Author's unit: 1. Guangzhou University of TCM, Guangzhou 510405. 2. Guangdong Gaoming Medical and Medicinal Institute of Encephalopathy, Gaoming, Guangdong 528500.

number: 970722); Hydergine was the product of Tianjin Huajin Pharmaceutical Factory (lot number: 981196); malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), cholinesterase (AChE) and total protein kit were products of Nanjing Jiancheng Bioengineering Institute.
Instruments A round plexiglass avoidance reaction case, 30 cm in diameter, was equipped with copper grid at the bottom of the case, the copper grid could be electrified, the voltage being 30 v, there was an insulation rubber stopper, 5 cm in diameter and height, in a corner of the case, which served as safety area. 722 grating spectrophotometer was the product of Shanghai Third Analytical Instruments Factory.
Methods
Administration by Grouping The 9-month-old mice were divided, randomly and according to body weight, into 6 groups: group of early senility, group of D-galactose-induced simulated senile mouse pattern, group of Hydergine and groups of low, moderate and high dosage of Naolizhibao; the 3-month-old mice served as group of young mice. Mice in the group of simulated senile mouse pattern and in the treatment groups were daily given intraperitoneal injection of 0.5 ml of 5% D-galactose for 6 successive weeks; (2) mice in the young group and in the early senility group were given equal volume of physiological saline by intraperitoneal route. Mice in the Hydergine group were given Hydergine in the dose of 0.6 mg·kg－1. Mice in the groups of low, moderate and high dosage of Naolizhibao were given Naolizhibao in the dosage of 2,4 and 8 g·kg－1, respectively. Mice in the young group, early senility group and group of simulated senile mouse pattern were given equal volume of physiological saline. Animals in all groups were administered by intubation feeding in accordance with 20ml·kg－1, once a day.
Test of Learning Capacity and Memory Six weeks later, jumping-platform training was carried out 2 hours following administration, at the beginning of the training mice were put in the round avoidance reaction case to adapt the environment for 2-3 minutes. Then the mice were gently put on the platform and when the animals jumped down from the platform and contacted the copper grid an electric stimulation of 30 v was given. The latency period for the mice to escape to the platform and the frequency for the mice to get an electric shock in 5 minutes (the number of mistakes) were recorded and served as targets of assessment of learning capacity. A test of memory was made 24 hours later. The mice were put on the platform and the time (latency period) of the first electric shock and the frequency of getting an electric shock in 3 minutes (the number of mistakes) were recorded and served as targets of assessment of memory. If any mouse remained on the platform for more than 3 minutes in the test, its latency period was counted by 180s.
Determination of Exponents of Brain, Spleen and Thymus Gland After the test of jumping platform, the mice were weighed and killed by decapitation. The brain, spleen and thymus gland were quickly removed and weighed, the exponents of brain, spleen and thymus gland were calculated, respectively.
Exponent of brain (spleen, thymus gland)=weight (mg) of brain (spleen, thymus gland)/body weight (g)
Detection of Biochemical Criteria The brain tissue after weighing was made into a 1: 10 (w/v) homogenate with precooling physiological saline and then subjected to centrifugation at 3 000 rpm for 10 minutes. The supernatant was aspirated and tested for SOD activity by the method of xanthine oxidase (3), for MDA level by thiobarbituric acid reaction, for GSH-Px activity by the direct method of DTNB (5), for AChE activity by the modified alkaline hydroxylamine colorimetric method (6), and for tissue protein content in the brain homogenate by Lowry's method . (7)
Statistical Method Experimental data were expressed by ±S, and t test was used in comparison among groups.
Results
Effect of Naolizhibao on the Appearance and Learning and Memory Capacity of D-Galactose-Induced Simulated Senile Mouse Pattern The results obtained showed that compared with the young group and the early senility group, mice in the D-galactose group had lusterless, withered and yellow hair, acted slowly and had obviously reduced autonomous activity and that those in all administration groups had obvious improvement. It can be seen from Table 1 that the learning and memory capacity of D-galactose mouse pattern declined obviously, as manifested by the prolonged latency period of escaping to the platform in training and the shortened latency period of remaining on the platform in testing, with increased frequencies of mistakes, the difference being significant or highly significant as compared with the young group and the early senility group and that mice in all administration groups had their learning and memory capacity improved, the difference being significant or highly significant as compared with the D-galactose group.

Table 1 Effect of Naolizhibao on learning and memory capacity of simulated senile mouse
pattern ( ±S, n=10)

Dosage Learning capacity Memory capacity
Group (g·kg－1) Latency Frequency Latency Frequency
period(s) of mistakes period(s) of mistakes
Young － 17.5±11.3** 3.8±2.1 128.4±52.0*** 1.8±2.0*
Early senility － 50.9±25.2* 4.5±2.8 88.9±32.1* 1.9±0.9**
D-galactose － 120.1±109.8 4.6±3.2 48.4±34.4 5.3±2.7
Hydergine 6×10－4 59.6±47.1 4.5±2.1 80.8±51.2* 2.4±1.8*
Naolizhibao 2 36.8±20.5* 3.3±1.7 98.0±67.0* 1.7±1.8*
Naolizhibao 4 36.9±25.0* 3.3±2.2 117.8±65.7** 1.4±1.7**
Naolizhibao 8 29.7±15.8** 3.7±2.6 108.9±66.1** 1.4±1.5**
Note: * p<0.05, ** p<0.01, *** p<0.001 compared with D-galactose group

Effect of Naolizhibao on Exponents of Brain, Spleen and Thymus Gland of D-Galactose-Induced Simulated Senile Mouse Pattern As shown in Table 2, compared with the young group and the early senility group the exponents of brain, spleen and thymus gland of mice in D-galactose group declined significantly and those of all administraion groups picked up to varying degrees.

Table 2 Effect of Naolizhibao on exponents of brain, spleen and thymus gland
of D-galactose- induced simulated senile mouse pattern

Dosage Exponents of viscera (mg/g of body weight)
Group (g·kg－1)
Brain Spleen Thymus gland
Yong － 13.5±1.6*** 5.2±1.2* 1.8±0.6***
Early senility － 12.1±1.2* 5.1±1.9 1.4±0.8*
D-galactose － 10.4±1.2 4.0±1.3 0.8±0.4
Hydergine 6×10－4 12.0±1.0* 5.4±1.7* 1.0±0.1
Naolizhibao 2 11.9±1.0 4.2±0.8 1.2±0.6
Naolizhibao 4 12.2±1.3* 5.2±1.1* 1.3±0.7
Naolizhibao 8 11.8±0.6* 5.3±1.5* 1.3±0.3**
Note: * p<0.05, ** p<0.01, *** p<0.001 compared with D-galactose group

Effect of Naolizhibao on MDA Level and Activities of SOD, GSH-Px and AChE in Brain Tissue of D-Galactose-Induced Simulated Senile Mouse Pattern Table 3 shows that compared with the young group and the early senility group the brain tissue of mice in D-galactose group had markedly elevated MDA level, markedly reduced activities of SOD and GSH-Px and markedly elevated activity of AChE, the difference being significant or highly significant and that the brain tissue of mice in all administration groups had reduced MDA level, enhanced activities of SOD and GSH-Px and reduced activity of AChE, the difference being significant or highly significant.

Table 3 Effect of Naolizhibao on MDA level and activities of SOD, GSH-Px and AChE in
brain tissue of simulated senile mouse pattern
Dosage MDA SOD GSH-Px AChE
Group (g/kg－1)
(n mol/mg) (n U/mg) (U/mg) (U/mg)
Young － 6.9±0.6 6.2±0.2 3.7±1.2** 1.5±1.0**
Early senility ＿ 7.1±0.1 6.2±0.2 3.0±0.4 2.4±0.6
D-galactose ＿ 7.4±1.2 6.1±0.2 2.4±0.8 3.1±1.1
Hydergine 6×10－4 5.5±0.8** 6.5±0.2** 3.3±0.7* 2.0±0.7*
Naolizhibao 2 4.1±2.0** 6.6±0.2** 3.6±0.8** 1.8±0.6*
Naolizhibao 4 3.6±1.2** 6.9±0.3** 4.2±1.0** 1.6±0.7*
Naolizhibao 8 2.8±1.3** 6.9±0.4** 4.3±0.8** 1.9±0.5*
Note: * p<0.05, ** p<0.01 compared with D-galactose group


Discussion
Subacute intoxication caused by D-galactose leads to production of large amount of superoxide anion free radicals in the body, they produce peroxidation injury to tissue, and the change in biochemical and tissue structures tallies basically with manifestations of natural senility, and it has been demonstrated experimentally that subacute intoxication caused by D-galactose leads to obvious injury in the aspects of learning, memory and behavior, similar to senile dysmnesia. (8)
The results of the present study showed that Naolizhibao significantly improved learning capacity and memory of D-galactose-induced simulated senile mouse pattern, suggesting that Naolizhibao may have the effects of enhancing immunity of the organism, increasing the activity of anti-oxidase, scavenging free radicals, reducing the products of peroxidation lipids, inhibiting AChE activity, reducing ACh decomposition and increasing ACh level in the brain, thus exerting the effect of improving such brain functions as learning capacity and memory.
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