MATERIALS AND METHODS:

Test material:

· Proayurveda Neurotip Capsule

Animals:

· Albino mice in the range of 25-30 gm

· Wistar rats in the range of 120-200 gm

Experimental study was carried out using adult animals of either sex of Wistar rats and Albino mice. Animals were procured from Lacsmi Biopharm Pvt. Ltd., Alephata CPCSEA No. 1277.

Chemicals and Drugs:

All the chemicals used in this investigation were of analytical grade, and obtained from Maximaa Proyurveda, B Joshi Agrochem Pharma Mumbai, S.D. Fine chemicals, Sigma, Merck, and Ranbaxy chemicals.

Preparation of soluble form of Neurotip Capsule:

The Neurotip capsule is soluble in distilled water with 1% w/v gum acacia. Hence distilled water with 1% w/v gum acacia was selected as a solvent for preparation of the doses.

Experimental animals:

The pharmacological investigations were conducted on albino mice and wistar rats of either sex.

· Approval of Experimental protocols:

Ethical clearance was obtained for procuring of animals and for “Pharmacological evalution of adaptogenic and immunomodulating potential of neurotip in rodents” (Approval No. IAEC/03/2020/11).

· Animal maintenance (Housing and feeding condition):

Experimental studies were carried out using normal adult Albino mice (25-30 gm) and Wistar rats (150-200 gm) of either sex. Animals were housed in clean and sanitized polypropylene cages under standard environmental conditions. All animals were acclimatized and habituated to laboratory conditions, for seven days prior to experiment, to minimize nonspecific stress conditions.

Acute toxicity study:

Six adult albino mice in the age group of 90±10 days were used for the study. Single dose of the drug was administered orally to each animal for the determination of gross behavior and median lethal dose (LD₅₀), which can be expected to cause death in fifty percent of animals ^{6, 7}.

Principle:

An acute toxicity study was conducted for Neurotip capsule as per guidelines set by Organization for Economic Co-operation and Development (OECD guideline No. 423) received from CPCSEA.

Limit test at 5000 mg/kg body weight was considered to determine acute toxicity study of test substance. After the survival of the first animal, then two additional animals were administered with the extract at the dose of 5000 mg per kg body weight. Test was terminated after the survival of additional animals. Observation of animals was carried for 14 days.

Preparation of stock solutions:

Fresh solution of Neurotip capsule was prepared by suspending 50 gm of test substance in 100 ml of distilled water using 1% w/v gum acacia.

Administration of test substance:

Oral administration of test substance into the mice was done by using oral feeding needle.

Observation:

Individual mice were observed continuously for the first 30 min after the administration of test solution. Then mice were observed after every 30 min during the first 4 hr and observation was continued on daily basis thereafter for a period of 14 days for delayed toxicity. Parameters of observation were mortality, moribund status and gross behavior. Gross behavior observations were behavior, neurological, autonomic responses and biochemical parameter.

Sub-chronic oral toxicity:

Repeated dose oral toxicity study was carried out according to OECD guideline 407. The animals were divided into three groups of 10 animals each (5 males and 5 females). Group 1 received 10 ml/kg body weight of distilled water and served as a control. Groups 2 and 3 received doses of 500 and 1000 mg/kg body weight, respectively. The drug was administered daily for 28 days on the same time daily and observed at least twice daily for morbidity and mortality. Body weights of the animals were evaluated weekly⁸.

Haematological and biochemical analysis:

During the sub-chronic oral toxicity, on the 29^th day, after an overnight fast, the rats were anaesthetized with ether and blood sample for haematological and biochemical analysis were collected into tubes with and without EDTA, respectively. Biochemical analysis was performed on serum obtained after centrifugation of total blood (without anticoagulant) at 2500 rpm for 15 min. Standardized diagnostic kits were used for spectrophotometric determination of the following biochemical parameters: alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, alkaline phosphatase, glucose, total proteins and urea^{9, 10}.

Effect of extracts of neurotip test substance on swimming endurance of mice:

In this method, the antistress effect of neurotip test substance was evaluated by determining the improvement in swimming endurance period and overall performance of the animals, when subjected to swim in restricted space like water vessel ^{11, 12}.

Animals:

Experimental investigation was carried out using albino mice of either sex, weighing between 25 to 30 gm.

Drugs:

Testing drugs were neurotip test substance suspended in distilled water using 1% w/v gum acacia. Geriforte was used as standard drug suspended in distilled water using 1% w/v gum acacia.

Dose selection:

The dose of neurotip test substance selected in this investigation was 500 and 1000 mg/kg body weight of animals. The dose of Geriforte was 100 mg/kg body weight of animal, selected from previous reported studies of Geriforte as antistress agent. The dose of vehicle was 10 ml/kg body weight of animal ¹³.

Treatment protocol:

Albino mice of either sex were randomly assigned into six groups, and each group consisted of six mice as follows:

Group 1: Animals of this stress control group were administered with vehicle.

Group 2: In this standard group, animals were administered with Geriforte at the dose of 100 mg/kg body weight.

Group 3: Albino mice of this group were administered with neurotip at the dose of 500 mg/kg body weight.

Group 4: This group of animals were administered with neurotip test substance at the dose of 1000 mg/kg body weight.

Method:

Albino mice of all groups were treated with vehicle, neurotip test substance and Geriforte by oral route in the above order. The treatment was given for a period of 10 consecutive days. On tenth day, swimming stress was given to all the mice, by allowing for swimming in the transparent tub containing water at room temperature. The swimming of these mice was not restrained and they allowed for swimming till they got exhausted. The end point of this swimming endurance was drowning and death of the mice ¹⁴.

Effect of Neurotip test solution on chronic stress model (CS) in Rats:

Animals:

Experimental investigation was carried out using wistar rats of either sex, weighing between 150 to 200 g.

Drugs:

Testing drugs were Neurotip capsule test substance suspended in distilled water using 1% w/v gum acacia. Omeprazole (10mg/kg) was used as standard drug suspended in distilled water using 1% w/v gum acacia. Vehicle was 1% w/v gum acacia prepared in normal saline.

Dose selection:

The dose of test substance selected in this investigation was 500 and 1000 mg/kg body weight of animals. The dose of Omeprazole (10 mg/kg) body weight of animal, selected from previous reported studies. The dose of vehicle was 10 ml/kg body weight of animal.

Treatment protocol:

Wistar rat of either sex were randomly assigned into six groups, and each group consisted of six rats as follows:

Group 1: Animals of this control group were administered with vehicle and not subjected for chronic stress.

Group 2: In this disease control group, animals were administered with vehicle (distilled water using 1% w/v gum acacia) and then subjected for chronic stress.

Group 3: Animals of standard group were administered with standard substance i.e. Omeprazole at the dose of 10 mg/kg body weight and subjected for chronic stress.

Group 4: Wistar rats of this group were administered with neurotip test substance at the dose of 200 mg/kg body weight and subjected for chronic stress.

Group 5: Wistar rats of this group were administered with neurotip test substance at the dose of 400 mg/kg body weight and subjected for chronic stress.

Method:

Adult male rats of 150- 200g were selected and divided into five groups of six animals. The treatment was given as stated above for 10 days, 1 hour before the exposure of stress. Stress induced by immobilizing rats with head down supine position by fixing fore limbs and hind limbs to a wooden board daily for 2 hour (12pm to 2pm) for a period of 10 days. The anterior abdominal wall muscle of rat was incised using a sharp scalpel. The stomach was carefully dissected out from the body of rat. Glandular portion of the stomach was opened along greater curvature and gently rinsed with water. The weight of organs, such as liver, spleen, adrenal gland, and testes after washing phosphate buffer recorded per 10 % weight of organs.

Assessment of ulcer:

Stomach mucosa was examined under microscope with 10X magnification for the numbers of ulcer and severity of ulcer ^15.

Statistical analysis:

The date of this investigation was subjected to one way analysis of variance, followed by Dunnet΄s test for comparison between the groups of treatment. Difference with p<0.05 was considered as significant.

RESULTS AND DISCUSSION:

Acute oral toxicity:

During acute toxicity studies, it was found that the animals were safe up to maximum dose of 5000 mg/kg body weight and as per OECD guidelines which falls under class five values. There were no changes in normal behavior pattern and no signs and symptoms of toxicity and mortality were observed. Also it was noted that no change in alertness, visual placing, grooming, reactivity, spontaneous activity, touch response, pain response, posture, limb tone, grip strength and abdominal tone. Pupil size, corneal and pinna reflex were normal. There were no signs of CNS or autonomic stimulation, such as vocalization, restlessness, aggression, fearfulness, tremors, convulsion, Straub tail phenomenon, writhing and piloerection. Animal showed normal body weight gain during 14 days. There was no significant change in body weight as compared to control group. There was no noticeable toxicity and no deaths till the dose tested (up to the dose of 5000 mg per kg body weight of animal).

Table No.2: Effect of neurotip on body weight of Wistar rats

Sr No.	Group	Mean body weight of rats (g)± SEM
Sr No.	Group	Before dosing (1^st Day)	After 1^stweek (7^th Day)	After 2^nd week (14^th Day)
1	Control	123.67±0.71	127.67±.49	131.33±0.61
2	Neurotip test substance	126.17±1.11^ns	132.33±1.12^ns	139.33±0.76^ns

Values shown for each group as the mean ± S.E.M. obtained from six observations. Where ^nsp>0.05 represent non-significant, compared to control group.

Table 3. Acute toxicity study of Neurotip test solution

Parameters		Time (min) on 1^st Day								2 nd	3 rd	4 th	5 th	6 th	7 th	8 th	9 th	10 th	11 th	12 th	13 th	14 th
Parameters		30	60	90	120	150	180	210	240	2 nd	3 rd	4 th	5 th	6 th	7 th	8 th	9 th	10 th	11 th	12 th	13 th	14 th
Behavioral observation
	Alertness	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4
	Stereotypy	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Awareness	Visual placing	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4
	Passivity	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Vocalization	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Grooming	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4
Mood	Restlessness	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Irritability	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Fearfulness	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Spontaneous	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4
	Activity
Motor activity	Reactivity	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4
	Touch response	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4
	Pain response	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4	4
Neurological observation		0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Central Excitation	Straub΄s response	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Tremor convulsions	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0	0 0

Table 3. Acute toxicity study of Neurotip test solution

Parameters

Time (min) on 1^st Day

120

150

180

210

240

Motor

inco- ordination

Body position Limb position Righting reflex

Muscle tone

Limb tone Grip strength

Body tone

Abdominal tone

Reflexes

Pinna reflex

Corneal reflex

Autonomic observation

Optical signs

Pupil size

Secretory sign

Urination

Salivation

General signs

Writhing Piloerection

Skin color

Toxicity

Mortality

Acute

Delayed

Moribund status

Acute

Delayed

The base score for normal signs or effects is 4. Score below or above 4 indicates subnormal or supernormal respectively. The base score for abnormal signs is 0 and the maximal score is 8. N indicates normal

Sub-chronic oral toxicity:

In sub-chronic oral toxicity studies (the doses chosen on the basis of 1500-3000 mg of the drug per 60 kg body weight of human beings and which may be 10 to 20 times for the sub-chronic toxicity studies. In this regard, here, in our experiment for chronic toxicity studies the dose after calculation was taken as 500 mg/kg and 1000 mg/kg body weight of rats) it was found that there were no changes in normal behavior pattern and no signs and symptoms of toxicity. Similarly, no significant differences in body weight were observed between control and treated groups during this period (P > 0.05) and data is presented in table.

There was small increase in the weight of liver in the treated group at dose level 1000 mg/kg but is not statistically significant (P > 0.05). A slight decrease in the weight of testis in treated group was found in comparison to control group and may be considered as variations close to or within the normal range. Weights of other organs are not changed significantly and are presented in graph.

Although a small decrease in RBC counts in comparison to control group were noted but was found within the limits. Other hematological parameters were also within the normal range. Neurotip test substance is not showing the toxicity in the circulatory system even at high dose. The serum enzyme levels, total protein levels and creatinine remain close to the control values indicating that there were no deterioration in the function of liver and kidney which further suggests that on chronic administration of the test substance neither impaired the physiology of the kidneys and liver nor the cellular structures of the liver and kidneys. The level of urea and blood sugar are statistically insignificant (P > 0.05). The biochemical parameters for rats after 28 days of treatment with two different doses are presented in table.

So, in a conclusive way it may be said that the Neurotip capsule are found to be well-tolerated by rats. Acute oral toxicity studies in rats using the highest attainable dose of 5000 mg/kg for 14 days did not cause any lethal effect indicating that LD 50 if any, should be higher than this dose. Organ weight and hematological parameters also remained close to or within the normal range suggesting no side effect.

Table no. 4: Effects of Neurotip test substance on organ weight

No.	Group	Organ weight in gm after 28 days of treatment
No.	Group	Heart	Liver	Kidney	Testis
1.	Control	0.51 ± 0.12	2.5± 0.16	0.36 ± 0.24	1.36 ± 0.71
2.	500 mg/kg test substance	0.52 ± 0.24	2.7 ± 0.34	0.36 ± 0.41	1.37 ± 0.69
3.	1000 mg/kg test substance	0.53 ± 0.19	2.7 ± 0.39	0.38 ± 0.21	1.39 ± 0.87

Results were expressed as mean SEM (n = 6) of rats after 28 days treatment with neurotip test substance. Neurotip test substance treated groups showed non-significant changes as compared with control rats (P > 0.05).

Table 5. Hematological parameters for rats after 28 days treatment with Neurotip test substance at different doses

Parameters	Control	500mg/kg	1000mg/kg
Hemoglobin (g/dl)	11.30 ± 0.91	11.50 ± 0.88	11.62 ± 1.08
RBC (×10⁶/µl)	8.65 ± 0.65	8.35 ± 0.35	7.94 ± 0.78
WBC (×10³/µl)	7.31 ± 0.48	7.65 ± 0.35	7.68 ± 0.65
Haematocrit (%)	36.85 ± 1.52	36.85 ± 1.65	36.95 ± 1.34
MCV (fl)	42.14 ± 2.32	42.52 ± 3.40	43.21 ± 2.21
MCH (pg)	14.5 ± 0.42	15.10 ± 0.45	15.1 ± 0.32
MCHC (%)	35.03 ± 0.32	35.06 ± 0.12	35.23 ± 0.32
Platelet (×10³/µl)	852.10 ± 34	935.26 ± 0.21	981.32 ± 0.34

Results were expressed as mean SEM (n = 06). Neurotip test substance groups showed non-significant changes as compared with control rats (P > 0.05).

Table 6. Biochemical parameter for rats after 28 days treatment with Neurotip test substance at different doses

Parameters	Control	500 mg/kg	1000 mg/kg
Urea (mg/dl)	16.12 ± 0.98	16.32 ± 0.24	16.54 ± 0.29
Sugar (mg/dl)	118.16 ± 3.12	119.52 ± 3.32	119 ± 0.12
Alkaline Phosphate(U/L)	260.12 ± 41	223.21 ± 0.14	232.34 ± 0.28
AST (U/L)	234.28 ± 12	209.36 ± 0.16	218.14 ± 0.23
ALT (U/L)	65.31 ± 5	59.32 ± 0.61	57.24 ± 0.42
Total Proteins (g/dl)	5.81 ± 1.2	5.92 ± 1.64	6.43 ± 1.51
Creatinine (mg/dl)	8.21 ± 0.5	7.82 ± 0.42	7.32 ± 0.52

Results were expressed as mean SEM (n = 06). Neurotip test substance groups showed non-significant changes as compared with control rats (P > 0.05).

Twenty eight days toxicity studies with 500 mg/kg and 1000 mg/kg also revealed no lethal action and no adverse effect on the organs as indicated by no change of organ weight. Hematological parameters and some specific serum enzyme levels remain also close to the control values.

Effect of Neurotip test substance on swimming endurance in mice¹⁶

Improvement in swimming endurance period of mice was used as a parameter to evaluate antistress activity of extracts.

The swimming endurance or survival time of mice in stress control group without any drug treatment was found to be 187.33± 7.3 min.

The swimming survival time of mice in standard drug treated group with Geriforte was found to be 248±12 min. Compared to stress control group value, Geriforte exhibited highly significant (p<0.001) increase in swimming survival time.

Group of mice (n=6) treated with Neurotip test solution at the dose of 500 mg per kg body weight of animal, exhibited survival time of 227.33 ± 5.4 min.

In group no. 4 response study, Neurotip test solution at 1000 mg per kg body weight of animal, exhibited survival time of mice with 239.5±9.2 min. Neurotip test solution showed highly significant (p<0.001) increase in swimming survival time.

Table 7. Effect of Neurotip test substance on swimming endurance

Sr No.	Group	Treatment	Dose/kg Body weight	Survival time (min) Mean ± S.E.M.
1	Control	Vehicle + Swimming	0.25ml/mice	187.33 ± 7.365
2	Standard	Geriforte+ Swimming	100 mg/kg	248 ± 12.056^**
3	NT 500 mg/kg	Neurotip test solution + Swimming	500 mg/kg.	227.33 ± 5.469^*#
4	NT 1000 mg/kg	Neurotip test solution + Swimming	1000 mg/kg	239.5 ± 9.291^**#

Values shown for each group as the mean ± S.E.M. obtained from six observations. Where *p<0.01 and **p<0.001 represent very significant and highly significant respectively, compared to the control group. Whereas #p>0.05 represent non-significant compared to standard (Geriforte) group.

Effect of extracts of Neurotip test substance on chronic stress model (CS) in rats¹⁷:

In this method, the level of Ulcer index in the stomach of albino rats was used as a parameter to evaluate extent of gastric ulceration due to stress, and the effect of neurotip test substance on this Ulcer index was determined to evaluate antistress activity.

In group No. 2 immobilized stress produces extremely significant (p<0.0001) increase in mean Ulcer Number, mean Ulcer Severity score, Ulcer Percentage and mean Ulcer index with the values of 7.66±0.55, 2.66±0.20, 100 and 11.03 respectively, as compared to the control group value. The pH was found to be extremely significant (p<0.0001) with the value 1.85 ± 0.09.

In group no. 3 Mean Ulcer Number, mean Ulcer Severity score, Ulcer Percentage and mean Ulcer index in the albino rats of Omeprazole (standard drug) treated group was found to be 0.66±0.66, 0.16±0.16, 16.66 and 1.74 respectively. The pH was found to be extremely significant (p<0.0001) increased to 3.23±0.15. Thus extremely significant (p<0.0001) decrease in ulceration was observed, when compared to the stress control group value. The ulcer protection by Omeprazole (standard drug) was found to be 84.22 %.

In Group no. 4, Animals are (n=6) pretreated with neurotip at the dose of 500 mg per kg body weight of animal, exhibited decrease in mean Ulcer Number, mean Ulcer Severity score, Ulcer Percentage and mean Ulcer index with the mean values of 0.83±1.04, 0.66±0.49, 31.56 and 3.48 respectively. Compared to the stress control group value, Neurotip test solution showed statistically significant (p<0.01) decrease in the ulceration under same stressful condition. The pH was found to be extremely significant (p<0.0001) increased to 2.79±0.07. The ulcer protection by Neurotip test solution was found to be 68.44 %, thereby showing antistress effect of neurotip test solution.

In group no 5 of dose response study, neurotip test solution at 1000 mg per kg body wt. of animal exhibited decrease in mean Ulcer Number, mean Ulcer Severity score, Ulcer Percentage and mean Ulcer index with the mean values of 0.76±1.09, 0.5±0.5, 24.14 and 2.25 respectively. This value was found to be statistically non-significant compared to the value of standard Omeprazole treated group. Compared to the stress control group value, Neurotip test solution showed statistically significant (p<0.01) decrease in the ulceration under same stressful condition. The pH was found to be extremely significant (p<0.0001) increased to 3.04±0.08. The ulcer protection by Neurotip test solution was found to be 83.86 %, thereby showing antistress effect of Neurotip test solution.

Ingredients	Composition	Scientific name	Part used	Solubility
Guduchi	40 mg	Tinospora cordifolia	Stem	Water
Ashwagandha	50 mg	Withania somnifera	root	Water
Shankapushpi	30 mg	Convolvulus Pluricaulis Linn	leaves the roots	water
Jatamansi	12 mg	Nardostachys jatamansi DC	Rhizomes (underground stems)	Water
Pippali	41.67 mg	Piper longum	roots and fruits	Water
Shilajit	20 mg	-	blackish-brown powder or an exudate from high mountain rocks,	water
Satksheera Powder	73 mg	Probiotics		Water

Group	pH	Mean Ulcer Number	Ulcer Severity score	Ulcer %	Mean Ulcer index	Percentage Protection
Control	3.10±0.04	0	0	0	0	100
Stress Control	1.85±0.09^a	7.66±0.55^a	2.66±0.2^a	100	11.03	0
Standard + stress	3.23±0.15^b	0.66±0.66^b	0.16±0.1^b	16.66	1.74	83.34
NT 500 mg/kg + stress	2.79±0.07^bf	0.83±1.04^bf	0.66±0.4^df	31.56	3.48	68.44
NT1000 mg/kg + stress	3.04±0.08^bf	0.76±1.09^bf	0.5±0.5^df	24.14	2.25	75.86