Formulation Of Semisolid Dosage Form Using Euphorbia Hirta and Silver Nanoparticles for Diabetic Wound

Nanotechnology is an emerging field focused on the synthesis and enhancement of nanomaterials. Recent studies have applied silver nanoparticles across diverse sectors, including medicine, textiles, food packaging, and the disinfection of drinking water. Silver nanoparticles (AgNPs) are typically produced using traditional techniques, such as chemical and physical methods, which can be costly and detrimental to the environment [1]. Plant extracts might require less time to convert silver ions compared to microbes, as the phytoconstituents in plants rapidly reduce silver to form silver nanoparticles and also serve as capping agents to produce biogenic silver nanoparticles [10].  The biologically green synthesis of metal nanoparticles is an economical, one-step process involving bio-reduction. This method is used to synthesize silver nanoparticles, providing a safe and straightforward technique for production. Silver nanoparticles perform a variety of functions in areas such as diagnostics, drug delivery, and material coatings. Due to their antibacterial properties, these nanoparticles are utilized in wound healing treatments. Prior research indicates that ointments containing silver nanoparticles enhance skin wound healing by reducing the wound size and the count of cells, including neutrophils and lymphocytes [1]. 

Diabetic wound:

The WHO census indicates that 830 million individuals are impacted by diabetes. Diabetes mellitus is a chronic metabolic condition characterized by high blood glucose levels [3]. The primary factor contributing to the rise in morbidity and mortality associated with diabetes is believed to be the onset of macro- and micro-vascular complications, which leads to an impaired wound healing process [4]. Diabetic foot ulcers (DFUs) are a significant complication associated with diabetes mellitus, stemming from a complex interplay of neuropathy, weakened immune response, poor blood circulation, and chronic wounds. Treating these ulcers can be particularly challenging, potentially resulting in the amputation of the lower limbs [3]. Peripheral neuropathy plays a major role in the development of diabetic wounds, as long-term high blood sugar levels result in nerve damage due to oxidative stress and glycosylation [4].

In individuals with diabetes, elevated blood sugar levels lead to sustained chronic inflammation, which is accompanied by increased matrix metalloproteinases (MMPs). MMPs are a group of zinc-dependent endopeptidases that can break down components of the extracellular matrix and play a role in tissue remodeling and restructuring. Previous research indicates that MMP-9 is associated with hindered wound healing, while MMP-8 is implicated in the process of wound repair [20]. Plant-based medicines enhance wound healing thanks to their numerous phytochemical compounds, which are readily accessible, have fewer side effects, and serve as an alternative to synthetic medications [2].

Figure 1: Diabetic wound

Plant profile:

Euphorbia hirta Linn. is a significant medicinal plant from the Euphorbiaceae family. It is a small, upright or ascending annual herb that can grow up to 50 cm tall and has hairy stems [4]. In Tamil, it is referred to as “Ammaan pacharisi,” and the different parts of Euphorbia hirta have been used in traditional medicine to address ailments such as asthma, bronchitis, coughs, colds, ulcers, and skin infections [6].

Synonyms-

5. asthma weed
5. snake weed

Figure 2: Euphorbia hirta

Taxonomical classification:

Table 1: Taxonomical classification

Phytochemical constituents:

This plant is composed of several phytochemical constituents, including alkaloids, steroids, tannins, glycosides, cardiac glycosides, terpenoids, proteins, saponins, anthraquinones, fats, oils, gums, coumarin, flavonoids, mucilages, and phenolic compounds [7]. The existence of flavonoids in the plant extract demonstrates properties that reduce inflammation and promote wound healing [4].

Pharmacological activity:

Antimicrobial activity, Antioxidant activity, Anticonvulsant, Anxiolytic, Anti-inflammatory, Analgesic, Antipyretic, Antihistaminic, Antiasthmatic, Antidiabetic, Anticancer, Wound and burn healing, Diuretics, Anti-parasitic, immunological effects, hepatoprotective effects, galactogenic effect, ACE inhibiting, and anti-dipsogenic activities [7].

The goals of this research were to create a semisolid dosage form of Euphorbia Hirta extract and silver nanoparticles (AgNPs) and to investigate its effectiveness for healing diabetic wounds.

2. MATERIALS AND METHODS:

2.1. Materials:

Euphorbia hirta leaves, ethanol, white soft paraffin, liquid paraffin, beeswax, distilled water

2.2. Methods:

2.2.1. Preparation of Euphorbia hirta ethanolic extract:

Fresh leaves of Euphorbia hirta were gathered and air-dried in the shade. They were then ground into a fine powder. This powder was weighed and subjected to extraction using ethanol as a solvent in a Soxhlet apparatus at a 1:10 ratio at 40?C. The extraction process was continued until the solvent in the thimble appeared clear or colorless. Afterward, the heat source was turned off, and the distillation flask was removed and allowed to cool. Finally, the extraction was filtered using Whatman filter paper [8,9].

2.2.2. Screening of Euphorbia hirta extract:

The phytochemical analysis of Euphorbia hirta was performed using established methods, including tests for flavonoids (Shinoda test, alkaline reagent test, and zinc hydrochloride test), alkaloids (Dragendroff’s reagent, Hager’s reagent, Mayer’s reagent, and Wagner’s reagent), glycosides, saponins (Froth test), tannins (Ferric chloride test), as well as steroids and triterpenoids (Liebermann-Burchard test and Salkowski test) [16].

2.2.3. Fourier transmission infrared spectroscopy (FTIR):

The FTIR spectrum of the ethanolic extract of Euphorbia hirta (EHEE) was acquired, and the sample was dried at 45?C. The dried EHEE sample was combined with KBr to create a fine powder, which was then pressed into a thin pellet. FTIR spectra were recorded by placing the pellet into the holder of the spectrophotometer [10].

2.2.4. Green synthesis of silver nanoparticles:

Accurately weighed 0.1698g of silver nitrate and dissolved it in distilled water within a 50 ml beaker. This solution was then transferred to a 250 ml volumetric flask, and the volume was adjusted to the mark to create a 1mM solution. Next, 50 ml of plant extract was combined with 1 mM of the 250 ml AgNO3 solution. To prevent photochemical activation of AgNO3 and to promote the bio-reduction of silver, the conical flask was placed in a dark room at 27?C. A color change to dark brown signifies the formation of silver nanoparticles. The dispersion of Euphorbia Hirta silver nanoparticles (EH-AgNPs) was centrifuged at 5000 RPM for 30 minutes, followed by decantation. The EH-AgNPs were suspended in distilled water and re-dispersed four times to remove any unreacted phytoconstituents, resulting in purified EH-AgNPs. These nanoparticles were then dried in a hot air oven at 45?C [10,11].                                            

2.2.5. Characterization of EH- AgNPs:

2.2.5.1. UV-Vis spectrophotometric analysis:

The EH-NPs that were synthesized were examined within the wavelength range of 400-800 nm to determine the size of various metal nanoparticles. A UV-Vis spectrophotometer was used, with distilled water serving as a blank [10,11].

2.2.5.2. Particle size analysis of AgNPs from EHEE:

Nanoparticle tracking analysis (NTA) is a relatively recent technology that has gained popularity in recent years. The Nanoparticle Tracking Analyzer (NTA) - Particle Metrix Zetaview-PMX 130-Mono laser is utilized for determining the particle size and zeta potential of liquid samples. The samples were exposed to a laser with a wavelength of 405 nm. The duration of each measurement was 30 seconds, with the shutter speed and gain settings for each sample adjusted individually to achieve the best signal-to-noise ratio in the recorded image of the Brownian motion of particles. To ensure optimal concentration for studies using the NTA method, the samples were diluted with distilled water before measurement [21].

2.2.6. Preparation of ointment:

Ointments are made using the fusion technique. Specific quantities of components like beeswax, white soft paraffin, and liquid paraffin were placed into a porcelain dish, which was then heated in a water bath until it reached 70?C before being taken out. When the temperature decreased to 50?C, an extract of varying concentration was incorporated into the previously mentioned ointment base [12].

An ointment formulated with Euphorbia hirta extract and EH-AgNPs was created using the formula outlined below:

Table 2: Composition of ointment

Figure 4: Ointment formulation

2.2.7. Characterization of ointment:

2.2.7.1. Physical examination:

The color, odour, and texture of the prepared ointment were assessed through visual inspection [14].

2.2.7.2. Determination of pH:

2.5 grams of ointment were incorporated into 50 milliliters of water in a 100-milliliter beaker and then placed into a water bath heated to between 60?C and 70?C for a duration of 10 minutes. Afterward, it was cooled and centrifuged at 3000 RPM for 10 minutes. The pH was measured using a pH meter [14].

2.2.7.3. Measurement of viscosity:

The ointment's viscosity was assessed with a Brookfield viscometer set at 10 RPM, and the measurement was taken in centipoises [14].

2.2.7.4. Spreadability:

The prepared ointment was positioned between two slides, and a certain force was exerted to assess its spreadability. This is measured in seconds. If the duration required for the two slides to separate is shorter, it indicates good spreadability [14]. It is calculated by using the following formula,

S = M. L/T

Where,

M = weight tied to the upper slide

L = length of glass slides

T = time taken to separate the slides?

2.2.7.5. Diffusion study:

To investigate the diffusion of ointment, agar medium was created and poured into a petri dish to solidify. Next, a hole was made in the center of the medium, where the ointment was deposited. The duration for the ointment to diffuse was recorded after 60 minutes [22, 23].

2.2.7.6. Loss on drying:

The loss on drying was assessed by placing the formulation in a petri dish and drying it in a water bath at 105? [22, 23].

2.2.7.7. Solubility:

The substance was dissolved in distilled water and an organic solvent to assess its solubility [22, 23].

2.2.7.8. Washability:

The mixture was applied onto the skin and then rinsed off with water [22, 23].

2.2.7.9. Skin irritation test:

The ointment was smeared onto the affected skin area and monitored for different negative reactions such as redness, irritation, swelling, and itching [15].

2.2.8. Molecular docking study:

A silicon-based protein-ligand docking software named Autodock Vina 1.5.6 was used to evaluate the binding affinities and interaction patterns between the compound CG and its proposed targets. The molecular structure of Rutin (PubChem Compound CID 5280805) was retrieved from the PubChem database. The 3D coordinates for the target proteins MMP 8 and MMP 9 were sourced from the PDB website. The molecular and protein files were converted into PDBQT format by eliminating water molecules and adding polar hydrogen atoms. Grid boxes were created to cover the protein domains and permit the free movement of the molecules. Autodock Vina 1.5.6 was utilized for conducting the docking studies. We extracted the lowest binding energies and estimated inhibition constants (pKi) from the docking log files (dlg) [24].

3. RESULTS AND DISCUSSION

3.1. Screening of herbal extract:

Assessment of Euphorbia hirta ethanolic extract was conducted through different phytochemical tests to verify the existence of flavonoids, polyphenols, alkaloids, and glycosides.

Table 3: Phytochemical screening of EHEE

3.2. Fourier Transform Infrared Spectroscopy (FTIR):

FTIR is a method used to acquire infrared spectra for the absorption or emission of liquids, gases, or solids. It is an effective technique for identifying phytoconstituents in herbal extracts. The FTIR spectrum of the ethanolic extract of Euphorbia hirta is displayed in Fig.4. within the wavenumber range of 400-4000 cm?¹. In the figure, the wavenumber 3306.38 cm?¹ corresponds to the stretching of the OH group, indicating the presence of flavonoids and polyphenolic compounds. The wavenumbers 2923.98 cm?¹ and 2854.13 cm?¹ are indicative of CH stretching, while the wavenumber 1707.64 cm?¹ signifies C=O stretching, pointing to the presence of terpenoids. Additionally, a prominent band at 3306.38 cm?¹ for the OH group and wavenumbers 1203.24 cm?¹ and 1031.26 cm?¹ for aromatic C-O groups suggest the presence of phenols and flavonoids.

Figure 5: Graph of IR spectrum

3.3. UV-vis spectrophotometer analysis:

Spectrophotometric absorption in the 400-450 nm wavelength range was utilized to characterize AgNPs [1]. The UV analysis of the extract is shown in Fig.6. We determined the presence of flavonoids through UV spectrophotometry, which is suitable for wound healing, with absorbance peaks appearing at 350-380 nm, while phenolic acids show absorbance in the 250-300 nm range. Additionally, the synthesis of AgNPs using Euphorbia hirta extract may result in a polydispersed product, with absorbance observed at 430 nm.

Figure 6: Graph of UV absorbance of EHEE

Figure 7: Graph of UV absorbance of EH-AgNPs

3.4. Particle size analysis of EH-AgNPs:

NTA is primarily used to evaluate the size of particles, zeta potential, and concentration in liquid samples. The separate particles influenced by Brownian motion were identified and observed. The NTA results showed that most particles are within the size range of 50 to 200 nm.

Table 4: NTA results

Figure 8: Particle size of silver nanoparticles using NTA

Figure 9: Zeta potential of silver nanoparticles using NTA

3.5. Characterization of ointment:

This research aimed to create both a herbal ointment and an ointment containing silver nanoparticles. The herbal extracts were obtained through Soxhlet extraction, yielding a high amount of extract and ensuring that the chemical components and their efficacy were preserved. Silver nanoparticles were synthesized using a green method involving the plant extract. The ointment was formulated via the fusion technique to guarantee a uniform mixture of the herbal extract with the ointment base, maintaining stability during storage. The phytochemical properties were evaluated. The ointment demonstrates positive results in terms of spreadability, washability, drying loss, and other factors.

Table 5: Physicochemical results of ointments

3.6. Molecular docking analysis:

Molecular docking studies of the phytoconstituent rutin from Euphorbia hirta were conducted with the wound healing-related enzymes MMP-8 and MMP-9 using Autodock. The docking results revealed a binding energy of -4.88 Kcal/mol for MMP-8 and -4.49 Kcal/mol for MMP-9, suggesting a moderate interaction between rutin and the active sites of these enzymes. The inhibition constant (Ki) values were found to be 224.54 μM for MMP-8 and 264.57 μM for MMP-9, indicating a moderate binding affinity for the respective active sites.

Figure 10: Docking studies using MMP-9 as the target protein

Figure 11: Docking studies using MMP-8 as the target protein

Table 6: Docking results with target protein

Table 7: Results of binding interaction with target protein