inhibition of human blood acetylcholinesterase and butyrylcholinesterase by some alkaloids
TRANSCRIPT
0022-0930/02/3801-0035$27.00 © 2002 MAIK “Nauka/Interperiodica”
Journal of Evolutionary Biochemistry and Physiology, Vol. 38, No. 1, 2002, pp. 35—39. Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii,Vol. 38, No. 1, 2002, pp. 28—31.Original Russian Text Copyright © 2002 by Kuznetsova, Nikol’skaya, Sochilina, Faddeeva.
Inhibition of Human Blood Acetylcholinesteraseand Butyrylcholinesterase by Some Alkaloids
L. P. Kuznetsova, E. B. Nikol’skaya, E. E. Sochilina, and M. D. Faddeeva
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences,St. Petersburg, Russia
Received December 26, 2000
Abstract—A comparative study has been carried out on effects of berberine (diisoquinoline alkaloid) andsanguinarine and chelidonine (benzophenanthridine alkaloids) on erythrocyte acetylcholinesterase andserum butyrylcholinesterase from human blood. The studied alkaloids have been shown to be strong revers-ible inhibitors of the cholinesterase activity. Acetylcholinesterase is more sensitive to their action, thanbutyrylcholinesterase. The type of reversible inhibition was determined, and inhibitor constants were cal-culated. It is revealed that the character of inhibition is identical for the both cholinesterases. Berberine andsanguinarine are competitive-noncompetitive inhibitors, whereas chelidonine, a competitive inhibitor.
COMPARATIVE AND ONTOGENICBIOCHEMISTRY AND IMMUNOLOGY
INTRODUCTION
Isoquinoline alkaloids are known to have a widespectrum of biological activity. Some of them are usedin medicine for drug therapy [1, 2]. Of special inter-est are alkaloids isolated from plants, which have awide spectrum of pharmacological action, includingantitumor activity. Study of mechanisms of the cyto-toxic effect has revealed some molecular and intrac-ellular targets of their action. Specifically, alkaloidssanguinarine, chelethrine, and berberine, constituentsof the juice of the known medicinal plant, celandineChelidonium majus [3], are DNA-intercalators, whilesanguinarine and chelethrine also impair ATP syn-thesis in mitochondria. Apart from this, these alka-loids are also able to modify thiol groups of organiccompounds, including SH-dependent enzymes. Ac-tivity of these enzymes, particularly of SH-dependentmembrane-bound cation-transporting ATPases, isinhibited [4]. Among other cellular systems sensitiveto such agents, cholinesterase systems are to be takeninto account [5, 6]. Inhibition of acetylcholinesterase(AChE) of the rat brain [7] and butyrylcholinesteras-
es (BuChE) of human blood serum [8] by sangui-narine, chelethrine, and berberine, isolated from theplant material, was shown earlier. There are no dataon effects of such alkaloids on the AChE activity ofhuman blood erythrocytes.
Studied in the present work is the action of ben-zophenanthridine alkaloids, sanguinarine and cheli-donine, and of diisoquinoline alkaloid, berberine, onthe activity of erythrocyte AChE and serum BuChEfrom human blood. The studied alkaloids are presentin the celandine juice [3].
MATERIALS AND METHODS
Used in the work were acetylcholinesterase (AChE;acetylcholine : acetylhydrolase, EC 3.1.1.7) fromerythrocytes of human blood (HuAChE) with specif-ic activity of 3.2 U/mg, production of Perm NPO“Biomed,” and butyrylcholinesterase (BuChE; acyl-choline : acylhydrolase, EC 3.1.1.8) isolated from se-rum of human blood (HuBuChE) by the method de-scribed earlier [9], with specific activity of 7.4 U/mg.Acetylthiocholine iodide (AThCh; Chemapol, Cze-
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KUZNETSOVA et al.
chia) was used as substrate. Sanguinarine nitrate (Al-drich, USA), berberine sulfate (Sigma, USA), andchelidonine hydrochloride (Nowicky Pharma, Aus-tria) were used as inhibitors. Structural formulae ofthe studied compounds are presented below:
Sanguinarine and chelethrine are known to haveability to react with SH-groups of some organic com-pounds [4]. Therefore, to confirm correctness of theuse of the Ellman’s method, control experiments werecarried out and showed that the studied compoundsdid not react with thiocholine, the enzymatic reac-tion product.
The type of reversible inhibition of the enzyme wasdetermined by Lineweaver–Burk graphical method[11] in coordinates l/v vs 1/[S ], where v—initial rateof the enzymatic hydrolysis, [S ]—the substrate con-centration. Efficiency of the inhibitory effect of thecompounds was evaluated from the value of the gen-eralized inhibitory constant Ki related to the compet-itive Ki and the uncompetitive Ki inhibitory constantsby the equation 1/Ki = 1/Ki + 1/Ki ([5], p. 129). TheKi values were found by Dixon graphical method [12]in coordinates: 1/vi vs 1/[I ], where vi—initial rate ofthe enzymatic hydrolysis in the presence of the in-hibitor, [I ]—the inhibitor concentration. The Ki val-ues were determined by Cornish–Bowden graphicalmethod [12] in coordinates [S ]/v vs [I ].
RESULTS AND DISCUSSION
All studied alkaloids are reversible inhibitors ofHuAChE and HuBuChE. The degree of inhibitionreaches practically instantaneously a certain level anddoes not change with further increase of time of theenzyme interaction with inhibitors for more than30 min. Besides, the activity of both cholinesterasesis restored almost completely as a result of filtrationof the inhibited enzyme solution through a SephadexG-100 column.
To elucidate the type of reversible inhibition, thedependence is studied of the initial rate of the AThChcholinesterase hydrolysis on its concentration in thepresence of different concentrations of the alkaloids.It has turned out that the character of effects of thestudied alkaloids on the substrate hydrolysis underaction of HuAChE is similar to that under action ofHuBuChE. The arrangement of experimental straightlines in Lineweaver–Burk coordinates indicates thatberberine and sanguinarine are reversible inhibitorsof the mixed type for both cholinesterases. Figure 1presents an example of dependence of the initial rateof the AThCh hydrolysis under HuBuChE action inthe presence of sanguinarine on the substrate concen-tration: the experimental straight lines are intercrossed
Catalytic activity of cholinesterases was determinedby the method of Ellman [10] at pH 7.5 ± 0.1 andtemperature 25 ± 0.1°C. The rate of increase of opti-cal density of the reaction medium was measured us-ing a Specol-221 spectrophotometer (Germany) at412 nm. The enzymatic hydrolysis of substrate oc-curred in 0.02 M phosphate buffer in the presence of0.1 mM Ellman’s reagent (5,5´-dithiobis[2-nitroben-zoic acid]) at AThCh concentrations from 0.1 to2.0 mM. The reaction was triggered by addition of theenzyme to the sample containing the substrate andalkaloid.
´´
CH3ON
+
O
O
CH2
OCH3
Berberine
CH3N
+
O
CH2O
H2C
Sanguinarine
CH3N
O
CH2
O
H2C
HO
Chelidonine
´
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INHIBITION OF HUMAN BLOOD ACETYLCHOLINESTERASE
in the fourth quadrant. Chelidonine, unlike berberineand sanguinarine, is a competitive inhibitor of HuA-ChE and HuBuChE. As an example, the dependence,similar to that presented in Fig. 1, for HuBuChE andchelidonine is shown in Fig. 2. It is seen that the ex-perimental straight lines are intercrossed on the ordi-nate axis.
The values of constants of reversible inhibition ofthe cholinesterase hydrolysis of AThCh by alkaloidsare shown in Table 1. Based on values of the general-ized inhibitor constant, the studied alkaloids can beascribed to strong reversible inhibitors of cholinest-erases: the Ki values vary in the range from 0.23 to2.0 µM for HuAChE and from 3.2 to 76.9 µM forHuBuChE. For comparison, the tetramethylammo-nium cation considered a typical reversible inhibitorof cholinesterases inhibits AChE 150–1400 timesweaker: Ki = 300 µM, while HuBuChE, 50–1000 times weaker: Ki = 4500 µM [5].
Action of alkaloids on the HuAChE activity isstronger than on the HuBuChE activity. Sanguinarineacts on HuAChE 14 times more strongly than onHuBuChE, while berberine 20 times, and chelido-nine, 38 times. Out of the studied compounds, san-guinarine and berberine, which contain quaternaryatom of nitrogen, produce the strongest inhibitoryeffect on both enzymes. Chelidonine, free of the qua-ternary nitrogen atom, acts much more weakly, thiseffect being more pronounced in HuBuChE, than inHuAChE. Thus, chelidonine inhibits the HuAChE
activity approximately 9 times more weakly, than san-guinarine and berberine, while the HuBuChE activi-ty, approximately 20 times more weakly.
Comparison of values of competitive Ki and un-competitive Ki inhibitory constants indicates that thereversible mixed inhibition of HuAChE andHuBuChE in the presence of sanguinarine and ber-berine is of the competitive-noncompetitive type (α =Ki /Ki < 1), i.e., the competitive component makesthe chief contribution to the inhibition.
Comparison of the data obtained for berberine andsanguinarine with the data published earlier is diffi-cult, as in the works [7, 8], acetylcholine and bu-tyrylcholine were used as substrates, whereas effectsof the alkaloids on cholinesterase was estimated fromthe I50 value: an alkaloid concentration, at which theenzymatic reaction rate is reduced twice. This valueat reversible inhibition of enzyme is well known todepend on the substrate nature, whereas at compet-itive and mixed types of reversible inhibition, alsoon concentration of substrate, which is used for de-termination of this value. Taking this into account,we will try to compare our results with the publishedones. Thus, the I50 values presented in Table 2, indi-cate unequivocally that the action of berberine andsanguinarine on the BuChE activity is weaker, thanon the AChE activity. HuAChE is more sensitive,than the rat brain AChE, this effect being the mostexpressed for sanguinarine (approximately 40-fold),than for berberine (approximately 2-fold). The dif-
Fig. 1. Dependence of 1/v on 1/[S ] for human blood serumbutyrylcholinesterase at concentrations of sanguinarine(µM) 0 (1 ), 2.5 (2 ), 5.0 (3 ), 10.0 (4 ).
Fig. 2. Dependence of 1/v on 1/[S ] for human blood serumbutyrylcholinesterase at concentrations of chelidonine (µM)0 (1 ), 64 (2 ), 128 (3 ), 256 (4 ).
0.3
0.2
0.1
1/[ ] (Ì )S �1
1000 5000 10 000
4
3
2
1
0.3
0.2
0.1
1/[ ] (Ì )S �1
1000 5000 10 000
4
3
2
1
´
´
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KUZNETSOVA et al.
ferences in the type of the reversible inhibition bythe alkaloids of two AChE (noncompetitive for ratbrain homogenate and competitive-noncompetitivefor HuAChE) seem to be due both to the nature ofenzyme and substrate, and to conditions of perfor-mance of the reaction. Differences in the I50 valuesfor the purified and not purified HuBuChE are mostlikely to be due to that the reported data use differ-ent substrates and their different concentrations.Nevertheless, it might to suggested that the proce-dure of purification leads to an increase of sensitivi-ty of HuBuChE, probably owing to a decrease of theamount of ballast proteins present in the humanblood serum.
Thus, the performed investigation has shown thatberberine, sanguinarine, and chelidonine actively in-hibit cholinesterases of the human blood, whichshould be taken into account when studying thera-
peutic effects of these alkaloids to prevent their toxicaction.
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Table 1. Values of constants (mM) of reversible inhibition by some alkaloids of acetylthiocholine hydrolysis under action oferythrocyte acetylcholinesterase and serum butyrylcholinesterases from human blood (х ± Sx , n = 4–5)
Inhibitor Ki
Ki
Ki
Inhibition type
Human acetylcholinesterase
Berberine Mixed
Sanguinarine Mixed
Chelidonine � Competitive
Human butyrylcholinesterase
Berberine Mixed
Sanguinarine Mixed
Chelidonine � Competitive
0.26 ± 0.05 2.0 ± 0.3 0.23 ± 0.04
0.25 ± 0.05 3.1 ± 0.5 0.23 ± 0.04
2.0 ± 0.3 2.0 ± 0.3
5.2 ± 0.1 41.7 ± 0.8 4.6 ± 0.8
3.8 ± 0.08 19.7 ± 0.4 3.2 ± 0.4
76.9 ± 8.0 – 76.9 ± 8.0
Table 2. I50 values (M) of inhibition by some alkaloids of human blood serum butyrylcholinesterase and acetylcholinest-erase of human blood erythrocytes and rat brain
Alkaloid
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inhibition
type
inhibition
type
inhibition
type
inhibition
type
Berberine
Competitive-
noncompeti-
tive
Competi-
tive
Competitive-
noncompeti-
tive
Competi-
tive
Sanguinine
Competitive-
noncompeti-
tive
Competi-
tive
Competitive-
noncompeti-
tive
Competi-
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2.6 × 10–5 > 1 × 10–4 5 × 10–7 9.8 × 10–7
1.0 × 10–5 2.4 × 10–5 8 × 10–7 3.5 × 10–5
1 × 10–3 Macetylcholine
1 × 10–3 Mbutyrylcholine
1 ×10–3 Macetylcholine
1 × 10–3 Macetylcholine
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INHIBITION OF HUMAN BLOOD ACETYLCHOLINESTERASE
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