https://doi.org/10.29070/n3eggx09
Development and Evaluation of Nutraceutical Tablets from Selected Plant Extracts: A Novel Approach to Functional Medicine
Jayashri Behera1*, Dr. Ashish Sarkar2
1 Research Scholar, School of Pharmacy, YBN University, Ranchi, Jharkhand, India
Email: jayashri.b89@gmail.com
2 Professor, School of Pharmacy, YBN University, Ranchi, Jharkhand, India
Abstract - The potential of a seed powder blend made of Cucumis melo, Punica granatum, and Linum usitatissimum in a 1:1:1 ratio is investigated in this study. In order to guarantee a consistent particle size distribution (10–30 µm), the seeds were ground into a fine powder. The parameters of bulk powder showed strong cohesion and poor flow ability. Photo microscopy, however, showed fibrous, capsular structures that were perfect for formulation. Significant growth of Lactobacillus acidophilus and Bifidobacterium bifidum indicated that the mix had superior prebiotic potential compared to individual seed powders and the conventional chicory powder. It also showed remarkable antioxidant activity. Studies on animals verified the blend's effectiveness and safety. A 40-day feeding trial in Albino Wistar rats demonstrated its haematinic effects, raising haemoglobin levels from 12.1% to 16.5% and red blood cell counts from 7.46 to 9.55 million/mm³. Acute toxicity trials revealed no negative effects. Additionally, the combination decreased VLDL and triglycerides and raised serum protein levels, suggesting possible cardiovascular advantages. Its safety for those with diabetes is supported by the fact that blood glucose levels stayed within the usual range.
The product, which was made as capsules, complied with pharmacopoeial requirements for disintegration time and weight variation. A shelf life of nine to twelve months is suggested by stability studies conducted under accelerated settings. Over the course of three months, the blend's prebiotic activity stayed steady, maintaining its capacity to promote probiotic growth. This nutraceutical formulation is a good option for treating anemia, malnutrition, and metabolic disorders since it provides a promising blend of antioxidant, prebiotic, and health-promoting qualities.
Keywords: Plant extracts; Prebiotic activity; Microbiological quality standards; Bioactive compounds; Nutraceutical tablets
INTRODUCTION
Dr. Stephen L. DeFelice came up with the phrase "nutritraceuticals," which combines the words "pharmaceutical" and "nutrition" to refer to non-toxic food ingredients that have health benefits, such preventing or treating disease (De Felice, 1989). Based on the idea that "food is medicine," as proposed by Hippocrates, this quickly expanding multidisciplinary discipline is based on human nutrition and seeks to improve health by research and creative formulations. All mental and physical functions are fueled by nutrition, with vital nutrients such as fiber, vitamins, minerals, proteins, carbs, and fats playing important roles. Maintaining health requires both macronutrients (proteins, fats, and carbs) and micronutrients (vitamins and minerals). Millions of people worldwide suffer from malnutrition, which the World Health Organization defines as an inadequate, excessive, or unbalanced nutrient consumption.
1.9 billion people are overweight, and 462 million are underweight. Of children under five, 15 million are stunted and 41 million are overweight. Furthermore, iron supplements help more than half of the 528 million women of reproductive age who suffer from anemia. However, compared to inorganic sources of iron, which can result in gastrointestinal distress, plant-based iron is frequently better absorbed (Khera et al., 2017; Singh et al., 2018).
The significance of dietary supplements, prebiotics, probiotics, and plant-based nutrients in supporting health is highlighted by the fact that unhealthy eating patterns and nutritional deficiencies are contributing factors to diet-related disorders. While nutraceuticals hold promise as possible medications, antioxidants are essential in preventing oxidative damage to biomolecules. To maximize their use, more study is necessary (Reddy et al., 2019; Patel et al., 2020).
There has been a modest but steady decline in the percentage of children suffering from malnutrition in India, in contrast to other nations. However, women, the primary caregivers of children, often suffer from poor health and limited access to clean water and sanitation, contributing to inadequate nutrition. India has the highest incidence of underweight children globally, with rates nearly double those of sub-Saharan Africa, particularly in states such as Maharashtra, Madhya Pradesh, Rajasthan, Orissa, Uttar Pradesh, and Bihar. Melghat in Maharashtra is especially affected by chronic malnutrition. As dietary and health changes impact children's physical development, growth assessment remains the gold standard for determining their nutritional and health status (Khera et al., 2017; Singh et al., 2018). About two billion people worldwide suffer from vitamin and micronutrient deficiencies, with one-third of them living in India, which is home to one-sixth of the world's population. Although trace levels of these micronutrients are necessary for many body functions, new research indicates that shortages in certain of these nutrients may be at an all-time high (Reddy et al., 2019; Patel et al., 2020).
REVIEW OF LITERATURE
The Function of Nutraceuticals in Health
Nutraceuticals are compounds that provide health advantages by preventing and treating a variety of illnesses. Dr. Stephen L. DeFelice, the term's creator, claims that nutraceuticals serve as a functional food to cure medical disorders or preserve health, bridging the gap between pharmaceutical therapy and nutrition (DeFelice, 1994). These chemicals contain bioactive molecules that have therapeutic properties, are generally produced from natural sources, and are non-toxic. The potential of plant-based extracts to enhance public health outcomes is demonstrated by their growing application in the creation of nutraceuticals (Wang et al., 2015).
Nutraceuticals Using Plant Extracts
For generations, people have used plants as a source of therapeutic treatments. Bioactive substances with potential health advantages, such as alkaloids, flavonoids, polyphenols, terpenoids, and essential oils, are found in many plant species. The pharmacological effects seen in the plants are caused by these chemicals (Patel et al., 2018). For example, because of their anti-inflammatory, antioxidant, and immune-modulating qualities, Curcuma longa (turmeric), Withania somnifera (ashwagandha), Ocimum sanctum (holy basil), and Camellia sinensis (green tea) are frequently utilized in the creation of nutraceutical formulations (Pandey et al., 2016).
Nutraceutical Tablet Formulation
There are several steps involved in making nutraceutical tablets from plant extracts, such as choosing the right plant material, extracting the bioactive ingredients, and formulating the tablets with the right excipients. To separate the active ingredients from plant materials, extraction methods like maceration, Soxhlet extraction, and ultrasonic-assisted extraction are frequently employed (Chaudhary et al., 2019). Following the extraction process, the formulation of tablets is the next stage, which calls for careful consideration of variables like dose, stability, and bioavailability. The effective delivery of bioactive substances to the intended areas in the body is ensured by the use of natural binders and carriers in tablet formulations (Gurav et al., 2020).
Assessment of Nutritional Tablets
Both in vitro and in vivo investigations are used to evaluate the safety, effectiveness, and therapeutic potential of nutraceutical tablets. While in vivo research focuses on how the tablet affects animal models or human clinical trials, in vitro testing evaluate antioxidant activity, antibacterial qualities, and enzyme inhibition (Gupta et al., 2017). One important factor affecting the effectiveness of the nutraceutical tablets is the bioavailability of the active ingredients. According to research, some formulations can increase the bioavailability of plant-based substances, such as those containing nanoparticles or improved absorption strategies (Mohan et al., 2018).
Uses of Nutraceutical Tablets in Therapeutic Settings
Nutraceutical tablets made from plants have demonstrated potential in the treatment and prevention of a variety of illnesses. Nutraceuticals made from ginseng, for instance, have been shown to improve cognitive performance and lessen fatigue (Lee et al., 2019), while extracts from ashwagandha have shown anti-stress and anti-anxiety properties (Choudhary et al., 2017). Additionally, anti-inflammatory nutraceuticals, including those made from green tea and turmeric, are frequently used to treat long-term illnesses like cancer, heart disease, and arthritis (Ghosh et al., 2018).
OBSTACLES AND PROSPECTS
Although there is a lot of promise in the creation of plant-based nutraceutical tablets, there are a number of obstacles to be addressed. These include addressing concerns about the stability and shelf life of the tablets, standardizing plant extracts, and guaranteeing the constant strength and quality of the active substances. Furthermore, the absence of precise standards for the safety and effectiveness of plant-based nutraceuticals makes regulatory approval difficult in many nations (Singh et al., 2020). To determine the therapeutic effects of these products, future research should concentrate on advancing extraction techniques, increasing bioavailability, and carrying out additional clinical trials.
MATERIALS AND METHODOLOGY
The first step in creating nutraceutical tablets from plant extracts is choosing plants such as Ocimum sanctum (holy basil), Curcuma longa (turmeric), and Withania somnifera (ashwagandha) for their anti-inflammatory and antioxidant qualities (Patel et al., 2018). These plants undergo cleaning, solvent extraction, and powder concentration (Chaudhary et al., 2019). HPLC and GC-MS are used to identify and quantify the bioactive chemicals (Gupta et al., 2017). Extracts and excipients are combined to create tablets, which are then made by wet granulation or direct compression (Gurav et al., 2020).
The physicochemical characteristics of the tablets, including their hardness and rate of dissolution, are assessed (Patel et al., 2020). Their effectiveness is evaluated using in vivo animal experiments and in vitro bioactivity tests (Mohan et al., 2018). Monitoring the absorption of bioactive compounds allows for the measurement of bioavailability (Mohan et al., 2018). For their nutritional and practical qualities, essential ingredients such as milk powder, cocoa powder, sodium glycolate, and tomato and ginger powders are utilized (Patel et al., 2020; Weier et al., 1982). GC, HPLC, and drying muffle furnaces are among the apparatus utilized (Gurav et al., 2020). The stability of the tablets is safeguarded by appropriate storage conditions (Chaudhary et al., 2019).
Prebiotic Activity (Direct Inoculation Method)
The direct inoculation method was used to evaluate the prebiotic activity of seed powders derived from Linum usitatissimum, Cucumis melo, and Punica granatum. The medium was solidified using plain agar, and the seed powder samples were inoculated with either Lactobacillus acidophilus or Bifidobacterium bifidum. The apparatus consisted of a standard chicory powder, a positive control (MRS medium for Lactobacillus acidophilus and specialized nutritional media for Bifidobacterium bifidum), and a negative control (plain agar without seed powder). To assess bacterial growth and prebiotic potential, all petri dishes were incubated anaerobically for 48 hours at 37°C. Colony counts were then reported (Patel et al., 2020; Mohan et al., 2018).
Microbiological Load Determination
In accordance with I.P. 2014 recommendations for herbal products, microbiological quality standards were evaluated. The material was diluted in sterile distilled water and incubated on Soybean Casein Agar (TAC) and Sabouraud Dextrose Agar (TFC) at 37°C for 24 hours in order to measure the total aerobic count (TAC) and total fungal count (TFC). When Salmonella, Shigella, and Escherichia coli were examined for their presence in the samples, no growth was found (I.P., 2014). In order to ensure compliance with the permissible limits—TAC < 10⁷ CFU/g, TFC < 10⁵ CFU/g, and absence of pathogens—the findings were examined by averaging duplicate counts.
RESULT AND DISCUSSION
Reduction in size
The seeds of Cucumis melo, Punica granatum, and Linum usitatissimum were cleaned, dried, and crushed to guarantee uniform weight and particle size. In accordance with the United States Pharmacopoeia National Formulary (2000), which defines powders with this mesh size as fine, the resultant powders were filtered through an 85 mesh screen. For nutraceutical formulations to behave consistently, including uniform dissolving and sedimentation rates, homogeneous particle size distribution is necessary to avoid segregation. To make a homogenous mixture for the investigation, the powders were combined.
Photographic microscopy
Using a Motic optical polarizing microscope at 4X magnification, photomicroscopy photographs of the nutraceutical powder blend (in a 1:1:1) were obtained. The particles had a fibrous, capsular structure and ranged in size from 10 to 30 micrometers.
FEATURES OF BULK POWDER
The individual powders of Cucumis melo, Punica granatum, and Linum usitatissimum, as well as their combination, showed poor flow and compressibility features when compared to bulk powder characteristics. The limited flowability and high cohesiveness were indicated by the angle of repose exceeding 40 degrees and the Carr's index above 25%.
Table 1 : Properties of Punica granatu, Cucumis melo, Linum usitatissimum, and a combination of the three as a powder
Seed powder | Mesh size | % Compressibility | Angle of repose (˚) | Flowability |
Linum usitatissimum | 85 | 25.55 | 44.15 | Poor |
Punica granatum | 85 | 22.3 | 45.00 | Poor |
Cucumis melo | 85 | 28.58 | 40.95 | Poor |
1:1:1 mixture | 85 | 23 | 43.36 | Poor |
Proximate analysis
Table 2: Estimating the distance between two points in the seed mixture
Parameter | Units | Result | Test method |
Proteins | g/100g | 35.5 | AOAC 920.152 |
Crude fibre | g/100g | 17.75 | IS 2234.2011 |
Fat | g/100g | 22.44 | IS 12711.2010 |
Carbohydrates | g/100g | 33.42 | IS 1656.2012 |
Calcium | mg/100g | 1620 | 944.02,320109 and 999.10 |
Iron | mg/100g | 22.67 | AOAC |
Soluble dietary fibre | g/100g | 4.1 | IS 11062.2010 |
Fatty acid profile Saturated fat | g/100g | 1.93 | AOAC 996.01 |
Trans fat | g/100g | Not detected | |
Polysaturated fat | g/100g | 14.82 | |
Monosaturated fat | g/100g | 5.69 | |
Omega 3 fatty acids | g/100g | 34.13 |
Antioxidant Activity Determination
The information is displayed as percentage inhibition, IC50, and quercetin equivalent. All three of the active nutraceutical ingredients demonstrated extremely high antioxidant potential, and the synthesized nutraceutical product exhibited remarkable antioxidant activity. The outcomes of the three seed powder combinations are shown in the following table.
Table 3 : The DPPH assay of nutritional powders as a percentage of inhibition
Name | % inhibition | Equation (Squared co relation coefficient) R2 | IC50 (µg/ml) | ||||
Concentration in µg/ml | |||||||
10 | 20 | 30 | 40 | 50 | |||
Quercetin (standard) | 59.02 | 76.23 | 81.4 | 87.01 | 90.23 | Y=53.666x+2.9925 R2=0.9925 | 7.51 |
Cucumis melo | 70.54 0.83 | 80.74 0.94 | 85.02 0.95 | 91.20 0.95 | 94.74 0.95 | y = 57.438x +3.696 R² = 0.9749 | 6.3322 |
Punica granatum | 51.78 1.13 | 65.30 1.16 | 78.75 1.03 | 88.95 0.95 | 93.82 0.96 | y = 61.584x - 11.562 R² = 0.9847 | 4.2087 |
Linum usitatissimum | 61.49 0.95 | 68.36 1.11 | 75.76 1.07 | 84.41 1.03 | 91.52 0.98 | y = 42.198x + 16.564 R² = 0.9351 | 6.1995 |
Seed Powder mixture Quercetin equivalent | 45.35 1.08 | 62.21 1.22 | 74.35 1.09 | 86.25 1.00 | 92.65 0.97 | Y=68.436x -24.729 R² = 0.9902 | 2.3402 |
It is more effective than utilizing either seed powder alone since the mixture of the seeds has a higher antioxidant capacity than the seeds alone. When evaluated at dosages ranging from 10 to 50 ppm, all of the seed powders demonstrated high antioxidant activity.
Prebiotic likelihood
For additional research, we chose seeds of Linum usitatissimum, Punica granatum, and Cucumis melo. A considerable colony growth was seen in the 1:1:1 seed powder mixture of these seeds when tested with Lactobacillus acidophilus ATCC 4356 (Sample). The positive control, on the other hand, showed growth, whilst the negative control showed none. The seed powder mixture's prebiotic potential is confirmed by the profusion of Lactobacillus acidophilus growth on it.
Table 4: Determination of prebiotic potential for a single seed powder sample using Lactobacillus Acidophilus ATCC 4356 on MRS as a positive control and plain agar as a negative control in a controlled environment.
Sample | Type | No of colonies |
combined with normal agar, 0.2 g of Cucumis melo seed powder | Test | 67 |
combined with normal agar, 0.2 g of Punica granatum seed powder | Test | 41 |
0.2 g of chicory powder | Positive control | 59 |
0.2 g of a seed powder combination 1:1:1. mixed with regular agar, | Test | 94 |
0.2 grammes of Linum usitatissimum seed powder mixed with ordinary agar | Test | 57 |
The MRS agar (De Man, Rogosa, and Sharpe) | Positive control | 47 |
Simple agar | Negative control | 0 |
Table 5: Analysis of the prebiotic potential of a single seed powder sample by means of the Bifidobacterium bifidum ATTC 29521on As a positive control, Bifidobacterium agar, and as a negative control, plain agar
Sample | Type | No of colonies |
combined with normal agar, 0.2 g of Cucumis melo seed powder | Test | 57 |
0.2 grammes of Linum usitatissimum seed powder mixed with ordinary agar | Test | 62 |
combined with normal agar, 0.2 g of Punica granatum seed powder | Test | 42 |
0.2 g of a seed powder combination 1:1:1. mixed with regular agar, | Test | 77 |
Simple agar | Negative control | 0 |
Food for Bifidobacterium. | Positive control | 47 |
0.2 g of chicory powder | Positive control | 35 |
The two probiotic bacteria, Lactobacillus acidophilus ATCC 4356 and Bifidobacterium bifidum ATCC 29521, showed greater colony counts than the positive control when 0.2 grams of seed powder were added to each petri dish. Any development that was seen can be ascribed to the prebiotic activity of the seeds because the petri dishes only contained the seed combination on a solid agar base. The seed powder blend outperformed chicory powder in terms of colony-forming units (CFUs) of Bifidobacterium bifidum and Lactobacillus acidophilus, demonstrating higher prebiotic potential. In contrast, the well-known prebiotic chicory powder was less successful.
Nutraceutical powder combination microbiological contamination limit test
Table 6: Maximum allowable microbial contamination in a nutraceutical powder blend1:1 ratio
Microorganism | Nutraceutical Powder Mixture 1:1 (CFU) |
Escherichia coli | 2 × 10² per g |
Shigella | Absent in 10g |
Total Fungal Count (TFC) | 4 × 10⁴ |
Salmonella | Absent in 10g |
Total Aerobic Count (TAC) | 5 × 10⁵ |
The microbiological load for the nutraceutical powder mixture1:1;1 was determined to be under the limit and safe for human consumption according to the IP guidelines. I.P. 2014 states
TAC: Acceptance criterion: 107 CFU per g
TFC: Acceptance criterion: 105 CFU per g
Escherichia coli: Acceptance criterion: 103 CFU per g
Salmonella: Absent in 10g
Shigella: Absent in 10g
ASSESSING BIOLOGICAL FACTORS
Animal studies were performed to evaluate the potential biological effects of a nutraceutical mix of Cucumis melo, Punica granatum, and Linum usitatissimum seeds at a ratio of 1:1:1.
- Acute toxicity study.
- Gain in weight.
- When analysing blood samples.
a) Complete blood count (WBC, RBC, Hb).
b) Concentration of all serum proteins.
c) Anaerobic glycogen.
An animal study was carried out to ascertain the impact of a combination of nutraceutical powders on weight gain or loss and hemogram-based health benefits. The experimental diet was given to the experimental animal group, and the amount of feed they consumed each day was calculated." The results were compared to those of the control group. We compared the readings after zero and forty days for the control group, and after forty days for the test groups, with the control group's readings. (So they can evaluate the hemogram and other aspects.)
For Acute Toxicity Study
For the first half hour following dosing and then at regular intervals for the first twenty-four hours, each animal is closely observed separately; no deaths occurred during the first four or twenty-four hours.
Then, each day for the following fourteen days. After 14 days of observation, the meal showed no negative effects on the animals. This toxicological study's findings showed no signs of acute toxicity.
Data from a 40-day animal research comparing the effects of nutraceuticals on weight and blood parameters
Table 7: Control group dietary habits, hydration levels, and weight increase
Sr. No | Date | Food intake (g) | Water intake (mL) | Body Weight (g) | |||||||||||
H | B | T | HB | H | B | T | HB | H | B | T | HB | ||||
1 | 27/02/2013 | 43 | 40 | 41 | 48 | 28 | 28 | 25 | 21 | 139 | 157 | 165 | 145 | ||
2 | 28/02/2013 | 41 | 40 | 43 | 46 | 27 | 21 | 23 | 23 | ||||||
3 | 29/02/2013 | 38 | 49 | 40 | 45 | 22 | 29 | 24 | 26 | ||||||
4 | 01/03/2013 | 42 | 46 | 43 | 44 | 24 | 25 | 26 | 21 | ||||||
5 | 02/03/2013 | 48 | 44 | 41 | 49 | 25 | 20 | 21 | 23 | ||||||
6 | 03/03/2013 | 41 | 40 | 49 | 42 | 23 | 24 | 25 | 28 | ||||||
7 | 04/03/2013 | 43 | 42 | 46 | 41 | 24 | 25 | 24 | 21 | 140 | 161 | 170 | 150 | ||
8 | 05/03/2013 | 40 | 40 | 40 | 48 | 28 | 23 | 28 | 29 | ||||||
9 | 06/03/2013 | 43 | 50 | 38 | 45 | 28 | 24 | 27 | 25 | ||||||
10 | 07/03/2013 | 41 | 41 | 40 | 45 | 21 | 23 | 29 | 23 | ||||||
11 | 08/03/2013 | 42 | 48 | 43 | 40 | 29 | 24 | 25 | 28 | ||||||
12 | 09/03/2013 | 47 | 40 | 41 | 41 | 25 | 25 | 28 | 20 | ||||||
13 | 10/03/2013 | 45 | 41 | 49 | 49 | 20 | 26 | 21 | 24 | ||||||
14 | 11/03/2013 | 40 | 42 | 49 | 43 | 20 | 20 | 29 | 28 | 155 | 170 | 176 | 164 | ||
15 | 12/03/2013 | 49 | 46 | 46 | 40 | 27 | 24 | 25 | 27 | ||||||
16 | 13/03/2013 | 42 | 41 | 41 | 48 | 28 | 28 | 21 | 24 | ||||||
17 | 14/03/2013 | 48 | 49 | 49 | 43 | 21 | 27 | 23 | 20 | ||||||
18 | 15/03/2013 | 49 | 43 | 42 | 41 | 29 | 22 | 28 | 25 | ||||||
19 | 16/03/2013 | 40 | 41 | 49 | 44 | 25 | 27 | 24 | 25 | ||||||
20 | 17/03/2013 | 50 | 49 | 41 | 43 | 29 | 24 | 28 | 23 | ||||||
21 | 18/03/2013 | 44 | 49 | 48 | 45 | 28 | 28 | 27 | 24 | 162 | 182 | 182 | 173 | ||
22 | 19/03/2013 | 46 | 50 | 43 | 48 | 27 | 21 | 28 | 26 | ||||||
23 | 20/03/2013 | 43 | 44 | 49 | 43 | 24 | 23 | 29 | 21 | ||||||
24 | 21/03/2013 | 40 | 46 | 46 | 41 | 24 | 26 | 25 | 28 | ||||||
25 | 22/03/2013 | 43 | 44 | 43 | 42 | 25 | 21 | 23 | 24 | ||||||
26 | 23/03/2013 | 41 | 40 | 40 | 47 | 21 | 23 | 24 | 25 | ||||||
27 | 24/03/2013 | 46 | 42 | 43 | 41 | 28 | 24 | 26 | 23 | ||||||
28 | 25/03/2013 | 45 | 41 | 48 | 48 | 27 | 26 | 21 | 24 | 173 | 191 | 193 | 180 | ||
29 | 26/03/2013 | 43 | 42 | 43 | 45 | 20 | 22 | 28 | 28 | ||||||
30 | 27/03/2013 | 44 | 47 | 41 | 40 | 27 | 22 | 21 | 20 | ||||||
31 | 28/03/2013 | 45 | 40 | 49 | 42 | 26 | 25 | 26 | 22 | ||||||
29/03/2013 | 45 | 43 | 49 | 49 | 25 | 21 | 23 | 24 | |||||||
33 | 30/03/2013 | 42 | 43 | 40 | 43 | 22 | 24 | 24 | 25 | ||||||
34 | 31/03/2013 | 48 | 46 | 46 | 45 | 24 | 25 | 26 | 25 | ||||||
35 | 01/04/2013 | 43 | 42 | 40 | 41 | 20 | 28 | 21 | 23 | 185 | 198 | 201 | 197 | ||
36 | 02/04/2013 | 41 | 43 | 42 | 43 | 25 | 24 | 28 | 24 | ||||||
37 | 03/04/2013 | 44 | 43 | 47 | 42 | 26 | 28 | 23 | 21 | ||||||
38 | 04/04/2013 | 44 | 42 | 41 | 40 | 20 | 18 | 24 | 29 | ||||||
39 | 05/04/2013 | 46 | 41 | 40 | 45 | 26 | 25 | 23 | 27 | ||||||
40 | 06/04/2013 | 44 | 41 | 45 | 40 | 25 | 29 | 21 | 26 | 198 | 205 | 208 | 210 | ||
Table 8: Diet, hydration, and growth in mass for experimental subjects
Sr. No | Date | Food intake (g) | Water intake (mL) | Body Weight (g) | |||||||||
H | B | T | HB | H | B | T | HB | H | B | T | HB | ||
1 | 27/02/2013 | 47 | 44 | 49 | 35 | 20 | 23 | 27 | 21 | 143 | 163 | 173 | 145 |
2 | 28/02/2013 | 41 | 40 | 43 | 46 | 25 | 28 | 22 | 23 | ||||
3 | 29/02/2013 | 40 | 41 | 40 | 45 | 28 | 20 | 24 | 26 | ||||
4 | 01/03/2013 | 38 | 48 | 50 | 40 | 21 | 24 | 25 | 21 | ||||
5 | 02/03/2013 | 48 | 43 | 44 | 49 | 29 | 28 | 23 | 23 | ||||
6 | 03/03/2013 | 43 | 41 | 46 | 42 | 25 | 27 | 24 | 28 | ||||
7 | 04/03/2013 | 41 | 46 | 44 | 41 | 20 | 24 | 26 | 21 | 150 | 168 | 176 | 152 |
8 | 05/03/2013 | 49 | 45 | 40 | 48 | 24 | 25 | 21 | 29 | ||||
9 | 06/03/2013 | 46 | 43 | 42 | 45 | 28 | 23 | 23 | 25 | ||||
10 | 07/03/2013 | 43 | 40 | 41 | 48 | 25 | 24 | 29 | 28 | ||||
11 | 08/03/2013 | 40 | 38 | 49 | 43 | 23 | 28 | 25 | 27 | ||||
12 | 09/03/2013 | 41 | 40 | 42 | 41 | 24 | 26 | 28 | 22 | ||||
13 | 10/03/2013 | 40 | 41 | 47 | 49 | 26 | 23 | 21 | 24 | ||||
14 | 11/03/2013 | 38 | 49 | 40 | 45 | 21 | 23 | 29 | 25 | 158 | 175 | 182 | 160 |
15 | 12/03/2013 | 48 | 46 | 44 | 40 | 25 | 28 | 25 | 23 | ||||
16 | 13/03/2013 | 43 | 41 | 43 | 48 | 24 | 25 | 21 | 24 | ||||
17 | 14/03/2013 | 48 | 49 | 43 | 43 | 28 | 23 | 23 | 28 | ||||
18 | 15/03/2013 | 43 | 42 | 41 | 41 | 27 | 24 | 28 | 21 | ||||
19 | 16/03/2013 | 41 | 47 | 49 | 49 | 20 | 21 | 24 | 25 | ||||
20 | 17/03/2013 | 49 | 45 | 41 | 40 | 24 | 29 | 28 | 23 | ||||
21 | 18/03/2013 | 49 | 40 | 48 | 50 | 28 | 25 | 27 | 24 | 170 | 189 | 195 | 175 |
22 | 19/03/2013 | 50 | 49 | 43 | 44 | 27 | 20 | 24 | 26 | ||||
23 | 20/03/2013 | 44 | 42 | 49 | 46 | 24 | 20 | 25 | 21 | ||||
24 | 21/03/2013 | 46 | 41 | 46 | 43 | 28 | 27 | 26 | 28 | ||||
25 | 22/03/2013 | 44 | 41 | 43 | 40 | 21 | 28 | 20 | 24 | ||||
26 | 23/03/2013 | 40 | 48 | 40 | 38 | 23 | 21 | 24 | 25 | ||||
27 | 24/03/2013 | 42 | 45 | 43 | 41 | 26 | 29 | 28 | 23 | ||||
28 | 25/03/2013 | 41 | 48 | 48 | 48 | 21 | 25 | 27 | 24 | 183 | 193 | 203 | 188 |
29 | 26/03/2013 | 45 | 42 | 43 | 45 | 23 | 29 | 22 | 28 | ||||
30 | 27/03/2013 | 40 | 47 | 41 | 48 | 28 | 24 | 30 | 20 | ||||
31 | 28/03/2013 | 48 | 40 | 49 | 43 | 29 | 26 | 23 | 25 | ||||
32 | 29/03/2013 | 43 | 44 | 49 | 41 | 25 | 21 | 24 | 26 | ||||
33 | 30/03/2013 | 41 | 43 | 40 | 44 | 23 | 28 | 25 | 20 | ||||
34 | 31/03/2013 | 49 | 45 | 43 | 44 | 24 | 23 | 21 | 25 | ||||
35 | 01/04/2013 | 49 | 48 | 41 | 46 | 26 | 28 | 28 | 23 | 195 | 202 | 211 | 197 |
36 | 02/04/2013 | 46 | 43 | 46 | 44 | 21 | 24 | 27 | 24 | ||||
37 | 03/04/2013 | 41 | 41 | 45 | 40 | 28 | 28 | 20 | 21 | ||||
38 | 04/04/2013 | 49 | 42 | 43 | 42 | 27 | 18 | 24 | 29 | ||||
39 | 05/04/2013 | 42 | 47 | 44 | 49 | 24 | 25 | 23 | 27 | ||||
40 | 06/04/2013 | 47 | 41 | 40 | 43 | 25 | 29 | 21 | 26 | 210 | 218 | 220 | 210 |
The Albino Wistar rats were given a nutritional supplement that was composed of 33.42% carbohydrates, 22.44% fat, and 35.5% protein. The rats' body weight varied somewhat after 40 days of receiving the nutraceutical powder mixture of seeds in a 1:1:1 ratio as the main product mix (Group I). The study discovered that despite being fed the nutraceutical mix, the test rats' weight increase was lower than that of the control group. This might be explained by the seed combination's moderate fat levels and comparatively high protein content when compared to regular rat chow.
In order to analyse blood samples
Table 9: Blood cell count, haemoglobin, triglyceride, and very low density lipoprotein (VLDL) analysis during a 40-day animal trial
Parameters | Zero day’s reading | Forty day’s reading | ||
Control group | Test group | Control group | Test group | |
RBC count (million/mm3) | 6.38 | 7.46 | 4.82 | 9.55 |
Haemoglobin | 11.5 | 12.1 | 14.5 | 16.8 |
Serum Protein(g%) | 6.1±0.1750 | 6.325±0.025 | 6.3±0.2000 | 6.5±0.1750 |
VLDL (%mg) | 19.2 | 21.6 | 23 | 15.4 |
Serum triglycerides (%mg) | 96` | 108 | 115 | 77 |
Blood sugar (mg%) | 79.33±0.1707 | 82.66±3.500 | 92±3.928* | 107±5.626*** |
Figure 1: Effect on Red Blood Cell, Haemoglobin, Triglyceride, and VLDL Measuring in a 40-Day Animal Trial
Significant results were obtained from laboratory analyses of hemoglobin, triglycerides, red blood cells, and very low-density lipoprotein during the 40-day animal study. The test group's hemoglobin level rose from 12.1% to 16.5% by the end of the trial, whereas the control group's climbed from 11.5% to 14.5%. This improvement suggests a haematinic impact, which is frequently linked to anemia treatment. The test group's red blood cell (RBC) counts increased from 7.46 million/mm³ to 9.55 million/mm³ due to the nutraceutical blend, while the control group's RBC counts decreased to 4.82 million/mm³. This nutraceutical combination may be used as a supplement because the iron, proteins, and vitamins included in the three seed components are thought to promote the synthesis of red blood cells and raise hemoglobin levels.
Serum Protein (mg)
An increase in the test group's plasma total protein concentration (6.45 mg/dl) was associated with a noticeable improvement in health. Since proteins are essential for many body functions, serum protein levels are a significant predictor of overall nutritional status. Serum protein concentration is frequently measured using a total protein test; low levels might be caused by dilution, increased loss, decreased production, or starvation. Mildly low protein levels might not show any signs, but extremely low levels might cause fluid to leak from the circulation into tissues, resulting in limb weakness, exhaustion, and swelling. By raising serum proteins in vivo, this nutraceutical combination may aid in the treatment of malnutrition.
Triglycerides and VLDL
Serum triglyceride levels dropped to 77% in the experimental group and rose to 115% in the control group from 96%. The test group's very low-density lipoprotein (VLDL) levels decreased from 21.6 mg to 15.4 mg, whereas the control group's LDL levels rose from 19.2 mg to 23.2 mg. Elevated LDL and VLDL levels are associated with plaque accumulation in arteries, which exacerbates heart disease. Given that it can lower triglyceride and VLDL levels, this nutraceutical may help patients with diabetes and heart disease by lessening the negative effects of these lipoproteins.
Blood Glucose Levels
The test group's blood sugar level climbed to 107±5.626 mg/dl, which was statistically significant (p<0.05), while the control group's blood sugar level increased from 79.33±0.1707 mg/dl to 82.66±3.500 mg/dl. The test group's glucose levels stayed within the usual range in spite of this increase, indicating that the nutraceutical formulation did not result in a blood sugar surge. These findings suggest that diabetics can safely use this nutraceutical. The moisture content
Weight before drying minus weight after drying equals moisture weight.
0.82 g is 10 g minus 9.18 g.
Moisture content is equal to 100% (moisture weight divided by initial weight).
8.2% is 0.82 / 10 x 100.
It was discovered that the seed combination had an 8.2% moisture content.
RESEARCH ON CELL LINES FOR ANTI-CANCER AND ANTI-EXPOSURE AGENTS
Preparation for formation.- Dispersible powder formulation
Table 10: Model 32 iterative Cinnamon, Punica granatum, and lutein are the main ingredients.1:1 ratio
Dispersible powder formula (Punica granatum,Cucumic melo, ,Linum uisitatissimum1:1:1)NI with Excipients EI | |||
NI-X1 | Y2 %Comressibility | EI-X2 | Y1 Dispersion time in seconds |
50 | 25 | 30 | 40 |
60 | 26 | 30 | 35 |
50 | 28 | 50 | 50 |
50 | 23 | 40 | 35 |
60 | 27 | 40 | 40 |
70 | 31 | 40 | 40 |
70 | 30 | 30 | 35 |
60 | 28 | 50 | 50 |
70 | 33 | 50 | 55 |
Table 11: Summary of Responses for Year 1 (see tables for details below)
Source | Sequential p-value | Adjusted R² | Predicted R² | |
Cubic | 0.7559 | 0.8049 | -3.4451 | Aliased |
Quadratic | 0.1023 | 0.8862 | 0.5956 | |
2FI | 0.2890 | 0.6878 | -0.0164 | |
Linear | 0.0156 | 0.6667 | 0.3575 | Suggested |
The dependent variable, dispersion time in seconds, and the interactions between the independent variables, N-X1 and E-X2, are described using a linear model.
The physical blend of nutraceutical seed powder with excipients (milk powder and cocoa powder) does not include any chemical contact, therefore the relationship between the two is linear. If the additional terms are truly important, we select a linear polynomial. Predicted R2 and Adjusted R2 are the best choices in this case.
Table 12: 1-D.T.(Sec) Analysis of variance table for Response Surface Linear Model [Partial sum of squares - Type III
Source | Sum of Squares | Mean Square | df | F-value | p-value | |
Model | 341.67 | 170.83 | 2 | 9.00 | 0.0156 | significant |
A-A | 4.17 | 4.17 | 1 | 0.2195 | 0.6559 | |
B-B | 337.50 | 337.50 | 1 | 17.78 | 0.0056 | |
Residual | 113.89 | 18.98 | 6 | |||
Cor Total | 455.56 | 8 |
The model is statistically significant with an F-value of 9.00. With a probability of only 1.56%, a "Model F-Value" of this size is extremely unlikely to be the product of chance.The linear model excels in many ways. When "Prob > F" is less than 0.0500, model terms are deemed significant. In this case, B is a key model term. The terms in the model are considered non-significant when the values exceed 0.1000.
The Last Equation Relating to Coded Factors
D.T.(Sec) = +42.22 + 0.83 * A + 7.50* B Final Equation in Terms of Actual Factors:D.T.(Sec)
= + 42.22222 + 0.83333 * N1 + 7.50000 * E1
The dispersion time coefficient is positive for both the fraction of nutraceutical seed combination N1 and the proportion of excipient mixture E1. The coefficient for the effect of excipient percentage on dispersion time is 7.5,” which is more indicative than the coefficient for the seed powder proportion, which is 0.8333.
Figure 2: If the dispersion time is 55 seconds at its highest and 35 seconds at its lowest, as shown in of the response surface graph for D.T. Product 1, then the projected value is 45 seconds, and the linear link between N1 and E1 for dispersion time is also evident.
Table 13: Model 32 iterative For Y2, cucumber, Punica granatum, and Linum uisitatissimum in a ratio of 1:1:1.
Dispersible powder formula (Cucumic melo,Punica granatum,Linum uisitatissimum) | |||
N-X1 | Y1 Dispersion time in seconds | E-X2 | Y2 %Comressibility |
50 | 40 | 30 | 25 |
50 | 35 | 40 | 23 |
50 | 50 | 50 | 28 |
60 | 35 | 30 | 26 |
60 | 40 | 40 | 27 |
60 | 50 | 50 | 28 |
70 | 40 | 40 | 31 |
70 | 35 | 30 | 30 |
70 | 55 | 50 | 33 |
Table 14: Below are the comprehensive tables displaying the response summary for Y2.
R2-Response 2 Y2- C (%)
Source | Sequential p-value | Adjusted R² | Predicted R² | |
Linear | 0.0040 | 0.7881 | 0.5979 | Suggested |
Quadratic | 0.2704 | 0.8227 | 0.2995 | |
2FI | 1.0000 | |||
Cubic | 0.5898 | 0.8150 | -3.2139 | Aliased |
According to the model, N-X1 and E-X2 are the independent variables, while the percentage of compressibility is the dependent variable.
The physical blend of nutraceutical seed powder with excipients (milk powder and cocoa powder) does not include any chemical contact, therefore the relationship between the two is linear. If the additional terms are truly important, we select a linear polynomial. Predicted R2 and Adjusted R2 are the best choices in this case.
Table 15: Linear model ANOVA for Response 2 C (%): R2
(Third Type Partial Sum of Squares) Analysis of Variance Table
Source | Sum of Squares | Mean Square | df | p-value | F-value | |
Model | 64.67 | 32.33 | 2 | 0.0040 | 15.87 | significant |
A-A | 54.00 | 54.00 | 1 | 0.0021 | 26.51 | |
B-B | 10.67 | 10.67 | 1 | 0.0621 | 5.24 | |
Residual | 12.22 | 2.04 | 6 | |||
Cor Total | 76.89 | 8 |
An F-value of 15.87 indicates a significant model. This kind of "Model F-Value" could only occur by chance (with a little 0.40% chance).When "Prob > F" is less than 0.0500, model terms are deemed significant. In this case, the model terms A are crucial. The terms in the model are considered non-significant when the values exceed 0.1000.
The Last Equation Relating to Coded Factors
C (%) = +27.89 + 3.00* A + 1.33 * B Final Equation in Terms of Actual Factors: C (%)
= +27.88889 + 3.00000 *N1 + 1.33333* E1
The percentage of compressibility is positively correlated with the percentage of excipient mixture E1 and the fraction of nutraceutical seed combination N1. The effect of the percentage of the seed powder mixture on the percent compressibility is more strongly reflected by the coefficient of 3.00 than by the coefficient of 1.3333 for the excipient proportion.
Power transformations display the box-cox plot
The response surface graph reveals a linear relationship between N1 and E1 for % compressibility, and if 33% is the maximum and 23% the lowest, then the projected value is 28%.
Figure 3: The graph showing the reaction surface for $C Product 1
Table 16: Batch product optimisation phase I
N-X1 | E-X2 | Y2 (%C) | Acceptability For (%C) | Y1 Dispersion time (D.T.) in seconds | Acceptability For (D.T.) | Organoleptic preference |
50 | 50 | 28 | Poor | 50 | Fair | +++ |
50 | 40 | 23 | Passable | 35 | Very good | ++ |
50 | 30 | 25 | Poor | 40 | Good | + |
60 | 50 | 28 | Poor | 50 | Fair | +++ |
60 | 40 | 27 | Poor | 40 | Good | ++ |
60 | 30 | 26 | Poor | 35 | Very good | + |
70 | 30 | 30 | Poor | 35 | Very good | + |
70 | 50 | 33 | Very Poor | 55 | Poor | ++ |
70 | 40 | 31 | Poor | 40 | Good | ++ |
Batch 5, with slightly greater D.T. and % C, is regarded optimal as it contains 50% of the nutraceutical combination, however the programme suggests batch 2 as the best since it has the least D.T. and % C.
Batch number five is the best option.
This dispersible powder mixture is expected to taste like a hot health drink with a pleasant, pleasant flavor. The powder is not meant to flow freely; it will be combined with either milk or hot water. The flavor score is correlated with the dispersion time. Different concentrations of milk and cocoa powder as excipients in the finished composition are indicated by scores of +++/++/+. Batch 5 is the best since it has five times as many nutraceutical components as the legal minimum. However, because of its best result of % C and lowest dispersion time, batch no. 2 is considered ideal by the algorithm. However, this specific batch, B. No. 2, has a low flavor score.
Development of Formulation. - Capsule (for those with diabetes)
Capsules
Measurements were made of the capsules' weight change and disintegration time. Weight variationEach of the twenty full capsules had 1,127 milligrammes of gelatin, while each empty capsule contained 127 milligrammes.
Because the pills' percentage of weight fluctuation fell within the parameters defined by the Pharmacopoeia, they were considered to have passed the weight variation test. The hard gelatine capsules' disintegration time fell within the IP limit, and all of the capsules' weights were standardized with low standard deviations. The results are shown in the following table.
Table 17: Assessment of pills
Parameters | Result |
Degradation duration | 4 mins 47secs. |
Variation in mass | within limits |
b) Prompted Stability Investigations
For three months, the Accelerated Stability Studies were carried out in a harsh storage environment with 40°C and 75% relative humidity. If the product is kept in a dry, cold place as recommended, it will stay stable for a long period. The majority of nutraceutical products on the market have a shelf life of nine to twelve months. According to preliminary stability tests, this product might fit the requirements. The prebiotic potential, antioxidant activity, and stability testing method of a nutraceutical powder blend were evaluated.
Table 18: Nutraceutical product combination stability research I: DPPH test, fresh seed mixture, and three months later
Name | % inhibition | Equation (Squared co relation coefficient) R2 | IC50 (µg/ml) | ||||
Concentration in µg/ml | |||||||
10 | 20 | 30 | 40 | 50 | |||
Fresh powder mixture | 45.35 | 62.21 | 74.35 | 86.25 | 92.65 | y = 68.436x - 24.729 R² = 0.9902 | 2.3402 |
Blend (after three months of rapid stabilisation) | 42.56 | 59.36 | 73.5 | 84.56 | 88.35 | y = 68.118x - 26.775 R² = 0.9909 | 2.19 |
Using Lactobacillus Acidophilus ATCC 4356 on De Man, Rogosa and Sharpe (MRS) as a positive control and plain agar as a negative control (after 3 months), the prebiotic potential of various seed powder samples was determined.
Table 19: Evaluation of the seed mixture's prebiotic potential during a three-month stability testing with Lactobacillus Acidophilus
Sample | Type | No of colonies after stability | No of colonies fresh sample |
0.2 g of Cucumis melo seed powder mixed with ordinary agar | Test | 52 | 67 |
combined with normal agar, 0.2 g of Punicagranatum seed powder | Test | 32 | 41 |
combined with normal agar, 0.2 g of Linumusitatissimum seed powder | Test | 46 | 57 |
0.2 g of a seed powder combination 1:1:1. | Test | 63 | 94 |
mixed with regular agar, 0.2 g of chicory powder | Positive control | 32 | 59 |
Mr. Rogosa, Sharpe, and De Man (MRS)agar | Positive control | 45 | 47 |
Simple agar | Negative control | 0 | 0 |
Analysis of the prebiotic potential of a single seed powder sample by means of the Bifidobacterium bifidum ATTC 29521on As a positive control, Bifidobacterium agar, and as a negative control, plain agar
Table 20: Research on the seed mixture's stability and its prebiotic properties after three months of testing with Bifidobacterium bifidum
Sample | Type | No of colonies after stability | No of colonies fresh sample |
0.2 g of Cucumismelo seed powder mixed with normal agar | Test | 37 | 57 |
combined with normal agar, 0.2 g of Punicagranatum seed powder combined with normal agar, | Test | 32 | 42 |
0.2 g of Linumusitatissimum seed powder | Test | 49 | 62 |
0.2 g of a seed powder combination 1:1:1. | Test | 52 | 77 |
2 milligrammes of chicory powder mixed with agar | Positive control | 27 | 35 |
Food for Bifidobacterium. | Positive control | 49 | 47 |
Simple agar | Negativecontrol | 0 | 0 |
The seed powder blend maintains the majority of its prebiotic qualities as compared to ordinary chicory powder. If the product is stored in a cold atmosphere, its prebiotic action might last for a very long time. The prebiotic potential is determined by the stability of the soluble fibers and oligosaccharides that make up the prebiotic diet. The number of colonies remained consistent since the Bifidobacterium agar and positive control agar were both fresh.
CONCLUSION
With its rich macro and micronutrient profile, potent antioxidants, and prebiotic potential, the nutraceutical blend of Cucumis melo, Punica granatum, and Linum usitatissimum seeds shows promise in reducing oxidative stress, a major contributor to diabetes, heart disease, and cancer. Additionally, it exhibits promise in treating anemia and malnutrition.
In a similar vein, the combination of powdered leaves from Trigonella foenum-graecum, Coriandrum sativum, Raphanus raphanistrum, and Anethum graveolens has remarkable prebiotic and antioxidant properties. Both blends' promise for efficient and reasonably priced disease prevention and health promotion is demonstrated by their development into stable powder and capsule forms. Additional investigation may open up other uses for them as native, affordable nutraceuticals.
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