|Year : 2015 | Volume
| Issue : 1 | Page : 13-19
Screening of novel polyphyto formulations, a natural remedies for learning and memory enhancing properties in rat
Shibnath Kamila1, Nookala Venkata Satheesh Madhav2, Chandra Nath Sarkar3
1 Department of Pharmacology and Pharmacy Practice, Research Scholar, Uttarakhand Technical University, Uttarakhand, India
2 Department of Pharmacy, Dehradun Institute of Technology University, Faculty of Pharmacy, Dehradun, Uttarakhand, India
3 Department of Nephrology, Fortis Hospital, Anandpur, Kolkata, West Bengal, India
|Date of Web Publication||27-Jan-2015|
Department of Pharmacy Practice, Bharat Institute of Technology, Ibrahimpatnam, Mangalpally, Ranga Reddy - 500 010, Telangana
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The present study objective is to find out the effectiveness of the two medhya (polyphyto) formulations for its learning and memory activity. Materials and Methods: There is a hypothesis is to slow down the brain degeneration caused by different pathophysiology with natural treatments and it has shown the promising effect in the treatment of memory loss. The two medhya formulation test drugs are FM01 (Habiscus rosasinensis, Oscimum sanctum, Santalum album, Phyllanthus Emblica, Convolulus pluricaulis, Bacopa monnieri), and FM02 (Convolulus pluricaulis, Abelmoschus esculentus, Terminalia Arjuna, Phyllanthus Emblica, Avena sativa) at a dose of (50 mg and 100 mg/kg p.o.) studied against standard drug Bacopa monnieri having potential effect to improving memory, evaluate it using three different animal model like Elevated plus maze (EPM), Morris water maze (MWM) and Pole Climbing apparatus (PCA) for the effect of nootropic action. Results: On administration of polyphyto formulations FM01 and FM02 showed significant (P < 0.001) reduction in transfer latency in EPM, MWM and escape latency in PCA test as compared with the control; whereas FM01 is comparatively better than FM02 and Bacopa monnieri. Conclusion: It was concluded that the nootropic polyphyto FM01 formulation composed of (Habiscus rosasinensis 30%, Oscimum sanctum 20%, Santalum album 15%, Phyllanthus Emblica 15%, Convolulus pluricaulis 10%, Bacopa monnieri 10%) having promising significant effect for enhancing learning and memory properties can be use for memory loss or dementia and prophylactically can be use to prevent neurodegeneration.
Keywords: Dementia, elevated plus maze test for memory, learning and memory, medhya or intelligence, pole climbing test for memory, Santalum album, Terminalia arjuna
|How to cite this article:|
Kamila S, Madhav NV, Sarkar CN. Screening of novel polyphyto formulations, a natural remedies for learning and memory enhancing properties in rat. Int J Nutr Pharmacol Neurol Dis 2015;5:13-9
|How to cite this URL:|
Kamila S, Madhav NV, Sarkar CN. Screening of novel polyphyto formulations, a natural remedies for learning and memory enhancing properties in rat. Int J Nutr Pharmacol Neurol Dis [serial online] 2015 [cited 2020 Jun 1];5:13-9. Available from: http://www.ijnpnd.com/text.asp?2015/5/1/13/150067
| Introduction|| |
Herbal remedies for Alzheimer's disease or use to improving memory have become more and more popular in the recent years and not without a reason that there is a possibility to slow down the brain's degeneration caused by different pathophysiology with natural treatments and it has shown the promising effect in the treatment of memory loss. The researched on natural herbal medicines have been shown the benefits derived from using herbal treatments for Dementia have been very promising and effective for improving learning and memory. All the nootropic medicinal herbs may hold the key to the cure to this severe disease and second, the herbs are inexpensive and can be easily obtained. Also Clinical research is being conducted to test the efficacy of herbal medicines vis-a-vis prescription medicines in treating Alzheimer's or dementia patients. The results are promising, as herbal products have been found to be not only as effective as prescription drugs but also with fewer side effects. Herbal supplements may be used as a substitute for pharmaceutical drugs or can be used in conjunction with the latter.
The loss of memory in Dementia or in Alzheimer's disease is the main challenges of treatment. Memory is the ability of an individual to record sensory stimuli, events, and information; retain them over short or long periods of time; and recall the same at a later date when needed. Poor memory, lower retention, and slow recall are common problems in today's stressful and competitive world. The drug use for treatment of Alzheimer's disease is effective for short term and symptomatic only. Another way, Cholesterol-lowering drugs called statins, anti-oxidants, and anti-inflammatory drugs prevent formation of beta-amyloid plaques, nerve growth factor to keep neurons healthy.  There are several studies and alternative therapies that offer ways to slow the onset and progression of Alzheimer's disease in some patients.  Important plants that act on the nervous system and improve memory include: ''Japapushpa'' (Habiscus rosasinensis),  ''Tulsi'' (Oscimum sanctum),  "Chandana" (Santalum album),  ''Shankhapushpi'' (Convolvulus pluricaulis),  ''Brahmi'' (Bacopa monniera),  and "Jai" (Avena sativa).  The plants that act by anti-inflammatory activity is "Amla" (Phyllanthus Emblica),  and act by reducing free radical and antioxidant property are "Bendakaya" (Abelmoschus esculentus),  "Arjuna" (Terminalia arjuna). 
| Materials and methods|| |
Plant material and preparation of formulation
Different parts of all plants were obtained from local sources and were identified and authenticated by department of botany, Sri Venkateswara University, Tirupati. Various Plant parts were air dried in the dark, and grounded into a fine powder and passes it through a # 100 sieve, then prepared two formulations FM01 and FM02 according to their composition shown in [Table 1].
Standard drug as Bacopa monnieri
Bacopa monnieri has been shown to be very useful in improving learning and memory. , Bacopa monnieri, a member of the Scrophulariaceae family, is a small, creeping herb with numerous branches, small oblong leaves, and light purple flowers.  It has been used in Ayurvedic medicine and traditional treatments for a number of disorders, particularly those involving anxiety, intellect, and poor memory.  The plant has prominent action on the central nervous system, where it improves understanding, memory, intellect, and speech, and corrects aberrations of emotions, mood, and personality of an individual. Animal studies have found Bacopa monnieri attenuates scopolamine-induced dementia, and anticholinesterase activity has been demonstrated.  Preclinical and clinical studies have shown that Bacopa monnieri improves memory and mental function.  The plant, plant extracts and isolated bacosides have been investigated for nootropic activity. A recent study reveals B. monnieri extract is able to reduce amyloid levels in PSAPP mice which is a transgenic mice expressing the "Swedish" amyloid precursor protein and M146L presenilin-1 mutations. 
Adult albino wistar strain rats (120 ± 20 Gms) of either sex were procured and were grouped randomly. The male and female rats were separated and were acclimatized for one week in the animal house facility. They were housed in polypropylene cages in an ambient temperature of 25 ± 1°C with a natural dark-light cycle. The animals had been provided standard pellet diet and water given ad libitum. All experiments were conducted in the daytime (9:30 AM to 5:00 PM). The study was approved by the institutional ethics committee (CPCSEA registration no. - 1156/ac/07/CPCSEA) on 26 th May' 2011.
All the groups received the vehicle, standard drug and the test drug one hour prior to each experiment. Animals were selected and divided into groups (n = 6). It was studied for Elevated Plus Maze test (EPM), Morris Water Maze Test (MWM), Pole Climbing Test (PCT). Group classifications are shown in [Table 2].
Acute toxicity study
To evaluate the acute toxicity of drugs after a single oral dose, Swiss albino rats were fasted for 6 hours with only water provided ad libitum. Rats were divided into experimental groups (n = 6) and were treated orally at doses of 300, 1000 and 2000 mg/kg. The animals were then allowed free access to food and water. The animals were observed for any abnormal behavior, changes of body weight and mortality was noted for 14 days after the oral administration of formulation for the acute toxicity. The control group was treated with normal saline (1 ml/kg, i.p.). FM01 and FM02, the formulations were found to be safe. 
Morris water maze test
The Morris water maze consisted large circular pool, 1.50 m across and 0.60 m high filled with water, which was made opaque by adding milk. Water provided a uniform intramaze environment, thus eliminating any olfactory interference. A 28 x 10 cm rectangular escape platform was constructed of water resistant material and covered with material that allows the animal to remain on top when it is submerged. The platform was 28 cm in height so that it could be submerged 2 cm below the level of water surface. The water temperature was maintained at 26 ± 2°C. The animals were given a daily session of three trials per day. Latency time to reach the platform was recorded in each trial. Significant decrease in latency times from that of the first session was considered as successful learning.  The group 1, 4, 7, 10, 13, 16 and 19 were used as control, standard (50 mg/kg), standard (100 mg/kg), FM01 (50 mg/kg), FM01 (100 mg/kg), FM 02 (50 mg/kg), FM02 (100 mg/kg) respectively for MWM test.
Elevated plus maze test
The elevated plus maze served as the exteroceptive behavioral model (wherein the stimulus existed outside the body) to evaluate learning and memory in rats. The apparatus consisted of two open arms (50 cm × 10 cm) and two covered arms (50 cm × 40 cm × 10 cm). The arms extended from a central platform (10 cm × 10 cm) and the maze was elevated to a height of 50 cm from the floor. On the first the day, each rat was placed at the end of open arm, facing away from a central platform. , With little modification transfer latency (TL) was taken at the time taken by the rat to move into any one of the covered arms enter with all its four legs where opposite gender of rat is placed in any one of the covered place to observe retentive memory of test rat to come faster toward that area. TL was recorded on the first day for the each animal. The rat was allowed to explore the maze for another 2 min and returned to its home cage. Retention of this learned task was examined 24 h after the first day trial.  The group 2, 5, 8, 11, 14, 17 and 20 were used as control, standard (50 mg/kg), standard (100 mg/kg), FM01 (50 mg/kg), FM01 (100 mg/kg), FM 02 (50 mg/kg), FM02 (100 mg/kg) respectively for EPM test.
Pole climbing test
Cook's Pole Climbing Apparatus use to study cognitive function, mainly a response to conditioned stimuli during learning and its retention. The apparatus has an experimental chamber (25 × 25 × 25 cm) with the floor grid in a soundproof enclosure. Scrambled shock (6 mA) is delivered to the grid floor of the chamber composed of stainless steel rods. A pole, 2.5 cm in diameter, hangs inside the chamber through a hole in the upper center of the chamber. The study rat was placed in the chamber and allowed to explore the chamber for 45 seconds. Conditioned stimulus (CS) i.e buzzer signal was turned on and unconditioned stimulus (US) i.e electric shock delivered through grid floor for 45 Sec. Animal learned to associate the buzzer with the impending foot shock and was capable of avoiding the foot shock by climbing the pole after buzzer signal. Avoidance response was defined as climbing reaction time <10 sec only; and escape response was climbing after applying reaction time >10 sec. Every rat was subjected to maximum 05 trials on 1 st day, and 24 hrs later, rat was subjected to Relearning trials (2 nd day 3 trials and on 3 rd day one trial) and transfer latency was noted to check the retention of Conditioned Avoidance Response (CAR) and escape response. Animals were screened by using this model and those who demonstrated at least one escape response either on day one or two were included in the study. , The group 3, 6, 9, 12, 15, 18 and 21 were used as control, standard (50 mg/kg), standard (100 mg/kg), FM01 (50 mg/kg), FM01 (100 mg/kg), FM 02 (50 mg/kg), FM02 (100 mg/kg) respectively for PCT test.
Data was analyzed using one way ANOVA and two way repeated measures followed by Tukey's multiple comparisons and student's unpaired t test using the graph padprism statistic software. P < 0.05 was set as statistically significant.
| Results|| |
Water maze test
The transfer latency on a water maze test was studied using a circular pool (diameter 70 cm; height 28 cm) and a platform (diameter 3.8 cm) was placed 1.5 cm below the water level in the middle of a fixed quadrant. The differences in the transfer latency were noted in control, standard, and test group. Results indicated that test group animals at 50 mg and 100 mg doses of FM01 and FM02 showed lesser transfer latency time in seconds during the study and found to be an extremely significant decrease in transfer latency (P < 0.001) when compared to respective control groups and at 100 mg dose of test group found to be much better than standard (Bacopa monnieri) per oral route [Figure 1].
|Figure 1: The transfer latency of polyphyto formulations in rat in secs using MWM, (a) FM01 50 mg, (b) FM01 100 mg, (c) FM02 50 mg, and (d) FM02 100 mg dose; Statistical significance test was analyzed by one way ANOVA and two way repeated measures followed by Tukey's multiple comparison test (n = 6) and students unpaired t Test; *** denotes P <0.001 was considered as statistically significant vs control; Test groups FM01, FM02 were extremely significant comparing control and better than standard for its learning improvement ability at 50 mg and 100 mg dose per oral route|
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Effect of transfer latency using elevated plus maze
Transfer latency was defined as the time (in seconds) taken by the animal to move from the open arm into one of the covered arms with all its four legs. A little modification has been made that an opposite gender of rat is placed in any one of the covered place to observe retentive memory of all group of animals tested for whether it come faster toward that area where a different sex of animal kept. Significant decrease in transfer latency (TL) value of retention indicated improving memory. Test formulation both FM01 and FM02 at 50 mg and 100 mg dose showed decrease in TL on second day when compared to control groups indicating significant (P < 0.001) memory improvement [Figure 2]. FM01 and FM02 observed better than Standard drug in improving learning and memory.
|Figure 2: Bar graph of mean Transfer Latency in elevated plus maze using rat (1) EPM 50 mg, and (2) EPM 100 mg dose; Statistical significance test was analyzed by one way ANOVA and two way repeated measures followed by Tukey's multiple comparison test (n = 6) and students unpaired t Test; *** denotes P <0.001 was considered as statistically significant vs control; Test group at 100 mg dose FM01, and FM02 highly significant for its improving learning and memory ability|
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Pole clmbing test
To study the escape latency in seconds - rat placed inside the Pole climbing apparatus, a shock for controlled duration of 200V AC 50 Hz single phase - 0.2 mA was applied. The Test group FM01 at 50 mg doses revealed a statistically significant (P < 0.01) and at 100 mg dose highly significantly (P < 0.001) decrease in escape latency in pole climbing test as compared to the control animals and the effect was found to be very similar to Standard (Bacopa monnieri) treated group [Figure 3]. In two formulations, it was found that FM01 comparatively better than FM02.
|Figure 3: Bar graph of escape latency of rat in secs using Pole Climbing Apparatus (1) PCT 50 mg, and (2) PCT 100 mg dose; Statistical significance testing was analyzed by one way ANOVA and two way repeated measures followed by Tukey's multiple comparison test (n = 6) and students unpaired t Test; *** denotes P < 0.001 statistically highly significant vs control; Test group FM01 and standard were highly significant on day 3. FM02 * P < 0.05 denote test formulations are significant as compared with control|
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| Discussion|| |
Poor memory, lower retention, and slow recall are common problems in today's stressful and competitive world. Herbal drug has shown the promising effect in the treatment of memory loss. The natural medicinal plants or herbs. Acting on the brain are called as Nootropic drug of natural resource ("Nootropic" is derived from Greek and means acting on the mind) and their isolated constituents referred to as smart drugs. Memory enhancer herbs enhance the memory and increase blood circulation in the brain. The herbs/shrubs/plants act either by improving memory or preventing neurodegeneration by antioxidant and anti inflammatory activity.  Morris Water Maze is a traditional tool in assessing learning and memory performance in laboratory animals. Originally designed to evaluate the antianxiety agents, elevated plus maze has also been recently extended to measure the spatial long-term memory in animals. Passive avoidance behavior is used to examine the long term memory based on negative reinforcement.  Elevated plus maze, Morris water maze (MWM) and Pole Climbing apparatus (PCA) were used to evaluate the effect of learning and memory improvement properties in rat.
Dose selection for in-vivo study was made on the basis of acute toxicity studies (300, 1000 and 2000 mg/kg body weight) result and in consideration of estimating the human equivalent dose (HED) for treating the patient. The dose 50 mg/kg in rats, means 8 mg/kg in humans calculated by division method:
mg/kg animal dose χ [Km human/Km animal]
The drug dose 50 mg/kg or 100 mg/kg was found to be safe as well as effective for two novel polyphyto herbal formulations, so it can use clinically at a dose range of 500 mg to 1000 mg to a 60 kg adult human.
In MWM test observed that test group animals treated with 50 mg and 100 mg dose p.o of FM01 and FM02 showed less transfer latency time (in seconds) during the study and found to be an extremely significant (P < 0.001) when compared to control groups and much better than Bacopa monnieri at 100 mg dose for its nootropic action. In an Elevated plus maze test two nootropic agent FM01 and FM02 at 50 mg and 100 mg dose showed decrease in transfer latency on second day when compared to control groups indicating significant (P < 0.001) memory improvement. In an another test model like pole climbing test revealed that the test group treated with FM01 (50 mg doses p.o) found to be statistically significant (P < 0.01) and at dose (100 mg, p.o) was highly significantly (P < 0.001) compared to the control groups, while FM01 found that comparatively better than FM02.
The phytoformulation FM01 (Habiscus rosasinensis, Oscimum sanctum, Convolvulus pluricaulis, Bacopa monnieri, Santalum album) act on the nervous system and improve memory whereas Phyllanthus Emblica act by anti-inflammatory activity imparting nootropic formulation- FM01, a natural remedies found to be effective after in vivo pharmacological screening on different animal model.
| Conclusion|| |
All of the above test result suggested that the nootropic polyphyto FM01 formulation composed of (Habiscus rosasinensis 30%, Oscimum sanctum 20%, Santalum album 15%, Phyllanthus Emblica 15%, Convolulus pluricaulis 10%, Bacopa monnieri 10%) having promising significant effect for enhancing learning and memory properties can be use for memory loss or dementia and prophylactically use to prevent neurodegeneration.
| Acknowledgment|| |
The Chairman of The DIT, University, Faculty of pharmacy, Dehradun, Uttarakhand, India supports me to conduct a work and providing a lab facility and infrastructure.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]
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