Rutin

RUTIN Taha I . Khalifa, Farid J . Muhtadi, and Mahrnoud M.A. Hassan 1.

2.

3. 4. 5. 6. 7.

8.

Description 1.1 Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Taste, and Odour Physical Properties 2.1 Crystal Properties 2.2 Melting Point 2.3 Solubility 2.4 Optical Rotation 2.5 Spectral Properties Stability and Incompatibility Isolation 4.1 Industrialition Synthesisof Rutin Biosynthesis of Rutin Biological Properties 7.1 Phannacological Activity 7.2 MicrobiologicalActivity 7.3 Therapeutic Uses 7.4 Metabolism of Rutin Methods of Analysis 8.1 IdentificationTests 8.2 Quantitative Determination 8.3 UV Spectrophotometry 8.4 PMR Spectrometry 8.5 Fluorimetry 8.6 Polarography 8.7 Densitometry 8.8 Gravimetry 8.9 Other Analytical Uses 8.10 Chromatography References

ANALYTlCAL PROFILES OF DRUG SUBSTANCES VOLUME I2

623

624 624 625 626 626 626 626 626 626 626 621 627 639 639 639 642 65 1 65 1 65 1 654 656 656 658 658

660

664 664 666 666 667 668 668 668 675

Copyright by the American Pharmaceutical Associalion. ISBN 0-12-260812-7

TAHA I. KHALIFA ETAL.

624

1.

Description 1.1. Nomenclature

1.1.1.

Chemical Names 3-[[6-0-(6-Deoxy-L.mannopyranosyl) -~-D-glucopyranosyl]oxy]-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-Ibenz op yran-4 -one.

5

/

, 4 , 5-7-pentahydroxy, Flavone, 3, 3(6(0(6-deoxy-L-mannopyranosyl)) $-D-glucopyranoside. 2-(3, 4-Dihydroxyphenyl)-3, 5 , 7 trihydroxy 4-ox0-4H-chromen-3-yl rutinoside. /

/

3, 3 , 4 , 5-7-pentahydroxyflavone3-rutinoside. Quercet in-3-rut i n o s i d e .

1.1.2.

Generic Names (1-8) Rutin; R u t i n o s i d e ; Rutoside; Vitamin P, Melin; Phytomelin, E l d r i n ; E l i x a t h i n ; Sophorin; G l o b u l a r i c i t r i n ; P a l i u r o s i d e ; O s y r i t r i n ; O s y r i t i n ; M y r t i c o l o r i n ; Violaq u e r c i t r i n ; B i r u t a n ; Rutabion; Rutozyd; Tanrutin.

1.1.3.

Pharmacopoeias Rutin i s o f f i c i a l i n t h e f o l l o w i n g pharmacopoeias (8) : Hungarian; Japanese; P o l i s h ; Roumanian; Russian and S w i s s .

1.1.4.

Pharmacopoeia1 P r e p a r a t i o n s Th& f o l l o w i n g p r e p a r a t i o n s are o f f i c i a l i n t h e r e s p e c t i v e pharmacopoeias ( 9 ) : (a)

R u t i n I n j e c t i o n (Japanese Pharmacop o e i a ) , A s t e r i l e aqueous s o l u t i o n of r u t i n . No s t r e n g t h s p e c i f i e d . I t should be p r o t e c t e d from l i g h t .

625

RUTIN

(b)

1.1.5.

1.2.

T a b u l e t t a e R u t i n i ( R u s s i a n and German pharmacopoeias), Each t a b l e t c o n t a i n s 20 mg (Russian) o r 50 mg (German) of r u t i n .

P r o p r i e t a r y N a m e s (8, 10).

B i r u t a n (E. Merck, Germany) ; R u t i n o n (Rheinpharma, Germany) ; R u t a c i d (CID, Egypt). I n j t e n s ( D a g r a , H o l l a n d , Rutaminal Schenely, USA). Formulae 1.2.1.

Empirical '27

1.2.2.

H30 '16

Structural

OH

R u t i n o s e is: 6-0-(6-Deoxy-'DC-L-mannopyranosy1)-D-glucose w i t h e m p i r i c a l f o r m u l a C12H22010 mol. wt.326.30 and s t r u c t u r a l f o r m u l a as f o l l o w s : H

H

OH

TAHA I. KHALIFA ETAL.

626

T h i s s t r u c t u r e w a s proposed by Zemplen and Gerecs (11) and confirmed by t h e t o t a l s y n t h e s i s of r u t i n achieved by Shakhova e t a 1 ( 1 2 ) .

1.3.

1.2.3.

Chemical A b s t r a c t R e g i s t r y Number[CAS No] ( 1 ) [153-18-41

1.2.4.

Wiswesser Line Notation

T 66 BO EVJ CR-CQ DQ & DO A GQ IQ Molecular Weight

610.51

1.4.

Elemental Composition C , 53.11%; H,4.95%; 0,41.93%

1.5.

Appearance, Color, T a s t e , and Odour P a l e yellow n e e d l e s from water which g r a d u a l l y darkens on exposure t o l i g h t , tasteless and odourless.

2.

Physical Properties 2.1.

Crystal Properties

2.1.1.

Water of C r y s t a l i z a t i o n The c r y s t a l s from water c o n t a i n 3 H20, and become anhydrous a t l l O O C and 10 mm Hg

.

2.2.

Melting P o i n t Anhydrous r u t i n browns a t 125OC, m e l t s a t 188.7'C, becomes p l a s t i c a t 195-197OC, and decomposes w i t h e f f e r v e s c e n c e a t 214-215OC ( 1 )

2.3.

Solubility One gram r u t i n d i s s o l v e s i n about 8 l i t e r s water, about 200 m l b o i l i n g water, and 7 m l b o i l i n g methanol. It i s s o l u b l e i n p y r i d i n e , formamide and a l k a l i n e s o l u t i o n s ; s l i g h t l y s o l u b l e i n a l cohol, a c e t s n e , e t h y l acetate; p r a c t i c a l l y insol u b l e i n chloroform, carbon b i s u l f i d e , e t h e r , benzene and petroleum s o l v e n t s ( 2 , 3 ) .

RUTIN

627

2.4.

Optical Rotation

[ d l i 3+

13.82'

(ethanol);

[dli3 -

d i n e ) ; Deca-methyl d e r i v a t i v e [=I1' D nol. 2.5.

39.43'

-

33'

(pyri(etha-

Spectral Properties 2.5.1.

U l t r a v i o l e t Spectrum The UV spectrum of a u t h e n t i c r u t i n i n 95% methanol was scanned u s i n g Pye Unicam SP 800; from 200-500 nm. 2-4 d r o p s of 2 M NaOH s o l u t i o n were added t o t h e c e l l s o l u t i o n and t h e spectrum w a s measured i n presence of a l k a l i . Other s p e c t r a l s h i f t s were recorded by scanning d i f f e r e n t 95% e t h a n o l s o l u t i o n s of r u t i n t o which w a s added s u c c e s s i v e l y powdered sodium a c e t a t e and b o r i c a c i d and by adding two d r o p s 5% a l c o h o l i c aluminium c h l o r i d e s o l u t i o n and t h e n dil.HCl(13-13. The W and V i s i b l e S p e c t r a l maxima and s h i f t s f o r r u t i n are shown i n Table I and F i g . 1.

Table I! W and V i s i b l e S p e c t r a l Maxima and S h i f t s f o r Rutin.

bH

0

628

Ethanol S o l u t i o n

Alone

* h)

S p e c t r a l Maxima (nm)

Spectral effect

Structural diagnosis

3

Band I

Band I1

Rand 111

259

266,S, 299,s

363

1 2 nm hypsochromic s h i f t (band 111)

-

OH substituted

/

- OH f r e e

Plus 2 drops 2 M NaOH s o l u t i o n

272

327

415

52 nm bathochromic

P l u s 2 d r o p s 5% A 1 C1 s o l u t i o n 3

275

303,s

433

7 0 nm bathochromic s h i f t (band 111)

5 - OH f r e e

P l u s powdered NaOAc

271

325

393

1 2 nm s h i f t (band I)

7 - OH f r e e

P l u s NaOAc and

262

298

387

2 0 nm bathochromic s h i f t (band 111)

H3B03

s h i f t (band 111)

S = Shouder

These f i n d i n g s are i n agreement w i t h t h e r e p o r t e d d a t a (14, 15 and 16).

4

/

3

/

,4

, di

OH f r e e

TAHA I. KHALIFA ETAL.

630

2.5.2.

I n t r a r e d Spectrum The I R spectrum of r u t i n as K B r been determined on a Perkin-Elmer I n f r a r e d Spectrophotometer (Fig. s t r u c t u r a l assignments have been t e d f o r t h e c h a r a c t e r i s t i c bands t e d i n Table 2.

Table 2.

d i s c has 580 B 2 ) . The correlaas lis-

I n f r a r e d band f r e q u e n c i e s of Rutin and i t s c o r r e l a t i o n t o s t r u c t u r a l assignment.

Frequency (Cm'l)

Assignment

3330

OH (bonded)

2920

CH s t r e t c h

1660 1620

c=o c=c

1600

Aromatic s t r u c t u r e

1510

C = C aromatic

1460 1360

c - 0 - c

1295

c-0-c c-0-c c-0-c

1200 1060 8 10

S u b s t i t u t e d aromatics

These f i n d i n g s are i n agreement w i t h r e p o r t e d d a t a (16). Other f i n g e r p r i n t bands c h a r a c t e r i s t i c t o r u t i n

are: 970, 880, 730 and 700. 2.5.3.

Nuclear Magnetic Resonance S p e c t r a 2.5.3.1.

Proton

Spectra

The p r o t o n NMR S p e c t r a of f l a von i d s have been e x t e n s i v e1y s t u d i e d (13). A t y p i c a l PMR s p e c t r a of r u t i n are shown i n Fig. 3 & 4. The sample w a s d i s s o l v e d i n DMSO-D6 and TFA r e s p e c t i v e l y , and run on a Varian T 60A, 60-MHz NMR Spectrometer. All chemical s h i f t s

632

TAHA I. KHALIFA ETAL.

fig. 3 NMR Spectrum o f R u t h in DMSO-D,

I

I

I

I

.

I

80

I

TO

I I

I

I

6.0

1

I

54

I 1

300

400

500

200

I

4.0

PPM (6)

I

I

-

. I

3.0

I

1

I +

100

I

I

2.0

fig. 4 NMR Spectrum of Rutr'n in TFA.

I

in

1

01

RUTIN

633

reported a r e i n reference t o t e t r a m e t h y l s i l a n e (TMS) a t 0 ppm. The PMR s p e c t r a l a s s i g n ments of r u t i n are given i n T a b l e 3. Table 3.

Chemical S h i f t s of R u t i n i n DMSO-D6 and TFA

Group

Position

Chemical S h i f t s (6) DMSO-D5

TFA -

103(d)

1.45 (bS)

Rhamnoglucosyl

3.20;3.40 (bS)

3.90;4.20 (bS)

H-1 Rhamnosyl

4.40 ( S )

5.00 (S)

5.30 (bS)

5.00 (S)

CHM4

Rhamnosyl Me

H- 6

Aromatic

6.17 (SL)

6.72 ( S )

H-8

Aromatic

6.36 (SL)

6.83 (SC)

Aromatic

6.80 (d)

7.10 (d)

Aromatic

7.50 (bS)

7.85 (d)

H-2

Aromatic

7.50 (S)

8.00 (S)

(S) = S i n g l e t ;

(d) = d o u b l e t ;

/

H- 5 /

H- 6 /

(bS) = broad s i n g l e t ;

(SL) = S i n g l e t showing long range c o u p l i n g .

2.5.3.2.

Carbon-13 Magnetic Resonance Spectroscopy Proton-noise and o f f - r e s o n a n c e decoupled 13C-NMR s p e c t r a were measured on a V a r i a n FT-80 A80 MHz F o u r i e r t r a n s f o r m NMR Spectrometer o p e r a t i n g a t 23.5 m z . Samples were p r e p a r e d i n 1 0 mm 0.d. t u b e s i n approxtmately 10% s o l u t i o n i n D?'fSO-D6 w i t h tetramethylsilane a s i n t e r n a l r e f e r e n c e . S p e c t r a were r e c o r ded w i t h 8 K d a t a p o i n t s a t a probe t e m p e r a t u r e of 23OC. For an average s p e c t r a l width of

TAHA I. KHALIFA ETAL.

634

5000 Hz., a 1 0 ps p u l s e w i d t h corresponding t o a t i l t angle of 30° w a s employed w i t h 2 s i n t e r v a l between p u l s e s . 13CNMR c o m p l e t e l y decoupled and o f f - r e s o n a n c e of r u t i n a r e p r e s e n t e d i n F i g s . 5 and 6 and t h e carbon chemical s h i f t s a s s i g n e d on t h e b a s i s of t h e a d d i t i v e l y p r i n c i p a l s and t h e o f f r e s o nance s p l i t t i n g p a t t e r n are shown i n T a b l e 4 . R e c e n t l y 1 3 C NMR d a t a of f l a v o n o i d s w e r e r e p o r t e d ( 17-24) Chang ( 2 5 ) determined 1% NMR of t h e aglycone of r u t i n amongst o t h e r f a l v o n o i d s by t h e g a t e d decoupling t e c h n i q u e . The s p e c t r a l i n t e r p r e t a t ion s we re based on t h e i n f o r m a t i o n from 13C-lH c o u p l i n g p a t t e r n s .

.

OH

Rutinose

635 4600 H z

2doo

Fig. 5

13

C

NM R

OP Rutin, Noise Decoupled S p c t f U m .

4 1 i

I

3h

fig.6

N

C NMR OF Rutin, off Resonance Spectrum.

n n n n n n n

z z z w- z z z b

e m

n

v

n

w

n

N

U)

\* \m

rl rl

0

m e

rl

. . . . . . .

m r l m o N r l e m b m * m o b m r l r l U ) m e o

m N N r ( r ( O 0

cd

e

r l r l r l r l r l r l r l

W\U)\d\N\Ul

3 N O 3 A 1 3 V

n w

m m c n m

w

. . . . .

r l U ) U l m * U ) m U ) e e

cd

03

r l r l r l r l r l

Ul

3 N 0 3 1 1 3 V

v)

a,

n

1

0 U M

rl W

u

I

U)

8 V

3ns

8 V 3 f l S

a

*d cd

I-r

U

G

aJ U

0

m

h

.d U

.d

a

a, 0 Ll (d

: M

0

w

G

0 P Ll cd U .K

RUTIN

637

2.5.4.

Mass Spectrum The mass spectrum of r u t i n o b t a i n e d by d e s o r p t i o n chemical i o n i z a t i o n (DCI) u s i n g ammonia as a r e a c t a n t g a s shows a molecular ion a t m / e 611 amu. The prominent fragments and t h e i r r e l a t i v e i n t e n s i t i e s are shown i n Table 5 . OH

A

Table 5 .

m/e 303 611 628 164 180 3 04 308 320 32 6 449 465

B

C

Mass Spectrum of R u t i n Relative i n t e n s i t y (2’) 100.00

28.88 2.22 57.77 66.66 44.44 22.22 24.44 42.22 11.11 33.33

Fragment

A + 2 H M+

M+

-

+ NH3

C A + 3 H

-

!

J

164

197 212

180

431

-

-

,430 ,4[0 449

,

290

I .

-

;I0

1

.

1

530

550

570

463472482

Fig. 7

590 611

Mass Spectrum

of

Rutin

628

.

RUTIN

3.

639

S t a b i l i t y and I n c o m p a t i b i l i t y Rutin is more s t a b l e than q u e r c e t i n i n t h e presence of low c o n c e n t r a t i o n s of a l k a l i . I n a c i d s o l u t i o n s , r u t i n is hydrolysed t o q u e r c e t i n which is r e l a t i v e l y s t a b l e under t h e s e c o n d i t i o n s ( 2 6 ) . It should be p r o t e c t e d from l i g h t . Rutin i s incompatible w i t h a c i d s and s a l t s of heavy metals ( 8 ) .

4.

Isolation R u t h i s undoubtedly t h e most widespread of a l l quercet i n g l y c o s i d e s and probably o c c u r s i n up t o 25% of any given l o c a l f l o r a ( 1 4 ) . It h a s been found t o occur i n many p l a n t s , e s p e c i a l l y t h e buckwheat p l a n t (Fagopyrum esculentum Moench., Polygonaceae) up t o about 3% (1) l e a v e s of tobacco ( N i c o t i a n a tabacum L . , Solanaceae) (1) Ruta g c a v e o l e n s Rutaceae, flower buds of Sophora japonica ; teguminosae up t o 18% (27) t h e s t a l k s of tomato,Solanum persicum s o l a n a c e a e , l e a v e s of Eucalyptus spp., f l o w e r s of c e r t a i n Mangolia ( 28 ) and many o t h e r p l a n t s . A g e n e r a l procedure f o r t h e i s o l a t i o n of r u t i n comprises drying of p l a n t material, followed by e x t r a c t i o n wtth a l c o h o l , t h e s o l u t i o n c o n c e n t r a t e d and t h e g l y c o s i d e is l e f t f o r c r y s t a l i z a t i o n (29). A d e t a i l e d procedure f o r i s o l a t i n g r u t i n from small quant i t i e s of p l a n t m a t e r i a l i s o u t l i n e d i n Fig. 8 ( 30 ) .

-

4.1.

Industrialization Due t o t h e h i g h p e r c e n t a g e s of r u t i n c o n t a i n e d i n t h e f a m i l y of Eucalyptus known as Myrtaceal [4?4% ( 2 7 ) ] ; thesc. s p e c i e s a r e processed now i n A u s t r a l i a f o r t h e commercial p r o d u c t i o n of r u t i n (27 6 31). Although many s o l v e n t s f o r t h e e x t r a c t i o n s t a g e have been i n v e s t i g a t e d i n c l u d i n g 95% e t h a n o l , and hot d i l u t e i s o p r o p y l a l c o h o l ( 3 2 ) , a s i n g l e - s t a g e b a t c h e x t r a c t i o n w i t h b o i l i n g water is recommended. (Fig. 9) r e p r e s e n t s a flowsheet diagram f o r t h e production of 50.000 l b l r u t i n p.a from Eucalyptus l e a v e s a c c o r d i n g t o Humphrey's method (31).

TAHA I. KHALIFA ETAL..

Ground p l a n t mate f i a l

I

I

Discard

4

Extract with boiling 80% Ethanol (2x200 m l )

Alcoholic Extract

Ether Extract

I

Aqueous Solution

S o l i d mate-

r,+ G , 1

I

Cry st a l l i n e Solid

Wash w i t h water,followed by e t h e r

iscard

Crude Rut i n P u r i f i c a t i o n on Magnesium s i l i c a t e column Pure Rut i n F i g~. . 8.

Procedure O u t l i n e f o r I s o l a t i o n of Rutin.

RUTIN

641

I

,

H a r v e s t e d Leaves of E u c a l y p t u s

1

Moi;;ye.

f

Comminution

aS3<44 mesh I

U s i n g hammer Ffill

Extract ion usinE b o i l i n g water

Retention t i m e 1 hour

Filtration >0.7.5% R u t i n

C o n s t r u c t i o n Material (wood o r ceramic)

Crystalization temp. 4n0c

4 hours I R u t i n r e c o v e r y 95-97%

I

-

F i l t e r cake n r v i n p: Crush i n g

I F i g . 9:

Packing

Rutin p u r i t y

P r o t e c t from l i g h t ,

Flow s h e e t f o r t h e commercial p r o d u c t i o n of R u t i n .

642

5.

TAHA I. KHALIFA ETAL..

Synthesis of Rutin The synthesis of rutin can be achieved according to the following three schemes. These schemes differ in the synthesis of ouercetin (the aglycone moiety of rutin). Scheme 1: Kostanecki -et al. 1904 (33 ) . Based upon the Claisen reaction between 2-hydroxy4, 6-dimethoxyacetophenone [l] and 3, 4-dimethoxybenzaldehyde [2] to give the intermediate [3] which upon treatment with HC1, cyclization occurs to give 5, 7, 3 , 4 ' -tetramethoxyflavonone [4]. Oximination affords [5] which upon treatment with F2SO4 enolisation occurs to give 5, 7, 1'34', -tetramethoxyflavonol [6]. Demethylation with HI affords quercetin [7].

,

Scheme 2: Robinson et al. 1926 (34 ) . Condensation ofw-methoxypholoroacetophenone [I] with veratric acid anhydride [2] in the presence of the potassium salt of veratric acid to give the diarylester [3]. On hydrolysis with alcoholic KOH affords 5, 7-dihydroxy-3,/3 ,4' -trimethoxyflavone [ 41 , which on demethylation with HI gives quercetin [5]. Scheme 3:

Shakhova et al. 1962 (35), complete synthesis of rutin.

W-methoxyphloroacetophenone [2] was condensed with 0-benzylvanillinic acid, anhydride [ 13 in triethylamine to give 5 , 7-dihydroxy-4 -benzyloxy-3, /3 -dimethoxyflavone [3]. On treatment with AcOH-HC1 mixture gave 5, 7,4' -trihydroxy-3,'3 -dimethoxyflavone [4]. Demethylation of the latter with HI yielded (about 802) quercetin [5]. Ouercetin potassium salt [6] was produced upon treating [5] with AcOK in ethanol. Levoglucosan [7] was acetylated with Ac20 in the presence of AcONa to give 2, 3, 4-triacetyllevoglucosan [8] which with TIC14 gave 1-chloro-2, 3, 4-triacetyl Dglucose [9]. L-rhamnose tetraacetate [lo] treated with TiBr4 in CHC13 gave 1-bromo-2, 3, I-triacetyl-L-rhamnose [ll]. [lo] + [11] heated with Hg (OAC)~in C6H6 gave (53x) CC acetochloro-f3-l-L-rhamnosido-6-D-glucose [12]. [12] was treated with AgOAc and acetylated with Ac20 to prodilce (68.703 B-heptaacetyl-f3-1-L-rhamnosido-6-D-glucose [13]. This with 33% HBr in AcOH gave (61%) d acetobromo-~-l-L-rhamnosido-6-D-glucose [14]. [14] and quercetin potassium salt [6] were dissolved in NH40H which was evaporated and treated with methanol and

-

RUTIN

Scheme 1 : Synthesis of Quercetin By Kostanecki e t al.

643

TAHAI. KHALIFA E T A .

644

H3C0

OCH3

HO

0

I

HI

Querc etin

645

RUTIN

Scheme 2 :

Synthesis of Quercetin By Robinson bt al. 0 0 II

I1

-c

c-0

1

1 +

COCH20CH3 OH

[11

ArCOO

0ch3

0

ArCOO

[31

KOH

EtOH

TAHAI. KHALIFA ETAL..

646

HO

OH

0

[41

HO OH

OH

0

[51

647

RUTIN

Scheme 3 : Synthesis of Rutin

-

OH

co

O t

OH O

O

H

COCH20CH3

FOCH2 OCH3

HO

OH

0

HO

I

[31

TAHA I. KHALIFA ETAL.

648

OH

[51

RUTIN

649

CH2 -0

OH

H

-4

[71 Tic14 CH20H

0

0

+

TiBr4

0

-

650

TAHA I. KHALIFA ETAL.

Ho(o$l

OH

OH

AgOAc Ace0

I

OH

0cch3 II 0

[i21

HO

0

651

RUTIN

p u r i f i e d o v e r a chromatographic column packed w i t h polycaprolactum r e s i n t o g i v e r u t i n [151. 6.

B i o s y n t h e s i s of R u t i n P o s t u l a t i o n of t h e b i o s y n t h e t i c pathway of f l a v o n o i d s s t a r t e d i n 1936 w i t h t h e s u g g e s t i o n o f Rohinson ( 3 6 ) t h a t t h e C15 s k e l e t o n of f l a v o n o i d s t o b e composed of two p a r t s c 6 and Cg as f o l l o w s :

'6

c-c I

cq ( cg+c3 )

B i r c h e t a l . (37 ) proposed t h a t r i n g A of t h e f l a v o noid s t r u c t u r e i s produced by t h e a c e t a t e pathway i . e . 3 a c e t a t e u n i t s condensed h e a d - t o - t a i l . Grisebach ( 38) f e d l 4 C H 3 COOH and CH314COOH and proved t h a t r i n g A i s b i o s y n t h e s i z e d from a c e t a t e . Neish and o t h e r s proved t h a t r i n g B i s formed from d i f f e r e n t r o u t e ( 3 9 ) i . e . cinnamic acid.The b i o s y n t h e s i s of r u t i n i s p r e s e n t e d i n Scheme 4 .

7.

Biological Properties

7.1.

Pharmacological A c t i v i t y R u t i n a s w e l l as i t s aglycone, q u e r c e t i n , have a d i r e c t c o n s t r u c t o r a c t i o n on t h e c a p i l l a r y bed and d e c r e a s e t h e p e r m e a b i l i t y and f r a g i l i t y of t h e v e s s e l s (40). It h a s been s u g g e s t e d t h a t t h e s e s u b s t a n c e s could b e c l a s s e d a s v i t a m i n s , p a r t i c u l a r l y of t h e "Vitamin P" t y p e . R u t i n h a s been found t o relax t h e i s o l a t e d i n t e s t i n e (41). Administered i n t r a v e n o u s l y t o t h e dog and r a b b i t , i n d o s e s of 5, 20 and 100 mg/kg r e s p e c t i v e l y , r u t i n i n v a r i a b l y produces a lowering of t h e blood pressure (42). Experimentally r u t i n p r o t e c t s a g a i n s t c a p i l l a r y i n j u r y (43-44) and d e c r e a s e s t h e erythematous r e s p o n s e t o l o c a l chloroform i r r i t a t i o n (43 & 4 4 ) . T h i s action may be due t o t h e a n t i a c i d a n t a c t i o n of r u t i n on adrenal i n e , thus r e s u l t i n g i n a s l i g h t increase i n its l e v e l and so i n c r e a s i n g t h e t o x i c i t y o f t h e p r e c a p i l l a r y s p h i n c t e r s and d e c r e a s i n g t h e t o t a l number of t r u e c a p i l l a r i e s f i t t e d w i t h f l o w i n g blood (43 & 4 4 ) . An i n t e r e s t i n g e x t e n s i o n

TAHA I. KHALIFA ETAL.

652

Scheme

4

: Biosynthesis of Rutin

Shikimic a c i d k -

Prephenic a c b id -.

653

RUTIN

OH

HO

OH

OH

0

Quercetin UDP-D-glucose

OH

Quercetin 3-glucoside

UDP-L-rhamnose

HO

Rutin

OH

OH

OH

TAHAI.KHALIFA ETAL.

654

of t h i s h a s been t h e r e s u l t s o b t a i n e d from t h e a d m i n i s t r a t i o n of r u t i n t o r a b b i t s s u f f e r i n g from s t a n d a r d i z e d experimental p r o s t h i h e . A dose of 50 t o 100 mp;/kg p e r day by stomach t u b e produces a marked acminution i n t h e loss of t i s s u e gangar e n e following p r o s t b i t e of r a b b i t f e e t , b u t is i n e f f e c t i v e i n p r e v e n t i n g loss of t i s s u e f o l l o wing p r o s t b i t e of r a b b i t ears (45) Quercetin is less e f f e c t i v e i n t h i s regard t h a n r u t i n ( 4 6 ) Rutin p r o t e c t s a g a i n s t h i s t a m i n e shock i n an ind i r e c t way h u t i s n o t a t r u e a n t i h i s t a m i n i c (47).

7.1.1.

LD 0 LD50 determined i n mice by i n t r a v e n o u s

-5

a d m i n i s t r a t i o n of propylene g l y c o l s o l u t i o n is 950 mg/kp hody weight (48).

7.2.

Microbiologlcal Activity Naghski, Copley and Couch(49-50) r e p o r t e d i n 1947 t h a t q u e r c e t i n , t h e aglycone of r u t i n , e x c r e t e d some i n h i b i t o r y e f f e c t on t h e growth of S t a o h y l o coccus aureus and o t h e r organisms, w h i l e r u t i n and q u e r c e t r i n w a a inacrive t5l), A recent a n t i m i c r o b i a l s c r e e n i n n of r u t i n and a u e r c e t i n was Derformed bv c u v - d a t e agar d i f f u s i o n method a g a i n a t Gram-positive, Gram-negative b a c t e r i a and y e a s t - l i k e fungus (52). The results of a c t i v i t v are p r e s c r i b e d i n Table 6 . The minimum i n h i b i t o r v c o n c e n t r a t i o n of r u t i n a g a i n s t Pseudomonas a e r u n i n o s a and P r o t e u s vulgaris were 10 mg and 10 mg/ml r e s p e c t i v e l y . Querc e t i n minimum i n h i b i t o r y c o n c e n t r a t i o n a g a i n s t

--

tively.

0

0

hl d

r-l

cr)

Kl

c

QI

0

l-i

0

0

0

c

*rl

U

PI

u

&

PI

7 0

656

TAHA I. KHALIFA ETAL..

7.3.

T h e r a p e u t i c Uses C o n s i d e r a b l e i n t e r e s t h a s been evinced i n t h e p o s s i b l e c l i n i c a l a p p l i c a t i o n of r u t i n t o t h e medical t r e a t m e n t of t h e s i c k (53 - 5 7 ) . R u t i n w a s f o r m e r l y used i n t r e a t m e n t of d i s e a s e s t a t e s c h a r a c t e r i s e d by c a p i l l a r y f r a g i l i t y , b u t evidence of i t s v a l u e i s i n c o n c l u s i v e . It h a s been claimed t o b e e s p e c i a l l y of v a l u e i n t r e a t ment of r e t i n a l haemorrhage. Though t h e r e i s no evidence t h a t c a p i l l a r y s t r e n g t h i s s p e c i f i c a l l y a s s o c i a t e d w i t h v i t a m i n C , some workers have claimed b e t t e r r e s u l t s from t h e u s e of r u t i n and a s c o r b i c a c i d s t h a n from r u t i n a l o n e ( 8 ) . R u t i n from tobacco l e a v e s w a s found e f f e c t i v e i n t r e a t i n g p a t i e n t s w i t h h y p e r t e n s i o n complicated by i n c r e a s e d c a p i l l a r y f r a g i l i t y ( 2 7 T 5 8 ) . R u t i n can i n h i b i t t h e a c t i o n of h y a l u r o n i d a s e , p a r t i c u l a r l y when combined w i t h a s c o r b i c a c i d . T h i s l e d t o t h e t e s t i n g of r u t i n w i t h a s c o r b i c a c i d as an o r a l c o n t r a c e p t i v e b u t i t s e f f i c a c y i n t h i s r e s p e c t h a s n o t y e t been confirmed. R u t i n h a s been used w i t h s u c c e s s i n t r e a t i n g some t y p e s of h e r e d i t a r y haemorrhagic d i s o r d e r s , s u c h as haemophilia, and a l s o b l e e d i n g gums, m i g r a i n e headaches, toxaemia i n pregnancy, e t c . ( 27 I n t h e US, r u t i n i s o f t e n i n c o r p o r a t e d i n v i t a m i n p r e p a r a t i o n s because of i t s e f f e c t i v e "vitamin P" f u n c t i o n ( 27 ) . Reports t h a t r u t i n a s w e l l as o t h e r "vitamin P" l i k e flavonoids decreased m o r t a l i t y o r hastened t h e r e c o v e r y of Roentgen-ray i r r i d i a t e d a n i m a l s were c i t e d (59,60). R u t i n and g e n e r a l l y b i f l a v o n o i d s s t i m u l a t e t h e p r o d u c t i o n of blood p l a t e l e t s , which a r e import a n t i n c o a g u l a t i o n , and a r e recommended i n t r e a t m e n t of thrombopenia ( 61

>.

>.

7.3.1.

T h e r a p e u t i c Dose (8) 50

7. 4 .

-

300 mg d a i l y .

Metabolism of R u t i n R u t i n and o t h e r f l a v o n o i d s are r e p o r t e d (62-67) t o be r a p i d l y absorbed f o l l o w i n g o r a l a d m i n i s t r a t i o n and c o n v e r t e d i n t o a v a r i e t y of hydroxy a r o m a t i c a c i d s which are r a p i d l y e l i m i n a t e d i n t h e u r i n e . The metabolism of r u t i n i s p r e s e n t e d i n Scheme 5.

657

RUTIN

Scheme 5 : Metabolism of Rutin

m-hydroxy phenyl acetic acid

3-rnethoxy, 4-hydroxyphenyl a c e t i c a c i d

658

8.

TAHA I. KHALIFA ET AL.

Methods of A n a l y s i s 8.1.

I d e n t i f i c a t i o n Tests

8.1.1.

Spot Appearance Daylight : grenish yellow : deep p u r p l e U V / N H ~ : yellow

w 8.1.2.

Chemical Tests a. b. c.

8.1.3.

D i l u t e s o l u t i o n of r u t i n g i v e s g r e e n c o l o u r w i t h f e r r i c c h l o r i d e T.S. ( 1 ) . R u t i n i s coloured brown by t o b a c c o enzyme under e x p e r i m e n t a l c o n d i t i o n s ( 2 ) . On a p p l i c a t i o n of t h e modified indophen o 1 method f o r t h e d e t e c t i o n of phenols ( 68 ) , r u t i n (> 10 pg) changes f i r s t t o b l u e then t o green. The c o l o u r f a d e s on t h e a d d i t i o n of 2 d r o p s 0.02% aqueous s o l u t i o n of N a C l O ( 6 9 ) . Among r e l a t e d compounds, h e s p e r i d i n ( > l o p g ) , h e s p e r t i n ( > 2 u g ) , and a c a c e t i n (> 5 pg) g i v e a similar c o l o r a t i o n which becomes more i n t e n s e on a d d i t i o n of NaOCI.

M i c r o c r y s t a l Tests R u t i n as w e l l a s i t s a g l y c o n e s q u e r c e t i n (0.2% m e t h a n o l i c s o l u t i o n ) gave c h a r a c t e r i s t i c golden c r y s t a l s ( F i g . l o ) , w i t h 2 , 4-dinitrophenylhydrazine H C 1 r e a g e n t ( l g i n 30 m l methanol 2 m l H2S04) ( 7 0 ) . T h i s t e s t could b e u t i l i z e d f o r t h e r a p i d d i f f e r e n t i a t i o n of r u t i n and q u e r c e t i n .

+

Fly, 10

Micfocf’sfals

of

Rutin

A 1 and Quercetin

(

0 ) with 2,4 Dinitrophenylhydraline

TAHA I. KHALIFA E T A .

8.2.

Q u a n t i t a t i v e Determination 8.2.1.

Colorimetry Use i s made of t h e c o l o u r e d d e r i v a t i v e s having h i g h molar a b s o r p t i v i t y i n UV and v i s i b l e r e g i o n , formed e i t h e r by c h e l a t i o n w i t h metals o r by r e a c t i o n w i t h s u b s t i t u t i o n reagents. 8.2.1.1. Che l a t ion

i.

With ALC13

A c c u r a t e l y weighed 5 g p l a n t sample& of t h e material were transferred into extraction t h i m b l e s and e x t r a c t e d w i t h abs o l u t e alcohol f o r 8 hours i n a Soxhlet a p p a r a t u s . The a l c o h o l e x t r a c t i o n w a s allowed t o c o o l to room t e m p e r a t u r e and made up t o 250 m l volume u s i n g a b s o l u t e a l cohol. A 25 m l p o r t i o n of t h i s s o l u t i o n w a s made up t o 100 m l volume w i t h isoamyl a l c o h o l and thoroughly mixed; a 20 m l a l i quot w a s t h e n t r a n s f e r r e d t o a s e p a r a t i n g f u n n e l and shaken w i t h f i v e s u c c e s s i v e p o r t i o n s of 25 m l of 0 . 1 M AlCl3 s o l u t i o n , a f t e r each s h a k i n g t h e s e t t l e d aqueous l a y e r b e i n g r u n o f f i n t o 1000 m l v o l u m e t r i c f l a s k and mixed t h o r o u g h l y , p l a c e d i n 1 cm c e l l and t h e a b s o r p t i o n a t 416nm n o t e d . The p e r c e n t a g e r u t i n w a s t h e n c a l c u l a t e d from a s t a n d a r d curve p r e p a r e d w i t h p u r e r u t i n (71). A s t h i s method does n o t measure t h e rutin-AlC13 complex alone but everything i n the s o l u t i o n d e r i v e d i n t h i s way which a b s o r b s a t 416nm, t h e p r e sence of o t h e r s u b s t a n c e s must be checked by chromatography and an examination of t h e a b s o r p t i o n curve i n t h e r a n g e 35Onm-5OOnm. Other flavonoid-AlC13 complexes u s u a l l y have a b s o r p t i o n m a x i m a

661

RUTIN

which d i f f e r from t h e r u t i n A l C 1 3 complex. An a l t e r n a t i v e p r o c e d u r e ( 72 ) f o r t h e d e t e r m i n a t i o n of r u t i n overcoming t h e f o r e mentioned d i s a d v a n t a g e s i s t o pap e r chromatograph t h e m e t h a n o l i c e x t r a c t of t h e p l a n t m a t e r i a l w i t h e t h y l acetate-anhydrous a c e t i c acid-water (50 :15 :18) a s developer. After drying t h e r u t i n s p o t was c u t o u t and ext r a c t e d w i t h nleefianol (2 m l ) , t h e n w i t h anhydrous a c e t i c a c i d (0.6 ml) and 20% aqueous p y r i d i n e (10 ml) and 12% m e t h a n o l i c A1C13 r e a g e n t (2.5 m l ) were added. The r e s u l t i n g s o l u t i o n was d i l u t e d t o 25 m l and t h e abs o r p t i o n was measured a t 42Onm (5-cm c e l l s ) a g a i n s t water. Beer's l a w was obeyed w i t h up t o 250 pg of r u t i n .

ii. With

Beryllium N i t r a t e

To a sample c o n t a i n i n g 0.1 + 1 . 2 u moles of r u t i n ( o r i t s aglycone q u e r c e t i n ) , an e q u a l amount of B e (NO3)2 d i s s o l v e d i n methanol was added, t h e n 2 N Na a c e t a t e s o l u t i o n (1.5 ml) w a s added, and t h e m i x t u r e was d i l u t e d t o 25 m l w i t h methanol. A f t e r 10 minutes t h e absorbance a t 465 nm was measured and t h e r e s u l t s were r e f e r r e d t o a s t a n d a r d curve ( 7 3 ) . R u t i n a s w e l l as i t s aglycone q u e r c e t i n , r e a c t w i t h Be2+ g i v i n g r e s p e c t i v e l y , yellow and orange I:1 complexes ( 7 3 ) . i i i . W i t h Quadrivalent Titanium Salts The c o n c e n t r a t i o n s of r u t i n an a l c o h o l i c e x t r a c t could be measured by t h e i n t e n s i t y of c o l o r of an orange yellow complex

TAHAI.KHALIFA ETAL.

662

formed w i t h Ti0 S04. The pH of r u t i n containing solution was a d j u s t e d t o 5.8 w i t h 3 N Na a c e t a t e . The c o l o u r , which i s s t a b l e f o r 30 minutes, w a s meas u r e d and t h e r e s u l t s were r e a d from a graph o b t a i n e d from r e a d i n g s of s t a n d a r d s o l u t i o n s ( 7 4 1. T h i s method e n a b l e s low c o n c e n t r a t i o n s (17 ppm) of r u t i n t o be determined i n pharmaceutic a l p r o d u c t s as well as i n p l a n t m a t e r i a l ( 7 4 1. iv. With Uranyl S a l t s The c o n c e n t r a t i o n of r u t i n i n a l c o h o l i c e x t r a c t s was d e t e r mined a b s o r p t i o m e t r i c a l l y by measuring t h e i n t e n s i t y of t h e colour of an orange complex formed by r u t i n and Uranyl acet a t e . The maximum a b s o r p t i o n was o b t a i n e d w i t h equimolar s o l u t i o n s i n a 1:l r a t i o (75 ) . Conc e n t r a t i o n s i n ppm range can be determined by t h i s method ( 75 ) . v.

With Cupric S a l t s

Rutin (10-M) was determined by t h e absorbance measurement of r u t i n CU (IT) complex i n s l i g h t l y a l k a l i n e methanol medium ( 76 & 77

1-

Other i n o r g a n i c i o n s produc i n g coloured complexes w i t h r u t i n e.g. Gallium (111) ( 78 and Antimony I11 ( 78 ) were a l s o used f o r i t s q u a n t i t a t i v e d e t e r m i n a t i o n In p l a n t material and pharmaceutical p r e p a r a t i o n s . 8 . 2 1.2.

Electrophilic Substitution i.

With p-aminobenzoic a c i d

A methanolic s o l u t i o n of r u t i n ( 4 ug) w a s a p p l i e d t o a Whatman No. 1 paper, and developed f o r 10 hours by t h e

RUTIN

663

a s c e n d i n g t e c h n i q u e w i t h n-butan o l - a c e t i c acid-water (20:5:11). The r u t i n zone w a s l o c a t e d on d r y chromatogram under UV o r by t r e a t m e n t w i t h ammonia fumes. The chromatogram w a s s e c t i o n e d and e l u t e d i n a t e s t t u b e by shaking w i t h 5 m l of acid-methano1 ( 1 : l ) . For t h e c o l o r i m e t r i c d e t e r m i n a t i o n 0.5% p-aminobenz o i c a c i d (0.4 m l ) , 10% H 2 S O 4 ( 0 . 4 m l ) , 0.2% NaN02 s o l u t i o n ( 2 ml) and 10% NaOH s o l u t i o n (5 ml) were added and t h e m i x t u r e w a s shaken. The a b s o r p t i o n a t 420 nm, was measured immediately ( 79 ) . T h i s method c o u l d be a p p l i e d t o o t h e r f l a v o n o i d compounds 6 i s claimed c o n v e n i e n t f o r samples w i t h a low r u t i n c o n t e n t

>.

( 79 ii. With E-aminocaproic a c i d

Rutin condensation product w i t h <-aminocaproic a c i d w a s determined c o l o r i m e t r i c a l l y a t 410 nm u s i n g Al2(SO4)3 a s chromogen i c a g e n t ( 8 0 ) . No i n t e r f e r e n c e w a s shown by u n r e a c t e d r u t i n (80 >. Other e l e c t r o p h i l i c s u b s t i t u t i o n r e a g e n t s used f o r t h e e s t i m a t i o n of r u t i n and o t h e r flavonoids i n plant e x t r a c t s i n c l u d e d i a z o t i s e d amines, 4aminophenazone, 2 , 6 dibromoquinone c h l o r i m i d e , n i t r o u s a c i d ( 81 ) , and b o r i c a c i d i n d r y a c e t o n e ( 82 ) . Generally t h e s u b s t i t u t i o n methods s u f f e r from s e v e r a l d r a w backs. Not o n l y are t h e m a j o r i t y of t h e r e a g e n t s r a t h e r u n s t a b l e , b u t t h e r e a c t i o n s are c a r r i e d o u t i n a l k a l i n e s o l u t i o n , where many p h e n o l s are r a p i d l y o x i d i z e d . Furthermore, t h e p r e s e n c e of c a r b o n y l groups i n s e v e r a l f l a v o noids reduces t h e i r a c t i v i t y Again, t h e p r e s e n c e of c o l o r l e s s

.

TAHA I. KHALIFA ETAL.

664

8.3.

p h e n o l i c compounds makes i t a l most i m p o s s i b l e t o u s e such methods f o r t h e d e t e r m i n a t i o n of t h e f l a v o n o i d components i n crude plant extracts. W Spectrophotometry R u t i n h a s been determined i n pharmaceutical prepar a t i o n s by measuring i t s a b s o r p t i o n a t 256 nm a f t e r being e l u t e d w i t h a 1:l m i x t u r e of 0.1 N h y d r o c h l o r i c a c i d and methanol from s i l i c a g e l p l a t e s ( 83 ). R u t i n (Ca. 15 mg) i n compound p r e p a r a t i o n c o n t a i n i n g a e s c u l i n ( C a . 5 mg) i n a d d i t i o n was d e t e r mined by a p p l y i n g t h e drug sample t o t h e t o p of a column of 3 g of polyamide. Aesculin w a s e l u t e d w i t h water, then r u t i n w i t h methanol. The r u t i n c o n t e n t w a s determined s p e c t r o p b o t o m e t r i c a l l y a t 360 nm ( 8 4 ) . The recovery of r u t i n is s a i d t o be 95% ( 84 ) . The q u a n t i t y of r u t i n and o t h e r f l a v o n o i d of orange j u i c e of high pulp c o n t e n t determined by e x k r a c t i n g w i t h e t h y l a c e t a t e , and t h e f l a v o n o i d s of t h e e x t r a c t were s e p a r a t e d on a polyamide l a y e r w i t h ljenam~-dioxan-form~ca c i d ( 4 : 5 : 1) as developing s o l v e n t . The chromatogram was i r r i d i a t e d w i t h l i g h t of wavelength 254 nm where r u t i n zone was i d e n t i f i e d by i t s d a r k brown f l u o r e s c e n c e . R u t i n zone w a s e x t r a c t e d w i t h methanol and t h e abs o r p t i o n measured a t 358 nm ( 85 ) .

8.4.

PMR Spectrometry

A r a p i d and simple PMR procedure f o r t h e e s t i m a t i o n of r u t i n i n b u l k d r u g s and i n p h a r m a c e u t i c a l p r e p a r a t i o n s h a s been r e c e n t l y r e p o r t e d ( 86 1. The peaks a t 1.03 ppm and 7 . 5 0 ppm a s s i g n e d t o t h e t h r e e p o r t i o n s of rhamnose methyl group and t h e two 2 , 6 p r o t o n s of t h e a r o m a t i c r i n g of t h e aglycone moiety were chosen f o r t h e q u a n t i t a t i v e a n a l y s i s of r u t i n . Acetamide, e x h i b i t i n g t h r e e methyl p r o t o n s s i n g l e t a t 2 . 0 0 w a s used as an i n t e r n a l s t a n d a r d ( F i g . 1 1 ). DMSO-D6 h a s been used a s a s o l v e n t i n t h e a s s a y . The method proved t o be r e l i a b l e and a c c u r a t e bes i d e s i t a l s o f u r n i s h e s a s p e c i f i c means of ident i f i c a t i o n of r u t i n a s w e l l as simultaneous d e t e c t i o n of any h y d r o l y t i c p r o d u c t s of t h e assayed r u t i n v i z . q u e r c e t i n , rhamnose and g l u c o s e . T h i s f i n d i n g h a s c o n t r i b u t e d g r e a t l y t o t h e method.

TAHA I. KHALIFA ETAL.

666

8.5.

Fluorimetry A method based on measuring t h e i n t e n s i t y of r u t i n aglycone, q u e r c e t i n , as w e l l as o t h e r f l a v o n o i d s complexes w i t h A 1 d i r e c t l y on p a p e r chromatograms w a s d e s c r i b e d by Tyukavkina e t a 1 ( 87 ) . R u t i n and o t h e r f l a v o n o i d s were s e p a r a t e d by a s c e n d i n g chromatography on slow paper w i t h CHCl3-acetic a c i d (1:2) a s a s o l v e n t . The chromatogram w a s t r e a t e d w i t h 0.02 M A1C13 - 0 . 1 M Na acetate i n 50% e t h a n o l . Pieces of t h e chromatogram (3x4 cm) were a t t a c h e d t o t h e w a l l of a c e l l i n a f l u o r i meter, and t h e f l u o r e s c e n c e of t h e complexes were e x c i t e d w i t h a mercury lamp through a USF-3 f i l t e r a t an a n g l e of 45O ( 8 7 ) . The r e c t i l i n e a r p a r t o f t h e c a l i b r a t i o n graph l i e s i n t h e range of 1 . 6 3 iJg f o r q u e r c e t i n . The method w a s proved f o r various flavonoids i n prepared mixture with a d e t e c t i o n l i m i t of 0.05 I.rg and 0.8 I.rg f o r quercet i n and d i h y d r o q u e r c e t i n r e s p e c t i v e l y . Another f l u o r i m e t r i c - p l a n i m e t r i c method f o r t h e e s t i m a t i o n of r u t i n and f l a v o n o i d compounds w a s d e s c r i b e d by J e r z y e t a 1 ( 88 ) . The f l a v o n o i d compounds were s e p a r a t e d by two-dimensional p a p e r chromatography a s d e s c r i b e d by Glotzbach and Rimpler ( 89 ) and t h e n t h e c o n t e n t s of conpon e n t f l a v o n o i d s were determined by p l a n i m e t r y a f t e r d i r e c t measurement of t h e f l u o r e s c e n c e a t 365 nm ( 89 ) .

8.6.

Polarography

A method based on t h e d e t e r m i n a t i o n of n i t r o d e r i v a t i v e s of r u t i n (and q u e r c e t i n ) i n p h a r m a c e u t i c a l p r e p a r a t i o n s w a s d e s c r i b e d by Davidek and Manousek ( go ) The drug ( 0 . 1 g) w a s d i s s o l v e d i n met h a n o l (25 m l ) , and an a l i q u o t of t h i s s o l u t i o n (0.5 ml) w a s mixed w i t h methanol (0.5 m l ) , 0.2 N H2SO4 (5 ml) and 3 M KN02 (2 ml) i n a p o l a r o g r a p h i c v e s s e l . A f t e r bubbling t h e mixed s o l u t i o n w i t h n i t r o g e n f o r 2 . 5 minutes, 2.5 M N a a c e t a t e (2 ml) was added and t h e b u b b l i n g w a s c o n t i n u e d f o r a f u r t h e r 4 minutes. The p o l a r o g r a p h i c c u r v e w a s recorded and t h e f i r s t s t e p measured. The h e i g h t of t h e s t e p w a s e v a l u a t e d by means of a c a l i b r a t i o n curve o r by s t a n d a r d a d d i t i o n . It i s r e p o r t e d t h a t even 10-6M s o l u t i o n of r u t i n may b e analyzed; t h e h e i g h t of t h e waves are independent of t i m e and t h e e r r o r is f 4% ( g o ) . A s c o r b i c a c i d and o t h e r compounds l i k e l y t o be p r e s e n t i n p h a r m a c e u t i c a l s are s a i d t o have no i n t e r f e r e n c e ( 90 1.

.

667

RUTIN

Another p o l a r o g r a p h i c d e t e r m i n a t i o n of r u t i n and q u e r c e t i n i n c o n c e n t r a t i o n of Ca 1 0 - 6 ~a f t e r n i t r o s a t i o n by means of t h e f o u r - e l e c t r o n wave produced by t h e r e d u c t i o n of t h e n i t r o s o group w a s a l s o mentioned by t h e same a u t h o r s ( 91 ). 8.7.

Densitometry Cine e t a 1 ( 92 ) d e s c r i b e d a d e n s i t o m e t r i c method € o r t h e d e t e r m i n a t i o n of r u t i n and q u e r c e t i n i n m i x t u r e s . S e p a r a t i o n of r u t i n from q u e r c e t i n w a s done on s i l i c a g e l G p l a t e s u s i n g a 72:18:10 benzene-pyridine a c e t i c a c i d system. The two compounds were determined a t 370 nm and 400 nm respectively. The d e t e r m i n a t i o n e r r o r was 5%.

8.8.

Gravimetry The g r a v i m e t r i c methods by Rodwell ( 9 3 ) , Naghski ( 9 4 ) , based on Sando and B a r t l e t t ' s ( 95 ) method of i s o l a t i n g r u t i n were used i n t h e p r e l i minary work. An estimate of t h e v a r i a t i o n i n t h e r e s u l t s o b t a i n e d by t h e s e methods w a s made, t h e s t a n d a r d d e v i a t i o n b e i n g about 0.5% f o r r e s u l t s v a r y i n g from 5-20% r u t i n .

et a1

8.9.

Other A n a l y t i c a l Uses 8.9.1.

A s Chrcmogenic Reagent R u t i n and i t s aglycone, q u e r c e t i n , were found t o b e s e n s i t i v e r e a g e n t s € o r d e t e c t i n g i n o r g a n i c c a t i o n s on paper chromatog r a p h s ( 9 6 ) . The t e s t e d c a t i o n s i n c l u d e Ag, Hg, Cu, B i , Sb, Sn, Fe, A l , N i , Co, Mg, L i , Mo, Be, Ga, G e , I n , P r , N e , Sm, U, V , W, T i , L a , Th, Z r , and a r s e n a t e ( 96 & 97 1 From t h e a b s o r p t i o n c u r v e s i t w a s concluded t h a t e a c h atom of a b i , t e r and q u a d r i v a l e n t metal combines w i t h 2 , 3 and 4 m o l e c u l e s of r u t i n o r q u e r c e t i n r e s p e c t i v e l y ( 9 6 & 92).

8.9.2.

A s A n A n a l y t i c a l Reagent Oka and Matsuo ( 98 ) r e p o r t e d a s p e c t r o p h o t o m e t r i c method f o r t h e determin a t i o n of microgram q u a n t i t i e s of Germanium u s i n g q u e r c e t i n , t h e aglycone of

668

TAHAI. KHALIFA ETAL.

r u t i n . Q u e r c e t i n w a s allowed t o react with G e i n n e u t r a l s o l u t i o n (PH 6.4 - 7 . 1 , phosphate b u f f e r ) t o g i v e a y e l l o w i s h compl e x (A max 410 nm) which i s s o l u b l e i n water c o n t a i n i n g > 40 % methanol. The abs o r p t i o n spectrum of q u e r c e t i n i t s e l f h a s X max of 258 nm and 375 nm and h a s l i t t l e i n f l u e n c e on t h e l i g h t a b s o r p t i o n a t 410, 420, 430 and 440 nm f o l l o w s Beer's l a w f o r 0 . 5 pg of G e p e r m l i n t h e p r e s e n c e of an e x c e s s q u e r c e t i n (> 1 4 t i m e s t h e equiv a l e n c e of Ge) ( 98 ) . Other n e t h o d s were r e p o r t e d u s i n g r u t i n o r i t s aglycone q u e r c e t i n f o r t h e d e t e r m i n a t i o n of c a t i o n s e . g . Sn ( 99 ) , Z r ( gg 1, B ( gg 1, and V ( 100 ). 8.10. Chromatography 8.10.1.. Paper Chromatography 8.10.1.1.

One-Dimensional Descending PC The chromatographic d a t a of r u t i n u s i n g one-dimensional descending PC under d i f f e r e n t c o n d i t i o n s i s g i v e n i n Table 7

.

8.18.1.2.

Two-Dimensional Descending PC R u t i n i s r o u t i n e l y used as a s t a n d a r d marker i n s c r e e n i n g a l coholic plant e x t r a c t s f o r t h e i r flavonoid p a t t e r n s using t h e s o l v e n t s n-butanol-acetic acidwater (4: 1:5; t o p l a y e r ) BAW, and 5% a c e t i c a c i d ( 1 4 ) . R u t i n i s u s e f u l s i n c e i t occup i e s a p o s i t i o n approximately i n t h e middle of t h e chromatogram and a l s o i s , i t s e l f , v e r y common i n p l a n t s and t h u s one of t h e most l i k e l y compounds t o be found d u r i n g s u r v e y work.

Table 7.

Paper Chromatography of Rutin

Technique

One-dimensional descending PC

Paper

Whatman No. 2.

Solvent Detection

sl W;

45

hRf Reference

s2

s3

s4

s5

‘6

s7

51

23

Brownish Yellow Fluorescent s?ot I

46

15

a3

45

(101-1l92X( 101-102) (1031 (lo$) 0 0 3 )

S1

BAW (4:1:5, upper phase).

S2

Acetic acid-conc. HC1-water (30:3:10).

S3

Ethyl acetate-water (saturated).

S4

150-propanol-water (6: 4 )

S5

n-Heptane-n-Butanol-water (29:14:57).

s6

Acetic acid-water (15:85).

S7

Water.

.

(103) (102 )

TAHAI.KHALIFA ETAL.

670

8.10.1.3.

P r e p a r a t i v e PC P r e p a r a t i v e PC i s such a w e l l known t e h c n i q u e and i t s u s e i n t h e f l a v o n o i d f i e l d h a s been s o well-reviewed r e c e n t l y (13, 1 4 , & 104 ) t h a t a b a r e o u t l i n e of recommended t e c h n i q u e s should b e sufficient The u s u a l p a p e r used f o r l a r g e s c a l e s e p a r a t i o n (1-100 mg) i s Whatman No. 3 o r i t s e q u i v a l e n t . The s o l u t i o n t o b e s e p a r a t e d (Ca. 1 0 ml) i s a p p l i e d as a cont i n u o u s even narrow s t r e a k o r band a l o n g t h e s t a r t l i n e by succ e s s i v e a p p l i c a t i o n s . For r u t i n and t h e m a j o r i t y of f l a v o n o i d s s e p a r a t i o n i s f i r s t e f f e c t e d by t h e use of BAW m i x t u r e s ( e . g . 6:1:2). It i s convenient t o l o c a t e t h e s p o r t s by t h e i r f l u o r e s c e n c e i n W. A f t e r l o c a t i o n , t h e bands are c u t o u t , t h e compounds e l u t e d s e p a r a t e l y , u s u a l l y w i t h 70% aqueous methanol, and he s o l u t i o n s c o n c e n t r a t e d f o r r e p u r i f i c a t i o n i n a second s o l vent.

.

8.10.2.

L i q u i d Column Chromatography (LC) Tomas e t a1 ( 1 0 5 ) s e p a r a t e d r u t i n , querc e t i n and o t h e r f l a v o n o i d s on sephadex G25. Glyzosides were r e a d i l y e l u t e d w i t h water, w i t h good s e p a r a t i o n of r u t i n and q u e r c e t r i n : t h e accompanying aglycones w e r e r e t a i n e d a t t h e t o p of t h e column and could b e s u b s e q u e n t l y e l u t e d w i t h 0.1% aqueous ammonia s o l u t i o n . A 16~0.9cm column of Amberlite XAD-2 (200-400 mesh) maitltained a t 95OC w a s used f o r t h e s e p a r a t i o n of many f l a v o n o i d s ( 1 0 6 ) . The column w a s f i r s t e q u i l i b r a t e d w i t h 20% e t h a n o l a t a f l o w r a t e of 60 ml/hour and a s o l u t i o n o r s u s p e n s i o n of f l a v o n o i d s i n 20% e t h a n o l (each c o n t a i n i n g l e s s t h a n 500 pg/ml) w a s placed on t h e t o p of t h e column. A 100 m l volume of 20% e t h a n o l i s run f i r s t , followed by l i n e a r g r a d i e n t e l u t i o n w i t h a t o t a l volume of

RUTIN

671

1000 m l , t h e e t h a n o l c o n c e n t r a t i o n i n c r e a si-ng from 20 t o 9Oc. The r u t i n group f l a v o n o i d s w e r e e l u t e d from t h e column i n t h e o r d e r r u t i n , q u e r c i t r i n , and t h e n querc e t i n (106 ) . T h i s p r o c e d u r e w a s a l s o a p p l i e d t o t h e d e t e r m i n a t i o n of f l a v o n o i d s i n c r u d e m e t h a n o l i c e x t r a c t s from p l a n t s ( 106 ) The chromatographic behaviour of r u t i n u s i n g LC under d i f f e r e n t p a r a m e t e r s i s summarized i n T a b l e 8.

.

8.10.3.

Thin Layer Chromatography (TLC) Although a n a l y t i c a l TLC of r u t i n and o t h e r f l a v o n o i d compounds on m i c r o c r y s t a l l i n e c e l l u l o s e , s i l i c a g e l o r polyamide i s cons i d e r e d a r a p i d method of i n i t i a l s c r e e n i n g o r checking t h e p u r i t y of i s o l a t e d compounds ( 1 3 , 1 4 and 81 ) , t h e t e c h n i q u e is n o t f a v o u r i t e l y used f o r i n i t i a l examination of c r u d e p l a n t e x t r a c t s because t h e r e s o l v i n g power i s g e n e r a l l y i n s u f f i c i e n t . However, TLC is o f t e n t h e method of c h o i c e f o r f i n a l p u r i f i c a t i o n of r u t i n and o t h e r f l a v o n o i d s , e s p e c i a l l y using s i l i c a g e l s i n c e here contamination i s less t h a n on p a p e r ( 109 ) . The chromatographic d a t a of r u t i n u s i n g d i f f e r e n t TLC t e c h n i q u e s a r e d e p i c t e d i n T a b l e . 9. The chromatographic behaviour of f l a v o n o i d s on t h i n l a y e r s i s sometimes m i s l e a d i n g s i n c e some of them w i l l g i v e t h e same Rf v a l u e s even w i t h more t h a n one s o l v e n t system. T h i s l e d H u r s t and Harborne t o develop a method based on r e d u c t i v e c l e a v a g e of t h e s e compounds t o give rise t o phenols, phenolic alcohols and p h e n o l i c a c i d s which c o u l d b e more r e a d i l y i d e n t i f i e d (110). Q u e r c e t i n , t h e aglycone of r u t i n , g i v e s r i s e t o Dhloro$ u c i n o l (A-ring fragment) and 3,4-dihydroxyphenylpropionic a c i d and 3,4-dihydroxyphenylpropanol (B-ring f r a g m e n t s ) (110).

Table 8 .

Column Chromatography of Rutin

Packing

Sephadex G-25, medium particle size 35 cm long

Column Material Solvent Flow rate

H20 25mlllw1.r

I

X

2.5

0.05 M NaCl 25 ml/hour

grade

Sephadex LH-20

cm diameter

45 cmx2.5 cm

0.1 M NH40H

0.01 M Sod. Molybdate

Methanol

25 ml/hour

25 ml/hour

3-5 ml/min.

Temperature Detect ion

9 N

Kd

Kd

*

=

5.60

6.50

3.90

0.26

Ve/Vo*

(107)

(107)

(107)

(107)

( 108)

Dsstribution coefficient Under these conditions Ve/Vo

=

2.2 Kd

+

1; Ve = elution volume, Vo = intersitial volume.

4.00

$4

i

a, P

cu a u

a 1

(d $4

m

u v) ..L

(d

t

a c M

.d

c .r( a

!i

u

v)

(d

3 0 rl

a,

M

rl

0

(d

.d d

rl v)

I

a

(d

a

3 h

PI

0

rl

a

0

u LA

u c a c)

a

(I)

1

0

N F

rl

3

0

*a

rl rl

% $4

0

-2 a rl

673

n

rl 4 rl

W

a, CJ

!ll al

N

d

w

TAHA I. KHALIFA ETAL.

674

8.10.4.

Gas L i q u i d Chromatography (GLC)

The a g l y c o n e s of r u t i n and o t h e r f l a v o n o i d s could be s e p a r a t e d as t r i m e t h y l s i l y l (TMS) e t h e r s by GLC ( 1 1 2 ), b u t l i t t l e u s e h a s been made of t h i s t e c h n i q u e c o n s i d e r i n g i t s s e n s i t i v i t y ( t o t h e nanogram l e v e l ) , r a p i d i t y and t h e f a c t t h a t t h e compounds can be r e a d i l y e s t i m a t e d q u a n t i t a t i v e l y (113). I n almost r e p o r t e d c a s e s f l a v o n o i d e t h e r s have been s e p a r a t e d on columns cont a i n i n g t h e s i l i c o n e t h e r polar phases e . g . OV 1, OV 1 7 o r SE 30 ( 1 1 2 ) . 8.10.5.

Electrophoresis E l e c t r o p h o r e s i s on e i t h e r paper o r t h i n l a y e r s i,s a r e p o r t e d t e h c n i q u e (:114) which h a s been s c a r c e l y used i n t h e f l a v o n o i d f i e l d . R u t i n , and q u e r c e t i n amongst o t h e r f l a v o n o i d g l y c o s i d e s and a g l y c o n e s could be r e a d i l y s e p a r a t e d u s i n g b o r a t e b u f f e r s on TLC c e l l u l o s e l a y e r s ( 114 ) . However, e l e c t r o p h i l i c examination of e x t r a c t s cont a i n i n g charged flavonoid-compounds of a l l t y p e s , which may be more widely d i s t r i b u t e d t h a n h i t h e r t o b e l i e v e d , may pay handsome d i v i d e n d s ( 8 1 and 115).

RUTIN

675

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The Merck Index, 9th Edn. Martha Windholz, Merck Inc., Rahway, N . J . , U.S.A. (1976).

Co.

2.

W. Karrer, "Konstitution and Varkommen der Organischen Pflazenstoffe", Birkhauser Verlag, Basel und Stuttgart, (1958).

3.

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4.

L.P. Miller, "Phytochemistry" Vol. 11, Van Nastrand Reinhold Co., (1973).

5.

Pharmaceutical Codex , 11th Edn. Press, London, (1979).

6.

J.F. Couch, J . Naghski, and C.F. Krewson, U.S.A. Bar Agric. and Industr. Chem. Rpt. AIC-52, (1944).

7.

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8.

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9.

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10.

Index of Specialities, The Egyptian General Organization for Pharmaceuticals, Chemicals and Medical Appliances, Cairo, (1969).

11.

G. Zemplen, and A. Gerecs, Ber.,

&

The

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103,

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12. Dictionary of Organic Compounds, 4th Edn., Vol. V, Eyre and Spottiswoode Publishers Ltd., (1965).

13. T.J. Ma:bry, K.R. Markham, and M.B. Thomas, "The Systematic Tdentification of Flavonoids", SpringerVerlag, Berlin, (1970). 14.

J.B. Harborne, "Phytochemical Methods", Chapman and Hall, London, (1976).

15.

J.Q. Griffith, Jr., "Rutin and Related Flavonoids'', Mack, Easton, Pa., (1955).

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676

16. J.G. Grasselli, and W.M. Ritchy (Eds.), "Atlas of Spectral and Physical Constants of Organic Compounds", Vol. I, 2nd Edn., CRC Press, Inc., Ohio, (1975). 17. J.C. Chang, J. Org. Chem.

2,1881, (1976).

18. P. Joseph Nathan, J. Mares, M.C., Herandez, and J.N. Shodery, J. Magn. Reson., 16,447, (1974). 19. C.A. Kingsbury, and L.H. Looker, J. Org. Chem., 1120, (1975).

40,

20. F.W. Wehrli, J. Chem. SOC. Chem. Commun., 663, (1975). 21. H. Wagner, V.M. Chari, and J. Sonnenbichler, Tetrahedron Lett., 1799, (1976). 22. A. Pelter, R.S. Ward. and T.I. Gray., J. Chem. SOC. 2475, (1976). Perk.

r,

23. K.R. Markham, and B. Ternai, Tetrahedron, 32, 565, (1976). 24. K.R. Markham, and B. Ternai, Tetrahedron, 32, 2607, (1976).

41, (l), 17, (1978). 25. C.J. Chang, LLyodia, 26. E.B. Dechene, J. Am. Pharm. Ass. Sci. Edn., (1951).

40,

495,

27. F.Malyneux, Process Biochem., 23, (1971). 28. R. Plouvier, Compt. Rend.,

216, 459, (1943).

29. E. Ramstad, "Modern Pharmacognosy", McGraw-Hill, Book Co., Inc., New York, (1959). 30. E.W. Underhill, J.E. Watkin, and A.C. Neish, Can. J. Biochem. Physiol, 35, 219, (1957). 31. F.R. Humphreys, Economic Bot.,

18,(3),

195, (1964).

32. J.F. Couch, Dept. US Agric. AIC-202, (1948). 33. S.V. Kostanecki, V. Lampe (1904).

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