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Introduction
Khamira Gawzaban Ambari Jadwar Ood Saleeb Wala (KGAJOS) is a compound Unani Pharmacopoeial formulation. KGAJOS is considered as Muqawwi-e-Aza-e-Raeesa (tonic for brain, heart, liver and stomach)1) and is used in epilepsy, ummus-subiyan (infantile epilepsy) and ikhtenaqur-reham (hysteria)2. KGAJOS is reported to possess anxiolytic3 and antidepressant activity in mice4. Most of the herbal ingredients of KGAJOS have been reported to possess cognitive improvement potential5-17. However, no data is available regarding efficacy of this valuable Unani formulation on cognitive function. The present study is designed to evaluate efficacy of KGAJOS on learning and memory function in mice.
Materials and Methods
KGAJOS was evaluated for cognitive function improvement activity using Morris water maze (MWM) test in C57BL/6 mice. Piracetam was used as positive control for comparison. Anymaze video tracking software was used for tracking the path of mice in pool as per standard protocol.
Preparation of the formulation
Test formulation (i.e., KGAJOS) was prepared in the GMP certified Pharmacy Section of National Research Institute of Unani Medicine for Skin Disorders, Hyderabad as per the composition and classical methodology mentioned in National Formulary of Unani Medicine (NFUM) Part-V2. The composition of KGAJOS is given in Table 1:
Table 1. Composition of KGAJOS.
| S.No. | Ingredients (Unani Name) | Scientific Name | Quantity |
|---|---|---|---|
| 1. | Abresham Muqarraz | Cocoon of Bombyx mori L. | 25 g |
| 2. | Badranjboya | Melissa officinalis L. | 175 g |
| 3. | Burada Sandal Safaid | Santalum álbum L. | 125 g |
| 4. | Berg Gaozaban | Borago officinalis L. (Leaf) | 150 g |
| 5. | Behman Surkh (Neem Kofta) | Salvia haematodes W. | 100 g |
| 6. | Tukhm Balango | Lallemantia royleana Benth. | 125 g |
| 7. | Tudri Surkh | Cheiranthus cheiri L. | 50 g |
| 8. | Kishneez Khushk (Dhania) | Coriandrum sativum L. | 150 g |
| 9. | Gul Khatmi | Althaea officinalis L. | 50 g |
| 10. | Gul Gaozaban | Borago officinalis L. (Flower) | 50 g |
| 11. | Shakar Safaid | White sugar | 10 Kg |
| 12. | Sat Leemu | Citrus aurantifolia (Christm.) | 20 g |
| 13. | Natroon Banjawi | Sodium benzoate (as preservative) | 6 g |
| 14. | Ambar Ashhab | Ambra grasea | 2.645 g |
| 15. | Warq-e-Nuqra | Silver leaves | 33 g |
| 16. | Warq-e-Tila | Gold leaves | 55 Nos. |
| 17. | Jadwar Saeeda | Delphinium denudatum Wall. ex Hook & T | 106 g |
| 18. | Ood Saleeb Saeeda | Paeonia emodi Wall. ex Royle | 137 g |
Experimental mice
C57BL/6 Mice (25-30 g, 8-9 weeks old) used for the present study were procured from Hylasco Bio-Technology (India) Pvt. Ltd., Hyderabad (A Charles River Licensee). Mice were group housed in polysulfone cages in the temperature-controlled room maintained at the temperature of 22°C ± 3°C and relative humidity of 30-70%, with a 12:12 h light/dark illumination cycle. CPCSEA guidelines of laboratory animal care was followed throughout the experiment18. Study protocol was presented before the Institutional Animals Ethics Committee and approved vide protocol no. CRIUM/IAEC/2018/01/P03 dated 21.07.2018. Mice were maintained on standard diet (SDS diet, England) and water ad libitum, unless mentioned otherwise. Only male mice were used in order to avoid the influence of the estrus cycle on drug metabolism and/or efficacy. Mice were acclimatized to the laboratory conditions for one week before using them for experiment.
Dose selection and study design
Therapeutic dose of KGAJOS in human is reported as 05 g per day. Therapeutic Equivalent Dose (TED) in mouse is about 1000 mg/kg bw per day as per body surface area conversion method19. Accordingly, present study was performed at three dose levels of KGAJOS i.e., 500, 1,000 and 1,500 mg/kg bw/day to observe any dose dependent activity. A total of 40 male mice were randomly divided into five groups containing 08 mice in each (n=8). Group-I served as vehicle control. Group-II served as positive control and was administered with piracetam (400 mg/kg bw, i.p.)20. Group III-V were treated with three dose levels of KGAJOS (i.e., 500, 1,000 and 1,500 mg/kg bw/day) for seven consecutive days before recording the Morris water maze performance and treatment was continued throughout the procedure till probe trial. KGAJOS was administered as an aqueous suspension which was freshly prepared each day and orally administered using stainless steel gavage by calculating the dose for individual mouse as per body weight. Mice of piracetam group were subjected to behavioural recording 60 min after piracetam injection (i.p.) while MWM performance of vehicle and KGAJOS treated mice was observed 90 min after oral gavage administartion20.
Morris water maze test
Morris water maze (MWM) method consisted of six-day trials of mice in circular pool. The basic paradigm requires an animal to swim in a pool until it finds a hidden escape platform. The mice learn to find the platform using extra-maze cues and, after several training trials, are able to swim directly to platform from any starting location. Memory for the platform location is assessed by examining swimming pattern with the platform removed from the maze21.
A metallic circular pool with a diameter of 150 cm and a depth of 50 cm and wall height 20 cm above the water level was used. Non-toxic white tempera paint was added to make the water opaque so that black mouse will be captured by software on an otherwise white background22,23. A circular platform of about 10 cm diameter was hidden 2 cm below the water level. Pool was filled with water until the platform was 2 cm above the water surface. The water was kept at about 23°C during the experiment. Room was arranged such that the mouse being tested cannot see the experimenter during testing and no change in arrangement of instrument and other objects was allowed throughout the experiment duration. High contrast spatial cues were placed in the room and on the interior of the pool at a location which were above the water surface.
The pool was calibrated using computerized software (Anymaze, Stoelting, USA) so that the camera can create physical distance information from captured pixel-based information. Pool was virtually divided into 4 quadrants and platform zone was specified as a variable zone which can change with each trial. 5 platform subzones were specified viz. one in each quadrant, and one in the centre of the pool. Calibration was saved and used for the remaining test days. The maximum trial duration was set as 60 sec. If the mouse finds the platform before this time, software was programmed to stop the trial. Program protocol was specified to begin tracking automatically, when the experimenter exits the testing area. Time spent in each quadrant and time to reach the platform quadrant was tracked using Anymaze video tracking system.
Procedure: Mice were trained for five consecutive days, and each mouse was subjected to five trials per day21, followed by a probe test on Day 6.
Day 1 (Visible Platform): Five trials were conducted with an inter-trial interval about 5-10 minutes. The platform location and starting direction was different with each trial.
Testing procedure: To begin testing, mouse was lifted from the home cage by the base of the tail. The mouse was gently placed into the water, facing the edge of the pool. If the mouse finds the platform before the 60 seconds cut-off, mouse was allowed to stay on the platform for 5 seconds and then returned to home cage. If any mouse did not find the platform within 60 seconds, mouse was guided to the platform and allowed to stay there for 20 seconds before returning to home cage. Each subsequent trial was initiated with a different platform location and starting direction. When testing was complete, mice were returned to their housing facility. Mice were dried off properly with towel and normothermia was assured prior to returning to the cage.
Days 2-5 (Hidden Platform): Five trials were conducted with an inter-trial interval of about 5-10 min. Platform location was programmed to remain in the same position throughout all trials and days, but the starting direction was different with each trial, each day.
Testing procedure: Platform was submerged in water by adding additional water. Platform was not visible from the surface of the water. Rest of the procedure was same as for Day-1.
Day 6 (Probe Trial): Only single probe trial was conducted on Day-6 with platform removed from the pool, and one starting direction for all mice. The starting direction farthest from the platform quadrant used on days 2-5 was used. Maximum trial duration was kept as 60 seconds. For day 6, escape latency (time to reach platform quadrant), and time spent in the platform quadrant for each mouse was recorded. A higher percentage of time spent in the platform quadrant is interpreted as a higher level of memory retention21,24,25.
Statistical Analyses
Data from the experiments was expressed as mean ± standard error of mean (SEM). The mean difference between the control and treatment groups was analysed by one-way Analysis of Variance using Graph Pad prism (version 5) Graph Pad Software, Inc., CA, USA. p value < 0.05 was considered as statistically significant.
Results
During probe trial (i.e., day-6), average time spent by mice in target platform quadrant of MWM is depicted in Figure 1. There was a significant increase (p<0.01) in time spent in platform quadrant in piracetam treated group compared to vehicle control group. No significant difference was observed at KGAJOS 500 mg/kg bw compared to vehicle. KGAJOS significantly increased the time spent in the target quadrant at 1000 and 1500 mg/kg bw as compared to vehicle control (p<0.01 and 0.001, respectively). Latency to reach the platform quadrant (escape latency) was significantly reduced (p<0.001) in piracetam and KGAJOS group at 1000 and 1500 mg/kg bw compared to vehicle control (Figure 2). No change in escape latency was observed at 500 mg/kg bw of KGAJOS. Further, average distance travelled by mice in platform quadrant is significantly higher (p<0.001) in piracetam group compared to vehicle control. KGAJOS significantly increased distance travelled by mice in platform quadrant at 1000 mg/kg (p<0.01) and 1500 mg/kg (p<0.001) while no difference was observed at 500 mg/kg bw compared to vehicle (Figure 3).
Figure 1. Time spent in platform quadrant in MWM test in seconds; data presented as mean ± SEM (n=8); One-way ANOVA; **=p<0.01 vs. Vehicle Control; ***=p<0.001 vs. Vehicle Control
Figure 2. Latency to enter platform quadrant (escape latency) in MWM test in seconds; data presented as mean ± SEM (n=8); One-way ANOVA; ***=p<0.001 vs. Vehicle Control
Discussion
Morris Water Maze was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memory and long-term spatial memory26. MWM test has become one of the most frequently used research tools in various aspects of learning and memory27. The robustness/simplicity and reliability make this paradigm as one of the ‘gold standards’ of behavioural neuroscience. The main advantage is the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions24-26,28. In addition, the MWM testing environment reduces odour trail interference29. Therefore, considering these advantages, MWM test is extensively used in the study of the neurobiology and neuropharmacology of spatial learning and memory.
In the present experiment, mice treated with 1000 and 1500 mg/kg bw of KGAJOS and piracetam (400 mg/kg bw) spent much longer time in platform quadrant compared to vehicle control group, suggesting that these mice gathered the clue from spatial arrangement in the room and pool regarding the possible location of the platform and explored the same area of pool (quadrant) for the platform repeatedly which is also supported by longer distance travelled in platform quadrant by mice of these groups compared to vehicle group. Further, mice treated with 1000 and 1500 mg/kg bw of KGAJOS and piracetam took significantly less time to reach the platform quadrant compared to vehicle treated mice as they slowly learn the escape platform location using objects or symbols placed outside the maze as cues, and progressively swim to the platform in a shorter time29,30, which is again an indicator of improvement in spatial memory.
Most of the herbal ingredients of KGAJOS or their phytoconstituents have been extensively reported to exhibit improvement in cognitive function. Cocoon of Bombyx mori which is composed of two proteins i.e., fibroin (72-81%) and silk gum or sericin (19-28 %)1. The silk fibroin protein enzymatic hydrolysate is reported to significantly improve memory in healthy adults at the daily doses of 280 mg and above for three weeks2. Oral administration of Melissa officinalis (lemon balm) extract (200 mg/kg) significantly augmented learning and memory of naïve rats and significantly ameliorate scopolamine-induced learning deficit3. Similarly, repeated administration of a 50% ethanol extract of M. officinalis leaves (200 mg/kg, p.o. for 28 days) showed an improvement in long-term memory in rats and a decrease of Acetylcholinesterase (AChE) mRNA level by 52% in the cortex and a strong inhibition of BACE1 mRNA transcription (64% in the frontal cortex; 50% in the hippocampus) was also observed4. A randomised, placebo-controlled, double-blind, balanced-crossover study investigated the acute effects of standardised extract of M. officinalis on cognition and mood. There was improvement in Accuracy of Attention following 600 mg of M. officinalis and time- and dose-specific reductions in both Secondary Memory and Working Memory factors5. Protective effects of Borago officinalis extract has been reported on Amyloid β (Aβ)-induced memory impairment in rats6. It is also reported that that Aβ (25-35) can effectively inhibit long term potentiation in the granular cells of the dentate gyrus in hippocampus, and B. officinalis supplementation reverse the synaptic plasticity in dentate gyrus following Aβ treatment and may lead to an improvement of Alzheimer's disease -induced cognitive dysfunction7. Hydro-alcoholic extract of Salvia haematodes root possesses protective effect on cognitive functions in scopolamine-induced amnesia in rats and may prove to be a useful memory restorative agent in the management of cognitive dysfunctions8. Dose-dependent improvement in memory scores was reported in young as well as aged mice following dietary administration of Coriandrum sativum leaves for 45 days. It also reversed the memory deficits induced by scopolamine and diazepam. Brain cholinesterase activity was also considerably reduced9. Oral administration of C. sativum seed extract for 12 weeks is reported to ameliorate age induced memory deteriorations in the senescence-accelerated SAMP8 mouse model10. Phytochemical investigation of Delphinium denudatum resulted in the isolation of isotalatazidine hydrate in crystalline form which is a potent dual cholinesterase inhibitor and may be used as a target drug in Alzheimer diseases11. Norditerpenoid alkaloids of D. denudatum were also reported as cholinesterase inhibitors12. Oral administration of ethanolic root extract of Paeonia emodi (300 and 600 mg/kg) is reported to effectively improved impaired learning and memory performance in Morris water maze in pentylenetetrazole kindled mice13.
Efficacy of KGAJOS observed in MWM test confirmed the traditional claims and usage of this formulation. The observed efficacy of KGAJOS in MWM test is in agreement with the reported pharmacological activities of individual phytoconstituents of KGAJOS and their synergistic effect on memory and cognition.
Conclusion
Morris water maze experiment conducted in mice revealed improvement inlearning and memory function at the dose levels of 1000 and 1500 mg/kg bw of KGAJOS whereas 500 mg/kg bw was not found to be effective. Observed efficacy of KGAJOS confirmed the traditional claims and usage of this formulation in conditions associated with cognition and memory.
