Formulation and Evaluation of Gel Containing Anthocyanin of Punica Granatum L. Extract for Anti-Inflammatory and Anti-Microbial Activity

Introduction

Anthocyanin is a plant’s pigment of blue, red, or purple color that can be found in its flowers, fruits, and tubers. Anthocyanins emerge as a red pigment in acidic surroundings and as a blue pigment in alkaline situations. It has an appositive charge on the oxygen atom in its C-ring, which is characteristic of basic flavonoids.Among the anthocyanins in pomegranate (2-phynylchromenylium), the flavylium ion makes up a significant part. This fruit’s seed coverings have been processed and used to make juice and wine. In pomegranate juice, delphinidin-3,5-diglucoside is a significant anthocyanin. However, the pigment composition of the fresh fruit has not been described.^1,2 Flavonols and anthocyanins show anti-carcinogenic, antimicrobial, anti-inflammatory, and antioxidant activities.³

Pomegranate, the fruit of the Punica granatum L. has been utilized to treat a range of ailments since ancient times. Pomegranate rind extract (PRE) is derived from the pericarp (rind) of the pomegranate, which has a high concentration of phytochemicals, particularly polyphenolic flavonols and ellagitannins such as ellagic acid and punicalagin. Herpes Simplex Virus (HSV) type 1 and 2 lesion eruptions cause a lot of inflammation in the localized area, which causes erythema, edema, and pain, especially in the later stages of infection. In addition, other HSV-related diseases such as herpetic stromal keratitis, have been linked to increased COX-2 expression.⁴

Topical administration allows medications to have localized effects on the place of their application by passing through the skin or mucous membrane and bypassing the first-pass metabolism. Topical formulations have the advantage of avoiding the risks and inconvenience of intravenous therapy, as well as the various absorption conditions such as pH changes, enzymes, and gastric emptying times, although foams, sprays, medicated powders, and even medicated adhesives are available, almost all topical formulations are semi-solid. In pain management, when other methods of drug administration fail, topical drug delivery systems are frequently employed for contraception. Throughout the last few decades, various methods of administering drugs to the body have been used, including oral, sublingual, rectal, parental, topical, and inhalation. Drugs delivered topically are meant to treat cutaneous diseases (e.g. acne) or chronic cutaneous disorders (e.g. psoriasis) directly, effectively confining the pharmacological or other effects to the skin’s surface. Depending on the topical activity, intracutaneous penetration or deposition may be needed. Topical delivery methods use a variety of pharmaceutical dosage forms, including semisolids, liquid preparations, sprays, and powders. The most often used semisolid formulations for topical medication administration are gels, creams, and ointments.⁵

Local inflammation is a response (reaction) to endogenous and exogenous insults in living tissues. It comes from the Latin word “inflammare” which means “burn”. Inflammation serves to localize and eradicate the causing factor while also limiting tissue damage. Inflammation is therefore a physiologic (protective) response to an injury. Inflammation is not considered a disease but rather a positive physiological process that is triggered either by violence or by disease.⁶

As the main global health challenge, bacterial-resistant infections pose a threat to society’s health. This is due to the growth of resistant infections, conventional antibacterial medications have become less effective or even ineffective, necessitating the creation of new antibacterial drugs. The resemblance of structural and antibacterial mechanisms to the old ones has prompted worries regarding the quick evolution of resistance through clinical use.

Antibacterial medicines are still in the early stages of development as alternatives. It is vital to avoid the development of resistant bacteria to retain the efficiency of present treatments for common and life-threatening illnesses. Understanding the mechanisms of bacterial resistance will aid in the development of more effective antibiotics. All possible alternatives include antibiotic efflux, antibiotic inactivation, biofilm formation, and target alteration.⁷

The goal of this study is to create a novel herbal topical formulation that contains anthocyanin. The present study attempted to formulate a stable topical gel with anthocyanin extract to reduce inflammation.

Materials and Methods

Materials

Methanol, tri-ethanolamine, and hydrochloric acid (HCl) were obtained from SDFCL Mumbai. Carbopol-940, Miller Hilton Agar, and HPMC K100M were obtained from Himedia, Mumbai. Merck SCL, Mumbai provided the sodium CMC while Loba Chemie Pvt. Ltd. provided the xanthan gum and propylene glycol. Changshu Hongsheng FC Co. Ltd. in Mumbai provided ethanol, Genuine Chemical Co. in Mumbai provided propylparaben, and NR CHEM in Mumbai provided methylparaben. All the chemicals obtained were of an analytical grade.

Methods

Extraction of Anthocyanin from Punica granatum L.

Pomegranate fruits were picked from a farm near Kaladagi, Bagalkot. Anthocyanin was extracted from fresh arils of pomegranate by placing methanol containing 1% v/v HCl in a beaker. The lid of the beaker was sealed with aluminum foil and maintained at room temperature for 24 h. After 24 h, filtration was carried out using filter paper.⁸ The filtrate was evaporated in a steam bath at 40° to get viscous anthocyanin. Dry anthocyanin was obtained by freeze drier (Lab gene RKVY/C-PHT/2015-2016) at -135° temperature and 2.58 mbar pressure as shown in Figure 1.

Confirmatory Test for Anthocyanin ^9,10

 Using methanol as a blank, the presence of anthocyanin in the extract was confirmed using a UV-visible spectrophotometer. A spectrum was taken in the UV-visible range i.e. 200 nm-800 nm.

 To the Punica granatum L. extract, aluminum chloride was added which gave a shift of 12 nm in the spectrophotometer that confirms the anthocyanin.

 One ml of the Punica granatum L. extract was combined with 2M HCl and heated at 100°C for 5 minutes. The presence of anthocyanin was confirmed when the extract retains its stable purple (magenta) color.

 The presence of anthocyanin was indicated by one ml of Punica granatum L. extract combined with 2M NaOH and the production of green color.

Phytochemical Tests for Extract ^11,12

Punica Granatum L. extracts were screened for photochemical using the procedures outlined by Khandelwal and Firdouseet et al.

Gel preparation methods^13,14,15

For different gel formulations, the concentrated Punica granatum L. extract (Anthocyanin) was used as the active ingredient, and Carbopol-940, HPMC K100M, Sodium CMC, and Xanthan gum were used as gelling agents (polymers). Gels were prepared by adding a specific amount of methyl paraben and propyl paraben (as a preservative) was dissolved in purified water by using Carbopol-940 at 40°C, HPMC K100M at 80°C, Xanthan gum, and sodium CMC at 50°C (Table 1). They were mixed individually until homogenous using a magnetic stirrer at 1200 rpm for 30 min. In a separate beaker, anthocyanin extract was dissolved in propylene glycol and ethanol. The above mixture was slowly added to the polymeric solution to get a gel and tri-ethanolamine was added to adjust the pH of the gel.

Evaluation of Formulations

Extrudability test¹⁶

A clean, lacquered aluminum collapsible tube was filled with around 5 g of the gel formulation on the crimped end, and a clamp was attached to prevent any rollback. The cover was then taken off, and the gel was extruded. The amount of gel extruded through the tip was then used to calculate the extrudability. The extruded gel was collected and weighed, and the percentage of extruded gel as well as grades was calculated.

(>90% Extrudability: Excellent)

(>80% Extrudability: Good)

(>70% Extrudability: Fair)

Viscosity¹⁷

By using the Brookfield viscometer, the viscosity of formulated gel was determined. The gels were rotated at 50 rpm using spindle no.64. At each speed, the reading was recorded. The viscosity determination of samples was repeated three times.

Spreadability¹⁸

The equipment, which consists of a glass plate block with a pulley at one end, was used to determine spreadability. Spreadability was measured using this method based on the slip and drag properties of gel compositions. The ground slide was used to keep an excess amount of gel under observation (approximately 2 g). The gel was then sandwiched between this slide and a 2nd glass slide with the same dimensions as the fixed ground slide and a hook was attached. For 5 mins, a 20 g weight was placed on top of the two slides to expel any air and generate a consistent gel coating between the two slides. The excess gel was scraped away from the edges. After then, a 20 g pull was applied to the top plate. The time (in s) required for the top slide to travel a distance of 6.5 cm should be recorded with the help of a string tied to the hook. Better spreadability is indicated by a shorter interval. The following formula was used to determine spreadability: S = M× L/T

Where, S = Spreadability,

M = Weight tied to the upper slide

L = Length moved by the glass slide (6.5 cm) and

T = Time (in S) taken to entirely separate the slides.

In-vitro release studies ¹⁹

Using a modified diffusion testing device, the in vitro release of anthocyanin from the produced formulations was investigated., Freshly prepared phosphate buffer (pH 6.8) was employed as the diffusion medium. A semipermeable membrane was connected to the bottom end of a specially built glass cylinder (open at both ends) with an inner diameter of 3.4 cm after being immersed in the diffusion medium overnight. 2 g of prepared gel was carefully pipetted into the donor chamber, a glass cylinder. The cylinder was suspended in a beaker (acceptor chamber) filled with 50 ml of diffusion medium, with the membrane just touching the medium’s surface. A magnetic stirrer was used to maintain a temperature of 37.2°C and a stirring rate of 50 rpm in the receptor chamber. At 1 h intervals, 4 ml of aliquot was taken and replaced with an equivalent volume of fresh diffusion media. A UV spectrophotometer was used to examine the aliquots at 532 nm.

Anti-microbial activity study^20,21

Antibacterial activity of the anthocyanin extract was determined using the Kirby- Bauer disk diffusion method. Standard strains of Escherichia coli ATCC25922 and Staphylococcus aureus ATCC29213 were used and a disk of amikacin was used as a control for comparison.

The organisms were inoculated in peptone water, and the turbidity obtained was matched with 0.5 Mc Farland standards. This was implanted as a lawn culture on Muller Hinton agar. A sterile filter paper disk impregnated with the anthocyanin extract (250 µl) and a commercially available amikacin disk were placed on it. Both plates were incubated for 24 h at 37°C. The plates were inspected for the zone of inhibition (in mm) after 24 h.

In-vivo Anti-inflammatory activity study^22,23,24

Animals

Wistar albino rats (165-220 g) were employed for the anti-inflammatory activity investigation. Hanagal Shri Kumareshwar College of Pharmacy and Research Centre’s central animal house in Bagalkote, Karnataka provided the animals. All of the animals were kept under standard husbandry conditions at 22-28°C, relative humidity 65±10% for 12 h dark and 12 h light cycles respectively in standard propylene cages. Standard food (Pranav agro-industries Sangli, Maharashtra) and ad libitum water were fed to the experimental animals. All the experiments were conducted following the direction of trials by the Institutional Animal Ethics Committee (IAEC/HSKCOP/MARCH 2020/PG9).

After over-night, starved rats were classified into 6 groups each group containing 6 animals as follows:

Group-I: Carrageenan (1%) Induced Paw Edema (control)

Group-II: Carrageenan + 1% diclofenac gel (standard)

Group-III: Carrageenan + 1% Anthocyanin gel containing 1.5% Carbopol-940 [F₁ ]

Group-IV: Carrageenan + 1% Anthocyanin gel containing 2% HPMC K₁₀₀M [F₃ ]

Group-V: Carrageenan + 1% Anthocyanin gel containing 2% Xanthan Gum [F₅ ]

Group-VI: Carrageenan + 1% Anthocyanin gel containing 2% Sodium CMC [F₇ ]

Edema was induced in all of the grouped animals by injecting 0.1 ml of carrageenan solution (1.0% w/v in normal saline) into the left hind paw’s sub-plantar area. Following the applications of carrageenan gels to the paw for 20 m at a time, the paw volume was measured using a digital Plethysmometer (7140 UGO Basile, Italy) at various time intervals of 0, 0.5, 1, 2, 3, 4, and 5 h. The following calculation was used to compute the % inhibition of paw edema volume in each of the treatment groups:

Percentage of inhibition = Volume of control - Volume of test/ Volume of control × 100

Results

Drug-excipients interactions were studied by ATR, by the interaction studies of anthocyanin concentrate and excipients, it was observed that there was no significant interaction. Hence ingredients in the gel were compatible as shown in Figure 2. 

ATR-FTIR Studies:

Confirmation test for anthocyanin

Anthocyanins were confirmed at 532 nm by UV-Visible spectrometer as shown in Figure 3. Phytochemical confirmation of anthocyanin was confirmed by observing pink color with 2M HCl and by addition of 2M NaOH to the extract gave a green color that confirmed the presence of anthocyanin in the extract as shown in Figure 4.

Evaluation of Formulations

pH

Formulated gels were subjected to the measurement of pH which was fairly constant between 6.4 to 6.8 in 3 months shown in table 2.

Visual appearance and Homogeneity

Formulated anthocyanin gels were evaluated for their homogeneity by visual inspection and it was found that all the preparations were clear, translucent without aggregation, and free from residual particles.

Temperature change test

Anthocyanin gels were exposed to different temperature conditions and no visual changes were observed.

Centrifuge test

There was no visible sediment in centrifuge testing, and the gels remained homogenous.

In-vitro drug release studies

All formulations were evaluated for cumulative percentage of drug release, where F7 and F8 formulations showed 94.41% and 90.62% drug release, respectively at the end of 5 h. Results are shown in Figure 5. 

Anti-inflammatory studies:

The anti-inflammatory efficacy of F1, F3, F5, and F7 gels was evaluated using the rat paw edema method. When compared to commercially available diclofenac gel, formulation F7 significantly reduced the paw edema as depicted in Figure 6.

In comparison to the control group, all values are expressed as mean SEM, n=6, *p<0.05, **p<0.01,***p<0.001. Multiple comparisons Dunnett’s test was followed by one-way analysis of variance (ANOVA).

Antimicrobial activity:

The antimicrobial efficacy of anthocyanin concentrate showed a good inhibitory effect against both grampositive and gram-negative micro-organisms (Figure 7). 

Discussion

The current study aimed to develop gel from anthocyanin concentrate of Punica granatum L. by maceration method using methanol containing 1% HCl. The gel was formulated by dispersion method and subjected to various evaluation parameters and the optimized formulation was subjected to in-vivo anti-inflammatory activity by the rat paw edema method. Among the gelling agents used, Carbapol-940 had gelling properties at low concentrations because Carbapol-940 is a cross-linked poly acrylic acid polymer whereas other gelling agents are linear polymers.

ATR-FTIR Studies

Compatible studies were performed using ATR. The IR spectrum of pure freeze-dried anthocyanin concentrate, polymers, and physical mixture of extract and polymers were performed. The characteristic peak found in the anthocyanin concentrate were also found in the physical mixture of anthocyanin concentrate and polymer which indicates no interaction of anthocyanin concentrate with other excipients in the gel.

Confirmation test for anthocyanin

The maximum absorbance of the anthocyanin extract was at 532 nm in UV-visible spectrophotometer. The addition of aluminum chloride to the Punica granatum L. extract gave a shift of 12 nm in the spectrophotometer due to the substitution or solvent effect. It showed a bathochromic shift on the spectrophotometer, and it contained cyanidin, delphinidin, or petunidin. Blue shift refers to the change of absorption towards a shorter wavelength. Punica granatum L. extract was boiled for 5 mins at 100˚C with 2M HCl. Because of the dominant type of anthocyanin, the extract retained its purple hue. The shift of absorption towards a shorter wavelength is called a blue shift. Punica granatum L. extract was mixed with 2M HCl and heated for 5 mins at 100˚C. Extract remained purple, due to the dominant form of anthocyanin. On mixing Punica granatum L. extract with 2M NaOH, a green color appeared as shown in Figure 4 due to the formation of a quinoidal base.^9,10

Phytochemical investigation of Extract

The phytochemical screening is a significant parameter in determining the phytoconstituents in the extract. Phytochemical study of Punica granatum L methanolic extract contained anthocyanin, flavonoids, flavanoidal glycosides, phenolic compounds, and glycosides as active phytoconstituents.^11,12

Evaluation of Formulations

pH

Topically applied gel pH should have the same pH as that of the skin, if not it will lead to irritation on application hence formulated anthocyanin gel was subjected to pH studies. The pH of the gels was within the skin pH and hence gels were considered non-irritants and can be used for topical application.²⁵

Visual appearance and Homogeneity

Formulated gels were investigated for the presence of a particle, improper mixing aggregation, and another residue. So prepared gels were filled in a transparent container and visually inspected. All the formulations were found to be homogeneous and there was no aggregate formation of particles.²⁶

Temperature change

Formulated gels should be stable in different environmental conditions along with different temperatures which should not affect the gel characteristics. Thus all the formulations of gel containing anthocyanin were evaluated for different temperatures and all formulations retained gel properties at various temperatures.²⁷

Centrifugation

To study the uniformity and sedimentation of the gels, a centrifuge test was carried out at 10000 rpm. No apparent residue was observed and the gels maintained their homogeneity.²⁷

Extrudability

Extrudability is an important parameter for any topical semi-solid dosage form which reveals the formulation can be easily removed from the packing container and can be applied topically. Formulated gels showed good extrudability. Among these, formulation gels prepared with sodium CMC showed excellent extrudability compared to the gels prepared with Carbopol-940, xanthan gum, and HPMC K100M.²⁸

Viscosity

The viscosity of gel composition is crucial. The formulation’s viscosity influences drug release from the gel. If the gel has a higher viscosity, the drug is released slowly, but if the gel has a lower viscosity, the drug diffuses quickly into the diffusion medium. The various anthocyanin gel formulations were subjected to viscosity studies, all the gel formulations showed good viscosity and they were capable of remaining at the site of application for a prolonged time. Among these, formulation gels prepared using HPMC K100M were more viscous compared to gels prepared with Carbopol-940, sodium CMC, and xanthan Gum.¹⁷

Spreadability

Spreadability was measured in terms of the time it took for the two slides to slip away from a gel put in between the slides under specified stress. The less time it takes to separate the two slides, the higher the spreadability. It is important for patient compliance because it ensures consistent application of gel to a greater region of skin. The gel having low viscosity showed better spreadability. The various anthocyanins gel formulations were subjected to spreadability studies. The spreadability of the gel decreases with increases in the concentration of polymer. The values of spreadability revealed that the gel was easily spreadable by a small amount of shear. Because of less viscosity, gels prepared using sodium CMC were more spreadable than gels prepared using Carbopol-940, xanthan gum, and HPMC K100M.¹⁸

In-vitro drug release studies

This test is used to investigate the drug profile. Drug-release behavior is a critical element in polymer topical administration since it is directly proportional to drug stability and therapeutic outcomes as well as formulation development. The various anthocyanin gel formulations were subjected to in-vitro drug release and diffusion profiles. All of the gel formulations demonstrated prolonged release for a set amount of time. Up to 5 h, the percentage release between time intervals was generally stable. All the gel formulations displayed a good percentage release. Among these formulations, the F7 (94.41%) and F8 (90.62%) showed better release than F1(89.55%), F2(83.02%), F3(88.26%), F4(82.96%) and F5(90.42%), and F6(96.64%). Viscosity is an important physical property that affects the rate of drug release. In general, an increase in viscosity decreases the rate of drug release. In our study, formulations with less viscosity such as gel F7 and F8 showed a better rate of drug release than formulations with more viscosity such as F1, F2, F3, F4, F5, and F6.¹⁹

Anti-microbial studies:

The most essential property of pharmaceutical businesses is antibacterial activity, which protects against a variety of pathogens, biological fluids, and aerosols, as well as disease transmission. A product’s shelf life can be increased by adding artificial preservatives or implementing hygienic steps throughout the production process. Every part of the Punica granatum L. tree is used for medicinal and cosmetic purposes.²⁰ Here anthocyanin extract was tested for antibacterial activity against both gram-positive and gram-negative organisms using the Kirby- Bauer disc diffusion method. Standard strains of E. coli (ATCC25922) and S. aureus (ATCC29213) were utilized, as well as an amikacin disc was used as a control for comparison. At a concentration of 400mg/ ml, anthocyanin extract demonstrated anti-microbial activity against both gram-positive and gram-negative microorganisms with a zone of inhibition of 18mm and 16mm against S. aureus and E. coli, respectively.²¹

Anti-inflammatory studies

The optimal formulations (F1, F3, F5, and F7) from the in-vitro release were subjected to anti-inflammatory activity investigations. Carrageenan is a general name for a group of gel-forming, viscous polysaccharides extracted commercially from particular kinds of red seaweed (Rhodophyceae) and it is composed of a linear galactose backbone with a varying degree of sulphation (15%–40%). Edema develops after carrageenan inflammation that stimulates the release of arachidonic acid from pleural macrophages. This leads to the activation of phospholipase A2 and produces carrageenan action then PGE2 and LTC4 may mediate inflammatory response to carrageenan. The acute phase (30 mins) is attributed to the release of serotonin, and leukocytes. The edema maintained (1 h) is delayed- release at that time histamines releases. In the intermediate phase (3 h), at the time, Bradykinin releases and the final phase chronic inflammation (5 h) takes place where PGF2 releases. Acute inflammation is a short-term process that is characterized by the topical signs of inflammation like swelling, pain, and loss of function as a result of plasma and leukocyte infiltration of the tissues. Edema is one of the main characteristic of acute inflammation and is an important criteria to consider when assessing drugs with anti-inflammatory activity.²²

In the present study, the control group showed a significant increase in the paw volume after carrageenan induction in the sub-plantar region of the right hind paw. This is because inflammatory mediators such as histamine, serotonin, kinins, PGs, and others are released. After inducing carrageenan to all groups of animals, gels were applied to respected groups and after 30 mins, edema volume was measured. The result showed that marketed diclofenac (Voveran) gel is more active against serotonin mediator with (12.57%) edema inhibition followed by F7 (11.98%) and F1 (15.30%) and F7 (12.00%) showed good edema inhibition compared to marketed diclofenac gel (10.42%) at 1 h. This indicates that formulated gels F1 and F7 are more active against histamines compared to diclofenac gel. At the intermediate phase, diclofenac gel (42.08%) showed a good edema inhibition rate followed by F7 (38.04%). In the final stage of inflammation at the end of 5 h, marketed diclofenac gel (61.03%) showed excellent inhibitory activity followed by F7 (55.54%). Our study revealed that formulated gel F1 and F7 have good inhibitory activity on histamines (delayed stage) and marketed diclofenac is more active against mediators like serotonin, bradykinin, and PGF2 (initial, intermediate, and chronic phase). The marketed diclofenac Gel (Voveran 1% gel) and anthocyanin gel F1, F3, F5, and F7 showed significant inhibitory activity against carrageenan-induced paw edema inflammation.²³ Among these groups, gel F7 showed the best percentage inhibition (55.54%) and marketed diclofenac gel showed (61.03%) and gel F1, F3, and F5 showed (53.95%), (46.99%) and (41.71%) inhibition, respectively. This may be due to the inhibition of the cyclo-oxygenase enzyme which leads to the inhibition of PG synthesis and shows significant activity.²⁴

Conclusion

Anthocyanin gel formulations can be conveniently prepared using different gelling agents such as Carbopol-940, HPMC K100M, xanthan gum, and sodium CMC at different concentrations. Among the used gelling agents, formulations of 1% and 2% showed better results as compared to 1.5% and 2.5%. Our observations and results suggested that there is a potential and remarkable protective role of anthocyanin gel F7 in the acute inflammatory model as compared to other formulations. Anthocyanin extract showed good microbial protection against microorganisms such as Staphylococcus aureus and Escherichia coli.

Conflicts of Interest

None