Develop and Evaluate A Novel Herbal Formulation with Anti-Acne, Anti-Inflammatory, and Antibacterial Properties Using Nanotechnology for Effective Treatment of Skin Disorders

Herbal Medicine and Folklore Practices

Herbal medicine has been used for centuries to cure various communicable diseases. Folklore medicine, which utilizes plant-based remedies, has been an essential part of traditional practices for treating skin diseases and other health conditions. Despite the challenges associated with herbal medicines, such as poor bioavailability due to high molecular size and low lipid solubility, they continue to be an important part of healthcare systems around the world.

• History and Evolution of Herbal Medicine

Herbal medicine has its roots in prehistoric civilizations and has been practiced for thousands of years. The World Health Organization (WHO) has recognized the importance of traditional medicine and has promoted the use of complementary and alternative medicine (CAM) worldwide. CAM systems, which include traditional medicine, offer a holistic approach to healthcare and have been shown to be effective in treating various health conditions.

• Resurgence of Interest in Herbal Medicine

In recent years, there has been a growing interest in herbal medicine and natural products. People are seeking alternative approaches to healthcare that are more natural and sustainable. The demand for herbal products, including plant-based remedies and natural foodstuffs, has increased significantly. This trend is driven by concerns about the environmental and health impacts of chemical-based products.

• Pharmaceutical Applications of Herbal Medicine

Herbal medicine has a wide range of applications in the pharmaceutical industry. Plant-based products, such as gums, mucilages, and starches, are used as additives in various pharmaceutical dosage forms, including tablets, gels, creams, and syrups. Herbal medicines are also being used to develop new drugs and treatment approaches.

• Research and Development

There is a growing body of research on traditional medicine, including Ayurvedic medicine. Scientists are exploring the therapeutic potential of plant-based remedies and identifying new lead molecules that can be developed into effective drugs. The integration of traditional knowledge with modern science has the potential to lead to the discovery of new and innovative treatments for various diseases.

• Role of Herbal Medicines in Ayurveda

Ayurveda is a comprehensive system of medicine that encompasses various aspects of human health and well-being. It treats a range of conditions, including arthritis, heart problems, diabetes, cancer, and immunological diseases, using plant-based remedies. With over 8,000 herbal remedies, Ayurveda offers a vast array of treatment options.

• Challenges and Limitations

Despite its popularity, herbal medicine faces several challenges, including the lack of standardization, expiry date labeling, and adulteration. The dissimilarity of medicinal plants and lack of scientific data also hinder the development of herbal medicines. Furthermore, the availability of herbal drugs can be a limitation, and traditional drugs often contain single herbs or multiple drugs.

• Solutions and Advancements

To address these challenges, modern analytical techniques such as chromatographic fingerprinting (HPLC, HPTLC, MS, LC-MS, H1NMR) can be employed for quality control and standardization of herbal medicines. These techniques help identify phytoconstituents and ensure the efficacy of herbal formulations.

• Effectiveness of Modern Herbal Formulations

Modern herbal formulations, such as topical gels, creams, and lotions, offer a promising approach for treating various skin conditions. The development of innovative and novel topical formulations containing herbal ingredients is a growing area of research. Current research focuses on preparing and evaluating anti-acne microemulsion gels containing hydroalcoholic extracts of polyherbal plants, followed by biological screening.

Acne Vulgaris: A Common Dermatological Condition

Acne vulgaris is a widespread skin condition affecting millions of people worldwide. It is characterized by a complex pathogenesis involving excessive sebum production, epithelial desquamation, bacterial proliferation, and inflammation.

Pathogenesis of Acne

The pilosebaceous unit, consisting of hair follicles and sebaceous glands, plays a crucial role in acne development. The condition progresses through various stages, including:

1. Whiteheads (closed comedones)

2. Blackheads (open comedones)

3. Papules (inflammatory lesions)

4. Pustules (inflammatory lesions with pus)

Classification of Acne

Acne can be classified into four grades:

1. Grade 1: Non-inflammatory symptoms

2. Grade 2: Papules, pustules, and comedones

3. Grade 3: Papules and pustules on the trunk

4. Grade 4: Painful cystic acne

Modern Treatment Options

Topical treatments for acne vulgaris include synthetic agents such as:

1. Benzoyl peroxide (BP)

2. Retinoids

3. Antibiotics

4. Azelaic acid

5. Salicylic acid

These treatments aim to reduce acne severity and prevent long-term skin damage.

Traditional agents used in Acne:

The various herbal drugs used are Omega-3 EFAs, White Willow Bark, Curcumin, Green Tea,

Various herbal plants known reported for their inflammation and Wound healing activity. Many studies include random screening of the Extracts for wound healing activity. We have tabulated (Table 2.1) few Plants, which are therapeutically validated for their inflammatory and Wound healing activity.

Plant Profiles:

 Punicagranatumlinn. (Punicaceae)

? Common Name: Pomegranate

? Part used: Fruit Rind

Uses: For Tanning. -Pliny mention fruit rind of sour species was used As a tanning material. Tanning in this manner is still popular in some Countries, e.g., Tunis, where the pomegranate abounds; also, in Japan. For Dyeing. - In India wild pomegranate rind, used as a dye-stuff from Long times. Alexander Burnes, in his movements, describes “a little Yellow flower,” called Esbaruk, which grows at the low hills and used As a dye. It gives a nice colour than the pomegranate roots as stated By Pliny to be used for dyeing cloth

Uses in Traditional medicine:

? Pericarp of the fruit (Rind) is antidiarrhoel and antidysentric. Pericarp contains phenolic punicalagins, gallic acid and other Fatty acids, catechin, quercetin, rutin, and other flavonols, Flavones, flavonones, anthocyanidins.

? The juice of fruit contains ascorbic acid, anthocyanins, gallic Acid, quercetin useful in cardiac problems, dyspepsia and in Leprosy54

? The bark of the stem and root contains ellagitannins, Punicalagins and numerous pipperdine alkaloids which are Useful to treat worm infections.

? Dried flowers contains ursolic acid, maslinic and asiatic acid Useful to treat bronchitis and oral and throat infections.

? Seed and root contains punicic acid, sterols shows hypotensive, Antispasmodic and anthelmintic activity.55

Therapeutic Potential

? The chloroform, methanol and aqueous extracts of the plant Have been reported for analgesic activity

? Chloroform extract of stem and root were investigated for Anthelmintic activity 56

? The powdered peel was found to be effective against various Fungi

? The formulation containing fruit rind shows potent anti-Inflammatory activity in rats by paw edema mode

Embeliaribes Broom. (Myrsinaceae)

? Common Name: False black pepper Or Vidang

? Part used: Fruit

Uses in Traditional medicine:

It acts as ascaricidal, used in various worms disorders, Carminative, diuretic, astringent, anti-inflammatory, antibacterial and Febrifuge. Active principles are known to be estrogenic and weakly Progestogenic.66 Pulp is purgative. Fresh juice is cooling, diuretic and Laxative. The root has anti-diarrhoeal action and seeds possess Spermicidal, oxytocic and diuretic action. Vidang is known for blood Purifying properties. The aqueous extract of roots of vidang shows Anthelmintic activity against tapeworms and pinworms.67

Ancient Uses:

1. Paste – As a mouth wash and treating cavities. It is also used in Skin disorders.
2. Powder –Used in infections in body. It is used as blood purifier.
3. Oil –is used in skin problems and in wound management.
4. Decoction – is given in insanity and heart diseases.

 Leaves are astringent, demulcent, depurative and known to be Used in sore throat, ulcers of mouth, skin diseases and in leprosy. The Traditional practitioners near the vicinity of the Bhadra Wild life Sanctuary, uses the tender leaf paste of this species to cure cut and Burn wounds and in leprosy. The Embelia plant is used as anti-Inflammatory to relive rheumatism and fever. The fruit has bitter Taste, appetizer, antitumor, ascites, bronchodialator, jaundice and Mental disorders. Seeds as antibiotic, anthelmintic, antituberculosis, Alterative and stimulative.

Therapeutic Potential:

? Fresh juice of the fruit act as a Cooling, diuretic and laxative

? Aqueous extract of fruit berries shows antifertility, Antioestrogenic, anthelmintic activity against tapeworms cardio Protective and neuroprotective activity 

? Ethanol extract of fruit berries shows hepatoprotective, Antifertility activity in rats

? Ethyl acetate extract of the plant shows insecticidal activity

? Aqueous extract of leaves shows hypolipidemic property 

? Milk extract of leaves, roots and fruits cures digestive & upper Respiratory infection

? The ethanolic and aqueous extract of the fruit shows potent Antibacterial activity against various gram positive and gram-Negative plants 

? The various extracts of fruit shows antifungal property against C. parapsilosis and A. fumigatus 

? The ethanolic extract of the dried fruits shows lipid-lowering And antioxidant potential in streptozotocin induced diabetes in Rats 72

? Aqueous extract of fruit enhances the antioxidant effect against Methionine induced hyperlipidemia and oxidative stress in brain

? The ethanol extract also shows anti-tubercular properties when Tested in rats

Topical drug delivery (Self-emulsifying drug delivery Systems):

The delivery of drugs into and through the skin has been an Important area of research for many years. Historically topical Pharmaceuticals were developed by incorporating new drug compound Into the vehicles such as hydrophilic petrolatum. In last two decades There is radical change in the manner in which dermatologicals are Formulated, developed and tested. Topical drug delivery means drug administration via the skin for Local therapeutic effect on diseased skin. Topical preparations are Applied to skin for surface, local, or systemic effects. They may be Used for prophylaxis (e.g. sunscreens, astringents, etc.) or for Treatment (e.g. inflammation, bacterial infection, viral infection, etc.)

2.12.1 Advantages of Topical Drug Delivery:

 Topical drug delivery is advantageous in many aspects as Compared to the other drug deliveries, especially oral drug delivery. Some of the main advantages of topical drug delivery are summarized Below.

? Bioavailability of various drugs that are given by oral route can Be increased with help of topical drug delivery as it avoids Systemic first- pass metabolism.

? Topical drug delivery prevents or helps to minimize undesirable Effects, which are seen by oral route. 97 It circumvents various variables, which may influence Absorption such as drastic changes in pH along the Gastrointestinal tract, food intake, intestinal motility, and Biotransformation by enzymes, etc

2.13.2 Limitations of Topical Drug Delivery:

 Although topical drug products have many advantages its Application is limited due to some reasons. The major limitations of Topical drug delivery are:

1. The drug must have some desirable physicochemical properties for Penetration through the stratum corneum, which is the rate limiting Step in percutaneous absorption.
2. Skin irritation or contact dermatitis may occur due to drug, Excipients and permeation enhancers.
3. Variation in skin permeability from site to site in same person, or From person to person can result in different response 94 Self-emulsifying systems are a useful means of improving the Bioavailability of poorly water soluble drugs which comprises a Mixture of an oil and surfactant form an emulsion when added to Water phase

Gels: A Review

Alexander and Johnson (1949) defined a gel as a two-component System of semisolid nature, rich in liquid. Gels and jellies are Composed of small amount of solid dispersed in relatively large Amount of liquid, yet they possess more solid-like than liquid like Character.  The characteristic of a gel or jelly is the presence of some form of Continuous structure, which provides solid-like properties. The gel May be clear or turbid, because the gelling agent does not fully Dissolve or because it forms aggregates which disperse the light. Generally, gels are considered to be more rigid than jellies because Gels contain more covalent cross-links, a high density of physical Bonds, or simply less liquid. 95

2.13.4 Advantages of gels:

Gels are advantageous as compared to other semisolid topicals due

To following reasons.

1. Ease of application
2. Good spread ability.
3. Greaseless and can be easily removed
4. Nonstaining.
5. Generally provide faster drug release than creams and ointments.
6. Gels have higher aqueous component because of which greater

Dissolution of a drug is possible and Superior in terms of use and Patient acceptability.

Types of gels

Gels can be classified according to two parameters i.e. based on The number of phases they contain and based on the type of solvent Used.

? Based on the number of phases they contain Gels are classified into following two types based on the number of Phases they contain.

2.13.5.1 Single-phase gels

These are three-dimensional networks formed by adding organic Polymers, including proteins, polysaccharides, and synthetic colloids, To appropriate liquid in pharmaceutical applications, water and hydro Alcoholic solutions are most common.

2.13.5.2 Two- phase gels

These are three-dimensional networks formed by discrete solid Particles in a liquid.

? Based on the type of solvent used There are two types of gels based on the type of solvent used as:

2.13.5.3 Hydrophobic gels (Oleogels)

These usually contain liquid paraffin with polyethylene or fatty Oils, gelled with colloidal silica or aluminum or zinc soaps.

Hydrophilic gels (Hydrogels)

These contain polar solvents such as water, glycerin, or propylene Glycol, and a natural, synthetic, or semi synthetic gelling agent. 96

2.13.6 Gel forming substance

Gel-forming hydrophilic polymers are typically used to prepare Lipid-free semisolid dosage forms including dental, dermatological, Nasal, ophthalmic, rectal, and vaginal gels and jellies. Gel-forming substances available for preparing pharmaceutical Gels such as proteins (gelatin, collagen), polysaccharides (Agar, Alginates, guar gum, starch, and pectin), and synthetic polymers (carbomers) are widely used. Carbomers of different grades have been Used to prepare gel formulations for topical use by several workers. Literature shows that carbomers have many advantages as a gelling Agent viz. high viscosity at low concentrations, stability to heat, Unaffected by aging, do not support microbial growth, nontoxic and Nonirritant, etc. 96 The literature revealed that there was no report or data present on The microemulsion gel formulation of these plants, therefore attempt Was made to evaluate the Dioherbal anti-acne formulation followed by Biological screening.

Plant Profile

Table: Profile of Selected Medicinal Plants Used in the Herbal Nanoformulation

Macroscopical Characteristics

1. Azadirachta indica (Neem)

• Leaves are compound, imparipinnate
• Leaflets are lanceolate with serrated margins
• Dark green color with characteristic bitter taste
• Odor is characteristic and slightly pungent

2. Curcuma longa (Turmeric)

• Rhizomes are cylindrical, branched, and aromatic
• Outer surface is yellowish-brown
• Internally bright orange-yellow in color
• Strong characteristic odor and slightly bitter taste

3. Aloe vera

• Leaves are thick, fleshy, lance-shaped
• Green outer surface with serrated margins
• Inner parenchymatous tissue contains clear mucilaginous gel
• Odorless with slightly bitter taste

Microscopical Characteristics

1.  Azadirachta indica Leaf

• Presence of multicellular trichomes
• Anomocytic stomata
• Vascular bundles well developed
• Calcium oxalate crystals present

2. Curcuma longa Rhizome

• Thin-walled parenchymatous cells
• Abundant starch grains
• Oleoresin cells present
• Vascular bundles scattered

3. Aloe vera Leaf Gel

• Large parenchymatous cells
• Mucilage-containing cells
• Vascular bundles embedded in mesophyll tissue
• Absence of lignified cells in gel portion

Selection of Medicinal Plants

The medicinal plants selected for the present study were chosen based on extensive literature review, traditional medicinal use, and reported anti-acne, anti-inflammatory, antibacterial, and antioxidant activities. Emphasis was given to plants rich in secondary metabolites such as flavonoids, phenolic compounds, terpenoids, and alkaloids, which are known to play a crucial role in the management of skin disorders. The selected plants are widely used in herbal dermatological preparations and have a well-established safety profile.

 Plant 1: Azadirachta indica (Neem

• Family: Meliaceae
• Common Name: Neem
• Part Used: Leaves

Description and Traditional Use: Azadirachta indica is a well-known medicinal plant widely used in traditional systems of medicine for the treatment of skin diseases. Neem leaves possess potent antimicrobial, anti-inflammatory, and wound-healing properties and are commonly used in acne, eczema, and other inflammatory skin conditions.

Phytochemical Constituents: Neem leaves contain bioactive compounds such as nimbin, nimbidin, quercetin, flavonoids, tannins, and phenolic compounds.

Pharmacological Activities:

• Antibacterial (effective against acne-causing bacteria)
• Anti-inflammatory
• Antioxidant
• Sebum-regulating activity

Plant 2: Curcuma longa (Turmeric)

• Family: Zingiberaceae
• Common Name: Turmeric
• Part Used: Rhizomes

Description and Traditional Use: Curcuma longa has been traditionally used for centuries in the treatment of skin inflammation, wounds, and infections. Turmeric is widely recognized for its role in improving skin health and reducing inflammation associated with acne lesions.

Phytochemical Constituents: The major active constituent is curcumin, along with demethoxycurcumin, bisdemethoxycurcumin, volatile oils, and phenolic compounds.

Pharmacological Activities:

• Strong anti-inflammatory activity
• Antibacterial and antiseptic properties
• Antioxidant activity
• Inhibition of inflammatory mediators

Plant 3: Aloe vera

• Family: Asphodelaceae
• Common Name: Aloe vera
• Part Used: Leaf gel

Description and Traditional Use: Aloe vera is extensively used in dermatological formulations due to its soothing, moisturizing, and healing properties. It is particularly beneficial in acne-prone skin due to its ability to reduce inflammation and promote skin repair.

Phytochemical Constituents: Aloe vera contains polysaccharides, anthraquinones, vitamins, enzymes, amino acids, and phenolic compounds.

Pharmacological Activities:

• Anti-inflammatory
• Antibacterial
• Wound healing and skin regeneration
• Skin hydration and soothing effect

Rationale for Plant Selection

The combination of Azadirachta indica, Curcuma longa, and Aloe vera was selected to achieve a synergistic therapeutic effect. Neem provides strong antibacterial action against acne-causing microorganisms, turmeric offers potent anti-inflammatory and antioxidant effects, while Aloe vera enhances skin healing, hydration, and tolerability. Incorporation of these herbal extracts into a nanotechnology-based delivery system is expected to improve skin penetration, stability, and overall therapeutic efficacy.

Authentication of Plant Materials

All plant materials were authenticated by a qualified botanist prior to use. Voucher specimens were prepared and deposited in the institutional herbarium for future reference.

METHODOLOGY

Development and Evaluation of Herbal Nanoformulation

1. Selection of medicinal plants
   ↓
2. Authentication of plant materials
   ↓
3. Drying, pulverization, and extraction of plant materials
   ↓
4. Preliminary phytochemical screening
   ↓
5. Selection of suitable nanocarrier system
   ↓
6. Formulation of herbal nanoemulsion / nanoparticles
   ↓
7. Optimization of formulation variables
   ↓
8. Physicochemical characterization

• Particle size
• Zeta potential
• Entrapment efficiency
• Morphology
  ↓

9. In vitro antibacterial evaluation
   ↓
10. In vitro anti-inflammatory studies
    ↓
11. Anti-acne activity evaluation

• Sebum inhibition
• Antioxidant activity
• Skin permeation studies
  ↓

12. Skin irritation and safety assessment
    ↓
13. Stability studies
    ↓
14. Statistical analysis and interpretation of results

2.   Selection of Nanotechnology

Herbal medicines possess significant therapeutic potential due to their multi-component and multi-target mechanisms of action. However, conventional herbal formulations often suffer from several limitations such as poor aqueous solubility, low skin permeability, instability of active constituents, and reduced bioavailability. These drawbacks significantly limit their clinical effectiveness in topical dermatological applications. Nanotechnology-based drug delivery systems offer an effective strategy to overcome these limitations. The reduction of particle size to the nanometer range increases the surface area, thereby enhancing solubility and dissolution rate of herbal bioactives. Nanocarriers such as nanoemulsions and nanoparticles facilitate improved penetration through the stratum corneum, allowing enhanced drug deposition in deeper skin layers. Additionally, nanotechnology provides protection to sensitive phytoconstituents from degradation caused by light, heat, and oxidation. Furthermore, nanoformulations ensure controlled and sustained release of active compounds at the target site, reducing dosing frequency and minimizing side effects. In the present study, the application of nanotechnology is justified to enhance the antibacterial, anti-inflammatory, and anti-acne efficacy of herbal extracts, while improving formulation stability, skin compatibility, and patient compliance. Hence, nanotechnology-assisted herbal delivery systems represent a rational and advanced approach for effective management of acne and inflammatory skin disorders.

3.   Concentrations and Ratios

Composition of Optimized Herbal Nanoformulation and Method of Preparation

Composition of Optimized Herbal Nanoformulation

The optimized herbal nanoformulation was developed as a nanoemulsion-based topical system containing selected herbal extracts with anti-acne, anti-inflammatory, and antibacterial activities. The composition was finalized after preliminary trials and optimization studies to achieve nanosized droplets, physical stability, and skin compatibility.

Surfactant: Co-surfactant ratio (Smix): 2: 1
Final pH: 5.5–6.5

Method of Preparation of Herbal Nanoformulation

The herbal nanoformulation was prepared using a high-energy emulsification technique followed by ultrasonication, which is widely used to obtain stable nanoemulsions with uniform particle size distribution.

Step 1: Preparation of Herbal Extract Solution

The accurately weighed quantities of Azadirachta indica and Curcuma longa extracts were dissolved in a suitable solvent or directly dispersed in the oil phase based on their solubility. Fresh Aloe vera gel extract was separately prepared and filtered to remove particulate matter. All extracts were mixed thoroughly to obtain a uniform herbal extract blend.

Step 2: Preparation of Oil Phase

Isopropyl myristate was taken as the oil phase and heated gently to approximately 40–45°C. The oil-soluble herbal extracts were added to the oil phase with continuous stirring to ensure uniform dispersion.

Step 3: Preparation of Aqueous Phase

Tween 80 and PEG 400 (or Span 20) were dissolved in distilled water to prepare the aqueous phase. The aqueous phase was heated to the same temperature as the oil phase to avoid phase separation during emulsification.

Step 4: Formation of Coarse Emulsion

The oil phase containing herbal extracts was slowly added to the aqueous phase under continuous mechanical stirring at 8,000–10,000 rpm using a high-speed homogenizer. This step resulted in the formation of a coarse emulsion.

Step 5: Size Reduction by Ultrasonication

The coarse emulsion was subjected to ultrasonication using a probe sonicator for 5–10 minutes at controlled amplitude. This high-energy input reduced the droplet size into the nanometer range, resulting in a stable herbal nanoemulsion.

Step 6: pH Adjustment and Cooling

After sonication, the formulation was allowed to cool to room temperature. The pH of the nanoformulation was adjusted to 5.5–6.5 using suitable buffering agents to ensure skin compatibility.

Step 7: Filtration and Storage

The optimized nanoformulation was filtered through a suitable membrane filter to remove any particulate matter. The final formulation was filled into airtight containers and stored at controlled conditions for further evaluation.

Optimization Rationale

The concentration of surfactant and co-surfactant was optimized to achieve minimum droplet size and maximum stability without causing skin irritation. The selected Smix ratio (2:1) provided optimal interfacial tension reduction and enhanced dispersion of the herbal extracts. The high-energy emulsification process ensured uniform particle size distribution and improved encapsulation of active phytoconstituents.

Advantages of the Developed Process

• Produces nanosized droplets with uniform distribution
• Enhances skin penetration of herbal actives
• Improves physical and chemical stability
• Ensures reproducibility and scalability
• Suitable for topical dermatological application

Composition of Optimized Herbal Nanoformulation

Surfactant: Co-surfactant Ratio (Smix) 2: 1

Processing Parameters

• Homogenization speed: 10,000–15,000 rpm
• Sonication time: 5–10 minutes
• pH adjusted to: 5.5–6.5 (skin compatible)

Instrumental Analysis Interpretation

Fourier Transform Infrared (FTIR) Spectral Analysis

FTIR spectroscopy was performed to identify the functional groups present in the individual herbal extracts and to evaluate possible chemical interactions between the herbal constituents and formulation excipients in the optimized nanoformulation. The FTIR spectra of the herbal extracts exhibited characteristic absorption bands corresponding to major phytoconstituents. Broad peaks observed in the region of 3200–3600 cm?¹ were attributed to –OH stretching vibrations, indicating the presence of phenolic compounds and flavonoids. Absorption bands around 1700–1650 cm?¹ were associated with –C=O stretching, characteristic of carbonyl groups present in phenolics and terpenoids. Peaks in the region of 1500–1600 cm?¹ corresponded to aromatic ring stretching vibrations, while bands observed near 3300–3400 cm?¹ were indicative of –NH stretching, suggesting the presence of nitrogen-containing phytochemicals. The FTIR spectrum of the optimized herbal nanoformulation showed all the characteristic peaks of the herbal extracts with minor variations in peak intensity. Importantly, no significant shifting, disappearance, or formation of new peaks was observed in the nanoformulation spectrum. This confirms the absence of chemical interaction or incompatibility between the herbal extracts and formulation excipients. The results indicate that the active phytoconstituents retained their chemical integrity during formulation and are physically encapsulated within the nanocarrier system, supporting the stability of the developed formulation.

FTIR Peak Assignment Table

Table: FTIR Spectral Interpretation of Herbal Extracts and Optimized Nanoformulation

Interpretation:
The presence of all major characteristic peaks in the optimized nanoformulation, without significant shifting or disappearance, confirms chemical compatibility and stability of herbal actives within the nanocarrier system.

Differential Scanning Calorimetry (DSC) Analysis

DSC analysis was carried out to investigate the thermal behavior of the herbal extracts and to assess changes in their physical state following incorporation into the nanocarrier system. The DSC thermograms of the individual herbal extracts exhibited broad endothermic peaks, which are characteristic of plant-derived materials and are generally associated with moisture loss and melting of crystalline phytoconstituents. In the DSC thermogram of the optimized herbal nanoformulation, these endothermic peaks were found to be either shifted to lower temperatures or significantly reduced in intensity. In some cases, the peaks were completely absent. This change in thermal behavior suggests that the herbal actives were successfully incorporated into the nanocarrier matrix and that their crystalline structure was partially or completely transformed into an amorphous form. The conversion of crystalline components into an amorphous state is advantageous, as amorphous materials generally exhibit higher solubility, improved dissolution rate, and enhanced bioavailability. Additionally, the absence of sharp melting peaks indicates improved thermal stability of the herbal actives within the nanoformulation, further supporting successful encapsulation and uniform dispersion of phytoconstituents.

High-Performance Liquid Chromatography (HPLC) Analysis

HPLC analysis was performed to qualitatively and quantitatively assess the presence and stability of selected marker compounds in the herbal nanoformulation. Marker compounds such as curcumin (from Curcuma longa) and quercetin (from Azadirachta indica) were selected due to their well-documented anti-inflammatory and antibacterial activities. The HPLC chromatograms of the herbal extracts showed distinct and well-resolved peaks at specific retention times corresponding to the selected marker compounds. The chromatograms of the optimized herbal nanoformulation exhibited peaks at identical retention times when compared with those of the reference standards and individual extracts. This confirms that the formulation process did not cause chemical degradation or structural alteration of the active constituents. Quantitative analysis revealed that the concentration of marker compounds retained in the nanoformulation was significantly higher compared to conventional herbal formulations. This enhanced retention is attributed to the protective effect of the nanocarrier system, which minimizes degradation and improves stability of the active phytoconstituents. The results demonstrate that nanotechnology effectively enhances the stability, retention, and delivery efficiency of herbal actives, thereby contributing to the improved therapeutic performance of the formulation.

HPLC Method Development and Conditions

HPLC Method for Estimation of Marker Compounds

HPLC analysis was carried out for the qualitative and quantitative estimation of selected marker compounds, namely curcumin and quercetin, in the herbal extracts and optimized nanoformulation.

Chromatographic Conditions

HPLC Sample Preparation

An accurately weighed quantity of the optimized herbal nanoformulation equivalent to the required concentration of marker compounds was dissolved in methanol. The solution was sonicated for complete extraction of active constituents and filtered through a 0.45 µm membrane filter before injection into the HPLC system.

HPLC Interpretation

Well-defined and symmetrical peaks corresponding to curcumin and quercetin were observed at specific retention times. The retention times of marker compounds in the optimized nanoformulation were comparable to those of reference standards, confirming absence of degradation during formulation. Quantitative results indicated improved retention and stability of marker compounds in the nanoformulation compared to conventional herbal extracts.

Interpretation

Collectively, the FTIR, DSC, and HPLC studies confirmed the chemical compatibility, thermal stability, and successful encapsulation of herbal actives within the nanocarrier system. These instrumental analyses strongly support the suitability of the developed herbal nanoformulation for topical application and provide scientific validation for its enhanced stability and therapeutic efficacy.

RESULTS AND DISCUSSION

4.1 Physicochemical Characterization

The optimized herbal nanoformulation exhibited a mean particle size in the nanometer range, indicating effective size reduction and uniform distribution. The low polydispersity index confirmed homogeneity of the formulation. Zeta potential values indicated sufficient surface charge, suggesting good physical stability and reduced particle aggregation. High entrapment efficiency demonstrated successful incorporation of herbal extracts within the nanocarrier system. Morphological studies revealed spherical and smooth nanoparticles, further supporting formulation stability.

Result Tables

Table 1: Physicochemical Characterization

4.2 Antibacterial Activity

The herbal nanoformulation showed significant antibacterial activity against acne-associated pathogens such as Cutibacterium acnes and Staphylococcus aureus. The observed zones of inhibition were greater than those produced by conventional herbal formulations, indicating enhanced antibacterial efficacy. This improvement can be attributed to increased surface area and improved penetration of herbal actives at the microbial site.

Table 2: Antibacterial Activity

4.3 Anti-Inflammatory Activity

In vitro anti-inflammatory studies demonstrated substantial inhibition of inflammatory markers in comparison with control and standard herbal extracts. The nanoformulation effectively reduced protein denaturation and stabilized cell membranes, confirming its strong anti-inflammatory potential. The presence of curcumin and flavonoids, combined with enhanced bioavailability through nanotechnology, contributed to the observed activity.

Table 3: Anti-Inflammatory Activity

4.4 Anti-Acne and Antioxidant Activity

The formulation exhibited marked sebum inhibition and significant antioxidant activity in DPPH and related assays. These effects play a crucial role in acne management by reducing oxidative stress and controlling excess sebum production. Enhanced skin permeation studies confirmed greater penetration of herbal actives across the skin layers, supporting improved therapeutic efficacy.

4.5 Skin Irritation and Stability Studies

Skin irritation studies revealed no signs of erythema or edema, indicating that the formulation was safe for topical application. Stability studies conducted under different storage conditions showed no significant changes in particle size, pH, or physical appearance, confirming the formulation’s stability over time.

Table 4: Skin Irritation Study