The present study aimed to determine acute toxicity, the protective effect, and underlying mechanism of PM52, a combined extract of
Aging is a phenomenon leading to the dysfunction of normal cellular regulation including cognitive function. As the age advances, the cognitive capability is declined. Since the cognitive decline is the most costly, in terms of the financial, personal, and societal, it is regarded as a major health and social issue burden. Therefore, age-related cognitive memory impairment is one of the important health problems that should be concerned.
Current studies demonstrate that cognitive impairment in both aged human and rodents is correlated with the accumulation of oxidative damage to lipids, proteins, nucleic acids [
In traditional practices of medicine, numerous plants have been used to enhance cognitive function both in healthy individuals and those with diseases states such as mild cognitive impairment (MCI) and Alzheimer’s disease (AD). The herbal medicine can be used either by single herb or by polyherbal formulation. The concept of polyherbalism is very peculiar to Oriental Medicine such as Ayurveda and Traditional Chinese Medicine (TCM). It is believed that the polyherbalism can provide high efficiency partly due to synergism. Therefore, the herbal ingredients in these formulations are selected based on their healing property with respect to the disease condition such as antioxidant and acetylcholine inhibitory (AChEI) effects. In order to assure the safety and therapeutic efficacy, this study aimed to determine the acute toxicity and cognitive-enhancing effect of combined extract of
All plants materials used for the preparation of extract were purchased from organic farms of Srithat District, Udon Thani province. They were identified morphologically, histologically and authenticated by Associate Professor Panee Sirisa-ard, Faculty of Pharmacy, Chiang Mai University. Voucher specimens were kept at Integrated Complimentary Alternative Medicine Research and Development Group, Khon Kaen University. Powders of the
Total phenolic compounds of combined extract of
Total flavonoid content in extract of
Healthy male Wistar rats (180–220 grams, 8 weeks old) were obtained from National Laboratory Animal Center, Salaya, Nakhon Pathom. They were housed in group of 4 per cage in standard metal cages at
All rats were randomly assigned to 7 groups of 8 animals each.
Group I Vehicle + ACSF: rats were treated with vehicle at a period of 14 days before and 7 days after the administration of artificial cerebrospinal fluid (ACSF) via intracerebroventricular route bilaterally.
Group II Vehicle + AF64A: rats had been treated with vehicle for 14 days before and 7 days after the administration of AF64A, a cholinotoxin, in order to induce a cholinergic deficit as observed in MCI and early phase of AD.
Group III Donepezil + AF64A: animals were treated with Donepezil, a cholinesterase inhibitor which used as standard treatment for cognitive impairment. This group was used as positive control in this study.
Group IV Vitamin C + AF64A: animals were treated with Vitamin C (250 mg/kg BW), a well-known antioxidant which previously showed the neuroprotective and cognitive-enhancing effects. This group was also used as positive control in this study.
Group V–VII combined extract of
The animals determined the spatial memory at 7 days after AF64A administration.
Then, they were sacrificed and determined the density of survival neurons and in various subregions of hippocampus.
AF64A was prepared as an aqueous solution of acetylethylcholine mustard HCl (Sigma, St. Louis, MO, USA) and was adjusted to pH 11.3 with NaOH. After stirring for 30 min at room temperature, the pH was lowered to 7.4 with the gradual addition of HCl and stirred for 60 min. The amount of AF64A was then adjusted to 2 nmol/2
The Morris water maze test is one of the most important paradigms used for testing spatial navigation task, which is thought to be dependent on the proper functioning of the hippocampus. The testing apparatus for all tasks used in this study was a stainless-steel circular pool that 147 cm in diameter and 47 cm in depth. The interior of the pool was flat and the pool was placed on the steady floor. The pool was filled with water to a depth of 12 cm. The water was maintained at
The pool was divided into four quadrants (NE, NW, SE, and SW) by two imaginary lines crossing the center of the pool. For each animal, the invisible platform was placed in the center of one of the quadrants and remained there for a training period of 4 days. Each rat was gently placed in the water facing the wall of the pool from one of the four starting points (N, E, S, or W) along the perimeter of the pool, and the animal was allowed to swim until it climbed onto the platform. When an animal could not reach the platform in 60 s, it was gently placed on the platform by the experimenter. In either cases, the animal was left on the platform for 10 s and removed from the pool. Then, it was quickly dried with a towel before being returned to the home cage. The time which animal spent to find the immersed platform was regarded as escape latency. The 24 hr after the determination of escape latency, rats were reexposed to the same condition except that the immersed platform was removed and the time which the animal spent in the quadrant previously located in the immersed platform was recorded as retention time.
Following anesthesia with sodium pentobarbital (60 mg/kg BW), fixation of the brain
was carried out by transcardial perfusion with fixative solution containing 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.3. The brains were removed after perfusion and stored over a night in a fixative solution that used for perfusion. Then they were infiltrated with 30% sucrose solution for approximately 4°C. The specimens were frozen rapidly and 30
Five coronal sections of each rat in each group were studied quantitatively. Neuronal counts in hippocampus were performed by eye using a 40x magnification with final field 255
Hippocampus was isolated and prepared as hippocampal homogenate and the determination of the malondialdehyde (MDA) level and acetylcholinesterase (AChE) activity in hippocampus was performed. Malondialdehyde was indirectly estimated by determining the accumulation of thiobarbituric acid reactive substances (TBARS) while the activity of AChE was determined using Ellman method.
In order to determine the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), the brain tissues were weighed and homogenized with a buffer consisting of 10 mM sucrose, 10 mM Tris-HCl, and 0.1 mM EDTA (pH 7.4). Then the brain homogenates were centrifuged at 3000 g for 15 min at 4°C. The supernatant was used for bioassays. The activity of SOD was determined using a xanthine/xanthine oxidase system for the production of superoxide radical and subsequent measurement of cytochrome
Data are presented as mean ± standard error of mean (S.E.M). One-way analysis of variance (ANOVA), followed by Tukey post hoc test. A probability level less than 0.05 was accepted as significance.
To date, the use of plant-based formulations is leading to a fast growing market for Ayurvedic, nutraceutical, and polyherbal formulations. The development of polyherbal formulation has been regarded as a challenging task because of the large number of varied chemical compounds present in the different medicinal plants can possibly provide more benefit. However, during the formulation of new drugs or the reformulation of existing products, the interaction between active markers of various plant extracts also occurs resulting in changes in the chemical nature and therapeutic response. Therefore, the characteristic of PM52 has been developed and determined in this study. It was found that the extract contained the total phenolic compounds at concentration of 582.09 ± 8.72 mg of GAE/100 g of plant extract and contained total flavonoids at concentration of 18.80 ± 0.25 mg of QE/100 g of plant extract. Therefore, a combined extract of
Total phenolic compounds and total flavonoids content in
Tested substance | Total phenolic compounds | Total flavonoids |
---|---|---|
(mg of GAE/100 g of plant extract) | (mg QE/g of plant extract) | |
|
462.80 ± 4.59 | 10.90 ± 0.11 |
|
404.56 ± 3.06 | 8.38 ± 0.01 |
PM52 | 582.09 ± 8.72 | 18.80 ± 0.25 |
In the acute toxicity study, it was found that PM52 up to the level of 5000 mg/kg BW failed to exhibit the lethality and toxic symptoms. No behavioral change and macroscopic changes of histology of vital organs were observed. Further dosing to evaluate the LD50 of PM52 had not been performed. According to the Organization of Economic Cooperation and Development (OECD) guidelines for acute oral toxicity, an LD50 of 2000 mg/kg BW or above is categorized as unclassified and hence the product is found to be safe. Therefore, PM52 is safe especially for short duration application.
Figure
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on the escape latency and retention time evaluated using Morris water maze test in memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
Since PM52 exerted the cognitive-enhancing effect, the effect of PM52 on the neuron density in hippocampus was investigated. The results were shown in Figures
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on neuron density in CA1 of hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on neuron density in CA2 of hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on neuron density in CA3 of hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on neuron density in dentate gyrus of hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
Since acetylcholine has been reported to play the crucial roles on cognitive function especially learning and memory, this study also focuses on the alteration of the mentioned transmitter and the activity of AChE was used to indicate the alteration of acetylcholine. The effect of PM52 on the activity of AChE in hippocampus was investigated and the results were shown in Figure
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on the acetylcholinesterase inhibitory activity in hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
The data obtained from previous part had revealed the neuroprotective effect of PM52. Based on the crucial role of oxidative stress on the pathophysiology of neurodegeneration, this part was focused on the effect of PM52 on oxidative stress markers including MDA level and the activities of scavenger enzymes including SOD, CAT, and GSH-Px.
Figure
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on level of malondialdehyde (MDA) in hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
The effect of PM52 on the activities of SOD, CAT, and GSH-Px activities was shown in Figures
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on the superoxide dismutase (SOD) activity in hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on the catalase activity in hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
Effect of Donepezil, Vitamin C, and various doses of PM52 ranging from 2, 10, and 50 mg/kg BW on glutathione peroxidase (GSH-Px) activity in hippocampus of memory impairment rats induced by AF64A. Values are expressed as mean ± SEM. (
Medicinal plants have long been used in traditional folklore in various cultures throughout the world. Recently, a scientific interest for phytotherapy has increased in various aspects especially the researches targeting at justifying the reputations of medicinal plants in traditional folklore and at their possible underlying mechanism.
PM52 showed not only cognitive-enhancing effect but also neuroprotective effect. PM52 could attenuate the neurodegeneration and could disturb the function of the affected areas. It has been reported that neurodegeneration occurs as the result of various factors including oxidative stress. Previous study demonstrated that oxidative stress is strongly scavenged by polyphenolic compounds including flavonoids which are found in herbal extracts [
Hippocampus is regarded as a brain region essential for intact cognitive abilities and appears to be particularly vulnerable to the oxidative stress during aging [
Therefore, our data suggested that the cognitive-enhancing effect of PM52 might occur via 2 main mechanisms: (1) the suppression of AChE leading to the elevation of ACh, a neurotransmitter playing an important role on learning and memory and (2) the enhanced neuron density in hippocampus via the decreased oxidative stress induced by the increased antioxidant enzyme activities as shown in Figure
Schematic diagram of the possible underlying mechanism of PM52 to improve memory impairment in animal model of age-related cognitive decline condition such as mild cognitive impairment (MCI) and early phase of Alzheimer’s disease.
In this study, PM52 failed to show dose-dependent manner in both cognitive-enhancing effect and neuroprotective effect. The possible explanation might be related to the nonsimple relationship between the concentration of PM52 and the interested parameters such as spatial memory and neuron density. Since both the memory and survival of neuron were under the influence of numerous factors, it was not possible to observe the simple relationship between the concentration of PM52 and the interested parameters. In addition, PM52 is the combined extract of
The results obtained from this study confirm that PM52, a polyherbal formulation of ethanolic leaves extracts of
This study was supported in part by the National Research Council of Thailand, The Royal Golden Jubilee, Ph.D. Program, Research Affair Division, Faculty of Medicine, and the Integrative Complementary Alternative Medicine Research and Development Group, Khon Kaen University, Khon Kaen, Thailand.