Formulation and Evaluation of a Transdermal Reservoir Patch Containing Tridax procumbens Extract

Historically, Tridax procumbens has played a pivotal role in traditional medicine systems worldwide. Indigenous communities have utilized it for generations, harnessing its therapeutic potential to alleviate various health issues. Inflammation, in particular, has been a primary target of Tridax procumbens-based remedies due to its well-documented anti-inflammatory properties. Transdermal patches have emerged as a modern drug delivery system, offering numerous advantages such as sustained release, improved patient compliance, and non-invasive administration. This innovative approach holds significant promise for delivering the bioactive constituents of Tridax procumbens, particularly for addressing inflammatory conditions effectively. Against this backdrop, this study endeavours to formulate and evaluate transdermal patches containing Tridax procumbens extract specifically targeting anti-inflammatory activity. By harnessing the traditional knowledge surrounding Tridax procumbens and leveraging modern pharmaceutical technology, this research aims to develop a novel.

PLANT PROFILE :

Fig : Tridax procumbens

Introduction :

• Plant name :-  Tridax procumbens
• Common name:- Coat Button or tridax daisy
• Family:- Asteraceae
• Chemical constituents:- Flavonoids, alkaloids, carotenoids, tannins and saponins
• Pharmacological actitvity:- anti-microbial, anti-inflammatory and antioxidant activities

Taxonomical Classification:

Kingdom: Plantae

Subkingdom: Viridiplantae

Superdivision : Spermatophytina

Class: Magnoliopsida

Superorder: Asteranae

Order: Asterales

Family: Asteraceae

Genus: Tridax L-tridax

Species: Tridax procumbens L.

Chemical Constituents:

The phytochemical study revealed presence of flavonoids, carotenoids, alkaloids, tannins and saponins. The adjacent profile shows that the plant is rich in sodium, potassium and calcium [8]. Leaf of Tridax procumbens mainly contains proteins, fiber, carbohydrates, and calcium oxide. Whereas the fumaric acid and tannin has also been reported in the plant.[9] Oleanolic acid was obtained in good amounts from Tridax and found to be a potential antidiabetic agent when tested against a glucosidase[10]. A number of chemical constituent were reported from the plant that are alkaloids, flavonoids, carotenoids, fumaric acid, lauric acid, tannins etc. The medicinal values of the plants depend on the presence of certain chemical substances (secondary metabolites) that are involved in production of different kinds of effects on human body. Some compounds are responsible to give plants the its Specific odors and others are responsible for imparting different colours to plants.[11]

Pharmacological activity:

1. Wound Healing

2. Hepatoprotective

3. Immunomodulatory

4. Antidiabetic Activity

5. Antimicrobial Activity

6.Anti-Cancerous Activity

7. Hypotensive

8. Repellency Activity

9. Anti-Urolithiatic Activity

10. Hypoglycemic and Antihyperglycemic Activity

11. Anthelmintic Activity

Inflammation:

Inflammation is a defensive response which causes the different physiological adaptations which limits the tissue damage and removes pathogenic insult. This type of mechanism involves a complex series of events which includes dilation of arterioles, venules and capillaries with increased vascular permeability, exudation of fluids which includes plasma proteins and the migration of leukocyte into the inflammatory area. In the inflammation, there is immediate infiltration of a specific site or lesion with PMN followed by monocytes and lymphocytes.

1) Acute inflammation: Acute inflammation is of short duration and its duration is from minutes to few days. The main characteristics are:

a) Exudation of fluids

b) Plasma protein (edema)

c) Emigration of leukocytes specially neutrophils

2) Chronic inflammation: Chronic inflammation is having longer duration than acute inflammation. It is associated histologically with the presence of lymphocytes, macrophages, proliferation of blood vessels, fibrosis, and tissue necrosis. It is the processes of active inflammation and tissue destruction occurs. It is followed by acute inflammation and it starts from the low grade, smoldering asymptomatic response. It may also arise due to the persistent infection by certain organisms such as tubercle bacilli or Treponema pallidum, prolonged exposure to highly toxic agents, either exogenous like silica or endogenous like plasma lipid component resulting into atherosclerosis, autoimmune like rheumatoid arthritis. Acute inflammation response is the initial response after the infection or trauma. It is non- specific in nature and is the first line of defence of body after the danger (9,10). In acute inflammation, there is increased level of copper, decreased level of zinc. There is occurrence of leukocytosis, thrombocytosis, negative nitrogen balance, increased BMR, increased lipogenesis and lipolysis. There is decrease in plasma protein level, increased C reactive protein level

Fig : Difference between acute and chronic inflammation

Mechanism of Inflammation:

1. Vasodilation and Increased Permeability:

In response to inflammatory mediators such as histamine and prostaglandins, blood vessels dilate (vasodilation) and become more permeable, allowing immune cells and fluid to move from the bloodstream into the tissues.

2. Leukocyte Recruitment:

White blood cells, particularly neutrophils and macrophages, are recruited to the site of inflammation to eliminate pathogens and cellular debris.

Chemotactic factors guide the migration of leukocytes to the inflamed tissue.

3. Phagocytosis:

Phagocytic cells, such as neutrophils and macrophages, engulf and destroy foreign invaders, dead cells, and debris through a process called phagocytosis.

4. Release of Inflammatory Mediators:

Various molecules, including cytokines, chemokines, prostaglandins, and leukotrienes, are released during inflammation.

These mediators regulate immune cell activation, inflammation intensity, and tissue repair processes.

Regulation of Inflammation:

1. Resolution Pathways:

Inflammation is tightly regulated by specialized pro-resolving mediators (SPMs), which promote the resolution of inflammation and tissue repair.

Lipoxins, resolvins, protectins, and maresins are examples of SPMs that help terminate the inflammatory response.

2. Anti-inflammatory Mechanisms:

Several mechanisms counteract inflammation to prevent excessive tissue damage.

Anti-inflammatory cytokines, interleukin-10 such as (IL-10) and transforming growth factor-beta (TGF-β), inhibit pro inflammatory pathways.

Endogenous inhibitors, such as glucocorticoids, regulate the activity of inflammatory mediators.

Transdermal drug delivery systems (TDDS):

Transdermal drug delivery system has been in existence for a long time. In the past, the most commonly applied systems were topically applied creams and ointments for dermatological disorders. The occurrence of systemic side-effects with some of these formulations is indicative of absorption through the skin. A number of drugs have been applied to the skin for systemic treatment. In a broad sense, the term transdermal delivery system includes all topically administered drug formulations intended to deliver the active ingredient into the general circulation. Transdermal therapeutic systems have been designed to provide controlled continuous delivery of drugs via the skin to the systemic circulation. Moreover, it overcomes various side effects like painful delivery of the drugs and the first pass metabolism of the drug occurred by other means of drug delivery systems. So, this transdermal drug delivery system has been a great field of interest in the recent time. Many drugs which can be injected directly into the blood stream via skin have been formulated.

Fig : How does Transdermal Patches Actually Deliver Drug To Our Body

TYPES OF TRANSDERMAL PATCH

Transdermal patches were categorized into four main types :

1. Matrix type.

2. Reservoir type

3. Membrane matrix hybrid.

4. Micro reservoir type.

Matrix type

Matrix type of transdermal patch was designed in the year 1990s. Here, the patch is very slim and less visible when sticked on the skin. In this type, the film controls release of medication from the patch. In this method, the drug and polymer dissolved in solvent (soluble) or insoluble drug homogeneously dispersed in hydrophilic or lipophilic polymer. To the above, add required quantity of plasticizer and permeation enhancer. Medicated polymer formed is then molded into the ring with defined surface area and controlled thickness over the mercury on horizontal surface followed by solvent evaporation at an elevated temperature. Thus, film formed is separated from the ring and mounted onto the occlusive base plate in a compartment fabricated from a drug impermeable backing. Then, an adhesive polymer is then spread along the circumference of the film. The adhesive layer and backing are integrated into one layer. A few examples of matrix type transdermal patches are Nitro-glycerin transdermal patch-Nitro Dur®.

Reservoir type

It is different from single layer drug in adhesive and multilayer drug in adhesive systems. It is having separate drug layer. In this, the drug reservoir layer is a liquid compartment which is consisting of drug solution or the suspension where the drug particle is suspended in silicon fluid gives paste such as suspension/gel/clear solution in ethanol, which is separated by the adhesive layer. The rate controlling membrane is prepared by solvent evaporation or compression method. This patch is also consisting of backing layer, as shown. Advantage of this type is it follows true zero order release to achieve a constant serum drug level, for example: Duragesic®, Estraderm®, and Androderm®.

Mechanism of Action:

Structure of skin: The skin can be considered to have four distinct layers of tissues including non-viable epidermis (stratum corneum), viable epidermis, viable dermis and hypodermis (subcutaneous connective tissue). The epidermis is the relatively thin, tough, outer layer of the skin. The epidermis has keratinocytes. They originate from cells in the deepest layer of the epidermis called the basal layer. New keratinocytes slowly migrate up toward the surface of the epidermis. Stratum corneum (Non-viable epidermis) is the outermost portion of the epidermis, relatively waterproof and, when undamaged, prevents most bacteria, viruses, and other foreign substances from entering the body. The epidermis also protects the internal organs, muscles, nerves, and blood vessels against trauma. The outer keratin layer of the epidermis (stratum corneum) is much thicker. Viable Epidermis layer of the skin resides between the stratum corneum and the dermis and has a thickness ranging from 50-100 µm. The structure of the cells in the viable epidermis is physiochemically similar to other living tissues. Cells are held together by tonofibrils. The water content is about 90%. The dermis, the skin's next layer, is a thick layer of fibrous and elastic tissue (made mostly of collagen, elastin and fibrillin) that gives the skin its flexibility and strength. The dermis contains nerve endings, sweat glands and oil glands, hair follicles, and blood vessels. The subcutaneous tissue also known as hypodermis is not actually accepted as a true part of the structured connective tissue. It is composed of loose textured, white, fibrous connective tissue containing blood and lymph vessels. Most investigators consider the drug permeating through the skin enter the circulatory system before reaching the hypodermis where the fatty tissue serve as a depot of the drug (Barry 1983; Alexander et al., 2012).

Pathways of Skin Permeation: Drug molecules permeate through skin surface by the different potential pathways including through the sweat ducts, through the hair follicles and sebaceous glands or directly across the stratum corneum (Flaynn et al., 1985; Kasting et al., 1992; Potts and Guy 1992). Since the last few years there is a point of debate among scientists for the relative importance of the shunt or appendageal route of transport across the stratum corneum and is further complicated by the lack of a suitable experimental model to permit separation of the these pathways (Scheuplein et al., 1969; Scheuplein and Bank 1971). A recent review by Menon (2002) provides a valuable resource. In-vitro experiments tend to involve the use of hydrated skin or epidermal membranes and the lipid phase behaviour is different from that of other biological membranes (Scheuplein 1967). The stratum corneum consists of 10 to15 layer of corneocytes (Andersion and Cassidy 1973; Holbrook and Odland 1974; Bouwstra et al., 1991).

Components of transdermal patch:

The basic components of transdermal patch consists of polymer matrix / Drug reservoir, active ingredient (drug), permeation enhancers, pressure sensitive adhesive (PSA), backing laminates, release liner, and other excipients like plasticizers and solvents (Aggarwal 2009).

1. Polymer matrix

Polymers are the backbone of a transdermal drug delivery system. Systems for transdermal delivery are fabricated as multilayered polymeric laminates in which a drug reservoir or a drug–polymer matrix is sandwiched between two polymeric layers: an outer impervious backing layer that prevents the loss of drug through the backing surface and an inner polymeric layer that functions as an adhesive and/or rate?controlling membrane.

2. Drug

The most important criteria for TDDS are that the drug should possess the right physicochemical and pharmacokinetic properties. Transdermal patches offer much to drugs which undergo extensive first pass metabolism, drugs with narrow therapeutic window, or drugs with short half-life which causes non? compliance due to frequent dosing.

3. Permeation enhancers

To increase permeability of stratum corneum so as to attain higher therapeutic levels of the drug permeation enhancers interact with structural components of stratum corneum i.e., proteins or lipids. The enhancement in absorption of oil soluble drugs is apparently due to the partial leaching of the epidermal lipids by the chemical enhancers, resulting in the improvement of the skin conditions for wetting and for trans-epidermal and trans-follicular permeation.

4. Pressure sensitive adhesive (PSA)

A PSA maintains an intimate contact between patch and the skin surface. It should adhere with not more than applied finger pressure, be aggressively and permanently tachy, and exert a strong holding force.

5. Backing laminate The primary function of the backing laminate is to provide support. Backing layer should be chemical resistant and excipients compatible because the prolonged contact between the backing layer and the excipients may cause the additives to leach out or may lead to diffusion of excipients, drug or permeation enhancer through the layer. They should have a low moisture vapour transmission rate. They must have optimal elasticity, flexibility, and tensile strength.

6. Release liner During storage release liner prevents the loss of the drug that has migrated into the adhesive layer and contamination. It is therefore regarded as a part of the primary packaging material rather than a part of dosage form for delivering the drug. The release liner is composed of a base layer which may be non?occlusive (paper fabric) or occlusive (polyethylene and polyvinylchloride) and a release coating layer made up of silicon or teflon. Other materials used for TDDS release liner include polyester foil and metalized laminate.

7. Other excipients Various solvents such as chloroform, methanol, acetone, isopropanol and dichloromethane are used to prepare drug reservoir. In addition plasticizers such as dibutyl pthalate, triethylcitrate, polyethylene glycol and propylene glycol are added to provide plasticity to the transdermal patch.

Extraction: Material and Methods

Collection of plants:

• The whole plant of T. procumbens (Linn.) were collected from different places such as Pravara rural college of pharmacy campus loni bk, Maharashtra , India and Nursery near campus .
• The whole plant  was shade-dried for 2 weeks After drying, the homogenate was transformed into a fine powder by using an mixer .

Preparation of Plant Extract:

• The plant material was shade-dried and finely powdered. Approximately 40 g of plant powder was placed in a thimble and subjected to Soxhlet extraction using petroleum ether as the solvent for defatting.
• The extraction was carried out at a temperature of approximately 60°C for about 2 hours, allowing multiple extraction cycles.
• After completion of the defatting process, the residue remaining in the thimble was collected and air-dried. The dried residue was then treated with a small amount of distilled water to obtain the aqueous extract.
• The mixture was subjected to magnetic stirring at 150–200 rpm to ensure proper homogenization.
• Petroleum ether was used for defatting, while water was used for preparing the aqueous extract.

                               Fig : Soxhlet apparatus                                  Fig : Magnetic Stirring

Isolation:-

Thin Layer Chromatography (TLC):

TLC = separation + identification using silica (stationary phase) and mobile phase solvent movement.

Rf =Distance travelled by substance

• Rf values:-

• Using Toluene :-0.80
• Using Ethyl Acetate:-0.82

Phytochemical Screening :

Figure: Scanning absorbance maxima of Tridax procumbens (240, 284  and 330 nm)

Table: Calibration curve of Tridax procumbens

Figure: Calibration Curve of Tridax procumbens

Figure: FTIR Spectra of Tridax Procumbens Extract

Figure: FTIR Spectra of HPMC K15

Figure: FTIR Spectra of gellan gum

Figure: FTIR Spectra of physical mixture

Figure : FTIR spectra of extract

Figure : FTIR spectra of physical

Analytical Data :

Graph (UV Spectrum)

• Shows absorbance vs wavelength for Tridax procumbens
• Three peaks at 240, 284, and 330 nm (λmax)
• Indicates presence of active phytochemicals
• Used to select wavelength for analysis

Calibration Curve (Graph + Table)

• Table shows concentration vs absorbance
• As concentration increases, absorbance also increases (direct relationship)
• Slope = 0.0119, Intercept = 0.0039
• r² = 0.9996 → shows excellent linearity and accuracy
• Used to determine unknown drug concentration

FTIR Graph/Table

• Shows functional groups present in extract and polymers
• Peaks confirm groups like O-H, C=O, C-H, C-O
• No major shift → indicates no interaction (compatible mixture)

Overall: Graphs identify compounds and tables help in quantification and compatibility study.

(A) Formulation of transdermal patch :

Formulation of transdermal patch (Solvent casting Method)

1. Preparation of Polymeric Solution

• Weigh polymers e.g ( HPMC ,Eudragit )
• Dissolve in suitable solvent (methanol/ chloroform or ethanol or water )
• Stir until clear solution is obtained

2. Addition of drug

• Dissolve drug (Tridax procumbens extract) in polymeric solution

3. Addition of Excipient

• Add Plasticizer (PEG 400, propylene glycol) for flexibility
• Add permeation enhancer if required

4. Mixing

• Stir solution until to get uniform mixture
• Remove air bubbles

5. Casting

• Pour the solution  into a petri dish or mould

6. Drying

• Allow solvent evaporate (room temperature or oven~24hrs)

7. Cutting

• Cut dried film into required patches

8. Storage

• Store in desiccator to prevent moisture absorption.

Formulation table :

Fig : Transdermal patch formulation

(B) EVALUATION OF TRANSDERMAL PATCHES:

1. Physical Characteristics:

• Visual Inspection: Inspect 10 patches for uniformity in size, shape, and colour.
• Thickness Measurement: Measure the thickness of each patch using a micrometre. Ensure the thickness falls within a specified range (e.g., 0.2 - 0.3 mm).
• Weight Variation: Weigh 10 patches individually and calculate the average weight to assess uniformity. Ensure weight variation does not exceed a certain percentage (e.g., ±5%).

2. Drug Content Uniformity:

• Extraction and Analysis: Extract the drug from 5 patches using 50 mL of ethanol.
• Analyze the drug content using HPLC.
•  Ensure the drug content is within the specified range (e.g., 90 - 110% of the labelled amount).

3. Mechanical Properties:

• Tensile Strength: Test 5 patches using a tensile tester. Measure the force required to break the patches. Ensure the tensile strength meets a specified minimum value (e.g., >2 N).
• Elastic Modulus: Measure the elasticity of 5 patches using a texture analyzer. Ensure the elastic modulus falls within a specified range (e.g., 100 - 200 MPa).

4. Adhesion Properties:

• Peel Adhesion Test: Test 5 patches using a peel tester. Measure the force required to peel them off from a substrate surface. Ensure the peel strength meets a specified minimum value (e.g., >0.5 N/cm).
• Skin Adhesion Test: Apply 5 patches to human or animal skin and assess adhesion using a skin adhesion tester. Ensure the patches adhere well to the skin without causing irritation or discomfort.

5. In Vitro Drug Release Studies:

• Franz Diffusion Cell: Conduct in vitro release studies using 5 patches and Franz diffusion cells. Measure drug concentration in the receptor compartment at specified time intervals (e.g., 1, 2, 4, 6, and 24 hours). Calculate cumulative drug release over time.

6. Skin Irritation and Sensitization Studies:

• Skin Irritation Test: Apply 3 patches to rabbit skin for 24 hours. Assess any signs of irritation at 24, 48, and 72 hours according to OECD guidelines.
• Skin Sensitization Test: Apply 3 patches to guinea pig skin for 24 hours. Observe for any signs of allergic reactions at 24, 48, and 72 hours.

7. Moisture content

• Prepared films were taken and then weigh them individually and then keep them in the desiccator which is containing calcium chloride. After completion of 24 h at the room temperature, all the films should again weigh. Using the formula given in Eq. 1, the percentage moisture content is determined.

% Moisture content =

{Initial weight-Final weight}×100

Final weight

8. Moisture uptake

• Weigh the films first and then these films were kept in the desiccator at the room temperature for 24 h. The relative humidity of 84% is exposed to those patches using the saturated solution of potassium chloride in the desiccator until the constant weight is achieved. The moisture uptake is determined by the formula given in Eq. 2

% Moisture uptake =

{Final weight-Initial weight}×100

Initial weight

9. Stability Studies:

• Accelerated Stability Testing: Store 5 patches at 40°C and 75% relative humidity for 3 months. Assess changes in physical characteristics, drug content, and release kinetics at 1-month intervals.
• Long-Term Stability Testing: Store 5 patches at room temperature (25°C) for 12 months. Assess stability at 3, 6, 9, and 12 months.

10. Folding endurance

• Evaluation of folding endurance involves determining folding capacity of films after that subject them to the extreme conditions of folding. Folding endurance can be determined by repeatedly folding the film at the same place without breaking. That is the folding endurance value of that patch.

11. In vitro release studies :

• The amount of drug available for the absorption into the blood is depends on the release of the drug from the polymeric transdermal film. The drug that reaches the surface of the skin is passed to usual permeation studies, which were performed by attaching the transdermal patch to the rat skin or to the synthetic membrane which is present in between the receptor and donor in vertical diffusion cells. The transdermal system is applied to hydrophilic side of the membrane and the lipophilic side is in contact with the receptor fluid. The receiver compartment is maintained at specific temperature usually 32°C and stirred continuously. The samples were withdrawn at equal time intervals and an equal amount of buffer is replaced at each time. The samples were diluted, and the absorbance is determined using UV spectrophotometer. The amount of the drug permeated per square centimetre at each interval is calculated. Drug release is depending on design of the system, patch size, surface area of the skin, thickness of the skin and temperature, etc.

12. In vivo studies

• In these evaluation studies, the drug performance can be depicted truly. In this study, the TDDS can be carried out using.
  • Animal models.
  • Human volunteers or human models

Animal models

Animal studies can be preferred mainly by the time and the resources which were required to carry out the human studies. Most common animal species used for the evaluating the transdermal patches on mouse. Hairless rat, hairless dog, hairless rhesus monkey, rabbit, guinea pig, etc., various experiments conducted on these animals lead us to the conclusion that the conclusions the hair less animals were preferred over the hairy animals in both in vivo and in vitro experiments. The most reliable model for performing this experiment is rhesus monkey.

Human models

It is the final stage for the development of the transdermal device involving the collection of the pharmacokinetic and pharmacodynamic data from the human volunteers by the application of the patch. The clinical trials were conducted by various parameters such as efficacy, risk involved, side effects, and patient compliance. Phase 1 clinical trials are conducted to determine the safety of the volunteers and the Phase 2 clinical trials were mainly to determine the short-term safety and the effectiveness in the patient. Phase 3 trails indicate the safety and effectiveness in the large number of patients. Phase 4 trials at the post marketing surveillance are done for the marketed patches to detect the adverse drug reactions. These are the best to assess the performance of the drug.

(1) Evaluation table:

2) Evaluation parameters for Transdermal patch :