Simple densitometric method for estimation of cyanogenic glycosides and cyanohydrins under field conditions

Biochemtil Systematics Printed in Great Britain.

0305-1978/86 $3.00+0.00 Pergamon Press Ltd.

and Ecology,Vol. 14, No. 1. pp. 97-103.1986.

Simple Densitometric Method for Estimation of Cyanogenic Glycosides and Cyanohydrins Under Field Conditions LEON BRIMER and PER MBLGAARD Department

Key Word kwdleri.

of Pharmacognosy and Botany, Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen 0, Denmark

Index-Cyanogenic

glycosides;

cyanohydrins:

detection;

quantitative

determination;

field method;

Thalictrum

Abstract-In this new design of the picrate test (Guignard Method) for small scale cyanide determination under field conditions, hydrolysis of the cyanogenic compounds is catalysed by ‘@glucuronidase’, a commercial enzyme preparation from Helixpomatia. The method described facilitates the quantification of small amounts of cyanogenic compounds in plant material, with a detection limit about 60 ng of released HCN (equivalent to 1 pg of amygdalin). False positive tests produced by hydrolysis products of glucosinolates could be excluded by inhibition of the myrosinae enzyme system present in plants that contain glucosinolates. The total weight of equipment needed for a total of loo0 determinations (in groups of eight) does not exceed 500 g, thus making the method convenient for field work.

microdiffusion based picrate (Guignard) test, with the use of ‘j3-glucuronidase’ from Helixpomatia as a source of hydrolytic enzymes [5]. In the modified picrate test, released HCN is detected as red to brown spots on transparent hydrophobic picrate reagent sheets (Fig. 1). This system permits densitometric recording of colour intensities corresponding to quantities of HCN as low as 60 ng released from 50 mg, or less of

Introduction Only a few reports of the design of methods for phytochemical screening in the field are found in recent literature. Usually, the methods employed are only qualitative, or, at best, semiquantitative, and they often involve a visual comparison with standards and the results are expressed as + to ++++ [I], or unusually, by a scale O-9 [2]. Cyanogenic constituents (i.e. cyanogenic glycosides, cyanogenic lipids and cyanohydrins) are often included in such screening programs. Detection of cyanogenesis, as well as the qualiand quantitative estimation of cyanogenic compounds in plants and insects, is traditionally based on reactions specific for HCN released after cleavage of the cyanogenic constituents [3]. However, enzyme preparations able to hydrolyse all types of cyanogenic glycosides have not been readily available [4,51 and quantitative release of HCN from the matrix of organic compounds may be incomplete [31. Until now, nearly all known methods for quantitative determination of HCN have been inconvenient for use in the field. Thus, Kaplan et a/. [6] recently stated the need for a more reliable field test system. As a possible way to solve these difficulties, we explored the combination of a modified

d

FIG. 1. SKETCH OF MICRODIFFUSION OF

RELEASED

together

CYANIDE.

(a)

(c) Picrate impregnated

(di Part of reagent sheet after exposure

(Received 25 January 1985)

welldefined

densitometer.

97

ground

with a drop of CHCI, (to promote

agent sheet cover; to violet

CHAMBER

Material

FOR DETERMINATION in enzyme

solution

cell lysis); (bl Transparent layer of the reagent

to HCN, showing

spot. to be rated visually

re-

sheet;

an orange-red

or measured

with

a

LEON BRIMER AND PER M0LGAARD

I5 r

plant material. This corresponds to about 1.2 pg HCN/g. From a chemotaxonomic point of view, plants are normally considered cyanogenic if they release more than IO-20 frg of HCN/g fresh material [7]. Results and Discussion Two different designs (1 and 2, see Experimental) were developed for qualitative and quantitative determination of liberated HCN, respectively. The two designs are satisfactory for a set of basic demands for accuracy and reproducability and still the equipment is easy to use for field trials. A large number of samples can be screened simultaneously with a detection limit as low as 60 ng of liberated HCN, permitting use of small samples (50 mg or less) and study of weakly cyanogenic specimens. For field tests it is important that part of the equipment is reusable, sturdy and easy to clean, although of light-weight material. The advantage of the quantitative method is the dual rating of the reagent strips, in the laboratory with a densitometer and in the field by comparison with a set of standards on reagent sheets. The densitrometrically determined continuous calibration curve (Fig. 2, Design 2) was obtained by means of standard solutions of amygdalin, and compared with the curve obtained, when

15r

.

.

:

.

8

I

1; , , ,:,

i

loo

50

0

Amount FIG. 2. CALIBRATION AMOUNTS

GRAPH

OF RELEASED

of substrate

(n ml)

FOR MICRODIFFUSION

CYANIDE-CLOSED

tion 2% v/v, 24 h, 21”. The dots represent deviation.

with standard

OF INCREASING

DOTS.

picrate sheets have been read with a densitometer. standard

2bo

means

The developed

Enzyme

concentra-

of three replicates

+

Open dots present results from a single experiment

solutions

with a 6% vlv enzyme

applied

to silica gel plates, which were sprayed

solution and covered with the reaction

sheet.

Amount

of substrata

FIG. 3. EFFECT ON THE CALIBRATION PLANT of

MATERIAL.

50 mg

of fresh

Conditions leaves

(n mol)

GRAPH

OF THE PRESENCE

OF

as in Fig. 2 (filled dots) and with addition of

Geranium

grevekanum

Plantego major (open dots). The curve represents

(squares)

the calibration

and graph

from Fig. 2.

solutions were applied to silica gel plates, sprayed with a 6% v/v enzyme solution and covered directly with the reaction sheet. This later method has previously been shown to cause total hydrolyses under these conditions [5] and since the two calibration curves are identical, the reaction was considered quantitative in the case of the microdiffusion model also. However, experiments including addition of different kinds of non-cyanogenic plant material clearly proved that the matrix could impede the release of HCN, as previously described [3]. Species of Geranium proved to be very efficient in this respect, and G. grevelleanum Wall. (among other species) was used for the investigation of incubation parameters, in order to establish a set of standard conditions which permit the quantitative determination of most cyanogenic plant material (Fig. 3, Table 1). The assay was checked both qualitatively (Table 2) on different plant material (low-, highand acyanogenic) and quantitatively on leaves from Prunus laumcerasus L. cv. schriphaensis. Quantitative HCN-determination with Prunus leaves was performed according to the established ‘Recommended standard assay (2)’ (Experimental) with 50 mg of fresh material and compared with a traditional calorimetric determination of HCN from 200 mg of fresh material collected by aeration in NaOH (2 ml, 0.1 M) [81. The results from four replicates of the densitometric and the calorimetric determination of

ESTIMATION

OF CYANOGENIC

99

COMPOUNDS

Finally, two standard strips were produced by placing a piece of silanized filter paper (J. C. “mol)* Binzer, Hatzfeld/Eder, GFR) between the picrate reaction sheets and the reaction chamber. In Time of Colour response Enzyme exposure Temperature equivalent to concentration one of these experiments, the rack was placed released HCN (nmol) (% vlv/F.u.ml ‘) (‘C) (hl upside-down, which without the silanized filter paper would have completely soaked the reac2/2oQo 16 21 12fl 2/2goo 40 21 21f3 tion sheet. Both of these strips were found 4/4000 16 21 25k3 nearly identical with strips obtained by the stan2/2000 16 40 51f4 dard procedure when read densitometrically. Thus, a protective layer of this type may be ‘65 nmol of amygladi” with addition of Geranium grevelleanum leaves (50 mg) as HCN retaining material. Results of four replicates *standard introduced, in order to protect the reaction sheet, deviation. when transported during development. The methods described seem to be well suited for determination of total content of liberated cyanide were 76f4 f.tg HCN/g and cyanogenic glycosides and/or cyanohydrins in 77f 5 pg HCN/g, respectively. plant material under field conditions as well as in However, due to a possible retaining of liberthe laboratory. When rated visually, experiments ated compounds in the complex matrix of plant material and enzyme solution in the reagent jars, in our laboratory showed that all the standards a proper quantitative analysis must comprise at below 150 nmol used for the calibration graph shown in Fig. 2 could be arranged in the proper least two independent but identical determinations of HCN under different conditions in order by five individuals aged 2B-54. However, respect of enzyme concentration, temperature or one disadvantage should be mentioned, namely the relatively short dynamic range and the resulttime. ing difficulties encountered with strongly cyanoAuthentic sinignn as well as selected plant genie materials, since very small samples must material known to contain glucosinolates that be used in order to obtain quantitative results, yield steam volatile products of hydrolysis, were i.e. to stay below 100 nmol of released HCN. investigated by this standard assay. Degradation products from these compounds are reported also to form orange red products with picrate Experimental (Table 2). All species previously reported to be &sign 1 IF&.4).Consistsof a number of screw cap vials procyanogenic gave the expected fast colour reac- vided with the corresponding open-top caps and placed in a tion, while sinigrin (with addition of myrosinase) light-weight rack. Circular discs of reagent sheet are made by means of a hollow punch or cork borer and placed into caps and some of the species known to contain only glucosinolates showed a slow but definite reac- from which the septa have been removed. Colour reactions may be observed during exposure, due to the transparency of tion. In order to eliminate false positive tests, a the sheet, and developed discs can be stored easily in small qualitative confirmatory test was developed, tubes for subsequent comparison with standards. Design 2 (Fig. 5). Consistsof microtest tubes (35 X 12.5 mm, based on extractive acid denaturation of any polypropylene lymphocyte tubes) placed in a block so that the myrosinase that may be present in the plant material, prior to incubation. This procedure is a open ends of the tubes are level with the plane of the upper surface of the block, allowing a rectangular strip of reagent modification of a method used for extractions sheet to be placed as a close cover on top of the tubes. The and determination of cyanogenic compounds in sheet is pressed against the surface by means of a transparent cover (e.g. Perspex”). The spots on the developed reagent cassava roots [9]. Results for selected species strip may be rated in the field and/or measured with a denproved this test valuable (Table 2), and warranted sitometer after return to the laboratory. Developed strips the rejection of the noncyanogenic species should be protected from light and corrosive vapours in a found positive in the standard assay, while all closed container or mounted between two glass plates. A cyanogenic species remained positive. standard strip for comparison in the field may be preserved in Thalicbumfkndleri Engelm., which has not pre- an envelope of transparent heat sealed plastic and kept proviously been reported cyanogenic, appears to tected from light. Preparation of reagent sheets. Precoated ion-exchange contain at least two cyanogenic compounds, sheets (Polygram lonex 25SE-AC, Macherey-Nagel, D&en, when examined by thin-layer chromatography. Germany) were impregnated by consecutive immersion in

TABLE

1. COLOUR

AND TEMPERAlURE

RESPONSE

TO ENZYME

IN STANDARD

CONCENTRATION,

INCUBATION

OF AMYGDALIN

TIME

(65

100

LEON BRIMER A N D PER M O L G A A R D

TABLE 2, QUALITATIVE HCN DETERMINATION, BY MEANS OF 'RECOMMENDED STANDARD ASSAY' AND 'CONFIRMATORY TEST' Species and origin*

Part

Amount

Colour reaction:l:

tested1

(mg)

after (h) 24

Constituents as compiled from literature cyanogeniet

48

120

glucosinolatest

compounds

Confirmatory

Reference

test ( / + colour reaction) 24 h

Barbarea intermedia Bor. (4851/6)

I

50

0

0

0

Barbarea rntermedia Bor. (4851/6)

I

200

0

0

0

gluconasturtiin §,s

s

10

0

0

s

20

0

0/I

0

sinigrin§,l

10

sinigrin§,l.

10 10

Brassica ]uncea (L.) Zern. et Coss. (commercial)

Brassicaluncea (L) Zern. et Coss. (commercial)

Brassicajuncea (L.) Zern. et Coss. 100

2

3

3

Brasslca nigra (L.) Koch. (4949/19)

50

0

O

0

Brasstca nigra (L.) Koch. (4949/19)

200

0

0/1

0

Dl#lotaxls tenuifo#a {L.) D.C. (4955/5)

50

0

0

0

Diplotaxts tenuifolia (L.) D.C. (4955/5)

200

0

0

0

glucoerucin§,l.

1126)

50

0

0

0

--

Isatls tmctoria L. (4938/4)

50

0

0

0/1

Isatis tinctona L. (4938/4)

200

0

0/1

0

IsatJs tmctona L. (4938/4)

50

0

0

0

/saris tinctoria L, (4938/4)

200

0

0/1

2

50

4

(commercial)

--

Geramum grevelleanum Wall. (S 1971 /

L iriodendron tulipifera L. (4740/1)

gluconapin§,s 4,sh

+,1

t0, 11

triglochinin

11

taxiphyllin, i

Lunana redJvlva L. (4863)

50

0

0

Lunana redlviva L. (4863)

200

0

0

0

Lunaria rediviva L. (4863)

50

0

0

0

Lunaria rediviva L. (4863)

200

0

0

0/1

0

0

0 glucoberteroin§,s

tO

Malus baccata Borkh. var. oblonga (6342F/2)

50

0

Malus domestlca Borkh. (commercial)

50

;>5

+, s

--

11

4, s

-

11

Nandina domestma Thunb. ($1971/ 0577)

50

4/5

nandinaglucosid, I

Passiflora quandrangularis L. (5098/19)

50

2

f, t/s.

Ptantago major L (2170/182)

50

0

Schripkaensis (P1974/5112)

50

> 5

Sambucus ebulus L. (RDSP)

50

0

Sambucus mgra L. (own)

50

5

0

0

--

0

0

--

1! --

~

11

Prunus lauraceracus L cv. prunasin, I

11

sambunigrin, prunasin,

t

11

+

12

zierin, holocalin, I.

Taxus baccata L. (~ (18168/1)

50

4

taxiphyllin, t.

Thahctrum aquilegifolium L. (4772/3)

50

5

triglochininmono

-o

methylester, proteacin, nandinaglycosid, I.

Thalictrum fendleriEngelm, (4772/21)II

50

3

3

3

-~, I

Thahctrum foetidum L. (4772/7)

50

0

0

0

7halictrum glaucum Desf. (4772/8) Thalictrum polygamum MQhl. (4772/20)

50

0

0

0

50

1

--

11

--

11

triglochinin, I

11

*The samples were either: collected August 3rd in Botanical Garden, University of Copenhagen, parenthesis indicates plant n o , obtained through normal commerce (commercial), collected in the garden of Royal Danish School of Pharmacy (RDSP}, or in the author's o w n garden {own) tl, leaves; s, seeds; fr, fruit; gl, green leaves; sh, shoot. :l:Colour reaction rated visually against five standards equivalent to 1 ,= 60, 2 - 180, 3 §Steam volatile products of hydrolyses. II Not previously reported cyanogenic.

600, 4

1800, 5 -- 6000 ng of HCN.

101

FIG. 4. MICRODIFFUSION SYSTEM, DESIGN 1. Light weight rack for 15 vials in a plastic box. An open-top cap and a vial ere seen to the right (arrow). A number of exposed discs may be seen on top of vials in the rack. In front, a number of discs have been exposed to HCN from different amygdalin standards.

102

FIG. 5. MICRODIFFUSION SYSTEM, DESIGN 2. Rack made of PVC. Reagent strip (arrow) exposed to different amygdalin standards. Stabilizing aluminium-profile cover is seen to the right of the perspex cover.

ESTIMATIONOF CYANOGENICCOMPOUNDS three solutions: (1) a saturated soln of picric acid in H20, followed by air drying; (2) a 1 M aq. Na2CO 3 soln, followed by air drying; and (3) a 2% w/v ethanolic 1-hexadecanol soln, followed by air drying [5]. Calibration graphs should be produced for each batch of impregnated sheets due to small differences among batches. Assays. A suitable amount of material (based on preliminary tests) was placed in a vial (Design 1) or tube (Design 2). One drop of CHCI3--to promote cell lysis--and 500 p.I of an aq. soln of I~-glucuronidase from Helix pomaga (Sigma G-0876) were added. The material was ground in the liquid with a glass pestle to form a homogeneous mixture. Finally the reaction chamber was closed by means of the reagent sheet, and left until complete reaction had occurred, in general 24 hours. Absorbance of the spots may be rated visually by comparison with standards or measured by the transmission technique with a densitometer [Vitatron TLD 100; light source: tungsten; secondary filter; 540 nm; mode: --log; aperture: 2.0 mm (circular)]. Spectrophotometric determination of total HCN released. HCN, released from material ground in a 2% aq. soln of I~glucuronidase Helix pomatia was collected in 2 ml of NaOH by aeration with N2 [3]. Total cyanide released after 24 h at 35° was determined according to Epstein as modified by J~rgensen [8]. Confirmatory test. Plant material (500 mg) was ground in a mortar with orthophosphoric acid (0.05 M, 2.5 ml). The homogenate was filtered by pressure through silica gel 60 (400 mg) placed on top of glass wool in a 2 ml syringe. Phosphate buffer (0.2 M, pH 7.0, 1.0 ml) and a 20% v/v aq. soln (50 p.I) of I}-glucuronidase Helix pomatia were added to the filtrate (0.51.0) in a vial/tube. The resulting pH was about 6.0-6.8. The reaction chamber was closed by means of the reagent sheet. Recommended standard assay. (1) Qualitative determination: amount of material, 50 mg; enzyme concentration, 2% v/v (corresponding to about 2000 Fishman units per ml); time and temperature, about 20 h for temperatures above 10-15°.

103 (2) Quantitative determination--laboratory conditions: amount of material, decided after the colour response from a preliminary qualitative determination; enzyme concentration, 3-4% v/v (about 3-4000 Eu. per ml); time and temperature, 20 h at 40°. (3) Quantitative determination--field conditions: amount of material, as above; enzyme concentration, 8% v/v (about 8000 F.u, per ml); time and temperature, 20 h at temperatures above 15-20%. Acknowledgements--The authors thank Dr. S. Brogger Christensen and Dr. F. Nartey for critically reading the manuscript. References 1. Farnsworth, N. R. (1966) J. Pharm. Sci. 55, 225. 2. McGregor, D. I. and Downey, K. (1975) Can. J. Plant Sc~ 55, 191. 3. Nahrstedt, A., Erb, N. and Zinsmeister, H.-D. (1981) in Cyanide in Biology (Vennesland, B., Conn, E. E., Knowles, C. J., Westley, J. and Wissing, F. eds) p. 461. Academic Press, London. 4. H6sel, W. (1981) in Cyanide in Biology (Vennesland, B., Conn, E. E., Knowles, C. J., Westley, J. and Wissing, F., eds) p. 217. Academic Press, London. 5. Brimer, L., Bragger Christensen, S., Molgaard, P. and Nartey, F. (1983) J. Agric. Food. Chem. 31, 789. 6. Kaplan, M. A. C., Figueiredo, M. R. and Gottlieb, O. R. (1983) Biochem. Syst. Ecol. 11, 367. 7. Hegnauer, R. (1977) Plant Syst. Evol. Suppl. 1, 141. 8. Jergensen, K. (1955) Acts Chem. Scand. 9, 548. 9. Cooke, R. D. (1978) J. Sci. FoodAgric. 29, 345. 10. Kje~r,A. (1960) Fortschr. Chem. Org. NatursL 18, 122. 11. Tjon Sie Fat, L. A. (1979) Contribution to the Knowledge of Cyanogenesis in Angiosperms, p. 18. Dissertation, University of Leiden, The Netherlands. 12. Hegnauer, R. (1959) Pharm. Weekbl. 94, 241.