Formulation, physicochemical Characterization and preliminary evaluation of a polyherbal hydrogel incorporating clitoria ternatea and annona squamosa

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Morphological characteristics:

The five-petaled pea blossoms measure roughly 4 cm in length and 3 cm in width. Two wings, two keels, and a bright yellow symbol in the center of the banner. One such is the pea plant. The extremely thin leaves of this climbing legume plant measure 1.5–3.5 cm in length and 2.5–5 cm in width. Each and every width. This plant has fibrous roots and is evergreen. Its enormous nodules may fix nitrogen so that other plants can use it. Bacteria are called rhizobia. The perennial plant Clitoria ternatea reproduces by using black seeds. The vibrant pods are between 7 and 11 cm long. The root and leaves are used in the food business to make herbal and therapeutic drinks. Instructing The most popular peas in the world are butterfly peas [2].

Chemical constituents

Flavonoids: Plant antioxidants called flavonoids aid in lowering inflammation and shielding cells from oxidative damage. Additionally, they enhance wound healing and promote tissue restoration.

Anthocyanins :they are naturally occurring pigments with potent antioxidant properties that give blue and purple hues. They support skin renewal and aid in lowering oxidative stress.
Alkaloids: they  are bioactive substances that have analgesic and antibacterial qualities. They may lessen discomfort at the wound site and aid in the prevention of infection.

Tannins:they are polyphenolic substances that have antibacterial and astringent properties. They lessen bleeding, aid with wound contraction, and guard against infections[16].

2] Plant profile of annona squamosa

The Annonaceae family includes the little tropical fruit-bearing tree Annona squamosa. It is extensively grown in India and other warm climates and is also referred to as sitaphal or custard apple. The plant is prized for both its therapeutic qualities and its sweet, creamy fruit. Traditional medicine makes use of the plant's leaves, bark, seeds, and roots, among other elements. Its medicinal properties are attributed to a variety of bioactive substances, including tannins, alkaloids, and flavonoids. The antibacterial, anti-inflammatory, and antioxidant properties of Annona squamosaare well-known. It is extensively researched for its potential in wound healing and other medicinal uses because of these characteristics [17,18 ]

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Taxonomical classification:

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Morphological characteristics:

Annona squamosais a tiny, semi-deciduous tree or shrub with an uneven, spreading crown

that can reach heights of 3 to 8 meters. The bark is light brown and the stem is woody. The simple, alternating, thin, oblong-lanceolate leaves have a smooth surface, a distinct smell, and a length of around 5 to 15 cm.
The fragrant, greenish-yellow blooms have three outer and three inner petals and are typically carried individually or in tiny groups. The fruit is a distinctive round or heart-shaped structure with a scaly or segmented outer surface, green in colour when unripe and turning slightly yellowish on ripening. Inside, the pulp is soft, white, and sweet, containing numerous shiny black seeds[20,21].

Chemical Constituents

• Annonaceous Acetogenins: These are the most significant compounds found primarily in the seeds and leaves (e.g., Annonacin, Squamocin, and Bullatacin). They are potent mitochondrial inhibitors known for their significant cytotoxic (anti-cancer) and insecticidal properties.
• Alkaloids: The plant contains various isoquinoline alkaloids such as Anonaine, Roemerine, and Isocorydine. These contribute to the plant's antimicrobial and analgesic (pain-relieving) effects.
• Flavonoids & Phenolics: The leaves and fruit pulp are rich in Quercetin, Kaempferol, and Rutin. These polyphenols provide strong antioxidant and anti-inflammatory benefits, which are crucial for skin health and wound healing.
• Diterpenoids: Specific kaurane-type diterpenes (like kaurenoic acid) are present in the bark and leaves, offering potential cardiovascular and anti-inflammatory protection.
• Essential Oils: The leaves and fruit peel contain volatile oils such as $\alpha$-pinene, $\beta$-pinene, and Limonene, which give the plant its characteristic aromatic scent.
• Nutritional Components: The edible pulp is high in Vitamin C (Ascorbic Acid), dietary fiber, and minerals like Potassium and Magnesium.[22]

MATERIALS AND EQUIPMENTS:

1. Materials:

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2. Instruments:

EXPERIMENTAL METHODS

1. COLLECTION OF PLANTS

High-quality plant materials and pharmaceutical excipients must be gathered in order to manufacture a hydrogel including clitoria ternatea and annona squamosa extract for the treatment of wounds. For the final hydrogel formulation to be effective, safe, and stable, the choice and acquisition of these materials are essential.
Gathering of Plant Materials (annona squamosa leaves and Clitoria ternatea flowers)
To guarantee the maximum concentration of bioactive chemicals, plant materials will be gathered methodically[23].

clitoria ternatea flowers:

• Medicinal clitoria ternatea was collected from natural habitats, botanical gardens, or authorized herbal farms in the Kolhapur district.
• Preference was given to clitoria ternatea   species known for their antimicrobial, antioxidant, and skin-soothing properties.
• Flowers was hand-harvested early in the morning to retain maximum bioactive content.
• Collected flowers was washed, shade-dried, and stored properly to prevent degradation[11].

Annona squamosa leaves:

• Selected mature, fully developed green leaves. Avoid young buds (low active content) and yellow, senescent leaves (degraded metabolites).
•  Collected in the early morning (before the sun is fully up). This minimizes transpiration and degradation of heat-sensitive alkaloids and acetogenins.
• Picked only disease-free, clean, and intact leaves.
• Collected leaves was washed, shade-dried, and stored properly to prevent degradation[23].

PLANTS EXTRACTION:

The Maceration Extraction Process

1. Extract reparation
To maintain the phytochemical integrity of the active chemicals, the harvested clitoria ternatea blooms and annona squamosa leaves were dried in the shade. After drying, a mechanical grinder was used to finely grind the plant materials in order to enhance their surface area and boost extraction efficiency. For extraction, the powdered material was precisely weighed.

2. Solvent Selection

Choosing an appropriate solvent is crucial for efficient extraction of bioactive compounds.  Ethanol (70%) was used as they effectively extract flavonoids, alkaloids, terpenoids, and phenolic compounds—essential for wound healing treatment.  A solvent-to-sample ratio of 10:1 (v/w) was maintained to ensure optimal extraction.

3. Maceration Process

 The powdered plant materials were soaked in the selected solvent inside a closed glass container or maceration vessel.  The mixture was agitated occasionally to improve solvent penetration and diffusion of bioactive compounds. The maceration process was carried out at room temperature for 48-72 hours to ensure maximum extraction of phytochemicals.

4. Filtration and Solvent Recuperation
To get rid of solid residues, the extract was filtered using Whitman filter paper or a Buchner funnel following maceration. A concentrated extract was produced by utilizing a rotary evaporator to evaporate the solvent at low pressure.

5. Drying and Storage of Extracts

The obtained extracts were air-dried or freeze-dried to form a semi-solid or powdered form, ensuring long-term stability.  Extracts were stored in amber glass vials under controlled temperature conditions to prevent degradation of bioactive compounds.

This maceration-based extraction ensures high bioavailability of clitoria ternatea and annona squamosa  extracts, making them suitable for incorporation into the hydrogel formulation for wound healing treatment.[21]

Study of Preformulation

To guarantee the creation of a stable, efficient, and secure hydrogel dosage form, preformulation studies are crucial. During this phase of development, the drug compounds' physicochemical characteristics are described, and their interactions with different formulation components are evaluated.
Preformulation Study Objectives
• To ascertain a new medicinal substance's essential physicochemical characteristics.

• To determine whether the formulation's excipients are incompatible.[24]

EXPERIMENTAL DESIGN

Procedure for the Hydrogel Formulation

The formulation of an annona squamosa and clitoria ternatea Extract Hydrogel involves the preparation of a stable hydrogel base, the incorporation of bioactive plant extracts, and the adjustment of physicochemical properties to ensure optimal therapeutic efficacy

1) Preparation of the Hydrogel Base

A stable hydrogel requires a gelling agent to create the 3D network. Carbopol 934 and HPMC (Hydroxypropyl Methylcellulose) are the most common polymer choices for herbal topicals.

• Step A (Polymer Dispersion): Disperse Carbopol 934 (approx. 1–1.5% w/v) in distilled water. Allow it to hydrate overnight to ensure complete swelling and a lump-free consistency.
• Step B (Neutralization): Since Carbopol dispersions are acidic, add Triethanolamine (TEA) dropwise while stirring. This neutralizes the pH and triggers immediate gelation, transforming the liquid into a thick, transparent gel

2) Incorporation of Active Ingredients

• Active Phase: Dissolve the weighted extracts of both C. ternatea and A. squamosa (typically 0.5% to 5% each, depending on the study) in a small amount of Propylene Glycol. This acts as a humectant and aids in the uniform distribution of the herbal extracts .
• Mixing: Gradually add the extract- propylene glycol mixture to the gel base with continuous mechanical stirring to ensure homogeneity.
•  Preservation: Add Methylparaben or Propylparaben (0.1–0.2%) to prevent microbial growth.

3) PH Adjustment and Finalization

The pH of the hydrogel was measured using a digital pH meter and adjusted to a skin-compatible range (5.5–6.5) by the dropwise addition of triethanolamine under continuous stirring. The extract dispersion was incorporated into the hydrated Carbopol base and stirred until a smooth, uniform, lump-free hydrogel was obtained. [25,26,27].

FORMULATION TABLE

The hydrogel is developed to topically deliver bioactive compounds from clitoria ternatea extract (anti-inflammatory) and annona squamosa leaves extract (antimicrobial) using a biocompatible gel base that ensures stability, controlled release, and user acceptability. Formulating a hydrogel incorporating clitoria ternatea extract and annona squamosaextract involves both pharmaceutical science and cosmetic formulation theory. The goal was develop a stable, bioactive, and biocompatible hydrogel with effective release of active compounds from the clitoria ternatea and annona squamosa  extracts.

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PRELIMINARY PHYTOCHEMICAL INVESTIGATION

Following the extraction of bioactive chemicals from Annona squamosa leaves and Clitoria ternatea flowers, a preliminary phytochemical screening was carried out to determine the presence of important secondary metabolites that are responsible for their medicinal qualities. Numerous qualitative assessments are employed to identify various phytochemical classes.

A. Alkaloids Detection

  Mayer's Test: Mayer's reagent (potassium mercuric iodide) is applied to the filtrate.

The presence is indicated by the formation of a creamy white precipitate

. ● Wagner's Test: Wagner's reagent (iodine in potassium iodide) is applied to the filtrate. Alkaloids are confirmed by a reddish-brown precipitate.

B. Detection of Flavonoids

• Shinoda Test: Concentrated hydrochloric acid (HCl) and magnesium turnings are applied to the extract. Flavonoids are indicated by a magenta or scarlet red tint that develops after a few minutes.

• Alkaline Reagent Test: The extract was treated with 2 ml of 10% sodium hydroxide solution, yielding an intense yellow coloration. Upon the addition of dilute hydrochloric acid, the solution became colorless, confirming the presence of flavonoids.

C. Detection of Phenols and Tannins

• Ferric Chloride Test: A 5% ferric chloride (FeCl3) solution is applied to the extract. Phenols and tannins are indicated by a bluish-black or brownish-green hue.

• Gelatin Test: A white precipitate forms when a 1% gelatin solution containing sodium chloride (NaCl) is added to the extract.

D. Detection of Saponins

• Froth Test: The extract is violently shaken after being diluted with distilled water. Saponins are indicated by the development of foam (froth) that lasts for at least fifteen minutes

E. Glycoside Detection

• Keller-Kiliani Test: Particularly for glycosides in the heart. FeCl3, concentrated H2SO4, and glacial acetic acid are used to treat the extract. A deoxysugar feature of cardenolides is shown by a brown ring at the interface [21]

EVALUATION OF THE HYDROGEL

 1) Organoleptic Evaluation:

 The gel is visually inspected for color (typically deep blue to greenish-blue depending on the extract ratio), odor, and clarity .

2) PH Determination:

 To ensure skin compatibility, the pH should be between 5.5 and 7.0. A digital pH meter is used on a 1% aqueous solution with the gel.

3)Spreadability:

 This is determined using the parallel plate method. A known weight is placed on the gel sandwiched between two slides, and the diameter of the spread is measured. Higher spreadability (typically 20–25 g.cm/s) indicates easier application.

4) Stability Studies:

The formulation is kept in a stability chamber at 40°C/75% RH (accelerated conditions) for 1 months to check for phase separation, colour fading, or changes in viscosity.

5) Skin Irritancy Test:

Often performed as an open patch test on human volunteers or animal models to check for redness, edema, or itching. A score of “0” indicates a non-irritant, skin-friendly product.

6) Phytochemical Screenings:

Following the extraction of bioactive components from Annona quamosal and Clitoria ternatea flowers, a preliminary phytochemical screening was carried out to identify the presence of important secondary metabolites that are responsible for their medicinal qualities. These bioactive chemicals are identified by a variety of qualitative testing[28,29,30].

RESULT :

The formulated clitoria ternatea and annona squamosa hydrogel was subjected to various physicochemical, antioxidant, antimicrobial, and stability evaluations to determine its suitability as a natural wound healing formulation. The results obtained from different evaluation parameters are discussed in detail below.

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a) Organoleptic Evaluation

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b) PH Measurement:

The pH of the hydrogel is an essential factor in ensuring skin compatibility and preventing irritation. The measured pH of the hydrogel ranged between 5.5 and 6.5, which is close to the natural pH of human skin (4.75-5.75). This confirms that the formulation is mild, non-irritating, and safe , preventing excessive Dryness or disruption of the skin barrier.

PH of hydrogel: 6.0

C) Viscosity and Rheological Behaviour:

For topical formulations to be stable, applied, and retained, viscosity is essential. A Brookfield viscometer was used to determine the hydrogel's viscosity. For topical hydrogels, this is a perfect feature that guarantees appropriate consistency and skin adhesion.
Pseudo plastic (measured in cps at 25°C)

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d) Spreadability:

Spreadability determines how easily a formulation spreads over the skin surface, ensuring effective application and absorption. The spreadability of the hydrogel was 20.62g.cm/sec, indicating good application properties. The hydrogel was neither too thick nor too fluid, allowing smooth application without excessive friction. The results confirm that the hydrogel spreads evenly, ensuring effective skin coverage and absorption.

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E) Stability Study:.

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f) Skin Irritation Test :

The absence of adverse effects suggests that the hydrogel is well-tolerated and suitable for application on sensitive skin.

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g) Phytochemical Screenings:

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