1. INTRODUCTION:

1.1 Sport Person:

The demand for sports Nutraceuticals is increasing day-by-day due to the greater consumer awareness and increasing acceptance of Nutraceutical supplements in the market. This increasing consumer demand for supplements geared towards sports nutrition and will continue to see strong sales in coming years. Global market for sports nutrition products is expected to grow at a rate of 24.1%, and is projected to reach $91.18 billion.¹

The major reasons for explosive growth of such types of athlete performance supplements are due to changing product market focus. Traditionally, sports endurance supplements mainly target towards body-building and other professional athletes. However, sales of sports nutrition products have now been promoted and have been classified into multiple fitness categories.

The sports industry has expanded to include the individuals that also participate in recreational workouts and other physical activity. Although athletes’ performance products have been geared towards male consumers but now-a-days, the sports performance Nutritional supplements are popular in both the genders. There has also been growth within the youth segment for athletes’ performance Nutritional supplements. A survey performed by the National Health Interview Survey (NHIS) shows that 1.6% of kids, are now users of sports performance formulations.¹

For 2–3 hrs of physical loads on a daily basis, athletes must consume the recommended amount of carbohydrates (7–12g/kg of body weight), the recommended protein intake is 1.2–1.6g/kg of body weight, and the energy value supplied by fat should not be higher than 35%. In addition, athletes must consume the recommended amounts of vitamins and minerals.²

1.2 NUTRACEUTICAL TYPES FOR ATHLETES³:

Organic Fat Burners:

Fat burners enhance basal metabolic rate and energy levels of the body, suppress appetite, and reduce excess water levels. Athletes typically use fat burners to improve their energy levels to enhance their performance.

Muscle Building Supplements:

These supplements are required to stimulate growth hormone, testosterone and maintain metabolic rate. Some of the important muscle building supplements are:

Whey proteins (WP):

As per recommended dietary allowance (RDA), the normal daily intake of a person should be 1-1.5 g of protein per kg body weight. This amount cannot be recovered from the diets alone. To build and repair muscle that is broken down during exercise protein is essential for athletes

Branched Chain Amino Acids (BCAA):

Branched chain amino acids (BCAAs) are the most widely used supplements among natural bodybuilders. BCAA helps to stimulate protein synthesis and promote muscle building and faster recovery from exercise.

Essential Fats:

The fats can be synthesized by body from the diet. But fatty acids like linolenic and linoleic acid cannot be synthesized in the body, so must be obtained from food. These basic fats, found in plant foods, are used to produce specialized fats in the body called omega-3 and omega-6 fatty acids.¹

1.3 NUTRACEUTICAL⁴:

The term 'Nutraceutical' was coined from 'Nutrition' and 'Pharmaceutical' in 1989 by DeFelice and was originally defined as ‘a food (or part of the food) that provides medical or health benefits, including the prevention and/or treatment of a disease’. A Nutraceutical may be a naturally nutrient- rich food such as spirulina, garlic, soy or a specific component of a food like omega-3 oil from salmon. They are also known as medical foods, nutritional supplements and dietary supplements.

2. MATERIAL AND METHODS:

Spirulina procured from NB lab Nagpur Pvt. Ltd. Ashwagandha powder, Safed musli, milk powder, cocoa powder were collected from local market. Safed musli powder were processed according to the procedure described by Ravi Agrawal (2013)⁷.

2.1 Pre-formulation study:

2.1.1 Preliminary Phytochemical Screening:

As per the WHO Guidelines for quality standardization of Herbs and Herbal Formulation. The preliminary Phytochemical screening of herbs including Safed musli, ashwagandha and spirulina were carried out.⁵

2.1.2 Standardization of Herbs:

1] Ash value:⁵

Total Ash:

Procedure:

Weighed 2 g test portion into porcelain crucible and placed in temperature controlled furnace preheated to 600ᵒc. Hold at this temperature for 2 hr. and transferred crucible directly to desiccators, cooled and weighed immediately. Reported percent ash to fiest decimal placed and total ash.

Calculation:

value calculated by the given formula.

Weight of empty dish = x

Weight of drug taken = y

Weight of dish + Ash (after complete incineration ) = z

wt. of ash = (z-x)

‘y’ g of crud drug gives (z-x) g of the ash

100 g of crud drug gives 100 × (z-x)g of the ash

Total Ash value of the sample = 100 (z-x ) %

a) Acid insoluble Ash:⁵

Boiled the total ash for five minutes with 25 ml of dilute hydrochloric acid, collected the insoluble matter in a Gooch crucible or on an ash less filter paper, washed with hot water, ignite, and weighed. Calculated the percentage of acid- insoluble ash with reference to the air dried drug.

Calculation:

Similar to previous experiment

Weight of the residue (step vi) = ‘a’ g

g of the air dried drug gives ‘a’ g of acid insoluble ash

100 g of the air dried drug gives 100 × a g of acid insoluble ash . y

Acid – insoluble ash value of the sample = 100 × a %

y

b) Water- soluble ash:⁵

Boiled the total ash for five minutes with 25 ml of water, collected the insoluble matter in a Gooch crucible or on an ash less filter paper, washed with hot water, ignite, and weighed. Calculate the percentage of water soluble ash with reference to the air dried drug.

Calculation:

Similar to previous experiment

Weight of the residue (step v) = ‘a’ g

g of the air dried drug gives ‘a’ g of acid insoluble ash

100 g of the air dried drug gives 100 × a g of water soluble ash . y

Acid – insoluble ash value of the sample = 100 × a %

2.1.3 Moisture Content by Gravimetry Method (Loss on Drying):

Weighed about 1.5g of the powdered drug into a flat and thin porcelain dish. Dry in the oven at 100ᵒc or 105ᵒc, until two consecutive weighings do not differ by more than 0.5 mg. Cooled in a desiccator and weighed. The loss in the weight is usually recorded as moisture.⁵

2.2 Processing of Safed Musli powder:

The processing of Safed Musli was carried out by making modification in the method described by Ravi Agrawal (2013)⁷. After washing the roots of Safed Musli, all dirt was removed. The drying conditions were followed by hot air oven, temperature at 40-60ᵒc.

During drying of the samples, the observations like time and temperature were recorded with respect to moisture loss. Crushed the sample and collected the fine powder and then packaged in a translucent or colored polythene bag and kept in a plastic container with cover and stored at room temperature at 30°C ± 2 for chemical analysis. The preparation of the Safed Musli has been depicted in Figure.2⁶

2.3 Preparation and formulation of Nutraceutical rejuvenator for sport persons:

Nutraceutical powder was prepared by mixing of safed musli, spirulina powder, ashwagandha powder with other ingredients (cocca powder, milk powder). The prepared powders were then sealed in translucent or colored polythene bag and used for chemical analysis and sensory evaluation. For shelf life study, prepared nutraceutical powder was also sealed in colored polythene bags and stored up to 4 months at room temperature. The preparation and formulation of the product has been depicted in figure no.3

2.4 Physicochemical parameter⁴:

Determination of pH:

Calibrated the pH meter by 4 pH buffer and 7 pH buffer. Accurately weighted 10 g of sample in 25 ml beaker. Dip pH meter in sample and determined the pH of sample.

2.5 Sensory Evaluation⁶

Determination of the sample size.

(StatCal ) V 1.1 to satisfy Likert’s Test

· The best estimation of the population size = 400

· The best estimation of the rate in population = 10%

· Maximum acceptable difference = 10%

· Desired confidence level in outcome = 93%

· Required sample size = 27

2.6 Estimation of Nutritional Value^6,7:

A. Protein Content:

The food sample to be analyzed is weighed into a digestion flask and then digested by heating it in the presence of sulphuric acid (an oxidizing agent which digests the food), anhydrous sodium sulphate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction). Digestion converts any nitrogen in the food (other than that which is in the form of nitrates or nitrites) into ammonia, and other organic matter to CO₂ and H₂O. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH4+). After the digestion has been completed, the digestion flask is connected to a receiving flask by a tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide, which converts the ammonium sulphate into ammonia gas. The ammonia gas that is formed is liberated from the solution and moves out of the digestion flask and into the receiving flask - which contain standard acid. This standard acid is titrated with known concentration of alkali.

Calculations:

Calculate the protein content of the sample with the following formulations:

%Protein = Blank – Reading * 1.4 * N of Acid / Wt of sample *100

B. Carbohydrate:-^6,7

Calculate the carbohydrate content of the sample with the following formulations:

% Carbohydrate= 100 – (Moisture+ Ash+ Protein + Fat)

C. Fat: ^6,7

A known weight of sample(M) added in extraction flask, added 50 ml of solvent and water mixture and extract solvent layer in pre-weighed empty dish (W1). Repeated the extraction process thrice times. Then evaporate the solvent & weigh the dish after complete evaporation (W2).

Fat = [(W2 – W1) /M] *100

D. Energy:-^6,7

Calculate the Energy content of the sample with the following formulations:

Energy (Kcal/Kg) = (Carbohydrate+ Protein)*4 + (Fat)*9

2.7 Metal content ⁶

Scope of metal – cd, pd, Ni, Al, As, Se, Ag, Hg, Cu, Fe, Mn, Zn

Procedure for sample preparation and analysis:

A sample of food cleaned with distilled water and dry out at room temperature and then grinded the sample and homogenized in homogenizer. Added 5gm sample directly in centrifuge tube and shake for 5 min on vortex vibrator for better mixing. If sample is semisolid then opt for digestion as under.

Put sample direct in clean round bottom flask or assembly in heating metal and attached to closed assembly heating Y tube with on-off knob. Added 15-20ml of conc. Nitric acid and distilled water through Y tube, immediately closed it and kept for 5 min as to subside reaction slowly. Start constant heating 80-95˚C in heating mental connected with water condenser system and heating must be slow and steady until the sample matrix get cleared (approx 5 hrs). After completing all the reaction, cool the sample and settle down the fumes. Then rinse condenser with distilled water in flask and then filter sample through whatman (no 40) filter paper and make up the volume upto the mark 100 ml with distilled water. Ready sample in the analyser in ICP - AEC and AAS flame, AAS furnace, AAS cold vapour pressure technique.

2.6 Microbial Assessment:

In-vitro anti Microbial Activity (Well Diffusion Method):

Nutrient agar media plate was prepared with a depth of 4mm. Dried surface of nutrient agar plate was inoculated by spreading the swab 3 times over the entire agar surface. After standardization of bacterial suspension (100 µl), sterile cotton swap was immerged in it and the swap was rotated several times, with firm pressure on the inside wall of the tube to remove excess fluid. A sterile 5mm cork borer was used to punch holes after solidification of media. The wells formed were filled with different concentrations of the extract which were labeled accordingly; 50mg/ml, 37.5mg/ml, 25mg/ml, 12.5mg/ml. The plates were then left on the bench for 1 hour for adequate diffusion of the extracts and incubated at 37°C for 24- 48hours in upright condition. After incubation, the diameter of the zones of inhibition around each well were measured to the nearest millimeters along two axis i.e. 90° to each other and the mean of the four reading were then calculated included 5mm well.⁸

2.7 Initial accelerated stability study of formulation:

These studies were carried out on Nutraceutical formulation by using REMI SC-6 plus stability chamber according to ICH guidelines. The accelerated stability conditions of 40^oC ±2^oC with 70±5% RH for 90 days. The physical stability of powder formulation was observed periodically. The Nutraceutical formulation was evaluated after one month for the physiochemical parameter, flow property, antimicrobial study, biological evaluation.⁹

2.8 Evaluation method of animal study¹⁰:

Animals:

About 25 rats of either sex weighing between 130 -250 gms procured from disease free animal house of Aquarium Gupta, Nagpur. Animal had free access to food and water and maintained under standard laboratory condition with a natural light and dark cycle. The animals were acclimatized for at least five days before behavioural experiment.

Study design:

The animal were selected randomly for each experiment and divided into 5 equal group(n=5)

Experimental model¹¹:

1) Swimming Exercise Performance Test:

The swimming exercise performance test was followed by some modifications. Rats were pre-treated with vehicle, Nutraceutical Formulation-6mg, Nutraceutical Formulation-8mg, Nutraceutical Formulation-10mg mg for continued 14 days and one hour after the last administration, followed by an exhaustive swimming test. The rat was taken out from each treatment for swimming exercise and loaded the constant weight (attached to the tail) corresponding to 5% of individual body weight. The rats were individually carried out in a columnar swimming pool (65 cm tall and radius 20 cm) with 40-cm water depth maintained at 27 ± 1 °C. The endurance for each rat was measured as swimming times recorded from the beginning to exhaustion which was determined by observing loss of coordinated movements and failure to return to the surface within 7s. In the swimming period, it was considered the time spent floating, struggling and making necessary movements until exhaustion and possible drowning.

2) Determination of Blood Biochemical Variables:

The effects of nutraceutical formulation on creatine kinase (CK) activity were evaluated after exercise. After 1 h of the last administration, a 15-min swimming test was performed without weight-loading. Blood samples were immediately collected from the pre-treated rat after swimming exercise. The plasma was prepared by centrifugation at 1,500 × g, 4 °C for 10 min. glucose levels and CK activity were determined.

PROCEDURE FOR ORBITAL SINUS BLOOD SAMPLE COLLECTION⁹:

Requirements:

Animal, anesthetic agent, cotton, capillary tube and blood sample collection tubes.

1 This technique is used with recovery in experimental circumstances and this method is also called periorbital, posterior orbital and orbital venous plexus bleeding.

2 Blood sample is collected under general anesthesia.

3 Topical ophthalmic anesthetic agent is applied to the eye before bleeding.

4 The animal is scruffed with thumb and forefinger of the non-dominant hand and the skin around the eye is pulled taut.

5 A capillary is inserted into the medial canthus of the eye (30 degree angle to the nose).

6 Slight thumb pressure is enough to puncture the tissue and enter the plexus/sinus.

7 Once the plexus/sinus is punctured, blood will come through the capillary tube.

8 Once the required volume of blood is collected from plexus, the capillary tube is gently removed and wiped with sterile cotton. Bleeding can be stopped by applying gentle finger pressure.

9 Thirty minutes after blood collection, animal is checked for postoperative and periorbital lesions [steps: 3 & 4]

CREATINE-KINASE ACTIVITY:

The creatine-kinase CK] (adenosine 5'-triphosphate creatine-phosphotransferase, E.C. 2.7.3.2) catalyses the following reaction.

High CK enzyme activity is detectable in muscle, heart and brain. CK activity is also detectable in serum. The CK [MW.: 80,000.00 D] consists of two subunits. The subunits are signed as CK-M [M = muscle] and CK-B [B = brain]. The combination of two subunits results in the formation of three types of isoenzymes: CK-MM, CK-BB and the hybrid form of CK-MB are characteristic for muscle, brain and heart, respectively. CK-MB activity is characteristic for the heart. Figure shows the distribution of CK activities of the human body.

Determination of CK activity

CK activity is determined with the help of the following coupled enzyme reactions:

ATP - the product of CK - is utilised by hexokinase. The reaction results in the formation of glucose-6-phosphate. Glucose-6-phosphate serves as a substrate for the next enzymatic reaction: one of its products is NADPH. The NADPH shows absorption maximum at 340 nm. The generation of reduced NADPH can be detected photometrically.

Solutions:

1 50 mM TRIS-HCl pH 7.2 containing 1 mM ADP, 20 mM Glucose, 30 mM MgCl2, 5 mM Cystein, 1 mM NADP and 10 mM Creatine-phosphate

2 Hexokinase (60 U/ml)

3 Glucose-6-phosphate dehydrogenase (30 U/ml)

4. Serum of normal animal

Calculation

Δextinction

Minute

Serum Activity [IU//1]= --------------- ^* 10,400

6.22

3. RESULTS:

3.1 Preformulation study:-

3.1.1 Quality Standardization of Herbal drug.

A) Physicochemical parameter

Table no.:-1: %LOD, Total ash value, water soluble ash and acid insoluble ash

Sr. No	Parameter	Spirulina	Safed musli	Ashwagandha
1	%LOD Mean (n = 3) ± SD (% w/w)	0.23 ±0.5	0.16±0.20	0.39 ±0.25
2	Total ash Mean (n = 3) ± SD (% w/w)	5.26 ±0.15	6.01± 0.25	7.02 ± 0.60
3	Water-soluble ash Mean (n = 3) ± SD (% w/w)	3.23 ± 0.15	3.6 ± 0.21	3.07 ± 0.02
4	Acid-insoluble ash Mean (n = 3) ± SD (% w/w)	0.46 ± 0.30	0.36 ± 0.25	0.16 ± 0.25

*All values are average of three determine (n=3)

3.2 Evaluation of Nutraceutical powder

3.2.1 Physicochemical parameter

Table no.:-2: %LOD and Total ash value for Nutraceutical Powder formulations

Formulation	%LOD Mean (n = 3) ± SD (% w/w)		Total ash Mean (n = 3) ± SD (% w/w)
Formulation	Pre stability Mean±SD	Post stability Mean±SD	Pre stability Mean±SD	Post stability Mean±SD
F1	1.5±0.050	2.0±0.150	5.3±.0.18	5.06 ±0.15
F2	0.98±0.05	1.16±0.20	5.6±.0.15	5.01± 0.25
F3	1.0±0.15	1.39 ±0.25	6.3±.0.15	6.22 ± 0.60
F4	0.82±0.1	0.99 ±0.5	7.6±.0.15	7.3±.0.15

*All values are average of three determine (n=3)

Table no:-3. Water-soluble ash Mean and Acid-insoluble ash, Mean of formulations

Formulation	Water-soluble ash Mean (n = 3) ± SD (% w/w)		Acid-insoluble ash Mean (n = 3) ± SD (% w/w)
Formulation	Pre stability Mean±SD	Post stability Mean±SD	Pre stability Mean±SD	Post stability Mean±SD
F1	2.44±0.25	2.23 ± 0.15	1.90±0.24	1.46 ± 0.30
F2	3.90±0.15	3.6 ± 0.21	1.0±0.2	1.36 ± 0.25
F3	4.80±0.15	4.07 ± 0.02	1.01±0.8	1.16 ± 0.25
F4	2.15±0.18	2.19±0.25	0.99±0.28	0.92±0.24

*All values are average of three determine (n=3)

The physicochemical quality standardization like moisture content and Ash values of herbal crude drug and Formulation batches (pre and post stability) were determined, Moisture (loss on drying) is one of the major factors responsible for the deterioration of the drugs and formulations. Low moisture content is always desirable for higher stability of Herbal drugs. Moisture contents of the individual Herbal drugs (table no.1) were found in the range 0.16-0.39 %, w/w, and different Formulation batches F1, F2, F3, F4 pre and post stability (table no 2) in the range of 0.82 – 2.0 %w/w , all the values were less than 5%. Formulation F1,F2,F3 shows change in moisture content and F4 shows 0.82-0.99 %w/w moisture better stability, and high ash value is indicative of contamination, substitution, adulteration, or carelessness in preparing the drug or drug combinations for marketing. All the individual drugs and different Formulation batches F1, F2, F3, F4 Pre and post stability were determined. Ash values of the individual Herbal drug found to be Total ash value (table no.1) in the range 5.26 – 7.02 % w/w, Water-soluble ash (table no.1) in range of 3.07 - 3.6 % w/w and Acid-insoluble ash (table no. 1) in the range of 0.16 – 0.46 % w/w. and ash values of different Formulation batches F1, F2, F3, F4 pre and post stability (table no. 2) were found to be, total ash value in the range of 5.01-7.6 % w/w, water soluble ash (table no. 3) in the range of 2.15-4.80 % w/w, acid insoluble ash (table no. 3) in the range of 0.92-1.90 % w/w. These values were found to be reasonably low indicating low contamination. Moisture and ash values of the formulations matches with the average total ash values of the individual drugs. Formulation F1,F2,F3 shows change in values and F4 shows total ash 7.3-7.6, water soluble ash 2.17-2.19 and acid insoluble ash 0.99-0.92 having better stability.

Table no.4. pH of nutraceutical formulation

Formulations	PRE STABILITY	POST STABILITY (After 1 month)
Formulations	pH Mean±SD	pH Mean±SD
F1	4.66±0.15	4.92±0.24
F2	4.02±0.02	4.72±0.01
F3	4.5±0.20	4.97±0.02
F4	4.31±0.020	4.26±0.02

*All values are average of three determination (n=3)

Nutraceutical Rejuvenator formulation batches F1, F2, F3 and F4 combination were prepared and evaluated further by determining pH. Formulations F1, F2, F3 shows change in pH. Only F4 formulation shows pH range 4.26-4.31

3.3 Sensory Evaluation

· Determination of the sample for percent size to statistical calculation (StatCal ) V 1.1 to satisfy Likert’s Test

· The best estimation of the population size = 400

· The best estimation of the rate in population = 10%

· Maximum acceptable difference = 10%

· Desired confidance level in outcome = 93%

Required sample size = 27

Table no. 5: Sensory Evaluation

T1	Parameter	Very Poor	Poor	Good	Very Good	Excellance
	Flavor	16	11	-	-	-
	Taste	20	7	-	-	-
	Consistency	8	19	-	-	-
	Color	17	10	-	-	-
T2	Flavor	15	6	-	-	-
	Taste	14	13	-	-	-
	Consistency	14	13	-	-	-
	Color	18	9	-	-	-
T3	Flavor	17	6	4	-	-
	Taste	6	15	6	-	-
	Consistency	3	19	5	-	-
	Color	6	1	20	-	-
T4	Flavor	-	6	12	8	1
	Taste	-	5	9	9	4
	Consistency	-	4	13	8	2
	Color	-	6	12	7	2

Sensory evaluation is an essential concept in food product development as it reduces the risk of product failure and links the consumer perception about quality of food. Even though formulated food products are nutritious but without taste and flavour, the product cannot reach market successfully. The results of sensory evaluation different formulation batches F1, F2, F3, F4 are illustrated in the Table no. 5. The data reveals that, formulation F4 was found to be highly acceptable with significant difference. When compared to other formulation F4 having better taste, flavour, consistency and colour of soup. From this study we finalize the F4 powder formulation as optimized formulation.

3.4 Estimation of Nutrition Value

Table no.6. Estimation of Nutrition Value

Sr. No.	Nutritional Parameter	Nutritional Value (per 100 gm)
1.	Energy Value ( Kcalories/100gm)	340.63
2.	Carbohydrates	66.04
3.	Total Fat	0.91
4.	Protein	17.7

Nutraceutical Rejuvenator Powder formulation was prepared using performance enhancer herb’s, flavoured powder. Optimized formulation subjected for Nutritional analysis and it was found to highly acceptable. The nutrient analysis of the formulation is represented in the table no. 6. Protein content was found to be 6.54 gm/100gm so the formulation is high in protein content which is required for sport person, carbohydrates was found to be 66.04 gm/100gm which is found to be in safety range required for sports persons, total fat 0.90 gm/100gm shows low total fat essential for sport person. Energy Value (Calories) was found to be 340.63 Kcal/100gm shows acceptable calorie value. The above study shows prepared formulation is in superior and safe nutrition content.

3.5 Heavy Metal Content

Table no. 7: Heavy Metal Content

Sr. No.	Heavy Metal	Result ( mg/ kg )
1.	Lead ( Pb )	Absent
2.	Arsenic ( As )	Absent
3.	Cadmium ( Cd )	Absent
4.	Mercury ( Hg )	Absent

Heavy metals are harmful and become toxic for health if they are taken above the limit of daily allowance recommended. In this study, heavy metals were not found in this Nutraceutical formulation for sports persons which shows formulation is safe.

3.6 In-vitro anti Microbial Activity (Well Diffusion Method)

Table No. 8: Well Diffusion Method

Strain Used: S. Bacillus

Zone of Inhibition in mm

Standard drug

(Ofloxacin) 10mcg/ml

Concentration

50 mg/ml

100 mg/ml

150 mg/ml

200 mg/ml

Sample

12.50±0.577

14.50±0.577

16.00±0.816

18.50±0.577

31.25±0.957

*All values are average of three determination (n=3)

The standard drug ofloxacin and extract of Instant soup powder sample subjected for antimicrobial assessment for checking sustainability against S. bacillus. Different concentration of sample extract shows zone of inhibition as follows: 50 mg/ml shows 12.50 mm, 100mg/ml shows 14.50 mm, 150mg/ml shows 16.50 mm, 200 mg/ml shows 18.50 mm against standard Ofloxacin. Though sample was not superior than std but good susceptibility shown by sample againt S. bacillus.

3.7 Biological Evaluation:

Animal study:

Table No.9: Effect of creatine kinase in force swim test on rat

Duration	Control (Vehicle) (U/L)	Test(U/L)			Standard (U/L)
Duration	Control (Vehicle) (U/L)	6mg	8mg	10mg	Standard (U/L)
Day 1	226±40.45	651±51.73	712±36.58	812±35.64	326±41.66
Day 2	235.4±34.17	704±14.45	814.6±44.33	836.18±35.35	322.8±25.50
Day 3	256±61.09	724.2±16.51	804.6±96.44	874.4±18.35	342.66±20.42
Day 4	299.2±12.59	726.8±11.94	834.12±49.00	904.8±15.15	375.8±33.54
Day 5	227.8±21.75	768.4±31.48	851±28.14	969±20.65	397.6±33.24
Day 6	300±69.5	899±47.54	972.6±25.55	1066.42±36.49	446.44±37.89
Day 7	312±58.8	879±25.70	989.64±29.5	1098±32.12	459±12.34
Day 8	358 ±41.18	99.48±66.39	1020.78±75.78	1146.18±43.58	590±67.34
Day 9	258±34.67	1008±23.25	1077±21.23	1187±45.76	603±34.34
Day 10	298.23±43.23	1034±32.78	1127±34.21	1204.32±52.02	640±45.23
Day 11	312.09±43.12	1043.32±12.54	1148±62.12	1289.32±31.12	734±54.03
Day 12	353.6±32.28	1057.4±28.16	1166±65.89	1304±28.65	798.72±38.84
Day 13	305.2±21.63	1139.22±3.30	1234±33.89	1321.74±28.65	841.72±41.84
Day 14	317.70±19.43	1176.31±6.48	1249±51.04	1343.3±69,83	865.46±25.57

*All values are average of three determination (n=5)

The Nutraceutical powder formulation could increase the swimming time to exhaustion of test animals, as well as the muscular building and increase creatinine kinase. These results indicate that nutraceutical powder formulation has anti-fatigue activity and increased swimming time as well as creatinine kinase as compared to marketed formulation and day-by- day increasing the activity but 6 mg (table no. 9) concentration dose of formulation gives positive results within limits i.e 100-900 U/L and can elevate exercise performance.

4. DISCUSSION:

Throughout the experimental work experiments carried out under Standard and safety measures. The use of conventional rejuvenator and herballife etc. may have side effect in long run use. Nowadays Nutraceutical formulations is in trend to control various diseases. Nutraceuticals play important role to renew the energy and strength.

Nutraceutical Herbs like roots of Safed musli, roots of ashwagandha, spirulina powder play vital role in renew the energy and strength by use of this nutraceutical Herb. Nutraceutical Formulation was formulated in the form of powder. The formulation made by use of milk powder for sweetening, coca powder for flavor, all the ingredients are collected from local market having good quality.

The formulated Nutraceutical powder Formulation were evaluated for sensory evaluation initial accelerated stability study, pH, Viscosity, Moisture, Ash Values, physicochemical Properties , nutritional Values, Heavy Metal Content and Antimicrobial properties, and in vivo animal study. formulation showed satisfactory results. The stability studies were carried on the basis of ICH guideline for 90 days.

5. CONCLUSION:

Safe and effective Nutraceutical formulation of combined nutraceutical herbs and flavor in the form of powder was successfully developed. The present study revealed that on the basis of sensory evaluation and initial accelerated stability studies, Study F4 is superior than rest with least moisture content and taken for further analysis and it shows absence of trace heavy metals like arsenic, cadmium, lead and mercury. It is important to note that this Nutraceutical Rejuvenator is rich in protein, carbohydrates and low in fat. High energy value of the powder make it appropriate choice for fulfillment of nutritional value. Further study for antimicrobial shows the product is rich in Antimicrobial activity. The Study of animal testing was carried out and it was found that the Nutraceutical powder Formulation is superior than the marketed nutraceutical formulation.

6. ACKNOWLEDGEMENT:

We acknowledge the immense help received from authors, scholars and researchers whose articles are cited in this manuscript. We acknowledge Dr Nilesh Mahajan, Dr Vinod Thakre, and other faculty members of Dadasaheb Balpande College of Pharmacy Besa, Nagpur for their guidance and constant support to complete this study.

7. REFERENCES:

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Received on 13.02.2019 Modified on 02.03.2019

Research J. Pharm. and Tech 2019; 12(8):3903-3910.

DOI: 10.5958/0974-360X.2019.00672.3