O€>)

BAR.C-1360

I

ISOLATION, BIOSYNTHESIS AND BIOLOGICAL ACTIVITY OF ALKALOIDSOF TYLOPHORA ASTHMATICA, A VERSATILE MEDICINAL FLANT

by

N. B. MulchandaniBio-Organic Division

1987

B.A.R.C. . 1360

GOVERNMENT OF INDIAATOMIC ENERGY COMMISSION

aCJ

CD

ISOLATICJN, BIOSYNTHESIS AND BIOLOGICAL ACTIVITY OF

ALKALOIDS OF TYLOPHORA ASTHMATICA,

A VERSATILE MEDICINAL TLANT

by

N.B. MulchandaniBio-Organic Division

BHABHA ATOMIC RESEARCH CENTREBOMBAY, INDIA

1987

•. . ' BARC -.1360

INIS Subject Category * C4S.DQ

iDescriptors

MEDICAL PLANTS

ALKALOIDS

ANT1H1STAMINICS

BIOSYNTHESIS

ORNITHINE

TYROS1NE

ALBUMINS.

LYSOZYME

CATTLE

CARBON 14 COMPOUNDS

STRUCTURAL CHEMICAL ANALYSIS

X-RAY DIFFRACTION

INFRARED SPECTRA

ULTRAVIOLET SPECTRA

FLUORESCENCE

TOXICITY

RESPIRATORY SYSTEM DISEASES

RATS

BLOOD SERUM

PRECURSOR-

METABOLISM

TISSUE DISTRIBUTION

ALANINES

TRACER TECHNIQUES

- 1 -

P R E F A C E

Our oountry with it» varied ollmatio oonditions and

topography hat been considered at "Botanical Garden of the

Vorld" and its botanical wealth consists of several thousands

of known medicinal and essential oil bearing plants*

In spite of considerable advances taking place in

the pharmaceutical field especially in the synthetics, plants

and their derivatives have been able to maintain their

importance* In faot there is a growing trend even in the

highly developed oountrles of the world to ttake use of natural

drugs in preference to the synthetio ones wherever possible*

Pharmaeopeas of various oountriet desorib* numerous formula-

tions based on natural products*

Therefore, systematlo efforts to explore and exploit

the potential of valuable natural products is gaining great

deal of importance* At the Bio-Organic Division, inter-

disciplinary work related to some selected plants has been

aotively pursued and as a result significant findings have

been made whloh are desorlbed in this report*

Tylophorine and related new alkaloids have been •'.[ [y

Isolated from Tvlophora asthmatioa. Pargularia pallIda and

Pious hispida plants*

- 2 -

Biosynthesis of thia group of alkaloids hat baan

--' oarriad out using various labelled precursors for the first

time and fro* tha systematic degradation of the isolated

radiolabelled tylophorine, It has been oonoluded that these

alkaloids arise from one noleoule each of tyrosine, pheny-

lalanine and ornithine*

The interactions of Tylophora alkaloids particularly

tylophorinidine with bionoleoules such as lysosyme and

I bovine serum albumin have also been studied and binding oharao-V

teristioe determinedt

In our pharmacological studies it was found that

Tylophora alkaloid extract possesses antianaphylaotio aotivlty

as observed in passive peritoneal anaphylaxis in rats* The

drug also possessed mild antihistaminio and anticholinergio

activities* Studies of the extract on the bronohial smooth

muscle both In vivo and in vitro did not reveal bronchiodilator• • • ' • . r . , . _ . . . • • . - • • • - • • • • • • - - - - . • - • • • • •

potential of the drug.^ Furthermore this extract showed

similar action as the conventional drug, oromolyn; Hovevert

Tylophora drug has the advantage that it is effective in

mlcrogram quantities and also it is orally absorbed whereas

oromolyn is inhaled eaoh time to, the extent of 20-25 mg which

gets direotly deposited on the lungs. Therefore, the axtraot

developed by us should be considered superior* The prolonged

relief offered by this drug oould be due to its aotion on oell

- 3 -

mediated immunity sinee thif oould prevent experimentally

induoed anaphylaxis in rats* Additional pharraaoologioal

evaluation of Tylophora by others have substantiated the

above findings as signifioant improvement in lung function

tests, deoreaae in eosinophil and leuoooyte oounts and

inereate in 17-ketosteroid exoretion in the urine vis

observed with this drug* Aoute toxioity studies with Tylophora

drug were carried out at the Industrial Toxioologioal Researoh

Centre, Lucknov and the therapeutic dose has been found to

be highly safe*

In addition, the distribution and metabolism of the

drug was studied in vivo using C radiolabelled alkaloids

prepared by biosynthetic method* This study further revealed

its usefulness since the drug is absorbed by vital organs

and Also it is not metabolised into fragments which oould

cause some other damage*

Tylophora alkaloids have also been found to be

anti-nutagenic*

TYLOPHORA A8THMATICA WIGHT AND ARN. SYN. TYLOPHORA INDICA

(BURM»F«) MERR.

(1) Boabays

(2) Deooan:

(3) Kanarese:

(4) Marathi :

(5) Hindi t

(6) Bengal :

(7) Malaysian:

(8)

(9) Talugu:

(iO) Urya :

VERNACULAR NAMES

Anto-mul Antha-nulKharakira»na» Pit-mari

Pit-kari

Adunuttada> KiruManji-balli,Nepala, Nepalada-beru

Pita-kari

Anta-aul, Jangli-Pikvan

Anto-mul

Valli-pala

Kagittam, Kagittiraai, Kaludai-palai,Kodagam» Kondaohani, Kuravaran, Kurinja,Naoharuppan, Naoharupaynjan, Naohohmppan,Nanja-miriohohan « Nanja-ruppan, Nay-palal,Nirkkurinja( Peyppalai, Saraagaa, Unaattadi.

Kaka-pala, Kukka-pala, MattukunittukoniNelatarpire, Podapatramu, Tellavedavela»Tellayadala, Verri-pala, Vetti-pala*

Mendi, Mulini

(11) Gujarat Is Dwrnl v«l

TYLOPHORA ASTHMATICA WIGHT AND ARN.

- 5 -

TYLOPHORA ASTHMATICA WIGHT AND ARN. SYN. TYLOPHORA INDICA

(BURM.F.) MERR.

VERNACULAR NAMES

(1) Bombay: ^ W

(8) D«ooan

(3)

(10) Uryat

(11) Gujarat!: £H

U>(5) Hindi :

(6) B.ngal :

(7) M»l*yalap: OJ

(8) fa«il # ^ ^

(9) Ttlugu. ;

a3^^

- 6 -

ISOLATION, BIOSYNTHESIS AND BIOLOGICAL ACTIVITY OF

ALKALOIDS OP TYLOPHORA ASTHMATICA. A VERSATILE MEDICINAL PLANT

Tylophora asthmatloa plant belongs to the natural order

Aaolepiadeaa and family Asolepiadaoeae* It la a perenlal,

twining oreeper, growing abundantly in North and East Bengal,

Assaa, Kaohar, Chittagon, Western Ghats, Deooan peninsula,

Buraa and Ceylon* It Is known by the nane of Jangli-Plkvan

or 'Anta-aul' in Hindi* It Is also oalled *Country Ipeoaouanha1

by itc English name* The leaves are thiok and deep green

generally 5 to 12 ea long and 2 to 6 on wide* These are

soaewhat variable in outline, usually oordate «t the base

and aore or less downy beneath with soft siaple hairs. The

pedicle is 1 to 2 oa in length and is channelled* In dry

state the leaves are rather thiok and harsh and of a pale

yellow colour* In Indigenous systea of medicine, the leaves

are given three tiaes a day or oftener in five grain doses

for the treataent of asthaa, bronohitis, catarrh, early

stages of whooping oough and other respiratory diseases*

The leaves and roots are also used as cathartic, eaetio and

an expeotorant *

At the Blo-Organle Division, the work was initiated

to carry out the biosynthesis of tylophorine and related

alkaloids Isolated froa T. asth—tiea plants using radie-

labelled precursors and also to Isolate new blogenetioally

- 7 -

related alkaloids* Since thia plant was ont of the most

Important medicinal plants, the therapeutic action of the

compounds isolated against allergio rhinitis and bronchial

asthma was also evaluated* It beoame further neoessary to

study the acute toxioity of alkaloids/their distribution in

vital organs and also the mutagenicity of the potential drug*

!• Chemistry of the oompounda

" • 2

In this process we Isolated a new phenolic alkaloid

which was named as tylophorinidine. The ooapound (CggEngNOj),

m.p. 213-214* (decoap), @$fi£\ • 168« (o, 1.91 MeOH),

M* 365 differed from tylophorine (I) and tylophorlnine (II)

earlier isolated from this plant0. Its UV absorption at

260, 287, 313 and 340 nn (log€ , 4.64, 4.4i, 3.90, 3.17)

closely reseabled tylophorinine and indicated a 2,3,6-

trioxygenated phenanthrene skeleton* The presenoe Of c

phenolic hydroacyl function was inferred by a green oolouration

with ferric chloride and by the shift in it» UV maxima on

addltiou of alkali. The infrared spectrum (KBr) of the .

oompound showed absorption bands at 3448 (OH), 1200 (Phenolio

OH), 1613, 1538, 1515 (aromatio-C«C-) and 125C oa"1 (C^O-C,

-OMe). Its mass upectrum revealed a molecular ion at m/e

365 and intense base peak (M-69) at m/e 296. The latter1

it eharaoteristio of phenanthroindoliBidine alkaloids

arising from the cleavage of the pyrrolidlne ring (

- 8 -

OCH3

H3COOCH3

OCH3

H3CO

H3CO

+ rn

m/e 69

TYLOPHORINEm/e 393

Fig.1. Ms OF PHENANTHROINDOLIZIDiNE ALKALOIDS.

- © -

(Fig* l)* Thue the presence of pyrrolldlne moiety v u

evident* Thli a/e 296 ion underwent further fragmentation

indicating the poeeible preeenoe of two aethoxyls, one

phenolio and one alooholio hydroxyl, at C-14* A strong

peak at a/e 286 arising froa 106 by loss of CO was

indioative of the preeenoe of a hydroxyl at C-14*

Methylation of tylophorinidine with diazoaethane gave

a aonoaethyl ether (IV), c23Hji5N04 < M + 379> *hOi' NMR

speetniB showed the presenoe of three methoxyl groups, a

CH-OH funotion and five aroaatio protons* The fonation of

the aonoaethylether with dlasoaethane showed the presenoe of

only one phenolio hydroxyl group in tylophorinidine* The

methyl ether still had a hydroxyl group as seen in its IB

and also by the formation of an acetate (Vl)0?nax 1 7 2° OB~1*

Its Bass speotrun showed a very weak noleoular ion at a/e

421* The NMR speotruB of acetate was virtually identioal

with that of aoetyl tylophorinine and olearly revealed the

presenoe of an acetate group at C-14* Aoetylation of

tylophorinidine yielded a diaoetate (VII) whose IR speotrua

V^B6x 1760> 1 7 3° c""lf exhiblted the presenoe of a phenolio

aoetate and an alooholio aoetate group* Its NMR speotrua

also showed the presenoe of two aoetate groups at£2*39

and 2*12, the foraer being due to the phenollo aoetate

group* Treataent of o-aethyltylophorinidine (IV) with

perohlorio acid followed by reduction with NaBH^ afforded

:10t

• C H 3

H3CO

H3CO«

OCH3

1TYLOPHORINE

H3CO

CH3O'

OCH3II

(U-TYLOPHORININE

H3GO ORi

RO'

OCH3

mR-RpH

TYLOPHORINIDINE

H3GO

H3CO

DESOXYTYLOPHORININE

OGH3

;

O-METHYLTYLOPHORINIDINE3ZT R=CH3;R1=C-CH3

0AGETYL- 0 - METHYLTYLOPHORINIDINE

2 1 R ; R i = C - C H 3

TYLOPHORINIDINEDIACETATE

- 11 -

dl-desoxytylophorlnlne (v)« Thli leads to struoturo IV for

o-aethyltylophorinidine and VI for aottyl o-nethyltylo{>he~

rinldlne* Of the three positions 3,6 and T for the looation

of phenolio hydroxyl group In tylophorinidine, potltion 6"

Is preferred fro« a consideration of the NMR speotra of

dlaoetyl tylophorinidine (VII) and abetyl-o-aethyltylopho-

rinidine (VI)• The protons at 0-11 C-3» 0-4 and C-8 are

found at approximately the saae chemical shifts in both the

oompounds* The proton at C-5, however, occurs at £7*88 as a

sharp singlet in aoetyl-o-aethyltylophorinidine and is shifted

down field to £8.18 in the diaoetate VII* In ooaparison to

a methoxyl group, the aoetoxy group has a deshielding lnfluenoe

of approzljiately 0.22 ppa on the ortho proton and this leads

to position 6 for the acetoxy group in the diaoetate* Tylo-

phorinidine henoe has the gross structure (III), and

desoj^rtylophorlnlne being (V)«

The gross struoture (IV) assigned to o-aethyltylophorinidine

also represents tylophorinine (II)* That the two are not

ldentioal or antipodal is shown by their physioal oonstants

and those of their aoetyl derivatives*

Absolute configuration of tylophcyinldlne by X-ray analysis

An X-ray analysis was undertaken to establish the

struoture of tylophorinidine unaabiguously and for obtaining

information about the stereooheaistry of the aoleoule parti*

cularly around oarbons-13a and 14* It was found that there

_ 12 -

is one alooholio hydroxyl at 0-14, one phenollo hydroxyl at

C-6, and two methoxyls at C-3 and C-7*

For X-ray analysis, a heavy atom derivative namely,

tylophorinidine diacetate ••thiodide was prepared by refluxing

tylophorinidine diaoetate with methyl iodide in ohloroform*

for one hour and working up the reaotion mixture* The product

wai erystallised from a •ethanol-vater mixture, and the yellow,

needle-tfhaped oryttalf obtained were dried under vacuum. The

crystal! darkened slightly on long exposure to X-rays.

After X-ray work was over, the mass spectra of tylopho-

rinidine aonoaethyl ether and tylophorinidine diaoetate were

also available to us* These revealed molecular ions at 379

and 449 respectively* Also, the M-60 ion due to the loss of

acetic acid in tylophorinidine diaoetate oould be seen at

389, thus confirming the assignment of structure (III) to

tylophorinidine*

OH

HE-Diocetnt*methlodide

A re-examination of the IR speotrum of the methyl ether

of tylophorinidine showed the presenoe of a band at 3175 cm" *

This can be attributed to the C-14 OH, provided the latter is

- 13 -

postulated at Involved in hydrogen bonding* This is possible

if tht Methyl tthtr txlttt at a dlMtr* When thit work wat

ooMpleted, Govlndaohari et al • reported a reinvestigation

of tylophorinidine by non-X-ray Methods* Tht grots ttruoture

proposed by them agrees with that obtained by us froM X-ray

diffraotion* Also, their oonolusion about the trans diaxial

dispotlt ion of the C-14 OH aud C-13a H is confirmed by Our

study. But for tht orientation shown in III, tht C-14 OH

It below the plant of the molecule, and C-13a II above it*

Thit it jutt the opposite of that suggested by Govlndaohari

et al • Therefore, tylophorinidine has been finally assigned

struoture as III*

1*3 Isolation of tylophorine and related alkaloids fromFerguiaria pallIda plants closely related to Tylophoraastftnailea plantsp#

In search for compounds biogenetically related to

tylophorine, phytoohemioal investigation of the plant

Fergularla pallIda grown at Experimental Field Station, Troabay

was undertaken* Five phtnanthroindolizldlne alkaloid!, A

xo E were Isolated from the roots of thlt plant* Of these,

three were In Major amountt while the reHaining two fomed

minor constituents* Two of these three Major alkaloids were

Identified as tylophorine (I) and tylophorinidine (III)

reported earlier from Tylophora asthmatioa plant* Tht third

Major alkaloid C was idtntifled at tht Monomethyl ether of

tylophorinidine (obtained by Methylation of tylophorinidine

H3COOH

H

H3CO

OCH3

VIIIPERGULARININE

)CH3

H3CO

TYLOCREBRINE

H3CO

H

H3CO:

OCH3

IXDESOXYPERGULARININE

H

H3CO'OCH3

XIISOTYLOCRERRINE

with diesomethane) on tht batif of a*p., a.m.p., IR and TLC«

The fourth alkaloid Mol, wt« 379 was characterised a* a

3,6t7<*trimethoxyphenanthroindolisidihe derivative and vat

shown to be identioal with the ooapound obtained by hydro-

genolysis of o-methyltylophorinidine* The fifth minor

alkaloid contained 4 methOxyl groups in phenanthrene ring

and one alooholio hydroxyl at C-i4. This possessed 2,3,6,7

substitution pattern similar to that of tylophorine* Table

I summarises the main characteristics of these alkaloids.

Wherever necessary, the alkaloids were converted into their

acetate* and methiodides*

The absence of any bathoohromio shift in the UV spectrum

with alkali and presence of IR absorption at 3175 om"

indicated the presence of alooholio hydroxyl at C-14 In compound

D* The MS also showed a base peak at m/e 310 arising by

cleatage of the pyrrolidine ring by a retro Oiels-Alder

reaction oharaoteristir/ of phenanthroindolisidine alkaloids*

A strong peak at m/e 281 arising from that of 310 by loss of

CHO was! In oonflrmity with the loss of OH at C-14. The IR

spectruin of alkaloid D and of the monomethyl ether of tylo-

phorinidine (IV) were nearly superimposable except that the

latter revealed two additional absorptions at 871 and 735

cm while an extra peak at 851 cm" was seen in alkaloid D.

However', o-methyltylophorinidine possessed a (<•) rotation

but the: alkaloid D showed a (-) rotation* This led to the

\ ._ 16 -

Table I. Tylophorine and related alkaloids from

Pergularia pallIda plants.

Alkaloid MS(M)' Rotation

"esf-No» ofMethoxylGroups

No.of hydroxylgroups

(log? >Identification

B

D

E

3 6 3

3 9 3

<C23H25N 04 ) 3 7 9

409

+ 190.6 Two,i.O CH30H)

- 14.6 Three(c,0.25 CHC13)

- 21.45 Pour,l.l CHCI3)

- 27.42(c,1.05

- 9.4(o,0.04 CE

Three

Pour

One phenolicOne alcoholicat C-14

One alcoholicat C-14

One alcoholicat C-14

258(4.7),286(4.4)310(3.9),340(3.2)356(2.8);with

, alkali258^4.6),296(4.4)328(3.9),352(3.7)368(3.4)

Tylophorinidine

258(4.3'311(3.5360(2.0

,286(3.9),341(3.1)

259J4.6),291(4.4)340(3.2'),357(2.8)

3,6 ,7-Tri»eth-oxy-phenantbr-oindolizidine( Deoaypergul a-rintoe)

Tylophorine

260U.7) ,287(4.4) 3,6,7-Triaethoxy-313(3.9),341(3.2) phenantbroindo-357(2.8)

258302

(4.4),287(4.2)(3.6),339(3.1)

lizidine. ( - ) -O-Methyltylo-phorlnidine.2,3,6,7-Tetra-•ethO3ty-14-hyd-roxyphennnthro-indolizldine(e.g. 14-hydroxy-tylophorine)

- 17 -

F

oonolusion that alkaloid 0 and o-aethyltylophorinidine (IV)

were related to eaoh other as diastereoisomers. Since this

new stereoisoaer has been obtained from a natural souroe,

it h«t'been named as pergularinine.

The IR spectra of pergularinine and o-uethyltylopho-

rinidlne showed the presence of a band at 3175 on" • This

can be attributed to the C-14 OH where the OH group of one

•oleoule is bonded with the lone pair of electrons on the

nitrogen of the other* This was confirmed by the preparation

of the corresponding methiodides in whioh well defined OH

absorption oouldclearly be seen at 3460 om~ beoause the

lone pair of electrons was no longer available on the nitrogen

for bonding*

O-aethyltylophorinidine with (+) rotation has been shown to

exist a* (IV) in̂ Whioh the C-14 OH arid C-13a H are trans

diaxially disposed. The OH is below the plane of the

soleoule and C-13 a H above it* Therefore diasterec isomerio

pergularinine with a (-) rotation oould possess the structure? • ' • • • • • ' ; " ' • . ' • ' . - • • - 1 : 1 - • • • • • ; • . : • • • ' • ' • • . ' • • • . . • • • • ; '

(VIII) or its Mirror image* ORD studies of desoxypergula-

rinine (IX) in CHCln showed a negative Cotton effect of thesame order of magnitude in the region 270 nm as observed in. . . • • • ' • . ; ' : m ' • - • • . ' • • ' • ' • • ' . • • • • • ' • • • ' : - : . ; • • • ' • ' • . • ' • • • • • • • • • • ' . : . ; . ; ' " ' " • . • • . . • ( ' • . ; • • ' . •

tylophorlne (I)* This indicated that the two compounds

possessedthesaaeabsolute configuration at C~13a. In

tylophorine C-13a H has been shown to be above the plane;

- 18 -

Therefore pergularinine itself could be assigned the confi-

guration shown in (VIII). (-) Tylophorinine (II) having

the 'same substitution has been shown to be raeemic •

(tee interesting observation has been made during

hydrogenoly-is of (-)-pergularinine and (+)-o-methyltylopho-

rinidine* The latter gives the raeemic desoxy base while

(-) ptrgularinine furnishes the (-) desoxy base*

In alkaloid E, the alooholic OH was located at C-14

sinoe the loss of 2S9 amu (CHO) was observed at »/e 3ii in its

MS fron a fragment arising by the cleavage of pyrrolidine

ring (m/e 340). This group was not involved in interaoleoular

H bonding sinoe the OH absorption in its IR is quite pronounoed

unlike that in o-Hiethyltylophorinidine and pergularinine*

Alkaloid E contains four aethoxy groups and its UV values

resemble that of tylophorine (i) more than those of tyloorebrine

(X) and isotylocrebrine (Xl) the alkaloids with four OMe groups

already known • Tylophorine itself has been isolated froa

this plant. Alkaloid E haa been named by us as tylophorinioine*

Sodium borohydride reduction of tylophorinioine yielded

tylophorine (I) and thist together with the spectral evidence,

suggested that alkaloid E itself has structure (XII). This

compound has also been isolated from Tylophora asthmatioa

plant* If one assumes there is an analogy with the aooompanying

trimethoxy 14-hydroxy bases then the stereochemistry for

- 19 -

tylophorinioine can be written as shown in structure (XII)*

H3CO'

' ' . ' OCH3

XII

TYLOPHORINieiNE

1,4 Isolation of tylophorine and related alkaloidsfrom Ficua hiapida plants^. '

Of the six hundred and odd species of Fieus (Urtioaoeae)t

only two have been reported to contain alkaloids* The alkaloids

of the phenanthroindolizidine group have been isolated fro*

only Ficu* septioa .

Air»dried leaves of the plant vere extracted with alcohol-

aoetic aoid and the crude bases isolated as usual* Trituration

with dry alcohol gave an insoluble residue whioh on ooluan

chronatography over alumina and subsequent preparative TLC

yielded two alkaloids. One of these was characterised as

3,6,7-trinethoayphenanthroindolizidine and found identical in

all respects with (-) desoxypergularinine (IX) earlier isolated

by us from Pergularia pallida« The other alkaloid whioh

interestingly possessed a (+)-rotation was shown to be the

corresponding 14-hydroxyderivative* It was found to be identioal

in all respects with o-methyl derivative of tylophorinidine

(IV)2. This i» the first report of its Isolation from a

natural source*

- 80

tfhe aloohol soluble portion yielded one more alkaloid

whioh alter ohromatographie separation on an alumina column,

had m.p» 124-5* ̂ J j J * 2 7 6» 235 "• onaraottrlttlo °* • ••«<»derivative* The infra-red spectrum elosely resembled that

of •eptioin««* Its «/e ion at 365 was two aa«»es higher than

that of desoxypergularinin* (IX) whioh further confirmed

the ieoo nature of the bate* In NMR, A 2B 2 tyatea of four

protons at ̂ 6.8 and 6,63 vas attributed to H-3, H-5 and H-2,

H-6 re»peotively« A »ignal at^6.35 wat assigned to H-61

while the signal8 for protons, H-2' and H-5' appeared

respectively at ̂ 6.22 and 6.5. The three nethoxyls on the

aromatio rings were located at S$• 55, 3*48 and 3.3•

Thus seoo alkaloid >as been shown to be 6-(3*,4*-di«etb-

ozyphenyl)-7(4-aethozyphenyl)-ir2t3,5}8,8a-hezahydroindolizine

(XIII) and has been named by us as hispidine*- k . • , . . . . • • . • • • - . " . • ' • . • • • •

II. Biosynthesis of Tylophora alkaloids

II. 1 Preparation of radiolabelled tylophorine

In view of the important biological activities exhibited

by Tylophora alkaloids it was neoessary to prepare radiolabelled

alkaloids which in turn can be used to study metmbollaa and [

distribution in animals* These hate been prepared by using

biosynthetic method in which radiolabelled preoursors were

administered to the intact plants*

A possible hypothetical biogenetio soheae by whioh these

alkaloids could be formed in vivo is shown in fig* 2*

- 21 -

Phenylalanirie-2-€• • • • • • u

Cinnamlc Acid-2-C

f

Shikimic Acid

H

14Tyrosine-2-C

Tylophorine

3 . . 1 4 ' • • • ' 'Location of C

•I:

FIG. 2 . BIOGENESIS OF TYLOPHORINE.

- 22 -

3,4-Dihydroxybenioylaoetio aoid (XIV) by reaction with

^-pyrrolin* from ornlthine oould yield compound (XV) whioh

on condensation with 3,4-dihydroxyphenylpyruvio aoid (ZVl)

originating from tyrosin© would be expected to give compound

(XVII) and the latter by ozidatiye ooupling oould yield

tylophorine (l). Accordingly if oinnamio acid 2- C via

phenylalanine and tyrosine-2- C are the precursors then

tylophorine should be labelled at C«-8f and C-71 respectively*

3,4-Dihydroxybenzoylacetio acid (nv)oould also arise either

from prephenate via cinnamic aoid or by the way of aromati-

sation of shikiaio acid and its condensation with an acetate

unit. Such aroaatisatlon of shikiaio aoid does ooour in

Neurospora crasaa but has yet to be demonstrated in higher plants.

To verify the above hypothesis several radioactive

precursors11'12f13 v e y e administered to plants of Tylophora

asthmatica individually by wick technique and their relative

incorporation into tylophorine was studied*

In order to establish the position of the label at C-7*

and C-6*, tylophorine was degraded aooording to prooedure as

shown in fig* 3. Carrier tylophorine was added to the aotive

sample* Tylophorine was converted into its methiodidt bjr

treatment with methyl iodide in chloroform. The latter on

heating with sodium amalgam resulted in formation of Emde

baie (XIX) which when subjected to Kuhn-Roth oxidetlon with

chromium trloxide in pyridine yielded aoetio aoid possessing

- 23 -

carbon atoms*?and -7* of tylophorine* The aoid on sohmidt

degradation yielded methylamine and, oarbon dioxide whioh

was absorbed in barium hydroxide to give barium oarbona* .

carrying oarbon atom -9 of tylophorine* Methylamine was

converted into N-methyl benzamide (XX) by treatment with

bensoyl ohloride in alkali* Methyl of N-methylbenzamide

represented carbon atom -7* of tylophorine* Barium carbonate

was devoid of activity whereas N-methylben*amide was found .

to contain most of the activity as assayed in tylophorine*

The results are presented in Table II*

In order to explore the origin of ring A and oarbon

atoms-10 and -61 of tylophorine the role of various other

precursors was investigated* These oould arise * (Pig.2)

from 3,4-dihydroxybensoyl aoetio aoid (XIV)* The same in

turn oould be formed from phenylalanine via oinnamio acid,

p-ooumario acid and oaffeio aoid* Alternatively the shiklmic

acid-aoetate pathway oould be operating* With a view to

resolving these possibilities• phenylalanine-2- C, bensoio

aoid-i- C, bensoio aoid ring- C, veratrio aoid-i- C,

aoetate-2- C were administered to T* asthmatioa plants and

tylophorine isolated in each oase and assayed for its

radioactivity*

' •14 • • ' ' v12

Phenylalanine-2> C was found to incorporate efficiently

(Table I I I ) . The degradation (f ig. 3) of labelled tylophorine

|84:

OCH3

H3CO

H3CO/

OCH3

Tylophorine

Tylbphorine methiodideXVIII

' Pyridihel Kmno4

OCH3

H3CO

H3CO

COOH

COOH

CH3

H3CO

H3CO

H

Tylophorine methiodide

XVIII

)CH3

H3CO-

H3CO

OCH3XIX

Emde baseK.R.Oxidotioni

- 7 ' * .CHJ COOH

CH3

- 7 ' - 9CC

Schmidt IDegradation

- 7 ' - 9CH3-NH2+CO2

I Ba(OH)2BaC031

-7H3C-HN-C=d

XXN- Methyl benzamide

OCH3

XXII

FIG.3.DEGRADATION OF LABELLED TYLOPHORINE

Table II* Specifio activities of undiluted tylophorineand its degradation products*

Aotivity in -dpn/aM x 10 c

Tylophorine (I) 2*3Tylophorine aethiodide (XVIII) 2.1Bade base of tylophorine (XIX) 2*0Aoetio acid (sodium aoetate) 2*0N-Methylbenzamide (XX) 1.8Barium carbonate - .

Radioactive samples were counted on a Packard Model314 EX TRICARB liquid Solntlllation Speotrometer* Thescintillation mixture was prepared by dissolving 4 gof BBOT: 2,5-bis(2-5 tert butyl benzoxyazoyl) tbiophenein one litre of toluene*

Table III* Specific activities of undiluted tylophorineobtained from phenylalanine 2-**C and itsdegradation products*

Activity in-Kdpm/mM x 10 °

Tylophorine 4.5Tylophorine methiodide 4.5Eade base of tylophorine 4*4Acetic acid (sodium aoetate) -2,3,6,7-Tetramethoxy phenanthrenedioarboxylio4•4acidBaCOg 2*22,3,6,7-Tetramethoxy-phenanthrene -

revealed that oarbon atom-T1 was devoid of any aotivity*

This Immediately suggested that transformation of pheny-

lalanlne to tyrosine had not taken place during the

administration of .phenylalanine*

On the other hand, the incorporation of phenylalanine

rla oinnanio aoid would result in tylophorine with label at

oarbon aton -6». This was oonfirmed by degrading labelled

tylophorine to 2,3,6,7-tetramethoxy phenanthrene-9,10-

dicarboxylio aoid (XXI) which on deoarboxylation liberated

carbon dioxide* This was absorbed in barium hydroxide

solution to get barium oarbonate which possessed 50JJ of the

aotivity as that of tylophorine proving thereby that oarbon

atom -6» was mainly active as oarbon atom-7' was already

shown to be inaotive.

J ' • • • •" • • . .

2,3,6,7-Tetramethoxy phenanthrene (XXII) obtained

from the reaction mixture lacked any aotivity. This

oleariy establishes the relationship between phenylalanine

and ring A and oarbon atoms -10 and -6' of tylophorine*

Bensoio aoid-i- C, benxoio acid-ring- C and veratrio

acid-1- C did not incorporate into tylophorine* Aoetate

was found to be a poor preoursor as i t yielded tylophorine

with low activity* Had these three precursors been directly

involved, the degree of their Incorporation into tylophorine

would have been comparable to that of phenylalanine, i f hot

- 2T -

better* This vat obviously not the oase* The incorporation

of aoetate to the extent obferred oould hare easily resulted

through the glyoxalate oyole in whioh phosphoenol pyruvate,

a known preoureor of phenylalanine and tyrosine, if formed

from aoetate*

The above data thereforei offer strong support to

phenylalanine being an important precursor in the biosynthesis

of tylophorine and rule out the operation of shiklmio aoid-

aoetate pathway for this purpose*

0rnithine-5-i4C (Table IV) was also found to be

efficiently incorporated thus suggesting its participation in

the tylophorine biosynthesis via Apyrroline. To oonfirm the• • • ; 1 3participation of phenylalanine via olnnanio acid , the

latter with C label at-2 position was administered to

Tylophora plants as in oase of other precursors. It was found

to incorporate efficiently into tylophorine with more activity

in isolated material than in oase of phenylal&nine~2- C

experiments* ,

Biosynthesis of tylophorinidine (ill) containing phenolic

hydroxyl at C-6 and alcoholic hydroxyl at C-14 (C-61) was

also carried out using same precursors* The results (Table IV)

indicated that tylophorinidine isolated in each oase retained

•uoh less activity than the two main alkaloids* Therefore»

tylophorinidine might serve as a direot preoursor Of

tylophorinine*

- 26

Table IV* Remit a obtained fro* eacperlaent* With otherprecursors«

Precursor dp* dTylophorine

pm/mH $ incor- iporation

Tylophor in ineIpa dpm/mH % Incor-

porationdp«

lophorlnidnedp^KM i» Incor-

poration

Ornlthine-5-C14

2i42 8.4xiOS 0.030

24i? 9»5x10" 0.030

91 3.5x10* 0.001

1764

1857

48

7«

7 ,

1 .

2x10

SxiO5

3x10*

0

0

0

.015

.018

.001

767 2t8xl0** 0.002

534 l«9xlOS 0.001

25 0.9*10* O.OO1

The alltaloida isolated fro« benzole acid«l» C, benzotc acid ring- C and veratric acid-1- «experiments were found to be devoid of any aetivity*

- 29 -

Extended biosynthesis was carried out by other workers

taking lead from our view that oinna^lo aoid presumably is

first transformed into benzoylaoetio acid and its p-hydroxy

deriratiTO} both of which are also incorporated* In a

subsequent stage the keto acids ere converted to phenacyl

pyrrolldines and feeding experiments With doubly labelled

substances of the latter type have shown that not only the

unsubstituted compound and its p-hydroxy derivative, but also

the corresponding 4-hydroxy-3-m,ethoxy derivative, are

incorporated intact* These substances are thus key inter-

mediates and their role is underlined by the recent isolation

of phyllostenon (XXIII) from Cryptooarya Phvllostemon ,

a plant which also produces antofine (XXIV). Three alkaloids

with structures closely analogous to phyllostemon have been

isolated from the canthaceobs plant Ruapolia hypercraterformia.

OCH3HO*.

t XXIII

PHYLLOSTEMON

>CH3

H3CO

XXIV

ANTOFINE

- 30 -

The final steps shown in soheme (fig* 2) inolude

oxidative phenol coupling and other reactions analogous to

those which ooeur during aporphine biosynthesis* In addition

to the final products (I and II) indicated in scheme, any of

the other phenanthroindolisidine alkaloids so far isolated

could be produced by simple aodifioations of the soheme

similar to those that take place in the biogenesis of

aporphines. It is interesting that in spite of the apparent

symmetry in substitution pattern of rings A and B of tylo-

phorine (I), these are formed by separate pathways from

tyrosine and phenylalanine respectively*

III. Interaction of Tylophora alkaloids with theraotitins,proteins t lysogyme and bovine serua albuaih»

Tylophorine and related alkaloidsf including tylopho-

rinidine (TPD) froa the T^ indica plants have;been shown to

be active against leukemia, asthma and immunopathological

and inflammatory reactions in model experiments in laboratory

animals. These drugs do not affect the incorporation of

leucine In protein-synthesis* It is, therefore, possible

that these interact with enzymes involved in protein

synthesis, Inhibition and/or the regulation of other bio-

chemical reactions in the cell function* The investigation

of the interaction of drugs with proteins, particularly

serum proteins are also important from other aspects suoh

as transport across the bell membrane, bioavailability and

. - 3 1 ' - ••'• ••'.; • , • •••• ; • . -

the aode of drug action at the aoleoular level* So far

nothing is known about the interaction of tylophorinidine

and its analogues with biologloal aaoroaoleoules. Ve,

thereforef studied the interaction of the drug, tylophorinidine

with two serua proteins, vis. the transport protein, bovine

serua albumin (BSA) and the lytie enzyme, lysozyae, using

fluoresoenoe speotroscopy* In order to do so, initial

studies on the effect of pH and buffer molarity on the

fluoresoenoe of tylophorinidine were studied*

The fluoresoenoe oharaoteristios of tylophorinidine

were exaained in methanol, water and in increasing buffer

aolarity at varying pH, 5-9 . In methanol, the fluoresoenoe

aaxiaua was observed at 380 na, with a smaller peak at 365

na and a shoulder at 400 na* When the solvent was ohanged

froa aethanol to water the saae structural features were

observed with a 5 na shift in \uax *© longer wavelength.

In phosphate buffer of varying pH, 5-9, tylophorinidine

fluoresoed aaxiaally at pR 5*0, However, fluoresoenoe

eaission deoreased with increasing pH» The effect of buffer

concentration 0,06-0.2 M was also exaained at eaoh pH value

in the range aentioned above* Increase in buffer concentration

deoreased the fluoresoenoe intensity by 5-12 per oent, whiob

was reflected on the pKa value (Table V). Although the pKa

at 375 na is not altered significantly, a slight deorease

. 32 -

H3CO

H3CO

OCH3OCH3

of tylophorinidine.

Flft 5 STERN-VOLMER PLOT FOR CALCULATION OFASSOCIATION CONSTANT(KQ)FOR

TPD WITH BSA

2 4 6 8 W 12 U

TPC)[M]xtO"e

- 33 -

Table V: Effeot of Buffer concentration of pka

Buffer eoneenrrationM

0.06

0.1

0,3

300 mi

6.65

6.30

6.025

STB na

6.65

6.65

6.525

6.675

6.50

6.20

is observed at 365 na and 400 na as the buffer concentration

is lnoreased. Increasing buffer aolarity proaotes ionisation

of the C-6 bydroxyl group of TPD i.e. ionlsat'ion is brought

about by a higher lonio strength at low pH Values, irhereas

at a higher pH, a lower ionio strength is required (f ig.4).,

Proa the shore results, it is obvious that the

fluoresoenoe oharaoteristies of TPD oan be utilised for

identification purposes and oan also be advantageously used

for the Investigation of the aotion and lnvolveaent of

tylophorinidine in biologioal and oheaioal reaotlons.

III.l Interaotion of tylophorinidine with lysoiyao

- Fluoresoenoe speotrosooplo studies and the effeot

of tylophorinidine on the aetivity of lysoiyae indicated

that the drug assooiated with lysosyae at pH 9.2 efficiently

with an assooiatlon constant, Ka • 3.3 x 10* M " 1 at 26*C.

Xa inoreased with increasing teaperature in the range 26

to S5*C.

- 34 -

Th# calculated enthalpy change 4H was found to b«

2*3 X oal/»ol. Under the same oondltlon* a* abort, TPD

alto aaaooiated with the fr«« aaino aoid, tryptophan -with

a Ka of U7 x 10* M"1, indioatini halt tha «ffioi«ncy of

lt» aj#ooiaiioa with lytosyma* TPD aiflooiat«£

laaa active than th« unooMp2«x«<f *tinym* la the abo-r* '

te*peratur« range aithou^li beyond 45*C, the inhibition va«

nore significant, The resuite imply that TPD bind* lyaoxyme

outside- the cleft region in the temperature range studied*• • • • ' • • • , - ' " . • • •

However, vith inoreasing teaperature, the cleft region

widens and can aooonnodate part or whole of the molecule,

leading to the inhibition of lytio aotivity.

111,2 The interaction of tylophorinidine with bovineserua albumin ,

The interaction of TPD with BSA was examined at

different pH values viz* 5*4, 7.0 and 9.2. Here again, as

in the case of lysozyae, efficient binding as Indicated by

the quenching of fluorescence was seen at pH 9.2 showing

that ionised TPD bound better to the protein* There was

no ohange in the J\liax of BS4 emission spectra. Fig* 5

shows the Stern-Volmer plot for the quenching data, which

yield a Ka * 5 x 104 M"1. Change in ionio strength had

ho effect on the Ka indicating that the binding was not

electrostatic in nature* The stoiohioaetry of binding »

from Job's plot was 1:1, '

- 35 -

Ka waa found to Inorease with Increasing teaperature*

The enthalpy change, AH 3*4.5 x oals and the free energy

change, AQ and the entropy change A S were - 6*33 K oals and

33*3 esu respectively* The effeot of teaperature shows

that the predoainant interactive forces are hydrophobio in

nature* The TPD aoleoule perhaps binds to one of the drug

binding sites of BSA with ah affinity for negatively oharged

organio aoleoules, since at pH 9.2, TPD is negatively

oharged*

IV* Antiaathaatio activity of Tylophora indioa (Synonya TVasthaatioa, T* pubasoens. Vail. T. voaltoria I Might)'.

IV.1 First scientific report about the use of leaves In

the treatment of asthaa and allergic rhinitis appeared wherein

the details of the preliminary olinical trials were described*

The two striking features of this trial were (i) aarked

relief In 40 to 50 per cent patients after a snail dose of

3 to 6 leaves only (ii) a high inoidenoe of side effects

(sore aouth, loss of taste, voaiting etc in 75 per cent20 21

of patients)* Double blind cross over trials ' with

this aaterial were also carried out* It was therefore

thought worthwhile to isolate aotive principle froa this

plant* Eventually, it has been found out that the total

alkaloid extract containing tylophorine and related

alkaloids isolated froa this plant possessed the saae action

as observed with the leaves. This extract also offered

_• 3 6 -

long term protection against this disease. Moreover, this

extract was derold of any side effeots as witnessed with

leaves, alcoholic extraote and dried powders*

• 22

Later on, its mode of action was investigated by us

in collaboration with the Haffkine Institute, Bombay* In

these pharmaoologioal studies, it was seen that this extraot

possesses antianaphylaotio activity as observed in passive

peritoneal anaphylaxis in rats* The drug also possessed

•ild antihistaminio and antioholinergio activities. Studies

of the extraot on the bronchial smooth musole both in vivo

and in vitro did not reveal bronohiodilator potential of the

drug* Another important observation is that this extraot

has similar action as that of ororoolyn (disodium oromoglyoate)*

However. Tylophora drug has the advantage that it is effective

in microgram quantities and also it is orally absorbed

whereas cromolyn is inhaled each time to the extent of 80-25

mg whioh gets directly deposited on the lungs* Therefore,

the drug developed by us should be considered suporior*

The testing parameters used are as follows:

Preparation of rat antiserum: Male rats. 120-170 g, were

injected with egg albumin (10 mg/Kg, i.m.) and B. pertussis

organism (2 x 10 ) intraperitoneally* On the twelfth day

the animals were anaesthetised with ether and blood was

oolleoted by oardiao punoture* After clotting, the serum

- 3T -

was removed, oentrifuged and stored at 0»C,

Passive peritoneal anaphylaxia,: Rat anti-egg albumin Benin

was'diluted 1:2 with 0,15 M saline* One ml of the diluted

serum Was injected into the peritoneal oavity of male rats

(206-300 g). After a latent period of two hours, 5 ml of

heparinized (50 mcg/ml) Tyrode solution containing 2 mg

of egg albumin was injected intraperitoneally* The animals

were sacrificed tvfter five minutes, abdomen was opened, the

peritoneal fluid was collected and oentrifuged at 100 g, for

five minutes. Hie supernatant was assayed for histamine*

Test drugs, in the volume of 1 ml, were injected one minute,

prior to the injection of antigen* The results are given

in Table VI,

Table VI: Inhibition of Histamlne Release by Tylophora >; Alkaloids as compared to D.S.C.G.

Method: Passive Peritoneal Anaphylaxis

Dos« % Inhibition ofCompound mg/Kg histamlne release

D.S,C.G. 5.0 89

Tylophora alkaloid 5.0 45

The prolonged relief offered by the same oould be due to

its aotion on dell mediated Immunity sinoe this dould prevent

- 36 -

23

experimentally induced anaphylaxif in rata . Further T*\

extracts also reduced the Sohults-Dale reaction in sensitised

guineapigs* Additional pharmaoologioal evaluation of

Tylophora' by others has substantiated the above findings as

slgnifleant improvement in lung funotion tests, decrease in

leuooeytlo and eosinophil oounts and increase in 17-keto

steroid excretion in the urine was observed with this drug.

Lung function tests were oarried out by estimating tidal volumes,

vital oapaoity, timed vital capacity, compliance, maximum

ventllatory volume and peak expiratory flow rate (PFBR)*. . . ' • . • • • ' ' . • #

V. Aoute oral toxicity ot Tylophora pure totalalkaloid extract;

These studies were carried out at the Industrial

Toxicology Researoh Centre, Luoknow* Tylophora alkaloids in

13*0 gm amount was isolated on large soale*

Male albino rats 150-200 g body weight, were indivi-

dually caged and maintained on pellet diet and water adr : ' • ; • • • • • • ' • ; • • - ' •

libitum* The animals were acclimatised to temperature and

lightning conditions of the animal house*

The animals were fasted overnight* Single dose of

the test compound, suspended in the vehlole/or peanut oil

was administered to each rat orally with the help of 1*0 ml

syringe attached with a blunt 16 gauge needle* The oontrol

group of animals were similarly treated with tht vehlole/or

- 39 -

peanut oil only* The animals wera under olose observation

for a period of 15 days* Signs of poisoning and time of

death were reoord«d* The LDQ0 values were determined by

the Method of Veil (1952). Toxioity rating waa aooordlng

to Gleason et al« (1969).

Dosage< / 2 Dead/Dosed Death Signa of toxioity

Control(Peanut o i l )

12*5

25,0

50,0

0

0

1

3

4

4

4

4

100.0

1 on 6th day Dullness,ereotlonof hair,respiratory

2 on 5th and distress, nasal dls-1 on 6th day charge, diarrhoea,

salivation andAll on 5th deathd a y • : ... : . ; • '•

tAutopsy of the animals at the end of 15 days did not

indicate any significant change! in vital organs*

'• Single oral administration of Tylophora alkaloids seem

to be extremely toxio to male albino rats at the tested dose*

LDQ0 was found to be 35.32 ag/kg,

VI* Acute toxiolty studies pertaining to biochemical.

lora alkaloids,"

This waa Investigated after single and multiple dose

administration* Rats given Tylophora extraot orally dally

for 15 days at different doses (0, 1*85, 2.5, 5*0 and 10.0 mg/k*)

- 40 -

hat suggested mild to severe tcxioologioal effects* The

highest dose was toxio as oould be seen with the signs

of toxioity and mortality of animals* The lower doses

indicated varied biochemical and haematologioal ohanges

whloh are not very significant* It is expected that even

these ohanges would not be seen at the therapeutlo dose

whiqh is miorogram per kg* Results are summed up ai followst

Results

VI.1 Mortality and morbidity

Rats exposed to the plant extract of Tylophora (1.26

and 2*50 mg/kg/day) for 15 days did not produoe any signs

of poisoning or overt toxioity* However, animals dosed with

5,0 and 10*0 mg/kg/day exhibited mild to severe olinloal

signs of toxioity whioh included inactivity, dyspnea and

diarrhoea* All the animals treatedWithTylophora (10 mg/kg/

day) died within ••ven days of treatment whereas 2 animals

died after 5 mg/kg/day treatment*

VI«2 Organ body weight ratio

A comparison of the organ body weight ratio of rats

exposed to 1.25, 2.5 And 5.0 mg/kg/day revealed a marginal

Irortase (P<0.05- P<0.01) in the adrenal weight* Kidney

showed a marginal inorease (P<0.05) at 2.50 mg/kg/day whereas

testes weight was inoreased (P<0.08) at the highest dose

- 41 -

Table VII: Relative organ weights of Male rats after daily oral

administration of Tylophora alkaloid for 15 days*

Dose(•g/kg/D)

•Control(Peanut oi l )

1.25

2.50

5.00

Liver

4.11

07i3

4.27' • - ' . • + • : ' • • - .

07*4

07l9

3.93+

07i9

Kidney

0.69

0703

0.76• • • ' • • • . • ' • ' • • ' . .

0703

6.78d

• • - . " • •

0703

0.75+

0702

Adrenal

0.O19; • • • . - • / •

o7ooi

0.02°. : • • • •

o7ooi

0.03b

• < • • ' •

0.002

0.03d

• •

07004

Brain

0.97•

0703

0.99. ' +-

0.03

0.92•

0.04

0.93

0706

Spleen

0.58• • •

o7i0.49

.- • • •

07l3

0.33

0.02

0.33+

0.05

Testis

1.06+

0705

1.08• +

0.12

1.25

0.08

1.29°• •0.05

Epididyais

0.26+•

07004

0.30•

0706

0.48*+ •

o7oi

0.46b

d704

Values represent the nean + SE of 6 animals.

a = P<0.001; b .•'• P<0.001; c = P<0.002; d = P<0.05.

- 48 -

level* An increase in the weight of epididyvis both after

2.50 and 5.0 mg/kg/day treatment was also observed (Table Y H ) .

VI.3 Biooheaioal studies

A highly significant (P<0.OOi) fall in the activity

of liver GOT and OPT was observed at different dose levels.

However, the activity of senna GOT (P<0.0i,<0.02) and GPT

(P<0.00i) was relatively less significant. The activity of

alkaline phosphatase both in liver and serum showed a signi-

fioant increase (P<0.00i), but no change in liver protein

could be recorded. A significant increase (P<0,00i) of

serum protein was evident* A marginal fall (P<0.02) was

also Seen in level of blood sugar at 2*5 and 5.0 mg/kg/day

(fable •III).

VI»4 HaematolOjftioal studies

Except for a significant decrease in the level of

haemoglobin (P<0,O0i, P<0.0i) there was no other significant

change in blood picture of male rats exposed to different

doses of Tylophora extract (Table IX ).

Conclusion

Rats given Tylophora extraot orally daily for 15 days

at different doses (0, 1,25, 5.0 and 10.0 nfi/kg) has sugge-

sted mild to severetoxioologioal effects* The highest

dose was toxic as could be seen with the signs of toxicity

-43 -

Table Till: Biochemical changes in lire* and sens* of Male rats after daily

' ' ' oral administration of Tylophora alkaloid for 15 days

' : - Dose

Control(Peanut o i l )

• ' . - . . ' • ' - •

: : ::1.25;V--v : \

2 .50- . - ' " • • - . . ' • " ' ;

- ' • . - ' -

- • • - . • • • ' • •

5.00

Alk.Liver

0.088\ - . ' • • • ' • ' : ' . . • •

0~OO5

0.08. • + ' • ' . -

07005

0.195*

07012

0.151*" " * • • ' : ' • • • - '

PhosSerum

6.088

• ' • : • + •

o7b05

0*203*

07002

O.l2i*

GOT*Lirer

14*69. . • •• •

1739

3.48*. •" • + • • v • •

OTBI

2.28*. . • • - . + • - - •

0.7>

2.49*

0738

Serum

0.021

07004

0.017

0.007°

0.003b

+•07004

GPTLirer

17.22

1716

6.86*: • . + . • - ' • • .

0762

2.28*

07*3

2.49*

0*38

Serum

1.93•

0719

1.66• • •

0725

0 .007*

07001

0.003*

070004

ProteinLiver

92.79

4793

87.07

. 6716

89.63

2789

102.87

1731

• • »

Serum

22.49• ; .

0789

31.00*

0703

68.27*

1772

69.37*

2709

Bloodsugar

110.55•

37li

106.40

5756

97.40°

1786

88.42C

5729

Values represent the swan £ SB of 6 animals

a m F<PwOOij b » P<O.pl C * P<X>.02Actlrity expressed in */'•• molm p^ruvate released/g tissue or/ml seruft/min; **/umole phenol

released/g tissue or/ml serum/min; •*• mg/g tissue or/ml serus/min.

Table IX : Baeaatologlcal changes in Male rats aftar daily oral"adBintfstration o f Vylopfcora-alkaloid.fox.l5.4a.x»».,

Dose(.ag/kg/D)

Control(Peanut o i l )

1.25

2.50

5.00

RBC(lO6/»-3)

8,944

0743

10,594

0757

9,824

0736

8,384

0781

WBC(iO3A»3)

7.8374

0777

6.77

0721

9.684

o7ei

8,0754

0735

Hb(g/lOO «1

18,67

15.95b

4" '" ••'

0746

14.75*4

0738

13.2*4

0787

83,25

2792

81.5• 4 '"''".

1744

75.25

5754

79.75

2787

*P

12.254

2796

14.0• 4 '

2716

22.5

5786

19.0

3718

2,25

0725

0.75°.4

075

O.75b

' 4'O725

0.75b

40725

E

2.254' •

0725

3,75*

075

1.754 ' '

0785

0.50b

4

0729

Values represent the Mean + SB of 6 aniaals,a * P<0,001; b « P<0,01; c * P<0.02; d • P<0.05.

; • • • • • 4 5 - . :

and Mortality of animals* The lower dosesindicated varied

biooheaicel and haematoiogioal changes* This study suggests

the need tor long tern evaluation of the teat Material*

VII* *Biodistribution an<7 pharBaookinatio atudiaaof 14G-Tylophora alkaloids.

Introduction

14

C-Tylophora alkaloid mixture was prepared by admini-

stering U-C-tyrosine. The biological aotivity of these

ooapounds is already discussed in the report. In view of

this it was necessary to investigate the metabolism and

distribution of this drug. This aspect with theae ooapounds

is not yet known* The speoifio aotivity of the compound used

was 0.03jiol/ag* 14C-labelled ooapound (5 mg> was dissolved

in 2.0 al of 0.05 N HC1. Thf pH was adjusted to 6.5 with

dil NaOH* The solution was oentrifuged to reaove the

insoluble impurities* The clear solution was used for bio-

distribution studies*VII.i* Bio-distribution studies

Swiss mioe weighing 25-30 gms were administered,0.016

Jioi (500 .ug In 0.2 al), 14C-labell«d Tylophora drug either

i«v* or orally keeping an equivalent amount as standard* At

speoifio time intervals, the mice were saorifload and the

various organs were removed for assay of radioaotivity* The

Carried out in collaboration with Radiation MedioineCentre, Parel*

- 46 -

Table X : Organ distribution studies lit pice at different tiaeinterval: % Administered dose*

5 pin . i .v. Inj. 15 min.2 1 2 3 4

i hr.1

2 hr. Oral Ada.4 2 hr 2

BloodLiverSpleen

KidneyStomachS«l. IntLarge Int

Heart

LungsMuscles*Brain

1.093.79

0.24

3.25

0.88

I*.49

0.40

1.16

28.3

0.95

1.458.03

0.74

4.110.59

5.29

0.51

0.93

29.11

1.48

0.79 1.23 1.25 0.642.42 4.55 2.68 1.850.22 0.13 0.33 0.22

1.40 0.69 1.06 1.720.41 0.55 0.62 1.38

6.51 10.78 6.14 5.40

0.02 0.02 0.34 0,430.64 0.65 0.95 0,176.86 - 14.68 24.250.33 0.18 0.48 0.36

0.421.09

0.16

6.430.33

1^88

0.09

0.15

2,35

0.04

0 .491.27

0.21

0.86

1.57

4.59

0.06

0.06

6.41

0.10

0.421.49

0.09

1.22

2.03

1.39

0.04

0.06

4.69

0.08

0.490.58

0.28

0.31

20.34

2.092.69

6.16

0.24

5.98

0.24

0.860.73

0.23

0*26

8.64

6.700.49

0.22

0.09

4.99

0.46

* Blood : 1% of the body weight.* Ma«ele:40* of the body weight.

disseoted organs were out into pieces and extracted twice

with chloroform The chloroform vat evaporated completely*

To the evaporated residue, 10 ml of liquid scintillation

fluid was added and counted in a liquid sointillation oounter*

The counts were oorreeted for quenching by internal standard

method. The aotivity In eaoh organ is expressed as £ admini-

ttered dose* The activity in total blood and total muscles

were ooaputed at 1% and 40# of the body weight*

Table X. shows the organ distribution studies in

•ioe at various time intervals after administration of

C-labelled Tylophora drug* *Sinoe the patients are given

the drug orally, the distribution 2 hrt after oral adainie-

tration is also studied;

VII,2 Metabolisa studies

1 Two hours after the i.v, administration of C-drug

to the awiss aloe* the organs were disseoted, aatcerated and

extracted with ohlorofora* The chloroform extraott were

aubjeojbed to HPLC. Blood, Liver, Kidneya, Lunge and Urine

extracts were analysed, and compared with that of Tvlophora

standard; It was found that HPLC pattern of the control,total

alkaloid extraot wat nearly the tame as that of the extraots

derived from various organs indicating thereby that the drug

wat not metabolised into other metabolites whioh oould pause

a direot or indireot damage in vivo* This observation indeed

should be considered as a plus point In favour of Tvlophora

as a drug*

VIII; Antioarcinogenloity of Tylophora alkaloids

This was investigated by studying aiorosoae Mediated

binding of H-benzpyrene to DNA at 100 yui levels* It was

found that Tylophora alkaloids did inhibit the adduct' • - ' • ' • • • • • 3 . • : • • "

formation between H-DP and SNA wh:

of an anti-oarcinogenio substanoe*

' • - ' • • • ' • • • • • 3 : • • "

formation between H-DP and SNA whioh is velry obaraoteristio

- 4 9 -

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