Effect of Solanum melongena peel extract in the treatment of arsenic- induced Bowen’s disease

The aim of this study was to see the effectiveness of Solanum melongena peel extract in the treatment of arsenic-induced Bowen’s disease. In total 8 patients of arsenic-induced Bowen’s disease were selected from the two arsenic-endemic areas. The peel extract containing ointment was provided to each patient and instructed him/her to apply twice daily on the site of the lesion for 12 weeks. The measurement of the lesion size as well as photograph were collected both before and at the end of the study. The mean (± SD) diameter of the lesion was 3.8 ± 1.9 cm which reduced to 2.5 ± 1.7 cm (reduction 34%). The result was statistically significant.


Introduction
melongena peel and to prepare the extract (ethanol, chloroform and acetic acid); c) prepare the ointment containing extract; and d) brine shrimp cytotoxicity assay.

Enrolment of subjects
The present study was carried out on 8 patients (3 males and 5 females) of Bowen's disease. Four patients were from the Bhanga Upazilla and the rest 4 patients from the Sirajdikhan Upazilla. They voluntarily agreed to participate in this study. They were enrolled according to the inclusion and exclusion criteria. The inclusion criteria were a) subjects aged >18 or <65 years of both sex; b) consumption of high concentration of arsenic water (>50 µg/L) for more than 6 months; c) Bowen's disease; and d) arsenicosis patient voluntarily agreed to participate. The exclusion criteria were a) pregnant and lactating mother; b) any drug allergy, or food allergy specially S. melongena; and c) patient who received any of arsenicosis treatment within the last three months.
Each patient was advised to attend the Temporary Arsenic Clinic. Clinical examination of the patient and distribution of ointment containing extract were done at two weeks interval.

Participant's data collection procedure
Detailed history, clinical examination, photographs and tracing diagram of the Bowen's disease areas were collected. Drinking water and nail were collected for estimation of arsenic in the samples. The appropriate instruction was given to them, how to apply S. melongena extract containing ointment at the Bowen's disease lesion areas.

Measurement of the size of the lesion
The size of the Bowen's disease lesions was measured both before starting the treatment (day 0) and after completion of treatment (day 85).

Collection of samples
The water sample as well as the nails (grown in one month) were collected for the estimation of the total arsenic level once before starting the treatment. The purpose was to confirm the diagnosis.
One polyethylene container containing one drop of nitric acid (100 mL size) was supplied to each patient to collect the water sample that was used by himself/ herself. All these samples were collected and transported to the Pharmacology lab and stored until analysis.
The patient's hand and toe nails (growth in one month) were collected (at least 200 mg) and stored in a supplied leveled plastic bag. All the nail samples were collected and transported to the lab and stored until analysis.

Estimation of arsenic
The level of arsenic in the drinking water and nail was measured by silver diethyldithiocarbamate (SDDC) method (Bhuiyan et al., 2015).

Method of S. melongena extraction (Box 1)
There are three botanical varieties of eggplant-based on the shape of the fruit (Niño-Medina et al., 2017). These are a) egg-shaped (S. melongena var. esculentum), b) long and slender in shape (S. melongena var. serpentinum) and c) dwarf types (S. melongena var. depressum).
The fruit was identified and authenticated by Mr. Khandokar Kamrul Islam, Scientific Officer, Bangladesh National Herbarium, Dhaka, Bangladesh.
In this study, 12 kg of S. melongena (size: at least 8 inches long and 1.5-2 inches breadth; skin color: purple) were collected from the local market in different time interval. These were washed and peeled off the skin from the pulp using a vegetable peeler. The peels were cut into small pieces. The rest of the S. melongena were discarded. The pieces of peels of S. melongena were then air-dried at room temperature.

Procedure
Step 1: Collection of S. melongena (2 kg) from the local market Step 2: Separate the purple color skin form the pulp of the S. melongena, made them into small pieces and dry them at room temperature avoiding the sun light Step 3: Add 65% ethanol, 32% of chloroform and 3% acetic acid in an amber colored container (2.5 L) Step 4: Immersed the dried peel into the solvent for 72 hours Step 5: Separate the solid parts from the solvent by using cheese-cloth Step 6: The solvent was filtered using Whatman 102 filter paper Step 7: Filtrate was collected at round bottom flask and fitted it to vacuum rotary evaporator for the evaporation Step 8: Condensation of filtrate in a vacuum rotary evaporator, rotation about 100/min at 60°C Step 9: Collection of condensed extract in an amber color glass container and preserved at 4°C until ointment preparation

Precaution
Protect the extract from exposure to light. That is why aluminum foil was used to wrap the flask containing extract during the use of rotary evaporator The small pieces of peel (500 g) were obtained and immersed into an ambered color bottle (size 2.5 L) containing 2 L of the mixture of ethanol: chloroform: acetic acid (65: 32: 3) and keep them for 72 hours at room temperature. Then the solvent was separated from the solid parts by using Whatman 102 filter paper. The filtrate liquid was concentrated at 40-50°C under reduced pressure using a rotary evaporator until a dark brownish color semi-solid substance was obtained. Finally, the extract was stored in an amber color container and kept it into a refrigerator at 4°C until use. According to the above-mentioned procedure, S. melongena peel extract was prepared several times. From 4 kg of small pieces of S. melongena peel extract 2 g extract was obtained.

Brine shrimp lethality study (Sarah et al., 2017)
The eggs of Artemia salina (brine shrimp) were collected from the local market. A small amount of eggs (15 g) were poured into a square shaped glass vessel (capacity = 6 L) containing 4 L of artificial seawater for hatching up to 48 hours with continuous aeration and artificial light using a 60 watt bulb. Active nauplii were free from eggshells within this period.
The S. melongena peel extract of 1 mg was weighted by an analytical balance. Sterile test tubes were taken and leveled them. The extract (soluble in water) was dissolved in 1 mL of water to prepare the stock solution. Serial dilution of the stock solution was done to prepare the concentration of 100, 10, 1.0, 0.1 µg/mL. About 1 mL of the prepared solution was taken into five (I-V) labeled test tube contained actively moving 10 live nauplii in 1 mL of artificial seawater containing different concentrations of S. melongena extract.
All the test tubes were incubated at room temperature and observed the active movement of nauplii after 2 and 24 hours.

Preparation of ointment containing extract
One hundred gram of ointment contained 30 mg of S. melongena peel extract. All the ingredients were accurately measured by either an electronic balance or a measuring cylinder. A bowl containing 100 mL of water was heated up to boiling by a gas burner. A beaker containing 90 mL olive oil was placed into the boiling water (Block, 2000). When the temperature of the olive oil was 90°C, then 10 g of bee wax was added to it. After complete melting of the bee wax, the gas burner was put off and the beaker placed for cool down at room temperature. Then 10 mg of benzoic acid and 30 mg of S. melongena peel extract were added and contentiously beat until it became thick. Finally, it was transferred into the plastic screw cap ointment container (20 g each) and stored in a refrigerator until distributed to the patient.

Distribution of ointment
S. melongena extract ointment was distributed to each patient and he/she was instructed to apply the ointment to the affected area of the skin by clean fingertip twice daily (at the morning and bedtime). A separate printed sheet was given for checking the compliance by giving a tick mark at the appropriate place just after applying of ointment.

Periodic monitoring of patients
Each patient attended the Temporary Arsenic Clinic every 2 weeks interval. The researcher monitored the lesion, check the adherence and note down the improvement(s). A regular communication to the patient was maintained over cell phone.

Statistical analysis
Statistical analysis was done by paired 't' test to compare the Bowen's disease lesion size before and after the end of the treatment. Data were presented as mean ± SD.

Results
Fifty percent of the death nauplii were found in the artificial seawater containing 100 µg/mL of S. melongena extract ( Table I).
The mean (± SD) age of the patients was 53.1 ± 6.6 years ( Table II). The mean amount of arsenic in the tube-well water consumed by the patients was 677.3 ± 140.1 µg/L. The mean amount of arsenic in the nail sample was 28.3 ± 15.0 µg/g. The mean duration of exposure to arseniccontaminated water was 20.9 ± 5.8 years. The mean duration of the appearance of the Bowen' disease lesions was 8.8 ± 7.3 years.
The number of drop out patients was two due to loss to follow-up. The new lesions appeared on the chest of a patient of Bowen's disease who was a smoker.

Measurement of the Bowen's disease lesion size
The size (mean ± SD) of the lesions during the enroll- ment was 3.8 ± 1.9 cm which was reduced to 2.5 ± 1.7 cm after 12 weeks of the topical application of ointment and the total percentage of reduction was 34% (Table III). This change was statistically significant (p=0.0003). There was a reduction in the elevation of the lesions (Figure 1).
During the first two weeks of topical application of ointment, the lesion became erythematous and subside after the next two weeks.

Discussion
The present study revealed the significant improvement effect in reducing the lesion of Bowen's disease. The improvement of patient's lesion was assessed by measuring lesion size. The size of the Bowen's disease lesions was 2 to 7.6 cm in diameter. After topical application of S. melongena peel extract containing ointment for 12 weeks, the percentage of reduction was statistically significant.
This is the first study showing the effect of topical application of S. melongena peel extract in the treatment of arsenic-induced Bowen's disease. Only 34% improvement was shown.
The effect may be more if the a) higher concentration of the extract was used, b) modification of the ointment for better penetration of the extract through the skin, c) prolonged duration of action, and d) increased frequency of administration. However, in this study, the recurrence was not observed except one who was a smoker.
S. melongena is a vegetable. Its peel is violet in color which contains nasunin and chlorogenic acid. The  Paired t-test was between before and after study to estimate the level of significance of difference Figure 1: Photographs show the effectiveness of the extract of three patients of Bowen's disease. Left column represents before treatment and right column represents after treatment nasunin is an anthocyanin (from the Greek anthos = flower and kyano = blue), 700 mg per 100 g and is responsible for the purple color of this vegetable. Both show anti-oxidant effect and can be used in aging, inflammation, cardiovascular disease, cancer (Gallo et al., 2014).
The present study is similar to the findings of the topical application of low concentration (0.005%) of a standard mixture of solasodine glycosides to 29 squamous cell carcinoma (skin) patients (Cham et al., 1991). There was complete regression of lesions without recurrence. This component is more specific for the malignant cells than the normal cells and the new normal cells regrow at the place of the lesion.
The solasodine glycosides are cytotoxic substances present in the S. melongena peel extract and able to kill cancer cells. The plasma membrane of the susceptible cancer cells expresses specific endogenous endocytic lectins that bind to the sugar moiety of the solasodine glycosides and induces cell death by lysis of the targeted tumor cells. It is harmless for normal cells due to the absence of endogenous endocytic lectins.
A study explained the mode of action of solamargine as a potent cytotoxic activity against tumor cells by initiate a death process named oncosis. It also displays an unusual mode of action thus it avoids the common multidrug resistant mechanism (Sun et al., 2011).
Nasunin is an anthocyanin of the S. melongena peel extract showed biological abilities to prevent the production of free radicals, induces apoptosis, inhibits cell proliferation and angiogenesis.
One study was conducted in the two arsenic-induced Bowen's disease patients treated with acitretin (1 mg/ kg) daily (Yerebakan et al., 2002). The total cure was obtained in case of the first patient after 10 months but another patient discontinued the treatment after five months due to symptomatic adverse effects like hearing loss, insomnia and joint pain.
A patient with arsenic-induced keratoses and Bowen's disease responded well to acitretin (Watson and Creamer, 2004).
Acitretin is a retinoid analog (vitamin A). It works by inhibiting the excessive cell growth and keratinization. It, therefore, reduces the thickening of the skin, plaque formation and scaling. S. melongena peel extract containing solasodine glycosides which induced apoptosis of the cells by up-regulation of tumor necrosis factor and Fas receptor. Also decrease the Bcl2 expression concomitant increase protein level of Bax which collectively activation of the intrinsic and extrinsic pathway of apoptosis through caspase -8,-9 and -3.
Forty one patients of non-arsenic-induced Bowen's disease were treated with topical 5-fluorouracil for 4-12 weeks (Sturm, 1979). The concentration of 5-fluorouracil was 1% for the face area and 2.5-3% for other areas of the body. After treatment (three patients recur), 8% recurrence occurred which was retreated with the same medication. But the main limitation was the prolonged treatment period with the recurrent application of medicine and skin irritation with burning sensation and discoloration of the affected site.
5-Fluorouracil is a pyrimidine analog that irreversibly binds within a cell to thymidylate synthetase. This prevents the incorporation of uracil into nuclear RNA, which destroys abnormal cancer cells. It acts nonspecifically both in cancer and normal cells.
Another study conducted with 16 patients of Bowen's disease (not arsenic-induced) applied the topical 5% imiquimod cream (Mackenzie-Wood et al., 2001). In that study, showed clinical improvement in ten patients after 16 weeks of treatment and the rest of six patients discontinued the treatment due to irritation, skin rash and erythema of the local skin.
Imiquimod cream (5%) is an immunomodulator which stimulates the local cytokines thus regression and normalization of the normal keratinocytes (Tyring et al., 1998).
The study of comparison of photodynamic therapy with topical 5-fluorouracil in Bowen's disease showed that 88% initial complete clearance rate with photodynamic therapy and 67% complete cured with 5fluorouracil (Salim et al., 2003). Follow-up at 12 months, photodynamic therapy group recurrences were two and six in 5-fluorouracil group. No adverse reaction was observed in photodynamic therapy-treated patients. On the other hand, three patients develop widespread dermatitis, one patient develops ulceration with painful erosions during the medication period. The photodynamic therapy is costly and may need more than one session.
Another study with methyl aminolevulinate with photodynamic therapy (MAL-PDT) when compared with placebo-PDT or cryotherapy (Bath-Hextall et al., 2013) demonstrate statistically significant but the cryotherapy shows recurrence at one year with no statistically significant difference in clearance rate when compared with the 5-fluorouracil in Bowen's disease. Cryotherapy is painful and the skin may be uncomfortable for a few weeks.

Conclusion
S. melongena extract containing ointment has significant beneficial effect in the treatment of Bowen's disease.

Ethical Issue
The protocol was approved by the Institutional Review Board of Bangabandhu Sheikh Mujib Medical University (Reg. No. BSMMU/ 2017/ 3322). This work was registered on the clinical trial.gov (Registration number was NCT 03122561).
All the participants were informed about the objective, potential hazards, benefits and outcome of this study. All the information was explained in an easy and local language (Bengali). Informed consent was taken in a consent-form with either participant's thumb impression or signature.
All the participants were monitored and looked after carefully. Whether any complication arises during this treatment period, they had under careful observations and the researcher agreed to organize the symptomatic treatment.