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EJ52-I 957-1 12

[Contribution from the Chemical Drugs Control Laboratory, Ministry of Interior)

A Qualitative' Study on the Color

Reactions of Reserpine
CHENG SUN (G.3)

Reserpine ~ 3 tested
s for color reactions with twenty different chemical
reagents. Some reagents showed specific color reactions while others
responded negatively, All colorations were painted with colors to serve
as a record. Both the color changes and the rcagents used were des-
cribed and fisted in detaiI.

RESERPINE is the name of ;t weak alkaloid first izolated f r o m the

plant Rauti-ol.lia x r p e n t i n a Dcnth as a pure crystalline compound by MiilIer,
Schlittler, and Bein('). It has been described as a drug pos:es:ing pronounced
hypotensive activity toward high blood pressure.
T h e empirica1 and structural f o r m u h e of reserpine have -been well esta-
blichcd by SchIittlcr e t a!.(") and by Steenhaucr(5) as follows:

Empirical f o r n i u h St r u c t u r d f o r q u t a
c
33 H 4 DO, N*
moIeculnr weight =
608.4
CHj0

.D
CH300C
OCH3
OCO

Since most alkaloids react with many reagents to give distinct, definite
and spccific color rcnctions, it is espccted that reserpine will also follow the
cxnmpk. T h e prcsent paper a t t e m p t s to report some re:uIts obtained on the

112
 COLOR REACTIONS OF RESERPLKE 113
color reactions of reserpice.
T h e tests were performed in ;L manner similar to that of thc "dropping
method" technique.('ld) I n order to gain 3 little quantitative idea, a definite
weight of sample w a s extracted once with a known volume of chloroform.
T h c n , from the number of drops of chloroform extract used in thc experiment,
which was conducted in a :mall white porccInin evaporating di:ti, the !east
amount of reserpine which gnvc a po:itive color rcaction may be ectimatcd.
T h c rcscrpinz was tested in i t s bncic form.
T w e n t y reagents were employcd in the tests, they are, Frijhde's reagent
(in cold and in hot), Marquis' reagent (in cold and in hot), concentrated
sulfuric acid, iodic acid, Mandelin's reagent, Keilcr's rertgent, iodic acid
reagent, Meeke's reagent, Erdmann's reagent, Licbcrmann's reagent, fuming
nitric acid, ferric chloride ptass.ium permangate concentrated su!furic
acid reagent, p-dimethyl-amir.0-benzaldehydc sulfuric acid reagent, pataszium
permanagate Frijhde's reagent, vanillin reagent, concentric hydrochloric acid,
cerium euIfate, cilicotungstic acid reagent, and p o t a x i u m perrrinngate M a r -
quis' reagent.

Exper imen t a I

Samples of 1.5 to 2.0 nig of reserpine, either in ampoulc, powder or

tablet form, were used and tested. If the sampIe were in ampouIc form,
the contents were simply transferred into a 250-ml separatory f u m e 1 and
10 t o 20 ml of water was added if the voIume of the ampouIe soIution was
found t o be too small. T h e solution was then acidified with citric acid,
followed by making alkaline with ammonia. If i t werc in powder form,
20 nql of water was added t o dissolve i t in a separatory funnel, thcn
acidified with citric acid and aIkalinified with ammonia. If thc sarnFIz
wcrc in tabtet f o r m , i t was first ground into powder and an amount of the
powder reprcscnting 2.5 mg of rcscrpinc was weighcd out, triturated w i t h
wxtcr and transferred into a 2 j - m I volumetric ilas!;. Five ml of 0.3 N
citric acid was added, and the flask w a s :haken violently for a while, thcn
ditutrd with water up to the mark on the neck of t h e flask. It vias s e t
azide for 10 niizutcc. a d f i l t e d . An zliquot por:ion of 20 ml sf thz filtrate
 114 CHENG SUN

with hrnmonii. A f t e r
w a s rnc'azured into 'a sepnrxtory funnel and a1l;zlinifcd
the aqueous colclioa of the szmple had been alkalinified w i t h ammonium
hydroxide, 50 rnl of chloroform was sdded i n and the funnel wix shaken f o r
a little while. By titixtit?: 10 nl of the extract with standard acid and by
counting the nurnbe: of drops per in1 of chloroform solution, the amount
of reserpine presenting in each drop of thc chloroforrnic extract may be
detcrmined approximately. A dcfinite numbcr of drops of this extract was
collccted in a small, ivhI!e porcelain evaporating dish, and was cvaporzted t o
Jryness over a boiling wp.ter-bnth. T o the residue remained in the dish, I
or 2 drops of the reagent, which was chosen for such a t e s t , were added.
T h c color changes obccri-ed are described and fisted in T a b l e I. All reagents
chosen f o r these tests are listed and described i n T a b l e I I . , and their actiyity
toward reserpine is indicated.
In this report, the author u x d 2 sample of pure reserpine as standard.
A11 the color c h x g e s were based on the standard sampIe. F o r genera1
pharmaceutical prepzrations of reserpine as obtained from the market their
color changes with variou: reagents a r e highly cornparabIe with these results
reported hcre ,. providicd no other alkaloids a r e present.

TABLE I . T h e Color Rcactims of RescrFine with Various Reagrnts

Least , -

Reagent amount
of
-- ._ reserpine
Description of Color Change r Emper-
I
used in ature
Froducing
the color
~
change
- -

Fr iihdc's '1 drop I 9 Y Grayish blue-*green+gray-+gray- loom

reagent grayish iaint blue-+yellow-greei temp.
I 1I --grayish green
fr
reagent
i i green
Deep yellow+greenish yellow+ /I
faint yelIow
!
Iodic acid No color change Ir
I
Distinct yellow If
 COLOR REACTIONS OF RESEIWNE 115
.
L--2
drops
Grayish-green-blue --t.qrL‘m-*
~rayisti-~rass-green+grny
taint: yel!ow
-. I If .

Meclie’s 1 drop Blue+grny--*faint grass-green If

reagent
Erdmann’s If Yellow-green-blue -t f aint yellow II
reagent
Fuming nitric L drop KO color change /I
acid
Ferric chloridc ff I? If II If

Lieberrnann’ s If Yellow-gray-.dull brown-+yellow I/

reagent
K M n Q and 1 granulr Dull green+dull brown rf
conc. sulfu- Sr
ric acid 1 drol
KiLlnO.l and L drop No color c h q e due to s i m p l e If
Frohde’ s
reagent
KMMnOP and ff If If If !I fr
Marquis’
reagent
KMn04 a n d If If If If If tr
iodic acid
Sil ico- tungs t ic I/ No color change It
acid
Frahde’s If Charring occurred her
reagent (ho t: electric
stove a t
cooking
i temp.

Central region was charred, while I/

cii.cumicrentia1 region showed
deep blackish violet color.
Conc.. hydro- ‘1-2 Very faint yellow roam
chloric acid drops temp.
p--Dime t h y I- 1--2 Grayish grass green-+grass If
amino-ben- drops green-grayish brown-red-
zddehyde- grayish brown-purple red
sulfuric acid
Tvhndelin’s 1.2 Irnmed ia t e sky-blu is5 green --* red- I/
reagent drops purplc-taint gray and gremish i
Vn nillin
I

I1! Idrop
yellow
Distinct light. ,red I I f
reagent(’) i
I
-
 Name of Reagent Composition of Reagent Remarks

Friihde’s reagent Positive result

Marquis’ reagent ir I/

Conc. sulfuric acid. II If

Iodic acid negative result

Cerium s u l h te positive result
Keller’s reagent If II

Mecke’s reagent II If

Erdmann’s reagent I? I?

Fuming nitric acid negative result

Ferric chloride II I1

Lieber rn 3nn ’s I? If positive result

reage :it
KMnO., 3nd c r ~ n c , A crys’xl 02 E(,Mn04 was added to the If If
suLuriC: acid s:mpk, t!ien followed by adrop 01
c m c , k12S02, a n d the whole miss
was mixed and rubbcd with aglass
rod.
RMn04 and A crystal of 10dnOgwas added to the negative result
Frohde’s rgt, s3mple, then follxvea by a drop o t
thc named rcagcnt, and the whole
miss was mixed and rubbcd with
a glass rod,
KMn04 and T h e pracess and operation were sini- II If
hkrquis‘ rgt . 1 x to the above one.
K M n 0 4 a n d iodic Similar to above one. If II
acid
Silica-tungs t ic acid See Re’.ercnce(J) II rt
Frijhde’s rengeat Same as Friihde’s reagent I/ If
(hot)
h.I ar qui s ’ re ag en t Same ns Marquis’rcagent positive result
(hot)
CXX. hydrochI3ric h n c . 1iCI acid (sp. g r . 1.18) II If
acid
PDimethyl-amino- If II
be nz a1deh y cic
suliuric acid
h.lanclclin’s rw!:cnt If If

Vaniilin rcaKent ir II
 Most oE the reagents used in this cxperirnont were ChGsen from the book
'Laboratory Manual for the Detection of Poisons and Powerhl Drugs"(<).

Discussions

1. T h e color reaction of reserpine with Frijhde's reagent may be

considered as a characteristic one. T h e coloration is quite different from
those of marly other kiilJs of alkaloidsc~>. T h i s test is scnzitive and specific
to pure reserpinc. I-lowevcr, for tot31 alkaloid of Rauwolfia scrpcntina, the
coloration is different f r o m that of pure reeerpinc. T h e m x i t i v i t y of the
test is high, 9 y of pure reserpine may give a very distinct, positive result.
T h u s , it is useful in detecting the completeness of reserpine extraction with
organic ~ 0 1 v e n t s ( ~ ~ ~ ~ ~ )
2. KelIer's reagent g i v s a color reactiw Liniilar to that reported in
the literaturd6), but its sensitivity is much less than FrijhlJc's reagent.
Reserpine is orily sensitive to f r e h t y prepilrcd Iieller's reagciit, and t h e
amount of reserpine needed for such a test i s much greater than t h a t of the
others .
3, It is found that those reagetits cuntainirlg concentrated sulfuric acid
produce color changes to blue, grecn,pellow arid gray (or black) only, w i t h
the exception of Wlandelin's reagent a i d p-dimethyt -amino-benzaldehyde
sulfuric acid reageiit, in which cases the color change is to rcd and violet.
Nitric acid produces no coloration. Hydrochloric acid devsiopes only faint
yellow color, b u t when vaniLlin is added, distinct red color is produced,
This is due to the fact that reserpine is an indole derivative.

Acknowledgement

The 'author is much indebted to prof. T . Y . Chia, Director of Chemical

Drugs Control Laboxttory, f o r his valuable help a i d advice.

Literature Cited
 118 CHENG SUN
(1955) ; 617---18(1954) ; (C. A . , 48, 14122r (1954)
(4) Cheng, S . S . , J . A . P h u r m . Ass., 43, 768 (1954)
(5) U.S.P. 14ih. ed. , p . 368 and p. 419
(6) Lee. C , P . , ''Chemi~try" (Printed in Chixese), No. 4,.351 , Note of table I,
(1955)
(7) Banes, D . , J . A . Pharin. Axj., 44, 40s-411 (1355)
(8) Autenrieth, W . , and Warren, L.E., "Labotratory Manual for the Oetection
of Poisons and l?ower:ul Drugs" through the American Translation Edition,
6 th. cd., P. Blakiston's Son Q Co. , Inc. , Philadelphia, 1928 pp. 641-43
(9) Autenrieth, W. , and Warren, L . E . , i b i d . , pp. 237-9
(lo) Banes, D . , J . A . Pharm. A S S . , 44, 4i38-411 (1955)
. ~. H, . , Pazdera, H, J., M i s s a n , S.R., G i a c c i o , L . L . ,
(11) M C M ~ I I -W and Cren-

fell, T . C . , ibid., 44, 446.453 (1955)

Received Mm. 5 ? 1957