Ameliorative effect of betulin from Betula platyphylla bark on scopolamine-induced amnesic mice

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disease induced by cholinergic neuron damage or amyloid-beta aggregation in the basal forebrain region and resulting in cognitive disorder. We previously reported on the neuroprotective effects of Betula platyphylla bark (BPB) in an amyloid-beta-induced amnesic mouse model. In this study, we obtained a cognitive-enhancing compound by assessing results using a scopolamine-induced amnesic mouse model. Our results show that oral treatment of mice with BPB and betulin significantly ameliorated scopolamine-induced memory deficits in both passive avoidance and Y-maze tests. In the Morris water maze test, administration of BPB and betulin significantly improved memory and cognitive function indicating the formation of working and reference memories in treated mice. Moreover, betulin significantly increased glutathione content in mouse hippocampus, and the increase was greater than that from betulinic acid treatment. We conclude that BPB and its active component betulin have potential as therapeutic, cognitive enhancer in AD.

Graphical abstract

Betulin and BPB Significantly Ameliorated Scopolamine-Induced Memory Deficits in Mice in the Passive Avoidance, Y-maze, and Water Maze Tests.

Key words:

• betulin
• passive avoidance
• Y-maze
• Morris water maze
• cognitive enhancement

Alzheimer’s disease (AD) is a neurodegenerative disorder of the brain common in the elderly.¹⁾ One of the most progressive symptoms of AD is memory loss followed by cognitive impairment.²⁾ Predominant causes of AD are cholinergic neuron damage, aggregation of hyper-phosphorylated tau protein, and oxidative stress in the basal forebrain region.^2–4) Among the neurotransmitter systems, cognitive functional impairment within the cholinergic system has been reported to be a main cause of, and an early marker for, AD.⁵⁾ Scopolamine, a cholinergic antagonist, can interrupt acetylcholine transmission in the central nervous system resulting in impairment of learning and memory.⁶⁾ Animal models of scopolamine-induced amnesia have been used to search for candidate agents with potential therapeutic value in AD treatment.⁷⁾ In our studies, we have used an animal model to search for and evaluate agents producing cognitive improvement.

In the course of searching for cognitive-enhancing agents from natural products, we observed that the total extract and fractions of Betula platyphylla bark (BPB) had significant cognitive-enhancing activity in a scopolamine-induced amnesic animal model. The bark of B. platyphylla, a birch tree widely distributed in Korea, is well known as a traditional medicine for arthritis, nephritis, and dermatitis treatment in China, Korea, and Japan.^8,9) Platyphylloside, aceroside VIII, and betulin are three of the main bioactive constituents of B. platyphylla and are reported to be responsible for a variety of biological activities of B. platyphylla including antioxidant, anti-inflammatory, anti-arthritis, and anticancer activities.^8,9) In our previous study, we reported on the neuroprotective effects of BPB and its major diarylheptanoids (platyphylloside and aceroside VIII) in vitro by using hippocampal cells and in vivo by using an amyloid-beta-induced amnesic mouse model.^10,11) However, till now any research about the effect of betulin on cognitive improvement in vivo had not been reported yet.

Betulin, a pentacyclic triterpene, is found predominantly in fruit peel, leaves, and Betula spp. stem bark. Betulin has been reported to have many pharmacologic properties including anti-inflammatory, anti-bacterial, and anti-HIV effects.¹²⁾ However, to the best of our knowledge, there is no widespread use of this easily isolable compound as a memory-enhancing agent. Because of their structural similarities, betulin, and its biologically derivative betulinic acid, have been studied simultaneously.^13,14) Betulin can be easily converted to betulinic acid and some reports have compared the effects of betulin with those of betulinic acid in a broad spectrum of biological and pharmacological activities.^13–15) In this study, via passive avoidance testing of mice, we examined whether betulin and betulinic acid could mitigate scopolamine-induced memory deficits. Furthermore, we evaluated the effects of betulin and betulinic acid on glutathione (GSH) levels. In addition, we undertook Y-maze and Morris water maze testing to investigate the reference and working memory-enhancing effects of betulin and BPB treatment in a scopolamine-induced amnesic mouse model.

Materials and methods

Experimental animals

Male ICR mice, obtained from the Experimental Animal Breeding Center of Seoul National University, Seoul, Korea, and weighing 25–30 g underwent passive avoidance, Y-maze, and Morris water maze testing after a one-week adaptation period (20–23 °C; 12 h light cycle from 09:00 to 21:00 local time; Agribrand Purina Korea food and water ad libitum). All experiments were conducted in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals of Seoul National University.

Sample preparation

The plant material (500.0 g) was ground and extracted with CHCl₃ at room temperature. The CHCl₃ extract was concentrated in vacuo to give a crude extract (52.5 g). The CHCl₃ extract was then suspended in H₂O and partitioned successively with n-hexane. After the removal of the non-polar organic constituents, the residues were loaded onto silica gel column chromatography and sequentially eluted with 150.0 mL of hexane-EtOAc (10:1). And then, the column was continuously eluted with 500.0 mL of hexane-EtOAc (4:1) with 500 drop fractions retained in each collection tube and betulin (10.5 g) was isolated from each tube by recrystallization and demonstrated to be pure (>97% purity) by performing high-performance liquid chromatography on the basis of its UV absorption at 200 nm. Donepezil (Aricept®) was used as a positive control. Betulin, betulinic acid, or BPB total extract were administered orally to mice 90 min before scopolamine treatment. Scopolamine was given subcutaneously 30 min before behavioral testing and was purchased from Sigma-Aldrich Chemical (St Louis, MO, USA).

Passive avoidance test

Training for and testing of passive avoidance performance were carried out in two identical light and dark square boxes (GEMINI, San Diego Instruments, San Diego, CA, USA) as described in our previous report.¹⁶⁾ Initially, a mouse was placed in the light chamber, and 10 s later, the door between the light and dark compartments was opened. When the mouse entered the dark compartment, the door automatically closed and a 2-s electrical foot shock (0.1 mA/10 g body weight) was delivered through stainless steel rods (one trial training). Twenty-four hours after that single training trial, the mouse was again placed in the light compartment. The escape latency, the time before entering the dark compartment, was recorded. If the mouse did not enter the dark compartment within 180 s, the experiment was stopped.

Y-maze test

The Y-maze test was performed as previously described.¹¹⁾ The Y-maze had three arms placed at equal angles. Each arm was 20 cm long and 5 cm wide with walls 12 cm high. Initially, a mouse was placed within one arm, and allowed to explore the Y-maze for 8 min. The sequence and total number of arms entered were recorded. Arm entry was considered complete when the hind paws of the mouse were completely within the arm. The percentage alternation among arms was calculated by using the following equation:$$\text{Alternation}(\%)=[(\text{number}\text{of}\text{alternations})/(\text{total}\text{arm}\text{entries}-2)]\times 100.$$

Morris water maze test

The Morris water maze test was performed as previously described.^11,17) The white pool was circular (90 cm in diameter and 45 cm in height) filled with water (20 ± 1 °C) in which 500 mL of milk was mixed. The pool was divided into four quadrants of equal area. A white platform (6 cm in diameter and 29 cm in height) was centered in one of the four pool quadrants. Each mouse was allowed to swim for training purposes, and in the days following training, the mice underwent two trial sessions each day for four consecutive days. During each trial session, the time taken to swim to the platform (escape latency period) was recorded. Once the mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 120 s, it was placed on the platform for 10 s and then removed from the pool by the experimenter. For each mouse, the initial trial on each day (trial 1) was followed by a second trial (trial 2) after an inter-trial interval of 20 min. The point of entry into the pool and the location of the escape platform remained unchanged between trial 1 and trail 2, but was both locations were changed each day. Water maze escape latency was averaged for each session of two trials and for each mouse. A day-to-day change in escape latency in trial 1 indicated a change in long-term or reference memory, while a change from trial 1 to trial 2 indicated a change in short-term or working memory. For the probe trial, a mouse was placed in the quadrant located diagonally from the target quadrant and allowed to swim to the quadrant from which the escape platform had been removed for a maximum of 60 s. Escape latency, escape distance, and swimming speed of each mouse were monitored by a video-tracking system (Smart 2.5, Harvard Apparatus, Holliston, MA, USA).

Evaluation of GSH content

Following completion of the passive avoidance test, each tested mouse was euthanized. The cerebral hippocampus was dissected rapidly under standard conditions at 4 °C and was homogenized in 0.1 M phosphate buffer (pH 7.4). The homogenate was centrifuged for 30 min at 3000 × g at 4 °C and the supernatant (cytosolic and mitochondrial fractions) was collected to determine GSH content. Total GSH in the supernatant was determined spectrophotometrically using an enzymatic cycling method.¹⁸⁾

Statistical analysis

All data were expressed as a mean ± SEM. The results of the behavioral tests and assays were analyzed by GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA, USA.). Statistical test results were considered statistically significant if the occurrence probability (p) was 0.05 or less.

Results and discussion

We previously reported on the neuroprotective effects of BPB in amyloid-beta-induced amnesic mouse model.¹¹⁾ In this study, we examined the cognitive-enhancing effects of BPB and its major component betulin in a scopolamine-induced amnesic mouse model by using passive avoidance, Y-maze, and water maze tests. Betulin, which comprised up to 37% of the total extract of BPB, was obtained by bio-guided fractionation and recrystallization from BPB.

The cognitive-enhancing effects of BPB and betulin were evaluated by using the passive avoidance test. In our light–dark box experiment, escape latency (25 ± 5 s) was well reduced in scopolamine-treated (1 mg/kg body weight, s.c.) mice compared to the latency in the control group (168 ± 15 s). At treatment concentrations of 50 and 100 mg/kg, BPB total extract and the CHCl₃ fraction of B. platyphylla showed cognitive-enhancing effects (Fig. 1S). The reduction in step-through latency induced by scopolamine was significantly reversed by treatment with betulin at concentrations from 1 to 10 mg/kg (Fig. 1). At a betulin dose of 5 mg/kg, the scopolamine-reduced step-through latency was significantly recovered to a latency (140 ± 25 s) comparable with that from donepezil treatment (145 ± 20 s), the positive control treatment. Our in vivo results indicate that betulin (5 mg/kg body weight p.o., 83%) to ameliorate reduced cognitive activity is comparable to that of donepezil (5 mg/kg body weight p.o., 86%), which is widely used as a treatment for AD. Moreover, the memory-enhancing effect of betulin was greater than that of betulinic acid (5 mg/kg body weight p.o., 57.5%), a widely used biological derivative of betulin.

Fig. 1. Effects of betulin and betulinic acid on the scopolamine-induced amnesic mice in the passive avoidance test.

Notes: Two hours before the training trial, mice received test samples (p.o.). After 90 min, amnesia was induced in orally treated mice with by scopolamine injection (1 mg/kg body weight, s.c.). Twenty-four hours after the training trial, the mice were again placed in the light compartment. The escape latency time from light compartment placement to entry into the dark compartment was recorded. Values shown are the mean latency ± SEM. Significant differences indicated by ###p < 0.001 vs. the control group and *p < 0.05 and ***p < 0.001 vs. the scopolamine-treated group.

The Y-maze test has been used to evaluate changes in short-term or working memory.¹⁹⁾ In our assessment of changes in spontaneous alternation behavior, the scopolamine-injected group exhibited a significantly lower alternation percentage (42 ± 5%) from that in the control group (61 ± 3%). Treatment with betulin at a dose of 5 mg/kg, which was the most effective dose in the passive avoidance test, resulted in recovery of the reduced alternation behavior in scopolamine-injected mice to 52 ± 1%, similar to the level obtained in the BPB total extract treatment group (Fig. 3(A)). Our results support previous reports indicating that betulin and BPB total extract can improve scopolamine-induced short-term or working memory impairment.^16,17)

Scopolamine has been used to induce central cholinergic blockade resulting in temporary cognitive impairment in amnesic mouse model.⁶⁾ Moreover, this muscarinic receptor antagonist interferes with memory and cognitive function and can subsequently result in impairment of working (i.e., short-term) and reference (i.e., long-term) memories.²⁰⁾ For simultaneous analysis of changes in working and reference memories, we used the Morris water maze test.^20,21) Scopolamine at a dose of 1 mg/kg was used to induce a memory deficit in the tested mice; a concentration reported to have no effect on swimming ability or acquisition latency and can be dissociated from hyperactivity caused by the drug.^20,22) The Morris water maze test results showed that the control group rapidly recognized the location of the submerged platform on their first training day and reached stable levels of escape latencies as days go by, whereas the scopolamine-injected group failed to arrive at the maze platform until the maximum time limit (120 s) on days 1 and 2 (Fig. 2(A)). The long escape latency in the scopolamine-injected group was significantly decreased in scopolamine-injected mice treated with BPB and betulin. The amnesic mice treated with betulin at a dose of 5 mg/kg exhibited a shorter escape latency than that in the scopolamine-injected mice during training, but a statistically significant reduction in escape latencies from trial 1 to trial 2 was not observed on days 1 and 2. The mouse group treated with betulin showed a marked reduction in escape latencies from trial 1 to trial 2 on days 3 and 4, suggesting that the betulin treatment of amnesic mice can significantly improve deficits in working or short-term memories. In addition, amnesic mice treated with BPB showed a gradual reduction in escape latency over the four training days. In our experiments, escape path length results were similar to the escape latency results, further indicating the cognitive-enhancing effects of betulin and BPB (Fig. 2(B)). In addition, the BPB-treated mouse group showed memory-improvement from days 2 to 4. We further investigated swimming speed (i.e., velocity) by dividing the escape path length by the escape latency period (Fig. 2S). The velocity results showed that neither scopolamine nor betulin treatments affected swimming speed, indicating the soundness of our experimental approach. The probe trails followed the acquisition tests (Fig. 3(B)). Compared to the scopolamine-treated group, the scopolamine-treated mice that were also treated with betulin and BPB remained significantly longer in the target quadrant, indicating that betulin and BPB treatment could improve spatial memory.

Fig. 2. Effects of betulin and BPB on the scopolamine-induced amnesic mice in the Morris water maze test.

Notes: During acquisition trials using a submerged platform in the Morris water maze test, escape latency (A) and escape distance (B) of each mouse were monitored by a video-tracking system. Each day, mice were treated with betulin (5 mg/kg body weight, p.o.) and BPB (100 mg/kg body weight, p.o.). After 90 min of treatment, amnesia was induced by scopolamine injection (1 mg/kg body weight, s.c.). All mice were tested for spatial memory 30 min after scopolamine injection. Results significantly different from the values in trial 1 are indicated by *p < 0.05 and ***p < 0.001.

Fig. 3. The enhancing effects of betulin and BPB on spatial memory impairment induced by scopolamine in mice.

Notes: (A) Effects of betulin and BPB on scopolamine-induced memory deficit in the Y-maze test. Samples were administered orally to mice 60 min before the tests. (B) Effects of betulin and BPB during the probe trial of the Morris water maze test. Probe trial sessions were carried out for 60 s. Values are mean ± SEM. Significant differences indicated by ^###p < 0.001 and ^#p < 0.05 vs. the control group and *p < 0.05 vs. the scopolamine-treated group.

To elucidate the effects of betulin on GSH levels in brain tissue, we measured cellular GSH levels in the hippocampus of mice euthanized after completion of a passive avoidance test (Table 1). Many studies have reported the linear relationship between reduced GSH levels and decreased cognitive status of AD patients.^23,24) The sulfur-containing tripeptide GSH is also known to be involved in several cellular functions in scopolamine-induced memory impairment which might decrease cognitive function in brain.^23,24) In this study, compared to the control group, scopolamine-treated mice had a lower GSH level in the hippocampus; however, the difference was not significant. Regardless, at all of the tested betulin concentrations, betulin-treated mice had significantly higher GSH levels than the scopolamine-treated mice.

Table 1. The effects of betulin and betulinic acid on GSH levels in the hippocampus of scopolamine-injected mice.

	Total GSH (nmol/mg of protein)
Control	7.264 ± 0.660
Scopolamine	6.840 ± 1.026
Betulin 1 mg/kg	8.779 ± 0.448
Betulin 5 mg/kg	11.800 ± 0.784*
Betulin 10 mg/kg	10.694 ± 0.676*
Betulinic acid 5 mg/kg	9.065 ± 1.888

Note: GSH content was measured as described in materials and methods. Values represent the mean ± S.D. Values significantly different from the scopolamine group value are indicated by *p < 0.05 or **p < 0.01.

Betulin, a triterpene (lup-20(29)-ene-3β,28-diol), is naturally occurring and abundant in various bushes and trees. Betulin has three structural positions; namely, a primary hydroxyl moiety at position C-2, a secondary hydroxyl moiety at positions C-3 and 28, and an alkene moiety at position C-20, where chemical modification can yield betulinic acid.¹⁵⁾ Several studies have compared the bioactivities of betulin and betulinic acid, because betulin is a precursor for betulinic acid and may have different pharmacological activity.^13,14) Betulin is lipophilic and poorly soluble due to its structural characteristics,¹⁵⁾ which might affect bioactivity assay results, mainly in vitro assays including cell cultures.^15,25) Our in vitro assay showed that betulinic acid was slightly more active than betulin in acetylcholine esterase inhibitory and anti-inflammatory effects (Figs. 3S, 4S). However, our in vivo assessment of scopolamine-induced amnesic mice demonstrated that betulin was more effective than betulinic acid in cognitive-enhancing activity. Although it is necessary to evaluate the antioxidant mechanism of betulin against scopolamine-induced oxidative stress in mouse brain, we showed the GSH increased potential of betulin in hippocampus. The difference in the cognitive-enhancing activity of these two pentacyclic triterpenes, despite their structural similarity, might be related, in part, to their different distribution constants.^25,26)

In this study, we show for the first time the memory-enhancing potential of betulin. Although more in vivo studies are needed to elucidate the mechanism of mammalian metabolites of betulin, we suggest that betulin has potential as a candidate as cognitive-enhancing therapeutic agent to attenuate the progression of AD. In addition, betulin and BPB have the potential, through their neuroprotective activity, to recover memory deficits. If those potentials are achieved, industrial-scale extractive isolation of betulin from BPB could be a source of a large supply of raw material as neuroprotective agent.^8,15)

Author contributions

Namki Cho and Sang Hyun Sung conceived and designed the experiments. Namki Cho, Hyeon Woo Kim, and Hee Kyoung Lee performed the experiments. Namki Cho, Byung Ju Jeon, and Sang Hyun Sung analyzed the data. Namki Cho, Byung Ju Jeon, and Sang Hyun Sung contributed reagents/materials/analysis tools. Namki Cho and Sang Hyun Sung wrote the manuscript.

Supplemental material

The supplemental material for this paper is available online at http://dx.doi.org/10.1080/09168451.2015.1072460.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the Forest Science and Technology Project [S121214L120100] with funding provided by the Korea Forest Service.