Poly-herbal hydrogel formulation for topical skin infection: Formulation development and characterization

Skin is the largest organ in human body. It has a variety of purposes, one of which is to cover and shield your body. It assists in preventing germs. However, occasionally the bacteria can result in a skin infection. This typically occurs when bacteria enter our body by a skin wound, cut, or break[1]. Wherever the skin scrapes against one another, further skin infections may develop, particularly if the area is damp. They can be brought on by fungus, bacteria, or viruses[2]. A group of illnesses known as acute bacterial skin and skin structure infections (ASSSIs), sometimes known as skin and soft tissue infections (SSTIs) caused by bacteria[3]. These infections require long term antibiotic treatment which may not be sufficient or without the side effects. Polyherbal hydrogel formulations have emerged as an alternative to conventional cream for topical skin infection management. It has eminence therapeutic benefit of herbal constituent with biocompatible matrix which help in absorption of them in the skin layers. The herbal extract present in the polymer matrix have application as anti-inflammatory, antimicrobial, wound healing properties. The gel matrix provides a three-dimensional network structure which provide better skin hydration with enhanced bioactive permeation[4-5].                                                                                                                                                                                                                                                                     The hydrogel formulation can easily spread and adhere to the skin layer to facilitate prolonged absorption and faster recovery of any skin condition compared to conventional formulations[6]. Polyherbal hydrogel contain multiple herbal extract (3 or more) so a broad-spectrum antimicrobial effect is expected. Even many researchers have reported enhanced activity of therapeutic molecule or a synergistic effect[1, 7]. These herbal components present in the hydrogel offer benefits to the normal skin due to their antioxidative and tissue regenerative properties. They are also more effective in reducing scar than the traditional gel and formulation[8-9]. Herbal extract which are most commonly used for skin preparation are aloe vera gel, clove oil, neem leaf extract, eucalyptus oil, turmeric powder etc. Multiple herbal extract when mixed together in a polymeric matrix of gel bring all the goodness of these herbs in the formulation. Now the single formulation exhibits broad spectrum antibacterial and antifungal activity. This could be a good approach for the treatment of antibiotic resistant skin infections[10] (Shetty et al., 2018). In the present research work, we have developed a formulation composition of polyherbal hydrogel loaded with Carica papaya leaf extract, clove oil and aloe vera. All of these ingredients have very good antibacterial and anti-inflammatory properties. In hydrogel formulation, all together they will act synergistically through various mechanisms to treat skin infections. Clove oil which contains eugenol responsible for broad spectrum antibacterial and anti-fungal activity and help in inhibiting the growth of pathogens such as Candida albican, causative organism of Candidiasis[10-11]. Papaya leaf extract also possesses antimicrobial and antioxidant activities that can help in healing skin infections. Extract consists of quercetin and kaempferol which have effective antimicrobial properties. It has also cell protective properties that help in reducing skin inflammation responses[7-12].  With their role in protection of extracellular matrix intrigrity, they become very crucial for tissue repair. Apart from these two important ingredients, presence of aloe vera gel provides skin moisturizing effect as well as antibacterial and anti-inflammatory activity. It also act as good skin penetration enhancer which further enhance the absorption of phytoconstituents[13-14]. The polyherbal hydrogel help in the controlled and localized release of these phytoconstituents in the layers of skin which increases the antibacterial and anti-inflammatory efficacy and also help in fast recovery to skin to normal condition.  Such combination of herbal ingredient will have potential to target multiple bacterial species especially gram-negative  bacterial, causative organism in most skin diseases [15].                                                                                                                                                                                                                                                                                                                                                                                                                                                                       In this research work hydrogel were developed using Carbopol gel base with various concentration of clove oil, papaya leaf extract, aloe vera gel. The formulation was characterized and antibacterial property was also analyzed.

MATERIAL AND METHODS

Carica papaya extract was collected from Kshipra biotech private limited, Mumbai. Carbopol 934 was collected from Loba chemie private limited. Most of the chemicals were obtained from MVN University, Palwal.  Aloe vera extract was obtained from Aloe vera leaves collected from herbal garden of the MVN University campus. Clove oil was obtained from Nice chemical (P) LTD. Polyethylene glycol 400 was brought from SDFCL(SD fine-chemical limited), Sodium hydroxide (NaOH) was collected from CDH Laboratory, India. Distilled water was used in the study was prepared fresh for each experiment. All other materials used were of analytical grade.

The main component of the polyherbal hydrogel is Carica papaya leaf extract. Therefore, to analyse it in the formulation, a calibration plot was prepared by using UV spectrophotometric method. For this a Carica papaya extract solution was prepared in distilled water in the renge of 20 µg/ml to 100 µg/ml. One of the dilutions was scanned for the determination of λmax (absorbance maxima). All the dilution’s absorbance were taken at the observed λmax and calibration plot was plotted and line equation was derived to find the unknown concentration. Carbopol 934 polymer is a cross-linked polyacrylic acid polymer was used for hydrogel. For blank hydrogel and Carbopol 934 was accurately weighed & transferred to distilled water and placed on magnetic stirrer set to 1200 rpm for 10-15 min at room temperature. The prepared hydrogel was collected in a beaker & stored for further use.  The polyherbal hydrogel was prepared by using a similar process as the blank hydrogel. Briefly in a beaker Clove oil, Aloe Vera gel was mixed together. In another beaker, Papaya extract (1 gm) was dissolved in 45 ml of distilled water & placed on magnetic stirrer to dissolve it completely. After 15min, dissolved papaya extract solution was filtered. The filtered papaya solution was placed on magnetic stirrer and required quantity of Carbopol 934 was slowly added to it. When it was completely dissolved, clove oil -aloe vera gel mixture was added                                             and stirred continuously to prepare a stable polyherbal gel. The final pH of the formulation was adjusted to 6.5 by adding dilute NaOH solution (0.1N) by using dropper. The various polyherbal gel were prepared by different proportion of components as given in table 1. The prepared hydrogel was collected in a beaker.

Table 1. Composition of various ingredient used for the preparation of polyherbal hydrogel fotmulations

Source: compiled by the authors

FTIR spectrophotometer (Bruker Optik GmbH, Ettlingen, Germany) was used for compatibility study of the ingredient. A precisely weighed 2 milligram sample was grounded with few mg of anhydrous KBr and compressed to a thin pellet manually by tablet presser. The sample pellet was placed in the disc and analysed at a rate of 4mm/s and resolution of 2 cm^-1 in Fourier transform infrared (FT-IR) spectra. Using indium as the standard reference material, the temperature and energy scale of the equipment were calibrated. Physical mixture and formulation were analyzed for any possible incompatibility issue [16].

Visual inspection was employed to assess the physical appearance of the Polyherbal hydrogel formulations to make sure that the observations aligned within the required limits[17]. A digital pH meter (Ajanta industries, India) was used to determine the gel's pH. Throughout the measurement, three readings were taken, and the overall average of the three was noted. The glass electrode was before reading by using pH buffer solution of pH 4, calibrated the pH of the hydrogels were measured by immersing the electrode into the hydrogel sample until the reading was stabilized[18]. The viscosity of the gel was determined using a Brookfield viscometer at Shree Guru Govind Singh Tricentenary (SGT) University Gurgaon. Rheological characteristics of gels were assessed at 25°C using a Brookfield viscometer (DV-III programmed rheometer). The entire range of speed settings from 10 RPM to 1000 RPM was measured, with 30 seconds elapsing between each setting. Optimization of hydrogel formulation were done on the basis of viscosity, appearance and pH[19] . Microorganisms used in the test organisms were isolated on EMB agar from polluted water sample collected from near to the sewage treatment plant in MVN University, by the method described by Aneja, 2006[20]. Bacterial strains were cultured and isolated. On the basis of staining characteristics, the strains were identified as Klebsiella species, Enterobacteracae species, and Pseudomonas species. Tetracycline was used as positive control and sterile water was used as negative control[21]. The microorganisms were inoculated into 20 ml nutrient agar broth and incubated at 37 ± 2°C for 24 hrs. The Kirby's well diffusion is widely used to evaluate the antimicrobial activity of plants or microbial extracts. The agar plate surface is inoculated by spreading, 100 µL of the respective bacterial inoculum over the entire agar surface. Then, a hole with a diameter of 6 mm is punched aseptically with a sterile tip, then 100 µL of the positive control (10 microgram/ml), negative control (sterile water) and test sample S1 (Blank gel) and S2 (Polyherbal hydrogel) (100 microgram/ml) were introduced into the well respectively. Then, agar plates are incubated at 37°C for 48hr[21]. After incubation, the zone of inhibitions were observed and measured by using ruler. The whole study was performed in triplicates and average zone of inhibition was recorded in mm.

RESULTS AND DISCUSSION

The melting points of A. vera, carica papaya, and clove oil were determined to be 33.5°C, 110° C, and 15.5°C respectively. It was compared to the reference samples reference melting points reported in authentic literature. Table 2 showed the specific melting point observed for various samples.

Table 2. Melting point observed for various extracts

Source: compiled by the authors

The visual inspection of the extracts of Aloe vera, carica papaya leaves and clove oil, were performed and results are shown in table 3 have a pale brown hue.

Table 3. Physical observation of herbal ingredient

Source: compiled by the authors

In distilled water, a calibration curve was plotted within the concentration range of 20 µg/ml to 100 µg/ml. The maximum absorbance (λmax) of CPLE in water was observed at 279 nm. Figure 1 presents the absorbance maximum obtained for CPLE.

Absorbance obtained for various dilutions of CPLE were plotted against concentration as x-axis. The calibration graph is presented in figure 2 displayed the linear line equation and regression's R² value.

Figure 1. Calibration curve of Carica papaya leaves extract (CPLEA) in distilled water

Source: compiled by the authors

The calibration curve was prepared in carica papaya in the concentration of range of 250-500 µg/ml. the absorbance of maxima (λmax) of CPLE in distilled water was found to be 279nm. The R² value of the linear regression was found to be 0.9983. This shows that most of the dilution’s absorbance lies on the same lines. The regressed line equation Y= 0.0016 x + 0.008 was used for the calculation of unknown concentration of drug.

Figure 2. Absorption maxima obtained for carica papaya leaf extract

Source: Obtained by instrument (UV spectrophotometer)

The polyherbal hydrogel were developed by using various concentration of Carbopol 934 (0.5 to 2% w/v) with other herbal extracts. Table 3 presents the physical appearance and consistency of the hydrogel formulations. It shown smooth & of appropriate consistency as shown in figure 3 B. It was translucent in appearance. The prepared then hydrogel was collected in a beaker was shown in figure 3.

Figure 3. Hydrogel formulation: A. Blank hydrogel; B. herbal extract loaded hydrogel

Source: Photo taken by author

Various formulations of hydrogel were prepared by using different compositions to optimize the formulation (Table 2). The formulation with the observed characteristics is presented in the table 3 and figure 3. The appearance and texture of the prepared hydrogel were sufficient to retain on the skin, feel of the hydrogel was also smooth. The colour of the hydrogel varied due to presence of herbal component in it.  Polyherbal hydrogel with clove oil, CPLE and aloe-vera gel are darker in appearance as shown in figure 3.

Table 3. Formulation with the observed characteristics

Source: compiled by the authors

The FTIR spectra of carica papaya leaf extract was shown in figure 4 A and table 4. Table 4 presents the characteristic peak obtained for various samples. The peak represented in the spectrum of carica papaya were at 3249.49cm^-1 is due to OH stretching , 2933.59cm^-1  is due to O=C=O stretching ,1598.18cm^-1 is due to N-O stretching ,1145.86cm^-1 is due to C-O stretching and 1015.20cm^-1 is due to C-O stretching of alcohol group respectively [25].

Table 4. Various peaks obtained for the samples

Source: compiled by the authors

Figure 4. FTIR spectrum of A) Carica papaya of extract, B) Blank hydrogel, C) Aloe vera +clove oil, and D) Polyherbal hydrogel formulation

Source: Image obtained from FTIR instrument

The FTIR spectra of blank hydrogel was shown in figure 4 B. The peak represented in the spectrum at 3757.80cm^-1 is due to the presence of OH stretching vibration of hydroxyl group in alcohol , 3314.10cm^-1 is due to OH stretching vibration ,2953.69cm^-1 is due to C-H stretching vibration,1639.82cm^-1 is due to C=O in hybrid stretching of carboxylate anion [26].

The FTIR spectra of hydrogel containing (Clove oil & Aloe-vera gel) was obtained at 3736.26 cm^-1, 3338.51cm^-1 , 1641.26 cm^-1 , and 2136.65 cm^-1 was due to presence of OH stretching vibration of hydroxyl group in alcohol, OH stretching vibration, C=O in hybrid stretching of carboxylate anion and C≡C stretching vibration respectively as shown in figure 4 C  [27].

The FTIR spectra of hydrogel loaded (Papaya leaf extract & Clove oil , Aloe vera gel) has showed peak at 3736.26cm^-1 was due to presence of OH stretching vibration of hydroxyl group in alcohol, 3348.57cm^-1 was due to OH stretching vibration ,2952.25cm^-1 was due to C-H stretching vibration,2135.21cm^-1 was due to C≡C is due to stretching vibration as shown in figure 4 D  [27].

The polyherbal formulation FTIR (figure 4) have all the characteristic peaks of Carbopol, clove oil and aloe vera gel. Disappearance of few peaks of carica papaya is due to the entrapment of drug in the hydrogel matrix.  A digital pH tester was used to determine the pH of the formulation. The average pH of the formulation was found to be 6.02. Human skin pH varies from 6.5 to 7. This pH is appropriate for application on the skin surface as it is near to 6.5 and will not cause any irritation on the skin after application. The irritation score of the final formulation must be ensured by animal trial before application to human skin.

Figure 5. Reading in digital pH meter

Source: Recorded by the author

The viscosity of the formulations was assessed at 25°C using a Brookfield viscometer (DV-III programmed rheometer) and is presented in the table 5. 1% Carbopol hydrogel had least viscosity compared to other hydrogel formulations. Formulation 6 with CPLE, clove oil and aloe vera gel had shown appropriate viscosity for topical application. The hydrogel component will spread easily and absorb in skin before without loss of content.

Table 5. Viscosity obtained for various hydrogel formulations

Source: compiled by the authors

The anti-microbial activity of the developed polyherbal hydrogel formulation was analysed for the strains of Klebsiella species, Enterobacteracae species, and Pseudomonas species. Tetracycline was used as positive control and sterile water was used as negative control. S2 was the final polyherbal hydrogel selected for the test which was compared with the S1, Blank hydrogel. Outcome of the study was presented in Figure 6 which represents the zone of inhibition obtained for A) Pseudomonas species B) Klebsiella species C) Enterobacteracae species respectively after incubation.

Figure 6. Zone of inhibition against various strains on agar plate after incubation; A1& A2 against Pseudomonas species; B1& B2 against Klebsiella species; C1& C2 against Enterobacteracae species

Source: Image of the anti-microbial study performed by author

The zone inhibition was also measured and presented in the table 6. The zone of inhibition was not found for blank hydrogel against Pseudomonas species & Klebsiella species but a very small zone of inhibition was observed against Enterobacteracae species but is considered enough to have any antibacterial effect. No zone of inhibition was observed for negative control sample while a strong inhibition of bacterial growth was seen for positive control sample.

Table 6. Zone of inhibitions obtained for herbal hydrogel against pathogenic bacteria

Note: Where (-) = No zone of inhibition, Positive Control = Tetracycline, Negative Control = Sterile water, S1= Blank hydrogel and S2 = Herbal extract hydrogel.

Source: compiled by the authors