopsis bhutanica, Ranunculus brotherusi,

Aletris pauciflora were also collected ITomthe

Lingshiregion.Aconitum orochryseum Stap'fhas

been collectedfrom alpineregions(3950-4720m)

fromChugalug,Lingshi.Herbariumspecimensfor

each of the plants collected for this study werehousedatthe PharmaceuticalandResearchUnitof

the Institute for Traditional Medicine Services, I

Thimphu,Bhutan.Theherbariumvouchernumber '

and the collection codes are noted in Table3. I

Alkaloid Test

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

129

Table 2. Selected medicinal plants with their indications

Botanical name Family

Aconitum Orochryseum Ranunculaceae

Distribution

EndemictoBhutan

4720 metres

Ethno -medical use

Fever,antidoteand otherinfections

Pneumonia,liverandlungdiseases

Gout, leprosyand otherinfections

Malaria andother infections

Antipyretic,wounds andpus

Arthritis,diureticsand

kidneydiseases

Table 3. Site of collection of the selected medicinal plantsMedicinal Plants Collection time

collected Herbarium No.Collection sites Parts

Aconitum orochryseum

Aletris pauciflora

Codonopsis bhutanica

Corydalis gerdaeRanunculus brotherusi

Tribulus terrestris

July/August

July/August

July/August

July/September

July/August

February

Jaradingthang

ZangpothangChewla

Chukhalung

Zangpothang

Wellington,NSW,Australia

Aerial

Whole

Whole

Whole

Whole

Aerial

83

47

71

07

18

NA

Tribulus terrestris was collected from near

Wellington(CentralWest,NSW) inFebruary2002

byDr.ChrisBourke,OrangeAgricultureInstitute,

Orange,NSW. The air-dried (room temperature)

plantmaterial (stems, leaves, flowers and fruits,

approx.8 kg) has been coarsely chopped using

petroldrivenplant-mulchingmachine.

3.3 Isolation, Structural Elucidation andIdentification of Alkaloids from the selected

medicinal plants of Bhutan

MethanolextractionwasdoneatthePharmaceutical

andResearchUnit in Bhutan andwas transportedto Australia. The extracts of two of the selected

medicinal plants, Aletris pauciflora and

Codonopsis bhutanica, were damaged on

transportation and as an alternative, the locally

available Australian naturalised plant, Tribulus

terrestris L42 was selected for the study. This plant

was known to have caused hepatopathy43,

Except for Aletris pauciflora and Codonopsis

bhutanica,all the plants collected here are rare

species41,and care was taken with respect to the

environmentalimpact. The collectedplants were

thenanalysedfor their alkaloidalcomponentsand

theresultsof the investigationsarediscussedhere.130

Aletris pauciflora Liliaceae Himalaya, 4100 metres

Codonopsis bhutanica Campanulaceae Endemic to Bhutan

4500 metres

Corydalis gerdae Fumariaceae Bhutan, 4100 metres

Ranunculus brotherusi Ranunculaceae Himalaya, 4100 metres

Tribulus terrestris Zygophyllaceae Australia and

Coastal areas

CH30H~ r

Figure 1. Structure and 3D-Model ofOrochrine (N-blue, O~fed, C=black, H-white).

staggers44and locomotor effects45in Australian

sheepbut the specific compound responsible forneurotoxicitywhose symptoms are related to the

symptomsof Parkinson's disease in humans,was

notknown.Theplant is alsoused inthe traditionalmedicineofBhutan.

For the extraction, separation and isolation ofalkaloids,generalmethodof acid-baseftactionation

methodwasused.Thepurificationwasdoneusing

preparative plate thin layer chromatography and

crystallization processes. Spectral data were

collected using Mass Spectrometer, Gas

ChromatographyMass SpectrometerandNuclear

Magnetic Resonance Spectrometry (1H-NMR,

13C-NMR, DEPT, gCOSY, gNOESY,TOCSY,

gHSQC and gHMBC). The structure elucidation

was done involving the technical experts and the

supervIsor.

Although,no alkaloidswereobtainedfromthreeof

the selectedmedicinalplants,twomedicinalplants

Aconitum orochryseum Stapf and Corydalis

gerdae gave interesting results both in terms of

phytochemical content as well as their biologicalactivities.Aconitum orochryseumfurnishedthree

new hetisane-type diterpenoid alkaloids named

orochrine,2-0-acetylorochrine,and lingshinaline,togetherwithtwoknownalkaloidsatisiniumchlorideand virescenine and also six other unidentified

alkaloids. One paper on these new alkaloids is in°t13~

17

CH2

HO"

'""P

theprocessof publicationin thejournal of NaturalProducts.

Corydalis gerdae, gave four known protopine type Iand protoberberine type isoquinoline alkaloids:

Protopine,scoulerine,cheilanthifoline,andstylopine

alongwithoneunidentifiedalkaloid. I

The crude methanolextracts, crude alkaloid~and

the pure alkaloids ofthese two plants were tested

for their antibacterialand antimalarialproperties. IWhile the sampleswere foundinactiveagainstthe I

Gram-positive human pathogenic bacteria I

Staphylococcus aureus and Enterococcus I

faecium, the samples gave very significantantimalarial activity against Plasmodiumfalciparum, TM4 and Kl strains when tested in

vitro.Anotherpaper,whichwillreportthe amazing

antimalarial activities of known protopine and

protoberberine type alkaloids especially that of

crudealkaloids,is also in theprocessofpublishingit in the PlantaMedica.

The in-vitro antimalarial assay was done by

Dr. Sumalee Kamchonwongpaisan and Miss

Roonglawan Rattanajak using a Microdilution

Radioisotopes Technique at the Protein-Ligand

Engineering and Antimalarial ScreeningLaboratories, National Centre for Genetic

EngineeringandBiotechnology,NationalScience

andTechnologyDevelopmentAgencyinBangkok,

Thailand. The antibacterial testing was done inAmrad,Australia. 0

131

17

CH2

Figure2. Structureand 3D-Model of2-0-acetylorochrine (N-Blue, O-Red, C-Black, H-White).

17

CH2

Figure 3. Structure and 3D-Model of Lingshinaline (N-blue, O-red, C-black, H-white).

4. Conclusions and Future Directions

This study concluded that Aconitum orochryseum

and Corydalis gerdae has the antimalarial activities

as indicated in the Bhutanese traditional medicine.

Therefore, anew formula with these two medicinal

plants as its major ingredients could be effective in

treatingPlasmodium malaria. Itwas also clear from

the study that the new alkaloids of Aconitum

orochryseum have the potential to be new

antimalarial lead molecules. Thus, the study provedforthe first time at the molecular level thatBhutanese

traditional medicines have clinical efficacy and that

this ithas lots of scope forbioprospecting that would

contribute to discovering new drugs as well as new

mechanism of drug action.

In future, since the extracts and the alkaloids of

Aconitum orochryseum and Corydalis gerdae

werefoundpotentiallyactiveagainst

132

Plasmodium falciparum strains, the immediate

follow up work should be focused on doing

antimicrobialassayswiththe otherminoralkaloids

present in these plants. In doing so, large scale

extraction would be needed to get a reasonable

amount of the minor alkaloids, especially from

Corydalis gerdae because the crude alkaloid of

thisplant showedhighestinhibitoryactivity.Sincethe high activityofthis crude alkaloid (Corydalis

gerdae) could alsobe due to synergisticeffectsof

total alkaloids present in the crude extract, the

antimalarial testing should be done on various

mixturesof commerciallyavailablealkaloidslike

protopine,scoulerine,cheilanthifolineandstylopineso asto findout ifthe activityof the crudealkaloid

wasbecauseof synergisticeffects.

The six unidentified alkaloids of Aconitum

orochryseumrequirefurtherstudyandfurtherbulk

extraction ofthe plant material will be needed in

ordertoobtainsufficientquantitiesfortheirstructureelucidation and the evaluation of antimicrobial

activities. Modifying the structures of the newalkaloidsaswellasoftheknownalkaloids(atisinium

chloride, virescenine, protopine, scoulerine,

cheilanthifolineand stylopine)which were active

against Plasmodium falciparum could improvetheirantimalarialactivities.Thiscouldleadultimately

to the development of candidates for new

antimalarialdrugs.

Also,sinceAconitum orochryseumis traditionally

used against colds, influenza and othermicrobialinfections, it would be worthwhile undertaking

antifungalandantiviraltestingonthealkaloidsfrom

this plant. Thenon-alkaloidal components ofthe

plantsstudiedheremaybebioactiveoratleastcould

alsobeactingsynergisticallywiththealkaloids.They

should also thus purified, identifiedand tested for

bioactivity.

...,...

The results obtained in this study indicated that the

combined ethno-medical-alkaloid search strategy

is an efficient and direct one for locating pot~ntial

new drug leads as well as scientifically validating

the Bhutanese traditional medicine. Such study

model or methodologies should be applied to study

and investigate other medicinal plants used in the

Bhutanese traditional medicine. This steps would

mean away forward in meeting the WHO standards

for such herbal drugs and most significantly, in

providing patients with better herbal medicines.However, the above research work could be carried

out only through collaborative approach. Since thereis no capacity both in terms oftechnical expertiseand equipments to conduct systematicallyexhaustiveresearch and development work at thePharmaceutical and Research Unit (pRU) in Bhutan,establishingcollaboratingpartners within and abroadisvital.PRU should colloboratewith similarresearch

institutions abroad by signing proper and suitableterms of referenceor memorandum of understandingso as to avoid exploitation.

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