The treatment of melasma by silymarin cream

Silymarin (SM), App-Chem-Bio; China prepared in cream base of different concentrations[11]. Placebo is a cream base free from active constituent.

Twenty-four healthy New Zealand rabbits Albino rabbits (weight: 1500 ± 500 g each), were individually housed in suspended cages, for 1 week before the experiment. The ethics committee, before the experiment, has approved the experimental protocol. They were kept in the same environmental and nutritional conditions (temperature 25 ± 2°C, relative humidity 40%-60%, and 12 hours in light and 12 hours in darkness cycles) in the animal house of the college of medicine. At the beginning of the study, animals were randomly divided into 4 equal groups (n=6); group [A] did not receive any treatment, [B] received placebo, [C] treated with SM (0.1 mg/ml.kg^-1) cream, and [D] treated by SM cream (0.2 mg/ml.kg^-1). 3 cm² of Albino rabbits' back were shaved. Then after 48 hours, the tested drugs were applied topically by cotton pad stick on the shaved area of all groups daily, 30 minutes before each UV sun light exposure. The rabbits were exposed to UV sunlight (11⁺ extreme) for 3 hours during each day of June (temperature 43 ± 2°C) for 30 days. All animals were painlessly killed by chloroform and samples were taken from the shaved area for tissue pathology study, the samples were put in formalin buffer 10%.

All samples were cut into small blocks. These blocks of tissues were then routinely processed. These paraffin blocks were sectioned into 5 μm thick and stained with hematoxylin & eosin. The stained tissues were examined for histopathological alterations. The histopathology departments' staffs were blinded to the tested drugs.

This is a double blind, randomized clinical study on patients with melasma attending the outpatient clinic of the Dermatology department of Medical City Hospital. The protocol was reviewed and approved by the ethic committee (University of Baghdad, College of Dentistry), and each participant signed a written informed consent. The inclusion criteria were adults with melasma without any topical, systemic, laser, and surgical treatment on face during the previous months. While the exclusion criteria were pregnant and nursing women, patients with history of hypersensitivity to some of the components of the formulas of the study, and coexistence of associate diseases and other pigmentation diseases, and concomitant use of other skin care products or systemic treatments. A history was taken from each patient, regarding age, gender, occupation, time of onset, history of pregnancy, contraceptive pills, and sun exposure. Patients were randomized in a double-blind manner to receive one treatment of the tested drugs; Group I (G I) SM (7 mg/ml) cream, Group II (G II) SM (14 mg/ml) cream, or Group III (G III) placebo, applied topically to the affected areas, twice daily for 4 weeks, also advised to avoid sun exposure and to use topical sunscreen with sun protection factor (SPF) of 15⁺ during the entire period of treatment and thereafter. The patients were seen regularly every week for one month for assessment; the response to treatment was rated by the size of lesions. Skin pigment evaluation by melasma area and severity index (MASI), physician global assessment (PGA); assessment of overall treatment of disease activity, used a scale from 0 to 10, by an independent observer blinded to the treated groups, and record the presence of any side effect. The Subjective assessment depending on recording improvement in patient satisfaction measures during the time course, and graded as follows: Grade 0 =not satisfied, Grade 1 =moderately or partially satisfied, Grade 2 =greatly but not fully satisfied, Grade 3 =fully or completely satisfied.

In order to analyze the data, Chi-square test, Student t-test and X 2 were used, P value of less than 0.05 was considered significant, data showed as mean ± SD (SPSS 18).

Some clinical features such as skin scaling, skin irregularity, erythema, skin hyperpigmentation, and edema were evaluated. The clinical observations are as follows:

Group B (G B); received placebo, dermal scaling, skin irregularity, erythema & hyperpigmentation, and edema were observed at the level (90%).

The significance between groups was; G A vs. B, C, & D groups; p=0.0001, G B vs. C, & D groups; p=0.0003, and G C vs. D; p>0.05, as showed in (Figure1).

Epidermal hyperpigmentation, epidermal hyperkeratosis, lymphocyte infiltration into epidermis, squamous cell proliferation, edema and dermal thickness increase, infiltration of lymphocytes; plasma cells, and eosinophils into dermis were noticed in all cases of G A (100%), in G B were (58%, 50.2%, 3%, 45%, 60.2%, 60.2%) respectively. While in G C, & G D all the microscopic observations were not seen. The level of significance was; G A vs. B, C, & D groups; p=0.0002, G B vs. C, & D groups; p=0.0001, and G C vs. D; p>0.05, as showed in (Figure2).

Ninety-six melasma patients were enrolled in this study, female {F} 80 (83.3%) and male {M} 16 (16.66%), the ratio of sex distribution {F: M} within the groups were [G I (26:6), G II (27:5), and G III (27:5)]. The patients' age ranged from 28 to 55 years (median, 41 years). The duration of melasma varied from 2 to 6 years (median 4 years). Family history of melasma was found in 46 (47.916%) patients. The most frequent precipitating factors were the sun exposure (90%), and pregnancy (10%).

The response to treatment was evaluated as per the size of the lesion at the end of each week till 4 weeks. G I & G II showed significant reduction in size of the lesion at the end of 1^st week compared to G III, complete clearing of the lesion was shown in G II after 3 weeks of treatment, while G I in the fourth week, (Figure3).

The average MASI score of G I before treatment was 17.1± 3.12. After treatment it changed into 12.3 ± 2.3, 6.6 ± 2.4, 0.2 ± 1.72, & 0 at the end of 1st, 2^nd, 3^rd, & 4^th week respectively. For G II before treatment was 16.5 ± 2.8. After treatment it changed into 10.4 ± 1.2, 3.5 ± 1.4, 0, & 0 at the end of 1st, 2^nd, 3^rd, & 4^th weeks respectively, and was statistically significant (P =0.0001). In G III there were no significant changes, as showed in Table 1.

The PGA rated the G I improvement as excellent in 5 patients, good in 21, moderate in 2, and mild in 1 at the end of 1^st week, excellent in 17 patients, good in 14, & moderate in 1at the end of 2^nd week, while at the end of 3^rd week; excellent in 28, good in 4 patients. While the G II improvement was excellent in 8 patients, good in 19, moderate in 5 at the end of 1^st week, excellent in 20 patients, good in 12 at the end of 2^nd week; and at the end of 3^rd week were excellent in 29, good in 3 patients. Data showed statistical significance for SM treatments compared to placebo, (P = 0. 002), (Figure4).

Patients' satisfaction was recorded as 100% were highly & completely satisfied (Figure5). During the period of treatment, no local or systemic adverse effects were recorded.