|Year : 2014 | Volume
| Issue : 2 | Page : 160-167
Low resource screening method of pre-cancerous lesions and its reversal by Triphala in teen-age Indian population
Anshula Deshpande1, Shobha Tandon2, Neeraj Deshpande3
1 Department of Pedodontics, K. M. Shah Dental College, Sumandeep Vidyapeeth, Vadodara, Gujarat, India
2 Department of Pedodontics, Babu Banarasi Das College of Dental Sciences, BBD University, Lucknow, Uttar Pradesh, India
3 Department of Periodontics, K. M. Shah Dental College, Sumandeep Vidyapeeth, Vadodara,Gujarat, India
|Date of Web Publication||5-Dec-2014|
Department of Pedodontics, K. M. Shah Dental College, Sumandeep Vidyapeeth, Vadodara - 391 760, Gujarat
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Cancer screening is the main weapon for early detection at a pre-invasive or premalignant stage. It has been reported that over 12 million people use some form of tobacco, which is one of the high risk factors and has hence become an alarming world-wide problem. Aim: To evaluate the effective diagnostic screening of disease in its early stage by inexpensive method and also to evaluate the effect of indigenous mouthrinse on reversal of pre-cancerous lesions. Materials and Methods: The screening for teenagers belonging to low socio-economic status was carried out. Suspected subjects were evaluated for the reversal of the lesions by use of Ayurvedic preparation as a mouthwash. From 13 to19 years working-child population of North India was selected for the study. Screening was performed by new method-visual inspection with acetic acid. The positive subjects were further investigated by pap smear and biopsy was done as a confirmatory histopathological report. In second phase, the subjects showing positive lesions were advised indigenous anti-cancer mouth rinse and its effect was evaluated after 6 month and 9 month of prescribing the rinse. Results: The total 1095 children were screened (831 boys and 264 girls). Out of total 34 teenager boys were diagnosed, as acetowhite positive lesion. All the acetowhite positive lesions were found exclusively in males. Histological findings after 9 month use of Triphala mouth rinse revealed no changes in cells in 23 (85.2%), hyperkeratinization in 2 (7.4%), hyperkeratinization and spongiosis was evident in 1 (3.7%), mild pleomorphism in 1 (3.7%) patient. Comparative evaluation from 0-9 month showed statistically highly significant test (P < 0.01). Conclusion: Use of different forms of tobacco and betel nut showed convincing relationship between developments of oral pre-cancerous lesions. Triphala was found to have great potential for reversal of these lesions.
Keywords: Alcohol, oral pre-cancerous lesions, screening, tobacco, Triphala and teenagers
|How to cite this article:|
Deshpande A, Tandon S, Deshpande N. Low resource screening method of pre-cancerous lesions and its reversal by Triphala in teen-age Indian population. AYU 2014;35:160-7
|How to cite this URL:|
Deshpande A, Tandon S, Deshpande N. Low resource screening method of pre-cancerous lesions and its reversal by Triphala in teen-age Indian population. AYU [serial online] 2014 [cited 2020 Feb 21];35:160-7. Available from: http://www.ayujournal.org/text.asp?2014/35/2/160/146233
| Introduction|| |
Oral cancer today is the fifth most common cancer seen in the human body. Ironically it is found in the most easily accessible part, which can be detected on the expense of least possible resources. It has been reported that over 12 million people use some form of tobacco, which is one of the high risk factors and has hence become an alarming world-wide problem.
Oral cancer is major problem in India and accounts for 50-70% of all cancers diagnosed when compared to 2-3% in UK and USA.  More than 80% of cancer patients in developing countries are diagnosed at an advanced stage and do not have access to early detection or screening for early-stage cancer. Keeping this in view India needs a safe, easy to perform and inexpensive screening method, which medical and dental health professionals can apply efficiently and giving results immediately by using equipment's which are available easily.
Over the last decade, there has been largest increase in the age-group of 8-17 years old in the consumption of tobacco in different forms especially smokeless tobacco.  This is an alarming sign for the health professionals and especially the dental surgeons, as the oral cancer can be, easily examined. The early detection and prompt treatment could be provided at the stage of pre-cancerous lesions, which can be one of the corner stone of cancer prevention.
There are different treatment modalities of cancer but the alternative method with herbs and diets is practically ignored. In our economic system, if there is no profit motive, a serious focus on prevention would wreck havoc on the present economic status quo and further, there is no incentive for a highly vested pharmaceutical company to conduct research on herbs or other low-tech cancer treatments like herbal medicine .  Triphala is the most popular Ayurvedic herbal formula of India, since it is an effective laxative, which also supports the body's strength, due to its high nutritional value. Triphala uniquely cleanses and detoxifies at the deepest organic levels without depleting the body's reserves. Triphala consists of equal quantities of Haritaki (Terminilia chebula Retz.), Amalaki (Emblica officinalis Gaertn.) and Bibhitaki (Terminilia bellerica Roxb.). All three fruits have proven to be antioxidant.  The cytotoxic effects of aqueous extract of Triphala, were investigated on human breast cancer cell line (MCF-7) and a transplantable mouse thymic lymphoma (barcl-95).  These results were suggested that, Triphala possessed ability to induce cytotoxicity in tumor cells but spared the normal cells. The differential effect of triphala on normal and tumor cells seems to be related to its ability to evoke differential response in intracellular reactive oxygen species generation.  Toxicity study done showed that triphala was non-toxic up to a dose of 240 mg/kg, where no drug-induced mortality was observed.  These results evidently show the promise of Triphala as a potential chemopreventive and/or anticancer drug. ,,
In the view of increased prevalence of oral cancer and pre-cancerous lesions with predisposition of teenagers to various habits, this study was undertaken with the following aims and objectives:
- Effective diagnostic screening by inexpensive method
- To curtail the disease in its early stage
- To evaluate the effect of indigenous mouthrinse on reversal of pre-cancerous lesions.
| Materials and Methods|| |
This study was undertaken by the Department of Pedodontics and Preventive Dentistry, Manipal College of Dental Sciences, Manipal.
In total, 1095 teenage (13-19 years) population from North India and South India was included for the study. The subjects were chosen from the government schools and slum areas of Kanpur, Uttar Pradesh, India and Polytechnic College, Manipal, Karnataka, India. An informed consent form was prepared to obtain consent from the principals of the schools, as well as, the parents of the children participating in the study. A questionnaire to gather information on their oral health status and some of the risk factors of pre-cancerous lesions and pre-cancerous conditions was formulated.
Equipment used for the study were
mouth mirror, explorer, tweezers, disinfectant for instruments, gauze pieces, acetic acid 1%, gloves, towel and soap, disposable glasses, kidney trays, torch with white light, slides and fixing agent, local anaesthetic agent (xylocaine 2%), disposable syringes and needle, biopsy instruments - punch biopsy forceps and B.P. blade no. 15. An Ayurvedic mouth rinse - Triphala was used as an anti-cancer mouthrinse for interventional study. Triphala (tri-three, phala-fruits) a combination of the fruit of the Chebulic, Belleric and Emblic myrobalan trees, respectively were used in equal proportions. The Triphala powder was prepared by the Pharmacy Manufacturing Department, KMC, Manipal.
The sample size for the screening population was determined by power analysis, where 0.1 prevalence of cancer as reported by World Health Organization for the year 1982-1999 in teenage group, was used. Consent forms were collected.
The schools and areas comprising of low-socioeconomic group of working teenage population were selected. Most of the working children were included for the study so equal sex distribution was not possible during screening. The screening was conducted at the initial 1 st month. The intervention study was terminated after 9 months with follow-up of the positive acetowhite lesions cases treated with triphala mouthrinse at 3 rd , 6 th and 9 th month.
The study was conducted in three phases
First phase of the study
All the aspects of the study were reviewed and approved by the Institutional Ethics Committee, MAHE, Manipal, India. A questionnaire to gather information on their oral health status and some of the risk factors of pre-cancerous lesions and pre-cancerous conditions was distributed among the study group.
Second phase of the study comprised of
The initial examination was done in natural light and the examiner was blinded from the information gathered by the questionnaires and the oral health status was documented. Screening was carried out by Visual Inspection with Acetic acid (VIA). Acetic acid is used to enhance and "mark" the acetowhite change of a pre-cancerous lesion or actual cancer, an approach suitable for lowest-resource settings. The study consisted of oral rinsing of mouth with 1% acetic acid for 1 min and then screening [Figure 1] and [Figure 2].
This method of screening was based on the concept of naked-eye visual inspection (VIA) of the cervix with acetic-acid wash. , The data collected in the questionnaires was analyzed and risk factors were correlated with the positive acetowhite cases.
The lesion recognition was based on the classification given by Scully and Porter for white lesions, based on the cause.  The positive acetowhite subjects were further investigated by Pap smear More Details and punch biopsy was done as a confirmatory histopathological report. A separate informed consent, from the parents of the children participating in the interventional study was obtained. The biopsy sites were selected on the basis of the clinical appearance of the lesion and the staining acetowhite "mark" result.
The sections were stained by hematoxylin and eosin and the histological diagnosis was recorded using a descriptive code. The lesions were histologically classified.  Histological diagnosis was done by two pathologists who were blinded from the type of the lesions. In the cases where the tissue analyzed presented different histologic appearances, by both the pathologists the most severe histologic diagnosis was considered.
All the subjects were counseled for cessation of harmful oral habits and reinforcement of instruction for maintenance of optimum oral hygiene was done. To ensure the stoppage of habit the subject were questioned and was cross-checked with their parent/guardians and peers.
Third phase of the study
The cases diagnosed of pre-cancerous lesion or pre-cancerous condition were advised Triphala mouth rinse.
The Triphala powder was dispensed in the containers with pre-weighed pack of 60 mg and measuring cups of 100 ml to the subjects to prepare fresh mouth rinse of 0.6% concentration. The mouthrinse was freshly prepared in lukewarm water prior to rinsing so as to avoid alcoholic content which is a known etiologic factor for lesions most of the mouthrinse have alcoholic. The children were advised not to eat or rinse for the next 30 min after mouthrinse. The subjects were examined again after 3 months, 6 months and 9 months of prescribing the rinse. The smear was taken after 6 and 9 months and biopsy was done at 9 months to evaluate the effect of Triphala cytologically as well as histologically.
The Statistical Packages for Social Sciences (SPSS version 10.0) was used for analysis in which the data were compared among groups using Chi-square test (or Pearson test or linear association as appropriate) for categorical variables. For all tests, P < 0.05 was considered to be statistically significant.
| Results and Observations|| |
The relation of "acetowhite" lesions, which were recorded during screening, was studied with risk factors associated with pre-cancerous lesions. The effect of indigenous mouthrinse on reversal of pre-cancerous lesions that were diagnosed during the screening was evaluated. A total of 1095 children with a combination of 831 boys and 264 girls were screened. Out of total 34 teenager boys were diagnosed, as acetowhite positive lesion. All the acetowhite positive lesions were found exclusively in males and none in females. Chi-square analysis indicated a very high statistically significant correlation (P < 0.001).
Of the total teenagers screened, 274 (25%) of teenagers were consuming tobacco, whereas 821 (75.0%) were not consuming tobacco. The results obtained showed high statistically significant relation between acetowhite positive and tobacco consumption (P < 0.001).
Odds Ratio for tobacco 25.11 and 95% confidence interval had lower limit of 8.76 and upper limit of 71.98. 27 (2.5%) of teenagers were consuming alcohol, whereas 1068 (97.5%) were not consuming alcohol. Only 1 of the 27 alcohol consumers showed acetowhite positive lesion. The results obtained showed no statistically significant value between acetowhite positive and alcohol consumption. The correlation was statistically significant (P < 0.05) to their low monthly family income. Out of 1095 screened 53 (4.8%) belonged to the family where total family members were 3, 250 (22.8%) were from family of 4 members, 405 (37.0%) were from 5 and 387 (35.3%) from more than 5. The acetowhite lesions to the number of members in the family was not statistically significant (P > 0.05).
Site of the lesion
Totally 34 acetowhite positive lesions were found on screening 1095 teenagers. 28 (82.4%) of it were present in buccal mucosa, 5 (14.7%) in vestibule and 1 (2.9%) in retromolar region. Of the total 34 teenagers diagnosed as acetowhite positive 29 (85.3%) were having leukoplakia, 2 (5.9%) cheek biting lesions, 2 (5.9%) had traumatic ulcer and 1 (2.9%) had oral submucous fibrosis.
The teenagers having acetowhite positive lesions were given the consent form again and 27 out of 34 subjects returned the filled consent form back and agreed to participate further for the study. The attrition in sample size was of 7 (20.6%) subjects. All 34 acetowhite positive cases were administered Triphala mouth rinse, whereas 27 acetowhite positive subjects whose parents gave the written consent, the laboratory investigations were done for them (cytological and histological investigations) and were subjected to statistical analysis to evaluate the effect of Triphala on these lesions. Baseline data - pre-rinse cytology and histology report was obtained as shown in the [Table 1] and [Table 2], [Figure 3] and [Figure 4]. Comparison of 1 st visit with 6 month after use of Triphala mouth rinse by Linear by Linear Association, Chi-square value is 0.822 and with 9 month Linear by Linear Association value is 1.731 both were found statistically insignificant (P = 0.364 and P = 1.731 respectively).
|Figure 4: Biopsy showing hyperkeratinization, acanthosis and parakeratin plugging|
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|Table 1: Smear report at baseline and after 6 and 9 month follow-up with Triphala mouth rinse|
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|Table 2: Comparison of biopsy report at 1st visit and 9 month after use of Triphala mouth rinse|
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Results after administering Triphala mouthrinse
Out of 34 positive acetowhite teenagers found in screening were administered Triphala mouth rinse, the written consent was obtained from 27 of their parents or guardians who agreed for the laboratory investigations (cytological and histological investigations) and took part in the study further. These 27 patients were evaluated after 3, 6 and 9 months [Figure 5] and [Figure 6].
Post-rinse cytology report at 6 month
The cytological report at 6 months after the use of Triphala mouth rinse showed normal cells in 19 (70.4%) of the total 27 cases, 5 (18.5%) showed anucleate keratin (keratosis), keratosis with pyknotic nucleus was seen in 3 (11.1%) cases. Linear-by-Linear Association by Chi-square test showed no statistically significant correlation (P > 0.05) [Table 1].
Post-rinse cytology report at 9 month
The cytological report after 9 months use of Triphala mouth rinse revealing normal cells in 23 (85.2%) of the total 27 cases, 4 (14.8%) showed anucleate keratin (keratosis) [Table 1] and [Figure 7].
Post-rinse histology report at 9 months
Histological findings after 9 months use of Triphala mouth rinse revealed no changes in cells (normal) in 23 (85.2%), hyperkeratinization in 2 (7.4%). Hyperkeratinization and spongiosis was evident in 1 (3.7%), mild pleomorphism in 1 (3.7%) [Table 2] and [Figure 8].
Linear-by-Linear Association by Chi-square test showed high statistically significant correlation with value 8.098 (P < 0.01) [Table 2].
| Discussion|| |
As there are very few studies done in the teenagers to assess the various risk factors causing oral pre-cancerous lesions the present study was undertaken in a small group of Indian teenage population. In this study, no significant association between age group and acetowhite positive lesion (P > 0.05) indicating lesion can occur in any age group even in the younger which may be also dependent on their socioeconomic status. One study reported that age of onset of oral cancer in India is falling and is significantly lower than reported in other countries.  Similarly, another study reported the age for initiation for Gutkha in India as low as 8-14 years, which is a known risk factor for pre-cancerous lesions.  All the acetowhite positive lesions were found exclusively in males and none in females, in a cross-sectional study of aging Germans for oral mucosal lesions found similar sex predilection for males as compared to females  [Table 2]. Of the total teenagers screened, 274 (25%) of teenagers were consuming tobacco, whereas 821 (75.0%) were not consuming tobacco. Researcher carried out survey of school children in a coastal village in the state of Kerala and showed 29% prevalence of tobacco chewing similar to the present study and in Mizoram showed a rate of 56.5%, which was slightly higher.  Out of the total 1095 teenagers screened, 27 (2.5%) of teenagers were consuming alcohol. Tobacco use and alcohol consumption by young people remain serious public health problem. Another study also reported that more than 51% of high school students in the US in 1995 had consumed at least one alcoholic beverage in the preceding 30 days.  Only 1 of the 27 alcohol consumers showed acetowhite positive lesion in this study. In contrast, researcher found alcohol consumption to be more implicated factor in etiology of oral cancer than tobacco.  The association was statistically significant (P < 0.05) in relation to the teenagers of lowest income family thus showing the increased prevalence of oral mucosal and pre-cancerous lesions in low socio-economic population. In agreement to this study, another study also have reported that oral problems are more common in lower socioeconomic group. 
Site of the lesion
The subjects detected with the lesions gave a history of the placement of tobacco in the same area where the mucosa was found altered. In previous study researchers also reported that 80% of the patients with oral cavity carcinoma developed their cancers in areas where the wad of tobacco was held.  Alcohol and tobacco has site specificity in the etiology of oral cancer and found alcohol consumption to be more in patients with highest cancer incidence in floor of mouth, tongue and buccal mucosa.  Out of 127 smokeless tobacco users, 29 (22.8%) had an oral lesion at the time of examination.  Of these lesions, 23 (79.3%) were hyperkeratotic and 6 (20.7%) were epithelial dysplasia. Researcher studied prevalence of oral lichen planus and oral leukoplakia in 112 patients with oral squamous cell carcinoma.  The tongue and floor of the mouth were the most common locations and oral squamous cell carcinoma was seen to be strongly associated with these lesions.
Similar to presented study, a previous study evaluated the use of exfoliative cytology for the early diagnosis of oral cancers and its role for in education and private practice.  Exfoliative cytology may be used as an important aid in the early detection of oral cancers. Researcher studied the use of biopsy in the diagnosis and management of oral soft-tissue lesions.  Study stated that immediate biopsy should be done as a confirmatory diagnosis. An international seminar in 1983 agreed that leukoplakia is strongly associated with the use of tobacco.  In presented study, out of 34 acetowhite positive, 30 were consuming some form of tobacco and 26 out of 27 acetowhite positive lesions showed alarming changes on histological staging [Graph 1] [Additional file 1]. Unfortunately, a major impediment to treating oral cancer is that diagnosis is not usually established until 3 months after the onset of symptoms, which affects the treatments selected and the prognosis.  Late diagnosis of oral cancer is attributable to numerous factors, including insufficient oral cancer screening examinations performed in the elderly, patient procrastination and improper diagnosis of oral lesions.  The biochemical changes in cell-surface glycoproteins and glycolipids that take place during malignant transformation which has been found of clinical utility of total and lipid bound sialic acid in patients with oral pre-cancer and oral cancer. Mean values of serum sialic acid (total and lipid bound) in oral cancer were significantly higher than control and the pre-cancer group (P < 0.001). Serum sialic acid levels can differentiate between patients with oral pre-cancer and oral cancer. It could be used as an adjunct to diagnosis, monitor response to therapy and assess the staging of cancer.  The focus on young adult population has to be established to accomplish the objective of tumor down staging. Consequently, at the time of diagnosis, patients are twice as likely to have an advanced tumor compared to a localized lesion.
Post-rinse histology report
Histological findings after 9 month use of Triphala mouth rinse revealed no changes in cells in 23 (85.2%), hyperkeratinization in 2 (7.4%). Hyperkeratinization and spongiosis was evident in 1 (3.7%), mild pleomorphism in 1 (3.7%) [Table 2] and [Graph 2] [Additional file 2]. Comparative evaluation from 0 to 9 month showed high statistically significant correlation on Linear-by-Linear Association by Chi-square test (P < 0.01) [Table 2]. A clinicopathologic study of 3256 oral leukoplakias showed that 80.1% of leukoplakias were varying combination of hyperorthokeratosis, hyperparakeratosis and acanthosis without evidence of epithelial dysplasia.  Mild to moderate epithelial dysplasias was noted in 12.2% of specimens and severe epithelial dysplasia or carcinoma in situ was found in 4.5%. Another researcher also described "smokeless tobacco lesions" histologically characterized by hyperkeratinization and vacuolization of the epithelium, acanthosis and proliferation of inflammatory cells. 
The results were quite evident and showed the reversal of the lesions with Triphala mouthwash. This can be attributed to its anti-oxidant property combined with various other beneficial properties like anti-bacterial properties,  exerts heart protective and cardiotonic effect, , anti-mutagenic properties, , anti-cancer properties.  Researchers in another study also proved that all three fruits have antioxidant property.  Each of the constituents of Triphala is equally efficient in scavenging superoxide and peroxide radicals, thus working synergistically to create a powerful antioxidant and detoxifier. , Thus the direct application of herbal medicine, Triphala and rinsing with it can definitely be of great help in downstaging of pre-cancerous lesions. Although it was a small group of population screened the percentage of lesions detected was 3.1% of the total 1095 teenage population, figure that develops apprehension for the health professionals. The 25% of the teenage population involved in some or the other form of tobacco is also quiet astonishing. Thus with the results discussed above it shows that India needs a simple and low resource screening method for early detection and downstaging of pre-cancerous lesion and cancers and also to create awareness in the young adults who are the future of our society.
| Conclusions|| |
- Significant number of children with positive acetowhite lesions (3.1%) indicates the importance of early prevention and intervention in adolescents
- Adolescents are highly vulnerable to injurious habits as 25.0% were tobacco users
- A definite association is present in these young adults with various risk factors
- Low resource screening method should be mandatory for all health personals to learn and be applied in all settings
- Triphala, the indigenous mouth rinse proved to be effective in reversal of the lesions and confirmed the promise of triphala as a potential chemopreventive and/or anticancer drug.
In the view of present findings in small population the increased percentage use of smokeless tobacco in young population is quite alarming. Thus, India being a developing country with limited funds needs such screening programs on a large scale to develop awareness, prevention and protection of this young population of India. The indigenous and cost-effective Triphala mouth rinse has proved to be a key tool in prevention and reversal of the pre-cancerous lesions, which is also another feather in the cap along with wide repertoire of treatment options available. However, the herbs need to be evaluated by long-term multicentric clinical trials.
| Acknowledgments|| |
The authors would like to thank Dr. Usha Arora, Senior Pathologist, Mariumpur Hospital, Kanpur, for her skillful assistance in preparing and diagnosing the histopathological slides.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2]