Development and physicochemical evaluation of a sugar-free polyherbal immunity booster powder

Microgreens are emerging as nutrient-dense functional foods that offer health benefits beyond basic nutrition. This review addresses the growing demand for sustainable and health-promoting dietary options by examining the nutritional potential, cultivation practices, and functional applications of microgreens. A comprehensive analysis of existing literature was conducted to evaluate their composition, including vitamins, minerals, and bioactive compounds such as polyphenols and carotenoids. The findings indicate that microgreens possess significantly higher nutrient concentrations than mature plants and exhibit antioxidant, anti-inflammatory, cardioprotective, and anti-diabetic properties. The study also highlights the influence of growing media on yield and quality, as well as the successful incorporation of microgreens into value-added functional food products. In conclusion, microgreens represent a promising component of modern diets and sustainable food systems, with considerable potential to enhance nutritional security and human health.

Illustrations explain the paper; charts are replotted from the paper's own table values.

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Antioxidant ComparisonClose ×

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Ascorbic acid and Batch 1: 0 vs 5 vs 10 vs 20 vs 40Close ×

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Ascorbic acid and Batch 2: 0 vs 5 vs 10 vs 20 vs 40Close ×

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Introduction

Our immune system defends our body against harmful microbes, diseases, infections, and cancer. To protect the body from foreign substances, the immune system creates an immune response to fight against foreign substances or harmful bacteria and disease. Major immune system components are macrophages and lymphocytes (B cells and T cells) which are responsible for protecting the body against infections [1] . Immune system is of two types, Innate and adaptive immunity, which provides different functions, while innate immunity provides a rapid, non-specific defence mechanism, adaptive immunity uses memory cell to provide long term, antigen-specific protection [2] . Boosting immunity is crucial for our health since the COVID-19 pandemic has brought difficulty for the entire world. The primary objectives of COVID-19 were elderly and young people with weakened immune systems. Our immune system contains various of useful bacteria in our body that protect our body from several diseases. When the immune system gets weakened, it causes infections like corona virus and diseases such as diabetes, heart disease, or blood cancer [3] . The most important way for improving the immune system is to adopt a healthy lifestyle which includes consuming nutritious food, getting enough rest. and regular physical workout. Further, research have shown that taking supplements containing specific vitamins, minerals, herbs, and other substances boost immunity and prevent disease [4] . Human beings have suffered severely in recent decades because of the rapid increase in the frequency of various infectious, inflammatory diseases, allergies, cancer, and autoimmune disorders. Immunity should be balance which is crucial for maintaining human health there are numerous benefits of using natural herbs with immune modulating properties to prevent or enhance the immune system. Plant based natural ingredients have fewer side effects and lower toxicity in the body compared to synthetic (organic) drugs, which makes them beneficial for human health, they have numerous of benefits, one of which is enhancing the immune system of the body. By boosting host immunity, plant based natural ingredients support health and help in the fight against illnesses the Phytoconstituents present in the plants-based ingredients responsible for various therapeutic benefits in body [11] .

A polyherbal formulation is a preparation that combines two or more herbs. Polyherbalism is the idea of synergism, which balance the combined therapeutic effect of each herb which is greater than the sum of individual effects. In Ayurveda, polyherbal solutions are frequently used to boost immune system and maintain energy balance. The immune system can be significantly strengthened and risk of disease by combining herbs with anti-inflammatory, antioxidant, adaptogenic and immune stimulatory qualities. Traditional herbal formulations often use sugar or honey as base ingredients to enhance flavour and preserve the composition; however, individuals' obesity, diabetes or metabolic disorders have significant difficulties as a result. Sugar intake can worsen pre-existing metabolic disorders in certain individuals, leading to insulin resistance and hypercalcemia. Due to increased awareness of dietary restrictions and lifestyles disorders, there is an increasing need for sugar-free alternatives that are both pleasant and medicinal. Safe, low-calorie sweeteners like stevia and mannitol which are suitable for all the individuals [51] . The best way to cope with health problems and the best substitute of energy drink is herbal immunity boosters. The name of selected plants for study: Amla (Phyllanthus emblica): family is Phyllanthaceae, Amla is a great source of vitamin C, which improves immunity, speedup metabolism and guards against bacterial and viral infections like cold and cough [25] . Tulsi (Ocimum sanctum): family is Liliaceae, Tulsi is commonly used to treat illness, such as the common cold, irritation, intestinal illness, cardiovascular disease, digestive issues and more [24] . Ginger (Zingiber officinale): family is Zingiberaceae, Ginger has a strong anti-bacterial and anti-inflammatory properties, which help to boost the immunity [39] . Turmeric (Curcuma longa): family is Zingiberaceae Turmeric is a well-known for its ability to boost immunity, its main constituent, curcumin has strong antioxidant and anti-inflammatory properties which enhances immune cell activity, reduces inflammation strengthen the body's defence against infections [39] . Cinnamon (Cinnamomum verum): family is Lauraceae, Cinnamon is used to increase the immunity because of its bioactive ingredients including cinnamaldehyde, cinnamic acid and cinnamyl alcohol. These ingredients, support the body's immune system response because of their antibacterial, anti-inflammatory properties [39] . Stevia (Stevia rebaudiana): family is Asteraceae Stevia leaf extract contain different bioactive ingredient like flavonoids, steviol glycosides, diterpenoids which gives S. rebaudiana its sweetness, its bioactive metabolites provide a variety of medicinal benefits including as immunomodulatory, antiinflammatory, antibiotic and neuro protective properties [52] . Flax Seeds (Linum usitatissimum): family is Linaceae flax seed is very rich in omega-3 fatty acids, fiber, protein, and antioxidants.

Plant Profile

Plant Profile

Family: Euphorbiaceae

Geographical source: It is most found in countries in like India, Pakistan, Uzbekistan, Sri Lanka, Southeast Asia, China and Malaysia.

Chemical Constituents: One of the Pants that has been studied the Most is amla. It may contain phenols, alkaloids, and tannins, according to several studies.28% of the tannins found across the whole plant have been discovered in fruits.it generally consist of carbohydrates, amino acids, and other substances. its fruit juice has the greatest vitamin C concentration (478.56mg/100mL). There is more vitamin C than oranges, tangerines, and lemons. The edible fruit tissue has much greater of most minerals and amino acids and is multiple times richer in proteins and 160 times more ascorbic acid than apple.

Uses: Amla, often known as Indian gooseberry, is a nutritious fruit with numerous health benefits. By boosting the body's immune system with its antibacterial properties and by creating more white blood cells, which help with detoxification, it improves immunity. Amla is also beneficial for hair care because of its high iron and antioxidant content.  Geographical Source: Ocuimum sanctum is found in Latin America, the southern hemisphere, and tropical and subtropical parts of Asia.  Chemical Constituents: Several phenolic substances (antioxidants), including cirsilineol, cirimaritin, isothymusin, apigenin, rosameric acid, and eugenol, were extracted from fresh leaves and stem of Ocimum sanctum. 0.7% of the volatile oil found in Ocimum sanctum leaves is made up of roughly 71% eugenol and 20% methyl eugenol. Additionally, the oil contains the sesquiterpine hydrocarbon caryophyllene and carvacrol. Two flavonoids, orientin and vicenin, have been identified from Ocimum sanctum's aqueous leaf extract. Ginger also includes raw fibres, lipids, organic acids, and polysaccharides; roughly 50-70% of its composition is madeup of carbohydrates Which shows antioxidant, anti-inflammatory, antimicrobial, and anticancer properties. Larger-scale cultivation takes place in Pakistan, Malaysia, China, India, and the East Indies.  Chemical constituents: Curcuma longa is the main constituent of turmeric, which contain different chemical constituents that contribute toits culinary and medicinal properties. Curcumin (around3-6%) make up curcuminoids, the main colouring compound. The essential oils include around 3-Zingiberene (25%), αphellandrene, sabinene, turmerone, and arturmerone are found in 7% of Turmeric. Cineole and borneol other substances found intuermeric include phenylpropene, diterpenes, sterols and alkaloid. These chemical constituents contain antioxidants, anti-inflammatory properties in it [14] . The active ingredient in the molecule is the volatile oil, 14-16% of the 90% alcohol-soluble extraction is produced by bark. 60-70% of cinnamonaldehyde,5-10% eugenol, benzaldehyde, cumin aldehyde are found in Cinnamon oil. These ingredients show carminative, stomachic, and mild astringent properties [24] . unistatissimum, is becoming increasingly well-liked because of its high α-linolenicacid, lignans, Fiber and Omega 3 fatty acids which act as a antioxidant.

Tulsi (Ocimum sanctum)

Tulsi (Ocimum sanctum)

Ginger (Zingiber officinale)

Ginger (Zingiber officinale)

Turmeric (Curcuma longa)

Turmeric (Curcuma longa)

Cinnamon (Cinnamomum zeylanicum)

Cinnamon (Cinnamomum zeylanicum)

Stevia (Stevia rebaudian)

Stevia (Stevia rebaudian)

Mannitol

Mannitol commonly known as D-mannitol, is a polyol or low-calorie sugar alcohol. it has a similar chemical structure to mannose and is an isomer of the medicinal excipient sorbitol. It is either a white, odourless, crystalline powder or free-flowing granules. It is similar to glucose and tastes half as sweet as sucrose. It functions as a tonicity agent, tablet, capsule diluent, chewable tablet excipient, sweetener. Five popular herbs-Amla, Tulsi, Ginger, Turmeric, Cinnamon, Stevia and Flax seed were employed in this study to make and evaluate a sugar free polyherbal mixture. These plants are well-known for their immunomodulatory, adaptogenic, and antioxidant properties. The main objective was to formulate a pleasant, sugar free supplement and assess its basic physiochemical characteristics.

Clinical investigation in selected herbs

In the present study, a selection of medicinal plants is clinically investigated in animal's models to assess their immunomodulatory, antioxidant, and health promoting properties. Many of them utilize herbs including Phyllanthus emblica, Ocimum sanctum, Zingiber officinale, Curcuma longa, and Stevia rebaudiana, Linum usitatissimum used to determine the activity on animals.  Toll-like receptor (TLR2, TLR4, and TLR7) gene expression was assessed in this study after amla fruit powder added to the diets of broiler chicks. The study showed that while TLR4 expression reduced in all treatment groups. Based on these studies, amla may strengthen broiler hens' immunity and serve as a natural antibiotic substitute.  The analgesic and anti-inflammatory properties of Ocimum sanctum leaves were assessed in this study.

Using the hot-plate method in mice and carageenaninduced paw edema in rats, the leaves' ethanol extract was tested for its analgesic and anti-inflammatory effects. The analgesic effect was proven by a significant, dose dependent increase in reaction time at doses of 250-500mg/kg. By reducing paw edema (43.33% inhibition at 500 mg/kg), the extract showed its anti-inflammatory. The Findings verify the traditional use of Ocimum sanctum refer to its potential as source of herbal medicine [54] .  The continued use of antibiotics in poultry production is no longer desirable because of the negative effects on animal and human health. Lengthened. In this study the production of poultry has led to the discovery and testing of numerous organic products. As well as turmeric. Curcumin, the active ingredient in turmeric, has demonstrated broad-spectrum antibacterial properties in this study it can also boost the chicken's immunity. These are also anti-inflammatory qualities to turmeric. That's studies have shown that turmeric can enhance gut health, boost feed intake, and hasten chicken growth, making it a very excellent organic product that can readily take the place of antibiotics in chicken production.  The current study claims that an aqueous extract from stevia leaves may lower blood sugar level, glycosylated haemoglobin (HbA1c), and fasting blood sugar haemoglobin, but the diabetic rats' levels of insulin and liver glycogen considerably rose in comparison with the control rats with and without diabetes after eight weeks' time for study. The aqueous extract of stevia was found to have a concentration of 500ppm/kg body weight in rats. Demonstrated the best outcomes of every parameter examined. It is clear from the findings that stevia extracts anti-diabetic effects in albino rats, so it could be used as a naturally occurring antidiabetic medication of diabetes and the problems it causes.  In this study they evaluated the effects of giving broiler chickens different amounts of ginger (Zinger officinale). A total of 1500 chicks were divided into five groups with varying amounts of ginger. The results showed that supplementing with ginger improved gut morphology (longer and wider villi), reduced the total bacterial count, and increased feed intake. Additionally, by increasing HDL and lowering LDL triglycerides, and cholesterol, it enhanced serum biochemistry. A0.6% ginger inclusion produced the best outcomes. 1. Hot air oven (for loss on drying) 2. Muffle furnace (for total ash and acid-insoluble ash determination)

3. Electronic weighing balance 4. Standard sieves.

Method of preparation 5.1 Cleaning and Drying

All herbs (Amla, Tulsi, Ginger, Turmeric, Flax seed, Stevia) were cleaned thoroughly removed dust and impurities. The material was shade-dried at room temperature.

Grinding

Each dried material was coarsely powdered using a mechanical grinder and passed through a 60 no of sieve to obtain uniform particle size. The powders were stored separately in airtight containers.

Mixing and Blending

All powdered herbal ingredients were accurately weighed according to the formula and mixed thoroughly in a clean, dry mortar and pestle to ensure uniformity.

Sieving and packaging

The final blend was passed again through a 60 no sieve to achieve a fine, homogenous powder. The polyherbal powder was then packed in airtight container.

Evaluation procedure 6.1 Organoleptic evaluation:

The product was evaluated based on its colour, taste, odour, appearance.

Physiochemical evaluation:

Numerous of physiochemical test was performed such as angle of repose, bulk density, tapped density, moisture content, etc.

Angle of repose:

To evaluate angle of repose a funnel is used and placed in such away so that the powder can flow easily after that a grid of paper was placed in a flat surface the before pouring the powder block the tip of funnel then pour the powder through the funnel on the paper then mark was made on the paper along the circumference of the pile. The radius(r) of the pile and the height(h) was measured. The angle of repose was measured by using:

Where, θ=angle of repose h= height of pile, r = radius of pile

Bulk density

To measure bulk density a 100ml measuring cylinder was used and the fine powder was poured into a clean and dry measuring cylinder ensuring the powder does not compact or tapped then note the reading volume occupied by the powder. Formula for calculating bulk density is as follows: 3. Tapped density: after measuring bulk density, the measuring cylinder containing powder was tapped using glass rod. The powder was tapped 100-200 times after tapping the volume was recorded to the closet graduated unit. To calculate the tapped density, we use the formula:

Where, ρ(tap)= tapped density, M=weight of sample, Vf = volume of sample.

Carr's Index (The Compressibility Index):

The tendency of a powder formulation is measured by an index. To be compressed, which is determined using the bulk and densities were tapped. It is better to have less compatibilities' a property of flow it measures the interparticulate interaction of the material, The disparity between bulk and tapped densities are far more significant. The formula used for calculation of Carr's Index is,

Where, ρ(b)= Bulk density, ρ(tap)= Tapped density.

Where, ρ(tap)=Tapped density, ρ (b) = Bulk density Very Very poor 6. Moisture Content: since the moisture content is high, which encourage the hydrolytic process that results in the development of microorganisms. As a result, herbal powder contains more moisture content compared to its peers, which is why it is important to know the moisture content, to select proper packaging for the product. The hot air oven was used to measure the moisture content by placing the measured amount of powder was placed in preheated porcelain dish and weigh the dish (W1) along with the powder and then the porcelain dish was placed in hot air oven for 60 C for 2 hours. After that porcelain dish was removed from the oven and weight again (W2). Then the moisture content was calculated:

Where, W =powder' weight(gm),W1 =weight of dish before drying(gm),W2 =weight of dish after drying(gm).

Ash Content:

It symbolizes the inorganic residue, including silicate, carbonate, and phosphate in herbal drugs.

It is significant because it demonstrates the quality as well as purity of herbal medicines. Total ash content -To a weighed crucible (W1) predetermined dry sample is added. The crucible is weighed again with the sample (W2). The crucible is then placed in a furnace until it burned off. Then again weight the crucible along with the residue (W3).

Total Ash(%) = [(W3-W1)/(W2-W1)]*100

Phytochemical Evaluation:

Phytochemical test was done using standard qualitative methods to detect the presence of phytoconstituents such as alkaloids, flavonoids, saponins, tannins, glycosides, and steroid/terpenoids.

Preparations of Extract

 Weigh 1-2 gm of the sample powder.  10ml of distilled water or ethanol is used depending on solubility.  The powder is mixed with distilled water, shake well and filtered. The filtrate was used for testing.

Test for Alkaloids (Mayer's Test)

 Take 1-2 ml of the extract.  Add 1ml of Mayer's reagent.  Creamy coloured precipitate was observed.

Test for Tannins (Ferric chloride Test)

 Take 1-2 ml of the extract.  Add few drops ferric chloride solution.  Black colour was observed.

Test for Glycosides (Keller-Killiani test)

 Take2 ml of the extract  Add few drops of glacial acetic acid, one drop of ferric chloride and sulphuric acid carefully down the side.

Reddish brown layer at the interface and bluish-green layer below was Observed.

Test for Saponin (Foam Test)

 Take 2 ml of the extract.  Shake 2ml of extract vigorously with 5ml distilled water in a test tube.  Allow to stand for few minutes.  Foam formation was observed.

Test for protein (Millon's Test)

 Take 3 ml of the extract.  Add 5ml of Millon's reagent.  Heat the solution in a water bath.  White precipitate turns red on heating was observed

Test for Carbohydrates (Molisch Test)

 Take 3ml of extract and add few drops of alcoholic alpha naphthalol  Shake well and add 2ml of conc. Sulphuric acid from the side wall of the test tube.  Purple colour was observed.

Results

Results

Organoleptic Evaluation Results

Organoleptic Evaluation Results

Physiochemical Evaluation Results

The result of the physiochemical evaluation is as follows: Thus, the Bulk density of powder was observed to be 0.465gm/ml. The Tapped density of powder is found to be 0.50 gm/ml. Thus, the flow property of powder is good. Therefore, the total ash value was found to be14%. The result showed that among the two batches, sample Batch 1 has a strong antioxidant activity with an IC₅₀value (3.62 μg/ml) nearly equal to ascorbic acid (3.60μg/ml), where as sample batch2 showed slightly lower activity (IC₅₀= 4.02 μg/ml). Overall, Batch1demonstrated stronger free radical scavenging potential compared to batch 2.

3.Tapped Density

3.Tapped Density

Carr's Index

Carr's Index

Hausner's Ratio

Hausner's Ratio

Phytochemical Evaluation Results

Phytochemical Evaluation Results

Discussion

The present study was carried out to evaluate the organoleptic, physicochemical, phytochemical, and antioxidant properties of the developed polyherbal powder formulation. The findings obtained from the evaluation confirmed the acceptable quality, stability, and therapeutic potential of the formulation. Organoleptic evaluation showed that all the trial formulations possessed a uniform powder appearance with fine texture, greenish-brown colour, and sweet odour. These characteristics indicate good blending of herbal ingredients and acceptable sensory properties, which are important for patient acceptability and product quality. The physicochemical parameters demonstrated satisfactory flow and handling properties of the powder formulation. The angle of repose value of 28.8° indicated good flowability of the powder blend. Similarly, Carr's index (11.6%) and Hausner's ratio (1.13) also confirmed excellent compressibility and flow characteristics, suggesting that the formulation can be easily processed during manufacturing. The bulk density and tapped density values indicated proper packing ability of the powder particles. Moisture content was found to be 6.9%, which is within acceptable limits and suggests lower chances of microbial growth and better stability during storage. The ash value reflected the presence of inorganic and mineral constituents in the formulation. Phytochemical screening revealed the presence of important secondary metabolites such as flavonoids, saponins, tannins, glycosides, and carbohydrates. These phytoconstituents are well known for their medicinal properties including antioxidant, antimicrobial, anti-inflammatory, and therapeutic activities. The absence of alkaloids and proteins in most formulations may be attributed to the nature of selected herbal ingredients and extraction methods used. The antioxidant activity assessed by DPPH radical scavenging assay showed promising results. Batch 1 exhibited stronger antioxidant activity with an IC₅₀ value of 3.62 μg/ml, which was almost comparable to the standard antioxidant ascorbic acid (3.60 μg/ml). Batch 2 also showed significant antioxidant activity but comparatively lower than Batch 1. The strong antioxidant potential may be due to the presence of flavonoids and phenolic compounds in the formulation, which are capable of scavenging free radicals and reducing oxidative stress. FTIR analysis further confirmed the presence of various functional groups such as hydroxyl, carbonyl, ether, and aliphatic compounds in the formulations. Trial 1 showed characteristic peaks corresponding to polyphenols and carbohydrate-related compounds, whereas Trial 2 exhibited an additional carbonyl peak indicating the presence of lipidrich constituents due to flaxseed incorporation. These findings support the phytochemical screening results and indicate the presence of bioactive compounds responsible for antioxidant activity.

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Amla

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Tulsi

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Ginger

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Turmeric

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Cinnamon

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Stevia

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Flax seed

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Value of angle of repose	Flow properties
<25	Excellent flow
25-30	Good flow
30-40	Passable
40	Bad flow

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Angle of repose values

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Carr’s Index(%)	Flowproperty
<10	Excellent
11-15	Good
16-20	Fair
21-25	passable
26-31	Poor
32-37	Very poor
>38	Very Very poor

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Compressibility Index Values

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Hausner’s ratio	Flow properties
1.00-1.11	Excellent
1.12-1.18	Good
1.19-1.25	Fair
1.26-1.34	Passable
1.35-1.45	Poor
1.46-1.59	Very poor
>1.60	Very Very poor

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Hausner’s Ratio Index

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Sr. No	parameter	Trial 1	Trial 2	Trial 3	Trial 4
1	Appearance	Powder	Powder	Powder	Powder
2	Texture	Fine	Fine	Fine	Fine
3	Colour	Greenish brown	Greenish brown	Greenish brown	Greenish brown
4	Odour	Sweet	Sweet	Sweet	Sweet

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Organoleptic Evaluation Results:

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Phytoconstituent	Trial 1	Trial 2	Trial 3	Trial 4
Alkaloids
Flavonoids	_ +	_ +	_ +	_ +
Saponin	+	+	+	+
Tannins	+	+	+	+
Glycosides		+	+	+
Carbohydrates	_ +	+	+	+
protein

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Phytochemical Testing of Trial Formulation (Trial 1toTrial4)

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Conc. (µg/ml)	Ascorbic acid	Batch 1
0	0.000	0.000
5	32.332	48.522
10	49.571	57.841
20	59.299	60.896
40	76.585	66.243
80	88.698	69.960
160	91.588	75.967
320	94.680	85.133

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Conc. (µg/ml)	Ascorbic acid	Batch 2
0	0.000	0.000
5	32.332	32.281
10	49.571	51.731
20	59.299	56.415
40	76.585	64.359
80	88.698	68.432
160	91.588	77.546
320	94.680	91.243

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Graph of DPPH assay Batch 1

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Phytochemical evaluation of plant extract

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Graph of DPPH assay Batch 2

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Graph of FTIR Trial 1

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Graph of FTIR Trail 2

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