DESC:FIELD
The present disclosure relates to topical formulations.
BACKGROUND
Aphthous ulcers, also known as Recurrent Aphthous Stomatitis (RAS), are characterized by repeated formation of benign and non-contagious ulcers on the mucous lining in healthy individuals. Aphthous ulcers may involve cheeks, gums, tongue, lips, roof and floor of the mouth. Aphthous ulcers are usually painful, cause redness, burning sensation, swelling and bleeding from the affected area. Aphthous ulcers can range from mild to severe and, in extreme cases, can even hinder an individual’s ability to ingest foods, and hence, make the individual susceptible to malnutrition. Aphthous ulcers usually first occur between the ages of 10 and 40 years. They then recur, but there can be days, weeks, months, or years between each bout of ulcers. Sometimes the ulcers can recur continuously, with new ulcers appearing before the previous ones have healed.
It is suggested that Aphthous ulcers are caused by T cell-mediated immune response triggered by a variety of factors, such as, nutritional deficiencies, local trauma, stress, hormonal influences, allergies, genetic predisposition and the like.
Various topical agents like corticosteroids, chlorohexidine, and systemic agents like antimicrobials, colchicine, and the like are used for the treatment of Aphthous ulcers. However, the anti-microbial agents are not capable of alleviating the symptoms of pain and burning sensation in the patients. Moreover, long term use of above mentioned medicines are associated with serious adverse reactions. Therefore, there is felt a need to provide a formulation that overcomes the drawbacks mentioned herein above.

OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
An object of the present disclosure is to provide a topical formulation which can be applied topically to treat/alleviate the symptoms of Aphthous ulcers.
Another object of the present disclosure is to provide a safe and stable topical formulation.
Still another object of the present disclosure is to provide a topical formulation which is safe, non-irritating and non-toxic.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure relates to a topical formulation comprising Curcuma longa, Piper betle, Terminalia chebula, Glycyrrhiza glabra, Syzygium aromaticum, and at least one pharmaceutically acceptable excipient. In one embodiment of the present disclosure, the topical formulation comprises ethyl acetate extract of Curcuma longa in an amount in the range of 0.1 % to 45 % with respect to the total weight of the formulation, hydroalcoholic extract of Piper betle in an amount in the range of 0.1 % to 20 % with respect to the total weight of the formulation, aqueous extract of Terminalia chebula in an amount in the range of 0.1 % to 20 % with respect to the total weight of the formulation, aqueous extract of Glycyrrhiza glabra in an amount in the range of 0.1 % to 15 % with respect to the total weight of the formulation, Syzygium aromaticum oil in an amount in the range of 0.1 % to 10 % with respect to the total weight of the formulation, and at least one pharmaceutically acceptable excipient in an amount ranging from 0.5 % to 40 % with respect to the total weight of the formulation. Typically, the topical formulation can be applied in the form of gel.
DETAILED DESCRIPTION
Aphthous ulcers are characterized by recurrent bouts of single or multiple shallow and painful ulcers in otherwise healthy individuals. Various factors, such as, nutritional deficiencies, local trauma, stress, hormonal influences, allergies, genetic predisposition and the like, can trigger the T cell-mediated immune response causing Aphthous ulcers.
Aphthous ulcer management includes topical and systemic antibiotics. However, these treatments just minimize the effect of the ulcers, and are unable to cure the ulcers and/or prevent the formation of ulcers. Also, these agents are not capable of alleviating the symptoms of pain and burning sensation in the patients.
The present disclosure therefore envisages a topical formulation having synergistic effect, that mitigates the drawbacks mentioned hereinabove.
In an aspect of the present disclosure, there is provided a topical formulation comprising Curcuma longa, Piper betle, Terminalia chebula, Glycyrrhiza glabra, and Syzygium aromaticum.
Curcuma longa (Zingiberaceae) grows in Pakistan, Sri-Lanka and Bangladesh.
Piper betle (Piperaceae) is native to central and eastern Malaysia. It is cultivated throughout Malaysia, Africa, West Indies, South Asia and South China.
Terminalia chebula (Combretaceae), also known as Chebulic Myrobalan is native to South Asia, southwest China (Yunnan), Sri Lanka, Malaysia, and Vietnam.
Glycyrrhiza glabra (Fabaceae) is native to southern Europe, and parts of Asia.
Syzygium aromaticum (Myrtaceae), also known as clove, is native to the Maluku Islands in Indonesia. It is harvested primarily in Bangladesh, Indonesia, Madagascar, Zanzibar, Pakistan, Sri Lanka, and Tanzania.
The plant parts used in the topical formulation of the present disclosure can be derived from the barks, roots, tubers, stigma, kernels, exudates, stolons, rhizome, leaves, seeds, nuts, berries, fruits, stems and flowers of the plants are commercially purchased from the market as value added products and therefore, the specific source of the product is unknown.
In an embodiment of the present disclosure, the topical formulation comprises extract of Curcuma longa, extract of Piper betle, extract of Terminalia chebula, extract of Glycyrrhiza glabra, and Syzygium aromaticum oil (clove oil).
The extract is selected from the group consisting of alcoholic, hydro-alcoholic, aqueous, ether, propylene glycol soluble, CO2, acetone, ethyl acetate, and hexane extract. Typically, the extract is prepared by using known techniques, such as percolation, decoction, maceration, soxhlet extraction, infusion and supercritical fluid extraction.
In one embodiment of the present disclosure, the topical formulation comprises:
a) ethyl acetate extract of Curcuma longa in an amount in the range of 0.1 % to 45 % with respect to the total weight of the formulation;
b) hydroalcoholic extract of Piper betle in an amount in the range of 0.1 % to 20 % with respect to the total weight of the formulation;
c) aqueous extract of Terminalia chebula in an amount in the range of 0.1 % to 20 % with respect to the total weight of the formulation;
d) aqueous extract of Glycyrrhiza glabra in an amount in the range of 0.1 % to 15 % with respect to the total weight of the formulation;
e) Syzygium aromaticum oil in an amount in the range of 0.1 % to 10 % with respect to the total weight of the formulation; and
f) at least one pharmaceutically acceptable excipient in an amount ranging from 0.5 % to 40 % with respect to the total weight of the formulation.
In accordance with the embodiments of the present disclosure, the pharmaceutically acceptable excipient is at least one selected from the group consisting of gelling agent, fluid medium, sweetening agent, flavoring agent, preservative, anti-oxidant, colorant, humectant, chelating agent, emulsifying agent, thickening agent, and combinations thereof.
The gelling agent can be selected from the group consisting of Carbomer (Acrypol 934P), hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose sodium and colloidal anhydrous silica.
The fluid medium can be selected from the group consisting of propylene glycol, isopropyl alcohol, water, and ethanol.
The sweetening agent can be selected from the group consisting of sucralose, acesulfame potassium, neotame, saccharine sodium, sucrose, aspartame stevia extracts, steviol glycoside and any sweetener derived from the stevia plant.
The flavoring agent can be selected from the group consisting of menthol, peppermint, lemon, fennel, honey, mix fruit, orange, pineapple, raspberry, strawberry, vanilla, chocolate and cardamom.
The preservative can be at least one selected from the group consisting of methyl paraben, propyl paraben, ethyl paraben, butyl paraben, bronopol, sorbic acid, benzoic acid and their salts thereof.
The anti-oxidant can be selected from the group consisting of tocopherol acetate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid, ascorbyl palmitate, citric acid, propyl gallate, methionine, sodium metabisulfite and methionine.
The colorant can be selected from the group consisting of Tartrazine, Sunset yellow, Carmoisine, Ponceau 4R, Brilliant Blue, Indigo carmine, Erythrosine and Allura Red.
The humectant can be selected from the group consisting of triacetin, glycerin, sorbitol, xylitol, maltitol, honey and propylene glycol.
The chelating agent can be selected from the group consisting of citric acid monohydrate, disodium ethylenediaminetetraacetic acid (EDTA), tetrasodium ethylenediaminetetraacetic acid (EDTA), maleic acid and tartaric acid.
The emulsifying agent can be selected from the group consisting of polyoxyethylene castor oil, Span 80, Span 20, Tween 80, Tween 20, sodium lauryl sulphate and Cremophor.
The thickening agent can be selected from the group consisting of povidone, hydroxylpropyl methylcellulose, ethylcellulose, hydroxypropyl cellulose, starch, gum acacia, xanthan gum, alginate, carboxymethyl cellulose sodium and carboxymethyl cellulose calcium.
In accordance with the embodiments of the present disclosure, the topical formulation can be in a dosage form selected from the group consisting of liquid, gel, lozenge and spray.
In an exemplary embodiment of the present disclosures, the topical formulation is in the form of gel.
In one embodiment of the present disclosure, the topical formulation in the form of gel involves the preparation of Curcuma longa slurry, a gel phase, an aqueous phase, and an oil phase.
The Curcuma longa slurry can be prepared by mixing the extract of Curcuma longa and the extract of Piper betle in a fluid medium/ humectant with stirring.
The gel phase can be prepared by dissolving at least one preservative and a chelating agent in water, followed by the addition of a gelling agent.
A slurry of a thickening agent in a humectant (emollient) is prepared and added to the gel phase prepared above, with continuous stirring.
The aqueous phase can be prepared by first heating water to a temperature in the range of 70 °C to 80 °C and dissolving extract of Glycyrrhiza glabra, extract of Terminalia chebula, a sweetening agent, an anti-oxidant, and an emulsifying agent to the heated fluid medium with stirring.
The oil phase can be prepared by dissolving an anti-oxidant and a flavoring agent in Syzygium aromaticum oil.
The emulsion can be prepared by adding the oil phase into the aqueous phase, with homogenization. The emulsion is mixed with the slurry of Curcuma longa, which is finally mixed with the gel phase with continuous stirring to obtain the topical formulation of the present disclosure.
The topical formulation of the present disclosure can be used for the prevention and/or treatment of Aphthous ulcers. The topical formulation provides an anesthetic effect and hence is capable of providing substantial relief from pain and burning sensation, when used by individuals suffering from Aphthous ulcers. The topical formulation exerts its action by a combination of mechanisms such as:
• anti-inflammatory activity of Glycyrrhiza glabra
• anti-microbial, antibacterial activity of Glycyrrhiza glabra and Curcuma longa;
• cleansing of wounds by Curcuma longa;
• subduing of bad odor and destroying oral pathogenic microorganisms by Piper betle; and
• analgesic activity of Syzygium aromaticum and Terminalia chebula
The topical formulation comprising Piper betle extract, Terminalia chebula extract, Glycyrrhiza glabra extract, Curcuma longa extract and Syzygium aromaticum oil, is in a homogenized form and the individual portions of the Piper betle extract, Terminalia chebula extract, Glycyrrhiza glabra extract, Curcuma longa extract and Syzygium aromaticum oil are value added products appearing in an unrecognizable and physically inseparable form.
The present disclosure is further described in light of the following laboratory scale experiments which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure. These laboratory scale experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial/commercial scale.
Experimental details:
Experiment 1: Topical Formulation in accordance with the present disclosure
A topical formulation in the form of gel was prepared by using the ingredients as given in Table-1. Curcuma longa extract, Piper betle extract, Terminalia chebula extract, Glycyrrhiza glabra extract, and Syzygium aromaticum oil were commercially purchased from the market as value added products and are in an unrecognizable form.

Table-1: Composition of the topical formulation of the present disclosure
S. No. Ingredients Part Used Type of extract Quantity (wt %)
1 Curcuma longa extract Rhizomes ethyl acetate 2.00
2 Piper betle extract Leaves Hydroalcoholic 1.00
3 Terminalia chebula extract Fruits Aqueous 1.00
4 Glycyrrhiza glabra extract Roots Aqueous 0.50
5 Syzygium aromaticum Oil Buds - 0.25
6 Excipients
i) Fluid medium/ Humectant Propylene glycol - 5.00
ii) Gelling agent Carbomer - 2.00
iii) Preservative Sodium methylparaben, sodium propylparaben - 0.20, 0.02
iv) Chelating agent Tetra-sodium EDTA - 0.10
v) Thickening agent Xanthan gum - 0.30
vi) Humectant/ Emollient Glycerin - 3.00
vii) Sweetening agent Sucralose - 0.02-0.10
viii) Anti-oxidant Propyl gallate - 0.05
ix) Anti-oxidant BHT - 0.10
x) Emulsifying agent Tween 80 - 2.00
xi) Flavoring agent Menthol - 0.05
xii) Water (Fluid medium) - - Qs up to 100

Experiment-2: Evaluation of the efficacy and synergistic activity of the topical formulation of the present disclosure
The topical formulation (AHPL/AYTOP/2014A: Group-I) of the present disclosure having the composition as summarized in Table-2 was used for the efficacy/clinical study. Topical formulations comprising only Curcuma longa extract (AHPL/AYTOP/2014B: Group-II), and comprising a combination of Glycyrrhiza glabra extract, and Curcuma longa extract (AHPL/AYTOP/2014C: Group-III) were used for illustrating the synergistic activity of the topical formulation of the present disclosure (the composition is summarized in Table-3 and Table-4, respectively). An Ayurvedic formulation (Table-5, Group-IV), and a conventional formulation (Table-6, Group-V) purchased from the market were used for illustrating the enhanced efficacy of the topical formulation of the present disclosure.
Table-2: Composition of AHPL/AYTOP/2014A gel (topical formulation of the present disclosure): Each gram contains:
S. No. Ingredients Botanical Name Part used Type of extract Quantity % (w/w)
1 Nagvalli Extract Piper betel Leaves Hydro-alcohol 1
2 Haritaki Extract Terminalia chebula Fruits Aqueous 1
3 Yashtimadhu Extract Glycyrrhiza glabra Stem/Roots Aqueous 0.5
4 Haridra Extract Curcuma longa Rhizomes Ethyl acetate 2
5 Lavanga oil Syzygium aromaticum Buds Oil 0.25
6 Gel base --- --- --- Q.S to make 100%

Table-3: Composition of AHPL/AYTOP/2014B gel (Curcuma longa): Each gram contains:
S. No. Ingredients Botanical Name Part used Type of extract Quantity % (w/w)
1 Haridra Extract Curcuma longa Rhizome Ethyl acetate 2

Table-4: Composition of AHPL/AYTOP/2014C gel (Glycyrrhiza glabra + Curcuma longa): Each gram contains:
S. No. Ingredients Botanical Name Part used Type of extract Quantity % (w/w)
1 Yashtimadhu Extract Glycyrrhiza glabra Stem/Roots Aqueous 0.5
2 Haridra Extract Curcuma longa Rhizomes Ethyl acetate 2

Table-5: Composition of Ayurvedic formulation purchased from market:
Sr. No. Ingredients Botanical Name Quantity
1. Khadir Extract Acacia arabica 134 mg
2. Irimed Extract Acacia farnesiana 134 mg
3. Tagar Extract Valeriana wallichii 3.2 mg
4. Rasana Extract Pluchea lanceolata 1.6 mg
5. Kushta Extract Saussurea lappa 4.8 mg
6. Lodhra Extract Symplocos racemosa 3 mg
7. Yashtimadhu Extract Glycyrrhiza glabra 40 mg
8. Karpoor Extract Cinnamomum camphora 1 mg
9. Sharkara Saccharum officinarum 150 mg
Table-6: Composition of conventional formulation purchased from market:
Sr. No. Ingredients Quantity (w /w)
1 Triamcinolone acetonide 0.1%

The efficacy and the synergistic activity of the present disclosure were evaluated by testing the formulation on subjects suffering from Aphthous ulcer. The trial was conducted at 12 centres in India. The trial was conducted in accordance with the guidelines given by the department of AYUSH (AYUSH GCP Guidelines, 2013). The trial was approved by Institutional Ethics committees and Independent Ethics Committee. The trial was registered on Clinical Trial Registry of India Platform (CTRI) on 21/04/2017. The registration number for the trial is CTRI/2017/04/008386.
The eligibility criteria for the same are mentioned below:
Inclusion Criteria:
- Subjects of both sexes of age between 18 years to 60 years clinically diagnosed with Aphthous ulcers;
- Minor mouth ulcers that have appeared less than a week prior to the enrollment;
- Ulcers at accessible locations (labial/buccal mucosa, tongue preferably);
- Subjects who can make all necessary visits in a 14 days period and comply with the study procedures; and
- Subject willing to participate in clinical trial and who have read, understood and signed informed consent form.
Exclusion Criteria:
- Subjects suffering from any other oral or dental disease or active skin disease;
- Subject under treatment of ulcers with systemic steroids, vitamins, antibiotics, antihistamines, oral Retinoids or immunomodulatory agents within three months before study entry;
- Subjects having any major systemic diseases, including genetic/endocrinal diseases, cancer, HIV/AIDS etc;
- Subjects known to have hematological disorders, ulcerative colitis or Crohn’s disease;
- Subject who had a history of probable sensitivity to any mouthwash or toothpaste;
- Subjects having Anemia or known iron, folic acid, Vitamin B12 deficiency;
- Subject having addiction of alcohol or smoking or tobacco chewing;
- Pregnant or lactating women; and
- Subjects having known hypersensitivity to ingredients used in the formulations.
The formulations were topically applied 4 times a day for 14 days or till complete resolution of ulcers, whichever was earlier. The subjects were requested to attend a total of 5 visits viz. Day 0, Day 3, Day 7, Day 10 & Day 14. The enrolled subjects were assigned one of the study Groups as per the computer generated randomization list, and the subject’s general and systemic examination was carried out. Pain from mouth ulcers was assessed on Visual Analogue Scale (VAS). The degree of burning sensation from mouth ulcers was also assessed. The number of mouth ulcers were counted and recorded. The largest Aphthous ulcer observed on the 1st visit was considered as the Index ulcer for future assessment and changes in this ulcer were recorded on next follow ups. Difficulty in consumption of solid and liquid foods was recorded on the basis of the graded scale. The subject was then given the formulation of the assigned Group. The subject was asked to apply the formulation and evaluate the formulation for taste, consistency and ease of applicability. Local tolerability of the formulation was also assessed by the subject. The timing of onset of action of the formulation and also duration of action of the formulation was also recorded. The subject was also asked to record the date of complete healing of the largest ulcer identified, in the next 14 days. The subjects were provided 1 lamitube (10g) of formulation on Day 0, 3 and 7 as per the requirement. All the subjects were advised to apply the formulation 4 times daily for 14 days or till complete healing of Aphthous ulcer, whichever was earlier. After the baseline visit, subject was called for follow up visits on 3rd day, 7th day, 10th day, and 14th day. If the Aphthous ulcers heal prior to 14 days, the subject was declared as completer.
On every follow up visit, the subject’s general and systemic examinations were carried out. Pain from mouth ulcers was assessed on VAS. The degree of burning sensation from mouth ulcers was assessed. The number of mouth ulcers were counted and recorded. The percentage healing observed in Index ulcer from the baseline visit was also recorded. The subject was asked for the day of complete healing of the Index ulcer if it had already healed. Difficulty in consumption of solid and liquid foods was recorded on the basis of the graded scale. Record of Adverse Event and Adverse Drug Reaction (AE/ADR) was maintained on each visit. On each follow up visit empty lamitube was collected from the subject and if required new lamitube was issued on Day 3 and 7. Drug compliance was assessed on the basis of number of times the subject has applied the formulation. The subject was considered compliant if he/she applied > 80% the given formulation over > 80% time in the whole study period.
On the last follow up visit, subject’s general and systemic examination was carried out. Pain from mouth ulcers was assessed on the basis of VAS scale. The degree of burning sensation from mouth ulcers was assessed. Number of mouth ulcers was counted and recorded. Difficulty in consumption of solid and liquid foods was recorded on the basis of graded scale, and AE/ADR, if any was recorded. The formulation compliance was assessed; assessment for overall improvement and tolerability of the formulation was assessed by the physician and subject. The subject was asked to stop use of the formulation and take advice for further treatment, if required.
Analysis was performed using SPSS (Statistical Package for Social Sciences 20.0) for Windows. All variables were analyzed descriptively. For descriptive statistical methods (mean, Standard deviation and frequency) analysis was performed using one-way ANOVA, Kruskal-Wallis and T-Test. The difference was considered significant only if P Value was < 0.05.
The Kruskal–Wallis test is a non-parametric method for testing whether samples originate from the same distribution. It is used for comparing two or more independent samples of equal or different sample sizes. The T-test is any statistical hypothesis test in which the test statistic follows a Student's t-distribution under the null hypothesis.
A total of 154 subjects completed the trial and were considered for the efficacy and the synergistic activity. The number of subjects in each Group is given in Table-7, and the age and gender wise distribution of the subjects is summarized in Table-8.
Table-7: Demographic details of the subjects
S. No. Groups Number of subjects
1 Group I 33
2 Group II 32
3 Group III 31
4 Group IV 34
5 Group V 24

Table-8: Age and gender wise distribution of the subjects
Groups Total Males Females Mean Age (Yrs.)
I 33 21 63.64% 12 36.36% 33.95
II 32 20 62.50% 12 37.50% 33.73
III 31 14 45.16% 17 54.84% 34.02
IV 34 20 58.82% 14 41.18% 34.11
V 24 15 62.50% 9 37.50% 33.73
Total 154 90 58.44% 64 41.56% -
The changes in pain as perceived by the subject was assessed using VAS (Screening visit /Baseline Day, Day 3, Day 7, Day 10 and Day 14 or complete remission of ulcer and symptoms, whichever was earlier). The pain assessment on Visual Analogue Scale (VAS) for all the subjects is provided in Table-9.
Table-9: Pain Assessment on Visual Analogue Scale (VAS)
Groups Day 0 Day 3 Day 7 Day 10 Day 14
Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value
I 65.45 ± 24.15 Statistic = 2.87

P value = 0.58 (Not Significant) 6.52 ± 15.13 Statistic = 13.986

P value = 0.007 (Significant) 1.06 ± 5.27 Statistic = 5.209

P value = 0.267
(Not Significant) NA Statistic = 3.607

P value = 0.462
(Not Significant) NA Statistic = 7.675

P value = 0.104 (Not Significant)
II 71.56 ± 23.71 25.63 ± 28.73 5.94 ± 18.11 2.19 ± 8.70 0.63 ± 2.46
III 72.58 ± 21.75 25.16 ± 28.27 1.13 ± 4.42 0.32 ± 1.80 NA
IV 74.41 ± 21.77 26.76 ± 27.82 4.41 ± 12.60 2.06 ± 9.14 NA
V 69.58 ± 22.55 16.25 ± 20.18 NA NA NA
By one way ANOVA- [Analysis of variance (ANOVA) is a collection of statistical models used to analyze the differences among group means and their associated procedures (such as "variation" among and between groups),]
S.D. represents the standard deviation.
In statistical hypothesis testing, the p-value or probability value is the probability for a given statistical model that, when the null hypothesis is true, the statistical summary (such as the sample mean difference between two compared groups) would be the same as or of greater magnitude than the actual observed results.
It is seen from Table-9 that the difference amongst the mean Pain score in all 5 Groups was found non-significant on Day 0. On day 3 all the test formulations significantly reduced pain. When compared among the Groups, the difference was found statistically significant. The mean pain score in Group-I was significantly (P = 0.007) lower than the mean pain scores of all other Groups. Day 10 onwards, no pain was observed in any subject of Group-I and Group-V. On Day 14 the mean pain score for Group-II was 0.63 ± 2.46 while no subject of other Groups suffered from any pain.
- The changes in the burning sensation were determined as perceived by subject using Likert scale (0- No burning, 1- Very Little, 2- Moderate, 3- Considerable, 4- Extreme);
- Changes in number of ulcers;
- Number of days required to heal the ulcers (Healing of largest ulcer considered as an Index ulcer);
- % healing of Index ulcer on each follow-up;
- Assessment of changes in ease of consumption of solid food. (0- No discomfort, 1- Very Little, 2- Moderate, 3- Considerable, 4- Extreme);
- Assessment of changes in ease of consumption of liquid food. (0- No discomfort, 1- Very Little, 2- Moderate, 3- Considerable, 4- Extreme);
- Assessment of onset of action (in minutes) and duration of action (in minutes);
- Assessment of overall improvement at the end of treatment by subject and physician;
- Assessment of test formulations in terms of:
o Consistency (excellent, good, unsatisfactory),
o Taste (excellent, good, unsatisfactory),
o Local tolerability (none, slight, moderate), and
o Ease of application (very easy, easy, difficult).
- Assessment of comparative safety of the topical formulation of the present disclosure in subjects suffering from Aphthous ulcers by assessing
o Adverse events on each follow up visit, and
o Changes in tolerability to the formulation (Adverse events) by subject and by physician at the end of the study.
The burning sensation on Likert Scale, for all the Groups is summarized in Table-10.
Table-10: Assessment of burning sensation on Likert Scale
Groups Day 0 Day 3 Day 7 Day 10 Day 14
Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value
I 2.76 ± 0.90 Statistic = 2.369
P value = 0.668 (Not Significant) 0.30 ± 0.68 Statistic = 13.543 P value = 0.009 (Significant) 0.06 ± 0.24 Statistic = 7.26
P value = 0.123
(Not Significant) NA Statistic = 3.251
P value = 0.517
(Not Significant) NA Statistic = 2.909
P value = 0.573 (Not Significant)
II 2.72 ± 0.92 0.94 ± 1.08 0.28 ± 0.81 0.06 ± 0.25 0.03 ± 0.18
III 2.84 ± 0.90 0.97 ± 1.08 0.06 ± 0.25 0.03 ± 0.18 0.03 ± 0.18
IV 3.00 ± 0.92 1.12 ± 1.12 0.24 ± 0.61 0.03 ± 0.17 NA
V 2.92 ± 1.02 0.88 ± 0.99 NA NA NA
By one way ANOVA-
A Likert scale is a psychometric scale commonly involved in research that employs questionnaires.
It is seen from Table-10 that all the formulations effectively reduced the burning sensation in mouth ulcers. On Day 0, the difference in Mean score of burning sensation was non-significant when compared among the Groups. All the Groups showed reduction in burning sensation on Day 3. When compared among the Groups, reduction in burning sensation by Group-I was statistically significant than all the other Groups (p= 0.009). For all other follow up visits there was no statistically significant difference observed in terms of burning sensation reduction among the Groups.
Table-11 summarizes the data regarding the subjects who reported completely healed ulcers.
Table-11: Number of completely healed ulcers
Groups Day 0 Day 3 Day 7 Day 10 Day 14
Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value
I 2.03 ± 1.07 Statistic = 0.278
P value = 0.991 (Not Significant) 033 ± 0.69 Statistic = 14.036 P value = 0.007 (Significant) 0.06 ± 0.24 Statistic = 5.208
P value = 0.267
(Not Significant) 0.03 ± 0.17 Statistic = 1.896
P value = 0.755
(Not Significant) NA Statistic = 3.968
P value = 0.410 (Not Significant)
II 2.09 ± 1.23 0.88 ± 0.87 0.13 ± 0.34 0.06 ± 0.25 NA
III 1.90 ± 0.91 0.94 ± 1.03 0.06 ± 0.25 0.03 ± 0.18 0.03 ± 0.18
IV 1.88 ± 0.91 1.15 ± 1.10 0.26 ± 0.71 0.06 ± 0.24 NA
V 1.88 ± 0.74 0.75 ± 0.90 NA NA NA
By one way ANOVA-
It is seen from Table-11 that the number of ulcers differed in every subject. On every follow up visit, the number of completely healed ulcers was counted and it was analyzed using one way ANOVA test. It was observed that number of ulcers healed were statistically significant in Group-I than the other Groups on first follow up visit (Day 3). Then onwards the difference in the number of completely healed ulcers was statistically insignificant when compared among the Groups.
The percentage healing of index ulcer was calculated on every follow up visit, and the results obtained are summarized in Table-12.
Table-12: Percentage Healing of Index Ulcer
Group DAY 0 DAY 3 DAY 7 DAY 10 DAY 14
M ± S.D. t S P v M ± S.D. t S P v M ± S.D t S P v M ± S.D t S P v M ± S.D t S P v
I NA NA NA 87.58 ± 23.85 99.09 ± 3.84 99.85 ± 0.87 NA
II NA NA NA 72.50 ± 28.06 2.337 .023 95.00
±
14.14 1.602 .114 98.13
±
7.38 1.333 .187 99.38
±
3.54 1.016 .314
III NA NA NA 72.90 ± 31.96 2.090 .041 96.94
±
13.82 .861 .392 98.39
±
8.98 .931 .356 99.03
±
5.39 1.032 .306
IV NA NA NA 71.76 ± 31.09 2.331 .023 93.82
±
18.26 1.622 .110 97.94
±
10.38 1.052 .297 97.06
±
17.15 0.985 0.328
V NA NA NA 67.50 ± 36.50 2.511 .015 NA -1.156 .253 NA -.851 .399 NA NA
Gp=Groups; M ± S.D.=Mean ± S.D.; t S=t Statistic; P v=P value
t Statistic is the ratio of the departure of the estimated value of a parameter from its hypothesized value to its standard error. It is used in hypothesis testing.
It is seen from Table-12, that the percentage healing of mouth ulcer in Group-I was significantly higher than Groups II, III, IV and V on Day 3. Later on the percentage healing was not significant on further visits, when compared among the Groups.
Difficulty in consumption of solid food due to mouth ulcers reduced with the treatment and the assessment of ease of consumption of solid food is summarized in Table-13.
Table-13: Assessment of Ease of Consumption of Solid Food
Groups Day 0 Day 3 Day 7 Day 10 Day 14
Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value Mean ± S.D. Statistic & P value
I 2.48
±
0.83 Statistic = 5.385
P value = 0.25
(Not Significant) 0.18
±
0.46 Statistic = 16.367 P value = 0.003 (Significant) 0.06
±
0.24 Statistic = 7.796
P value = 0.099
(Not Significant) NA Statistic = 5.448
P value = 0.244
(Not Significant) NA Statistic = 3.813
P value = 0.432 (Not Significant)
II 2.78
±
0.91 0.88
±
0.91 0.22
±
0.66 0.09
±
0.39 0.03
±
0.18
III 2.81
±
1.11 0.87
±
1.09 0.06
±
0.36 NA NA
IV 2.97
±
0.83 1.00
±
1.07 0.24
±
0.61 0.09
±
0.38 NA
V 2.92
±
0.93 0.92
±
1.18 NA NA NA

It is seen from Table-13, that on Day 3 the least difficulty was present in subjects of Group-I (0- No discomfort, 1- Very Little, 2- Moderate, 3- Considerable, 4- Extreme). The mean score of Group-I was significantly lower than any other Group (P value = 0.003) when compared between the Groups on Day 3. No significant difference in the score for ease of consumption of solid food was observed on next follow up visits.
Discomfort in consumption of liquid food resulting due to mouth ulcers, reduced with the treatment, and the results obtained are summarized in Table-14.
Table-14: Assessment of Ease of Consumption of Liquid Food
Gp DAY 0 DAY 3 DAY 7 DAY 10 DAY 14
Mean ± S.D. t Statistic P value Mean ± S.D. t Statistic P value Mean ± S.D. t Statistic P value Mean ± S.D. t Statistic P value Mean ± S.D. t Statistic P value
I 2.03 ± 1.07 0.24 ± 0.50 0.06 ± 0.24 NA NA
II 2.38 ± 1.01 -1.333 .187 0.69 ± 0.86 -2.560 .013 0.16 ± 0.57 -.880 .382 0.03 ± 0.18 -1.016 .314 NA NA
III 2.32 ± 1.05 -1.102 .275 0.65 ± 0.84 -2.348 .022 0.03 ± 0.18 .529 .599 NA NA NA NA
IV 2.41 ± 1.02 -1.491 .141 0.79 ± 1.01 -2.821 .006 0.15 ± 0.44 -1.000 .321 NA NA NA NA
V 2.25 ± 0.99 -.788 .434 0.67 ± 0.87 -2.327 .024 NA 1.222 .227 NA NA NA NA
Gp=Groups; M ± S.D.=Mean ± S.D.; t S=t Statistic; P v=P value
It is seen from Table-14, that on Day 3, the least difficulty was present in subjects of Group-I (0- No discomfort, 1- Very Little, 2- Moderate, 3- Considerable, 4- Extreme). On Day 3, the mean score of Group-I was significantly lower than any other Groups (P value = 0.003) when compared between the Groups. No significant difference in the score for ease of consumption of liquid food was observed on next follow up visits.
The time required for onset of action in seconds for all the Groups was determined, and the results obtained are summarized in Table-15.
Table-15: Time required for onset of action in seconds
Time required for onset of action in seconds Groups
I II III IV V
Below 60 Sec 21 15 15 14 14
% 63.6 46.9 48.4 41.2 58.3
60 to 120 Sec 8 9 9 11 6
% 24.2 28.1 29.0 32.4 25.0
120 to 180 Sec 1 5 4 8 3
% 3.0 15.6 12.9 23.5 12.5
180 to 240 Sec 3 1 3 1 1
% 9.1 3.1 9.7 2.9 4.2
240 or Above 0 2 0 0 0
% 0.0 6.3 0.0 0.0 0.0
Total 33 32 31 34 24
% 100.0 100.0 100.0 100.0 100.0

The frequency distribution of the subjects according to the time required for onset of action in seconds (Table-15) shows the onset of action to be within 60 seconds of application in 63.3% of subjects from Group-I, 46.9% from Group-II, 48.4% from Group-III, 41.2% from Group-IV, and 58.3% subjects from Group-V. The results obtained in Table-15 indicates faster onset of action of the formulation of Group-I as compared to the other Groups.
The duration of action in minutes for all the Groups was determined, and the results obtained are summarized in Table-16.
Table-16: Duration of Action in Minutes
Duration of Action In Minutes Groups
I II III IV V
Below 60 Min 11 17 18 12 11
% 33.3 53.1 58.1 35.3 45.8
60 to 120 Min 14 12 10 19 9
% 42.4 37.5 32.3 55.9 37.5
120 to 180 Min 5 2 3 3 4
% 15.2 6.3 9.7 8.8 16.7
180 to 240 Min 0 0 0 0 0
% 0.0 0.0 0.0 0.0 0.0
240 or Above 3 1 0 0 0
% 9.1 3.1 0.0 0.0 0.0
Total 33 32 31 34 24
% 100.0 100.0 100.0 100.0 100.0

The frequency distribution of subjects according to duration of action in minutes (Table-16) exhibits that the action of the formulation sustained for more than 240 minutes in 9.1% subjects from Group-I and 3.1% subjects from Group-II. For the other Groups the highest duration of action is seen up to 180 seconds only.
The days required for ulcer healing in the subjects were observed and are summarized in Table-17.
Table-17: Days required for Ulcer healing
Groups Mean ± S.D. Statistic & P value
I 4.39 ± 2.36 Statistic = 2.571
P value = 0.04* (Significant)
II 6.00 ± 2.27
III 5.48 ± 2.42
IV 6.12 ± 2.87
V 5.42 ± 2.15

Although the study period was scheduled as 14 days, the trial was an open ended trial. The use of formulations (Groups I to V) in the trial subjects led to faster healing and most of the subjects exhibited healing of the ulcers before 14 days. When the mouth ulcers were healed completely the subject was declared as completer and the days required for this complete healing of ulcers were recorded for every subject. When compared among all the 5 Groups, it was found that the least number of days were required in Group-I and this difference was statistically significant than all other Groups, as seen from Table-17.
The quality of all the test formulations (Groups I to V) was assessed on the basis of various parameters and the observations are summarized in Table-18.
Table-18: Quality of all test formulations
Parameters Groups Mean ± S.D. Statistic & P value
Consistency I 1.76 ± 0.44 Statistic = 1.684 P value = 0.794 (Not Significant)
II 1.84 ± 0.37
III 1.87 ± 0.43
IV 1.85 ± 0.36
V 1.88 ± 0.45
Taste I 1.64 ± 0.49 Statistic = 2.797 P value = 0.592 (Not Significant)
II 1.81 ± 0.47
III 1.77 ± 0.50
IV 1.74 ± 0.45
V 1.83 ± 0.56
Local tolerability I 1.27 ± 0.63 Statistic = 5.742 P value = 0.219 (Not Significant)
II 1.09 ± 0.30
III 1.19 ± 0.48
IV 1.26 ± 0.57
V 1.46 ± 0.72
Ease Of Application I 1.52 ± 0.51 Statistic = 2.594 P value = 0.628 (Not Significant)
II 1.69 ± 0.47
III 1.52 ± 0.51
IV 1.59 ± 0.50
V 1.58 ± 0.50
The analysis of these parameters was done using Kruskal-Wallis test. It is seen from Table-18 that there is no statistically significant difference in consistency, taste, local tolerability and ease of application for the Groups I to V.
The evaluation of the formulations by the physician and the subjects were recorded and the same is summarized in Table-19.

Table-19: Evaluation for Overall Improvement by Physician and Subject
Parameters Groups Mean ± S.D. Statistic & P value
Physician's Evaluation for Overall Improvement I 1.18 ± 0.46 Statistic = 11.893 P value = 0.018 (Significant)
II 1.75 ± 0.80
III 1.61 ± 0.76
IV 1.65 ± 0.85
V 1.58 ± 0.72
Subject’s Evaluation for Overall Improvement I 1.18 ± 0.46 Statistic = 10.615 P value = 0.031 (Significant)
II 1.63 ± 0.75
III 1.71 ± 0.82
IV 1.65 ± 0.95
V 1.58 ± 0.72

It is seen from Table-19 that the mean values of evaluation for overall improvement assessed by physician and subjects are significantly lower for Group-I than the values of the other Groups. This indicates that the formulation of the present disclosure has a synergistic effect and has enhanced efficacy as compared to other formulations in treating Aphthous ulcer.
The evaluation for the overall tolerability of the formulations (Groups I to V) were assessed by the physician and the subject, and the observations are summarized in Table-20.
Table-20: Evaluation for Overall tolerability of the formulations (Groups I to V) assessed by Physician and Subject
Parameters Groups Mean ± S.D. Statistic & P value
Tolerance to the formulation by Physician I 1.12 ± 0.42 Statistic = 0.572 P value = 0.966 (Not Significant)
II 1.09 ± 0.39
III 1.10 ± 0.30
IV 1.12 ± 0.41
V 1.13 ± 0.34
Tolerance to the formulation by Subject I 1.12 ± 0.42 Statistic = 0.572 P value = 0.966 (Not Significant)
II 1.09 ± 0.39
III 1.10 ± 0.30
IV 1.12 ± 0.41
V 1.13 ± 0.34

It is seen from Table-20 that there is no significant difference observed in the tolerability score for the formulations of the present disclosure (Group-I). Evaluation of the overall tolerability to the formulation, according to the physician and the subjects, in most of the subjects was good to excellent.
A single case of adverse event, i.e., fever was observed in one of the subjects, which subsided without stopping the study medication (formulation). Therefore, it was suggested that the fever was not related to the formulation. No other adverse reaction was observed in any of the subjects under study indicating that the formulations (Groups I to V) were safe for human use.
TECHNICAL ADVANCEMENTS
The present disclosure described herein above has several technical advantages including, but not limited to, the realization of:
? a topical formulation that is capable of bringing immediate relief from pain and burning sensation in individuals suffering from Aphthous ulcers;
? a topical formulation having anti-bacterial, anti-inflammatory, wound healing and analgesic, anesthetic activities; and
? a topical formulation that does not have any undesirable side effects.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Variations or modifications to the formulation of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this invention.
The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
,CLAIMS:WE CLAIM
1. A topical formulation comprising:
- Curcuma longa;
- Piper betle;
- Terminalia chebula;
- Glycyrrhiza glabra;
- Syzygium aromaticum; and
- at least one pharmaceutically acceptable excipient.
2. A topical formulation comprising:
- ethyl acetate extract of Curcuma longa in an amount in the range of 0.1 % to 45 % with respect to the total weight of said formulation;
- hydroalcoholic extract of Piper betle in an amount in the range of 0.1 % to 20 % with respect to the total weight of said formulation;
- aqueous extract of Terminalia chebula in an amount in the range of 0.1 % to 20 % with respect to the total weight of said formulation;
- aqueous extract of Glycyrrhiza glabra in an amount in the range of 0.1 % to 15 % with respect to the total weight of said formulation;
- Syzygium aromaticum oil in an amount in the range of 0.1 % to 10 % with respect to the total weight of said formulation; and
- at least one pharmaceutically acceptable excipient in an amount ranging from 0.5 % to 40 % with respect to the total weight of said formulation.
3. The topical formulation as claimed in claim 1 or claim 2, wherein said formulation is in a dosage form selected from the group consisting of liquid, gel, lozenge, and spray.
4. The topical formulation as claimed in claim 1 or claim 2, wherein said formulation is in the form of gel.
5. The topical formulation as claimed in claim 1 or claim 2, wherein said pharmaceutically acceptable excipient is at least one selected from the group consisting of gelling agent, fluid medium, sweetening agent, flavoring agent, preservative, anti-oxidant, colorant, humectant, chelating agent, emulsifying agent, thickening agent, and combinations thereof.
6. The topical formulation as claimed in claim 1 or claim 2, wherein said pharmaceutically acceptable excipient comprises a mixture of propylene glycol, Carbomer, sodium methylparaben, sodium propylparaben, tetra-sodium ethylenediaminetetraacetic acid (EDTA), water, xanthan gum, glycerin, sucralose, propyl gallate, butylated hydroxytoluene, Tween 80, and menthol.
7. The topical formulation as claimed in claim 1 or claim 2 for use in the prevention and/or treatment of Aphthous ulcers.