Effects of solasodine of Solanum melongena peel origin in the treatment of palmar arsenical keratosis

This study was conducted to confirm the effect of the ointment containing Solanum melongena peel extract in the treatment of palmar arsenical keratosis and to identify the compound responsible for the effect. In total, 30 patients with moderate to severe palmar arsenical keratosis were enrolled according to the inclusion criteria. Extract from S. melongena peel was obtained and a topical ointment was prepared from the extract and supplied at the field level at an interval of two weeks. Instruction was given to the patients about how to apply the ointment. Adherence and adverse effects of the treatment were monitored regularly through phone calls and during each visit. The mean (± SD) size of the keratotic nodules was 21.9 ± 10.0 mm2 before intervention and 6.6 ± 5.3 mm2 after intervention. The percentage of reduction was 69.8. Nuclear magnetic resonance spectroscopy, infrared spectroscopy, liquid chromatography-mass spectrometry and elemental analysis of the extract was done to identify the compound and solasodine, a steroidal alkaloid, was identified. Article Info Division of Arsenic Basic Research, Department of Pharmacology, Faculty of Basic Science and Paraclinical Science, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh For Correspondence: Mir Misbahuddin mmisbah@bsmmu.edu.bd Received: 8 January 2020 Accepted: 26 May 2020 Available Online: 16 June 2020 ISSN: 2224-7750 (Online) 2074-2908 (Print) DOI: 10.3329/bsmmuj.v13i2.47606 Cite this article: Sultana R, Misbahuddin M. Effects of solasodine of Solanum melongena peel origin in the treatment of palmar arsenical keratosis. Bangabandhu Sheikh Mujib Med Univ J. 2020; 13: 47-52. Copyright: The copyright of this article is retained by the author(s) [Atribution CC-By 4.0] Available at: www.banglajol.info A Journal of Bangabandhu Sheikh Mujib Medical University, Dhaka, Bangladesh Effects of solasodine of Solanum melongena peel origin in the treatment of palmar arsenical keratosis Razia Sultana and Mir Misbahuddin or nursing mother; b) patient who received any treatment of arsenicosis within the last three months and d) patient with any diagnosed skin diseases like atopic dermatitis, eczema, and psoriasis. All the patients were demonstrated how to apply the ointment containing extract at the lesion site The clinical examination of patient and distribution of ointment containing S. melongena peel extract were done at 2 weeks interval. Detailed history, clinical examination, and photographs of the lesions were collected. The size of the lesions was measured before and after completion of the study. Drinking water and nail samples were collected for estimation of arsenic. One labeled polyethylene container (capacity 100 mL) containing 1 drop of nitric acid was supplied to each patient in order to collect the water sample. All the collected samples were then transported to the Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University and stored until analysis. A labeled, sterile, air-tight, polyethylene bag was supplied to each patient and advised to collect finger and toe nails (growth of one month). About 200-500 mg nail samples of each patient were collected and transported to the laboratory and stored in the refrigerator until analysis.4 The amount of arsenic in water and nails was measured by modified silver diethyldithiocarbamate (SDDC) method.5 Specific species of S. melongena L was identified by Bangladesh National Herbarium. Then S. melongena was collected from the local market. The average length of S. melongena were six to eight inches, circumference were three to four inches and weight was ranged from 200-300 g. S. melongena was rinsed thoroughly with water and dried off. Peels were removed with a peeler and cut into small pieces. Peels were then air dried at room temperature avoiding direct sunlight for two days. Dried peels were immersed into an amber color bottle containing one liter of the mixture of ethanol: chloroform: acetic acid (65: 32: 3) and kept for 72 hours at room temperature. After 72 hours, the peels and solvent were filtered through a filter paper. Filtered liquid was concentrated using a rotary evaporator at 40-50°C temperature.3 Brine shrimp lethality study About three gram of brine shrimp (Artemia salina) eggs collected from the local market were poured into a glass tank (capacity = 6 L) containing artificial seawater (about 4 L) for hatching up to 48 hours with continuous aeration by air flow machine and artificial light using 60 watt bulb.6, 7 One milligram extract of S. melongena peel was weighted by the electronic balance and dissolved in 1 mL of water to prepare the stock solution. Serial dilution of the stock solution was done and taken in four sterile, labeled test tubes. The concentration of the solutions after serial dilution was 100, 10, 1 and 0.1 μg/mL. The brine shrimp larvae (nauplii) were collected with a Pasteur pipette from the hatching chamber in the test tubes. Different concentrations of the extract were poured into the test tubes containing ten nauplii and kept in room temperature. Motility of the nauplii was observed after 2 and 24 hours. Percentage of death = [Number of dead nauplii/ (Number of dead nauplii + Number of live nauplii)] × 100. The ointment (100 g) contained 30 mg of S. melongena peel extract, 3.97 g stearyl alcohol, 10 g white wax and 86 g white petrolatum.8 White petrolatum, bee wax and stearyl alcohol were measured by an electronic balance after calibration and taken in a glass beaker. The glass beaker was placed into the bowl containing water and heated up to complete melting of the ingredients using Bunsen burner. After complete melting of the ingredients, the burner was put off and the beaker was placed in a tray for cool down. After reaching at room temperature, S. melongena peel extract was added and stirred well. Ointment was poured into the high-density polyethylene container and stored in a cool and dry place until supplying to the patient. The ointment was distributed and each patient was demonstrated separately how to apply the ointment at the lesion site. Patients were instructed to apply the ointment twice daily in the morning and bedtime for 13 weeks. An adherence sheet was provided to each patient and advised to put a tick mark at the appropriate place after applying the ointment. The keratotic lesion was monitored. Any improvement was documented and check the adherence in every follow-up visit at two weeks interval. Paired t-test was performed manually to measure the differences between the keratotic nodular size changes before and after treatment. Steps of identification Compound was identified by nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectrophotometry, elemental analysis and liquid chromatography-mass (LC-MS/MS) spectroscopy.

or nursing mother; b) patient who received any treatment of arsenicosis within the last three months and d) patient with any diagnosed skin diseases like atopic dermatitis, eczema, and psoriasis.
All the patients were demonstrated how to apply the ointment containing extract at the lesion site The clinical examination of patient and distribution of ointment containing S. melongena peel extract were done at 2 weeks interval.
Detailed history, clinical examination, and photographs of the lesions were collected. The size of the lesions was measured before and after completion of the study.
Drinking water and nail samples were collected for estimation of arsenic. One labeled polyethylene container (capacity 100 mL) containing 1 drop of nitric acid was supplied to each patient in order to collect the water sample. All the collected samples were then transported to the Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University and stored until analysis.
A labeled, sterile, air-tight, polyethylene bag was supplied to each patient and advised to collect finger and toe nails (growth of one month). About 200-500 mg nail samples of each patient were collected and transported to the laboratory and stored in the refrigerator until analysis. 4 The amount of arsenic in water and nails was measured by modified silver diethyldithiocarbamate (SDDC) method. 5 Specific species of S. melongena L was identified by Bangladesh National Herbarium. Then S. melongena was collected from the local market. The average length of S. melongena were six to eight inches, circumference were three to four inches and weight was ranged from 200-300 g.
S. melongena was rinsed thoroughly with water and dried off. Peels were removed with a peeler and cut into small pieces. Peels were then air dried at room temperature avoiding direct sunlight for two days. Dried peels were immersed into an amber color bottle containing one liter of the mixture of ethanol: chloroform: acetic acid (65: 32: 3) and kept for 72 hours at room temperature. After 72 hours, the peels and solvent were filtered through a filter paper. Filtered liquid was concentrated using a rotary evaporator at 40-50°C temperature. 3

Brine shrimp lethality study
About three gram of brine shrimp (Artemia salina) eggs collected from the local market were poured into a glass tank (capacity = 6 L) containing artificial seawater (about 4 L) for hatching up to 48 hours with continuous aeration by air flow machine and artificial light using 60 watt bulb. 6, 7 One milligram extract of S. melongena peel was weighted by the electronic balance and dissolved in 1 mL of water to prepare the stock solution. Serial dilution of the stock solution was done and taken in four sterile, labeled test tubes. The concentration of the solutions after serial dilution was 100, 10, 1 and 0.1 µg/mL. The brine shrimp larvae (nauplii) were collected with a Pasteur pipette from the hatching chamber in the test tubes. Different concentrations of the extract were poured into the test tubes containing ten nauplii and kept in room temperature. Motility of the nauplii was observed after 2 and 24 hours.
Percentage of death = [Number of dead nauplii/ (Number of dead nauplii + Number of live nauplii)] × 100.
The ointment (100 g) contained 30 mg of S. melongena peel extract, 3.97 g stearyl alcohol, 10 g white wax and 86 g white petrolatum. 8 White petrolatum, bee wax and stearyl alcohol were measured by an electronic balance after calibration and taken in a glass beaker. The glass beaker was placed into the bowl containing water and heated up to complete melting of the ingredients using Bunsen burner. After complete melting of the ingredients, the burner was put off and the beaker was placed in a tray for cool down. After reaching at room temperature, S. melongena peel extract was added and stirred well. Ointment was poured into the high-density polyethylene container and stored in a cool and dry place until supplying to the patient.
The ointment was distributed and each patient was demonstrated separately how to apply the ointment at the lesion site. Patients were instructed to apply the ointment twice daily in the morning and bedtime for 13 weeks. An adherence sheet was provided to each patient and advised to put a tick mark at the appropriate place after applying the ointment.
The keratotic lesion was monitored. Any improvement was documented and check the adherence in every follow-up visit at two weeks interval.
Paired t-test was performed manually to measure the differences between the keratotic nodular size changes before and after treatment.

Steps of identification
Compound was identified by nuclear magnetic resonance spectroscopy, Fourier-transform infrared spectrophotometry, elemental analysis and liquid chromatography-mass (LC-MS/MS) spectroscopy.

Results
The mean (± SD) age of the patients was 41.3 ± 13 years. The mean amount of arsenic was 244.1 ± 177.5 µg/L and 8.2 ± 7.4 µg/g in the tube well water consumed by the patients and nail sample respec-tively. The mean duration of exposure to arsenic contaminated water of the patients was 9.3 ± 2.0 years. The mean duration of the appearance of keratosis was 4.6 ± 2.1 years Seven patients were dropped out during the study period due to loss to follow-up.

Size of the lesions
The mean (± SD) size of the keratotic nodules was 21.9 ± 10.0 mm 2 before intervention and the mean size was reduced to 6.6 ± 5.3 mm 2 after intervention ( Figure 1). The percentage of reduction was 69.8. Statistical analysis was done by paired t-test and pvalue was 0.0001. The result was statistically significant.

Brine shrimp toxicity assay
A concentration-dependent death of nauplii was observed after 24 hours in the brine shrimp cytotoxicity test performed in artificial sea water. The net dead of nauplii was found 26, 48, 50 and 62% at 0.1, 1.0, 10 and 100 µg/ mL concentrations of the extract respectively ( Figure 2).

Characteristics of the extract
Total 510 g extract was obtained from 140 kg of S. melongena. The extract was dark brown in color, semisolid in consistency and pungent in odor. It was soluble in methanol, dichloromethane and chloroform but insoluble in butanol, n-hexane and acetone.

Discussion
The patients with moderate to severe palmar arsenical keratosis were treated with the ointment containing S. melongena peel extract and it was found to be effective in the reduction of the size of the keratotic nodules. Nuclear magnetic resonance, infrared spectroscopy, elemental analysis and liquid chromatography-mass spectrometry of the extract were done and solasodine, a steroidal alkaloid, was identified. This is the second study to evaluate the effects of S. melongena peel extract on arsenical keratosis. The first study was done at the same department where improvement of keratosis was found after application of extract containing ointment. 3 In the present study, improvement was also found after applying the ointment containing S. melongena peel extract. About 10% of the total patients mentioned about slight burning sensation during the time of ointment application and that was within their allowable limit.
The presence of solasodine in the S. melongena peel extract was based on data obtained from nuclear magnetic resonance, infrared spectra and liquid chromatography-mass spectrometry of the extract. Elemental analysis of the extract was also done but no conclusion could be reached due to failure to  S. melongena peels are reported to possess steroidal alkaloids: solasodine, solamargine and solasonine. 9 These compounds exhibited moderate to potent activities against carbon tetrachloride-induced hepatocellular carcinoma in rats. The killing effect of the cancer cells by solasodine glycoside may be due to a) lysis of the cells after expressing specific endogenous endocytic lectins. This component is more specific for the cancer cells than the normal cells and b) inducing apoptosis of the cells by upregulation of tumor necrosis factor and Fas receptor. 10 S. melongena peel also possess antioxidant activity as it contains anthocyanin; delphinidine-3-(p-cumaroylrutinoside)-5-glucoside that is nasunin. 11-12 Delphinidine has an inhibitory effect on human fibrosarcoma HT-1080 invasiveness in vitro. 13 A study conducted to isolate solasodine from Solanum xanthocarpum where Fourier transform infrared spectra of solasodine were nmax (3400, 2950, 2880, 1733, 16930 cm -1 ) ( Anti-cancer activity of the fruit peels of S. melongena was revealed in a study. 9 In that study, the 13

Conclusion
This study suggests that ointment containing S. melongena peel extract is effective in reducing the size of the arsenical keratotic nodules. The preliminary steps for identification of compound present in the extract revealed that solasodine may be the compound responsible for this effect.