Chewing Tobacco (Maraş Powder) Alkaloids

Home , Nicotiana rustica

of of

61 61

kül•

fos

eks-. eks-. our our

ana

analiz analiz

This This

18 18

Maraş Maraş

analizi analizi

rustica rustica

occurs occurs

0.1112, 0.1112,

nikotin, nikotin,

0.2 0.2

itching itching

reason, reason,

powder powder

stop stop

relaxing relaxing

pinch pinch

powder. powder.

and and

ve ve

Otu Otu

% %

a a

yapılan yapılan

0.0579 0.0579

Otu) Otu)

During During

Alkaloitleri, Alkaloitleri,

for for

this this

!hat !hat

Ankara'da Ankara'da

!hey !hey

nikotin, nikotin,

ters-faz ters-faz

yaprakların yaprakların

and and

antiaggressive, antiaggressive,

!ip !ip

kantitatif kantitatif

Maraş Maraş

Nicotiana Nicotiana

For For

tobacco tobacco

Tütün Tütün

kullanılarak kullanılarak

kültürü kültürü

greenish greenish

0.0341, 0.0341,

placing placing

Analizi Analizi

usage. usage.

ve ve

Habituation Habituation

when when

a a

L., L.,

1.1705 1.1705

L. L.

ayrınıı ayrınıı

ile ile

edilmiş edilmiş

lower lower

uneasiness, uneasiness,

by by

is is

informed informed

tamponlanmış tamponlanmış

humidification. humidification.

L.yapraklarının L.yapraklarının

Jips. Jips.

it it

Anadolu'da, Anadolu'da,

Toz Toz

0.1538, 0.1538,

analgesic, analgesic,

and and

Metana! Metana!

Hazırlanan Hazırlanan

chewing chewing

rustica rustica

for for

Analizi Analizi

YBSK YBSK

cigarettes. cigarettes.

used used

month's month's

Ankara'da Ankara'da

2.1221, 2.1221,

% %

tütiinündeki(Maraş tütiinündeki(Maraş

ve ve

alkaloit/erinin alkaloit/erinin

observed observed

to to

until until

is is

ve ve

addicts addicts

rustica rustica

L. L.

teeth teeth

one one

40'/ık 40'/ık

bruised bruised

pH=7.25'e pH=7.25'e

Turkey. Turkey.

study; study; are are

irritability, irritability,

yapraklarındaki yapraklarındaki

Güneydoğu Güneydoğu

7.7069, 7.7069,

used used

TF-YBSK TF-YBSK

% %

yapılmıştır. yapılmıştır.

çiğneme çiğneme

smoking smoking

and and

after after

Turkey Turkey

L. L.

rustica rustica

sprinkled sprinkled

tobacco tobacco

rustica rustica

nornikotin, nornikotin,

anabazin anabazin

studies, studies,

are are

Ankara, Ankara,

kneaded kneaded

bulunmuştur. bulunmuştur.

Nicotiana Nicotiana takiben takiben alkaloit/erin

is is

between between

up up

Alkaloit/erinin Alkaloit/erinin

another another

ve ve

içeren içeren

is is

gingiva, gingiva,

behaviour behaviour

Kayseri, Kayseri,

Trietilaıninle Trietilaıninle

ln ln

rustica rustica

Nicotiana Nicotiana kelimeler:

who who

Powder) Powder)

0.0401 0.0401

06100 06100

sırasıyla% sırasıyla%

Kahramannıaraş Kahramannıaraş

hazırlanan hazırlanan

kullanılarak kullanılarak

Nicotiana Nicotiana

asit asit

water water

dropped dropped

içerdiği içerdiği

chewing chewing

: :

giving giving

yapılan yapılan

the the

it2. it2.

powder powder

38039 38039

'dan 'dan

fraksiyonunu fraksiyonunu

YBSK YBSK

on on

lonunda lonunda

ture, ture, questionaire questionaire

bazin bazin

approximately approximately nornikotin nornikotin

otunun otunun

cotiana cotiana

0.0705, 0.0705, the the ing ing

and and türü türü

gressive gressive

mixture mixture

Özet Özet This This

L. L.

have have

people people

gerçekleştirilnıiştir. gerçekleştirilnıiştir.

Anahtar Anahtar

forik forik

in in

Pharrnacognosy, Pharrnacognosy,

and and

oak oak

7.25 7.25

pro

and and

(Ma

a a

mix-

Tech

meth

of of

1.1705 1.1705

Pharmacology, Pharmacology,

Nicotiana Nicotiana

(Maraş (Maraş

in in

o/o o/o

pH pH

on on

cultiva

The The

follo~ved follo~ved

widely widely

and and

Nicotiana Nicotiana

of of

anabasine anabasine

this this

Powder). Powder).

to to

40 40

is is

Ankara Ankara

L. L.

commonly commonly

cııltivated cııltivated

of of

ERENMEMİŞOÖLU** ERENMEMİŞOÖLU**

naturalized naturalized

tobacco tobacco

of of

tissue tissue

and and

2.1221, 2.1221,

"Hasankeyf" "Hasankeyf"

L. L.

nornicotine nornicotine

found found

and and

is is

and·Chewing and·Chewing

are are

Alkaloids, Alkaloids,

Department Department

Gaziantep Gaziantep

limited limited

or or

alkaloids alkaloids

%, %,

leaves leaves

(Maraş (Maraş

L. L.

powder powder

rustica rustica

bıiffered bıiffered

Aydın Aydın

L. L.

phase phase

Department Department

Alkaloids Alkaloids

.are .are

L. L.

m.illed m.illed

the the

chewing chewing

the the

7.7069, 7.7069,

Chron1atographic Chron1atographic

very very

1998 1998

leaf leaf

tabacınn tabacınn

acid acid

in in

of of

nicotine, nicotine,

the the

Anatolia Anatolia

Tobacco Tobacco

a a

0.0401 0.0401

Graham Graham

rustica rustica

around around

that that

nıobile nıobile

Pharmacy, Pharmacy,

L. L.

rustica rustica

of of

planı planı

To To proportions.

rustica rustica

L., L.,

tobacco tobacco

the the

and and

a a

Faculty, Faculty,

of of

Nicotiana Nicotiana

Powder) Powder)

Liquid Liquid

has has

61-64, 61-64,

nicotine nicotine

"Delitütün" "Delitütün"

Tobacco Tobacco

KARTAL*, KARTAL*,

1:3 1:3

BfLfMSELARAŞTIRMALAR BfLfMSELARAŞTIRMALAR

respectively. respectively.

L. L.

found found

with with

/ /

0.0705, 0.0705,

glauca glauca

23, 23,

Southeast Southeast

or or rustica rustica

Nicotiana Nicotiana

% %

Analysis, Analysis,

rustica rustica

.fron1 .fron1

phosphoric phosphoric

Nicotiana Nicotiana

Medical Medical

Faculty Faculty

Ankara, Ankara,

Anatolia Anatolia

lvas lvas

tobacco tobacco

(Mara~ (Mara~ in in

extraction extraction

quantitation" quantitation"

called called

% %

Murat Murat

Nicotiana Nicotiana

1:2 1:2

chewing chewing

of of

Pressure Pressure

quantitate quantitate

Sci., Sci.,

it it

Analysis Analysis

is is

in in

coluınn coluınn

contained contained

of of

and and

rustica rustica

these these

in in

the the

to to

0.2 0.2

of of

0.1112, 0.1112,

0.0579 0.0579

and and

it it

18 18

High High

18.4.1998 18.4.1998

12.1.1998 12.1.1998

prepared prepared

C C

Nicotiana Nicotiana

ART!CLES ART!CLES

RP-HPLC RP-HPLC

Nicotiana Nicotiana

Tobacco Tobacco

A A

used used

University, University,

Pharnı. Pharnı.

University, University,

levels levels

cııltivated cııltivated

Analysis Analysis

prepare prepare

Southeast Southeast

tobacco tobacco

mixed mixed

J. J.

leaves leaves

species species

0.0341, 0.0341,

among among

where where

L. L.

involved involved

KURUCU*, KURUCU*,

phase phase

to to

was was

in in

dried dried

Nicotiana Nicotiana

containing containing

triethylamine. triethylamine.

are are

words: words:

Powder) Powder)

nornicotine nornicotine

Correspondence Correspondence Ankara Ankara

Erciyes Erciyes

"separation "separation

HPLC HPLC

The The

. .

%, %,

versed versed

by by raş raş

Kabul Kabul

** **

* * 0.1538, 0.1538,

rustica rustica

with with and and

Geliş Geliş

Semra Semra 1

used used

atolia; atolia;

Alkaloids Alkaloids

Maraş Maraş Three Three

Sun Sun

Kahramannıaraş Kahramannıaraş

cedure cedure

INTRODUCTION INTRODUCTION

Düzelti Düzelti tion tion

chewing chewing

anabasine anabasine

nique nique

Key Key

RESEARCH RESEARCH ash ash cultivated, cultivated, anol anol HPLC HPLC

Chewing Chewing

FABAD FABAD Summary: Summary: Kurucu, Kartal, Erenmemişoğlu exploratory and locomotor effects of total alkaloid 10 µ (15 cm x 3.9 mm) eluted with an isocratic mo extract of Nicotiana rustica L. were compared with bile phase of 40 % (v /v) methanol containing 0.2 % those of nicotine. No difference was observed be (v /v) phosphoric acid buffered to pH 7.25 with tri tween the effects of Maraşpowder and buccal nic ethylamine7. Nicotine was determined at a flow otine use 3. rate of 0.3 ml/min in ali samples. Nomicotine and anabasine were determined ata flow rate of 0.2 mi/ Epidemiological and screening investigations have min in Nicotiana rustica L. samples and MaraşPow also been made on Maraşpowder users. We have der. evaluated cytological smears obtained from lower !ip mucosa lesions in eighty Maraşpowder users Ali solvents and samples were filtered through a and punch biopsies were performed. Oral cancer 0.45 µm. Millipore filler prior to use. Nicotine, nor among Maraşpowder users appeared to be related nicotine and anabasine were quantitated by a Wa to the length of use as it was only observed in sub ters 745 B Data module which automatically in jects with 15 or more years of exposure 4. tegrated peak areas and compared them with those of authentic standards. It is known that Nicotiana rustica L. leaves contain higher amounts of nicotine than Nicotina tabacum L. Chemicals 2,5. However, !here have been no reports on the quantitative determination of tobacco alkoloids in (s)-(-) Nicotine was obtained from Aldrich Chemical Turkish Nicotiana rustica L. and MaraşPowder. Company (Milwaukee WIS 53233 USA), while (±) Nor nicotine and (±) Anabasine were obtained from Sigma Many GC methods have been published for de Chemical Company (ST. Louis, Mü 63178 USA. termining alkoloids in tobacco; which although sen sitive, do not allow rapid analysis of alkaloids; tem Chromatographic solvents were HPLC grade from perature programming being required or replicate Merek Chemical Company (D-6100 Darmstadt, F.R. analysis of samples at a higher temperature. High Germany). pressure reversed phase liquid chromatography is an effective and dependable method for the separa Material tion of tobacco alkoloids 6. Samples subjected to alkaloid analysis were as fol In our research, we aimed to determine the amount lows: Nicotina rustica L. leaves cultivated in Kah of alkaloids in cultivated Nicotina rustica L. leaves ramanmaraşand cultivated in our experimental and in prepared chewing tobacco (MaraşPowder). garden in Ankara. Three chewing tobacco (Maraş The amount of nicotine, nornicotine and anabasine Powder) samples were supplied in commercial were determined in Nicotina rustica L. samples. In packets of Kahramanmaraşbazaars. recent years, there appears to be more interest in MaraşPowder because of its intensive use in South Nicotine analysis east Anatolia for giving up smoking. The milled samples were weighed into 0.5000 g lots EXPERIMENTAL and extracted with 10 rnl of 25 mM sodium phos phate buffer (pH 7.8) at 30°C for 24 h with constant Apparatus agitation. The aqueous extract was filtered under re duced pressure through a Whatrnan No. 2 filler paper The HPLC system consisted of a Waters (Milford, and diluted ten fold with water. Each extract was fil MA, USA) model U6 K injector, a M-45 solvent tered through a 0.45 µm Millipore filler and 1 µl sam pump and a model 440 detector equipped with a ples were injected7. 254 nm filler. Nornicotine and anabasine analysis The tobacco alkaloids were quantitatively separated on a Waters Novapak C18 reversed - phase column The milled samples were weighed into 5.0000 g lots

62 FABAD J. Pharm. Sci., 23, 61-64, 1998 and extracted lwice with 150 mi of lN HCI for 24 h analysis. The extraclion method is given in the ex maceration and 2 h conslant agitation; acidic aque perimental section. ous extracts were filtered and combined. The com bined acidic extracl was basified with 6 N NaOH to It is seen from our findings that leaves of Nicotiana pH 11 and extracted three limes with 150 mi dich rustica L. cultivated in Southeast Analolia conlain loromethane. The dichloromethane layer was re nicotine 7.7069 %, nornicotine 0.1112 % and anab moved and gently evaporated to dryness under ni asine 0.1538 % (fig.l). trogen8. The crude alkaloid fraction was dissolved in 10 mi methanol and 1 µI samples were injected However nicotine, nornicotine and anabasine con onto the colurnn. tents may vary due to the effect of climate and soil conditions. Thus, leaf samples from cultivated plants in our experimental garden in Ankara are Quantification found to contain low amounts of nicotine 2.1221 %, nomicotine 0.0705 % and anabasine 0.0341 % (fig.2) An external standard rnethod based on peak area during the flowering stage. This decrease may be was used for quantitative determinations. The cal due to various enviromental factors such as sun ibration curves were prepared by analysing five di light, soil, climate, fertilization and others9-H Sim lutions (n=5) of authentic samples of nicotine (0.4 - ilar results were observed previously for Nicotiana 0.05 mg/ml), nornicotine (0.5 - 0.025 mg/ml) and rustica L. where the nicotine content was found to anabasine (0.5 - 0.025 mg/ml). The linearity of nic decrease from 6.6 - 8.8 % in greenhouse plants, to otine, nornicotine and anabasine were confirmed by 1.54 - 2.64 % in field plantsl2. regression analysis. The correlation coefficients were found to be r2=0.9968 for nicotine, r2=0.9990 On the other hand chewing tobacco (MaraşPow 2 far nornicotine and r =0.9999 for anabasine. Results der) prepared from Nicotiana rustica L. leaves was are expressed as the mean of three determinations. found to contain low amounts of nicotine 1.1705 %, nornicotine % 0.0401 and anabasine 0.0579 % (fig.3) RESULTS AND DISCUSSION when compared with the leaves. This decrease de pends partially on the mixture of dried leaf powder This report describes a RP-HPLC analysis of al with oak ash in various unknown proportions (1:3 kaloids applied to Nicotiana rustica L. of Turkish or igin (Table 1). j, ' j Table 1. Alkaloid Content of Nicotina nısticaL. sam ples from Turkey

Sample % Nicotine± S.0. %Nornicotine ± S.D. % Anabasine± S.D. ! ! l~lJ':1 Leavesof ! t): Nicotinarustica L. ;JJ_j . ' cultivatedin 1 f Kahramanmaraş7.7069±0.1090 0.1112± 0.0063 0.1538±0.0074

Leavesof

wlıi..ıe.din K...... ,,.ıu fdikııolllllO) Nicotinarustica L. cultivatedin Ankara 2.1221±0.1039 0.0705± 0.0043 0.0341±0.0070 ChewingTobacco

(MaraşPowder) 1.1705±0.0337 0.0401±0.0042 0.0579±0.0134

Eachvalue is theaverage ol threeruns ± StandardDeviation (S.0.)

The extraction method of Saunders and Blume was used to determine nicotine, but this method was not sufficent for minor alkaloids. Different extraction methods were used for nornicotine and anabasine fig. l cı-ingTnıı.ı...ı CM.arQP"""3")

63 Kurucu, Kartal, Erenmemişoğlu

or 1:2) and partially on the preparation technique, Powder", Doğa- Turkish J of Medical Sciences, 16, 867- where nicotine rnay be lost during hurnidification 876, 1992. 4. Erenmemişoğlu,A., Üstün H., Kartal, M., Carcinoma after rnixing with the ash; nicotine presurnably of buccal mucosa in smokeless tobacco users: a pre evaporates partly in a rnild alkaline medium. liminary study of the use of cytology lor early de

tection; Cytopatlıology,6, 403-308, 1995. These findings are in accordance with the reports on 5. Shmuk, A A, The Gıemistıyand Tedınology of Tobacco in: Tobacco Alkaloids, Ed. N.L Gavrilov, Pish nicotine content in Nicotiana rustica L. leaves 0.7 47 chepromizdat Press, Moscow, voL 3, pp. 53, 1953. % s, 1.54 - 2.64 % 12, 3.60 - 5.83 % 13, 4.52 - 8.61 % 14 6. Watson, I. D., Rapid Analysis of Nicotine and Cotinine and 7 - 8 % 5 , The nornicotine !eve! was reported to in the Urine of Smokers by Isocratic High Per be 0.0069 % B, 0.18 - 0.48 % 14 and 0.51 - 0.89 % B formance Liquid Chromatogr.aphy, J of Chrom atography,143, 203-206, 1977. and anabasine !eve! was reported to be 0.0085 % Sin 7. Saunders, j. A, Blume D.E., Quantitation of Major To a single study. bacco Alkaloids by High Performance Liquid Chrom atography, J ofChromatography, 205, 147-154, 1981. Analgesic, antiaggressive, exploratory and loco 8. Saitoh, F., Nama, M., Kawashima, N., The Alkaloid Contents of Sixty Nicotiana Species, Phytoclıemistıy,24 rnotor effects of total alkaloid extract of Nicotiana (3), 477-480, 1985. rustica L. and carcinorna of buccal mucosa in Maraş 9. Sayed, M. D., Karawya M.S., Reda, FA, Effect of Light Powder users have been discussed in detail in our Intensity on Growth, Flowering and Alk:aloidal Content L. previous reports 3,4, of Nicotiana rustica Grown in Egypt, Egypt J Phann. Sci. 14(2), 117-128, 1973. Ref: C.A 80, 118365c, 1974. 10. Diez, M. A, Barcelo, )., Lopez-Belmonte, F., Effects of The carcinogenic effects of the various types of Nitrogen, Phosphorus, Potassium and Magnesium De srnokeless tobacco have been attributed to the pres ficiencies on Growth and Nicotine Content of Nicotina ence of N-nitrosonornicotine (NNN). NNN is pra rustica L., An. EdafoL AgrobioL 42, 1663-1667, 1983. Ref: C.A 101, 229331k, 1984. duced in tobacco by bacterial or enzymatic activity Dimitriev, M. F., Gubanova, N. M., Gubanova, S. I., ln and it has been reported to cause oral and pha creasing the Nicotine Content of Azetec Tobacco, Ta ryngeal cancer experimentally15.16. Our research bak (Moscow), 3, 36-39, 1987. Ref: C.A 108, 19372!, will continue on NNN content of N rustica L. leaves 1988. 12. Chopra, R.N., Na yar, S.L., Chopra, LC, Glossary of In and MaraşPowder. dian Medicinal Plants, Council of Scientific and In dustrial Research, New Delhi, pp.176, 1956. It is clear that using MaraşPowder is nota safe sub 13. Bhide, S.V., Nair, J.,Maru, G.B., Nair, U.J., Kameshwar stitute for srnoking cigarettes. it can cause cancer Rao, B.V., Chakraborty, MK and Brunnemann, KD., Tobacco-specific N-Nitrosamines (TSNA) in Green Ma and a nurnber of non-cancerous oral conditions and ture and Processed Tobacco Leaves from India, Beitrage also can lead to nicotine addiction and dependence. zur Tabakforschung Intemational, 14(1), 29-32, 1987. 14. Watt, J.M., Breyer-Brandwijk, M.G.: Medicinal and REFERENCES Poisonous Plants of Southern and Eastern Africa, E. & S. Livingstone Ltd., Second Ed., Landon, p.962-989, 1962. L Davis, P. H, Flora of Turkey and The East Aegean Islands, 15. Glover, E. D., Schroeder, KL., Henningfield, ). E., Se Edinburgh University Press, VoL 6, Edinburgh, 1978. versen, H. H., Chiristen, A. G., An Interpretative Re 2. Kartal, M, Phannacognostic Research on Nicotiana rustica view of Smokeless Tobacco Research in The United L.Alkaloids, M.S. Thesis, Ankara University, Faculty of States, Fart 1,J ofDrug Education, 18, 285-310, 1988. Phannacy, 1991. 16. Charnberlain, W. )., Schlotzhauer, W. S., Chortyk, O. 3. Erenmemişoğlu,A, Tekol Y., Kartal M. and S. Ku T., Chemical Composition of Nonsmoking Tobacco

rucu, Abuse of Smokeless Tobacco in Turkey "Maraş Products, J Agıic.Food Chem.,36, 48-50, 1988.