CHEMICAL INVESTIGATION ON THE LEAVES OF

•CHEMICAL INVESTIGATION

ON THE LEAVES OFZANTHOXYLUM BUDRVNGA

•

•

•

•

OCTOBER 1998.

•

SUBMITED BY

RAINY CHOWDHURY

Roll No. 9205 F Reg. No. 92705

Session: 1990 - 91 - 92 J,.

•

ORGANIC RESEARCH LABORATORY

DEPARTMENT HI<'CHEMISTRY

BANGLADESH UNIVERSITY OF

ENGINEERING & TECHNOLOGY

DHAKA-IODO •.1IIIIIIIIIImllllllllllllllili

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•

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11

1.2

1.2.1

12.2

13

14

2.1

2.2

2.3

24

2.4.1

2.4.2

2.4.3

24.4

2.4.5

2.4.6

CONTENTS

CHAPTER 1

INTRODUCTION

(page 1-45)

General

Review on the Plants of the Genus Zllnt1UJxybml

General

Chemical Studies on the Plants of the Genus ZamhoxylumReview on Zanthoxylum hadrunga

Objective of the Research

CHAPTER 2

EXPERIMENTAL(Page 46 - 62)

General

Extraction of the leaves of Zanthoxyhlm budrungaExamination ofEltract M

Examination of Fraction M

Minimum effort column chromatographic separation of

"'Taction M

Separation of the components of Fraction M1

Preparative piate chromatographic separation oIFractioD M20.

Preparative plate chromatographic separatlon ofFractioD M%b

Preparative plate chromatographic separation of Fraction M:!<

Study on Fraction MI.

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Page

1- 3

4

4- 6

7-41

42-44

45

46-48

48-49

49-50

50

50-51

51- 52

52-53

53

54

55

•

2.5

2.6

3.1

3.2

3.2,1

3.2,2

3.3

Separation ofthe component~ of Fraction MJ

Examination of ethyl acetate fraction, Fraction E

CHAPTER 3

RESULTS AND DISCUSSION

(page 63-122)

Study on EItract M

Study on Fraction M

Separation and characterizatIOn of compoumb of Fraction M1

SeparatIOn and characterization of compound~ a/Fraction MJ

SeparatIOn and characterization of compo/Jnd~ of Fraction E

CHAPTER 4

SUMMARY

(Page 123 -126)

CHAPTER 5REFERENCE

(Page 127-132)

56-59

60-62

63

6364 -88

89 -100

100-122

•

LIST OF FIGURE

Fig. n08. Pilge

1. Specimen of ZanthoxylRm bud'unga 44

2 IR spectrum of compoUlld ZBI in thin film 65

3. IH NMR spectrum of compoUlld ZBI in CDCb 66

4. TRspectrum of compound ZB2 in thin film 68

j III NMR spectrum of compound ZB:l in CDCh 69

6. IR s~trum of compound ZBj in thin film 74

7. UV spectrum of compound ZBJ in methanol 75

8. IH NMR spectrum of compound ZBJ in CDjOO 76-77

9. Mass spectrum of compound 2:BJ 78

10. IR spectrum of compound Z~ in thin film 83

11. UV spectrum of compound ZBt in methanol 84

12. IH NMR spectrum of compound Z~in CHlOH 85-86

lJ. Mass spectrum of compound ZIlt 87

14. IR spectrum of compound ZB5in thin film 90

15. IH NMR spectrum of compound ZB5 in CDC!) 91

16 IR spectrum of compound ZB~in KBr 93

17 UV spectrum of compound Zs. in methanol 94

18. IH NMR spectrum of compound Z~ in CDlOD 95-96

19. Mass spectrum of compound ZB" 97

20. IR spectrum of compound ZB7in KBr 101

21 IH NMR spectrum of compound Z)h in COlOO 102

22. IR spectrum of compound Z~ in thin film 105

2J. UV spectrum of compound ZBo in methanol 106

24. Mass spectrum of compound ZBa 107

25. IHNMR spectrum of compound ZBs in CDCb 108-109

26. 11CNMR spectrum of compound ZBsin CDCI) 110-110

27. UV spectrum of compound Zs, in methanol 117

28. IR spectnun of compound Zs, in KEr 118

29. IH NMR spectrum of compound ZUg in methanol 119-120

)0. Mass spectrum of compound ZB, 121

LIST OF TABLE

Tllble 00$.

I. Reported medicinal uses of some plants of Zanthoxylum rIenus

2, Chemical constituents of plants of Zanthoxylum Genus

3, Chemical constituents of Zanthoxylum budrunga

4. Minimum eUort column chromatographic separation of Fraction M

5. Column chromatographic separation of Fraction Mz6. Column chromatographic separation ofFractioD MZoI

7. Column chromatographic separation of Fraction ~

8. Column chromatographic separation of Fraction E

9, Comparison of IHNMR absorption ofPseudophrynarnine A-I and ZBs.

10. Comparison of IJC NMR absorption ofPseudophrynamine A-I and Z~,

Page

4-6

35-41

44

51

52

55

57

60112113

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•,

•••••~,~. r.,!/:~"rr1":£,P.fOi.tr:' .....~e:...... Ilt~ ' ..•.

:d 1(',11. 58}':1.1 GENERAL ~ I.. ~.-._ ••• - ••••••, 0 ~.• '--- ...•. ~-;:;.\_"r~.•~.'fj'~~~.•._- .,.,._~

Men and other animal arc dependent on nature for existence in one wa 'Or' cr to a great

degree and the plant kingdom is of vital importance for them. The plant kingdom supplies us

not only with food and shelter but also with medicine and raw materials for clothings.

In the early stage of the civilization investigation on natural product received tremendous

boost because of theIr therapeutic uses. Discovery of new isolates accelerated the search for

newer medicines. During this period these natural products not only constiMed the main

source of organic compounds but also provided the main problems of organic chemistry

At the beginning of the nineteenth century when modem chemistry and phannacy began to

develop the original impetus to study the natural products utilizing medicinal plants also

started to develop. Morphine, the hypnotic and anaesthetic principle of opium was one of the

early (1805) active principle isolated from plant bodies, Quinine from cinchona bark used in

the treatment of malaria, atropine from Atropha belladonna, cocaine from coca leaves,

nicotine from tobacco leaves were other examples. Subsequently a whole series of plants were

investigated to detect and isolate the active principles This led to the discovery of a group of

nitrogenous bases having complicated structures and physiological activities and were called

alkaloids. The alkaloids of Rauwolfia group SlIChas Reserpine is used in medicine in mental

conditions, including tension and anxiety as well as in the treatment of hypertension. The

alkaloid vinblastine from Vmca rasca Linn. has been subject of medical studies particularly in

field of cancer.

From the very ancient time various traditional system of treatment like Kavuaze, Ayurvedic

and Unani have developed based on the use of medicinal plants. A number of large scale

academic and industrial screening and evaluation programs are now in existence and many

thousands of new plants are studied in the belief that primitive man has not exhausted the

possibilities of the plant kingdom,

Modem chemotherapeutic drugs have progressed research works in the field of natural

products chemistry for the newer medicines. These drugs, unfommately have a large degree

of side effect but systematic research in the field of natural produets may lead te the discovery

CHI\PfER I, INTRODUCTION

2

of newer drugs having minimum or no side effect. Some traditional drugs are particularly

attractive for some aliments because suitable alternatives are not available in modem

chemotherapeutic drugs. General tonics which improve the body resistance and remedies for

liver disorder, arthritic conditions, asthma, degenerative and old age conditions are examples

to the point Besides these the modem chemotherapeutic drugs have not yet been able to

conquer cancer and AIDS. It is expected that in the near future the chemistry of natural

products will afford newer medicine for the therapy of these fatal diseases.

The people of the developing country like ours cannot afford to use expensive modem

chemotherapeutic drugs due to economic reason. But our country abounds with a vast

majority of medicinal plants and herbs. Plants found suitable as remedy for anemia like

AcaCia catechu' (Beng. Khoyer), Termmalla Gryunal(Beng.Arjun, Aijuna), Coccinia indlcal

(Beng. Telakucha), TermifUllia chebula! (Beng. Horitoki), Eclipta albal (Beng. Keysuria),

Hydrocotyl aSiatic I (Beng.Thunkuni), lpamaea turpethuml. (Beng. Dulkalmi), Plumbago

zeylanica'(Beng. Chita, Chitruk), Trigonella joenumgraecum1 (Beng. Methi), effe:etive as

antifertility agents e,g" Acacia catechu' (Beng. Khoycr), Abrus precalonus Linnl (Beng.

Kunch), Areca catechu l.mrl (Beng. Supari, shupari), Carica papayab (Beng. Papaya, Pipay),

Plumbago zeylanica Unn' (Beng. Chita, Chitruk), Datura jastuosa8 (Beng. Sada Dhutura),

Acacia arabiea4 (Beng. Babla) etc; possessing antiseptic properties like Trigonella

joenumgraeICum9 (Beng. Methi), Eucalyptus globu/us! etc; releiving asthma e.g" Alstonia

seholaris' (Seng, Chhatinm), Cae.salpinia cristia! (Beng. Nata, Nata Koromza), Eugenia

jambolanal (Beng. Jam, Kalajam), EuginiajambO!ll (Beng, Jamrul), Coecinia IndiCal (Beng.

Telakucha), Mimusops dengi! (Beng. Bokul, BakuJ), ,TermifUllia ehebulal (Beng. Horitoki),

Dalura metal Linnl (Beng. Dhutora, Dhutura), Datura stamonium Linnl (Beng. Sada

Dhutura), Ricinus communis Linn' (Beng. Verenda), Aegel marmelosl (Beng. Bel), Mangift-ra

mdlcal (Beng, Aam), Adhatoda vasia/ (Beng, Bashok), Calofropis giganfea1 (Beng.

Akondo), Hydrocofyl a.~lalie' (Beng.Thunkuni) etc; effective against bronchitis AcaCIa

jame.nanal (Beng. Guya babla), Acacia catechul (Beng. Khoyer), Euginia jambos! (Beng.

Jamrul), Eugenia jambolanal (Deug, Jam, Kalajam, Chotojam), AcaCia arabical (Beng.

Babla), Puniea granantum1 (Beng, Dalimgach), PSldllon gI{V<lva!(Beng. Peyara , Piyara),

Hydrocolyl asiallel (Beng.Thunkuw), Vernonia cinereal(Beng. Kalajira), Lawsoma inermis1

(Beng. Mehidi, Mendi), etc; effective against cancer Jatropha gnssypl/alialO, Vinca rosea

CliM'rER 1: INTRODUCTION

3

Linn']] (Beng. Nayantara), Xanlhllml $Irumoriwn Linnl(Beng. Bonokra), arc few examples

which grow In abundance in Bangladesh.

Phytochemical study on most of these plants have bel::n carried out but in large number of

cases the study is difinirely far from complete. Moreover, there are a host of plants which are

used as folk medicine but has not at all been sul:!jeetedto chemical investigation.

In the present investigation a systematic phytochemical study on the plant Zbudrunga has

been carried out In tbe following section a review on the genus Zanthoxylum followed by a

review on the species Zbudrunga are presented .The findings of the investigation are

presented in the subsequent chapters.

CHAPTER 1: INTRODUCTION

1.2 REVIEW ON THE PLANTS OF THE GENUS ZAN11IOXYLUM

1.2.1 GENERAL

The genera Zanthoxylum belongs to the family of Rutaceae. The family of the genus

Zanthoxylum has about sixty species, of which five are reported by Kirtikar 12 [0 possess

important medicinal properties. In India subcontinent there are 20 species ullder the genus

Zanthoxylum.A number of plants of this family are reported to have insecticidal properties

besides medicinal uses. Table 1 outlines the medicinal values of some Zanthoxylum plants

reported in the literature.

Table 1

Reported medicinal uses of some Plants of Zanthoxylum Genus

Botanical name P,rt .r tho Reported medieinal use References

oftbe plants plants

planll powerful inhibitor ofletuce seeds "appetizer; anthelmintic; abdominal

troubles, u,clUl '" 'f' M" -fruits disease',diseases of the lips, headache,

heavine •• , leucoderma, asthma, trouble.

Z.accallrhopeJiMm ofth. 'pleen

"DC (Iamhul) .••• d. and b..-ks """ • ..-omatic tonic '" fever, ;,dyspepsia and cbolera

fruit" branches """ • remedy Co< loothache;AI,o

and thom. deemed Slomactie ""d carminative and

employed to intoxiCllle fish.

'- ,oake bite


o

Boumical name of P.rt of Reported medicinal use Referencesthe plants lb.

plants

Ztdtllum

plants same as those of Z,accanJhopod1um "(Gaira, tun. nepald hamal

household grain prole(:Unl, mothproofing

agents; used as bacteriostatic ogent for

preservation, ""' • "'" liquid fu, 13,14,15,plants

clearing away lung-bean; used .u highly 16,40Zln<<i"mgtlinoect repelling.; chinese herbal medicine,

high gade protein additives for feed

,~,weak anlifuogalaetivity "leaves and insecticidal activity against kalla.ose and

Zdipda/ ••". "pericarps hawii." •••

planl'inhibit growth of Erlich ascitic, =cinoma

";;ell.Z."itUblm

= as DNA topoioomerase inhibitors.,~, 58,18treating withdrawal from drug addictions

Z,fwmjltani"ttum ftuits stimulant "ZarypltyU ••m plants same •.• thoso of Zaccanthopodium "

roots and ,"ow inhibitory lIClivily <0 platelet-Zplanispium "SIems aggregation ClIlIscdby PAF,- same •.• those of Zplamspillm nZlIChi"ifo/ium

used for gynecol inflection and vtnerealpeels "di'eM'leS


,

•Botanical name of p"" .r Reported medicinal use Referencesthe plants the plants

exhibit cytotoxic ~Clivjly; ,how

aotiplale!tl_aggll'gation activity •• d '"""" "induced terminal differentillt;oo ;0 cultuureZ.•i",,,la ••• HL-60 ceU,

roots wood,significant antiplateltl_aggregation

"activity

show neuromusculio blocking activity '"Z.tingui.ceb. .tern bark "the rectus mxwminol muscle offrogs

Z."S<JIffb_,,~ ,,~ u~ in pneumooia and rh ••.•mrni'm "posse •• antiparasitic • insecticidal

plant. acaricida; antifcedant "'" lUIlimicrobial '"activity

Z.Tflntiwx}'loide. plant, show insecticid.] and antifoodant activity "

CHAPTER 1: iNTRODUCTION,

,1.2.2 CHEMICAL STUDIES ON THE PLANTS OF THE GENUS

ZANTHOXYLUM

Zanthoxylum is one of the most important genera of the.family Rutaceae. There are more than

sixty species in then Zanthoxylum genus. In the following sections a review of the chemical

investigations carried out on the various plants of the genus Zanthoxylum and that of

Zanthoxylum budrunga in particular is presented.

Z accanthopodium

In 1975 Ahuja and his coworkers12 worked on the seeds of Z accamhopodium _They isolated

a flavonoid and established ils structure as 3,5-dihydroxy-7,8,4' - trimethoxyflavone I, later

named as \ambu!in.

•

I

Chemical study on the fmits by ChatteJjee and her coworker? led to the isolation of two

llavonoids !ambulin and tambulol. Structure oftambulin I suggested by Ahuja ef ol.12was

confinned by chemical and spectroscopic studies. Further work on the fruits proved that

tambulo1 belonged to the gossypetin series and identified it to be glucosyl 7,4-dimethylether

of gossypetin II.

ORMoO

OR

n

CHAI'fER 1: INTRODUCTION

OM.

R'R- ~~:RO/\L'(

OR

, R'~OM~

.~. ,"

•A systematic study on the bark of Z accanthopodium has been done by Pal and Basu

Chowdhury.24They isolated two penmcyclic leipe1loids and a Iignan from the bark, The

terpenoids were identified as l3-amyrenone ill and l3-amyrin IV, on the basis of chemical and

spectral evidences However they could not identitY the lignan, a minor isolate,

,M.

M.

,m,RR =0

,IV,R=OH,R =H

In 1975 Petter and his group2Salso worked on the stem barks and were able to isolate a

number of lignans. They studied the structure of the lignans by spectroscopic means;

employed IJC NMR shifts for determining the nature and configuration of the aryl group.

Their study led to the structures V, VI, vn for (+)-methylpiperitol, (+ )-epicudesmin and (+)-

episasamin respectively for the lignans.

. 'V > R = VeratryJ, R =Piperonyl, R ~ H, "VI R=R =Ver'tn.] R =H, ~.,, ,

Vll, R =R = Piperonyl, R =H


,The plants Z accunthopodmm was also chemically investigated by Chakraborty el ai.2~ in

India, They reported the isolation of yet one more lignan which they called podotoxin and

established its structure as VIII, It was found to be a powerful inhibitor ofletuce seeds.

VIII

Z, Qcutifofium

The leaves of Z aculifohum were shown to oontain quinoione alkaloids by Anuda and his

coworkers2;. Their work afforded five new quinolone alkaloids, which were identified on the

basis of spectral analysis as acutifolin IX. acutifolidin X, O-methylaculifolin XI, acutifolin

palmilllle and neoacutifolin XU In addition, the known oompounds (E,E}N-{2-

methylpropyl}2,4-tetradecadienamide, (E)-3,4-dimethoxycinnamic acid XIII and 13-sitostero\

XlV were isolated and characterized.

IX,R=Me,R' =HX R=R' =H,Xl R=R' ~Me,

CHAPTER I: INTRODUCTION

XII

•

•

("'M.

XIVxm

Z. ailllflihoida

The stem woods of Z mlamhmdcs were chemically investigatedby Sheen and his

coworkersl8. From the chloroform solution fraction they were able to identify the presence of

one new nor-lignan, aJ.lanthoidiol and one new phenyl propanoid ailanthoidiol, together with

moe alkaloids, four coumarins and one sterol. The structure of the new compounds were

elucidated by spectral methods.

The barks of the plant of Z adanthoidC$ were chemically examined by Ishii el 01.29 They were

able to isolate ailantboidine, a novel benzo[Clphenanthridine alkaloid with cyanopyridine

pendant. The structure of ailanthoidine XV has been deduced from its spectral data including

2-D NMR spectroscopy and X-ray analysis ofXVL

MoO

XV, R =Me, R' = 4-Cyano - 2- pyridin)

. 'XVI, R = 4-Pyndmyl, R =Me

Z. a/alum

Non polar constituents of the hexane extract of the seeds of Z alatum (armatum) have been

identIfied as 6-hydroxynonadec-(4Z)-enoic acid, 8-hydroxypentadec-{4Z)-enoic acid, 7-


o

"hydroxy-7-vinylhexadec-(4Z)- enoie acid and hexadec-{4Z)-enoic acid by Atecque and his

coworkersJO, In addition the essential oil has been found to contain mainly linalool XVII

(58.3%), limonene XVIII (24.46%) and Me-cinnamate XIX (8.92%) besides several minor

constituents,

Z. aroorcscens

XVll XVIII

oIIr?'Y CH==eH- C -OC!)

VXIX

Chemical investigation of the seed husks of Z. arborescens by Dreyer and Brennei'l resulted

in the isolation of two quinazolone alkaloid content namely I-methyl_J_ (2'. phenylethyl}-

IH,JH-quinazoJine-l,4-dione XX and l-methyl-3-(2'-{ 4'-methoxyphenyl)ethyIJ-IH,3H-

quinazoline-2,4-dione XXl

Z. arnotlial'/Um

The roots of Z arnollianum were chemically investigated by Ishii el af. J2 They obtained a

non-phenolic dihydrofurano coumarin as a minor component and designated the compound as

xanthoranol and indicated it to be a derivative of6,7-disubstituted coumarin XXU In the year

1976 Ishii and his coworkers11,J. reported a more thorough chemical Investigation on the root

CHAPrER 1: INTRODUCTION

f

barks and the stems of Z arnollianum. They obtained six known alkaloids, namely

chelerythrine chlonde XXIII, N-desmethylchelerythrine XXIV, oxychelerythrine XXV,

decarine XXVI, amottianamide XXVII, skimmianine xxvm, along with a new alkaloid

iwamide XXIX. They were also able to isolate two lignans,l-asarinin XXX, and I-sesamin

XXXI, two cownarins, l-marmesein XXXII and columbianetin XXX"'; eight neutral

compounds as well as a mixturoof sesquiterpenes.

HO

o MoO

XXII xxrn0

0) )0 0

N

OM,

XXIV XXV

0 0) )0 0,

HOMoO

OM, OM,

XXVIIXXVI

N-M,

IH"-

XXVIn XXlX

CHAPTER I, INTRODUCTION

'" 0)'" 0

0'--9-0H

<:0 0O..J

XXX XXXI

XXXll

Z. avlcennae

OH

xxxm

The bishordenyI terpene alkaloid culantraramine XXXIV was isolated from the plants of Z.

avicennae by Miao and his coworkers.35 Selective long range DEPT IJC NMR was used to

identify nuclei and to connect spin systems separated by quaternary carbons and hctero atoms

in the compound. Total assignment of IH and 11C NMR spectra of the compound were

achieved.

M,

XXXIVZ bungeanultl

In 1981 LiJuan and Fengzhi.16 worked on the roots of Z. bungeanum and found the presence of

a number of alkaloids. They isolated six alkaloids in which the major alkaloid


zanthobungeanine was shown to be a ~o-2..quinoline type alkaloid XXXV, which is

responsible for weak antifungal activity. The"other five alkaloids were identified as 11-

methoxychelerythnne XXXVI, des-N-methy1ehelerythrine XXIV, amottianamide XXVTI,

skimmianine XXVUI, l-N-acetylanonaine XXXVII.

xxxv

XXXVII

xxxv.

N-L,H

Liu and WeiJ7 studied the chloroform extract of dried fruit peel skin of Z. bungeanum. From

thIS extract he was able to isolate skimmianine XXVm. zanthoxylin XXXVTII and bergapten

XXXIX

~----eH20H

M"'WH

YOM,

XXXVIU XXXIX

Xiong and his coworkersJ8 examined the pericarp of Z bungeanum. They isolated two new

flavonol glucosides viz, quercetin-3',4'-dimethylether-7-glucoside XL and tamarixitin-3,7.

bisglucoslde together with hyperin XLL quercetin XLll, foeniculin XLrn, isorhamnetin -7.

glUCOSide,rutin-3,5,6-trihydroxy-7,4'-dimethoxyflavone XLIV, arbutin XLV, sitosterol XIV,


~-glucose XLVI, L-sesamin XXXI and palmitic acid XLVU Their structures were

established by spectroscopic methods. Study on the effects of extracts of Z bungeanum fruit

and their constituents on spontaneous beating rate of myocardial cell sheets in culture19

exhibited inereased beating rate. Z bungei proved to be high grade protein additives forfeed'o.

MoO

'0

XL

HO o

xu

MoO 0

H M.'" '" I CH20H

'" '""

XUIlXLII

XLIV

HOH

HOXLVI


H

LXV

XLVII

'. "•

Z Caribacum

Dreyer and Brenner31 isolated the known alkaloid skimmianine XXVIUfrom the leaf a/racl

ofZ. caribacum.

Z davoherculis

Rao and Richard'i carried out chemical investigation on the barks of Z clavaherculis. They

obtained five icthyotoxic compounds and identified them as (-)-asarinin xxx, (-)-sesamin

XXXI, (-)-pluviatilol XLVID, y-y-dimethylether neoherculin XLIX and 1-N- acetylanonaine

XXXVII.

o

XLVIII

Z conspers;punclQ1ultl

XLIX

A group of Australian cbemists4l reported the isolation of known alkaloid allocryptopine L,

the amide (+)-N-benzoyl-2-hydroxy-(4-methoxyphenyl) ethylamine and hesperidine LI from

tbe barks of Z conspempunc/alum. Besides these alkaloids they were able to isolate a new

alkaloid isomeric With protopine which was shown to be LII. The structure of the alkaloid

was later confirmed by Sotelo and Giacopl104J by synthesis and also suggested the name

pseudoprotopinc for the natural base.

CHAPTER 1: tNTRODUCTION

"

OM,L LI

LIT

Z. coriaeeUltl

The roots of Z conaceum were chemically investigated by Swinehart and Stermitz44• They

were able to identify the presence of a number of alkaloids, chelerythrineLlll,

dihydrochelel)1hrine LlV, N,methylisocorydine LV, (-)-N-methylcanadine LVI and aegiline

LVII. Further work on the leaves of this species led to the isolation of four major alkaloids

and a number of minor ones which however were identical to the alkaloids separated from the

roots. They were also successful in isloating two other alkaloids hordenine LVIII and

aIfilcramine LIX along with two other alkaloids bishordeninyl terpene type,

,

')o

""M'OM

LIV

M,

LIII

M,

CHAPfER I: INTRODUCTION

"--0

"

LV

LVII

M,

HO

I iM,-1'oM,

CH2CH2NMc2

LIXZ. culantrillo

LVI

LVID

Chemical study on the stems of Z culanmfio by Swinehart and his group 44 enabled them to

identity the presence of (-)-N-methylisocorywne LV and candicine LX, synephrine LXI

skimmianine XXVllI, magnoflorine LXII and tembetarine LXIII and also isolate 2,3,10,11-

dihydroxydimethoxy protoberberine LXIV.

LXII


OH

LXIII LXIV

From the non polar fraction of the leaves of Z culantrilfo the same workers were able to

Isolate (+)-eudesmin LXV and epieudesmin VI. The alkaloid content nfthe leaves was low

compared to that of the stems and none of the stem alkalOids were detected in the leaves.

They were also able to isolate hordenine LVm and a new alkaloid culantraramine LXVI. The

structure of culantrnmmine LXVI was shown to be closely related 10 aIfileramine LXllI

which has also been from the leaves of Z punctatum61.

LXVI

",I

MoOOM.

LXV

Z deeary;

Vaquette and his collcagues4' isolated four alkaloids from the stem barks of Z decaryi which

included dictamnine LXVI, skimmianine XXVIII, 4-methoxy -]-methyl-quinolone LXVII,

decarme XXVI (9-methoxy-l O-hydroxy-2,3-methylenedioxybenzophenanthridine),

CHAITER 1: INTRODUCTION

o

Z. dipetaJum

LXVII

IM.LXvn

In 1975 Fmncis el aI,'" investigated the chemical constituents of Z dipe/alum. The root barks

of Z dlpe/alum were shown to contain the alkaloids canthin..(i.-one LXVllI ,chelerythrine

LUI, ni!idioc LXIX and tembetarine LXIll; the 'pyranocoumarins avicenol LXX and

l;anthxyIetin LXXI; the terpene lupeol LXXll and the flavonoid hesperidin LI. A third

coumann designated as ZD!J was tentatively suggested to possess the structure LXXID. Most

of these compounds along with sitosterol XN were also found from the stem barks and root

woods of Z dipe/alum, On the other hand magnoflorine LXII was isolated from the root

woods. Further work on the root barks of Z dipetalum by this group 4:; led to the isolation of

two pyranocoumarin, dipetalolactone (2-ox0-6,6, I0, 1Q..tetTamethylbenzo[1 ,2-b:3,4-b':5 ,6-b])

tripyran LXXIV, dipetaline [6-(J,3-dimethylallyl)-5-methoxy-l,2-dimethyl.2H-benzo[I,2-

b:3,4-b'] dipyran-8-onc LXXV.

M,O

MoO

LXVlllLXIX

OHLXX

CHAffER 1: INTRODUCTION

LXXI

H••H.••

LXXII LXXIll

LXXIV LXXV

o

Three new and one previously knO\\lllnatural aeyl histamines along with the rare protopine

type alkaloid thalictricine LXXVI were isolated from the leaves of several Z. dipetalum from

the Hawaiian Islands by Arslanin and his coworkers47.

LXXVI

Z. efeplumtiasi$

The root barks of the plant Z. elephantiasis were chemically investigated by Gray and

Watennan 48, Tbey were able to isolate a new pyranoeoumarin from its root bark and

identified it as CIS'3vicenolLXXVII [6-{J-hydroxy-J-methyl-cis-but -1-enyl-5-methoxy-2,2-

dimethyl-2H-benzo [1,2b:3,4b') dipyran-8-one»),on the basis of comparison of spectral data

with that of tran.\'avicenol and conversion to tetrahydroavicenol LXXVDl

CHAPTER I: INTRODUCTION•

LXXVII

Z. jagara

H

LXXVIII

!n 1980 Dreyer and Brenner)l were able to identity the presence of skimmianine XXVID and

scopo1cin from the leaf extracts of Z jagara. In the following year Synder el al.49 reported

the Isolation of biisocoumarin castanaguyone LXXIX from the fruits of Z fagan, and

establi,hcd the structure by spectral analysis_

LXXIXZ. jlavum

In 1976 Waterman,l" studied the petrol, CHCb and MeOB extracts of the root barks of Z

jlavum, From the petrol exlme! he was able to isolate imperatorin LXXX ,From the CHCh

and MeOH extracts the same workers were able to isolate ehelerythrine chloride xxm,canthin.6-one LXVIII, nitidine chloride LXXXI and dihydrorutaecarpin Lxxxn and all

these were IdentIfied by UV, Mass, PMR and IR spectroscopy.

LXXXCHAPTER 1: INTRODUCTION

LXXXI

Z. hem.;j

The barks of Z heitzii were chemically investigated by Ngoucla and his coworkers'l. They

were able to identity the presence of two new natural Iignans, meso-2,3-bis(3,4,5-

trimethoxyben7,y1)-1,4-hutanediolLXXXllI and 4-aeetoxy-2,3-bis(3,4,5- trimethoxybenzyl)-

I-butanol, along with four known Iignans, two alkaloids and triterpenes.

OHH

LXXXID

Z. integrifolium

The plants of Z mlegrifolium were chemically examined by Jen Chih Min and his

coworkl.:rs'l. They were able to isolate two new compounds, tridecanonchelerythrine

LXXXIV and conifegerol LXXXV and their structures were elucidated on the basis of

spectroscopic data including 2DNMR

M.

LXXXIV


LXXXV

Z. lemairei ami Z leprieurii

Waterman and his grouplJ investigated the samples of Z lemairei and found to contain BITQ

(bcnzyltetrahydroisoquinoline) alkaloid. Further work on the same samples by Watennan and

his groUplJ led to the isolation of a number of alkaloids and dihydrochelcl)'thrine LIV, lupeol

LXXII and sesamin XXXI in the samplels of Z lepriellrii

Z. linwncilfo

The leaf extract ofZ limoncillo were chemically investigated by Dreyer and Brennerll who

reported the presence of known alkaloid skimmianine XXVIII.

Z. microcarpum

Chemical study by Jimenz and his coworkersl4 on the bark of Z mlcrocarpum led to the

isolation ofa new chromone LXXXVI, 5-hydroxy-7-methoxy-2-pentylchromone.

LXXXVI

Z. myriacanthum

]n 1975 Watermanl5 carried out the extraction of the root barks of Z myriac:anthllm with

petrol (b.p. 40_600 C), CHCh and MeOH. Upon column chromatography of the extract he

was able to isolate sitosterol XIV, dlhydronitidine LXXXVII, nitidine nitrate LXXXVIII and

the quaternary alkaloids magnotlorine LXII and tcmbetarine LXIII.

LXXXVII

CHAffER I: INTRODUCTION

LXXXVID

z. nitldun

Arthur and his group56reported the bases isolated from Z nitidun which were identified as

oxynitidinc LXXXIX and nitidine LXVID respectively. In 1981 Shihl7 reported the isolation

of some more alkaloids, 6-rnethoxychelerythime LXL, nitidine chloride LXXXI,

oxychelerthrine XXV and skimmianine XXVIII from Z mtidwn. 6. methoxychelerythime

LXL inhIbIted the growth of Erlich ascities carcinoma cells, whereas the other compounds

were found mactive.

LXXXIX

MoO

OM,LXL

Later work of Fang, Sheng Ding and his coworkers~ion the roots of Z. nilidum enabled them

to isolate topoisomerase LXLI, which acts as DNA topoisomerase inhibitors.

OM,

LXLl

A methanol extract prepared from the roots of Z. mfidum inhibited topoisomerase LXLI

mediated DNA relaxation and established the covalent binary complex formed between

topoisomerase LXLI and DNA. Bioassay guided fractionation monitoring the biochemical

activities afforded three active principles in pure from. These were nitidine LXIX,

chelerythrine LIII, and a new betl7.ophenanthrindinealkaloid LXLI isomeric with fararidine,

for which the name isofararidine has been proposed. While all three of the isolated alkaloids

inhibited the relaxation of super COiledplasmid p5P64DNA by calf thymus topoisomerase

LXLI, only nitidine exhibited strong stabilization of the enzyme -DNA. Covalent binary

CHI\PTER 1: INTRODUCTION•

complex, isofararidine stabilized the binary complex weakly when employed at high (100

).1m)cone. while chelerythrine LIlI failed to stabilize the complell: at any tested concentration,

Z. oxyphyffum

Tiwari el (1/.'9 showed the defatted root barks of Z oxyphyllum to comprise of

7..antholl:yphyllineLXLII and corydine LXLIlI. From the petroleum ether extractW of the

defatted root harks the same workers found the presence of 3,4-bis (3',4'-

dimethoxyphenylmethyl)-dihydrofuran_2-one LXLIV.

LXUI LXIJII

MeO r"'~ OM.

Meo~ O1z...L...l...012--O-oMe

LXLIV

Z. plflnj.~pium

The roots of Z pkmi,~plUmwere chemically investigated by Hisashi el atl They reported the

presence of magnollorine LXIL xanthoplanine LXLV, skimmianine XXVIII, dictamnine

LXVI and y-fagarine LXLVl The root wood and bark of the plant was shown to contain

magnollorine LXII and lI:anthoplamne LXLV.

LXLV


LXLVI

{y

Further work of Sieb ct Zucc and his coworkers6l on the roots and stems of Z planispium

enabled them 10 isolate five compounds from the petroleum ether and methylene chloride

extracts The structure of these compounds were determined on the basis of physical constants

and UV, IR, IH NMR, MS spectral analyses, They are ~-amyrin N, ~-sitosterol XIV, L-

asarinin XXX, L-planine and zanthobungeanine XXXV. Compounds XXX and XXXV

showed inhibitory activity to platelet aggregation caused by PAF (platelet-activiting factors);

among them compound XXX was lcss active than L-planine; compound XXXV was the least.

Zanthobungeanine was isolated for the fIrst time from this plant.

Z. punctatum

Caolo and her colleaguelJ were successful to isolate an alkaloid from Z punctaturn and were

able to elucidate the structure of hordenine LVIII by the help of spectral analysis e.g, lH. BC

NMR, UV and mass spectroscopy. The structure has also been confirmed by X-ray analysis.

Z. rhetsa

Chemical study by Chatterjee and her groupM on th,e trunk bark of Z rhelsa led to the

isolatIon of a pentacyclic triterpenoid ketone, xanthoxylone LXLVII. The structure was

confinned by chemical as wen as spectral means, In the same pie>:e of work Chatterjee and

her group":; reported the isolation of quinazoline alkaloid rhetine, chelerythrine, LIII, rhetsine

LXLVJII, and rhetsinine LXLIX in addition to triterpene lupeol LXXI.

,\i-J

LXLVI

CHAPTER I, lNTRODUCfION

LXlJX

LXLVIII

" 1•

Z ru/)escens

Waterman and hIs coworkers51 investigated the chemical constituents of Z ruhescens and

were able to isolate benzyltetrahydroisoquinolinc (BTTQ) alkaloids. The pericarp of the

fruits, however, was shown to contain a new compound zanthosinamide LL along with 4-

lrans-cinanamoylarnides, dioxamin, dioxamide LLI, zanthomamide LLn by Adesina et at:'In 1989 this school of chemists on further work on this plant 67wereable to isolate two new

aromatic amides, i.e, rubenarnide LLm. and rubcamamide LLIV besides dioxamide LLI,

dioxamine LLV and zanthomamide LLII. They were also able to isolate lupeol LXXII and

amoninamide xxvn from the roots of this plant.

M,

MXrIP' N

",IM,

lL

LUI

LU

R1M:;croON~OM'P' P' 1

'" 1 '"M M,

u.m,R=MelLIV,R=H

<

Z. rhoifolium

",I

r""y0)~oH

lLV

The sterns, leaves and fruits of Z. rhoifo/ium were chemically investigated by Armda and his

coworkers"" and they reported the presence of six coumarins, four furoquinoline alkaloids,

one glycosyltlavonoid, two triterpenes and one steriod of which one furoquinoline alkaloid


"LLVI is a new cornlXlund. The structure of the compounds isolated were determined by

spectroscopic methods.

ILVI

Z $caruJem

From the roots of Z. scandens Brader and his coworkers69 isnlted zascanol epoxide LLVll,

prenyl sIde chain of which is characterised by an epoxide ring and a terminal hydroxyl group.

ILVD

Z. schir%Uum

Chemical investigation on the fruit peel of Z schinifolium by Sieb et Zuce and his

coworkers70 led to the isolation of five compounds from the Chinese traditional drugQinghuajiao (2 schmifolum). On the basis nfUV, IR,1H, 13C NMR, 1 H-1HCOSY, '1-1_ llC

COSY, NOESY and Mass spectroscopic analysis, four of them have been identified as

bergapten LXXIX, wnbelliferone LLVIII, skimmianine XXVID and schinifoline LUX,

H

LLVIII

IM,LUX

(CHZ)(;Me

Further work of this group'l on the stem of Z schmifolium enabled them to isolate

dictamlnlne LXVI, skimmlanine XXVlll, scoparone LLX, schinifolin LLXI, scopoletin

CHAPTER 1: iNTRODUCTION

•

LLxn, 7-hydroxy-8-methoxycownarin LLxm, N-methylflindersine LLXIV and 13-

sitosterol XIV, In the same year Meifang and his group70,?2 chemically investigated the roots

of Z schinifolium They could isolate two new ootnpounds schihifolin LLXI and

acetoxyschinifolin LLXV along with five known compounds aurapten, dictaminine LXVI,

scoparone LLX, skimmianine XXVID and l3-sitosterol XIV. The structure determination was

based upon spectroscopic analysis, i,e., UV,IR, MS, PMR, CMR, 2D NMR, In the test of

platelet aggregation caused by PAF (platekt-activiting factor), compounds LLXI, LLXV

aurapten, scoparone LLX showed inhibitory activity.

LLX

LLXII

OM,

LLXl

'"I

HO 0OM,

UXIII

MoO

M,O

'f"'('f"0OAo OMe

LLXIV LLXV

Later work of this groupW on roots of Z schinifolium led 10 the isolation of novel prenylated

schinifolinc LLXVI and N-methylschinifoline LLXVlL

R

lLXVI,R=H

ILXvn,R~Me

CHAPTER t: INTRODUCTION

"Z. simulans

Brader and his group69chemically investigated the roots and leaves of Z slrnulans. From the

roots of Z. simuluns the novel prenylated schinifoline LLXVI and N- metbylscbinifoline

LLXVII, and from the leaves predominantly N-acetoxymethylflindersine LLXvrn could be

isolated by them. The roots barks of Z .lImufam were investigated by Wu, and Chenn They

reported the isolation of three new 2-qwnolone alkaloids, zanthosimulin LLXIX,

huajiaosimuline LLXX, simulanoquinoline along with nine known compounds C.g. N-

acetylanonaine XXXVII, chelerythrine LID, norchelerythrine XXIV, bocconoline LLXXI,

8-methoxy-N-methylflindersine LLXXll, skimmianine XXVTII, N-acetylnornuciferine

LLXXIH, amottianamide xxvn and decanne XXVI. The structure of these compounds

were elucidated by spectral analysis. Simulanoquinoline is the first naturally occuring dimeric

alkaloid between dihydrobenzo[c] phenanthridine and 2-quinolone joined by a C.c linkage.

LLXVIU

LLXIX,R=CH=CMc;

u.xx, R = COCHMe2

A new dimeric two quinolone alkaloid, zanthobisquinolone LLXXIV, together with fifteen

knO\\mcompounds has been isolated from the root wood of Z simuJans by Chen Ih Sheng el

at'.4 On the basis of spectral analyses, the new alkaloid was elucidated as bis-(4-hydroxy-2-

keto-l-mcthyl-3"quinolinyl) methylene. Among the isolated compounds, several

furoquinoline alkaloids and N-acetylanonaine xxxvn showed Significant antiplateletaggregation actlvlty.

MoO

LLXXICHAPTER 1: INTRODUCTION

LLXXD •

0-'

ll.XXm

" OR

LLXXIV

o)

Further work of this group7~on the root bark of Z simwans enabled them to isolate two new

benzo[e]phenanthridine alkaloids, 6-Q-methyldihydrochelerythrine u..xxv and 6-

methylnorchelerythrine together with twenty three known compounds. Structures were

elucidated by spectral analyses. The pyranoquinoline alkaloids, zanthosimuline and

huajiaosimulme LLXX exhibited cytotoxic activity. LLXX showed antiplatelet aggregation

activity and induced terminal differentiation in culture HL-60 cells.

Z. tlngoasyulba

Bernhard and his collegaues76 chemically investigated the barks of Z lingoassuiha. They were

able to isolate (+)-tembamide LLXXVI. They also worked on the leaves of Z lingoassuiha

and were successful to isolate (-)-sesamin XXXI along Wlth the aporphinium alkaloid

LLXXVil The structure of these compounds were determined by spectral means.

M"'--O-CHCH2NHBZ- I

0"LLXXVI


Z. tinguiaceba

From the JXlwdered stem barks of Z linguiaceba Mehl and Nunon isolated a fagara base

LLXXVIIl The EtOH extract fraction of the powdered stem barks of Z Imguiaceba showed

neuromuscular blocking activity in the rectus abdominal muscle of frogs and in a rat phrenic

diaphragm preparation.

LLXXVIII

Z. usambarense

Kato and his coworkers711 studied the stems of Z usambarense. They reported the isolation of

eight quaternary alkaloids, onc of them was a new quaternary base of the

tetrahydmprotoberberine type, which was named H-usambarine LLXXIX,

ILXXIX

Z. utile huang

ReJiyuan el uL79 chemically investigated the roots of Z ulile huang and they were able to

isolate four pure compounds, one of them is a new aromatic amide named utilamide LLXXX.

The other three were fargesin LLXXXI, 6,7,8-trimethoxycoumarin LLxxxn and

skimmianine XXVllI.

CHAffER I, lNTRODUCfrON

•••

MeO~ CH'=CH-NHlli

LLXXX LLXXXI

!LXXXII

The chemical constituents of various plants of the genus Zanthoxylum reviewed above are

summarized inTable 2,

CHM'fER 1: INTRODUCTION

Table I

Chemical constituents of the nlants of Zantboxvlum enus.

Name of the plant Part of the Cbemical constituents References

plant

z. ~cc~"rllopoJ;um .eeds 3,5-di~.7,8,4'. trirnethoxyllavonc I

tambulin I22,23

fruit' umbulol(glueo,yl- 7,4-dimethyl_ethet

gO&Oypelinm

bark. I}-amyrenone III ,iJ.-.amyrin IV, a lignan '"•tern bark,

(+)-methy Ipiperilol V• (+}-cpiClldesmin

"VI, (+)-q>i •• samin VII

plant. podotoxin VHI "acutifoUn V<, aeutifolidin X O.•methylaeutifolin Xl, acutifolin palmitate

DCO.cutlfolln xn. (E,E}-N--(2-z.Q£uti[o/ium leaves "methylpropy l)-2,4-tetradecadienamide;

(E)-3,4-<:limethoxycinnamic acid XIII; p.

,itosterol XIV

ail.,uholdiol; phenyl propanoid,tem b.rI", ailanthoidiol; nine alkaloid.; four

Z..,;I~"thoitk. coumarin,; one sterol

"ailanthoidlne XV •OM', •benzol c]phenanlhridine alkaloid XVI

6-hydroxynonadec-( 4Z)-enoic acid, ••hydroxypentadec-( 4Z)-enoic acid; ,.

z.aJalIUtI """hydrOllJl-7-vinylhexadec-(4Z)- enoie acid,

"hexadec-(4Z}-enoic acid; Hnalonl XVH;

limoneno XVIll; Me-.:innamlte XV<.

he,ld"" several minor constituent'


•

• -Name orthe plant Part of the Chemical eon,tituentl! References

hmt

l,methyl_J_ (2'. pl>enyJethyI)-lH, m-Z"""'=M quioamline- 2,4-diooe xx t.methyl-3_

seed husk, • "[2' -{4'-mcthoxyphenyl) 1 ctbyl-1H,3H-

quinazoJine-2. 4-dione XXI

Z.amottianum ~antboranol (6,7-diii\lbstituted ~-,~ "XXIT)

chelerythrine chloride XXID; N-

desmcthylchelerythrine XXIV,

oxyche1erythrine XXV,decarine XXV>.

amottianamid. XXVll skimmianinerool barks and •

XXvm iwamide XXD< l_asarinin 33,34,tems

XXX, !_'lesamin XXXI; l-marmesein

XXXII; columbianetin XXXill eight•~tr.compounds • • mixture ofsesquilerpene,

culamraramine XXXN (bi •..hordclI)'lZ.avi"",,_ plants "terpene alkaloid)

zamhobungeanine XXXV; u_methoxycheltrythrine XXXVI; des.N_

~" methy lchelerythrine XXIV, "omottianamide XXVll; skimmlanine

XXVlIl; loN_acetyl anonaine xxxvn

skimmianino XXVID; zanthoxytinfullt, "Z.b""KM""'" XXXVllI; bergapten XXXIX

quercetin_3', 4' -dimethylcther _7-glucoside

XL; 1811W'ixitin_3,7_bisglucoside; hYJl<'rin

pericarpsXU; querctlin XLll: foeniculin XLIU,

"iwrhamnetin-7 -glucoside; rutin-l,S,6-

trihydroxy_ 7,4'_dimethoxy_ flaVOIle

XLW; arbutin XLV, sitollterol XIV;

CI/I\PrER I: INTRODUCTION

Name of the plant Part of the Chemical constituents Refereoces

plant

Zln<"~a,, ••mJ}-gluooside XLVI, L-sesamin XXXI,

palmitic acid XL VB

Zcaribac ••m Iellve. .Idmmianine XXVDI H

(-)-osarinin XXX; (-)-semnin XXXI;

Zcla",u,m: ••Ii.(-)-pluviatilol XLVDI; ,- ,-

barks "dimethylether neohen:ulin XLIX; 1- N-

acetylanonaine XXXVII

allOCIYPtopine L, (+)-N-benzoyl-2-

Z coIISl"",ipunctatllm b"", bydro"y-( 4-met/lo"yphenyl) etby tlmine,42,43

hellperidine U; a new alkaloid isomeric

with protopine LII

chelerythrinc un; dihydro-

chelerythrine LN, N-metbylisocorydineroot,

IN, (-)-N-methylcaoadine LVI; aegliine

Z coritu:eum LVll "leave, ,... hordenine LVIII; alfileramine UX; two

woo. alkaloid. ofbi,hordeninyl terpene type

(- j_N_methyl i,ocoryd ine LV; candicine

LX, synephrine LX>, skimmianine

xxvrn, magnoflorine LXn,,tem,

tembetarine unn, 2,3,10,11.Z, c••JantrilllJ dihydroxydimetho"y protoberberine 44,61

LXIV

Iellve. •• d (+)-eude,min LXV epieude.min V<,,stem. hordenine LVDI, culantraramine LXllI

dictamnine LXVI; .kimmianine

Z tkcaryi stem barks XXVIII; 4-methoxy -I-metbyl- "quinolo,," LXVB; decarine XXVI


Name of the P,rt of th, Chemicaloonstituents Referencesplant plant

c&nlbin-6-one LXVIll; ehe1erythrin. Lm:

root bark.nitidine LXIX; lernbetarine LXIII; Avioenol

LXX; and xanlhxyletin LXXI; lupeol LXXII;

he.peridin LI; a lhird coumarin LXXm

slem barksmost of lh. compound. is found in root bark. ;

sitoslerol XIV

major oflhe compouDds i. found in root barks ••Z. "'petal••,," root wood.

, magnoflorine LXn

dipetalolaetone (2'Qxo-6,6, W,ID-tetra

melhy lbemo[ I ,2-b:3, 441':5,6-b )) tripyran

root bark. LXXIV, diJl"laiine [6-{3,3-dimethyl a\lyl)-5_

melhox y_2,2-dimetbyl_ 2H_benz;o [1,2-b:l,4-b']

dipyran-S-orw LXXV

leave. a nalural .cyl hlSlamlnes thalietricine LXXVI "cis-avicenol LXXVll; ,,""'''''' avicenol

Z. 6qJ"antiQSv, Toot bark. ••LXXV'"

Zjagtm1. loaf skimmianine XXVlll, """polcin "fruits ca.stan.guyone LXXIX "

imperalorin LXXX; clleJerytllrine cbloride '"Z. j1trV1Ut1 root barks XXllL canlhin-6_one LXVIlI; nitidine

chloride LXXXI; dihydrorutaeelupin LXXXI

meso- 2,3-bis(3, 4,5-lrimelhoxybenzyl)- l,4-z.1rd4ii bUlaIIerliol LXXXDl; 4-acetoxy-l,3-bis(3A,S-

""h "trimctboxybenzyl}-I_bulano~ '00' ""O~lignans, two Ilk.loid. and lrilerpenes

Z. integrijo/iu", planl.tride<:llnoncllelerythrine LXXXIV; conifegerol

"LXXXV


.'.".

Name of tho P.rt .r ChemiClii constituents References

plant the plant

Z. felfUrirei lUfd Zbenzy lttlrahydrolsoquinoline alkaloid; dihydro-

samples chelerythrine UV; lupeoi LXXII; sesamin "kpri ••••riiXXXI

Z. IilfWncillo ,..r ,kimmianine XXVllI "Z.microctup"m barks

S_hydroxy_7_mMhoxy_2_penlylchromone

"LXXXVI

Z.m,yriaca"thum SItosterol XN, diltydronitidine LXXXVil,roots M'

niudine nitrate LXXXvm; ffitlglloAorine """", LXII: tembetarine LXID

oxynitidine LXXXIX; nitidine LXVID, .~plants

metho;rychelerythirne LXL; nitldine chloride56,57

LXXXI; oxychelerthrine XXV; skimmianine

Z. nin""n xxvrn

topoisomerase LXLI (benzopbenanthriruhne) ;

"~" mtidine LXIX: cbelerythrine LUI

Mdzanthoxyphylline LXLII; cnrydine LXLUI,~,

Z oxyplryll"m 3,4_bJS (3'A' -dimetltoxyphenyl methy l}-dihydro 59,60,,,'"furan-2-<>ne LXLIV

rnagooflorine LXII; XlIntltoplonine LXLV;

'OM' skimmilUline xxvm; dictomnine LXVI, y- "fagarme LXLVI

Z. pflUfispi"", 'OM' Md ~-"",yrin '" ~.,itosteml XN L-asarinin, ,",=, XXX, L.planine: zanthobungeanine XXXV

~ wOO<,magnoflorine LXII; Xlltltltoplanine LXLV "and bark

Z.punclillUm plants hOTdenineLvm "•


Name ,r tho P.rt .r Chemical constituents Reference!!lant the vlant

XlInlhoxylooe LXLVII, rhe1me; chelerythrine,

Zrftma trunk bark LlJI; rhetsine LXLVIU; rhetsinine LXLIX; 64,65

lup""l LXXI

plant. beTli)'Iletrahydroisoquinoline (BTTQ) alkaloid

pcricarp.U1ntho.inamide LL; 4-1mmdnanamoylamid •• ; 53,66

dioxamin; dioxamide LU; z.anthomamideand fruits

LW

Z. ""Mcensruben.mide LUll; rubelmamide LLIV;

plant, dioxamide LLI; dioxarnine LLV; "ZlUlIhomamide LLU

'00" lupeol LXXD; amollinamide XXVII

SiX coumarin,; four furoquinoline alkaloid.'tem, leave,

Z rlooifoli ••m LLV1; one g1ycnsylllavonoid; two lrilerpene, ••fruitsand o~ S1eriod

Z.• """dens ,=. =annl epoxide LL Vll "wgaple11 LXXIX; umbclHferone LLVllI;

Z <chi"i/O/ium fruit peels '",kimmianioe XXVlII; Ichinifolioe LLIX

prenylated lcilinifoline LLXVI; N- methyl-

=" schinifoline LLXVll"

leaves N.acetoxymelhylf1indlmine LLXVIIl

U11uhosimulin LLXIX; huajiaosimulineZ >itruJ"". LLXX ,imulanoquinol ine; N_ylanooaine,

XXXVll; chelerythrine LUI; norchelerythrine

root barks XX"" boccnnoline WOO, 8-melhoxy-N. nmethylflindersine LLXXU, skimmianine

XXVID; N-acetylnornuciferine LLXXllI;

amottianami(\e XXVll; decarine XXVI

CHAPrER 1: INTRODUCTION

Name of th, Port of Chemical constituents References

plllnt the plant

unthobi squinolOlIe LLXXIV; several

root wood. furoquinoline alkaloid., N-acetyllI1onaine ,.xxxvn

6-O-methyl dibydroche!erythrine LLXXV, •root barks methy lnorchelerythrine; twenly three known "

compound.

'"'''" (+}-tcmbamidc lLXXVI

Z fingoas""iiJa (-}-sesamin XXXI, an aporphinium alkaloid "leavesLLXXvn .

Z. tillgllitl£ciJa $Iem bark. fagara base LLXXVIll n

(-)-uoambarine lLXXlX; Md - otherZ. IlNam1uJnll'" ,~, "quaternary alkaloid

util.mide !LXXX; f"'lltsio !LXXXL 6,7,8_

trimethoxycoUllUlrio LLXXXll; skimrnianineZ.lltil ••hM""If root, xxvn, "

CllAPrER 1: INTRODUCTION

1.3 REVIEW ON ZANTHOXYLUM BUDRUNGA

Zanthoxylum budrun~a, locally known as Bawa is a small or moderate sized tree which is

widely found all over Bangladesh, specially in the districts of Dhaka, Chittagong, Ferri

Comilla, Faridpur and Kushtia. The tree (Fig. 1) with pale corky bark is covered with conical

prickles on stems and branches and sometimes a few small ones on the leafrhaclties; young

prickles u])Curved. Leaves are clustered toward the ends of the stout brancWets, eqllHlly or

unequally pinnate, 30-60cm. long, induding the petiole, long, leaflets 5-20 pairs, 7-15 em

long, oblong or laneeolate, caudate, entire or crenate, when crenate with a large gland in

sinus, very oblique at base, rounded on the upper side, with the lamina shorter, narrower and

acute on the petiole on the lower side. Flowers are in large terminal paniculate cymes often

more than 30 em. broad,' the branches opposite, angled; bracts minute, caduccus; peduncles

very long, sometimes prickly. Calyx-lobes are minute, triangular petals 4, elloptie, 25mm.

long, yellow valvate, ripe carpels 5 mm. diameter, solitary, spherical, tubercled, seeds

globose, bluish black, smooth, shining, tasting of black pepper.

Various parts of this plant are reported to have important physiolob<ical properties. According

to researchers the fruits of Z. budrnnga are hot and bitter, they can be used as digestive and

appetizer; as cure of asthma, bronchitis and for removal of pain. It is useful in heart diseases

liS well as mouth, teeth, throat disea.ies; relieves hiccough, piles. The fruit is used for its

aromatic and stimulant properties. The Mohammedan physicians consider it to be hot and dry

and to have astringent, stimulant and digestive properties. They prescribe it in dyspepsia

arising rrom atrahilis; also in some fonus of diarrhoea. The leaves arc used for cholera and the

root bark is reported to be purgative of the kidney.

Various parts of the plant Z budrunga have been subject of chemical mvestigallOn, specially

because of its reported medicinal value. Khastige?O reported the isolation of two alkaloids

budrungain and budrungainin in crystalline foons from the barks of Z budrunga, the former

as yellow rods which charred above 180°C but did not melt whereas budrungainin was

obtained as slender shiny oranb'l: needles shaped crystals from MeOH and CHCll, m.p. 155°C.

Khuda and his coworkersS1 chemically investigated the dried calyx of Z. budrunga fruit. They

isolated an essential oil named baznin by steam distillation along with the other fractions.

CHI\PTER t: INTRODUCTION

From the pet. ether extraction of the fruits as well as of the kernel, they were able to isolate a

mixture of stearic and palmitic acids. They obtained a mixture of carbohydrates and protein

from thc pulp of Z hudrunga, Khan and Mondal8l worked on thc oils ofthc pulp and seeds of

Z budrunga and rcported the presence of a ownber of fatty acids e.g" palmitic acid, stearic

acid, oleiCaCid,linoleic acid and linoenic acid in thc oil.

The fruits of Z budrunga were investigated by Abraham and his groupSl,They reported the

anti-inflammatory activity of the essential oil extraction. From the macerated bazna fruit in

EtOH and extraction with CHCb Ruangrungsi et af.M were able to isolate xanthoxylin and

two alkaloids arborine LLXXXlll and dictarnnine LXVI. Banerjee and Chowdhury"', for the

first time reported the isolation of the indopyridoquinazoline alkaloid, rutaecarpine

LLXXXIV from the seeds and fruits of Z budrunga. Chemical study 00 the barks of Z

budrunga by Joshi et at!' led them to isolate dihydroavicine LLXXXV aod rhetsinine

I.XUX. Ruanb'Tungsiet af.s7 worked on the stem barks of Z budrunga and were able to

isolate five compounds as lupeol LXXll, rutaecarpine lLXXXIV, xanthoxyletin LXXI,

osthol and scopoletin LLXII. The foregoing reports on the chemical investigation of

Zhudrunga is summarized in '!'able 3.

I)5N <,,0N '"

1-"llXXXlV

LLXXXV


T.ble3Cbrmk.1 roMtl11lm1i of Z. kbll'W'

hrl "r tltr o...mit.1 """n;lo •.••l. Rd.rrn«o

pllat>

budrungain; budnmgainin; dib)1IroIvicine lLXXXV. rbouinmebuh 80,86

I'xLiX.,." I.xXII;-~LLXXXIV; umlmy\elin LXXI;I1cm barl., "MlhoI; lC<IplIktinLUll

"'•.. clay<bullin, lteam.1Id ",Imili<: llCid,: CI!bohydl'lltes IIld procoi" "of I'ruit'

~Imilic: acid: Ileanc, Kid: oleic acid; linDleic acid; linoenic acid """', •••• untbo:<ylin; aborine u..UXnJ; dictamr:tine LXVI ••fruit,illdopyridoquilllUl!ine alkaloid: IIItI«&Ipine LWOOOV "

••

•

FIG. 1 SPEClMEN OF ZANTHOXYLUM BUDRUNGA

CHAPtER 1, Im-RODUcnON

J

1.4 OBJECTIVE OF THE RESEARCH

The plant Zanthoxylum hudrungu locally know as bazna belongs to the family of Rutaceae.

Zanthoxylum hudrzmga is one of the important medicinal plants of Bangladesh. Ellery part of

this plant is bItter and is reported to possess a variety of medicinal properties. The various

components oftlus plant are mainly used by Ayurvedists for the treatment of various diseases

in crude fonn. The actual active principle for the drugs is not known.

The plant Zanthmylum hudrunga has attracted the attention of the chemists for its medicinal

property. Different pants of Zanthoxylum budrunga have been subjects of chemical

investigation for isolation of the active principles. The plants of genus zanthoxylum have been

shown to oontain physiological actille alkaloids.

The barks of the plant have been reported to contain budrungainBO; budnmgainin; two

alkaloids, dihydroavicine and rhetsinine86 whereas the fruits to contain tluee alkaloids,

ruthacarpine,81arborine and dictaminineg4. Other parts of the plant e.g. seedsg2, stem barksS1

and dried clayx offruitsR1 have also been subjected to phytochemical investigation.

It appears that not much attention has been gillen to the leaves of Zanthoxylum hudrunga. The

present work was therefore pnmarily aimed at isolating the alkaloids and other different

compounds that may be present m the leaves of the plant. Study on pharmacological activity

of different extracts of the plants was also planned. The results of the phytochemical

investigations are dIscussed in this dissertation.

CHAPTER L INTRODUCTION

2.1 GENERAL

During the present work solvents were purified by distillation at the boiling point of the

respective solvents. Evaporation of the solvents were carried out on a rotary vacuwn

evaporator under reduced pressure at a temperature below 450C. The purity of the isolated

compounds were tested by analytical thin layer chromatography (TCL) llTIdthe spots were

made visible by exposure to iodine vapour llTIdUV light. For alkaloids and sugars the spots

were detected by spraying reagent, Draggendorff reagent llTId llTIiline-diphenylamine-

phosphoric acid reagent respectively on the TLC plates. The crudes were separated by using

column chromatography on silica geL

Melting point

Melting points were recorded by Fisher 10hn's electrothennal melting point apparatus by

using thin disc'method. The heating was done carefully so as to maintain a steady rise of

temperature, Melting'points reported are uncorrected.

Ultraviolet spectra (UV)

UltraVIolet spectra were recorded on a UV-J60J (PC) S, SHIMADZU, ULTRAVIOLET-

VISIBLE SPEClROPHOTOMETER in methanol solution at the Department of Chemistry,

Jahangimagar University, Savar, Dhaka.

Infrared spectra (IR)

Infrared spectra were recorded on a SHIMADZUFTIR DR_810l Spectrophotometer either as

solution in chlorofonn or as thin film or as KBr disc at the Department of Chemistry,

lahangimagar UniVersIty,Savar, Dhaka.

lH NMR Spectra

IH NMR spectra were recorded in CDCh, and CDJOD using 300 MHz spectrophotometer at

the Regional Sophisticated Instrumentation Center, Central Drug Research Institute,

Lucknow, India, and a 400 MHz Spectrophotometer at the Department of Chemistry,

Umversity of Kanazawa, Japan.

CHAPTER 2, l;XPl;R1Ml;NTAL

pi, '... ' --, ;...~

Mass Spcetllll

Mass spe<.::trawas nut on a Hewlett Packard mass spectrophotometer at the Regional

Sophisttcated Instrumentation Center, Centra! Drug Research Institute, Lucknow, India and at

the Chemistry Department, University ofKanazawa, Japan.

Column Chromatogllllphy (CC)

Column chromatographic method was used for the separation of the individual components

from a mixture having different Rr values. The colwnns were prepan:d by slurry method using

sihca gel (60 Kiesel gel 60, 70-230 mesh, ASTM, MERCK) as the stationary phase in freshly

distilJed solvents of the required polarity. The separation technique was carned out using

solvent and mixture of solvents selected by examining the behaviour of the crude on TLC

plates in vanous solvents.

Minimum effort column chromatography (MECC)

The method of minimum effort column chromatography was used for fractionation of crude

mixtures. The column was made by tightly packing dry silica gel as the stationary phase

(Kiesel gel 60, 230-400 mesh, ASTM, MERCK) in a heavy walled glass column equipped

with air tight connections of both sides and the crude absorhed in silica gel was placed on the

top of the column. The solvent as eluent was pumped on the top of the column by a FMI

pump and the e1uentswere collected at regular intervals.

Paper chromatography (PC)

Paper chromatography was carried out by descending technique. In a large cylindrical tank, a

trough containing mobile phase was supported near the top of the tank, On a 10 em wide and

50 cm long whatman filter paper no. 1 a pencil line was drawn about 5 cm from one edge.

Solution of the compound was placed along the line at regular intervals (1,5 em. apart) by

capillary tubes. The edge of the paper was held securely within the trough by means of glass

rod. The mobile phase was allowed to descend down the loaded paper for 16 hours, Finally

the paper was taken out from the tank and was developed using appropriate developing

reagents,

CHAI'fER 2: EXPERIMENTAL

t

Thin layer chromatography (TLC)

Thm layer chromatographic method was used for the analysis of the differem mixture of

comJXlunds and also to determine the purity of the isolated comJXlunds. Thin layer

chromatography was carried out on precoated silica gel plates (60 GF,>4 thickness 0.2 em. E

MERCK) and were activated hy drying before use. In some cases these plates (20 x 20 crn.)

were also used for preparative TLC

Regents

Draggeodorff rellgent

Bismuth nitrate (1,7 ml) was dissolved in distilled water (80 ml) and acetic acid (20 ml) was

then added to give solution A. Potassiwn iodide (32 g) was dissolved in distilled water (80

ml) to give solution B. The two solutious (solution A and solution B), 10 ml of each were

mixed with distilled water (20 m!) and acetic acid (4 mI) to give the reagent.

Mayer's reagent

Mercuric chloride (1.4 g) was dissolved in distilled water (60 ml) and was JXlured into a

solution of JXltassium iodide (5 g) in distilled water (10 rol). The volume of the solution was

made 100 ml hy adding required lIffiOlllltof water to give Mayer's reagent.

Spraying reagents:

Spraying reagents were prepared by dissolving 0.5g ofvaniline in 6 ml concentrated sulphuric

acid followed by dilution with 100 ml absolute ethanol.

Anilioe-diphcnyl amine-ph~phoric add reagent

2 g of diphenyl amine, 2 ml aniline and 10 ml 85% phosphoric acid were dissolved in 100 ml

of acetone to give aniline-diphenyl amine-phosphoric acid reagent.

2,2 EXTRACTION OF THE LEAVES OF ZANTHOXYLUM BUDRUNGA

The leaves of Zanthoxylum budrunga (Bang. Bazna) were collected from the Jahangirnagar

University Campus at Savar, Dhaka. The leaves were dried well in the sun and then powdered

in a grinding machine.

CHAPTER 2: EXPERtMENTAL

The dried powdered leaves of Z budrunga (1.75 kg) was extracted successively with

petroleum cther and methanol at room temperahm:: The solvents were removed from the

extracts on a rotary vacuum evaporator under reduced pressure at a temperature below 40-

45°C to yield a greenish gummy mass (390 g) and reddish brown gummy mass (546 g)

respectively. The pel. ether crude extract was denoted as E:ltmet P and the methanol crude

extract was noted as E:Jtract M,

2.3 EXAMlNATIONOFEXTRACT M

The Extract M (methanol extract) of the leaves of Z budrunga was reddish brown gummy

mass. It was almost insoluble in petether, diehloromethane but sparingly soluble in

chloroform, ethyl acctatc and soluble in methanol. TI.C examination of the extract was carried

out in ehlorofOm1,ethyl acetate and methanol and their combinations, but no good resolution

was obtained in any case, With Dragendorffreagent the methanol solution of Extract M gave

an orange red colour; it turned turbid ott addition ofMaeyer's reagent.

PARl1T10NlNG OFMETHANOLEXTRACT M

25 g ofmethanolic extract (Extract M) was taken in 60 m! of methanol. A major part of the

crude extract went into solution; the insoluble part was removed by filtration. The methanol

soluble part was diluted with 300 ml water; the clear solution was then extracted with pet.

ether, chloroform and ethyl acetate respectively. The extracts were then dried over anhydrous

magncsium sulphate and subsequently evaporated to dryness on a rotary evaporator to yield

three corresponding residues. The pel. ether residue was a greenish gummy mass (1.4'g) and

was denoted as Fraction P; chlorotboo residue was a greenish brown gummy mass (2.4 g)

and was denoted as "'raetion C; the brown gummy ethyl acetate extract (2.5 g) was denoted

as Fraction E. Water was removed from the remaining aqueous part by dIstillation under

reduced pressure (44° C); the residue so obtained was dissolved in methanol. The methanol

solutlon was then dried over anhydrous magnesium sulphate and then evaporated to dryness

on a rotary evaporator to yield a reddish brown gwnmy mass (9.6 g) and this was denoted as

Fraction M.

CH"'F'rER 2: EXPERIMENTAL

2.4 EXAMINATION OF FRACTION M

Fraction M, a reddish brown gummy mass, was indicated to be a mixture of several

compounds as revealed by TLC examination on precoated silica gel plates in different

solvcnts and mixture of solvents, The resolution of the fraction on TLC plates was best in

ethyl acetate: methanol 2:1 which exhibited seven distinCIspots al Rr 0.81, 0.76, 0.64, 0.53,

0.47, 0,32, 0.20 respectively with tailing from the base line,

2.4.1 MINIMUM EFFORT COLUMN CHROMATOGRAPHIC SEPARATION

OF FRACfION M

Fraction M (6.24 g) was dissolved in methanol and adsorbed in small quantity of silica gel.

The adsorbed mass was completely dried under reduced pressure and carefully poured on the

top of a colwnn of silica gel of a minimwn effort column chromatographic unit. The colwnn

was then eluted with pet ether, mixtures of pet. ether- ethyl acetate, ethyl acetate-methanol

and finally washed with methanol. A number of coloured bands e.g,; faint yellow and brown

were observed during the development of the co1wnn.Fractions of about 15 ml were collected

in each test tube at regular intervals and checked on TLC plates, In all 74 collections were

made, The detailed results of the chromatographic separation are shown in Table 4.

Collections showing similar or almost similar TLC behavior were combined together and the

total collections were combined to obtain four fractions, M]-~.

CHAPTER2: EXPERtMENiAL

Table 4

Minimum effort column chromatographic separation of Fraction M

[Eluting,nlventop"'. ether,grlIdientrruxturesof pel,ether-ethylacetate,ethylacetate-melhanol,methanolI

Collection nos. *TLC examination Behaviour with Yield and ohservation

Draggendorft' Reagent, " no 'pol' - -16_30 twospot, It<0.85,0.76

nocolouration17tog,mixtureof two

PE,EA,4,1 compounds.F•• etiOdM,

fourspotsIt<0,95,0.75,1,5g,mixtureoHour31_52 068,063 orangered

EA:MeOH;3;1 compound"FnIetiooM,

lhreespot.2 5g, mi"'urc, of three53_63 1t<0.63,056,0 52 orange red

EA, MeOH; 3:2 compounds.F•• etionMJ

twoSpolSwithtailing485mg,mixturesof two64-74 It<0.76, 0,74 No colouralion

EA: MeOH; 2,3compound.,FnIetiollM<

• PE ~ Pet. ether; EA =Ethyl acetate; MeOH =Methanol

2.4.2 SEPARATION OF THE COMPONENTS OF FRACTION M2

Fraction M1 (1.5 g) was a brownish yellow gummy material, soluble in methanol, but

sparingly soluble in chloroform and ethyl acetate, TLC examination of this fraction on

precoated plate showed four spots at R[ 0,48, 0.35, 0.30 and 0.23 with tailing in chloroform :

methanol 4:1, The brownish yellow crude (1.5 g) was dissolved in minimum volume of

methanol and adsorbed 10 silica geL It was then chromatographed over a silica gel coltunn

using chlorofonn and a mixture of chloroform and methanol as eluents. A total number of 131

portions each of about 7 m1 were collected. The portions were separately examined on TLC

plates and combined to give six Fractions M:ta - Mu. The chromatographic separation of

Fraction M2 is shown in Table 5.

CHAPTER 2: EXPERIMENTAL •

G1:'"'fe.

Table .5

Colwnn Chromatographic Separation ofFnlll:tion M:

[Eluent., ch1orofo~ mi><tureof chlorofonn- meihanol , methanol]

Collection nos. TLC examination Behaviour with Yield and observation

Dragendorff

Reagent, " "".port - -'00 ''"' wilh taii, Rr 0.66 "" 0"'" compound liightly16 -35 "" colourationPE.CHCl, 1:1 impur.l'ractinn M""

two spoil, Rr 0,95, 0.56 " •• mixture ill ,W>36~37 ..•.""CHCI,100% compounds. Fraction M••

,W> ,,",' '" "''' 0,53 'M •• mixture " ~38_42 orange redCHCi.,:M.OH;4:1 compowtd" Fractinn M",

two spoIs. RrO.53,0.42 '" •• mixture " ,W>43-53 orange red

CHCI,: MoOH 4:1 compounds. F"'dion M"

OM'pol with tail , RrO.42 m •• ~ compound ~"54_85 orangeredCHC!, MoOH; 4'1 impurity, Fr.etlon M",

86_95 no spot - -"'"' tailing fr,. b•• lin. ".. 00 resolution, Fnotion

96 - 131 No colouraUonCHel,: MoOH; 3;i M.

2.4.3 PREPARATIVE PLATE CHROMATOGRAPHIC SEPARATION OF

FRACfION Mla

Fraction MI. was a yellow gwnmy substance, which was soluble in dichloromethane,

chloroform, and sparingly soluble in ethyl acetate but insoluble in methanoL It was almost a

pure compound as indicated by its TLC. It (50 mg) was subjected to further purification by

preparative thin layer chromatography (PILC) using pet ether: chloroform 1:1 as developing

solvent Extraction of the relevant region of the plates with chloroform gave compound ZB,

(2.5 mg).

CHAPrER 2: EXPERIMENTAL

"Charae/erial/ion'ifZB1It was a yellow gummy substance, which on standing in the freeze for a long time yielded

needle shaped crystals. It was insoluble in pet. ether, ethyl acetate, methanol but soluble in

methylene chloride and chlorofann. Compound ZB1 melted at 51_530 C; with Draggendorff

reagent it did not show any colouration.

The compound exhibited tbe following spectral data:

IR(thin film) (Fig, 2): v..." 2920, 2851, 1707, 1655, 1508and 1259 em-I

lH•NMR(CDCh (Fig. 3): 1) 2,0 (5), 1.3 (5),1.09 (5), 0.89 (t)

2.4.4 PREPARATIVE PLATE CHROMATOGRAPHIC SEPARATION OF

FRACTION Mu.

Fraction M2b (25 mg) was a pale yellow gummy material. It was subjected to separation by

preparative thin layer chromatography using 100% chloroform. Extraction of the relevant

regions of the plates with chloroform gave two fractions, one of them (Rr 0,$4, 100% CHCh)

on further examination on TLC plates showed a single spot. It was denoted as ZB1. The other

fraction gave two spots.

Characleri:alion ojZIh.

Compound ZB1 melted at 63-65° C. It was soluble in dichloromethane, chloroform and ethylacetate but insoluble in methanol.

The compound exhibited the following spectral data:,IR(thin film) (Fig.4) : v""" 3649, 2963, 2177, 1670, 1361 and 1055 Cm"l

IH NMR (CDChXFil! 5):0 1.2(s); l.3(t); l.5(q); 2.5(m}; 2.7(s); 3.I(q); 4.0(m)

CHAPTER2: EXPERIMENTAL

2.4.5 PREPARATIVE PLATE CHROMATOGRAPIDC SEPARATION OF

FRACTION M%<

Fractioo Mk was a yellow gummy substance. It was soluble in methanol and sparingly

soluble in cthyl acetate but insoluble in chloroform. Examination on TLC plates showed two

spots with RrO.82 and 0.53 in 25% methanolinCHCl). The crude was subjected to preparative

plate chromatographic separation in MeOH : CHCI) (I :4). The process yielded two portions

(45 mg and 80 mg), both however contained two oompounds with RrO.82 (45 mg) and 0.53

(80 mg). These two were then separation, again subjected to preparative plate

chromatographic separatIOnin 1:9MeOH : CHCb. The process yielded one pure oompound

(Rr 0,82) from both the fractions; the other one (Rf 053) was obtained mixed ~th the one

with RrO.82.The pure compound was denoted as ZBJ.

CharacterizatIOn QfZBJ

The compound was a semi solid yellowish substance. It was insoluble in most of the organic

solvents but soluble in methanol and water. The compound gave orange red oolouration with

DraggendorfTreagent; with phenol sulphuric acid it gave a brown ring. The compound turned

liquid at 124_28°C.

The compound exhibited the following speetral data:

UV (CH)OH) (Fig. 6): A."".207,230.2, 281, 235 om

IR (thin film) (Fig 7): v""" 3584, 2900, t639 cm'l

I H NMR (COJOO) (Fig. 8): 0 0.81 (d); 0.88 (5); 0.9(s); 1.78(s, 3H); 3.l4(dd, lH); 3.227(dd,

IH); 3.36(m, IH); 3.64(dd, HI); 3.68(dd, IH); 3.86(dd, lH);

3.97(m, IH); 4.22(d, UI); 4.36(d, IH); 5.62(t, IH); 6.28(5, IH);

6.32(s,IH)

. ,Mass (FIg. 9): M. 757,594,552,433,402,387,279,278,233,149,148

CHAPTER 2: EXPERIMENTAL

2.4.6 STlJDYON FRACTION M2d

Fraction M2d was a yellow gummy substance, It was soluble in methanol and sparingly

soluble in cthyl acetate. With Drnggendorffreagcnt it gave orange red colouratlOn.

COI.U/vfN CHROMATOGRAI'lflC Sf..PARATJON OF FRACTION M1d

Fraction M1d (409 mg) \\IllS dissolved in minimmn quantity of methanol and adsorhcd in

silica gel. The mixture was made free from the solvent and it was then subjected to colwnn

chromatography over silica gel ..,...jib chiorofOlTIl,mixture of chlorofonn-methanol followed by

neat methanol as c1ucnts, Fractions of about 4 m! were collected in each test tube at regular

intervals and examined on TLC plates, In all 54 collections were made. The chromatographic

separations of Fraction M1dare shown in Table 6.

Table 6

Column chromatographic separation of fraction MZd[f;I"ents . chloroform,mixturelof chloroformlindmethanol,methanol]

Collection TLC examination Behaviour witb Yield and

""' Draggendorff reagent observation1_10 No spot - --10-14 one'pot RrO,79CHCI, 100% nocolounrtion I mg,onecompound

two 'poll R,0,79,0,55orangered

29mg,mixturesofi5-18

CHCI,' MeGH;4:1 twocompoundslwospots R,-0.54.0.48

orangered145mg,mixturesof19_25

CHCI,: MoOH:32 twocompound.onespo! R,0.48

orangered26-41 15mg,onecompoundCHC], MeOB;3'2

42~48One lpotR,-0,48

orangered21mg,onecompound

CHCI,,MoOH,3,2 with impurities

" " Long tatl noooloul1llion 4 mg,no resolution

Collectton nos 26-41 showed only one spot which corresponded to Rf 0.48 (CHC!] : MeOH;

3:2) whtch suggested these to contain a SIngle compound. They also gave poSitive test ..,...jib

Draggendorff reagent These collections were combined and the solvent was removed under

reduced pressure to give a compound, which on standing in the freeze gave needle shaped

CHAPrER 2: EXPERIMENTAL

crystals, 15 mg. It was denoted as compound ZB4. The other collections were obtained as a

mixture oftwo compounds or did not exhibit any distinct spot.

Characleri;:alion o[IB4

The compound exhibited the following spectral data:

IR (thin film) (Fig 10 ): v "",,,3500-3400,2934, 1647, 1635, 1589& 1565, 1540.1550, 1240

& 1209 em"]

UV (CHJOH) (Fig. 11):A.max345(sh), 305(sh), 286, 263, 250.5, 218, 207 mn

I H NMR (CHJOH) (Fig. 12): 0 1.00 (5, 3H); 1.03 (5, 3H); 1,22-1.27(d, 6H); 3.33 (s, 3H); 3.4

(m, lH); 3.6 (m, lH); 3.75 (s, 3H); 6,4(m, HI); 6.5 (m, IH);

6.75 (m, lH)

Mass (Fig. 13): nYz305 [M-r], 290, 273, 258, 242, 232, 220, 218

2,5 SEPARATlON OF THE COMPONENTS OF FRACTION MJ

Fractioo MJ was a gummy reddish brown material. It was insoluble in chloroform, sparingly

soluble in ethyl acetate and completely soluble in methanol. TLC examination of the gummy

mass on silica gel plates in different solvents and mixture of solvents indicated in to be a

mixture of compounds. The resolution of the components of the crude was best in ethyl

acetate: methanol; 4:1 in which the presence of three compounds was clearly revealed with Rr

values 0,54, 0.39 and 0.29 respectively along with a spot at the base line, The spot at Rf 0.39

and the one at the base line responded to Draggendorff reagent.

COLUMN CHROMATOGRAPHiC SEPARATION OF FRACTION MJ

Fraction 1\1.1 (25 g) was dissolved in minimum volume of methanol and was adsorbed on

silica gel in the usual way. The adsorbed mass was then transferred on the top of a silica gel

column in ethyl acetate. The column was then eluted with ethyl acetate followed by mixture

of ethyl acetate and methanol and finally by neat methanol. Fractions of about 3 ml were

collected in each test tube at regular intervals and examined on TLC plates, Detailed

chromatographic results are shown in Table 7

CHAffER 2, EXPERIMENTAL

Table 7

Column Chromatographic Separation ofFraetion ~

[Eluentl :Etilylacetate, ml"tures of ethyl acetate and methanol and methanol]

Collection TLC examination Behaviour with Yield and

'"' Draggendorff reagent observatioD

1_2500 """ - -

26-50OIlC spot with tailing

no colourlllion3 mg.,one compound with

R,-0,78EA 100% impurities, Fraetloll M""

51-75two spots

no colooration2 ms, mixture of two

RrO 78,0 75 EA 100"/0 compounds, F'nIotion M••

76-114on. 'pot with tailing

orange red21,1 mg, one compound,

R,-D.67MeOH EA, 1:9 FraotionM..,

115-125Two spots RfD,67,

orange red5 mg, mlldure of two

O,63M.OH,EA, 1:9 compounds, Fraction M",

126.142one spot with tail R,-0.63

orange red9 mg, one compound,

MeOH: EA; 1:4 Fraction M""

143-163two spots R,-0,63, 0,52

oflUlgered23 ms, mixture of two

MeOH:EA; 14 compound" Fnction Mo<

164-183on. 'pot with tail R,-0.60

no colouratioo84 ms, one major

MeOH' EA; 2:3 compounds, Fraction M••

184- 190two spots RrO,6D,0.42

no colouration650 mg, mlldure oftwo

MeOH 'EA2:3 compounds, Fractino M""

191-199two 'pol' R,-0.40,028

no colouration452 ms, mixtureoftwo

MeOH:EA,2:3 compounds, Fnction MJI200 _206 one spot with tail no colourarion 3 mg, no resolution

Collections no 126-142 and 143-163 were combined and evaporated to dryness, The total

mass was subjected to preparative plate separation in 30% MeOH in CHCh, Only 2 mg, of a

pure compound (Rt- 0.52) could be separated. It was denoted as ZB •. With Draggendorff

reagent this compound showed orange red colounrtion,

Characteri=ationofZB.


lR(thin film) (Fig 14): vm",3584, 1691, 1650, 1462 cm,l


1 n NMR (CDCh)(Fig. 15): 8 0.85 (t, 3H); 1.25 (s, 2H); 3.6 (s)

The TLC behavior of collections 164-183 and 184-190 were almost similar. Residue from

collection nos, 184-190 was subjected to crystallization from methanol, when white crystals

(146 mg) were obtained, Repeated crystallization from methanol yielded 126 mg of pure

white crystal, 11",a, regarded as IIpure compound and denoted as ZB", ill. p. 186_88°C.

CharactenzatlOn'ifZ~

Test for carbohydrate

Moliscb's TestR

A few mg of compound lB. was di~solved in .05 ml of water and 2 drops of a 10% of <X:_

naphthol in ethanol were added to it; 1.0 ml of concenlnlted sulphuric acid wa, poured along

the side of the test tube. A red violet ring appeared at the interface.

Tcst by Barfoed's Rcagcnt~8

Banoed's Reagent was prepared by dissolving crystals of neutral copper (II) acetate in 1%

acetic acid solution. 1.0 ml of feat,'CIlt and 1.0 ml solution of the compound in water was

heated in a test tube III a beaker of boiling water bath for 5-10 minute. The test tube was then

allowed to stand for few minutes, red residue of copper (1) oxide was found 10deposits at the

bottom of the test tube,

Test By Spraying Reagent

A spot of the compound on TIC plates was sprayed with vandine in sulphunc acid and then

dried in an oven al 110°; a black spot appeared.

IIydrolysis of compound Zs.20,8 mg of compound ZBt; was dissolved in 5.0 ml of water and 2-3 drops oftrifluoroacctic

acid (CFjCOlH) was added to the solutIOn. The acidified aqueous solution of the compound

was refluxed on a boiling water bath for 2 hours. Subsequently the conlents were evaporated

to dryness on a high vacuum pump eru;uring complete removal of water and triflouroacetic

aCid.

CHAPTER 2, EXPERIMENTAL

59

Paper Chromatography of compound ~

Paper chromatography of authentic compound Zs. was done against its hydrolysates, samplesof glucose, fructose, and sucrose. The paper chromatogram was run using descending method

in n-butanol pyridine: water (10:3:3) for 40 hours. The paper stnp was then dned and

subsequently washed several times in a solution of silver nitrate (AgNO)) in acetone. The

strip was then immersed in a 2% solution ofNaOH in water as a developing reagent. At this

stage spots on the paper slarted to appear gradually. Finally the paper strip was washed with

5% sodium thiosulphate solution and ZBG, glucose. fructose and sucrose each gave one spot

with spot for ZB6 corresp.:mding to that of sucrose. On the other hand the hydrolysate of ZBo

gave two spots winch corresponded to those of glucose and fnK:tose.

The compound ~ exhibited the following spectral data:

IR (KBr) (Fig 16): v",., 3563, 2942 & 2895, 1460 & 1346, 1288 & 1070 cm-'

UV (CH-,OH) (Fig. 17): A."."no absorption

1H NMR (CD,OO) (Fig. 18): 1)3.35 (t, IH); 3.48 (t, IH); 3.51(q, IH); 3.52 (t, IH);

3.53 (q, IH); 3.6 (d, Ill); 3.9 (t, lH); 4.1 (d, IH); 4.5 (s, IH);

4.35 (d, IH)

Mass (Fig. 19): m'z342 [rd!1311, 293, 275,163,133,73 (100%), 57

The mass from collection nos. 191-199 was dissolved in minimwn amount of methanol then

ethyl acetate was added drop by drop until turbidity appeared. On standing crystals separated

out; these were collected and denoted as ZB, m.p.190-9Io.

Charac/eriza/wn ofZB,


IR (KBr) (Fig 20): v"",,3564, 2944, 1279cm-1

I H NMR (COlOD) (Fig. 21): 0 1.7 (q); 1.9 (m); 3.37 (d); 3.6 (d); 3.8 (d); 4.9 (d); 5.9 (m)


2.6 EXAMINATION OF ETHYL ACETATE FRACfION (FRACTION E)

The ethyl acetate Fraction E was a brown gwnmy mass almost insoluble in pet ether but

sparingly soluble in chloroform and ethyl acetate and completely in methanol. With

Draggendorff rcagent the ethyl acetate extract gave orange red colour. TLC examination of

the extract was carried out in chloroform, ethyl acetate and methanol and their combinations,

and showed good resolution in all ofthem. The resolution was best in ethyl acetate: methanol;

9' I with three spots at Rl 0.72, 0,64, 0.45 respectively.

COU1MN CHROMATOGRAPHIC SEPARATION OF,FRACTION E

2.5 I! of cthyl acetate, Fraction E was dissolved in methanol and adsorbed in silica gel. The

mixture was evaporated to dryness and placed on the top of a column of silica gel made in

10% ethyl acetate in pet. ether; elution was carried out with 10% ethyl acetate in pet. ether

followed by mcreasing amount of ethyl acetate and finally mixtures of ethyl acetate and

methanol (9:1) Fractions of about 5 ml were collected at regular intervals and were examined

on TLC plates. The results are given in Table 8.

Table 8

Column Chromatographic Separation of Fraction E

[t:lu~nu ]0"/0ethylacetateinpetetherto 100%ethylacetate;mi;<tull:of ethylacetateandmethanol(9, I»)

Collection TLC examination Behaviour with Yield and

"" Draggendorff reagent observation1_ 14 nospot ~ ~

15.25onespotwithtail,

orangered114mg,onecompound,

1;0.66 EA-PE;3,] Fraction E,

26-43twospots

no colouration50mg,mi:<loreof at leastlWO

R, o 85,0.78EA-PE;6'1 omnpoond,Fracrlon E>twospols.

2.1gm,mimrn:oftwo44-6H 'ltrO78,0.64 orangered

EA-MeOH:g,1compound,F•.•dion E>

69-75onespotWIthlonglOll

""",," 3 mgnoresolutionRrO45EA:M.OH;9'1

CHArTER 2: EXPERIMENTAL

Acid. base Sel)aration of Fraction E,

Fraction E) was dissolved in chloroform and elttracted with 5% IICI. The acid layer was

separated and washed Wlth chloroform, which was then made alkaline with 5% NaOH

solution followed hy extraction with chloroform. The chloroform extract was then dned over

anhydrous Na1S04 and the solvent removed under reduced pressure. A yellow mass (24 mg)

was obtained. [t was a pure compound Rf 0,63 (CHCll : PE; 3:1) and was soluble in

chloroform, ethyl acetate and also methanol. It was denoted as~,

CharactenzatlOn QjZBs

Compound ZBs was yellow semi solid substance and turned liquid at 133_35°C. It showed

one spot on TLC plates in different solvents and had RrO.63 in CHCll : pet. ether (3:1). It was

soluble in chJorofonn, ethyl acetate and methanol and insoluble in less polar solvents. On

sprayiug a spot of eompound ZBawith Draggendorff reagent the spot turned orange red. On

treatment with Mayer's reagent, a solution of the compound turned turbid. Compound ZB8

exhibited the following spectral absorptions. The compound exhibited the following spectral

data:

IR (thin film) (Fig. 22): v""",3400, 3200, 2922, 2850, 1605 cm.1

UV (CH}OH)(Fig.B): Amnx209.20,237.20, 290.40 om.

Mass (Fig. 24): rniz258 [M~],24t, 227,199,173,144,130, t29, 103

lH NMR (CDCI) (Fig, 25): Ii 1.20 (unresol, s), 1.63 (s, 3H), 2.10 (d, 2H), 2.43 (impurity, s,

JR), 2,53 (m, 2H), 2.85 (m, 2B), 3.34 (b.s, 2H) 5.23 (s, lH)

5.48 (I, lH) 6.72 (d. lH), 6.8 (t, IH), 7.08 (IH), 7,08 (d, lH).

Be NMR (CDCh) (Fig. 26): Ii 14.1 (C-12), 34.6 (C-9), 35.5 (C-l), 35.8 (C.3), 52,2 (C-2),

58.6 (C.3a), 67,7 (C-13), 84.9 (C.8a), 110 (C-7), 118,I (C-IO),

120.3 (C.5), 122.7 (C-4), 128.7 (C-6), 133.5 (C-4a), 139.8 (C-

11),148.6 (C-7a)


•

Collection nos. 44 - 68, on removal of the solvent gave a deep yellow solid substance (2.1 g).

This was subjected to separation by preparative thin layer chromatography (PTLC) using

100% ethyl acetate as developing solvent. Extraction of the relevant regions of the plates with

methanol gave two fractions; one of the fracl10n gave a single spot with Rf 0.74 (EtOAc:

McOH; 8: I) and soluble in methanol. It was denoted as ZB.. With Draggendroff reagent it

gave positive test.

Characterization of compounds Z~.


lTV (CH]OH) (Fig. 27): A.m...309, 283, 207, 192, nm

IR(KBr) (Fig. 28): A.••••3400, 1640 em.!

IH NMR (CHJOH) (Fig. 29): 0 0.8 (d, 6H); 1.18 (d, 3H), L58 (s, 3H), 1.70 (m, IH), 1.98 (m,

IH), 2.13 (d, 2H), 15.82 (:>. IH), 6.10 (s, IH), 7.17 (m, IH), 7.5

(overlapping doublets, IH).

Mass (Fig. 30) ,m'z2S4 [M:} 256, 213,199,171,157,117,


The green leaves of zanthoxylum hUdrUIlgU were collected from the Jahangimagar University

campus at Savar, Dhaka, during November-December, 1995. The research work was designed

to investigate the polar constituents of the leaves.

1.75 kg of powdered leaves of Zanthoxylum budrUIlga was subjected to extraction

successively with pet ether and methanol at room temperature. The pet. ether and methanol

extracts yielded a greenish gummy mass (Extract P, 390 g) and a reddish brown gummy

mass (Extract M, 546 g) respectively.

t<)

~ 3.1 STUDY ON EXTRACT M

C'((j) The crude methanol extract, Extract M was a reddish broWll gummy mass and soluble in

methanol but almost insoluble in pet. ether, dichloromethane and sparingly soluble in

ehlorofonn and ethyl acetate. TLC examination of the extract was carried out in different

solvent system, but no good resolution was obtained in any case.

The methanol soluble part of Extract M was diluted with water and partitioned successively

with pet. ether (Fraction P, 1.45g), chlorofonn (Fraction C, 2.45g) and ethyl acetate

(Fraction E, 2.5g). The residua! aqueous methanol solution of the extract was evaporated to

dryne,s to give Fraction M (9.6g). The present work was concentrated on Fraction M and

Fraction E.

3.2 STlJDV ON FRACTION M

The erode Fraction M showed seven spots on the TLC plates at Rf 0.81, 0.76, 0.64, 0.53,

0.47,0.32,0.20 respectively in 33% methanol in ethyl acetate besides a long tailing from the

base line Jt (6.24g) was dIVided into fOUffractions (Ml-l\o4) by minimum effort column

chromatography ovef silica gel on the basis of their TLC behaviour. Fraction M, proved to

be a mixture of two compounds but the amount, 17 mg was not suffiCient fOf further

separation Fractions M1 aod M.J were obtained in relatively largc:rquantity and each ofthcm

appeared to contam at least three separate major compounds as revealed by TLC. The

fractions were thoroughly investigated and the results are discussed in the following section.

Because of time constraint no work could be done on FI1IctioDl\o4.

CHAPTER 3 : RESULTS AND DISCUSSION

3.2.1 Separatfun and characterization of compounds of FRACTION M2

Fl'lIction M: (1.5 g) was a brownish yellow gummy material and showed four spots at Rr0,48, 0.35, 0,30 and 0.23 in 20% methanol in CHCb. Fl'lIction M: was further

chromatographed on a silica gel column to give six subfractions M20 - M:r Fl'lIctions M1dand M:z. were almost similar; further work was carried out on Fraction Mld. Fraction Mlr

did not resolve well on TLC plates.

Fraction MIa was a yellow gummy substance and gave a major spot with a small tailing on

TLC plates. Preparative thin layer chromatographic separation of the fraction led to the

isolation of one pure compound, Rr 0.68 10 50%. CHC!) in pet. ethcr as a yellow gummy

substance. The latter on standing in the freeze for IIlong time yielded needle shaped crystals.

It was designated as compound ZB1•

The IR spectrum (Fig. 2) of the compound ZBj gave weak bands at 1707 and ]655 cm-I and

moderately sharp bands at 2920 and 2851 cm.l. The IHNMR spectrum (Fig. 3) indicated the

presence ofa teoninal methyl group ofan alkyl chain (6 0,89, t), methylene proton attached to

carbonyl group (8 2.0, s) and a number of methylene groups of alkyl chain (8 1.3, s) besides

some other peaks, The IR and InNMR spectrum although suggest it to be an aliphatic ketone,

the structure can not be suggested in the absences of mass spectra. Furthermore the IH NMR

spectrum does not completely corroborate with the structure of a long chain ketone.

CHAITER 3: RESULTS AND DISCUSSION

~ ~ ~c , :;;;: ~ N~ "'" ~ ~.-,. .~ - ,0 "'C ~ ~ ,~ -~ "

l:.oSll--

n:s£!

"",~.S(lSL,."g"a991

1.9591 ,l" Lei!,,

e

i~

N

1.19.82g 1)~6~ t

- -"' .,.

I ~ ~" .~ ~ ", ~.- 0 "~ ~ ~ ~,.,. ~ " ~'':;. ~ ~ - ~-

CHAPfER J ;RESULTS AND DISCUSSION

~U9'H _

9U.'9'--__

O~O~'l'-

<eO. i

~l-'

,•

_0

~UQU.,

• ~~==0~•0"•~--0,

• ="-""~•~Z

i9t. , =~

• t

o•

CHAPTER J : RESULTS AND DISCUSSION

I

Fraction M!b showed two sflOt~at RrO.95 and 0.56 in 100%CHCb which were separated by

preparative TLC. The compound corresponding to Rf 0.54 , ZB2 was obtained in reasonable

amount as a pale yellow gummy compound whereas the other was obtained in meagrc

, quantIty. Compound ZBl was soluble in dichloromethane, chloroform and ethyl acetate but

insoluble in methanol and turncd liquid at 63_650C.

The infrared absorptions at 3649 and 1670 em"] (Fig. 4) of ZB: indicated the presence of

hydroxyl group and unsaturated carbonyl group respectively, A sharp band 11.1 2177.9 cm"]

suggested the presence of C ==C or C ==N group. The proton absorptions (Fig. 5) of the

compound clcarly indicated it to be an aliphatic compound. However, the resolution was not

enough to interpret this spectrum with confidence. The mass spectrum uftbe compound is not

aVlUlableat present The compound remains to be characterized.

Fraction MI. yielded a yel10wgummy matenal, which was soluble in methanol, sparingly

soluble in ethyl acetatc but insoluble in chloroform. The fraction was comprised of at least

two compounds WIthRr 0.82 and 0.53 in 20% methanol in CHCh. The compounds were

separated by preparative thin layer chromatography but only one of the isolates proved to be

pure compound, ZBJ (30 mg),

Compound ZBJ was a semi solid yellowish substance which turned liquid at 124_128oCand

had Rf 0,82 in 10%metbanol in CHCb. It was insoluble in most of the organic solvents but

soluble in methanol and water, The compound ZB] responded to positive lests for alkaloid

and sugar and thus gave indications of it being a sugar alkaloid.


•

"

~ ~ 00

~ "' "' "', " "' C-O - 0~. ,~ -0

,Ia.ia~31

5"19Z1

i

Z'S5Ql

n5Pl

~,,~:}'~l

,

J ~ ~ ~- ~ ~ ~ "' ~..~ ~ ~ "0 ~ ~ - ,-.0 ~- ~,•• -

CHArTER J :RESULTS AND DISCUSSION

----------------

CHAPTER 3: RESULTS AND DISCUSSION

69

-J![tiltll

)f-~-3

, "-.~

"'c,---" '"-- U

QU.,iIN~•0000'1" • ••~•8••-~••2".-. ~

f•

~.=;GOO'z ~

.!!!l.~-.

Compound ZBl, a yellow semi solid substance, turned liquid at 124 - 2lf'C, gave positive

tests for both alkaloids (positive DraggendortT and Mayer's lest) and sugar (positive

Molisch's test). The compound was homogeneous on TLC plates and had Rf 0.82 in 10%

methanol in chlorofonn.

The UV spectrum (Fig. 6) of the compound showed bands at 207, 230.20 and 281 DIn with a

"shoulder at 235 run. Tbe compound thus possessed a conjugated system, most probably in the

from of a heterocyclic nucleus of the alkaloidal moiety. The IR spectrum (Fig. 7) of the

compound showed a sharp, slightly broad band at 3584 em-I for hydroxyl group and a band of

medium intensity at 1639 em-I probably due to a conjugated carbonyl system. The weak

bands around 2900cm.1 sugb'l:sted the low proportion of - Cfu and - CH) b'TouPSin the

molecule. This is however, contrary to the observations from lH NMR spectrum (Fig, 8) in

which the doublet at i50.81, tbe singlets at i50.88 and 0.9 which are attributable to the protons

ofCH) groups attached to spJ carbon atom and the singlet at i5 1.78 attributable to protons of

CH) group attached to Spl carbon atom. As a matter of fact the abSOTptions between 0 0,8-

2.0 tend to suggest the presence of protons belonging to sesquitelpenoid side chain attached to

a nucleus most probably of the alkaloid.

The presence of monosaccharide units is indicated by the doublet at i54.36 fOTthe anomeric

proton H-l, the doublet at 0 4,22 for H-6, doublet of doublets at Ii 3,86, 3.68, 3.64 for H-2, H.

3, H- 4 and multiplet at 0 3.97 for H-5 of a glucose unit. There is another set of doublet of

doublets at 0 3.14, 3.227, multiplet at i53.36 which may be from another monosaccharide unit.

The two singlets at i56,28 and 6.32 may be due to aromatic protons and the odd looking triplet

at Ii 5.62 may be overlapping peaks, containing vinylie protons. Since the intensity of the

peaks are difficult to be measured the relative proportions of the protons under each peak is

difficult to be determined. The quartet at i54.1 which is assignable to

,Io--rn--0i3 or

1fo--crr~CH3

may belong to the terpenoid residue, From the mass spectrwn (Fig. 9) of the compound, an EJ

spectrum, the molecular ion cannot be readily diagnosed. However, the mass fragments at

highest mass numbers indicated it to be a high molecular weight compound, A5 the amount of

CHAPTER J :RESULTS AND DISCUSSION

the compound available was not enough for hydrolysis, the sugar part and the aglycone part

could not be isolated and separately identified. Attempts to find out the removal of the

monosaccharide unit~ from the molecular ion could not be assessed either as the molecular

inn remained undetectahle. Attempts were therefore made to identify the more important mass

fragments exhibited by mass spectrum and build up the molecule. The more mtcnsc peaks c,

g. m/z 117, 149, 233,279,371,387 and 433 were considered and the partial structure X was

built up as shown in Scheme 1.

The partial structure X arrived however, as it appears, fails to explain important peaks at mlz

168,315 and mlz 330. The hydroxylic group of the benzene ring may be glycosidically linked

to a monosacchande and If it IS glucose, which is rather common the molecular ion should

appear at mlz (433 + 180 - 18) i.e, 595 where no mass ion is see. Now, if structure X is

attached 10 a disaecharide comprising of two glucose units the molecular ion should appear at

mlz (595 + 180 - Ill) i.e, mJz 757 where a weak mass ion is observable.

CHi\PTER 3 : RESULTS AND DISCUSSION

n

rn/z279

m!t.233

J

•

•

no

CO •H H

H

mh 117 m!..: 149

HOmJz 371

JH

m/z387

HO

H>C=~CH20HCHZ .••.•.•CHJH0;

H3C~H

m1z433

X

Scheme 1: Mass Ilrrangement or compound ZBJ


From above dIscussion the structure of compound ZBJ is tentatively suggested as

OH

lB,

However, it may be recalled that the structure does not explain the lH NMR absorptions, e.g.

doublets at 6 0.8t, the singlets at 0 0.88 and 0.90,

The confirmation of the structure needs (i) hydrolysis of the compound; separation of the

sugar and agiyoone unit and their identification and (ii) CI mass spectrum to determine the

molecular ion,

CHAffER 3 : RESULTS AND DISCUSSION

.._---~

CHAPfER 3, RESULTS AND DISCUSSION

-"

~=. .=,?O ~-

~

•

•••0 •~0 ••0 ••0

.S"- ~0 N~d ~•0 •., •0" ~

0 • ,n • •• "0" ~. • •0> ,~.,M" •"0 ~~

00 •0 0 0 I:;0 0 0 o.0 0 0 0"

n N " 0 ~

~

CHAI'TER J : RESULTS AND DISCUSSION

CHAPfER J: RESULTS AND DISCUSSION

of "" [-

Il: Q

Q.QU.,"•N~===.. ~

I •, =, 0

" ~'. =•="-, " ", .. ~••" ~..

-""- ~

t- ."..: ".

II

J

- ,~


n

i'e""

"

,'"

,'"

",", ..,, =

: '"~

'-'"- .5'" "N~•'" •0

L~50•

f" •0••, "-, ••~,

,~

,

=,, ~

.!1iJ~

• •

"'"

D' "ESULTS AND DISCUSSIONCHAPrE" "' : "

,"'"'

N

"' N"', "'NN"' ."'N"' 0"' "'"

m'z757

mlz552

<

mlz594

1- C6HIO04

•H0 "> CHzO"l.<W"'~<rn,I ""n

"" I_alpH

m'z433

'"t"

m'z402

mlz387

mlz 149

mlz 148

J

mlz233

Scheme 3: Ma5.!lfragmentation of compound ZBJ

CHAPTER 3 ,RESULTS AND DISCUSSION

t.. .'

Fraction Mld (409 mg) was fractionated into five subfractions by passing through a column

of silica gel. Of these subfractions, three subfractions (collection nos. 15 - 18, 19 - 25, 49 -

54) proved to he cither a mixturc of more than two compounds or did not resolve well on the

TLC plates. Attempts to separate pure compound from them proved unsuccessful. The other

two fractions (collection nos. 26-41 and 42-48) had almost identical TLC behaviour but one

of them (collection nos. 42-48) also showed a long tailing from thc base line. Fraction

containing collection nos. 26-41 on evaporation of the solvent gave a pale yellow pure

compound, ZB.; on standing in the freeze for a long time to yielded as needle shaped crystals.

Compound ZB4was a pale yellow needle shaped crystalline solid and melted at 86_890C.It

gave positive tests for alkaloid with both Draggendorff reagent and Mayer's reagent. The

infrared spectrum (Fig, 10) of the compound showed a broad absorption band at 3500-3400

em-l for hydroxyl group. The other IR bands at 1647, 1589& 1565, 1240& 1209 cm-l could

be attributed to N-eO, aromatic residue and ether linkage respectively. The band at 1635cm-l,

although not as sharp as expected, indicates a quinolone structure. Furthermore absence of

any absorption band around 1540-1550em-I suggested the presence of2-quinolone moiety in

the molecule 8~,The IN absorptins (Fig. II) at 207, 218, 250.5, 263, 286, 305(sh), 345{sh)

run are characteristic for quinolone type of chromophore.

The IH NMR spectrum (Fig. 12) of the compound showed absorptions for OCHJ, NCH1,

protons at S 3,75 and S 3.33 respectively. The doublet at S 1.27-1.22 for SIXprotons indicated

thc presence of an isopropyl system, Thus the pattial structures of thc compound can be

depicted as

In0 ' OCH), CsHll-IO(containingthe isopropyl group), OR~ '(,'

Quino10ne alkaloids both 2- and 4 - are known to occur in Rutaceae plants including the

plants of zanthoxylum genus. zanthoxylum budrUf/ga itself is known to contain 2-qllino1one

alkaloid like zanthobungeanine besides benzo{clphenanthridine alkaloid

(dihydrochelerythrine, dihydroavicineJ, quinazoline alkaloid (evodiamine), indoquinazoline

alkaloid (rhetsinine, rutacarpine), quinazolone alkaloid (arborine) and simple quinoline

alkaloid (dictamnine).90

CHAffER J: RESULTS AND DISCUSSION

(

"The highest mass peak in the mass spectrum (Fig. 13) of the compoWld is seen at mlz 273

suggesting the presence of odd munber of nitrogen atom in the moltx:ule If this mass ion is

consIdered as the molecular ion peak for the compound, the molecular formula, on the basis

of functlonal groups presumed to be present in the molecule is computed as C1JII9NOJ_

Considering the dIsposition of various groups prescnt on the 2-quinoJonc moiety the

following structure A may be attributed to compound with the isopropylidinc group at the end

of the alkyl chain.

A

The OCH1, NCH) protons occur at 0 3.75 and 3.33 and the two betUylic protons which are

non equivalent appear as two rnultiplets centred about /) 3.4 and 3.6, the three aromatic

protons appear as three multiplets centred about Ii 6.4, 6.5 and 6.75. Thus although most of

the absorptions in the lH NMR spectrum can be mtionalized the major objection is with the

chemical shIft of the methyl protons of the isopropylidene group. No doubt, the two methyl

groups may show up as two individual singlets but they would appear at much lower field e,g

01.5-1.7 than observed in this case (0 0.98-1.0). Furthermore the olefinic proton is not seen

and the mtensity for N-CHJ and OCH) are higher than expected

A survey of2-quinolone alkaloids present in Rutaeeae plants show that a number of alkaloids

with thc groups present in compound ~ do occur in nature, one of these being lunacridine,

B with molecular fOlTIluiaC17HnO,.N (M.W.305)91. The compound appeared important to us

as its reported melting point 86-81' is very similar to that of ZB. (86_89°). The compound

may give rise to a mass ion at mh 273, on loss ofa methyl group followed by a OH group.

B


".•

•

•

This structure of lunacridinc, B easily explains the doublet at 0 0.98 -1.0; as well as the

methine proton on the isopropyl group as a multiplet at 0 1.25. The other proton absorptions

are similar to those as shown by structure A.

A 2-qumazolone alkaloid, edulinine C, Cl(;I-bN04 (MW. 291, mp. 103_105°)is known to

occur in Zanthoxylum maya. Interestingly this compound shows an extremely faint molecular

ion peak (m1z 291) and a weak signal at m1z 273 due to ready loss of a molecule of water.

c

The analysis of IH NMR spectrum of ZB. clearly indicated that it was very similar to that

expected of edulinine. In edulinine two methyl groups are expected to exhibit two separate

singlets near 1\1.0 and is consistent with the absorption of ZB4 in the region. The other IH_

signals are simJlar to those for lunacridine as above. However edulinine has four aromatic

protons which cannot be justified by the lH_NMR spectrum. Therefore ooffipouud ZB. is

most likely lunacridine. The mass fragment of ZB4 c.g.; m1z 290, 273, 258, 242, 232, 220,218 can be explained ifit is lunacridine, Scheme 3.

CHAPrER 3 : RESULTS AND DISCUSSION

"'.;:;'

I1.'",''1:.. I_.\'l""7~,--~--n,p

-,,=,

8'5£01r 'J",:"l

9"r.~~

,'N~_. nmol

,l"Ct£!~'€S€l

.".,CIUIt'Ogp\

." ,'SO,!

S'~SSI.P"Lv9l

-.=-:t

-..~

''''; ,,,,'" ""~.:.;:

CHAPrER 3, RESULTS AND DISCUSSION

"

•••0 •-0 •0

,• •0 •~ ~0 N• ! ~0 =0 ••.< ~o. ,0 •0 = 8•o~ ~•0 , ••~ •N "0 'i.

•0 0 0 60 0 0 •0 0 0 0 0 -N 0 0 0 0 -N 0 N - 0 -t

CH•.••PfER 3 : RESULTSAND DISCUSSION

I.•..•

'10-0lI9~'O'00-'ItO "I-!OlKl'I- •~,I 1- 0

6~~-1-U~"I

""'I~ ,0

"11'16,6 -I =99Ed- 0•966-' =6.0-~ V•101 ~ • .,991"l "~'ri ::l~BI-~

w,~'!O'-~ ~c~I!O-~ - Q C

N C6BI (- _ 0Gl~"c •00(-( •0'6n; ~a,-( •• •l!l"-c- N

Ctl,'( •-om; t •Ot""f 0•w; c.

l0

~w,,-(

"6<:9"(-6'~-(- ZlI'I9'( =!01l1I"t

M

"l (~• -"~ 111 ",,,! ( : tIlfI",-I,n-:S:

99Br.----~, Q

(a-t~ •6a',~~Ir. ,-----...:OC,",---

II -II"'"' '-.......... iii16""' __." ••

CHAPTER J: RESULTS AND DlSCUSSION

,

;;;l: ,59-'

0"Ol( 9 •rec";!;t(-gIl[-9~~O~'9~ =00'-9-- 0~:::t ,

<;~u~< =U~19'9 .,"9-9;;9'9

1 '"~"9'9 N]'l"9- ~100' l ••0

'- ~In; l--

,"0• .; 0

"~ ~0,

• •"-"~•~

~,=M-it

~ -,~--

••'"' ,..•..,., •


,• J

"

g..

"N~0 •~ •••••u~•••u,

•••~~~.~~

~-

-CHAPrER 3: RESULTS AND DISCUSSION

,

, ••

-.---.

OCH) ,", -CH3 ~ '" - OH ",•" •1 0 000 1H,C l'H3H,C CH,

nYz305 [Mt] m'z290 ;/ mz273

mz284 [M"t] J -OCH,• -CzHz

, ,CH3

0

H,CH,C

miz232 rrVz 258 m'z242

Jm,onl3

rn'z218 mlzll8 miz220

Scheme3: Massfragmentation of compound ZB~


• •

3.2.2 Separation and characterization of compounds of FRACTION MJ

Frllclion MJ showed three spots at RrO.63, 0.56, 0.52 respectively in ethyl acetate: methanol;

2:3, besides a taihng from (he base line, Fraction MJ (2.5 g) was further chromatographed on

a silica gel column to give nine subfractions, Fractions MJ... Mn, MJ<, MJd either did not

resolve well on TLC plates or were obtained in 100 small an amount to carry out furlher

investigation.

Fractions Ml< and MJf had almost similar TLC behaviour; were combined together and then

subjected to separation by preparative TLC using 30% methanol in eBCb. The process

yielded 2 mg of a pure compound, ZB~

Compound ZB3 was a colourless semi solid substance and turned liquid at 104 - 105°C and

had Rr value 0.52 in 30% methanol in CHCh. 1t was partially soluble in dicblorometbane,

oompletely soluble in chlorofonn, ethyl acetate and methanol and insoluble in pet. ether. It

gave positive test for alkaloid, The IR spectrum (Fig. 14) showed it to be II. hydroxylic

compound (I,.",,, 3584 em-I), The IH NMR spectrum (Fig. 15) indicated the presence of

aliphatic chain (8 085, 1, teoninal methyl proton and 8 1.25 sharp S, CH1 group).ln thc

absence of mass spectra the compound cannot be characterized.

The TLC behaviour of Fractions MJg and MJh were almost similar. Crystallization of

Fraclion M", from methanol yielded globular white crystals. compound Zo.,

Compound Z~, a white crystalline substance melted at 186_88°. It was insoluble in common

organic solvents but soluble in DMSO and highly soluble in water. The compound did not

show any spot when exposed to iodine vapour but revealed II. spot when sprayed with vaniline

in sulphunc acid. The compound responded to positive Molisch's test indicating it to be a

carbohydrate. It also gave positive test for carbohydrates with Banoed's reagent when heated

for 5-10 minutes suggesting it to be a disaccharide mtber than a monosaccharide.

The infrared spectrum (Fig. 16) of the compound showed a sharp absorptIon !It 3563 cm-l for

hydroxyl group. The hands at 2942 & 2895, 1460 & 1346 em-I indicated the presence ofC- B

stretching and bendIng vibration respe>:lively. The band at 1070 cm-I and 1288 Cm"1were

CHAPTER 3: RESULn3 AND DISCUSSION

1-

."

.,," -.

o-.

,

~~-r- ;;".., t:.iI~ ~

-.

- ..,... ,~"-

CHArTER 3 : RESULTS AND DISCUSSION ,

\

•

•

•

CHAPTER J ,RESULTS AND DISCUSSION

•'''''''' g

Indicative of the -e-O- stretching of hydroxylic compound The compound did not absorb

UV (Fig. 17) light and was thus a non-eonjugated compound.

The In NMR spectrum (Fig.18) of the oompound showed a number of absorptions to the

region S 3,52- 5 6 which are characteristic for sugar molecules, In the proton NMR spectrum

of the compound Z~, the anomeric proton H-I appeared as a doublet at S 535 Comparing

the spectrum of the oompound with that of sucrose octaaeetate the doublet at S 4.1398. triplet

at Ii 3.9725, doublet at 0 3.6636, quartet at () 3.4765 and triplet at () 3.3890, oould be assigned

to B-3, H-4, B-3', H-2 and H-4' respectively. It is not possible to locate other prolon which

over lap each other 92

HOH

In order to idcntif)r the monomer units of the disacchande, the compound was hydrolysed

with trifluoroacetic acid. The hydrolysate on paper chromatographic examination showed the

presence of glucose and fructose, The compound was therefo~ assumed to be sucrose. The

melting point of the compound (m. p. 186-88°C) is also similar to that of sucrose (m. p.

I84°C).9' A mixed melting point of the compound with authentic sample of sucrose remained

undepressed. The compound is thus identical to sucrose.

The structure nicely accommodates the mass fragments with molecular ion at m/z 342. Mass

spectrum (Fig. 19) of the compound Z~ showed the highest mass peak at mJz 342. Other

important peaks at mlz 311, 293, 276, 220, 163, 133,73(100%),57,43 could be rationalized

as fragments from mJz 342.

CHAPTER 3, RESULTS AND DISCUSSION 1

"..

r.~r~~.~,';

..,'

•

~/.<r

<

" _." -,-.,..

.,. -

',~

.."~.

•"

.

CHAPTER 3, RESULTS AND DISCUSSION

'-,

••==•0 -•0 •0 =•0 :;l.00' ~M' ==0 =.0 ~O. •0 •-~ •~ ~0_., •

10'0' •C, " •-~0 ~

"0 0 0 0 0 0 >0 0 0 0 0 0 ~0 0 0 0 0 -M

_ _ -0 0 -t

CHAPTER J :RESULTS AND DISCusSION

• 1


, 1.'

,

I ~0, rS,

1u.,

I ••NI ~•••~,i •0~•, ,,i •0I ~I. ~••~zi -"I

~~:tI,I

I,i,III,i'

.1

,o•

oo

•

%

0.<" 0<" 0<00001,

", ,"".""~iiJ

~.J ••

o '.~~~~"fi,.Sf<" ,.0" " .. ", ,

•"," ~.. ,.0.

< '" ,<, C' "C, "-, '"

;~i~i~S;~~~:~R~~!!:~~l.~'~~~~Nn~.,,' ~~.,..,,, ~'~":':~":~":";'~,-, ': ': ':'~~~-:"~":,:-:'".., ., -" . ..',' .,~.-. .om~"~.,,._,.,,~••,('~I-.~""~"~~O""'--.M"4'" "'," ",.fuUH",""'~"'" -,_.,-.., ~~.O"","_" .,,, I.' ,.," _,' ,. ~p 'oc.' (,"~"., oJ ",.' 0," " ~ ""","'''''_'' -,'"_'"'"~"'''t"_,.,, -j ",,,,,,~"",,n, . , . . ' . . . . . . . . '" ...""".,-, """""","'~'"'''''' ">-',"'''''' .

1.00•.•0m•••ffi~~O~.NW.~.ON.mm~.O~"0_~.n.m",'O.,_..".~._~ft_Q~~"m.~~N••_~"_0~" ••.m~••".~om~~••".~••." "'" , .~"~.,_,._.,'".".,""',.O~.,••,..~.,"__"N.'.'.~~''''',' '" I ,'" 0 ,,'" ,"'" "",., ,,,n., _.•".•..,_"0"' ~~",,"., .. ->.00"'''''""'' ",,,., ",. ",,,. ""'''"'"'""""''''-_.~.,_ .._~

g0•• g• U• .,JN~

" =" =0" 0• ~• ,

00~0,00•• •0• ~•~Z.=

0 ~• ~• .~~

""

•


CHAPTER J, RESULTS AND DISCUSSION

"Mode 1:

Hl'

HHOHO

- CH20H

•mldll

mlz342

J =,,~"H

HO 0 HOIIHO ('J HO

••" CH

"0

H

Iff?; 293

JeoH

m'z275

H

mI,,311

j

oVz311

o!rH

/c++o

mlz220

Seheme 4: Ma.'lsfragmentation of compound ZSt;

CHAPI'ER 3 : RESULTS AND DISCUSSION

•\

Mode 2:

"

HO

m1z342

l'- G120H

•

niz 163

• - HmO

HO

mlz 133

1 rearragem:nl•o-rn-G120H

nYzl33

mlz 163

mlz73 (100%)! rcarragem:nt

mlz57

•

nYz73

Scheme4: Mass fragmentation of compound ZB,;


,,C •

wo

FraclioD MJ1 on crystallization from methanol and ethyl acetate gave pale yellow globular

crystals, compound ZIJ.r,

Compound ZR, melted at 190.9IoC. It showed one spot on tbe TLC plates at Rr 0.31 In

methanol in EtOAc; 2:3. It gave positive tests fOf steroid. The lR spectrwn (Fig. 20) of the

compound showed a broad absorption band at 3564 em-I which indicated the compound 10

contain a hydroxyl b'fOUP.The rather low intensity of the band is due to low content in the

mull. The band also indicated the presence nfOH group at 1209 cm-1 for C-O bond vibration.

Thus compound ZB7 is probably a hydroxyl steroid. HO\Vever, the lH NMR spectrum (Fig.

21) is not consistent with such results, Further work is necessary for the characterization of

the compound.

3..3 Separation and characterization of r:vmpollmls of Fraction E

The ethyl acetate fraction (Fraction E) was a brown gummy mass and proved to contain more

than three compounds as revealed by TLC. The crude was separated into four fractions by

colwnn chromoatograpby. Of these the second fraction (FractioD E1, 114 mg) on acid-base

separation gave a pure base, compound ~. Preparative thin layer chromotographic

separation of the third fraction (Fraction EJ, 2.1g) led to the isolation of another pure

compound, Z~. The other two fractions contained very small quantity of substance and no

attempt was made to separate the constituents of the fraction.

Compound Zu" was a light brown solid, melted at 133_35°and was an alkaloid as indicated by

its positive response to Draggendorff reagent and Mayer's reagent. It was homogeneous on

TLC plates when developed in different solvents and was regarded as pure compound. It had

RfO.63 in 3:1; chloroform: pel. ether.

The infrared spectrum (Fig. 22) of the compound showed a sharp and broad OH absorption at

3400-3200 cm'] and the IR absorption at 1605 em-I suggested the presence of aromatic ring.

The UV spectrum (Fig. 23) of compound Z~ showed absorptions at}.....", 237 and 290 om,

the ratio of the mtensities of the two bands being 2.43 which is regarded as characteristic forindohne chromophore. 94

CHAffER 3 : RESULTS AND DISCUSStON

,

,m

~'6~t, V m, O' 6L

CS .S\

II

---j

I-"I'

IiII,iI ,. S"Eza

." ,'-,i,I

i,,

0'9Ill'

;,,,!

i

,i,

,,,,.1 • _

~ ~ ~ ~ ~ -~ ~ ~ ~~ " ~ ~ ~ ~- ~ ~~

~ •0

-e ~ ~~ " ~- D -••


j

---------------

SOWfT > _

--------- ---

CHAffER J :RESULTS AND DISCUSSION

-.

,m

'0;

The mass spectrum (FIg. 24) of the compound provided a major clue for the nucleus of the

molecule. The hjghe~tmass fragment at mlz 258 was accompanied with the base peak at mlz

173and another major peak at mh 130. The base peak mlz 173 is readily assignable to an N.

methyltryptamine mOIetywluch fragment to 3-methylene indolenium ion (m/z 130)

m'zl1l

•r""Ir--fCH ,~y

H

mlz 130

The aromatic region of the lH NMR spectrum (Fig, 25) of compound ~ was similar to

those exhibited by induline alkaloids like chimonanthrine and pseudophrynaminc,91 It

exhibited 2H overlapping doublet and triplet at a 7.08, a lH triplet at a 6,84 and IH doublet at

o 6.77. It proved important 10 note that the mass spectrum of the compound showed the

hIghest mass peak at mlz 258 and if it is considered as the molecular ion peak then it is same

as that for pseudophyrnamine A.I with molecular formula CII;HnN)O, On further

examination of the lH NMR spectrum It became evident that besides the absorptions for

aromatic protons the other proton absorptions are also similar to those reported for

pseL!dophryanamineA-I. 95

PlIeudophryJlamine A-I

Two 3H singlets at 1il.63 and 2.53 were readily attributable to allylie methyl and N-methyl

protons respectively. A lH triplet was observed for an olefinic proton which is attached to a

mcthylenc group, the latter appearing as a 2H doublet at 0 2.53. A comparison of the IH NMR

absorption of compound ZBs with those ofpseudophrynamine A.I, (Table 9) clearly indicatc

them to be identical. However a number ofpcaks c.g. 0 3.49 (5, CHJOH), 1.26(s), 1.20-.80

CHAPTER 3 : RESULTS I\ND DISCUSSION

•

""region are observed in the spectrllrn of ~ which are due to impurities. Pseudophrynamine

has been also reported to be accompanied with similar impurities_

A comprehensIve examination of the mass speclrwn of the compound revealed that the

fragmentation parttcrn of compound ZRw was similar to those exhibited by pseudophyraminc

A-I, The mass fragments at m1z 241, 227, 199, 173, 130, 103,can be easily rationalized (Fig.

24, Scheme 5) with the structure of pseudophyramine A- 1. Comparison of the Be NMR

spectra (Fig. 26) of the compound (Table 10) oonfinn the identify of compound Z8B as

pseudophymmmc A-I. Some extra peaks are observed in the IJC NMR spectra of ZBs which

anse from the impurities present in it. It may be noted that similar impurities were indicated

by IH_NMRspectrum, CornJXlund~ is thus Identified as pseudophrynamine

,,

81 Nt

IC.,ZB,

CHAM'ER.3, RESULTS AND DISCUSSION "

,'028l!;'Um

I.," sn i9'~~r;d

'0;

-

~=~-c,

<l!N~==•~,•0~•,•0-~-- •• -_.- ~

M

.!!P•

';;;,

c_

"


= e_.c c

,. • - 0-co.,

•

'"'

~0 0

00 •-0 •" ,.,0 • ;;• 0 N00 ~0_ 0• •• 0

" ~

J~ ,

0 0 Bj w0_ -

1

oW 0

M> 8~ ••r -•0 ~•0 0 0 60 0 0

0 0 0 0 00 0 0 - M0 " Nrl 0 •~

CHAPTER 3; RESULTS AND DISCUSSION

,

o•

o-

."


00

'"N~

~••0 •; " ~" ,• 8" ~

• • •" " ••" 1l-•- ~•0 •• •"• ~•, ~0 M

" .~~

g•

'" ,..••.,.,

•o

o-

•,

o•

•-

,,.

-e•.-::l

AND DISCUSSIONCHAffER 3 : RESULTS

'"

no-.--~11-;--

l[~ .--~[-"--09' ""'Rlo-"~!lO~'"--I~<;'<;---«;1".---

91£E"0 • e- • QU----- ~

IOWO o!~ N~

• •l.g'g3\. • •ij!£-q •"- ~»'"~ •909"9~ w;,-~ 3E;,ij9~ C ~•lIGij-.-_ •• •".o.,.~ , "alO 17: f- •~

901" I •"--- ~

0000'1 ~~

~• ,~, ~

N

&••

•00 , •••• ,"1

CHAPtER 3, RESULTS AND DISCUSSION

n,

[;1-.1-."""'

[0-91.--.

."o9-l9-~

Z2'o<.-_~93"~~---

~~~-~~.""~

Af:"90~-~I. '6Z Ugg 6<.~ ••Q1' Of:-.......::' ,;;lfi-Or.--_' N8Il"lt-.-- ~19-.[~ 0

0<;' <;[-......:..:: 00

19~[--- ~9'1"9(:---- •lwili:/- 0u~['~( - 0 'Z99"Sf:------ • •I~-I~~ •10';;> /

u

"!Ifi<~- U~•69 i~~_

~0<;"9,----

0•81'<, ,UIn<;~~I"£~----- ~

NOl"O;<;_____~I[ -'><i-----

90"g<;______

1~"0<;--9i Og-___[£'19---


'"

'"'

I" ~l ----

•NE"G<i"S< ~G"l" __

9"~E ---

~';E-Y ••0"."

<"0. --~~~"gg---

900.;-- UoS

l"L9 -_

~9"9L

'''} ~<' LL -- • •• •,"Il •• ~O-- ~

S••~~

•,••

0"011- i•, 811____•

~[;"0<'1- - N-ro~I~-' ZL'O<I--' 9;'ffl-- -

• ~B&1- ..._.- , N

t9"0l'1---"

•~


Table 9

Comparison of IH-NMR absorption of Pseudophrynamine A.l and ZBs.

Pseudopbrynamioe A-I ZB,7.0S(d, JH) [4] 7.08(d, lH)

7.03 (t, IH) (6] 7,08 (1, lH)

6.73 (t, lH) IS] 6.8 (1, IH)

6.59 (d, IH) [7J 6,72 (d, IH)

S.58 (I x m, lH)[=C-H] 5.48 (t, lH)

4.47 (s, IH) S.23 (5, 1H)

3.96 (5, 2H) 3.34 (b, s,2H)

2.80-2.50 (m, 2H) [2] 2 8S (m, 2H)

2.51 (d, 2H) 2.55 (d,2H)

2.43 (s, 3H) [N-CH3] 2.52 (s, 3H)

2,3-2.0(m,2H) 2.10 (d, 2H)

1,64 (5, 3H) 1.63 (5, 3H)

Impurities

3.49 (s, CH30H)

1.26(s)

1.20-0.80

I.2S

1.25-0,80

CIIAITER J ;RESULTS AND DISCUSSION

Table 10

Comparison of IJCNMR llt'sorptJon ofpseudophrynamine A.l and ZBw

Pseudophi'ynllmine A.1 Z",14 1 C-12 14.1

36.9 C-9 34.6

37 C- 1 35.5

38.2 0.3 35.8

52.3 C- 2 52.2

57.9 C.3a 58.6

68.6 0.13 67.7

86.2 C- 8a 84.9

109.2 C- 7 110

120.9 C.I0 118

119.2 C-5 120.3

123.2 C-4 122.7

127.9 C- 6 128.7

134.9 C-4a 133.5

137.8 C-ll 139.8

149,8 C.7a 148.6

CHAM'ER J : RESULTS AND DISCUSSION

.,-f''l

'"

-m, •H

m'z 241

•H, ...,..rn2OHl./~C'cH

CH, '- OH '"•

miL 173 m1z 173 mlz 199

m/zl44 rroIL 130 llVz 130

j -n

r"'l----f"CH,~N"'"

m1zl03 mlz 129

Scheme 5: MailSfragmentation pattern of compound ZBs


Compound ZB" a deep yellow solid, m. p. 160 - 62°C, and showed a single spot on the TLC

plates With Rr 0.74 IIIEtOAc in methanol. Compound ZB, was almost insoluble in pet. ether,

dichloromcthanc, chlorofonn and sparingly soluble in ethyl acetate and completely soluble in

methanol. It gave positive tests for alkaloid with Draggendorffreagent and Mayer's reagent.

The UVabsorption (Fig. 27) bands a Am", 309, 283, 207 and 192 nm suggested the presence

of a highly conjugated system similar to those exhibited by quinazolonc ring system. It may

be noted that alkaloid arborine!l4 possessing such ring system has been reported to be present

in the fruits of Zanthoxylum budrunga.

Infrared spectrum, (Fig. 28) of the compound showed a slightly broad absorption band at

3400 em-I and band at 1640 cm"1indicating the presence ofO-H (or N.H) and conjugated

C = 0 groups in the compound respectively. HoWever a broad singlet at 0 5.82 in the IH

NMR spectrum (Fig. 29) of the compound indicated the IR absorption at 3400 em.] to be due

to NH group. The [H NMR spectrum of the compound clearly revealed that the compound

was not pure and mixed with compound Z~. The absorptions between 8 3.0 - 5,0 and 8 6.5 -

7.0 arc easily seen as the components of compound ~, Considering these as absorptions

from impurity the other absorptions are seen as three doublets at 8 0,8 (6H), 1.18 (3H),

2. 13(2H); two singlets at I) L58 (3H), 6.10 (IH); two multiplels at 8 1.70 (lH), 1.98 (lH), It

was obVIOUSthat these absorptions are due to substituents on quinazolone ring system.

Substitutions with isoprene units are quite common in n!ltural products including alkaloids,

Therefore attempts to rationalize the chemical shift of the protons led to the following

structure for the side cham.

The doublets at 8 0.8, U8 and 2.13 are auributable to the methyl groups a and e whereas the

doublet at 8 2.13 and multiplet at 1.5 fOf the fand c methylene protons respecttvely. The two

SInglets at I) 1.58 and 6.10 are appropriate for the methyl group (g) and vinylic proton (h); the

two multlplets at I) 1,70 and 1.98 can be ascribed to methine protons, band d respectively,


'"The absorptions in the aromatic region.s 7.31 (m) and 717 (m) indicated similar types of

aromatic protons U, k); on the other hand the overlapping doublets at 0 7.5 indicated another

pair of protons (i) on the quinazolone ring. On the basis ofUV and lH NMR data compound

Zs." is tentatively suggested to possess the following structure with molecular formula

CII,,H14N10 (M. W. 284).

The mass spectrum (Fig. 30) of the compound is in conformity with such molecular formula

wluch showed the highest mass IOn at mlz 284. The other mass fragments at mJz 256, 213,

199, 171, 157, 117 are nicely accommodated by this structure (Scheme 6). Therefore the

structure of compound ZB, is considered to be as above.


'00 ••0 ~0 •• s0 .S

~ •0~.,c, ~•0 •.~ 0

0" ~00 sc, •, •0, ~. , •0> S

I., •'" "-• ~0 •

0 0 0 0 0 50 0 0 0 "0 0 0 0rl

~" " ~ 0 N

t


'" ,..;~'"

•-,-

•0

"~0

c'~.G~l ;l~000~

0',00,.: '

~,0": "', -0•~•~00N

- ,!l>~

. G<~

, '

l'!,;f" >,

•

,'2-,

- -,

-

CHAPfER J :RESULTS AND DISCUSSION

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CHAPTER J :RESULTS AND DISCUSSION

LTS AND DISCUSSIONn3'RESUCHAPrE" .

~-

<"""WI

••

o,

o

•

'"

~~ ----_._ ..----


00M

~00N

--- NMN 0

N

-.,-

-w •

ID1.284 m'z256

m'z 171

lorn,

Q::: ",I N I ."'" ~CHP-t+

"mlz 157

mlz 199

QJ,H

mlzl!7

-eN Ct'•~ '"m'z91

mlz 213

Schem., 6: Mass frallmentalioD of compound Zo.

CHAI'I'ER 3, RESULTS ANDDISCUSSION

.'

•

L The planl Zanlh"xylum budrunga is reported 10possess a variety oftherapeUlic value,

Although much work on dillerent parts of the plant the appears to have been camed

out, hardly any work on the leaves is reported. The present work was undertaken to

isolate the alkaloids and other constituents of the leaves of Zamhoxylum budrunga

and study theIr pharmacological activity, The phytochemical investigation with

considerable amount of there and because of time constraint no pharmacological

work could be done.

The dried powdered leaves of Zanthoxylum budnmga (1.75 kg) were extracted

successively with pet. ether (3 x 48 hours) and methanol (3 x 48 hours) at room

temperature. The pel. ether extract yielded a greenish gwnmy mass (Extract p, 390 g,

22.3%) and methanol extract yielded a brown gummy mass (Extract M, 546 g, 31,2%)

respectIvely on removal of the solvent.

The methanol soluble part of Extract M (25 g) was partitioned with pet. ether,

chloroform and ethyl acetate to give three fractions, Fraction P (l,45 g), Fraction C

(245 g), and Fraction E (2.5 g) respe>:tivdyalong with Fraction M (9,6 g), the residual

part orthe extract. The present work was concentrated on Fraction M and Fraction E.

2. Fraction M showed thc presence of at least seven compounds on TIC plates. Fraction

M (6.24 g, 65%) was divided into four fractions (M. - Mo) by a method ofminimwn

effort column chromatographic separation. Most of these fractions were found to be

complicated mixtures. However, repeated column chromatographic separation followed

by purification of Fraction M2 yielded four pure compound, e, g. ZB], ZBl, ZBJ and

ZB.j.Similar treatment ofFractioD ~ led to the iSQlationof three pure compound, ZB5,

ZB,;and Z8?

Compounds ZBr, ZBl, ZB" ZB.~, Zs. and ZB, were characterized and identified by

extensive use ofUV, JR, IH NMR, BC NMR and mass spectral analyses.

2.1 Compound ZB] was a ycllow needle shaped crystals, m, p. 51 - 53° C. Infrared and IH

NMR spectra of the compound suggested it 10be a long chain aliphatic ketone but It

could not be fully characterized as the mass spectra of the compound was not available,

CHAPI'ER 4: SUMMARY

'"2.2 Compound ZBl was a pale yellow gummy substance and turned liquid at 63 _ 65°C. The

infrared spectrum of the compound suggested the presence of C-C or C" N

group. The lH NMR spectrum of the compound clearly indicated it to be an aliphatic

compound, however the resolution was not enough to interpret this spectrum with

confidence; the structure of the compound remains to be established.

2.3 Compound ZBJ was a semi solid yellowish substance and turned liquid at 124 - 12S°C.

It gave poSitive tests for alkaloid lIS well as for sugar. The UV, IR and IH NMR spectra

of the compound suggested it to be an alkaloid, the nucleus of which is connected to

both isoprenoid and sugar fragments. The molecular ion of the compound was not

readily distinguishable from other mass fragments. However, the mass peak atMt 757

if considered 10 be the molecular ion peak, then the following structure can explain more

or less all the spectral data

OH

2,4 Compound ZB4 was a yellow needle shaped crystalline solid, m, p, 86 ~ 89°C. It gave

positive tests for alkaloid. The UV and JR spectra of the compound suggested it to

contain a quinolone type of chromphore. The lH NMR spectra and melting point of the

compound are comparable 10 those of lunacridine (Mol. formula C17H1lN04, M W.

305) it truly be noted that the mass spectra (EI) of compound ZB4 gave the highest mass

ion at m1z 273 which is explained by loss of a molecule of water and a methyl group,from M' 305 oflunacridine. It may be noted that quinolone type of alkaloid acutifolm

has been shown to be present in the lives of Z acutifoliumU

CHAffER 4: SUMMARY

•

2.5 Compound ZB~ was a colourless semi solid substance and turned liquid at 104 - 105°C.

It gave (Xlsitive tests for alkaloid but the IR and lH NMR spectrum of the CQmpound

suggested it to be II long chain hydroxylic aliphatic compound. These findings are thus,

contradictory to each other. This problem remains to be sorted out. Moreover, the mass

spectnun of the compound is yet to be procured. Thus the compound could not be

thoroughly characterized.

2.6 Compound Zs. was IIwhite crystalline substance melted at 186 - 88°C. It gave positive

test for carbohydrates and more specifically for a disaccharide. Hydrolysis with

trifluoroacetic acid resulted in the formation of glucose and fructose. The prolon NMR

spel;trum, mass spectrum, IR spectrum and melting point of the compound and also the

results of hydrolysis of the compound confinn it to be sucrose,

H

HOH

H

2.7 Compound ZB, was obtained as pale yellow globular crystals, m. p. 190 - 91°e. It gave

positive tests for steroid. The IR and IHNMR spectrum of the compound suggested that

the compound most probably is a hydroxyl steroid Because of time constraint, further

work 011the compound could oot be done and the structure of the compound could not

be detennined.

CHAPfER 4: SUMMARY

'"3 Column chromatographic separation of Fnlction E (2.5 g, 100%) gave four fractions

(Et - E~). Because of time constraint only two of these fractions E2 and E3 were further

studied. Acid - Base separation of fraction "E:2gave one pure compound, ZBs; another

pure compound, Zs, was Isolated from fraction E3 by preparative TLC method.

3.1 Compound ZBs was a light bro'Ml solid, m. p. J33 - 35°C and was alkaloid as indicated

by its positive response to Draggedorffreagent and Mayer's reagent. The IR and UV

spectrum of the compound suggested it to contain a indoline cbromophore. The IH

NMR, l.lC NMR and mass spectra of the compound are identical to thosepseudophrynaminc_

3.2 Compound ZR.;,was a deep yellow solid, m. p. 160- 62°C It gave positive tests for

alkaloid. The UV spectnun suggests the presence of a highly conjugated system similar

to those exhibited by quinazolone ring system, The IR spectrum, lH NMR spectrum and,mass spectrum (M' 284) of the compoWld suggest it to possess the following structure.

CHAPrER 4, SUMMARY

••

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CHAPTER 5: REFERENCE

•

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Documents

CHEMICAL INVESTIGATION ON THE LEAVES OF