Development and Evaluation of Betacyanin Enriched Polyherbal Effervescent Tablet for Anti-Ageing Therapy

Ageing is an inevitable biological process associated with progressive deterioration of physiological functions. One of the major causes of ageing is oxidative stress generated by free radicals. Reactive oxygen species damage proteins, lipids, and DNA, resulting in cellular ageing and degenerative diseases. Antioxidants play a crucial role in neutralizing free radicals and protecting cells from oxidative damage. Natural plant-derived antioxidants are widely preferred due to their safety and additional therapeutic benefits. Polyherbal formulations combine multiple plant ingredients to achieve synergistic therapeutic effects. The use of medicinal plants in nutraceutical formulations has gained increasing attention due to their bioactive phytochemicals. In recent years, there has been a growing demand for herbal and natural products due to increased awareness Phyto-cosmeceuticals, derived from plant sources, offer multifunctional skin benefits such as moisturization, anti-ageing, antioxidant protection, and skin nourishment, making them safer and more acceptable alternatives to conventional formulations. Oxidative stress caused by free radicals is one of the major contributors to skin ageing, dryness, and loss of elasticity. Antioxidants present in herbal extracts play a crucial role in neutralising free radicals and protecting the skin from premature ageing. In addition, natural humectants and bioactive compounds help maintain skin hydration and improve overall skin texture. Incorporating such plant-based actives into solid dosage form formulations can provide multifunctional skin care benefits in a single product. Effervescent tablets are oral dosage forms designed to dissolve rapidly in water, producing carbon dioxide that results in a sparkling solution. This dosage form improves palatability, enhances dissolution, and increases patient compliance.

The present research focuses on developing a polyherbal effervescent tablet enriched with betacyanin and other antioxidants using four medicinal plants:

• Beetroot – rich in betalains, including betacyanin pigments with strong antioxidant activity.
• Ber fruit – contains vitamin C, flavonoids, and polyphenols beneficial for skin health.
• Neem leaves – known for anti-inflammatory, antimicrobial, and detoxifying properties.
• Amla -it is one of the richest natural sources of vitamin C and polyphenols, which strongly support anti-ageing and antioxidant activity. It also improves the nutraceutical value of the effervescent tablet.

The combination of these ingredients may provide synergistic antioxidant and anti-ageing effects.

1.1. Facial Skin Anatomy

The facial skin is a specialized part of the integumentary system that covers and protects the face. It is thinner, more sensitive, and contains more sebaceous glands than skin on many other parts of the body. Because the face is constantly exposed to environmental factors such as sunlight, dust, cosmetics, and pollution, understanding its anatomy is important in dermatology and cosmetic formulation.

Layers of Facial Skin

Human facial skin is composed of three main layers:

• Epidermis-the outer layer
• Dermis- the middle layer
• Endodermis- the inner layer

Figure no.1.1: Structure of skin

Facial skin anatomy is important for designing skincare formulations because:

The skin is composed of three layers,

Epidermis (50-100 µm)

It is composed of five layers the vascular thickness varies based on location. For example, it is thickest on the heels and thinnest on the eyelids. Areas that have increased use from friction or weight bearing can build up thicker layers of skin (e.g., where a pencil rubs your writing finger or a shoe rubs against your foot) It has no nerves, but free nerve endings from the dermis do extend into the mid layers of the epidermis. Five layers of the epidermis (most to least superficial):

Stratum corneum is composed of 15 to 30 layers of keratinocytes called square’s or corneocytes. These are dead keratinocytes. They contain a high concentration of keratin, which provides a waterproof barrier for the skin, hair, and nails.

This layer is continually being shed from the body. Shed cells are replaced via the process of skin cell migration from the stratum Basale. This process takes an average of 30 days, but this varies based on age and certain health conditions.

Stratum lucidum contains two to three layers of keratinocytes and is not living. It can be penetrated or shaved off without awareness. It is only found in areas of thick skin, like the palms of the hand and the soles of the feet. Present in calluses.

Stratum granulosum This layer contains the greatest concentration of free nerve endings that extend from the dermis. Free nerve endings are unencapsulated dendrites originating from a sensory neuron. They are the most common nerve endings in skin and provide sensory information about painful stimuli, hot and cold, and light touch. However, they are less sensitive to abrupt changes in stimulation. This is the most superficial layer of the epidermis which contains living cells.

Stratum spinosum Contains Langerhans cells and lymphocytes which play an important role in the immune system.

Stratum Basale The only layer that undergoes continuous mitosis to produce new cells. Keratinocytes are constantly being produced in the stratum Basale and they move up through the layers until they reach the outermost layer. Keratinocytes are the most dominant cell type in the skin. They play a critical role in wound healing as they are structural cells and they perform important immune functions. Melanocytes are also produced in the stratum Basale. They produce melanin, which contributes to the color of skin. Humans have approximately the same number of melanocytes. Therefore, skin color is based on the amount of melanin that these melanocytes produce in response to their environment. This layer also contains Merkel cells which can perform both nervous and endocrine actions. They can synthesize and store locally produced hormones and neurotransmitters. They function as mechanoreceptors for light and selective tactile perception, but not for hard touch and vibration; they are also involved in the transfer of nociceptive signals.

Dermis (1-2mm) Located deep to the epidermis Contains blood vessels and nerves which supply the epidermis via capillary loops and free nerve endings Composed of two layers Two layers of the dermis (most to least superficial):

Papillary layer

1. Interdigitates with the epidermis.

2. The ridges of this layer give rise to our unique fingerprints

3. Contains fibroblasts which are responsible for the production of collagen, elastin, and proteins. These qualities give skin strength and flexibility.

4. Contains mast cells which produce heparin and histamine, important factors in clot formation and the inflammatory response.

5. Contains macrophages which play an important role in the immune response, wound repair, cancer defence, salt balance, and hair regeneration. They are known for destroying foreign invaders through phagocytosis (the process by which a phagocyte, a type of white blood cell, engulfs and digests foreign cells and removes dead cells.

6. Contains leukocytes which are crucial to the inflammatory response following an injury to the skin. Leukocytes are essential for clearing infection and normal wound healing.

Reticular layer

1. Located between the papillary layer and the subcutaneous layer or hypodermis.

2. It is made up of collagen, blood vessels, nerve endings, T-cells, hair follicles and glands.

3. The hair follicles contain stem cells that produce keratinocytes that will become hair. They play an important role in wound healing by contributing epithelial cells for wound closure.

4. The T-lymphocytes are responsible for destroying pathogens and malignant cells.

5. Nerves located within the dermis detect sensations such as itching, touch, pressure,  vibration, pain, and temperature..

6. Injuries which reach into the dermis can result in pain due to nerve exposure and or damage. There will be an absence of pain if the nerves are completely destroyed and or severed by an injury.

Hypodermis (1-2mm) Located below the dermis and contains subcutaneous tissue. It is made up of loose connective tissue, adipose tissue. It is well vascularized and well innervated. It helps to attach the skin to the muscles and bones through superficial fascia, and provides insulation and cushioning through fat storage.

1.2. Ageing

Ageing is a multifactorial biological process characterised by progressive deterioration in physiological functions, leading to increased vulnerability to diseases and death. It is influenced by genetic, environmental, and lifestyle factors and occurs at molecular, cellular, tissue, and organism levels.

In pharmaceutical and cosmeceutical research, ageing is particularly significant due to its association with oxidative stress, inflammation, and degenerative disorders, making it a key target for therapeutic and herbal interventions.

Factors that influence Ageing

Figure no. 1.2 ageing

Beyond the usual culprits like poor diet and lack of exercise, several surprising lifestyle and environmental factors can accelerate the ageing process, both externally (such as skin wrinkles) and internally (like organ decline or cellular damage). Often overlooked, these hidden triggers include stress, pollution, and even certain habits we assume are harmless. They can contribute to premature ageing by increasing oxidative stress, reducing collagen production, and interfering with hormonal balance. Recognising and addressing these lesser-known contributors can help slow down the ageing process and support long-term vitality.

Factors that influence Ageing

1. Chronic stress

Ongoing stress raises cortisol levels, which can damage collagen, disrupt sleep, and impair your immune system. Over time, this accelerates cellular ageing and increases the risk of age-related diseases like heart disease and cognitive decline.

2. Lack of sleep

Consistently getting poor-quality or insufficient sleep impacts hormone regulation, weakens immunity, and hampers skin regeneration. It leads to puffy eyes, dull skin, and memory issues, all common signs of premature ageing.

3. Overexposure to screens and blue light

Spending hours in front of phones, tablets, or laptops exposes your skin and eyes to blue light, which can penetrate deeper than UV rays. This contributes to digital ageing, affecting skin elasticity and causing fine lines.

4. Excess sugar intake

Sugar accelerates a process called glycation, where sugar molecules bind to collagen and elastin fibres in your skin, weakening them. This leads to sagging, wrinkles, and loss of skin radiance over time.

5. Environmental pollution

Living in highly polluted areas exposes your skin and lungs to harmful particles and toxins. These free radicals trigger inflammation and oxidative stress, both of which speed up cellular damage and ageing.

6. Using straws or constant lip puckering

Frequent use of straws can create fine lines around your lips, much like smoking does. Repetitive facial movements break down collagen over time, leading to early wrinkles in those areas.

7. Skipping SPF indoors

Many believe sunscreen is only needed outdoors, but UV rays can penetrate windows and cause sun damage indoors too. Without protection, cumulative UV exposure breaks down collagen and leads to dark spots and wrinkles.

8. Too much alcohol

Excessive alcohol dehydrates your body and skin, depletes vital nutrients, and increases inflammation. This not only dulls your complexion but also disrupts liver function, a key organ in detoxification and ageing.

9. Negative thinking and isolation

Mental health plays a big role in how quickly you age. Studies show that chronic negativity or prolonged social isolation can shorten telomeres, the protective ends of your DNA, leading to accelerated biological ageing.

10. Overexercising or intense workouts without rest

While exercise is crucial for health, excessive intense workouts without adequate recovery can raise oxidative stress and cortisol levels. This leads to muscle breakdown, hormonal imbalance, and faster ageing of tissues.

By identifying and managing these often-overlooked ageing triggers, maintain better skin, brain, and body health.

2. Biological Basis of Ageing

2.1 Cellular Senescence

Cellular senescence refers to the irreversible arrest of cell division, which occurs due to accumulated cellular damage.

A major mechanism involved is telomere shortening

Telomeres progressively shorten during each cell division, ultimately leading to loss of replicative capacity and cellular ageing.

2.2 Oxidative Stress Mechanism

The Free radical theory of ageing explains ageing as a consequence of damage caused by reactive oxygen species (ROS).

ROS attack: DNA, Lipids, Proteins, results in Cellular dysfunction, Tissue degeneration

This mechanism is highly relevant in anti-ageing drug and herbal formulation development.

1. 3. Genetic and Epigenetic Factors

Ageing is partially genetically programmed, involving: Longevity genes, DNA repair mechanisms, Epigenetic modifications (DNA methylation, histone changes)

2.4 Protein Damage and Glycation

Non-enzymatic glycation leads to formation of advanced glycation end products (AGEs)

Causes collagen cross-linking Leads to Skin ageing, Loss of elasticity

2.5 Mitochondrial Dysfunction

Reduced ATP production, Increased ROS generation, Contributes to cellular ageing and apoptosis

3. Physiological Changes Associated with Ageing

1. 1. Skin Decreased collagen and elastin, Wrinkle formation, Reduced hydration
   2. Musculoskeletal System Sarcopenia (muscle loss), Bone demineralization → Osteoporosis
   3. Cardiovascular System Arterial stiffness, Increased risk of Hypertension
   4. Nervous System Cognitive decline, Neurodegenerative disorders such as:
   5. Alzheimer’s disease

4. Ageing and Disease Correlation

Ageing is a major risk factor for chronic diseases: Diabetes mellitus, Cancer, Cardiovascular diseases. These conditions are often linked to oxidative stress and inflammation.

5. Factors Influencing Ageing

1. 1. Intrinsic Factors Genetic predisposition, Hormonal regulation, Metabolic rate
   2. Extrinsic Factors UV radiation, Pollution, Diet and nutrition, Smoking and alcohol, Psychological stress

6. Anti-Ageing Approaches

1. 1. Pharmacological Approaches

Antioxidants, Hormone replacement therapy, Anti-inflammatory drugs

6.2 Herbal and Phyto-cosmeceutical Approaches

Plant-based compounds are widely explored due to their safety and efficacy.

Amla (rich in vitamin C) antioxidant, Neem anti-inflammatory and antimicrobial, Ber (Ziziphus mauritiana) skin rejuvenation; Beetroot (betacyanin) free radical scavenger

These ingredients act primarily by reducing oxidative stress, enhancing collagen synthesis, and protecting against cellular damage

7. Need for Anti-Ageing Intervention

Conventional therapies may cause side effects, hence there is increasing demand for:

Natural antioxidants, Herbal formulations, Nutraceutical delivery systems

8. Role of Selected Herbal Ingredients in Anti-Ageing

Amla (Emblica officinalis)

Rich in Vitamin C, Potent antioxidant, Promotes collagen synthesis, Helps reduce oxidative damage and improves skin elasticity

Neem (Azadirachta indica)

Anti-inflammatory, Antimicrobial, Detoxifying agent,  Protects skin and reduces ageing-related inflammation

Ber (Ziziphus mauritiana)

Contains flavonoids and saponins, Skin rejuvenating properties, Improves tissue repair and hydration

Beetroot (Betacyanin source)

Strong antioxidant pigment, Scavenges free radicals, Directly counteracts oxidative stress (core ageing mechanism)

9. Mechanism of  Formulation (Integrated Action)

The polyherbal tablet works via:

Antioxidant Action

Neutralises ROS, prevents cellular damage

Anti-ageing Cellular Protection

Reduces DNA and protein damage, Slows down cellular ageing

Detoxification

Removes metabolic waste, Improves overall skin health

Skin Health Improvement

Enhances collagen, Maintains hydration

10. Role of Effervescent Drug Delivery System

Effervescence provides a unique advantage: faster dissolution, rapid absorption, improved bioavailability, Better patient compliance, and enhanced stability of phytoconstituents. This makes your formulation more effective compared to conventional tablets.

1.3. Bottom of Form

Plants used:

• Beetroot (Beta vulgaris)
• Ber (Ziziphus mauritiana)
• Neem (Azadirachta indica)
• Amla (Phyllanthus emblica)

1.3.1. Drug profile of Beta vulgaris

1. Biological Source

Beetroot consists of the root of Beta vulgaris, belonging to the family Amaranthaceae.

2. Taxonomical Classification

Table no.1.1 Taxonomical classification of beetroot

Taxonomic Category	Classification
Kingdom	Plantae
Division	Magnoliophyta
Class	Magnoliopsida
Order	Caryophylalles
Family	amaranthaceae
Genus	Beta
Species	vulgaris

3. Synonyms

Beetroot, Red beet, Garden beet

Figure no.1.3. Beetroot

4. Geographic Source:

Native to Mediterranean region,Widely cultivated in Europe, North America, and Asia, Adaptable to temperate climates and well-drained soils

5. Macroscopical / Morphological Characteristics:

Type: Biennial herb cultivated as an annual,30–70 cm in height,  Edible, globular to conical root, red-purple in colour, Green, ovate to lanceolate leaves, petiolate, Small, greenish-yellow flower, clustered in an inflorescence

Microscopical Characteristics of Beetroot (Beta vulgaris L.) Root

Tissue Type: Typical dicot root structure with cortex, vascular cylinder (stele), and pith Epidermis a single layer of thin-walled, rectangular cells; may show root hairs in young roots Cortex Parenchymatous cells with large intercellular spaces; cells contain red-violet pigment (betacyanin) Endodermis: a Single layer of compact cells surrounding the vascular cylinder; cells have Casparian strips; gives rise to lateral roots )

Chemical Constituents:

Betacyanin, Betaxanthin (betalains), Vitamin C, folate, B-complex vitamins, Iron, potassium, magnesium, calcium, Phenolic compounds (ferulic acid, caffeic acid), Nitrates, saponins, carotenoids

Pharmacological Properties including Antioxidant, anti-inflammatory, hepatoprotective, cardioprotective, and anti-ageing, and is used in dietary supplements.

Mechanism of Action: Free radical scavenging, lipid peroxidation inhibition, collagen protection

1.3.2. Top of Form

Bottom of Form

Drug Profile of Ziziphus mauritiana

1. Biological Source

Indian jujube consists of the fruits, leaves, or seeds of Ziziphus mauritiana, belonging to the family Rhamnaceae.

2. Taxonomical Classification

Table no.1.2 Taxonomical Classification of Ber

Taxonomic Category	Classification
Kingdom	Plantae
Division	Magnoliophyta
Class	Magnoliopsida
Order	Rosales
Family	Rhamnaceae
Genus	Ziziphus
Species	mauritiana

3. Synonyms:

Indian plum, Ber, Badara, Desert apple

Figure no.1.4. Indian jujube (ber)

4. Geographical Source

The plant is widely distributed in tropical and subtropical regions, especially in India, Pakistan, Bangladesh, parts of Africa and Australia. It grows well in dry and semi-arid climates.

5. Macroscopical / Morphological Characteristics:

Leaves- Oval or elliptical, Smooth and shiny on upper surface, Hairy underside,Three prominent veins

Fruit- Round or oval drupe, Green when unripe, yellow to reddish-brown when ripens, Sweet and edible pulp

Seeds- Hard stone with kernels inside

6. Microscopical Characters

Epidermal cells with cuticle, Presence of trichomes, Calcium oxalate crystals, Vascular bundles well developed

7. Chemical Constituents

Major phytochemicals present in Ziziphus mauritiana include:

Flavonoids, Saponins, Alkaloids, Tannins, Glycosides, Phenolic compounds,Vitamin C, Terpenoids

These compounds are responsible for medicinal and cosmetic activities.

8. Pharmacological Activities

Antioxidant, Antimicrobial, Anti-inflammatory, Anti-ageing, Wound healing, Skin protective activity

9. Uses

Treatment of skin infections, used in herbal cosmetics and creams, Nutritional fruit, and traditional medicine for fever and digestive problems

1.3.3. Drug profile of Azadirachta indica

1. Biological Source

Neem is obtained from the leaves, bark, seeds, and oil of the tree Azadirachta indica, belonging to the family Meliaceae.

2. Taxonomical Classification

Table no. 1.3 Taxonomical classification of Neem

Taxonomic Category	Classification
Kingdom	Plantae
Division	Magnoliophyta
Class	Magnoliopsida
Order	Spindalce
Family	Meliaceae
Genus	Azadirachta
Species	indica

3. Synonym

Indian Lilac, Margosa, Nimba (Ayurvedic name)

Figure no.1.5. Neem leaves

4. Geographical Source

Neem is native to the India and grows widely in tropical and subtropical regions including Pakistan, Bangladesh, Sri Lanka, and parts of Africa.

5. Macroscopical / Morphological Characteristics:

• Color: Dark green,
• Odor: Characteristic, garlic-like
• Taste: Very bitter,
• Shape: Lanceolate leaflets with serrated margins,
• Arrangement: Pinnate compound leaves

6. Microscopical characters

1. Epidermis

Leaf is dorsiventral, Upper epidermis: single layer of rectangular cells with thick cuticle, Lower epidermis: single layer containing numerous stomata. Stomata are usually anomocytic type (surrounded by ordinary epidermal cells).

2. Mesophyll Tissue

Mesophyll is differentiated into:

a. Palisade Parenchyma

1–2 layers of elongated cylindrical cells, Located just below the upper epidermis.

Rich in chloroplasts.

b. Spongy Parenchyma

4–6 layers of loosely arranged cells, Large intercellular spaces present for gas exchange.

3. Midrib Region

Shows collenchymatous tissue beneath both upper and lower epidermis, Central vascular bundle present, Bundle is conjoint, collateral, and closed.

4. Vascular Tissue: Xylem towards the upper side, Phloem towards the lower side, Surrounded by parenchymatous cells.

5. Calcium Oxalate Crystals: Prismatic crystals of calcium oxalate present in mesophyll cells.

6. Trichomes: Unicellular covering trichomes may be present on epidermis.

7. Chemical Constituents

Major bioactive compounds include:

Limonoids: Azadirachtin, Nimbin, Nimbidin, Salannin

Flavonoids: Quercetin

Other compounds: Tannins, Steroids, Polysaccharides, Essential oils

8. Pharmacological Actions

Neem exhibits multiple biological activities: Antibacterial, Antifungal, Antiviral, Anti-inflammatory, Antimalarial, Antidiabetic, Immunomodulator, Anthelmintic

9. Therapeutic Uses

Neem is traditionally used for: Skin diseases (eczema, acne), Fever and infections, Dental care (chewing sticks), Blood purification in traditional medicine, Wound healing, Treatment of intestinal worms

 1.3.4. Drug profile of Phyllanthus emblica

 1. Biological Source

Amla consists of the fresh or dried fruits of Phyllanthus emblica (synonym: Emblica       officinalis), belonging to the family Phyllanthaceae.

2. Taxonomical Classification

Table no.1.4 Taxonomical classification of Amla

Taxonomic Category	Classification
Kingdom	Plantae
Division	Magnoliophyta
Class	Magnoliopsida
Order	Malpighiales
Family	Phyllanthaceae
Genus	Phyllanthus
Species	emblica

3. Synonyms

Indian Gooseberry, Amlaki, Dhatri

Figure no.1.6. Amla

4. Geographical Source

Amla is widely distributed in tropical and subtropical regions, mainly in India, Sri Lanka, Pakistan, Bangladesh, and China.

5. Macroscopical Characters (Fruit):

Nearly spherical in shape, Greenish-yellow colour when fresh, Smooth surface with 6 vertical furrows, Sour in taste, astringent, slightly bitter, Slight characteristic odor

6. Chemical Constituents

Major constituents present in amla include: Vitamin C (Ascorbic acid), Tannins: Emblicanin A, Emblicanin B, Punigluconin, Pedunculagin, Flavonoids, Gallic acid, Ellagic acid, Pectin and minerals

7. Pharmacological Actions

Amla shows several biological activities:

Antioxidant, Antidiabetic, Anti-inflammatory, Immunomodulator, Hepatoprotective, Antimicrobial.

8. Therapeutic Uses:

Used in digestive disorders, improves immunity, used in hair tonics and cosmetics, Helpful in hyperacidity and ulcers, Acts as a rejuvenating tonic (Rasayana) in Ayurveda, and is an important ingredient of Triphala and Chyawanprash.

Mechanism of action:

1. Ber (Ziziphus mauritiana) powder

Rich in vitamin C and polyphenols and supports collagen formation and antioxidant activity (anti-ageing)

2. Beetroot (Beta vulgaris) powder

Contains betalains and nitrates and improves circulation and reduces oxidative stress.

3. Neem (Azadirachta indica) leaf powder

Strong antioxidant and anti-inflammatory compounds and helps detoxification and skin health.

4. Amla (Phyllanthus emblica)

Acts as a rejuvenating tonic

1.4. Effervescent Tablet:

Oral dosage forms are the most common route of drug administration despite having several disadvantages such as poor patient compliance, slow absorption, etc. However, in liquid formulations, problems such as stability concerns may arise and even a slow onset of action. This gives an option for the use of effervescent tablets to overcome such problems. Effervesce is the evolution of bubbles in response to a chemical reaction. The most common reaction is an acid-base reaction between citric acid and sodium bicarbonate, giving us the evolution of carbon dioxide bubbles in the presence of water. There are several advantages of making an effervescent formulation, as they are already in liquid form. so they are easier to administer. They may also be flavoured to increase patient compliance. Due to less gastric irritation, the effervescent liquid can be very easily tolerated in the stomach and intestine.

The main components of the formulation include active ingredients, a mixture of acids or their salts, bicarbonates or carbonates or their salts, which on addition of water give CO2. The formulation also includes fillers, sweeteners, binders, flavouring agents and lubricants. The methods of preparation of effervescent tablets include wet granulation, fluidized bed granulation, fusion method and direct compression.

Formulation requires controlled conditions of temperature and humidity, i.e., a relative humidity of RH 25% or less and moderate temperature to prevent sticking of tablets or granules to the machine as a result of absorbed moisture. High moisture content may also cause pre-effervescence or moisture retention in the tablet and, hence a formulation problem.

The major limitation with the use and formulation of effervescent is the inability of excipients to prevent moisture absorption. Hence, the formulation scientists are focusing on exploiting different techniques, such as a direct compression method. The bubble- or gas-generating reaction of the effervescent couple in the granule is most often the result of the reaction of an acidic agent and an alkaline agent. The reaction of these two general classes of compounds produces effervescence upon contact with water.

Effervescence occurs due to a chemical reaction between an acid and a carbonate or bicarbonate in the presence of water, producing carbon dioxide gas. Citric acid + Sodium bicarbonate → Sodium citrate + Water + Carbon dioxide (CO?)The release of CO? causes bubbling or fizzing, helping the tablet dissolve rapidly.

1.4.1. Composition of Effervescent Tablets

1. Active ingredient (Beetroot, Neem leaves, Indian ber, amla)
2. Acid components: Usually organic acids such as Citric acid, Tartaric acid, Malic acid
3. Carbonate/Bicarbonate component Sodium bicarbonate, Potassium bicarbonate
4. Binders Polyvinylpyrrolidone (PVP)
5. Sweetening agents: Saccharin sodium, Aspartame, Mannitol
6. Flavouring agents: Lemon, orange, or other fruit flavours
7. Lubricants: (magnesium stearate, Talc)
8. Super disintegrating agent: sodium starch glycolate
9. Preservatives: Sodium Benzoate

1.4.2. Methods of Preparation

1. Dry Fusion Method

Citric acid monohydrate and tartaric acid are mixed with sodium bicarbonate. The mixture is heated slightly so that citric acid releases water of crystallisation. This moisture helps granule formation. The granules are then compressed into tablets.

2. Wet Granulation Method

Ingredients are mixed and granulated using a non-aqueous solvent such as alcohol. Water is avoided to prevent premature reaction. The granules are dried and compressed into tablets.

1.4.3. Evaluation of Effervescent Tablets

Important quality control tests include:

1. Appearance – tablets should be uniform and free from defects.

2. Weight variation test – ensures uniform drug content.

3. Hardness test – determines tablet strength.

4. Friability test – checks resistance to breakage.

5. Disintegration/Effervescence time – tablet should dissolve rapidly in water (usually within 1–3 minutes).

6. Moisture content test – low moisture is essential to avoid premature reaction.

1.4.4. Advantages

• Rapid drug dissolution and absorption.
• Pleasant taste due to flavours and effervescence.
• Easy to swallow (good for children and the elderly).
• Accurate dosage.
• Faster onset of action.

1.4.5. Disadvantages

• Highly sensitive to moisture.
• Require special packaging (airtight containers).
• Larger tablet size.
• Higher manufacturing cost.

1.4.6. Packaging and Storage

• Effervescent tablets are packed in moisture-proof containers, such as:
• Aluminum foil strips
• Plastic tubes with desiccants
• Airtight glass containers
• They should be stored in a cool and dry place.

1.5. Reason for selection of betacyanin-enriched polyherbal effervescent tablet

Fast onset of action - Effervescent tablets have a major advantage in that the drug product is already in solution at the time it is consumed. Thus, the absorption is faster and more complete than with a conventional tablet. Faster absorption means faster onset of action. Effervescent drugs are delivered to the stomach at a pH that is just right for absorption. Many medications travel slowly through the gastrointestinal tract or have absorption that is hampered by food or other drugs.

No need to swallow a tablet - effervescent medications are administered in liquid form so they are easy to take as compared to tablets or capsule. the number of people who cannot swallow tablet or who dislike swallowing tablet and capsule is growing. with an effervescent dosage form, one dose can usually delivered in just 3 or 4 ounces of water.

Good stomach and intestinal tolerance - effervescent tablet dissolve fully in a buffered solution. Reduced localized contact in the upper gastrointestinal tract leads to less irritation and greater tolerability. Buffering also prevent gastric acids from interacting with drug themselves, which can be a major cause of stomach. 

More portability - effervescent tablet is more easily transported than liquid medication because no water is added until it is ready to use.

Improved palatability - drugs delivered with an effervescent base, taste better than most liquids, mixture and suspensions. superior taste masking is achieved by limiting objectionable characteristics and complementing formulations with flavour and fragrances. the effervescent tablet essentially include flavouring so they taste much better than a mixture of non-effervescent powder in water. Moreover, they produce fizzy tablets, which may have better consumption appeal than the traditional dosage form.

More consistent response - drugs delivered with effervescent technology have predictable  and reproducible pharmacokinetics profile that are much more consistent than the tablets or capsule.

Accurate dosing - researchers have been shown that effervescent tablets enhance the absorption of number of active ingredients compared to conventional formulations. This is because the carbon dioxide created by the effervescent reaction can enhance active ingredient permeability due to an alteration of paracellular pathway. The paracellular pathway is the primary route of absorption of hydrophilic active ingredients in which the solutes diffuse into the intercellular space between epithelial cells.it is postulated that the carbon dioxide widens the intercellular space between cell which leads to greater absorption of active ingredients (both hydrophilic and hydrophobic). The increased absorption of hydrophobic active ingredients could be due to the non polar carbon dioxide gas molecules partition into cell membrane, thus creating an increased hydrophobic environment, which would allow the hydrophobic active ingredients to be absorbed.

Conventional tablets are often associated with slower onset of action and also undergoes first pass metabolism. Effervescent tablet avoid the first pass metabolism and also produce rapid onset of action. Oral liquid also provide rapid onset of action but required carefully handling. slower onset of action and also undergoes first pass metabolism. Effervescent tablet avoid the first pass metabolism and also produce rapid onset of action. Oral liquid also provide rapid onset of action but requires careful handling.

2. AIM AND OBJECTIVE

Aim: To develop and evaluate Betacyanin-Enriched Polyherbal Effervescent Tablet for Anti-Ageing Therapy.

Objectives

1) To select and authenticate herbal ingredients rich in betacyanin and other anti-ageing phytoconstituents.

2) To formulate a polyherbal effervescent tablet using suitable excipients.

3) To optimize the formulation for better effervescence, taste, and stability.

4) To evaluate pre-compression parameters such as angle of repose, bulk density, and tapped density.

5) To evaluate post-compression parameters like hardness, friability, weight variation, and disintegration time.

6) To determine physicochemical properties including pH and drug content uniformity.

7) To assess in-vitro antioxidant activity for anti-ageing potential.

8) To carry out stability studies as per guidelines.

2.1. Methods and Materials:

APIs like betacyanin from beetroot were extracted. Amla, ber, and neem leaves were collected from trees. All the raw materials used in the study were collected from natural plant sources. Effervescent agents like Citric acid, Tartaric acid, Sodium bicarbonate (anhydrous), excipients like Mannitol (diluent & sweetener), Aspartame (sweetener), Polyvinylpyrolidone K-30 (Binder), Talc (glidant), Magnesium stearate (lubricant), Sodium Benzoate(Preservatives) and Sodium starch glycolate  (Super-disintegrating agent) were taken from the lab of Rai University (Ahmedabad)

Formulation Table 1

Table no.2.1: Formulation of effervescent tablet 1

Ingredient	Quantity (mg/tablet)
Neem powder	40 mg
Ber powder	60 mg
Amla powder	80 mg
Beetroot betacyanin	10 mg
Citric acid	90 mg
Tartaric acid	60 mg
Sodium bicarbonate	120 mg
PVP K30	2 mg
Talc	2 mg
Magnesium stearate	2mg
Sodium benzoate	5mg
Sodium starch glycolate	14mg
Mannitol	205mg

Formulation Table 2

Table no.2.2: Formulation of effervescent tablet 2

Ingredient	Quantity (mg/tablet)
Neem powder	30 mg
Ber powder	50 mg
Amla powder	70 mg
Beetroot betacyanin	20 mg
Citric acid	90 mg
Tartaric acid	60 mg
Sodium bicarbonate	200 mg
PVP K30	2 mg
Talc	2 mg
Magnesium stearate	2mg
Sodium benzoate	5mg
Sodium starch glycolate	14mg
Mannitol	145mg

Formulation Table 3

Table no.2.3: Formulation of effervescent tablet 3

Ingredient	Quantity (mg/tablet)
Neem powder	50 mg
Ber powder	70 mg
Amla powder	90 mg
Beetroot betacyanin	20 mg
Citric acid	90 mg
Tartaric acid	60 mg
Sodium bicarbonate	160 mg
PVP K30	2 mg
Talc	2 mg
Magnesium stearate	2mg
Sodium benzoate	5mg
Sodium starch glycolate	14mg
Mannitol	135mg

Formulation Table 4

Table no.2.4: Formulation of effervescent tablet 4

Ingredient	Quantity (mg/tablet)
Neem powder	20 mg
Ber powder	40 mg
Amla powder	60 mg
Beetroot betacyanin	20 mg
Citric acid	90 mg
Tartaric acid	60 mg
Sodium bicarbonate	160 mg
PVP K30	2 mg
Talc	2 mg
Magnesium stearate	2mg
Sodium benzoate	5mg
Sodium starch glycolate	14mg
Mannitol	225mg

Each tablet weigh 700mg.

2.2. Preformulation:

Pre-formulation is a branch of pharmaceutical sciences that utilizes biopharmaceutical principles in the determination of physicochemical properties of a drug substance. the goal of pre-formulation studies is to choose the correct form of the substance, evaluate its physical properties and generate a through understanding of the material’s stability under various conditions, leading to the optimal drug delivery system. the preformulation study focuses on the physiochemical parameters that could effect the development of efficacious dosage form. these properties may ultimately provide a rationale for formulation design. also it will help in minimizing problems in later stages of drug development, reducing drug development costs and decreasing product’s time to market. it gives the information needed to define the nature of the drug substance and provide framework for the drug combination with pharmaceutical excipients in the dosage form. Objective: the overall objective of preformulation testing is to generate information useful to the formulation in developing desired, stable and bioavailable dosage forms. Scope: the use of preformulation parameters maximizes the chances in formulating an acceptable, safe, efficacious and stable product. preformulation encompasses at least following tests: -

1. Bulk Characterisation: crystallinity, polymorphism and hygroscopicity powder properties (flow, compaction, density, particle size, surface area etc.) microscopy (morphology, particle characteristics) molecular spectroscopy (ft-ir)

2. Solubility Analysis: pH, solubility profile, common ion effect, thermal effect on solubility, solubilization, dissolution

3. Stability Analysis: stability (heat, light, acid, base, oxidiser) solution stability

1. Solid-state stability

excipient compatibility consideration in effervescent tablet formulation: Several factors influence the release of drug from effervescent tablets. particle size, dose, solubility

2. Drug-excipient compatibility study:

For the drug-excipient compatibility study, the following excipients were studied, which are used in the experiments:

Table no.2.5: Ingredients & Function

Ingredient	Function
Amla powder	Antioxidant, vitamin C
Ber powder	Antioxidant, vitamin C
Beetroot powder	Antioxidant, color, nitrates
Neem leaf powder	Detox, anti-inflammatory
Citric acid (anhydrous)	Effervescent acid
Tartaric acid	Effervescent acid
Sodium bicarbonate	CO? release
Mannitol / sorbitol	Diluent, sweetness
Natural flavor (orange/berry)	Taste masking
Magnesium stearate	Lubricant
PVP or PEG	Binder
Sodium benzoate	Preservatives
Sodium starch glycolate	Super disintegrants

According to the functional category these excipients were mixed in the different ratio. these mixtures were kept at 40o c + 75% RH, and 45 degree celcius.

At the interval of 4 weeks, the sample was withdrawn and was subjected for analysis and related substances. at the interval of 2 weeks and 4 weeks, the samples were withdrawn and were tested for following parameters:

• Moisture content

• Assay

• Related substances

2.3. Evaluation of Granules

1. Angle of repose

2. Bulk density

3. Tapped density

4. Compressibility

I) Evaluation of granules: The ideal characteristics of a tablet that make it a popular and acceptable dosage form are compactness, physical stability, rapid production capability, chemical stability and efficacy. In general, the above characteristics of a tablet are dictated by the quality of the granulation from which it is made. Many formulation and process variables involved in the granulation step can affect the characteristics of the granulation produced. Therefore, various methods to measure certain granulation characteristics have been developed to monitor granulation suitability for tableting. The main characteristics required to be monitored in granulation are flow properties and compressibility.

i) Angle of repose: It was measured by fixed funnel method. The fixed funnel method employs a funnel that was secured with its tip at a given height H, above graph paper that was placed on a flat horizontal surface. Granules were carefully poured through the funnel until the apex of the conical pile just touches the tip of the funnel. Thus, with R being the radius of the base of the conical pile.

Tan α = H/R

Where, α = Angle Of Repose

ii) Apparent Bulk Density: (δu) An accurately weighed sample of granulation was carefully added to the measuring cylinder with the aid of a funnel. Then the volume was noted. The volume of the packing was determined in an apparatus consisting of a graduated cylinder mounted on a mechanical tapping device.

Apparent bulk density is determined by the following formula:- δU = M VU

Where, M = Mass Of Granulation In Gms Vu = volume of granulation (initial untapped volume)

iii) Packed Bulk Density: (δb) The above procedure was followed. The final volume was tapped till no further reduction in volume was noted.

Packed bulk density is determined by the following formula. b = m/vb

where, m = mass of granulation in gms Vb = volume of granulation (final tapped volume)

iv) Percent Compressibility: (%C) It is an important measure that can be obtained from bulk density measurements.

The following formula was used to compute the percent compressibility. c = δb - δu x 100 δb

where, δb = packed bulk density

δu = apparent bulk density

2.4. Evaluation of effervescent compressed tablets

 1. tablet shape & dimensions

 2. hardness

 3. thickness

 4. friability

 5.  weight variations

6. Measurement of Effervescence Time

7. Determination of Effervescent Solution pH

8. Determination of Effervescent Solution pH

9.  Measurement of Water Content 

1. Tablet Dimensions:

Thickness and diameter were measured using a calibrated dial caliper. Ten tablets of each formulation were evaluated.

2. Hardness:

Monsanto hardness tester was used to evaluate hardness of tablet. The tester consists of a barrel containing a compressible spring held between two plungers. The lower plunger was placed in contact with the tablet, and a zero reading was taken. The upper plunger was then forced against a spring by turning a threaded bold until the tablet fractures. As the spring compressed, a pointer rides along a gauge in the barrel to indicate the force. The force of fracture was recorded, and the zero force reading was deducted from it. Ten tablets of each formulation were evaluated.

3. Friability:

Roche friabilator was used to determine friability of the tablets. Twenty preweighed tablets were placed in the friabilator, which was then operated for 100 revolutions. The tablets were then dedusted and reweighed.

The friability was computed by following formula: F = 100 (1 – Wo ) w

where, f = percentage friability, wo = initial weight of 20 tablets, and w = weight after friability testing

4. Weight variation:

Twenty tablets were selected randomly. Tablets were weighed individually and average weight was calculated. Then deviation of each tablet from average weight was cacalculated and percent deviation was computed.

5. Measurement of Effervescence Time

Place one tablet in beaker of 100ml of distilled water at 20 °C ± 1 °C. The time was noted when a clear solution without any floating particles was obtained (21). The values are given in table 4.

6. Determination of Effervescent Solution pH

pH of the solution was determined by taking a single tablet in 200ml of distilled water at 20 ± 1 °C by using pH meter immediately after completing the dissolution time. Repeat this 3 times for each formulation. See table 4 (22, 23).

7. CO2 Content Measurement

Measure any weight changes that occur after the dissolution of one effervescent tablet in 100 ml of 1N sulfuric acid solution. The weight difference revealed the quantity (mg) of CO2 in each tablet. Table 4 shows the reported CO2 content, which is the average of three determinations (20).

8. Measurement of Water Content

10 tablets from each formulation were dried for 4 hours in a desiccator with activated silica gel.

9. Stability Study

The prepared tablets were stored in airtight containers at room temperature and evaluated periodically for changes in:

• Color
• Effervescence time
• Hardness
• Physical stability.

2.5. Methodology

1. Collection of Plant Materials

Fresh plant materials such as neem leaves, ber fruits, amla fruits, and beetroot were collected from local sources. The materials were washed thoroughly with distilled water to remove dirt and impurities.

2. Method of extraction of betacyanin from beetroot.

   

     

 

Figure no.2.1. extraction of Betacyanin from beetroot

Take  60-70 gm of beetroot and chopped it finely.

Mixed with 100 ml of distilled water.

Boiled for 30 min.

The boiled solution was filtered using Whatman filter paper.

And then transferred the filtrate to a petridish and concentrated it in hot air oven at 40-50 degree celcius.

Dried betacyanin powder was obtained.

3. Preparation of Herbal Powder

1. 1. 1. Neem leaves, ber fruit and amla fruit were cleaned and shade-dried for 7–10 days at room temperature to preserve their phytoconstituents.
      2. Dried neem leaves, ber fruit pulp, and amla fruits were ground using a mechanical grinder.
      3. The powders were passed through sieve no. 60 to obtain a uniform particle size.
      4. The powders were stored in airtight containers.

Figure no.2.2. drying of raw material in Hot air oven

4. Formulation of Effervescent Granules

Effervescent tablets were prepared by dry granulation method.

Step 1: Drying of Effervescent Components

• Citric acid
• Tartaric acid
• Sodium bicarbonate

These ingredients were dried separately at 40°C to remove moisture.

Step 2: Mixing of Herbal Ingredients

Neem powder, ber powder, amla powder and beetroot betacyanin powder were mixed thoroughly in a mortar and pestle and then mannitol was added to it and mixed well.

Step 3: Preparation of Effervescent Blend

Citric acid and tartaric acid were mixed together, followed by the addition of sodium bicarbonate.

Step 4: Granulation

Herbal powder mixture was blended with the effervescent mixture.

Binder PVP K30 was added to form granules.

The wet mass was passed through sieve no. 20 to obtain granules.

Step 5: Drying of Granules

The prepared granules were dried in a hot air oven at 40–45°C until moisture was removed.

Step 6: Lubrication

Dried granules were mixed with:

• Talc (glidant)
• Magnesium stearate (lubricant)

Figure no. 2.3 Mixing of Granules

5. Compression of Tablets

The lubricated granules were compressed using a tablet punching machine to obtain effervescent tablets of the desired weight.

Figure no. 2.4 Tablet punching Machine

 2.6. Observation

Table no.2.6: Observation of formulation

Formula	Dry compression
F1	Capping Problem, Less effervescence
F2	Tablets having very quick effervescence but the tablet surface found rough.
F3	The tablet gives good effervescence, no capping problem and the solution become found to be clear, hardness of tablet not good.
F4	The tablet gives good effervescence , no capping problem and the solution become found to be clear, hardness of tablet also good compaired to other batches

Evaluation of micromeritics of precompression blend formulation

Table no.2.7: Evaluation of micromeritics of precompression blend formulation

Formulation	Bulk density (gm/ml)	Tapped density (gm/ml)	Angle of repose	Carr’s index	Hausner’s ratio %	Compressibility	Flow property
F1	0.57	0.67	25.30	18.69	1.14	14.65	good
F2	0.49	0.56	23.40	17.57	1.12	12.23	good
F3	0.55	0.66	25.63	15.61	1.17	16.39	fair
F4	0.59	0.72	31.35	19.40	1.21	19.16	excellent

The homogeneous tablet thickness was achieved through uniform die filling, adequate flow characteristics, optimum pressure, and punch movement. Each formulation is within the weight variation limit (700 ± 15mg) as seen in Table 2.8. Friability was determined to be less than 1% for all formulations. A hardness tester was used to evaluate the tablets' hardness. The range of the values was 40 to 80 (N). The tablets' thickness ranged from 3 to 6 mm. The fusing process resulted in thicker tablets. Results can be seen in Table 3. The 100 ml of water was used for the effervescence test. All formulations had effervescence periods between 60 and 90 seconds as described in Table 2.9

Evaluation of Friability, Thickness, DT, Dissolution and Weight Variation

Table no.2.8: Evaluation of Friability, Thickness, DT, Dissolution and Weight Variation

Formulation	Thickness (cm)	Friability	Weight Variation	Disintegration time (sec)	% drug release
F1	3.78±0.017	0.512	700±7.86	45.3±2.30	95.60%
F2	3.81±0.018	0.768	700±8	42.8±2.42	96.20%
F3	3.80±0.013	0.536	700±6.4	46.6±2.12	94.80%
F4	3.77±0.015	0.569	700±5.3	34.6±1.15	98.12%

Evaluation of pH, effervescence time, % water content, taste and CO2 content

Table no.2.9: Evaluation of pH, effervescence time, % water content, taste and CO2 content

Formulation	CO2 Content	pH	% water content(w/w)	Effervescent time (sec)	Taste
F1	210±0.56	4.26±0.04	0.20±0.014	72±2.85	Too Sweet
F2	232±0.66	4.62±0.02	0.16±0.012	86±4.17	Satisfactory
F3	244±0.12	5.32±0.01	0.12±0.004	83±3.55	A bit bitter
F4	234±9.45	5.11±0.04	0.12±0.014	64±2.45	Excellent

In-vivo taste evaluation by analysis reveals that formulation F4 in table 1 is more popular and has a better taste than other formulations of varied effervescent compositions due to variations in the pH of the solutions. The dosage form's uniformity was confirmed by the content uniformity measured in accordance with USP standards. According to the stability research findings, even after 3 months of storage at varied temperatures and humidity levels, there was no discernible change in the physiochemical attributes. It can be assumed that the formulation was unchanged and did not degrade all the results of the zero-month study showed good percentage release within USP range and were mostly around 90-110%. After one month Real time and accelerated showed about the same results as average 96.8% and 96.43% respectively. Same behavior was seen until the 3-month stability studies. In real time stability data, the percentage on average was 95.57% and for accelerated study data results were 95.25% on average for all the formulations.

 

  

  

 

 

                          F1                                    F2                                      F3                               F4

Figure no. 2.5 Effervescent Tablets