Formulation and Evaluation of Polyherbal Aqueous Gel from Psidium guajava, Piper betel and Glycerrhiza glabra Extract for Mouth Ulcer Treatment

 

Nem Kumar Jain¹*, Rituparna Roy¹, Hero Khan Pathan¹, Aditi Sharma¹, Shakhi Ghosh¹, Santosh Kumar²

¹School of Pharmacy, ITM University, Gwalior, MP, India

²Department of Life Sciences, ITM University, Gwalior, MP, India

 

ABSTRACT:

Mouth ulcers often cause pain and irritation of the sores by salty, spicy and sour food items and may cause discomfort while healing occurs due to the use of chemical formulations. This project focuses on the preparation of a herbal mouth ulcer healing gel because of better cultural acceptability, better compatibility with human body and lesser side effects. The gel was prepared by using alcoholic extract of Psidium guajava, and betel leaves and aqueous extract of liquorice. Developed formulations were transparent, homogeneous and pH ranges from 6.8-7. Formulation showed acceptable rheological behaviour with applicable Spreadability and Extrudability properties. Anti-fungal studies of formulations showed excellent efficacy against Aspargilious aureus, Candida albicans. Therefore, developed formulations have potential to treat mouth ulcers. However, further clinical studies are required to establish clinical efficacy of prepared herbal gels.

 

 

 

INTRODUCTION:

Mouth ulcers are yellowish or whitish depression with red margination in mucus lining of mouth cavity, characterized by inflammation and pain. Based on clinical status, mouth ulcer patients can be categorized into three groups: minor, major and herpitiform. Etiology is unknown or often misunderstood; its diagnosis is largely based on clinical signs (1). However some factors like psychological stress, topical trauma, microbial infections, genetics, nutritional derangements, immunological, hormonal changes, allergies and medications are considered important factors as etiology of mouth ulcerations (2).

 

Various synthetic and semi-synthetic medicaments are suggested to treat mouth ulcers like antibiotics and antiseptics, local anesthetics, local analgesics, steroidal and non-steroidal anti-inflammatory drugs. Topical steroids viz. triamcenolon and prednisolon are most frequently used treatments but they have some serious side effects on continuous application like adrenal insufficiency, immuno-supression, osteoporosis, hyperglycemia, gastrointestinal disturbance, etc. Moreover commercially available formulations containing synthetic and semi-synthetic active agents are complained of local irritation, staining of teeth, burning sensation, etc, due to high alcohol concentration and presence of some organic compounds (3).

 

Considering these side effects and demand for better patient compliance the use of plant based medication is gaining popularity throughout the world. A number of studies have reported use of herbal plant’s part or extracts in the form of mouthwash, paste or muco-adhesive gels for treatment of oral ulcers namely Rosa damascene, Rurta graveolens, Ginger officinale, Cordia dichotoma, Glycerrhiza glabra, Anthemis nobilis, Myrtus communis, Melissa officinalis, Hypericum perforatum, Zataria mutiflora, Curcuma longa, Punica granatum etc (2-7). The present investigation deals with use of Piper betle, Glycerrhiza glabra and Psidium guajava leaves extract for the preparation of stable aqueous gel formulation which can be used as an alternatve treatment for mouth ulcers.

 

Betel leaves (Piper betle L.) commonly known as Paan belonging to family Piperaceae, have had several applications in Indian Folk and traditional medicine. Paan leaves have been used traditionally for treatment of various diseases like bad breath, boils, conjunctivitis, constipation, and headache, leucorrhoea, swelling of gum, cuts and injuries. Phytochemical investigations have reported chemical constituents like Alkaloids, Carbohydrate, Amino acids, Tannins and Steroidal components. Various studies on biological effects of Piper betle L. leaves indicated the presence of Anti-ulcer, Antibacterial, Anti-fungal, Antioxidant and Anti-inflammatory activities (8).

 

Another suggested herb is Liquorice root (Glycerrhiza glabra L.) belonging to family Leguminosae. Previous studies have reported the potential of licorice for controlling the pain and reducing healing time of mouth ulcers (9). Martin et al (2008) in a randomized, double-blind clinical trial using a dissolving oral patch containing a licorice extract for up to 8 days, observed an improvement in ulcer size and pain compared to the use of a placebo patch (10). In another study Das et al (1989) reported analgesic effect and shortening of healing period of mouth ulcers on administering mouthwash containing a deglycerinized Licorice extract to 20 subjects (11). Guava (Psidium guajava) belonging to family Myrtaceae containing Flavonoids, Alkaloids, Glycosides, terpenoids and terpenoids have shown to exhibit Anti-oxidant, Anti-ulcer, Anti-microbial and Anticancer effects. In Recent studies, aqueous gel of powdered Guava leaves, Aloe vera and Acacia leaves shown significant anti mouth ulcer activity comparable to synthetic control (12).

 

Since microbial infection, anti-oxidant activity and immunological activity disturbances are considered important factors in etiology of mouth ulcers and the disease is often accompanied by pain and inflammation. The selected herb can be used for amelioration of symptoms and decreasing disease period due to cumulative antioxidant, antimicrobial, anti-inflammatory, analgesic and wound healing properties. Also, Tannins one of the common constituent of the selected herbs, have been linked to astringent, antiseptic and protective activity in burns and wounds (13). Hence, reported biological activities of all the three selected herbs represent the same criteria provided by both topical and systemic drugs that have been reported for treatment of mouth ulcers. We prepare a stable mucoadhesive polyherbal formulation that can be used for treatment of mouth ulcers.

 

MATERIAL AND METHODS:

Collection of Plant material:

Guava leaves were collected from the university campus and paan leaves and liquorice roots were purchased from local market underwent a validation process by Pharmacognosy specialists in department of pharmacognosy; then the leaves were dried at room temperature in shade. The plant material were then powdered and prepared for extracting. In this study Carbomer 934, Triethanolamine, methyl paraben, propyl paraben and ethanol of analytical grade purchased from Loba Chemicals Mumbai.

 

Preparation of herbal Gel:

Specified amount of Carbopol 934 was dispersed in required amount of distilled water with continuous stirring. 5ml of distilled water was taken and required quantity of methyl paraben and propyl paraben were dissolved by heating on water bath. Further varying concentration of Guava leaves ethanolic extract, betel leaves ethanolic extract and liquorice root aqueous extract were mixed to the above mixture. Finally full mixed ingredients were mixed properly to the Carbopol 934 gel with continuous stirring and triethanolamine was added drop wise to the formulation for adjustment of required pH (6.8-7). Volume was made up to 100 ml with distilled water and few drops of Peppermint oil was added as flavourant. The composition of herbal gel prepared from the powdered guava leaves coded as F1, F2, and F3 is tabulated in Table 1.

 

Table 1: Composition of various gel formulations

Ingredients	F1	F2F2	F3F3
Guava Leaves AE	2%	1%	0.5%
Beetle leaves AE	2%	1%	0.5%
Liquorice root AqE	2%	1%	0.5%
Carbopol 934	1%	1%	1%
Methyl Paraben	0.0015%	0.0015%	0.0015%
Propyl Paraben	0.01%	0.01%	0.01%
Triethanolamine	q.s+pH 6.5-7	q.s+pH 6.5-7	q.s+pH 6.5-7
Peppermint oil	q.s	q.s	q.s
Distilled water	Up to 100 ml	Up to 100 ml	Up to 100 ml

 

Evaluation of Herbal Gel:

Physical Appearance:

Physical parameters such as appearance and colour were checked.

 

 

Table 2: in vitro evaluation parameters

Formulaition	Physical appearance	pH	Homogeneity	Spread ability (gm.cm/sec)	Viscosity (Pa.S)	Extrudability
F1	Greenish	6.8±0.9	Good	5.70±0.1	3.174±0.01	Good
F2	Greenish	7±0.9	Good	5.86±0.15	3.073±0.049	Good
F3	Greenish	6.9±0.5	Good	6.52±0.05	2.334±0.012	Good

 

 

Measurement of Ph:

The pH of herbal gel formulations were determined by using digital pH meter. 1gm of gel was taken and dispersed in 10ml of distilled water and keep aside for two hours. The measurement of pH of formulation was carried out in three times and the average values are reported. pH of gel formulation was reported in table 2.

 

Homogeneity:

All developed gel formulations were tested for homogeneity by visual inspection after the gels have been set in to the container. They were tested for their presence and appearance of any aggregates. Homogeneity of gel formulation was reported in table 2.

 

Spreadability:

Spreadability was determined by glass slide and wooden block apparatus. Weights about 20gm were added to the pan and the time were noted for upper slide to move to separate completely from the fixed slide. An excess amount of gel 2gm under study was placed on this ground slide. The gel was then sandwiched between this slide and another glass slide having the fixed ground slide and there is provided with the hook. A 1kg weighted was placed on the top of the slides for 5 minutes to provide a uniform film of the gel and remove air between the slides. Excess of the gel was removed off from the edges. The top plate was then subjected to pull with the help of string attached to the hook and the time in seconds required by the top slide to cover a distance of 7.5cm be noted. A shorter or less interval indicates better Spreadability. Spreadability of gel was calculated using the following formula Spreadability of gel was reported in table no 2.

 

S = M × L / T

 

Where, S = Spreadability, M = Weight in the pan which is tied to the upper slide, L = Length moved by the glass slide T = Time in second taken to separate the slide completely each other.

 

Viscosity:

Viscosity was determined by using Brookfield viscometer (DV-III programmable Rheometer). Formulated gels were tested for their rheological behaviors at 250C. The measurement was made over range of speed from 10rpm to 100rpm with 30seconds between 2 successive speeds and then in a reverse orders.

 

Extrudability:

The gel formulations were filled in standard capped collapsible aluminium tubes and sealed to the end. The extrudability was determined by pressing of the thumb.

 

Clarity:

The clarity of all the three batches was determined by visual inspection.

 

Gel strength:

Gel strength was determined by the time in seconds required by the weight to penetrate in the gel. A Sample amount of 5gm of each of the optimize batches was taken and 3.5gm weight was placed on the surface of gel. The time in seconds required by the weight to penetrate 0.5cm in the gel. The gel strength was then reported in table 3.

 

Bioadhesive Strength:

Bioadhesive strength was determined by using glass slide and wooden block apparatus. Bioadhesive strength used to measuring the force required to detach the formulation from cellophane membrane. Specified amount that is 1 gm of prepared gel was taken on glass slide wrapped with cellophane membrane. Intimate contact was provided by the movable glass slide was placed on fixed slide. Two minute contact time was given to ensure intimate contact between formulation and membrane. The weight was added in the pan which is provided to apparatus until slides got detached. The bioadhesive force, expressed as the detachment stress in dyne/cm2 was determined by the formula. Bioadhesive strength was reported in table 3.

 

Detachment stress = m·g/A

 

Where, m = Weight required to detach two glass slides from each other (gm). g = Acceleration due to gravity i.e 980 cm/s2. A = Area of membrane exposed (cm2).

 

Stability study:

Stability studies were done with open and close container. Here, by subjecting the product to room temperature for 1 month Stability study was reported in table 3.

 

Table 3: In vitro evaluation parameters

Formulaition	Bioadhesive strength (dyne/cm2)	Gelling Strength (Sec)	Open Container	Closed Container
F1	4422.22 ± 18.82	42±0.75	Unstable	Stable
F2	3525.31 ± 31.09	36±0.07	Unstable	Stable
F3	2873.48 ± 18.25	27±0.5	Unstable	Stable

 

Antifungal activity:

The antifungal activity of all developed batches of formulation and without drug containing gel formulation i.e. blank formulation were carried out by Cup-plate method in comparison with marketed antifungal formulation (Zolef). There are two different bacteria cultures used were Aspargilious aureusand Candida Albicans. The antifungal test was performed using the agar well diffusion Prepared nutrient brought and poured in to sterile petri plates and kept for drying and cooling. After that each bacterial culture were spread by micron wire loop. A sterile cork borer 6mm diameter was used to drill holes 4 mm deep. Then 0.5gm of gel from each batches add in to this holes. Plates were then incubated at 27⁰C for 48 hr. The zone of inhibition (diameter in mm) developed, if any, was then measured for the particular compound with each fungal strength. Antifungal studies were reported in table 4.

 

Table 4: in vitro Anti-fungal study

Formulation	Aspergillus aureus (mm)	Candida albicans (mm)
Blank	12	15
F1	22±0.4	20±0.4
F2	20±0.6	19±0.6
F3	19±0.4	18±0.5
Marketed Formulation	26±0.2	28±0.4

 

RESULT AND DISCUSSION:

From the result it is clearly shown that all the prepared gel formulations having good homogeneity and gelling properties. The pH of all gel formulations was in the range of compatible with normal pH range of the skin. The rheological behavior also indicates that the gels were neither too thick nor too thin. The spreadability shows that with increasing viscosity of formulation, spreadability decreases and vice versa. Extrudability study was done by pressing thumb and it’s easily extendable. The gelling and bioadhesive strength of all the batches was found in the suitable range. one Month stability study was done with open and close container and it’s showed that open container containing gel was not stable and close container gel was stable. Formulated gel containing open container when expose to ambient room temperature then syneresis was observed it means liquid exudates separating. Syneresis occurs when the interaction between particles of the dispersed phase becomes so great that on standing. In that dispersing medium is squeezed out in droplets forms and the gel shrinks. Syneresis it means the form of instability in aqueous gels. In syneresis system separation of a solvent phase is occur only because of the elastic contraction of the polymer means polymeric molecules. All the three batches of developed formulation showed antifungal activity against Aspargilious aureus and Candida Albicans this are main microorganism responsible for mouth ulcer and formulation it can also use to treat mouth ulcer infection.

 

CONCLUSION:

In present study, it was demonstrated that the developed herbal gel formulation possess significant, therapeutically efficacious, suitable vehicle for drug delivery in low cost but definitely with high potential. Developed new herbal gel formulation is suitable for mouth ulcer treatment. However, Clinical studies are in pipeline to establish clinical efficacy of prepared herbal gels.

 

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Received on 14.05.2020          Modified on 04.06.2020

Accepted on 15.07.2020  ©AandV Publications All right reserved