ABSTRACT

Oral sub mucous fibrosis (OSF) is a high-risk precancerous condition, associated with areca nut habit. The present study was conducted to evaluate the degree of improvement in clinical signs and symptoms of the disease before and after treatment.

Study Design: A pre- and post-treatment evaluation was completed by 80 patients suffering with oral submucous fibrosis. Total patients were divided in 4 groups of 20 each according to type of therapy received. Degree of mouth opening, tongue protrusion, cheek flexibility and burning sensation of mouth were assessed in every group of topical application, Aloe-Vera and Turmeric and/or therapeutic ultrasound.

Setting: All 80 patients with oral submucous fibrosis were selected from outpatient department of Oral Medicine and Radiology, Datta Meghe Institute of Medical Sciences (Deemed to be University), Sawangi (M), Wardha, Maharashtra; India. The study was conducted between January 2016-September 2017.

Results: Significant improvement was seen in all four groups of patients in clinical parameters of degree of mouth opening, tongue protrusion, cheek flexibility and burning sensation of mouth (p-Value=0.0001).

Conclusion: Topical use of Aloe-Vera and Turmeric, in OSF patients proved as safe, non-invasive and effective treatment modalities in improving mouth opening, tongue protrusion, cheek flexibility and reduction in burning sensation. Significant improvement in clinical signs and symptoms were achieved in Aloe- Vera and Turmeric with therapeutic ultrasound.

Keywords: Oral submucous fibrosis; Turmeric; Aloe-Vera; Therapeutic ultrasound.

INTRODUCTION

Oral submucous fibrosis (OSF) is a premalignant condition associated with the chewing of Areca nut (betel nut). [1] Approximately 600 million persons is areca nut habitual globally and fourth most consumed drug after nicotine, ethanol, and caffeine.[2, 3] Both Aloe-Vera and Turmeric (Curcuma longa) inhibit pro- inflammatory enzymes, histamines, leukotrines, Interleukins and has anti-inflammatory, immunomodulatory, antioxidant & anticancer properties. [4-7] Sterols in the Aloe-vera have similar action of cortisone without any side effects. [5] Ultrasound therapy increases the cell membrane permeability by altering sodium and potassium ion gradients. Ultrasound decreases inflammation, increases vasodilatation and waste removal, accelerates lymph flow, and stimulates metabolism the effect pf aloe. [8] Considering the effect of Aloe-Vera and Turmeric and Therapeutic ultrasound the present study was planned to evaluate its effect in the management of oral submucous fibrosis.

MATERIAL AND METHODS

The present study compared the different non-invasive treatment protocol for OSF patients. This study was conducted after approval from Institutional Ethics Committee, in department of Oral Medicine & Radiology, Sharad Pawar Dental College and Hospital and Dattatray Ayurvedic Rasashala, Mahatma Gandhi Ayurveda College and Research center, Salod, a constituent unit of Datta Meghe Institute of Medical Sciences (DEEMED TO BE UNIVERSITY), Wardha, Maharashtra, India.

Total 80 patients were included after obtaining an informed consent. Each patient detailed case history was recorded. All included subjects were explained the purpose of study. They were asked to discontinue the medicine & report immediately. Subjects reported with burning sensation, soreness of mouth or urticarial rashes on body surface were treated appropriately and excluded from study. Subjects had been divided into four groups.

Group 1: 20 OSF subjects were received ultrasound therapy for alternate days in a week for one month.

Group 2: 20 OSF subjects were received local application of Aloe-Vera thrice daily for one month.

Group 3: 20 OSF subjects were received local application of Turmeric gel, thrice daily for 1 month.

Group 4: It was divided in 2 subgroups:

A) 10 OSF subjects received local application of Aloe-Vera and Turmeric gel, three times in a day for 1 month.

B) 10 OSF subjects received local application of Aloe-Vera and Turmeric gel, three times in a day for 1 month along with ultrasound therapy alternate days for 1 month.

Inclusion criteria: Patients above the age of 18 years clinically diagnosed as oral submucous fibrosis.

Exclusion criteria:

1. Subjects having known allergic reaction to Aloe-Vera or turmeric.

2. Subjects who are undergoing treatment for oral submucous fibrosis in last 6 months.

3. Subjects with history of any systemic illness.

4. Pregnant or lactating women. No blinding was there. All the enrolled patients were aware of the type of medicament they received.

Preparation of Aloe-Vera and Turmeric gel for local application:

Aloe-Vera extracts gel: Freeze-dried Aloe-Vera powder was purchased from Aashna International manufacturing company (Batch no. AIFZ/15-10/G/200:1). Fifty grams of Aloe-Vera powder was mixed to 100 ml of distilled water and 150 ml of Aloe-Vera extract juice was prepared. Seven grams of carbopo^l9 weighed accurately by using electronic weighing machine and soaked in 630 ml of distilled water for 24 hours.70 ml of fresh Aloe-Vera juice was taken and added to the carbopol. The mixture was stirred until it gets mix uniformly. After it gets mixed uniformly, 1.4 gm of methyl paraben was added in the mixture. After that, 3.6 ml of triethanol amine was added in the mixture stirred until it gets distributed uniformly and get required consistency.

Turmeric extracts gel: Roots of curcuma longa purchased directly from the farmer cultivating the turmeric only on rain water without irrigation which might be containing optimal curcumin level. In the present study whole turmeric extract was used. 100 grams of turmeric powder was taken in sterilized vessel and 500 ml of water was added heated and stirred continuously unless it was reduced to 125ml. Seven grams of carbopol weighed accurately by using electronic weighing machine and soaked in 360 ml of distilled water for 24 hours. After that it sieved with the help through sterile cotton cloth (pore size = 1 nanometer). And the turmeric extract was collected in a cleaned glass beaker. Seventy ml of turmeric extract was taken and added in the corbopol (which was soaked for 24 hours).The mixture was stirred with the help of electronic stirrer. When it get stirred properly and mixed well, methyl paraben 1.4 grams was added in the mixture. After that, 3.6 ml of triethanol amine was added in the mixture and stirred until it gets in uniform consistency.

Combined Aloe-Vera and Turmeric gel: 35 ml of fresh Aloe-Vera juice and 35 ml of turmeric extract was taken and added in the carbopol. Seven grams of carbopol weighed accurately by using electronic weighimg machine and soaked in 630 ml of distilled water for 24 hours. The mixture was stirred until it gets uniform consistency with the help of automatic stirrer. After that, methyl paraben 1.4 gm was added in that mixture. After that 3.6 ml of triethanol amine was added in the mixture by drop count method and stirred until it gets homogenous required consistency. The pH of all 3 formed gels were checked and maintained at 6 to 6.5. The formed gels then filled in the 5 grams collapsible tubes with the help of automatic filler. 5 gm of prepared gels was dispensed to the patients and advised local application to the affected area three times daily for 4 weeks.

Therapeutic ultrasound was given in a dose ranging from 0.6 to 2w/cm2, pulsed 1:1, in the frequency of 3 MHz, using a 5 cm diameter transducer head for 3 to 4 minutes to each side involved over the cheeks, lips and submental region. All patients were evaluated for the degree of mouth opening, tongue protrusion, burning sensation on weekly basis till the end of 4th week. Baseline data was recorded before starting the therapy.

First author executed the group wise treatment protocol and second performed the measurements at weekly intervals. Each subject was questioned about what areca nut products consumed, e.g. locally prepared or commercially available. All patients were counseled for cessation of habit and assisted for habit cessation at every visit till the end of 4th week. Sample size was calculated using Lamorte’s power Calculations, Boston University Research, which was resulted as 20 patients in each group. Collected data was analyzed by using X2 and Pearson statistical test using SPSS (17.3) statistical package.

RESULTS

Present studies revealed that maximum number of the patients, 71 (88.75%) were in the age groups of 15-44 years; (Mean ± SD=30.78±7.22, Range=15-50 years) & male: Female Ratio was 8:1(Table-1). Mixed use of Kharhaa, Paan-Masala and Gutkha was found in 49 (61.25%) of the patients, 38(47.5%) used both right and left vestibule for the placement of quid. Mean frequency of consumption was found to be 9.8±5.06 in the range of 3-30 times per day while mean duration of the habit was 11.68±6.59 in the range of 5-30 years (Table-2).

All patients presented with blanching and fibrosis. Maximum patients 69 (86.25%) were presented with inability to blow cheeks on both sides; restricted tongue movement was seen in 66 (82.5%) patients. 34(42.50%) patients presented with normal uvula, 27(33.75%) patients presented with atrophic uvula, 11(13.75%) patients presented with hockey stick uvula, only 8(10%) patients presented with Shrunken uvula. 32(40%) patients presented with inter-incisal mouth opening less than 15 to 25mm, 29(36.25%) patients presented with inter-incisal mouth opening in the range of >25 to 35mm,16(20%) patients presented with inter-incisal mouth opening up to or less than 15mm. 3(3.75%) patients presented with inter-incisal mouth opening more than 35 mm.(Table-3) Post-treatment mean mouth opening ( Inter-incisal distance) & tongue protrusion in all four groups was found to be improved significantly with p =0.0001(Table-4). Post treatment Patients perception on VAS SCALE for mouth opening, tongue protrusion, cheek flexibility & burning sensation in all four groups was found to be improved significantly with p = 0.0001 (Graph :1- 4).

All the subjects included stopped the habit and there were no signs of again starting of habit in the studied subject. No patient was reported with any allergic or adverse effect included in the study.

Table 1: Demographic characteristics of study population (n=80)

Table 2: Characteristics of Areca nut and Tobacco chewing Habits of study population (n=80)

*BN-Betel nut, TL –Tobacco lime, K- Kharhaa, PM-Paan-Masala, G-Gutkha

Table 3: Clinical characteristics of study population (n=80)

Table 4: Pre and Post- treatment comparison of Mouth Opening (inter-incisal distance) and Tongue Protrusion (in mm).

(p =0.0001) A-Aloe-Vera, T-Turmeric, UST-Ultrasound

Graph 1: Pre and Post- treatment comparison of mouth opening (Inter-incisal distance) in all groups on VAS Scale

Graph 2: Pre and Post- treatment comparison of tongue protrusion in all groups on VAS Scale

Graph 3: Pre and Post- treatment comparison of cheek flexibility in all groups on VAS Scale

Graph 4: Pre and Post- treatment comparison of burning sensation in all groups on VAS Scale

FIGURE 1: Pre and Post treatment improved mouth opening at the end of 4^th week in

Aloe-Vera, Turmeric, & Ultrasound group.

FIGURE 2: Pre and Post treatment reduced upper labial mucosa fibrosis at the end of 4^th week Aloe-Vera, Turmeric, & Ultrasound group.

FIGURE 3: Pre and Post treatment reduced lower labial mucosa fibrosis at the end of 4^th week Aloe-Vera, Turmeric, & Ultrasound group.

FIGURE 4: Pre and Post treatment reduced buccal mucosa fibrosis at the end of 4^th week Aloe-Vera, Turmeric, & Ultrasound group

Discussion:

In the present study, maximum number of the patients (88.75%) were from the age groups of 15-44 years with (Mean ± SD=30.78±7.22, Range=15-50 years). This observation is different from Pindborg et.al. who reported the maximum number of OSF cases in the age group of 50-69 years in their study. [10] Increase in the chewing habit of betel nut with or without tobacco at the early age due to easily available commercial products containing areca nut may explain the decrease in the age of starting chewing habits. Age range in the present study was in accordance with the previous studies. [11-13] Male: Female Ratio was 8:1. Male predominance in our study can be correlated with the male dominance in OSF studies reported by various authors. [12, 14-17]

Ultrasound Therapy (Group 1)

Ultrasound therapy showed mean improvement in mouth opening and tongue protrusion of 2.6 mm and 1.7 mm respectively. Patient’s perception on VAS for all four parameters was improved at the end of 4th week. (p =0.0001). Increased local vasodilatation reduce the inflammation and burning sensation through removal of waste products. Desruption of collagen cross linkage may altered the elasticity of fibrous band thus improved mouth opening [18].

Aloe-Vera gel Therapy (Group 2)

Aloe-Vera therapy showed mean improvement in mouth opening and in tongue protrusion of 2.35 ± 0.47mm and 1.8 ± 0.02 mm respectively. Patient’s perception on VAS for all four parameters was improved at the end of 4th week. (p =0.0001) Soothing effect of Aloe-Vera prevents injury to epithelial tissues. [19]Accelerated vascular endothelial growth factor (VEGF) and TGF-1 along with growth factors and cytokines might be responsible for favorable alteration in inflammatory pathway which helps in reducing hyalinization of collagen tissues. [20, 21] There is increased synthesis of hyaluronic acid and dermatan sulfate in the granulation tissue of a healing wound following oral or topical treatment with Aloe-Vera. [22] Anti-genotoxic and antioxidant potentials of Aloe-Vera activate molecular mechanisms of DNA repair and effectively removes the damage. [20, 22, 23] Also sterol in the Aloe-Vera has strong ability to inhibit inflammation similar to the action of cortisone without any side effects. [5]

Turmeric Therapy (Group 3)

Turmeric therapy showed mean improvement in mouth opening and tongue protrusion was 2.85 mm and 1.8 mm respectively. Patient’s perception on VAS for all four parameters was improved at the end of 4th week. (p =0.0001). Turmeric is nontoxic and includes a type of therapeutic properties with active constituents of turmeric are the flavonoid, curcumin(diferuloylmethane) and varied volatile oils, together with tumeronea, atlantone, and zingiberone [24]

In the present study, whole extract of turmeric was used. The lack of efficacy of oral curcumin alone is because of the rapid metabolism of curcumin into its less effective conjugated forms of curcumin glucuronides and curcumin sulfates or, alternately reduced to hexahydrocurcumin .[25] In the experimental studies it was found that Turmeric had more effect on proinflammatory genes compare to curcumin.[26,27] Anti-inflammatory and antioxidant activities of curcumin are due to its capacity of inhibition of lipo-oxygenase and cyclo-oxygenase (COX) activities that can reduce inflammation by lowering histamine levels and increasing the production of natural cortisone [28] Thus helps in reduction of the burning sensation with normal and spicy food.

Curcumin lowers the activity of smokeless tobacco extract (STE) or NNK (nicotine derived

Nitrosamine ketone)-induced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and COX-2 in oral premalignant and malignant cells. Turmeric acts by increasing the number of micro nuclei in the circulating lymphocytes and by acting as an excellent scavenger of free radicals.

Curcumin also has a fibrinolytic property due to its ability to inhibit lipid peroxidation and decreasing cellular proliferation, which inhibit rate of collagen synthesis [29]In experimental studies curcumin down regulated cellular proliferation (COX2, cyclin-D1, and c-myc ), anti-apoptosis (IAP1, IAP2, XIAP, Bcl2, BclxL , Bfl1/A1, TRAF1, and cellular c-FLIP), and metastasis (VEGF, MMP-9, ICAM1).[30, 31] Thus its anti-inflammatory, antioxidant, anti-carcinogenic, immunomodulatory and anti-fibrotic properties are responsible for the improvement of clinical signs, symptoms & may be responsible for reducing malignant changes in OSF. [24, 25, 27-30]

Aloe-Vera and Turmeric Therapy (Group 4: A)

                Aloe-Vera and Turmeric group showed mean improvement in mouth opening and tongue protrusion of 3.1 mm and 1.8mm respectively. Patient’s perception on VAS for all four parameters was improved at the end of 4th week. (p =0.0001). Aloe- Vera and Turmeric (Curcuma longa) are most commonly used herbal medicines like liver tonics, anti-stress, antioxidants, antitoxic and growth promoting products. Apart from these benefits, these two herbs are used for various similar functions like antibacterial, antiseptic, anti-inflammatory, nematocidal and immunomodulatory properties. [4, 4, 20-30] Aloe-Vera and turmeric both has effect on nitrous oxide. Both reduces the nitrous oxide level which prevent or reduces the formation of cytokines, interlukin-1, interlukin-6, and interferons which will make the collagen more pliable.

Thus Aloe-Vera and turmeric might be responsible for degradation of collagen as it interferes with the synthesis of nitrous oxide. Turmeric inhibits lysyl oxidase pathways, reducing the chances of collagen deposition and elastin to insoluble extracellular matrix therefore reduces the chances of progression in fibrosis. [31] The role of antibacterial and anti-inflammatory agent in the wound healing process is to reduce the level of nitric oxide in the affected area.

Curcumin at 0.1 to 10μg/ml was founded to have the ability to decrease the NO level. [32] Topical delivery is an attractive route for drug delivery. As in the present study both the gels were used in combination, penetrating property of Aloe-Vera [33] had been helpful to deeper delivery of turmeric gel into the affected mucosa such way combined effects of both gels brought the desired results in OSF patients.

Aloe-Vera, Turmeric and Ultrasound group (Group 4: B)

This group showed mean improvement in mouth opening and tongue protrusion of 5.9 mm and 2.7mm respectively. Patient’s perception on VAS for all four parameters was improved at the end of 4th week. (p =0.0001).

In the present study increased cell permeability by therapeutic ultrasound [8] might responsible for increased local delivery of Aloe-Vera and Turmeric in deeper tissues, which achieved reduction in burning. In addition, increase extendibility of collagen fibers in ultrasound therapy resulted in improved check flexibility and mouth opening and tongue protrusion.

Maximum post-treatment mouth opening was found in combined therapy of Aloe-Vera, Turmeric and Ultrasound group (Group 4-B). (Mean improvement of 5.9 mm and on VAS by 5.2.) Maximum post-treatment tongue protrusion was seen in ultrasound therapy group. (Group 4) (Mean improvement of 1.7 mm and on VAS by 5.12.) Maximum post-treatment improved cheek flexibility was found in combined therapy of Aloe-Vera, Turmeric and Ultrasound group and mean improvement on VAS by 7.7. (Group 4-B). Maximum post-treatment reduction in burning sensation was found in combined therapy of Aloe-Vera, Turmeric and Ultrasound group (Group 4-B). (Mean improvement on VAS by 8.3). The mast cell response in oral sub mucous fibrosis was reported to be high in the early stages where the reaction of the tissue to the irritant is strong. As the disease advances tissue becomes less reactive, gets hyalinized and occasionally shows degeneration where the mast cells are reduced and are often even fewer than in the normal mucosa. The vascular response in this condition was observed to be complex consisting of persistent dilatation of blood vessels especially in moderately advanced cases. These investigations opined that the mast cell and the vascular response in this condition are similar to those described in certain disease of autoimmune origin. [34] Bhatt and Dholakia [35] reported abundant mast cells in Grade I and Grade II OSF were 4.5 and 4.9 respectively as compared to 1.02 in normal buccal mucosa. They noted abundant vesicle formation and symptoms of itching sensation to histamine released from the mast cells and suggested the concept of mast cell histamine chain.

The mast cell hyperplasia could probably attribute to some of the signs and symptoms of OSF. Mast cell mediators like prostaglandins and leukotrienes are potent secretogouge for the serous and mucous cells. This could attribute to the increased salivation seen in OSF. [36]

Curcumin inhibits histamine release and the secretions of tumor necrosis factor-α (TNF-α) and interleukin-4 (IL-4) from mast cells triggered by IgE, calcium ionophore A23187, or compound 48/80. [20-37] While Aloe-Vera treatment has accelerated the rising rate of expression in gene of vascular endothelial growth factor (VEGF) and TGF-1 in the area of wound. [38] Curcumin also suppressed COX-2 expression and attendant PGD2 generation. On the other hand, Alprogen, one of the chief components in Aloe-Vera gel, inhibit calcium influx into mast cells, thereby inhibiting the antigen-antibody-mediated release of histamine and leukotriene from mast cells. [37]Bautista et al stated that carboxypeptidase in Aloe-Vera inhibits prostaglandin synthesis and arachidonic acid, thus a potent anti-inflammatory agent. Salicylate magnesium lactate decarboxylase in aloe-vera inhibits histidine, thus preventing the formation of histamine from histidine in mast cells. [39]

Considering the close association between mast cells and fibroblast in pathogenesis of OSF, therapeutic implications of these findings include strategies directed toward mast cell endothelial cell axis. As mast cells play important role in initiation as well as in the chronicity of inflammation, the action of Turmeric and Aloe-Vera on inhibition of mast cells is the promising therapeutic modality.

Present study achieved the positive effects of Aloe-Vera and Turmeric along with therapeutic ultrasound in relieving the signs and symptoms of OSF.

Aloe-Vera and Turmeric inhibit the cyclooxygenese pathway, reduces prostaglandin E2 production. In addition, both contain natural sterol which has steroid like action without any side effects. All these contribute to anti-inflammatory effects. In Aloe-Vera and Turmeric therapy, effective inhibition of cell proliferation, induced apoptosis through suppression of NFkB, reduced fibroblastic proliferation, through lipid peroxidation reduce MDA level, which has the deleterious effect on cross linking of collagen. It inhibits hydroxyl radicals (OH) and nitric oxide formation, suppresses superoxide dismutase and glutathione peroxidase, and acts as radical scavenger. Thus, contributes to their antioxidant action. Ultrasound selectively raises the temperature in target areas, increases local drug delivery by increasing cell membrane permeability and thus optimal penetration of locally applied proven anti-inflammatory, antioxidant natural medicinal Aloe-Vera and Turmeric gels. [19-40]

CONCLUSION

Due to inherent properties of medicinal plants Aloe-Vera and Turmeric, their topical use on affected sites in OSF patients proved as safe, non-invasive and effective treatment modalities in improving mouth opening, tongue protrusion, cheek flexibility and reduction in burning sensation. There were no adverse effects of topical application of either gel during intervention period. Maximum utilization and enhanced drug delivery were achieved with the help of therapeutic ultrasound.

Conflicts of interest: There are no conflicts of interest.

Acknowledgement: Our thanks are due to Professor and Head, Dept. of Ras-Shala, Dr. Bharat Rathi, Ex- Assistant Professor Dr. Gokarna for their valuable assistance in preparation of Aloe-Vera and Turmeric whole extract gel.

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1. Tilakaratne WM, Klinikowski MF, Saku T, Oral Submucous fibrosis: Review on etiology and pathogenesis. Oral Oncol, 2006. 42: p. 561-568.

2. Pillai MR, Balaram P, Abraham T et al; Lymphocyte population in premalignant lesions and cancer of the oral cavity. Neoplasma, 1987. 34(4): p. 469-79.

3. Gupta PC, Ray CS, Epidemiology of betel quid usage. Ann Acad Med Singapore, 2004. 33(l): p. 31-36.

4. Monica.B, Monisha.R Aloe vera in dentistry- A review. IOSR Journal of Dental and Medical Sciences, 2014. 13 (B): p. 18-22.

5. Patil S, Halgatti V, Maheshwari S, Santosh BS, Comparative study of the efficacy of herbal antioxidants oxitard and aloe vera in the treatment of oral submucous fibrosis. J Clin Exp Dent, 2014; 6(3): e265-70.

6. H. Hatchera, R. Planalpb, J. Chob, F. M. Tortia, d, and S. V. Tortic, Curcumin: From ancient medicine to current clinical trials. Cell. Mol. Life Sci. 2008. 65(11): p. 1631-52.

7. Sa G., Das T, Anti cancer effects of curcumin: Cycle of life and death. Cell Div, 2008.

8. Vijayakumar M, Priya D, Physiotherapy for improving mouth opening and tongue protrution in patients with oral submucous fibrosis. International journal of pharmaceutical science and health care, 2013. 2(3): p. 50-57.

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