quercetin, kaempferol, isoquercitrin, baicalin, apigenin, lu- teolin, hesperedin, naringenin, myricetin, etc.), tanning agents, polysaccharides, triterpenoids, organic ...
Pharmaceutical Chemistry Journal, Vol. 45, No. 8, November, 2011 (Russian Original Vol. 45, No. 8, August, 2011)
AMINO-ACID COMPOSITION AND NOOTROPIC PROPERTIES OF POLYNOOPHYT I. G. Nikolaeva,1, 2 L. D. Dymsheeva,2 S. M. Nikolaev,1, 2 and G. G. Nikolaeva1, 2 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 45, No. 8, pp. 45 – 48, August, 2011. Original article submitted October 14, 2008.
The nootropic activity of the complex plant preparation polynoophyt (polynoofyt) has been established in animal experiments. The amino-acid composition of the phytopreparation has been studied. A total of 19 amino acids including seven essential ones was identified. The most significant concentrations were found for aspargic acid, asparagine, glutamic acid, glutamine, proline, cystine, methionine, leucine, g-aminobutyric acid, alanine, and arginine. Key words: polynoophyt, nootropic activity, amino-acid composition.
Polynoophyt is a complex substance of plant origin that exhibits nootropic activity. It contains a number of biologically active compounds including flavonoids (hyperoside, quercetin, kaempferol, isoquercitrin, baicalin, apigenin, luteolin, hesperedin, naringenin, myricetin, etc.), tanning agents, polysaccharides, triterpenoids, organic acids, gallic acid, vitamin C, etc., that provide a wide range of pharmacological activity [1]. The goal of the present work was to study the amino-acid composition and nootropic activity of the phytoextract polynoophyt.
EXPERIMENTAL PHARMACOLOGICAL PART Experiments were performed on Wistar white rats of both sexes (170 – 200 g). Polynoophyt extract from which the alcohol was removed was injected into the stomach through a catheter in doses of 0.1, 0.3, and 0.5 mL/100 g once per day for 4 d and a fifth time 1 h before testing. The reference drug was piracetam at a dose of 200 mg/kg [2]. Control animals in all experiments received distilled water in an equivalent volume according to an analogous scheme. The nootropic activity of the extract was studied in healthy animals and with an experimental amnesia model. The influence of polynoophyt on the development of a conditioned reflex with positive reinforcement in a T-shaped labyrinth was studied [3]. Rats that were fasted for 48 h were placed into the starting section of the T-shaped labyrinth with a feeder containing food placed in one of the arms. The door of the starting section was opened 30 sec later. The noise of the door opening acted as the conditioned stimulus. Animals were placed for 1 h in the labyrinth on the first day in order to eliminate the orienting reaction. Training was carried out on the next 4 d. Each animal was placed into the labyrinth for five times in a row. The time for the animal to move from the starting section to the feeder and the number of correct and incorrect movements were recorded on each day of training. The criterion for development of a reflex was eight correct movements out of ten attempted. Sunflower seeds were used as the food reinforcement. The following parameters were measured in the experiments: reaction time (time between
EXPERIMENTAL CHEMICAL PART The amino-acid composition was studied on an AAA 339 amino-acid analyzer (Czech Rep.). The starting plant material was exhaustively extracted with hot water (extract 1) and EtOH (40%, extract 2). Extracts 1 and 2 were filtered and evaporated to dryness in vacuo. Free amino acids in the dry solids (accurate weights) were determined by dissolution in lithium-citrate buffer (pH 2.2), precipitation of proteins by sulfosalicylic acid solution (30%), centrifugation, and analysis. Bound amino acids were determined after acid hydrolysis by HCl solution (6 N) at 110°C for 24 h. 1 2
Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, Ulan-Ude, Buryat Republic, Russia. Buryat State University, Ulan-Ude, Buryat Republic, Russia.
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leaving the starting section and taking the food); number of reactions made (number of times the animal found reinforcement during testing for 3 min); number of mistakes (number of entries into the section opposite the required one). The effect of the phytoextract on the development of a visual differentiation conditioned response (VDCR) was also studied in healthy animals using recognition of visual signals with negative reinforcement [electrical paw skin stimulus (EPS)]. The VDCR was induced by placing animals in one section of a Y-shaped labyrinth. After 5 sec, a light was switched on in one of the other two sections for 5 sec, after which an EPS was applied to the animal’s paw through an electric floor. A conditioned response was considered developed if the animal made five correct movements in a row into the lighted section of the labyrinth before the EPS was applied. The number of attempts required for training before the first correct response and before the training criterion and the times for finding the “safe” section and for responding in seconds were recorded. The influence of polynoophyt on the disruption of cognitive functions in rats was studied using the retention of a passive avoidance conditioned response (PACR) under model amnesia conditions induced by the maximum electrical convulsion shock (MES). The MES was applied through electrodes placed on the surface of the ear lobes (80 mA, 0.2 – 0.5 sec exposure). The current affected the control and test animals immediately af-
TABLE 1. Amino-Acid Content in Polynoophyt Amino acid
ter training (i.e., after development of the conditioned response). Rats of the healthy group were not subjected to the MES [4]. The retention of the developed reflex was checked in the animals after 1 and 24 h and 7 d. RESULTS AND DISCUSSION Tables 1 and 2 present the results from the study of the amino-acid composition of polynoophyt. Tables 1 and 2 show that the developed nootropic agent was rich in amino acids. Significant amounts of aspartic acid, asparagine, glutamic acid, glutamine, proline, cystine, methionine, leucine, g-aminobutyric acid, alanine, and arginine were noted. A total of 19 amino acids were detected, 7 of which were essential. The test results showed that the time to move from the starting section to the food decreased on the 10th day of training in a T-shaped labyrinth of white rats that received polynoophyt in doses of 0.1, 0.3, and 0.5 mL/100 g by 16, 33, and 56%, respectively (Table 3). Furthermore, most animals developed the food-finding habit with regular administration of the test extract at the same doses on the 4th and 10th days. Administration of piracetam caused a decrease of the time for showing a response by 37%, development of a reflex on the fourth day in 40% of the rats; on the tenth day, in all animals in the group. Retention of the trained response in animals that received extract at a dose of 0.5 mL/100 g upon reproduction of the reflex after a one-week interlude was observed in all animals. This parameter was 50% in all other groups.
Amino-acid content, mg/g Dried aqueous extract Dried alcohol extract
Aspartic acid Threonine* Serine Aspargine Glutamic acid Glutamine Proline Glycine Alanine Valine* Cystine Methionine* Iosleucine* Leucine* Tyrosine Phenylalanine* g-Aminobutyric acid Ornithine Lysine* Arginine
1360.42 348.25 403.37 2968.3 1294.33 3178.24 9347.5 80.9 787.5 513.3 3464.9 1449.8 319.1 704.3 146.8 216.3 519.1
404.49 277.38 333.38 1162.4 904.81 1550.60 9258.1 54.5 641.3 376.8 2436.2 1005.0 309.1 441.8 124.9 178.6 311.7
47.2 84.5 1737.5
42.3 81.7 1389.1
Here and henceforth, * are essential acids.
TABLE 2. Content of Bound Amino Acids (After Acid Hydrolysis) in Polynoophyt Amino acid
Amino-acid content, mg/g Dried aqueous extract Dried alcohol extract
*
Lysine Histidine Arginine Aspartic acid Threonine* Serine Glutamic acid Proline Glycine Alanine Valine* Methionine* Isoleucine* Leucine* Tyrosine Phenylalanine* g-Aminobutyric acid
365.5 268.5 2586.9 4252.6 693.2 931.2 5704.5 8143.3 1031.9 1139.6 895.8 107.4 583.8 488.1 440.3 583.2 963.0
130.1 71.4 2362.2 2721.9 175.1 388.87 3221.49 7231.7 419.8 674.5 863.0 107.0 291.3 309.6 177.6 280.8 568.1
Amino-Acid Composition and Nootropic Properties of Polynoophyt
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TABLE 3. Effect of Polynoophyt on Development of a Conditioned Response in Healthy Rats with Positive Reinforcement in a T-Shaped Labyrinth Animal group Parameter
Reaction time, s
Polynoophyt, mL/100 g
Control (n = 10)
0.1 (n = 10)
0.3 (n = 10)
0.5 (n = 10)
Pyracetam, 200 mg/100 g, (n = 10)
32.0 ± 3.6
26.9 ± 2.9
21.4 ± 2.5*
14.1 ± 1.3*
20.2 ± 1.8*
20 50 50
30 50 50
50 80 50
50 100 100
40 100 50
Number of animals, % trained on the fourth day trained on the tenth day with response retention
Here and henceforth: * are reliable differences compared with those for control animals with p < 0.05; n is the number of animals in a group.
TABLE 4. Effect of Polynoophyt on Development of a Conditioned Visual Differentiation Response in Healthy Rats Number of tries spent in training
Time, s
Animal group until the first correct response
until training criteria
search for safe sector in one try
reactions made
Control (H2O) (n = 10)
8.4 ± 0.75
30.8 ± 1.22
15.3 ± 0.98
8.7 ± 0.96
Polynoophyt, 0.1 mL/100 g
9.1 ± 0.55
30.0 ± 1.63
15.1 ± 1.39
8.4 ± 0.6
Polynoophyt, 0.3 mL/100g
7.3 ± 0.35
24.3 ± 1.3*
8.8 ± 0.9*
7.8 ± 0.5
Polynoophyt, 0.5 mL/100 g
5.0 ± 0.58*
16.9 ± 1.54*
7.5 ± 0.79*
6.8 ± 0.51
Piracetam, 200 mg/kg
5.4 ± 0.83
17.4 ± 1.08*
5.8 ± 0.66*
7.2 ± 0.66
TABLE 5. Effect of Polynoophyt and Piracetam on Retention of Passive Avoidance Response in White Rats with Amnesia Induced by Maximum Electrical Shock (MES) Sample No.
Animal group
Before training
After 1 h
After 24 h
After 7 d
Latent period, s 1
Healthy (H2O) (n = 10)
5.4 ± 0.6
164.4 ± 17.0
145.5 ± 17.6
108.2 ± 19.4
2
Control (MES + H2O) (n = 10)
8.4 ± 1.9
88.0 ± 0.5
83.4 ± 12.0
22.7 ± 11.0
3
MES + polynoophyt, 0.3 mL/100 g (n = 10)
8.1 ± 1.3
84.0 ± 2.7
55.25 ± 1.9*
40.6 ± 1.5 *
4
MES + polynoophyt, 0.5/100 g (n = 10)
9.0 ± 1.5
119.6 ± 4.2*
91.2 ± 4.0
52.3 ± 4.9 *
5
MES + piracetam, 200 mg/kg (n = 10)
142.9 ± 17.1*
102.9 ± 17.4
62.0 ± 10.6 *
1
Healthy (H2O) (n = 10)
187.1 ± 4.0
23.3 ± 3.4
42.8 ± 6.5
70.0 ± 17.1
2
Control (MES + H2O) (n = 10)
181.6 ± 6.3
103.3 ± 19.4
101.0 ± 17.1
167.0 ± 14.1
6.0 ± 0.7 Time spent in dark chamber, s
3
MES + polynoophyt, 0.3 mL/100 g (n = 10)
187.7 ± 1.9
102.0 ± 2.8*
104.3 ± 36.6*
144.3 ± 4.8 *
4
MES + polynoophyt, 0.5/100 g (n = 10)
161.1 ± 10.5
51.8 ± 5.4*
81.2 ± 4.4*
130.0 ± 5.1 *
5
MES + piracetam, 200 mg/kg (n = 10)
192.7 ± 1.6
34.3 ± 17.9*
68.6 ± 14.2*
116.6 ± 25.2 *
Thus, this extract at all tested doses had a positive action on training and memory processes in healthy rats. The most pronounced effect, which was comparable with that of piracetam, was noted with polynoophyt at a dose of 0.5 mL/100 g. The study of the effect of polynoophyt extract on development of a VDCR in healthy animals based on recognition
of visual signals (light) with negative reinforcement found that regular administration of the test agent at doses of 0.3 and 0.5 mL/100 g improved the development of a VDCR in them. Thus, the number of attempts required for the first correct behavioural response to the visual stimulus (by 13 and 40%, respectively) decreased in animals of this group. Also, the extract stimulated significantly the memory of the train-
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ing criterion (five correct movements). The number of attempts required decreased by 21 and 45%, respectively (Table 4). The time required to find the safe section on the first attempt decreased by 42 and 51% with administration of polynoophyt at these doses to the animals; the time to make a response, by 10 and 22%, respectively. This indicated that random movements were replaced by directed ones. Administration to rats of polynoophyt at a dose of 0.1 mL/100 g did not have a significant effect on the development of a VDCR. The time required to find the safe section in one attempt decreased by 62 and 22% in animals that received piracetam and polynoophyt, respectively, at doses of 0.5 mL/100 g. The number of attempts required for training did not differ significantly from that for the group that received polynoophyt at a dose of 0.5 mL/100 g. The results indicated that regular administration of polynoophyt improved the development of a VDCR in rats. This effect was comparable with that of piracetam. The test results showed that the action of the MES on rat brain had an amnesiac effect, causing a sharp degradation of the reflex reproducibility. Thus, the latent period in control rats during the whole observation period (1 and 24 h and 7 d) decreased by 47, 43, and 79% whereas their total residence time in the dark section increased by 56, 58, and 51%, respectively, compared with those parameters for healthy animals (Table 5). Regular administration of polynoophyt at a dose of 0.5 mL/100 g and piracetam improved the development of PACR. This was manifested as an increase of the latent period by 35 and 62% and a reduction of the residence time in the dark section by 50 and 67%, respectively, one hour after training. Administration of the test drug at a dose of 0.3 mL/100 g had no effect on the training process. Ad-
I. G. Nikolaeva et al.
ministration to animals of extract at a dose of 0.5 mL/100 g and piracetam caused the memory to be retained more strongly. Thus, the latent period in rats of these groups was longer than that of control rats after 24 h by 9.3 and 23.4%; after 7 d, by 2.3 and 2.7 times, respectively. Obviously a distinct reduction of the reflex was observed with administration of piracetam. The results indicated that the plant agent polynoophyt improved cognitive functions with amnesiac action of a MES. Its anti-amnesiac action was evident upon administration of it at a dose of 0.5 mL/100 g but less pronounced than that of piracetam for this type of memory impairment. Thus, the plant agent polynoophyt exhibited nootropic activity, had a positive influence on training and memory processes in healthy rats, and improved cognitive functions with amnesiac action of a MES. The significant content of amino acids enables it to be used as a source of amino acids that together with other biologically active compounds are responsible for the nootropic activity of the plant agent. REFERENCES 1. I. G. Nikolaeva, L. D. Dymsheeva, S. M. Nikolaev, et al., Khim.-farm. Zh., 40(10), 41 – 45 (2007). 2. A. Meister, Biochemistry of the Amino Acids, Academic Press, New York (1957). 3. J. Bures and O. Buresova, Psychopharmacological Meeting, Iesenik Spa (1979), p. 7 – 12. 4. T. A. Voronina and R. U. Ostrovskaya, Handbook of Experimental (Preclinical) Study of New Pharmacological Substances [in Russian], Moscow (2000), pp. 153 – 161.
