Accepted Manuscript

Clinico-biochemical evaluation of turmeric with black pepper and nigella sativa inmanagement of oral submucous fibrosis – a double blind, randomized preliminarystudy

Dr Pratik R. Pipalia, M.D.S, Dr Rajeshwari G. Annigeri, (Prof and Head), M.D.S, DrRanjeeta Mehta, M.D.S

PII: S2212-4403(16)30276-0

DOI: 10.1016/j.oooo.2016.07.023

Reference: OOOO 1568

To appear in: Oral Surgery, Oral Medicine, Oral Pathology and OralRadiology

Received Date: 13 April 2016

Revised Date: 8 July 2016

Accepted Date: 25 July 2016

Please cite this article as: Pipalia PR, Annigeri RG, Mehta R, Clinico-biochemical evaluation of turmericwith black pepper and nigella sativa in management of oral submucous fibrosis – a double blind,randomized preliminary study, Oral Surgery, Oral Medicine, Oral Pathology and Oral Radiology (2016),doi: 10.1016/j.oooo.2016.07.023.

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Title: Clinico-biochemical evaluation of turmeric with black pepper and nigella sativa

in management of oral submucous fibrosis – a double blind, randomized preliminary

study.

Name of Author

Author 1(Corresponding Author).

Dr Pratik R Pipalia , M.D.S., Department of Oral Medicine and Radiology, College

of Dental Sciences, Davangere, KARNATAKA, INDIA

E-mail: dr@pratikp.com

Phone-+91800787587

Author 2.

Dr Rajeshwari G Annigeri (Prof and Head), M.D.S., Department of Oral Medicine

and Radiology, College of Dental Sciences, Davangere, KARNATAKA, INDIA

E-mail: rajeshwariannigeri@rediffmail.com

Author 3:

Dr Ranjeeta Mehta M.D.S., Department of Oral Medicine and Radiology, Seema

Dental College, Rishikesh, UTTRAKHAND, INDIA

E-mail: ranjeetamehta20@gmail.com

Abstract word count: 197

Complete manuscript word count: 4748

Number of references: 58

Number of figures: 3

Number of tables: 4

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ABSTRACT

Title: Clinico-biochemical evaluation of turmeric with black pepper and nigella sativa in

management of oral submucous fibrosis – a double blind, randomized preliminary study.

OBJECTIVE :

To investigate the effectiveness of turmeric with black pepper and nigella sativa in Oral

submucous fibrosis(OSMF).

STUDY DESIGN: Forty OSMF patients were included and randomly divided into two

groups. Study was performed as double blind randomized design. Group A received turmeric

with black pepper and group B received nigella sativa, for 3 months. Clinical evaluation was

done at every 15 days. Patients’ serum superoxide dismutase (SOD) levels were assessed

before and after treatment and also compared with healthy controls. The response to

treatment was analysed using ANOVA, paired t-test, unpaired t-test.

RESULTS: After the treatment group A and group B showed 3.85±0.22 mm and and

3.6±0.07 mm improvement in mouth opening respectively (p<0.01); 87.90% and 78.91%

reduction in burning sensation respectively(p<0.01); and +0.62 U/ml and +0.74 U/ml

improvement in serum SOD levels respectively (p<0.05). The maximum MO achieved was

8mm and 7mm in group A and B respectively. The mean pre-treatment SOD level for the

controls and patients was 3.61 ± 0.24 U/ml and 2.63 ± 0.18 U/ml respectively.

CONCLUSION : Turmeric with black pepper and nigella sativa improved mouth opening,

burning sensation and SOD levels in the present OSMF study subjects, however further

investigations are needed.

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INTRODUCTION :

Most conditions affecting the oral mucosa are acquired through environmental and

lifestyle factors. In India and South-east Asia, use of smokeless tobacco in various forms is

very popular. This habit, which usually involves chewing of a betel quid (combined areca

nut, betel leaf, tobacco and slaked lime), has led to the development in a significant

proportion of users, of a unique generalized fibrosis of the oral tissues called Oral submucous

fibrosis (OSMF).1 In India, the prevalence increased over the past four decades from 0.03%

to 6.42%. 2, 3 Data published earlier reported an estimate of 5 million OSMF patients in

India.4 OSMF is characterized by inflammation and progressive fibrosis of the lamina

propria. Further it has characterized by juxta-epithelial fibrosis, along with atrophy or

hyperplasia of overlying epithelium and accumulation of hyalinized collagen beneath the

basement membrane with a progressive loss of vascularity. This potentially malignant

condition has reported malignant transformation rate of 7-13%. 5

The etiology of OSMF is multifactorial, but the most consistent factor identified

through epidemiological studies is areca nut consumption in form of quid, which is

associated with the generation of free radicals.6-9 Free radicals are usually inactivated and

scavenged by antioxidants before they can inflict damage to lipids , proteins or nucleic acids.

Superoxide dismutase (SOD), primarily an antioxidant present in the body, acts as a key

enzyme in the natural defense against free radicals. 10 Since the chronic OSMF patients are

under oxidative stress which has exhausted the ability of their antioxidative capacity to adapt

elevated levels of free radicals, we decided to estimate the activity level of antioxidant

enzyme SOD and correlation of these parameters in OSMF.

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Due to obscure etiology, there is no single definitive treatment for it. A plethora of

treatments have been advocated, hypothesized and justified by different workers. Steroids11,

antioxidants12, peripheral vasodilators13, placental extracts11, INF-γ14, turmeric15, lycopene16,

hyaluronidase17, collagenase18, pentoxifylline19, immune milk20, chymotrypsin11,

physiotherapies21, surgical treatments22-24 have been tried in OSMF but all are palliative and

have no curative value.

The advent of herbal renaissance has produced a profound effect on the system of

medicine. These include turmeric, black pepper and nigella sativa, which have been used in

treatment of various disease attributing to its extensive pharmacological activities.

Worldwide research has established the anti-inflammatory25, antioxidant26,

anticarcinogenic27-29, immunomodulatory26 and anti-fibrotic27, 30 properties of turmeric.

Another herb Nigella sativa also exhibits anti-oxidant31, anti- inflammatory32-34, anti-

carcinogenic31, 35, immunomodulatory33, 36, anti-fibrotic properties35, 37-39. All such properties

of turmeric and nigella sativa suggest possible benefits in OSMF. In addition, it is been

reported that black pepper prevents the metabolism of turmeric thereby increasing the

bioavailability of turmeric.40 So turmeric in combination with black pepper and nigella sativa

were tried as a novel therapeutic modality in treating OSMF. To the best of our knowledge,

this was the first study where turmeric with black pepper and nigella sativa was being used in

the management of OSMF.

MATERIALS AND METHODS

The study protocol was approved by Institution Review Board of College of Dental

Sciences, Davangere. The study was done in compliance with declaration of Helsinki. The

study was basically divided into two phases; phase I carried the interventional part and phase

II carried the SOD analysis of healthy controls.

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For the phase I, the sample size determination was based on similar reported studies

41, 42. The sample size calculated was 19 per group. Then the sample size was increased to

(n=) 23 per group, to account for refusal to participate, loss of follow up or other reasons.

The participants of either sex attending department were included based on following

inclusion and exclusion criteria.

Inclusion Criteria:

1. Age > 18 years

2. Clinically diagnosed OSMF patients.

3. Patients who have not undergone any other treatment for the same in the last 3

months.

4. Patients who are willing to quit the habit (gutkha, areca-nut, tobacco chewing).

5. Patients who are willing to attend the scheduled follow ups.

Exclusion Criteria:

1. Patients with any other oral lesion/condition except OSMF.

2. Patients with coexisting systemic illness.

3. Patient allergic to drug material.

4. Pregnant women and lactating mothers.

All the participants were explained about the study procedure and informed consents

were taken. The patients were counseled and advised to quit the habit completely. The

patients were randomly divided into two groups – group A and group B.

As the study was performed as double blind randomized study, the investigator and

patients, both were blinded by the supervisor. Randomization was computer generated.

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Group A received turmeric (400mg) with black pepper(100mg), 2 capsules TID for 3 months.

Group B received nigella sativa, 2 capsules of 500 mg TID for 3 months.

The mouth opening and burning sensation were the primary treatment outcome end

points. The interincisal mouth opening was measured using Vernier caliper from the

mesioincisal angle of upper central incisor to the mesioincisal angle of lower central incisor

and recorded in millimeters. The Visual Analogue Scale (VAS) of 0-10 was used to measure

intensity of burning sensation. Cheek flexibility was measured according to the method by

Mathur and Jha 43. In this method, two reference points were marked on both the cheeks at

one third the distance from the angle of the mouth on a line joining the tragus and angle of

the mouth. Patients were asked to blow cheeks fully and distance was measured between the

reference points. Then patients were asked to blow out and relax the cheeks; and the distance

was measured again. The difference between the two values gave cheek flexibility. Tongue

protrusion was assessed from mesioincisal angle of upper central incisor to the tip of tongue

when maximally extended with mouth wide open. All these parameters were measured and

recorded during patients’ every visit.

Based on the mouth opening, the patients were categorized into five groups,

Stage I: >40 mm

Stage II: 31 – 40 mm

Stage III: 21 – 30 mm

Stage IV: 11 – 20 mm

Stage V: < 10 mm

The phase II was carried out after the phase I, so that sample size of the healthy

individual can be determined. After the clinical intervention, it was found that 40 OSMF

patients had successfully completed the study, so the sample size of the healthy individual

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was kept 40, to match with the interventional group sample size. The healthy individuals

were included in the study, for SOD level analysis, based on following criteria.

Inclusion criteria :

1. Individuals having no adverse habits i.e. tobacco/areca nut chewing, alcohol

consumption.

2. Individuals without any oral lesion/condition.

3. Individuals without any systemic illness or taking any kind of medicine.

4. Individuals who are willing to give written informed consent.

The SOD levels were assessed and compared with healthy controls and also

correlated with clinical staging. Marklund and Marklund44 method was used for the

estimation of serum SOD level. Pre and post treatment SOD levels were measured and

compared.

The clinical parameters at first visit were considered as base line. After which patients

were recalled every 15 days for three months, which were considered as 1st to 6th visits, thus

making total of 7 values for each clinical parameter. Patients were asked about any GI related

discomfort after enrolling into the study, at each visit.

Statistical Analysis was done using software SPSS version 19.0 (SPSS, Chicago, IL,

USA). MS Excel 2010(Microsoft Corporation, Washington, DC, USA) was used to generate

tables and graphs. Descriptive data that included mean, numbers and percentages were

calculated for each group and were used for analysis. Tests applied were one way ANOVA

followed by Post Hoc Tuckey’s test, paired and unpaired t- tests. P values < 0.05 were

considered statistically significant.

RESULTS:

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A total of 131 patients were accessed for eligibility and on satisfying criteria

successfully, 46 patients were invited to take part in the study. Five patients (group A – 3,

group B – 2) did not turn up and one patient (from group B) discontinued the intervention due

to personal reasons. So the final sample was consisted of 40 OSMF patients for the

intervention. Demographic details of patients are shown in Table 1. The clinical and

biochemical parameters are tabulated in Table 2, 3 and 4 and Figure 2 and 3. Group A and

group B patients showed 14.37% (3.85mm) and 13.75% (3.65mm) improvement in mouth

opening respectively (p<0.01), 87.90%(5.45mm VAS) and 78.91%(5.05mm VAS) reduction

in burning sensation respectively (p<0.01), 7.98% (3.1mm) and 8.95% (3.2mm) improvement

in tongue protrusion respectively (p<0.01), 29.03% (0.45mm) and 44.12% (0.75mm)

improvement in cheek flexibility respectively (p<0.05) and +0.62 U/ml and +0.74 U/ml

improvement in SOD levels respectively (p<0.05).

The maximum mouth opening achieved was 8mm (p<0.5) and 7 (p<0.5) mm in group

A and B respectively. Depending upon the mouth opening, 1 patient was categorized in stage

I, 10 patients in stage II, 22 patients in stage III and 7 patients in stage IV. After the

treatment, 5 patients were in stage I, 11 patients were in stage II, 22 patients in stage III and 2

patients were in stage IV. (Table 2)

The pre-treatment SOD levels for group A and group B were 2.66 ± 0.19U/ml and

2.62 ± 0.17 U/ml respectively. After the treatment, the SOD level were 3.28 ± 0.19U/ml and

3.36 ± 0.26U/ml for group A and B respectively. The sample size of age and sex matched

healthy controls was 40. The mean SOD level of healthy controls was recorded as 3.61 ± 0.24

U/ml.

Further, no adverse effects were observed. The intra-group analysis was statistically

significant for both the groups (p<0.05) however intergroup analysis was not statistically

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significant between the groups (p>0.05) so there was no difference between group A and

group B regarding treatment outcome. (Table 3)

DISCUSSION:

OSMF is considered as major oral health problem with high degree of malignant

potential. Various etiological factors have been suggested for OSMF, which include local

irritant such as mainly areca nut use, capsaicin, pungent and spicy food. In addition to the

local factors, systemic factors have also been suggested to play a role in the development of

OSMF. These include anemia, chronic iron and vitamin B deficiency and genetic pre-

disposition. Chewing areca nut in its various forms is widely prevalent in the Indian

subcontinent, giving rise to increased prevalence of OSMF, from an estimated 2,50,000 cases

in 1980 45 to an estimated 5 million people in 2002.46

As the disease is still poorly understood and unsatisfactorily treated which is mainly

due to the fact that etiopathogenesis of the disease is not fully understood and the disease is

irreversible. Oxidative stress is implicated in the pathogenesis of various diseases including

oral cancer. OSMF being a potentially malignant condition, is reported to show increased

lipid peroxidation product and decreased antioxidant levels like SOD. Lack of a specific

treatment modality pose a greater challenge in treating this condition. Conventional therapies

include intralesional injections of corticosteroids, placental extracts, hyaluronidase,

physiotherapy and surgery have inconsistent outcome with several side effects .11, 14-18, 21-23

Thus management of OSMF has not been satisfactory till date and it is shifting focus

towards alternative medicine i.e. Ayurveda which is an ancient system of medicine.

Ayurvedic medicines are usually derived from the plant kingdom. Herbs like nigella sativa,

turmeric and black pepper have long been used in various diseases. Studies have shown that

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nigella sativa and its derivatives possess anti-oxidant, anti-inflammatory,

immunomodulatory, anti-cancer and anti-fibrotic properties. 31-39, 47, 48

Turmeric and its derivative curcumin also have anti-inflammatory, antioxidant,

anticarcinogenic, immunomodulatory and anti-fibrotic properties. 25-30 Studies reported in

literature using turmeric in the treatment of OSMF, have shown good results.49 Turmeric has

poor peroral bioavailability due to its rapid metabolism, hence it has been combined with

pepper which is known to increase the absorption of turmeric. 40, 50

In the present study, group A was given turmeric with black pepper in powder form.

Turmeric has already been tried in OSMF because of its anti-oxidant effect. However, there

were no case studies reported in the literature till date using combination of turmeric and

black pepper for OSMF. Studies have shown that combination of turmeric with black pepper

increases the bioavailability of turmeric by preventing its metabolism. To get this advantage

we combined black pepper with turmeric at 1:4 ratio (100 gm black pepper and 400 gm

turmeric). As black pepper inhibits hepatic and intestinal glucuronidation of turmeric, thereby

increases its bioavailability. Group B was given nigella sativa in powder form. Similarly, till

date there are no studies reported for the therapeutic use of nigella sativa in OSMF. However,

it has been tried in human studies for type II diabetes mellitus for glycemic control. In which

it was given at the dose of 2 or 3 gm/day for 3 months.51 So in our study patients were given

3 gm of nigella sativa in 3 divided dosage (2 capsules of 500 mg NS TID) for 3 months.

Patients were monitored on daily basis but none of the patients had reported any side effects.

In the present study, group A showed a better response by approximately 87.90%

reduction (p<0.05) in burning sensation compared to group B which showed improvement by

78.91% (Table 3, Figure 2). Both the groups showed better results than Lycopene which

showed 67.39% relief from burning sensation. 52 Another study in which Antoxid53 received

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patients and Pentoxifylline54 received patients showed 86.7% and 86.6% reduction in burning

sensation respectively which is similar to our results.

For mouth opening, group A patients showed improvement of 3.85 ±0.38mm while

group B patients showed 3.6±0.07mm improvement (Table 3, Figure 2). The maximum

mouth opening achieved was 8mm and 7mm for group A and B respectively. (p<0.01)

Percentage wise group A and B showed 14.37% and 13.75% improvement in mouth opening

respectively. In our study both the group has shown better results compared to the study in

which levamisole, antoxid and combination of both showed 0.3 ± 0.1, 0.2 ± 0.1 & 0.2 ± 0.3

cm improvement in mouth opening respectively. 53 Levamisole and Antoxid, both are

relatively expensive drugs compared to turmeric, black pepper and nigella sativa. In addition,

levamisole has more side effect compared to the herbs used in our study.

Group A and B showed 3.1 and 3.2mm improvement in tongue protrusion (Table 3,

Figure 2). It is similar to the preliminary study using topical aloe vera gel v/s antioxidant

capsules in which improvement in tongue protrusion was found to be 3.1mm and 1.7 mm

respectively.41 However, our study has shown better result than anti-oxidant group. It may be

due to the reason that anti-oxidant may have only anti-oxidant property while turmeric,

pepper and nigella sativa have other properties i.e. anti-inflammatory, immunomodulatory

also.

Group A and B showed 0.45 and 0.75 mm improvement in cheek flexibility (Table 3,

Figure 2). This can be correlated with preliminary study of topical gel v/s antioxidant

capsules in which the improvement in cheek flexibility was found to be 0.6mm and 1mm

respectively.41

In the present study, out of 40 patients, 1 patient was categorized in stage I, 10

patients in stage II, 22 patients in stage III and 7 patients in stage IV (Table 2). Out of 40

patients, all were males. Majority of the studies have reported a male preponderance.55, 56All

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patients in both the group have shown significant improvement in clinical parameters

irrespective of the clinical staging. Overall response was better in the stage II and III for both

groups. Overall correlation with the treatment outcome was not possible because of the

unequal distribution of patients belonging to different stages.

The serum SOD was assessed in 40 OSMF patients and age and sex matched controls

and the values were compared. SOD levels of controls and patients were 3.61 ± 0.24 and 2.62

± 0.18 U/ml respectively. The statistical analysis showed that SOD level were significantly

less in patients compared to the controls (p<0.05). It was in the accordance with the reported

study (SOD levels of Patients - 2.46 ± 0.26 U/ml; controls 3.46 ± 0.27 U/ml). 57

Comparison of SOD levels with clinical staging revealed that the SOD levels were

high at initial stages and decreased in the advanced stage of the disease. The SOD levels

(U/ml) were 2.9 (stage I), 2.8 ± 0.09(stage II), 2.65± 0.49(stage III) and 2.36 ± 0.09(stage IV)

(Table 4). It was in accordance with the previous reported study in which the mean level of

SOD for stage II, III and IV it was 2.51±1.99, 2.34±0.39 and 2.16±0.28U/ml respectively57. It

also showed similar results as the levels were decreased in the advanced disease57. This

decrease may be due to increased oxidative stress load as the disease progresses. In addition,

we found the mean SOD level for mild, moderate and severe burning sensation patients were

2.69 ± 0.19, 2.66 ± 0.69 and 2.62 ±0.18 U/ml. It also showed that SOD levels decreased as

the burning sensation increased. There is no previous reported study which correlated burning

sensation with serum SOD level. Similarly, severe burning sensation may also reflect the

increased oxidative stress load, so it could be reason for decreased SOD level.

Group A and B showed +0.62U/ml and +0.74 U/ml difference of SOD level from

base line respectively (Figure 3). It may be due to the anti-oxidative properties of turmeric,

black pepper and nigella sativa. However, stoppage of habit might also play a role. Other

studies, using MDA as a oxidative stress marker, supports anti-oxidative role of turmeric.58

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In the present study, overall treatment response was higher in group A compared to

group B (Table 3, Figure 2). As in group A patients, the black pepper would have potentiated

the action of turmeric, it could be the reason that group a showed more improvement in

burning sensation compared to nigella sativa.

LIMITATIONS

• There was short term follow up.

• No control/placebo group were considered.

• There was smaller sample size and unequal distribution of the patients among

different clinical stages.

• SOD level estimation method was manual which may result in certain errors.

• No pharmacokinetic study was done

FUTURE STUDY

• Large multicentric studies involving equal distribution of the cases in all the clinical

stages can be considered.

• Studies can be done making use of different dosages and combination of turmeric,

black pepper and nigella sativa; so that one standardized dose can be administered.

• To enhance effect, herbal preparation for topical application can also be tried.

CONCLUSION :

The findings of the present study demonstrated that turmeric with black pepper and

nigella sativa may have significant role to improve signs and symptoms of OSMF without

any adverse effects reported by the subjects. Further investigations (i.e., randomized double-

blind, placebo-controlled trials) are needed in order to determine the safety and effectiveness

of these affordable, easily available, and most importantly, non-invasive interventions for the

treatment of OSMF.

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Acknowledgement: We gratefully acknowledge Dr J Thimmasetty, Dr Garima J, Dr N

Narendra and Mr Parmeshappa for their contribution towards this study and special thanks to

Dr Parth Thakkar for statistical analysis.

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Figure 1: Flow diagram showing phases of this preliminary study

Figure 2: Intergroup comparison of overall treatment response

Figure 3: Group wise pre & post treatment comparison of SOD levels

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Table 1: Demographic details of the participants

Group A (n=20) Group B (n=20) Average Healthy

controls

Age(years) 29.60±7.58 26.80±6.36 28.2 ± 7.05 28.2 ± 7.05

Gender M M M

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Table 2: Clinical stage wise distribution of OSMF patients

Pre treatment Post treatment

Group A Group B Total Group A Group B Total

Stage I 1 0 1 2 3 5

Stage II 5 5 10 6 5 11

Stage III 10 12 22 11 11 22

Stage IV 4 3 7 1 1 2

Total 20 20 40 20 20 40

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Table 3 Clinical parameters before and after treatment

BURNING SENSATION MOUTH OPENING TONGUE PROTRUSION CHEEK FLEXIBILITY

(VAS) (mm) (mm) (mm)

Visit A B A B A B A B

Base line 6.2 6.4 26.8 26.55 38.85 35.75 1.55 1.7

1st 4.05 4.7 27.85 27.85 39.85 37.2 1.55 1.8

2nd 3.25 4.2 28.9 28.5 40.8 37.65 1.6 1.85

3rd 2.45 3.3 29.3 28.95 41.1 38.1 1.7 1.85

4th 2.1 2.65 30 29.45 41.75 38.55 1.9 2.15

5th 1.45 1.9 30.35 29.7 41.85 38.7 1.95 2.35

6th 0.75 1.35 30.65 30.2 41.95 38.95 2 2.45

Difference 5.45 5.05 3.85 3.65 3.1 3.2 0.45 0.75

in % 87.90% 78.91% 14.37% 13.75% 7.98% 8.95% 29.03% 44.12%

p Value

p < .01 p < .01 p < .01 p < .01 p < .01 p < .01 p < .05 p < .05

p > .05 p > .05 p > .05 p > .05

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Table 4 SOD level correlated with clinical staging

SOD (U/ml) Stage I Stage II Stage III Stage IV

Group A

N= 1 5 10 4 before 2.9 2.84±0.11 2.67±0.85 2.36±0.09 after 3.26 3.36±0.21 3.37±0.14 2.95±0.14

Group B

N= 0 5 12 3 before - 2.76±0.07 2.63±0.13 2.35±0.08 after - 3.39±0.14 3.44±0.22 3.03±0.06

Overall

N= 1 10 22 7 before 2.9 2.8±0.09 2.65±0.49 2.36±0.08

after 3.26 3.38+/0.18 3.41±0.18 2.99±0.1

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Turmeric with black pepper and nigella sativa may have significant role to improve signs and

symptoms of OSMF. Further investigations (i.e., controlled trials) are needed to determine the

safety and effectiveness of these affordable, easily available, and non-invasive interventions.