INTRODUCTION:

Alzheimer’s disease is a degenerative disease, meaning that it becomes worse with time. Alzheimer’s disease is thought to begin 20 years or more before symptoms arise^1,2 with changes in the brain that are unnoticeable to the person affected. Only after years of brain changes do individuals experience noticeable symptoms such as memory loss and language problems. Symptoms occur because nerve cells (neurons) in parts of the brain involved in thinking, learning and memory (cognitive function) have been damaged or destroyed^3-6. Alzheimer’s disease is the most common cause of dementia, accounting for an estimated 60% to 80% of cases. Although the complete sequence of events is unclear, beta-amyloid may begin accumulating before abnormal tau and increasing beta-amyloid accumulation is associated with subsequent increases in tau^7,8. The various indigenous systems such as Ayurveda, Siddha, Unani use several plant species to treat different ailments^9-12. Tyler defines herbal medicines as crude drugs of vegetable origin utilized for the treatment of disease states often of a chronic nature or of attain or maintain a condition of improved health. The treatment of injury or disease by plants or plant material either in the crude or processed state is known as traditional herbal medicine. The medicinal plants with ethno-medicinal values are currently being screened for their therapeutic potential^13,14. Herbal product has been used abundantly over the years in curing several diseases. Natural products and related structures are essential sources of new pharmaceuticals because of the immense variety of functionally relevant secondary metabolites of microbial and plant species⁶. Herbal combinations also known as Polyherbal therapy have been used in Chinese medicine practice for thousands of years, yet scientific evidence of their therapeutic benefits is lacking¹⁵. The concept of drug combination has been well established in Western medicine and remarkable success has been achieved over the decades. In recent years, drug combination therapies in cancer and infectious diseases have offered new hope to patients¹⁶. Naturally occurring herbs and herbal ingredients organized into certain formula has been shown to have potential interaction effects. These include mutual enhancement, mutual assistance, mutual restraint and mutual antagonism¹⁷.In Ayurvedic, system of medicine mainly Polyherbal formulation is used for treatment of various infections. The Unani system of medicine is also gaining global acceptance due to the amazing clinical efficiency of the formulations. Although Unani medicines have long been used, there is negligible documented evidence regarding their safety and effectiveness. The lack of evaluation has in turn, slow down the development of regulations and legislations¹⁸. In the present study we used polyherbal formulation was prepared by incorporating three traditional herbs i.e. Hibiscus vitifolius, Convolvulus pluricaulis, Michelia champaca and Hibiscus abelmoschus. These polyherbals were selected due to their uses as folk medicine in cognition enhancing and memory boosting effects. Hibiscus vitifolius Linn. is well known plant belong to the Family Malvaceae. It is also known as Hibiscus heterotrichus DC, Hibiscus jatrophaefolius, Hibiscus obtusifolius willed. Hibiscus vitifolius Linn (Malvaceae) is one of the largest genera in Angiosperms. The decoction is used to treat vaginal discharge and it is also used in the treatment of diarrhea, jaundice, inflammation and diabetes. Hibiscus vitifolius showed some pharmacological properties like Anti-hypercholesterolemic¹⁹, Anti-nociceptive²⁰, Anti-carcinogenic²¹, Hepatoprotective activity²². The Convolvulus pluricaulis is popularly known as Shankhapushpi^23,24. The plant contains amino acids, proteins, shankhpushpine, convolamine, convoline, convolidine, convolvine, confoline, convosine etc are present^25-28. The total plant formulations used as nervine tonic for enhancing intellect and memory^29-31. In Ayurveda it is a Majjadhatu rasayana [rejuvenates the nervous tissue]³². The Michelia champaca is one of medicinal plant belongs to the Magnoliaceae Family. It contains Iriodenine, Macheline, Lanuginosine, Polyisoprenoid, β-sitosterol, Liridenine, Champakin, Liriodenine, Micheliolide, Champacene, linalool³³, (-)-Anonaine³⁵, (-)-asimilobine, (-)-nuciferine, (-)-anolobine, (-)-romerine, (-)-N acetylanonaine, (+)-syringaresinol, scopoletin, 4-acetonyl-3,5-dmethoxy-p-quinol, vanillin, vanillic acid, syringic acid, stigmasterol³⁴. Michelia champaca leaves are used in brain disorders³⁵. The Hibiscus abelmoschus Linn belong to the Family Malvaceae. The seeds are used as diuretic, aphrodisiac, ophthalmic, cardiotonic, digestive, stomachic, constipating, carminative, pectoral stimulant, antispasmodic, deodorant, insecticidal and tonic^36,37. Seeds rubbed to a paste with milk are used to cure itch. Decoction of the seeds is used in stomach cancer. Mucilage made from root and leaves of the plant is recommended in gonorrhea and venereal diseases. The poultice of leaves and roots is used in cystitis, fever, headache, rheumatism, varicose veins and boils^36,38.

MATERIALS AND METHOD:

Chemicals

Ellman’s reagents, Epinephrine, Colchicines, 2-thiobarbituric acid were purchased from Sigma-Aldrich Co. Acetylthicoline iodide was purchased from Hi-Media. Follen’s reagent was purchased from Spectrochem Pvt. Ltd., Mumbai. Trichloroacetic acid and all other chemical were of analytical grade.

Animals

The Sprague dawley rats of either sex (200-280g) were obtained from Central animal house of H.S.K College of Pharmacy and Research Centre, Bagalkote, Karnataka. The animals were housed at room temperature (28±2°C) with 65±10% relative humidity for a 12 hr dark and light cycle and given standard laboratory feed and sufficient drinking water ad-libitum. The study was approved and conducted as per the norms of the Institutional Animal Ethics Committee of H.S.K College of Pharmacy, Bagalkote (Ref No. IAEC/HSKCOP/Aug 2021/UG2) and carried out in accordance with the CCSEA Guidelines for the use and care of laboratory animals.

Instruments

Refrigerated centrifuge (MPW-350R Poland), UV-Spectrophotometer (UV-1900i, Shimadzu Corporation, Kyoto, Japan), Homogenizer (Remi motor/RQ-127A/D, Mumbai, India), Mini Lyotrape (LTE Scientific Ltd, Oldman, Great Britain), Ultra low temperature deep freezer-86⁰C (OPERON/DFC-84CE, Korea), Stereotaxic instrument (STX-M No.1, Bengaluru, India), Analytical Balance (Shimadzu AUX2020), Nanojet Pump, 25µl Hamilton Microliter Syringe 702N series, 22 gauge.

Plant Material

In the present study the Hibiscus vitifolius, Convolvulus pluricaulis, Michelia champaca and Hibiscus abelmoschus were collected, identified and authenticated in Department of Botany, Basaveshwar Science College, Bagalkote Karnataka. The plant specimes were also deposited in the same college of herbarium for further future reference. The collected plants Hibiscus vitifolius (Flower), Convolvulus pluricaulis (Whole plant), Michelia champaca (Flower) and Hibiscus abelmoschus (Seeds) were shed dried and dried plant specimens were grounded to get a coarse powder. The coarse powder was subjected to successive extraction. The solvent was distilled and allowed to evaporate at room temperature to get concentrated extract then completely dried by lyophilization. The obtained residue was stored in an airtight container and kept under refrigeration at 2-4⁰C for further use.

Preparation of Polyherbal formulation

The formulation of herbs was prepared by mixing the 5 gm of Hibiscus vitifolius, 5 gm of Convolvulus pluricaulis, 5 gm of Michelia champaca and 5 gm of Hibiscus abelmoschus extracts followed by addition of tween 80 (0.1%, w/v) and uniformly triturated to form a smooth paste. The paste was rinsed with distilled water (100 ml) and vortexed using mechanical stirrer to get suspension. The formulation was found stable and used for the present activity³⁹.

Sl No	Content	Quantity
1	Hibiscus vitifolius	5gm
2	Convolvulus pluricaulis	5gm
3	Michelia champaca	5gm
4	Hibiscus abelmoschus	5gm
5	Distilled water	100ml
6	Tween 80	0.1%

Experimental protocol for Alzheimer’s disease activity

Spargue dawley rats of either sex (200-280 g) were divided into three groups of 6 rats in each group and treated as follows.

Group-I: Sham receives 5µl of ACSF and 0.2% of ascorbic acid by stereotaxic injection into the right lateral cerebral ventricle and vehicle orally for 28 days. (n=6)

Group-II: Control animals receive 15µg/5µl of colchicine in ACSF and 0.2% of ascorbic acid by stereotaxic injection into the right lateral cerebral ventricle and vehicle orally for 28 days. (n=6)

Group-III: Effect of PHF 2ml/kg group receives 15µg/5µl of colchicine in ACSF and 0.2% of ascorbic acid by stereotaxic injection into the right lateral cerebral ventricle and 2ml/kg PHF orally for 28 days. (n=6)

Acute toxicity Study

An acute toxicity study was performed for the Polyherbal formulation to determine the safest dose by the acute oral toxicity method of the Organization for Economic Co-operation and Development (OECD) as per 425 guidelines⁴⁰.

Surgery and intracerebroventricular administration of Colchicine

Animals weighing 220-270 g were anesthetized with Ketamine (75 mg/kg, i.p.) and positioned in a stereotaxic apparatus in which the frame was previously calibrated and cleaned with 70% alcohol. The head was properly positioned in a frame and a midline sagittal incision was made in the scalp and bregma was exposed⁴¹. One hole was drilled in the skull for the placement of the injection cannula into the right lateral cerebral ventricles. Co-ordinates for the intracerebroventricular (i.c.v) cannula implantation were 0.8 mm posterior to bregma, 1.8 mm lateral to the sagittal suture and 3.6 mm beneath the cortical surface. The scalp was then closed with a suture. Iodine was applied to the surgical area with help of cotton in order to prevent sepsis. Animals were housed individually in separate cages with soft bedding. Special care was provided during the post-operative period to provide food and sufficient water inside the cage of rats. Rats were infused with colchicines (15 µg/5µl/rat) dissolved in artificial cerebrospinal fluid (ACSF; in nM: NaCl 147, KCl 2.9, MgCl2 1.6, CaCl₂ 1.7 and dextrose 2.2) which was slowly injected into the cannulated right lateral ventricle using a 25 µl Hamilton syringe and the needle was held in place for 2-3 minutes for proper dispersal of the drug from the tip of the syringe. The Sham group was subjected to the same surgical procedure and received artificial cerebrospinal fluid (ACSF). Postoperatively, rats were kept in separate cages and monitored⁴².

Neurobehavioral Studies

Elevated Plus Maze Paradigm

The elevated plus maze consisted of two opposite black open arms (44×10 cm) crossed with two closed walls of the same dimensions with 40 cm high walls. The arms were connected with a central square of dimensions 10×10 cm. The entire maze was elevated to a height of 53 cm from the floor. Acquisitions of memory were tested after colchicine administration. Animal were placed individually at one end of the open arm facing away from the central square. The time taken by the animal to move from the open arm to the closed arm was recorded as the initial transfer latency (ITL). Animal were allowed to explore the maze for 20 seconds after recording the ITL and then returned to the home cage. If the animal did not enter the enclosed arm within 90 seconds it were guided on the back into one of the enclosed arm and the ITL were given as 90 seconds. Retention of memory were assessed by placing the rat in an open arm and the retention latency were noted on day 14 and day 28 of ITL and were termed as the first retention transfer latency (1st RTL) and second retention transfer latency (2nd RTL), respectively. During a 5 min test period the following measures are taken: the number of entries into and time spent in the open and enclosed arms; the total number of arm entries. The procedure is conducted preferably in a sound attenuated room. Motor activity and open arm exploratory time are registered. The values of treated groups were expressed as percentage of controls⁴³.

Spatial Navigation Task

Spatial learning and memory were tested in a Morris water maze. The maze consisted of a black circular pool (diameter 182 cm, radius 91 cm height 75 cm) filled to a depth of 30 cm with water (25 ± 2°C). On the pre-surgical day, rats received habituation (exposure in a water maze for 1 minute) in which there was no platform present. Then, on days 0, 7, 14 and 28, a circular platform (10 cm in diameter) was kept hidden 2 cm below the water level in the centre of one of the quadrants. At the beginning of each session, random sequences of four starting poles along the perimeter of the pool were generated. Each rat was placed in the water facing toward the wall at the start location and was allowed 90 seconds to find the hidden platform. The latency to reach the platform was recorded. If the rat was unable to locate the hidden platform, it was lifted out and placed on the platform for 20 seconds. The animal was allowed to remain 20 seconds on the platform. Two sessions of four trials each were conducted. Following that, the platform was removed and the probe trial (without the platform) was conducted 4 hours later. Each rat was placed in the pool at the same, randomly selecting the starting pole. The swimming time spent in the target quadrant (which initially contained the platform) of the pool was measured^42-46.

Locomotor activity

Locomotor activity was observed on days 0, 7, 14 and 28 following i.c.v colchicine administration. Animals were placed in a square (42 cm) closed arena equipped with infrared light-sensitive photocells using a digital photo-actometer and allowed to acclimatize or habituate for 5 min. The animals were observed for a period of 5 minutes and the values were expressed as counts/5 minutes. The chamber was swabbed with 10% ethanol every time to avoid interference due to animal odors⁴².

Open field test

The locomotor activity was carried out by open field in a sound attenuated room. The floor was white polyvinyl with a black grid divide the open field into 100 square (10 x 10). Illumination was provided with 60w bulb placed above centre of field, while rest of the room was darkened. The rat was initially placed at centre of the field and observed for 5 minutes in all parameters i.e. latency (sec), ambulatory movements (number), rearing (sec), grooming (number), rest (sec), rotations (clockwise and anti-clockwise). Every time the activity chamber was swabbed with 10% alcohol to avoid the interference due to animal odours. Locomotor activity was tested in each group of animals at beginning of experiment and 0, 7, 14 and 28 days after the lesion⁴⁷.

Statistical Analysis

All the data were expressed in Mean ± SEM. The significance of differences in a mean between control and treated animals for different parameters determined by one-way ANOVA followed by multiple comparisons Dunnett’s test and p<0.05 was considered as minimum statistically significant.

RESULTS:

Acute Toxicity Study

Oral administration of polyherbal formulation did not shown any signs of toxicity. The animals showed normal brething and there was an absence of convulsions, tremors, salivation and diarrhea. Observation of animals over period of 14 days did not shown any toxicity in animals.

Elevated Plus Maze Paradigm

The effects of Polyherbal Formulation (PHF) on Mean transfer latency on colchicines induced experimental AD in the elevated plus maze test are summarized in (Table 1.) In the present experiment, mean initial transfer latency (ITL) on Day 0 for each rat was relatively stable and showed no significant variation among different groups. All the rats entered into the closed arm within 90 seconds. sham-operated and PHF treated 2ml/kg rats entered closed arm quickly as compared to control group on Day 0. Mean retention transfer latencies [to enter into closed arm] (1^st RTL, 2^nd RTL and 3^rd RTL) on Days 7, 14 and 28 were shorter as compared to ITL on day 0 of sham group. In contrast, control group rats showed significant increase in the mean RTL on Days 7, 14 and 28 as compared to Day 0, demonstrating that colchicine-induced marked memory impairment in control group rats. In PHF treated groups was shorter (p<0.001) as compared to ITL on Day 0 of their respective group that shows the marked improvement in the impaired memory against colchicine toxicity in PHF treated groups. The effects of PHF on colchicine induced experimental AD in the elevated plus maze are summarized in (Table 1.). In elevated plus maze test, all the rats of control group showed significant increase in time spent in open arm and no. of entries in open arm (p<0.001) as compared to sham group on Day 7, 14 and 28. PHF treated group showed significant decrease in time spent in open arm and no. of entries in open arm on 14 and 28 day as compared to control group. Similarly, control group showed significant decrease in time spent in closed arm (p<0.001) and no. of entries (p<0.001) in closed arm as compared to sham operated group. PHF treated 2ml/kg group showed significant increase in time spent in closed arm and no. of entries in closed arm on 14 and 28 day as compared to control group.

Spatial Navigation Task

The results of Morris water maze are summarized in Table 2. In the first, second and third session of escape latency trail, the latency to locate the submerged platform was significant increased in control group (p<0.001) as compared to sham group at Days 7, 14 and 28. In contrast, PHF treated 400 mg/kg group shows significant decreased time to locate the submerged platform during all the three session on Days 14 and 28. In the probe trial a significant difference in special bias of animals towards the quadrant of the pool that contained the submerged platform (target quadrant) during the escape latency was observed. The animals of control group shows the less time spent in target quadrant on Days 7, 14 and 28 (p<0.001) as compared to sham group. In PHF treated (400 mg/kg) groups are significant (p<0.05 to p<0.001) spending longer time in target quadrant as compared to control group at Days 14 and 28 day. Similarly, In Time spent in platform quadrant showed a significant decrease in time spent in platform quadrant in control group as compared to sham group on Days 7, 14 and 28. In PHF treated group showed significant (p<0.001) increase in time spent in platform quadrant on Day 28 as compared to control group.

Locomotor activity

The results of spontaneous locomotor activity are summarized in Table 3. on the Day 0 control group did not show significant effect as compared with sham group and did not show significant effect when treatment group compared with control group. Whereas on 7, 14, and 28 Days control group shows a significant (p<0.001) increases in rest was recorded (counts) as compared to sham group. On PHF treatment on Day 7 shows significant (p<0.001) decrease in rest at a dose of 2ml/kg when compared with control group. Whereas in the treatment group on Day 14 at a dose of 2ml/kg shows significant (p<0.001) decrease in rest as compared with control group.

Open field test

The effect of PHF on locomotor activity on colchicine induced experimental AD in the open field test are summarized in Table 4. In locomotor activity, a significant (p<0.001) increase in latency, rest was observed in control group as compared with sham group and significant (p<0.001) recovery of latency was observed in PHF treated (2ml/kg) groups on 14 and 28 onwards as compared to control group. Similarly, significant (p<0.001) decreased in ambulatory movement, rearing and grooming was seen in control group as compared with sham group, whereas PHF treated 2ml/kg groups, showed significant (p<0.001) increase in ambulatory movement, rearing and grooming on Day14 and 28 as compared to control group. There is significant (p<0.001) increase in ipsilateral rotation in control group as compared to sham group but there is significant (p<0.001) decrease in ipsilateral rotation in PHF treated group as compared to control group.

Table 1. Effect of Polyherbal Formulation (PHF) on Elevated Plus maze test in Colchicine induced Alzheimer’s disease rat model.

Parameters	Groups	Day - 0	Day- 7	Day- 14	Day- 28
Mean transfer latency (in sec)	Sham	14.67±0.8028	20.17±1.195	15.00±1.033	14.67±0.5578
	Control	16.50±0.9220	79.83±2.257^a	75.83±4.028^a	72.33±2.261^a
	PHF (2ml/kg)	16.50±1.478	71.33±4.971	40.83±1.662***	26.50±2.045***
Time spent in open (in sec)	Sham	78.67±6.530	49.83±3.177	35.33±3.073	27.83±4.222
	Control	80.00±4.171	213.2±9.243^a	159.5±6.520^a	91.33±5.760^a
	PHF (2ml/kg)	81.00±6.039	201.0±3.642	132.7±10.83*	22.17±2.574***
Time spent in closed arm (in sec)	Sham	226.0±12.29	234.3±9.200	253.7±9.965	263.3±6.375
	Control	216.2±4.564	162.5±8.981^a	120.8±13.09^a	99.83±7.947^a
	PHF (2ml/kg)	218.0±5.627	183.3±13.03	213.5±9.570***	216.8±13.98***
Number of entries in open arm	Sham	2.500±0.2236	2.333±0.2108	2.000±0.3651	2.000±0.2582
	Control	3.500±0.2236	4.000±0.2582^b	4.000±0.2582^a	5.333±0.3333^a
	PHF (2ml/kg)	3.167±0.4014	3.167±0.3073	1.333±0.2108***	2.833±0.3073***
Number of entries in closed arm	Sham	2.833±0.1667	2.500±0.2236	4.500±0.2236	3.167±0.3073
	Control	2.667±0.2108	1.667±0.2108^c	1.500±0.2236^a	1.833±0.1667^b
	PHF (2ml/kg)	2.333±0.2108	1.500±0.2236	3.500±0.2236***	3.667±0.2108***

All the values are expressed as mean ± SEM, n=6, [One way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test] ^cp<0.05, ^bp<0.01, ^ap<0.001 as compared to sham group and ***p<0.001 as compared to control group. n=6

Table 2. Effect of Polyherbal Formulation (PHF) on Spatial Navigation Task in Colchicine induced Alzheimer’s disease rat model.

Days	Parameters		Sham	Control	PHF (2 ml/kg)
Day 0	Escape latency	L1	23.17± 2.786	23.50± 2.941	25.50± 2.754
		L2	26.50± 2.778	27.83± 2.227	28.33± 2.801
		L3	23.17± 2.822	27.83± 2.227	27.83± 0.833
	Probe trail	T1	26.67± 0.9545	27.50± 0.6191	25.67± 0.7601
		T2	29.00± 0.4472	29.67± 0.4216	28.50± 0.9574
		T3	26.17± 1.424	28.67± 0.4944	26.00± 1.438
Day 7	Escape latency	L1	36.67± 2.362	88.17± 1.641^a	77.00± 3.958^*
		L2	44.50± 3.304	88.67± 0.881^a	79.67± 2.836
		L3	42.50± 2.527	84.00± 1.862^a	78.83± 3.250
	Probe trial	T1	22.00± 1.414	18.00± 1.000	21.83± 0.8724
		T2	31.33± 0.9189	17.50± 0.7188^a	17.83±0.11
		T3	27.17± 1.515	17.33 ± 1.14^a	20.83 ± 1.35
Day 14	Escape latency	L1	27.00± 2.989	87.00± 2.295^a	70.50± 3.667**
		L2	27.67± 3.073	78.33± 1.667^a	45.17± 1.833***
		L3	29.00± 1.751	65.00± 3.386^a	43.17± 2.197***
	Probe trial	T1	27.17± 0.9098	17.50± 1.979^a	21.00± 0.9661
		T2	30.17± 0.5426	18.67± 0.8819^a	24.00± 1.862*
		T3	26.50± 0.8466	16.67± 2.108^a	23.00± 1.633*
Day 28	Escape latency	L1	22.67± 2.275	88.33± 1.085^a	52.83± 3.468***
		L2	23.83± 3.371	68.33± 5.426^a	44.50± 2.062**
		L3	21.17± 3.701	65.83± 3.506^a	38.33± 3.201***
	Probe trial	T1	27.17± 0.7491	16.00 ± 1.34^a	21.67 ± 1.34*
		T2	28.17 ± 1.86	17.67 ± 0.66^a	23.67 ± 1.38**
		T3	26.17 ± 1.30	14.67 ± 1.60^a	26.83 ± 0.79***

All the values are expressed as mean ± SEM, n=6, [One way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test] ^ap<0.001 as compared to sham group and *p<0.05, **p<0.01, ***p<0.001 as compared to control group. n=6

Table 3. Effect of Polyherbal Formulation (PHF) on Spontaneous locomotion in Colchicine induced Alzheimer’s disease rat model.

Treatment groups	Spontaneous locomotion (seconds)
Treatment groups	Day- 0	Day- 7	Day- 14	Day- 28
Sham	240.7±4.349	235.8±6.725	329.3±9.763	373.8±15.57
Control	244.8±5.558	74.50±6.371^a	176.3±12.96^a	224.2±24.87^a
PHF (2ml/kg)	228.5±2.604	151.7±6.912***	275.8±8.284***	259.8±29.12***

All the values are expressed as mean ± SEM, n=6, [One way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test] ^ap<0.001 as compared to sham group and ***p<0.001 as compared to control group. n=6

Table 4. Effect of Polyherbal Formulation (PHF) on Open filed test in Colchicine induced Alzheimer’s disease rat model.

Parameters	Groups	Day - 0	Day- 7	Day- 14	Day- 28
Number of Ambulatory movements	Sham	26.5±2.9	31.5±2.8	30.5±1.2	68.6±2.9
	Control	34.5±2.8	9.8±0.8a	12.8±1.4a	34.6±1.7a
	PHF (2ml/kg)	29.8±1.3	16.3±1.5*	31.1±3.3**	46.1±1.4***
Number of rearing	Sham	8.6±0.4	10.6±0.5	8.5±0.8	7.1±0.5
	Control	9.8±0.3	4.6±0.4a	3.1±0.4a	1.0±0.2a
	PHF (2ml/kg)	8.3±0.4	6.5±0.2*	5.8±0.6*	4.0±0.2***
Number of Grooming	Sham	77.0±6.5	50.6±2.5	38.3±3.1	47.3±2.9
	Control	58.1±6.5	24.5±2.0a	15.0±1.3a	13.5±0.5a
	PHF (2ml/kg)	70.8±3.6	37.6±3.5**	32.5±3.6***	22.5±2.1*
Rest (Seconds)	Sham	-	38.3±3.3	8.3±3.3	56.6±5.3
	Control	-	146.5±10.8a	153.2±8.4 a	132.7±2.7a
	PHF (2ml/kg)	-	121.2±11.5*	77.5±4.4**	91.6±6.1***
Number of Ipsilateral rotations	Sham	-	0±0	0±0	0±0
	Control	-	2.3±0.3a	3.8±0.4a	5.8±0.3a
	PHF (2ml/kg)	-	0±0***	0.5±0.3***	4.0±0.4*
Latency (Seconds)	Sham	-	0.6±0.3	0.3±0.2	0.0±0.0
	Control	-	6.3±0.3 a	3.6±0.4 a	3.1±0.3 a
	PHF (2ml/kg)	-	3.8±0.4***	1.6±0.2***	2.0±0.25*

DISCUSSION:

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and causes significant dementia in elderly. The neuropathological hallmarks of AD include deposits of amyloid β fibrils as a senile plaques and presence of abnormal tau protein filaments in the form of neurofibrillary tangles⁴⁸. Hippocampus, limbic system and cortex are the primary areas involved in the pathology of AD⁴⁹. The etiopathogenesis of AD is multifactorial and oxidative stress has been reported to play a significant role in the onset and progression of AD. Considering the mechanistic aspects, it has been recognized that β-amyloid aggregates cause oxidative damage by free radical generation⁵⁰. Centrally administered colchicine-induced cognitive dysfunction is a well-known model that represents sporadic dementia of Alzheimer’s type (SDAT)⁵¹. In the present study, we investigated the effect of Polyherbal formulation (PHF) in the prevention of sporadic dementia of Alzheimer’s type by using intracerebroventricular administration of colchicine in rats. Cytoskeleton disruption has been linked to neurodegeneration in AD⁵². Colchicine is an alkaloid derivative that binds irreversibly to microtubules and causes their depolymerization thereby inhibiting their assembly. This leads to impaired intracellular trafficking of neurotrophic factors, synaptic loss, and increased axonal excitotoxicity⁵³. In the present study, colchicines when given centrally resulted in significant memory impairment in Morris water maze tasks, Open field, Spontaneous locomotor activity and Elevated plus maze, which were attenuated by treatment with PHF. Rats treated with PHF showed shorter swimming latencies to the goal platform than control groups, indicating improved reference, i.e. spatial memory performance. PHF treated rats also showed enhanced working memory in probe trials, indicating consolidation of memory. Additionally, PHF treated rats also showed shorter mean retention transfer latencies in elevated plus maze. PHF treatment improved the memory inability and thereby increased learning ability. This indicates that PHF augments learning animal is able to learn a new aspect of the given task, resulting in a better performance. Behavioral studies in AD models can be used to characterize the extent of lesion or to detect therapeutic effects. In this study, significant improvement of locomotor activity was observed by increased ambulatory movements, grooming and decreased latency period, rest, in open field test PHF treated animals. These behavioral parameters reveal an enhanced motor function, which is usually disturbed in AD. Oxidative stress is a critical determinant in the stimulation of neuronal cell death and Aβ toxicity results in an increase in the reactive oxygen species (ROS) and superoxide radicals, which result in oxidative damage within the cell. The toxicity of Aβ is attenuated by treatment with antioxidant such as vitamin E, as well as agents that decrease intracellular superoxide levels⁵⁴. Central administration of colchicine produces marked destruction of hippocampus granule cells and septohippocampal pathways resulting in loss of cholinergic neurons and decreased activities of acetylcholinesterase and choline acetyltransferase.

SUMMARY AND CONCLUSION:

The present study includes the evaluation of neuroprotective activity of Polyherbal formulation against colchicine induced Alzheimer’s disease. The plants mainly contain terpenoids like chamazulene and various flavonoids are mainly responsible for neuroprotective property. Plants are extensively used to relieve neurodegenerative disorders e.g. Ginkgo biloba, Withania somnifera, Chaenomeles speciosa etc. In conclusion, the result of current study evidenced that the Polyherbal formulation (PHF) showed potent neuroprotective activity against colchicine induced Alzheimer’s disease in rats whereas further studies on the polyherbal formulation required to study to know exact mechanism of action of chemical constituents.

LIST OF ABBREVATIONS:

Aβ: Beta amyloid, AChE: Acetylcholinesterase, ACSF: Artificial cerebrospinal fluid, APP: Amyloid precursor protein, AST: Aspartate aminotransferase, CDK: Cyclin-dependent kinase, ChAT: Choline acetyl transferase, CP: Convolvulus pluricaulis, CPCSEA: Committee for the purpose of control and supervision of experiments on Animals, ERK: Extracellular- signal- regulated kinase, FST: Finite state transducer, GLUT: Glucose transporter, GSH: Glutathione, HMG- CoA: 3-hydroxy-3-methyl-glutaryl-CoA reductase, HRBC: Human red blood cells, IAL: Initial acquisition latency, ICV: Intracerebroventricular, IRS: Insulin receptor substrate, ITL: Initial transfer latency, LDLR: Low density lipoprotein receptor, MECP: Methanol extract of Convolvulus pluricaulis, NFT: Neurofibrillary tangles, NGF-TrKA: Nerve growth factor & Tyrosin kinase receptor, NSAIDs: Non steroidal anti-inflammatory drugs, OCD: Obsessive compulsive disorder

OECD: Organization for Economic Co-Operation and Development, PPAR: Peroxisome proliferator-activated receptors

PSEN: Presenilin, RTL: Retention transfer latency, SREBPs: Sterol regulatory element binding proteins, TST: Thiosulphate sulfurtransferase

ETHICS APPROVAL AND CONSENT TO PARTICIPATE:

The study was approved and conducted as per the norms of the Institutional Animal Ethics Committee of H.S.K College of Pharmacy, Bagalkote (Ref No. IAEC/HSKCOP/Aug 2021/UG2).

HUMAN AND ANIMAL RIGHTS:

No humans were used in this research. The study was carried out in accordance with the CPCSEA guidelines for the use and care of laboratory animals.

CONFLICT OF INTEREST:

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS:

The authors would like to thank the Principal and Management staff of Hanagal Shri Kumareshwar College of Pharmacy Bagalkote for providing the necessary facilities and their support for successfully completing this research work.

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Received on 06.02.2024 Modified on 03.04.2024

Asian J. Pharm. Tech. 2024; 14(3):199-207.

DOI: 10.52711/2231-5713.2024.00033

Neuroprotective Activity of Polyherbal Formulation on Colchicine Induced Alzheimer’s Disease in Rat Model

Instruments

Preparation of Polyherbal formulation

Mean transfer latency (in sec)

Time spent in open (in sec)

Time spent in closed arm (in sec)

Number of entries in open arm

Number of entries in closed arm

DISCUSSION: