INHIBITION OF MYROSINASE PREPARATION BY AN EXTRACT

FROM ARCTOSTAPHYLOS UVAE-URSI

Mykola S Mykytyn and Grygorij T Demjanchuk

Institute of Cruciferous Plants, Rozumovskogo 9a, Ivano-Frankivsk, 284028, Ukraine

ABSTRACT

The purpose of the work is to find the possible ways of rapeseed glucosinolates detoxification in animal intestines. Therefore screening of active in reference to glucosinolate extract or enzyme myrosinase of the water-soluble substances from the vegetative and generative parts of domestic and wild-growing plants including the medicinal ones has been carried out.

The search has been coordinated to find the nature inhibitors of the myrosinase and enzymes able to transform the glucosinolates to intoxic combinations too. Thirty three variants of plant extracts have been tested. Most of them have not acted at all or have changed the myrosinase action a little. The great inhibitory activity of the Arctostaphylos uvae-ursi and its foresee acting substance – hydroquinone, have been discovered. The quinone (0,1% concentration) has proved to be more active than hydroquinone. At the same time the phenol and resorcinol from phenolic row have turned out unactive at the same concentration. The direct dependence of the glucosinolates decompose degree from concentration of the hydroquinone and quinone has been determined. The cultivation of Brassica napus cut seeds by quinone in correlation 1:100 (m:m) stopped the glucosinolates decomposition by endogenous myrosinase for 50%.

As for some toxicity above mentioned substances, their practical usage in the nutrition is problematical. The mechanism itself of these matters action on the myrosinase reaction remains interesting and not solved yet.

KEYWORD: glucosinolates, rapeseed, bearberry, hydroquinone, quinone.

INTRODUCTION.

It is well-known that glucosinolates are not toxic in themselves. Only because of certain conditions (humidity, temperature, pH, etc) and influence of myrosinase there happens their hydrolysis and formation of toxic compounds in different extent (oxazolidinethiones, isothiocyanates, thiocyanates, nitriles), which have negative influence on the organism. That is why a practicable way to prevent negative influence of those compounds is natural agents, which can in a certain way disable glucosinolates' hydrolysis by myrosinase.

L-ascorbic acid is a known catalyst of myrosinase reaction (Ohtsuru M. and Hata T., 1979). As to specific enzyme inhibitors there is no available information. Therefore this research provides screening of water extracts from vegetative and generative organs of cultivated and wild plants, including herbs, which are active with respect to glucosinolates extracts and myrosinase enzyme.

MATERIALS AND METHODS.

To obtain water extract of glucosinolates, 1 g of ground seeds of 0-brand Jet-Nef were placed in a 50 ml retort and the retort was placed into a boiling water bath for 5 minutes. Then 20 ml of boiling phosphate buffer was added (pH 7.0) and the retort was kept in a water bath for more 20 minutes. After cooling down to the room temperature the solution was brought up to 50 ml by adding the same phosphate buffer, then shaken up and filtered. The filtrate was kept in the fridge for 24 hours. To obtain extract of plants, 5 g of ground dry plant material was pounded with pestle together with 50 ml of phosphate buffer (pH 7.0). After settling for 1 hour in room temperature suspensions were filtered. Fresh prepared filtrates were used. Myrosinase enzyme preparation was obtained from white mustard seeds (Neuberg C. und Wagner J., 1926).

For investigation of detoxication capacity plants' water extracts were poured in two test-tubes in the volume of 0.5 ml. 0.5 ml of myrosinase enzyme solution was added to the first test-tube and 2 ml of glucosinolates extract was added to the second test-tube. After settling for 2 hours contents of test-tubes were merged and after 30 minutes it was boiled in water bath for 5 minutes. The filtrate was analyzed apropos of containing of 5-vinyl-2-thiooxazolidone (Appelquist L. and Josefsson, E, 1967).

For the purpose of exclusion of possible influence on the analysis results on the side of plant extracts myrosinase reaction products and of chemical reagents, check experiments were carried out. For that 10% water extract of bearberry habitual (Arctostaphylos uvae-ursi) and hydroquinone and quinone (5% and 0,1% respectively) were added to glucosinolates hydrolysis products, content of which in the solution was known. Results obtained showed that water extract of Arctostaphylos uvae-ursi and also hydroquinone and quinone impede spectrophotometric content test of goitrine, and at the same time do not impede content test of glucose and isothiocyanates - other typical products of glucosinolates decomposition. Therefore in further investigation, in which influence of different extracts concentrations and reagents on the extent of glucosinolates decomposition by myrosinase were studied, these particular methods were used. (Youngs C. and Wetter L., 1967; Дем’янчук Г., 1985).

RESULTS AND DISCUSSION.

Due to carried out investigation concerning study of influence of water extracts from vegetative and generative organs of a number of plants it was ascertained that all the extracts, except for bearberry extract, did not appreciably influenced this indicator (table 1). In merged solutions of glucosinolates and bearberry absence of peak, which is typical of spectrophotometric content test of 5-vinyl-2-thiooxazolidone, can indicate either absence of goitrine or change of absorbing capacity of the solution in the same spectrum sector because of other compounds presence in volatile extracts.

Table 1

The content of 5-vinyl-2-thiooxazolidone (VTO) in the solutions of glucosinolates with addition of plants water extracts

Parts of plants	Botanical species of plants	VTO content
		mkg in the solution	% from the control
	Control (distilled water)	45.5 ± 1.0	100.0
Seeds	Hordeum vulgare L	46.5 ± 0.6	102.1
	Panicum miliaceum	48.0 ± 1.2	105.5
	Daucus sativus Hoffin.	55.3 ± 1.3	121.5
	Beta vulgaris L.	55.0 ± 0.8	120.8
	Petroselinum crispum Mill.	55.3 ± 1.1	121.5
Floscule	Tilia cordata Mill.	43.0 ± 1.0	94.5
	Helichrysum arenarium L.	42.8 ± 2.2	94.1
	Zea mays L.	43.2 ± 1.8	94.9
	Sambucus nigra L.	37.8 ± 1.4	83.0
	Taraxacum officinale Webb. ex Wigg.	46.6 ± 1.1	102.4
	Tussilago farfara L.	46.8 ± 0.2	102.8
Pine-needles	Pinus silvestris L.	44.9 ± 0.4	98.7
	Thuja occidentalis L.	43.2 ± 0.8	94.9
Herb	Calluna vulgaris L.	45.5 ± 1.5	100.0
	Mentha piperita L.	45.0 ± 0.3	98.9
	Hypericum perforatum L.	41.3 ± 0.6	90.8
	Polygonum aviculare L.	43.2 ± 0.6	94.9
	Arctostaphylos uvae-ursi (L.) Spreng.	0*	0
	Chelidonium majus L.	37.1 ± 1.7	81.5
	Bidens tripartita L.	38.5 ± 0.8	84.6
	Urtica dioica L.	42.0 ± 1.3	92.3
	Rumex crispus L.	49.0 ± 0.8	107.7
	Gnafalium uliginosum L.	45.5 ± 0.9	100.0
Fruits	Phaseolus vulgaris L.	46.7 ± 0.1	102.6
	Rhamnus cathartica L.	39.2 ± 2.3	86.2
	Capsicum annuum L.	46.8 ± 0.2	102.8
	Castanea sativa Mill.	53.9 ± 0.8	118.5
	Vaccinium myrtillus L.	46.0 ± 0.5	101.1
Bark	Quercus robur L.	39.2 ± 1.1	86.2
	Frangula alnus Mill	42.0 ± 2.2	92.3
Shoot	Salix fragilis L.	45.5 ± 1.4	100.0
	Solanum tuberosum L.	37.7 ± 0.3	82.8
Tissue juice	Aloe arborescens Mill.	49.7 ± 1.1	109.2

* Not measured spectrophotometrically (the typical peak is absent)

Carried out series of verifying experiments showed unfitness of quantitative detection of glucosinolates concentration according to 5-vinyl-2-thiooxazolidone content in bearberry extracts and hydroquinone and quinone solutions. At the same time for this purpose it was identified fitness of use of detection methods of glucosinolates decomposition extent by myrosinase according to content of glucose and isothiocyanates in the samples.

Thus analysis showed that in the bearberry solutions under study content of isothiocyanates was twice as low of the reference sample. Detection of isothiocyanates concentration in the solutions, in which basic water extract of bearberry was diluted 4 and 8 times, showed that there exists a certain interdependence between extract concentration and isothiocyanates content in merged solutions (table 2).

Table 2

The content of isothiocyanates in the solutions of glucosinolates depending on the concentration of water extracts of bearberry (A. uvae-ursi).

Concentration of the extract	General content of isothiocyanates
	Mkg in the sample	% from the control
Control (without extract)	41.5 ± 2.3	100
Basic solution, m : m (1 : 10)	19.9 ± 2.2	48
Diluted solution, m : m (1 : 40)	28.1 ± 1.3	68
Diluted solution, m : m (1 : 80)	27.8 ± 0.8	67

In our research we did not reveal inhibitory activity of exogenous myrosinase by Arctostaphylos uvae-ursi extract tannin. Thus after precipitation of the latter by gelatin stopping influence of the supernatant remained. Effect of the extract did not change also after boiling for 20 minutes.

The assumption that a possible inhibitor of myrosinase activity is hydroquinone, which is part of bearberry in bound (glucoside arbutin) and non-bound state, has proved to be true. After adding 5% concentration of hydroquinone to the reaction mixture there occurs myrosinase activity inhibition. In this case glucosinolates hydrolysis products are completely absent. There exists exact interdependence between hydroquinone quantity in the solution and glucosinolates decomposition extent, though its 0.01% concentration no longer affects enzyme activity.

While such compounds of phenolic series as phenol and resorcinol were absolutely inefficient in concentration 0.1%, quinone in the same concentration completely stopped glucosinolates hydrolysis, in concentration 0.01% it inhibited hydrolysis for 95% and in concentration 0.001% it did not virtually influence the process.

It should be pointed out that treatment of ground rape seeds with quinone in proportion 1:100 stops glucosinolates decomposition by endogenous myrosinase for 50%.

SUMMARY.

Taking into account chemical characteristics of quinone as well as hydroquinone (oxidizer, which is easily reducible and reducing agent, which is easily oxidised), and reagents system composition (raw glucosinolates extract, untreated preparation of myrosinase), we can state that the question of influence mechanism of these compounds on the process of myrosinase reaction is not cleared up yet and needs further investigation.

REFERENCES

1. Appelquist L. -A. and Josefsson E. 1967. Method for quantitative determination of isothiocyanates and oxazolidinethiones in diqest of seed meals of rape and turnip rape. Journal of the Science of Food and Agriculture.18:510-519.

2. Демьянчук Г. Т. 1985. Метод определения общего содержания глюкозинолатов в семенах крестоцветных культур. Селекция и семеноводство. 2:19.

3. Neuberg C. und Wagner J. 1926. Uber die Verschiedenheit der Sulfatase und Myrosinase. VIII. Mitteilung uber Sulfatase. Biochemische Zeitschrift. 174:457-463.

4. Ohtsuru M. and Hata T. 1979. The interaction of  L–ascorbic acid with the active center of myrosinase. Biochimica et Biophysica Acta. 567:384-391.

5. Youngs C. and Wetter L. 1967. Microdetermination of the major individual isothiocyanates and oxazolidinethione in rapeseed. Journal of the American Oil Chemists’ Society. 44:551-554.