Separation and HPLC Characterization of Active Natural Steroids in a Standardized Extract from the Serratula coronata Herb with Antiseborrheic Dermatitis Activity

Phytoecdysteroids are natural compounds with therapeutic benefits in both humans and animals. The effectiveness of natural products with health potential is based on the activities and potencies of their active ingredients. In this study, dominant ecdysteroids—ajugasterone C, 20-hydroxyecdysone and polypodine B—from the Serratula coronata (S. coronata) herb were separated by column chromatography, identified by spectroscopic data and quantified by high-performance liquid chromatography with a diode array detector (HPLC-DAD). The obtained concentration of ecdysteroids (approximately 23%) in the S. coronatae herb extract enhances the possibility of their use in pharmaceutical and cosmetic products with high levels of phytoecdysteroids. Moreover, this study has shown a positive effect of ecdysteroids-containing cream on changes in quality of life and a beneficial effect in reducing the symptoms of seborrheic dermatitis. It has been demonstrated that the application of the cream with phytoecdysteroids resulted in a statistically significant alleviation of symptoms (p < 0.05), especially in terms of itching, pain or burning sensations in the affected areas in comparison to previous symptoms.


Introduction
Phytoecdysteroids are a class of chemical compounds (triterpenoids) that are synthesized by plants for defense against insects. Over 300 plant ecdysteroid analogues have been identified so far with distinctive chemical, physical and biological properties. These compounds are derivatives of cyclopentane perhydrophenantrene. The vast majority of them have 27 atoms of carbon, with a double bond between C7-C8 and a keto group on C6. Ecdysteroids are characterized by methyl groups on C10 and C13 in the ß configuration and by the double bond between C7-C8. Ecdysteroids isolated from the Centaureinae subtribe plants are further characterized by two OH groups on C2

Serratula coronata Herb
Ajugasterone C (1) unclear. Thus, in this study, we focused particularly on the use of main phytoecdysteroids isolated from the Serratula coronate herb for skin disorders such as SD. The aim of this study was to develop and validate a simple, sensitive and precise method for the isolation and quantification of dominant ecdysteroids-ajugasterone C (1), polypodine B (2) and 20hydroxyecdysone (3)-from the S. coronata herb ( Table 1). The next step was to assess the clinical potential of a cream with a 2% concentration of ecdysteroids for reducing the symptoms of SD.

Extractionand Identification of Ecdysteroids
Dried herbs of S. coronata (330 g) were crushed and soaked in methanol (MeOH; Merck KGaA, Darmstadt, Germany) (1.32 L, three times). The MeOH extract was evaporated, and the residue was dissolved in distilled water (0.5 L). The aqueous extract was shaken with ethyl acetate (AcOEt; Merck KGaA, Darmstadt, Germany) (three times with 0.5 L). The AcOEt extract was evaporated, leaving 7.35 g of residue.
The compounds were separated by column chromatography on silica gel with a size of 0.063-200 nm (Merck Art. 7733, Darmstadt, Germany). Selected fractions were further rechromatographed on silica gel with a size of <0.063 nm (Merck Art. 7729, Darmstadt, Germany).
Dried and crushed herbs of S. coronata (620 g) was soaked three times in ethanol (EtOH) (2.85 L) for 24 h. The EtOH extract was evaporated and gave a residue-Extractum siccum (14 g)-which was used as the material for quantitative analysis and for the preparation of the standardized cream for application. 3

Plant Material
Herbs of Serratula coronata L. (Compositae/Asteraceae) were collected during the dry seasons of 2013-2016 (in May each year) from the Botanical Garden at the Department of Medicinal and Cosmetic Natural Products, University of Medical Sciences in Poznan (Poland), where the voucher specimens (No. 64/2009) are deposited. S. coronata seedswere provided by the Botanical Garden in Vacratot (Hungary). They were gathered from a natural habitat in the north of Hungary. The aerial parts and seeds of S. coronata were identified by Karol Latowski of Adam Mickiewicz University in Poznan.

Extractionand Identification of Ecdysteroids
Dried herbs of S. coronata (330 g) were crushed and soaked in methanol (MeOH; Merck KGaA, Darmstadt, Germany) (1.32 L, three times). The MeOH extract was evaporated, and the residue was dissolved in distilled water (0.5 L). The aqueous extract was shaken with ethyl acetate (AcOEt; Merck KGaA, Darmstadt, Germany) (three times with 0.5 L). The AcOEt extract was evaporated, leaving 7.35 g of residue.
The compounds were separated by column chromatography on silica gel with a size of 0.063-200 nm (Merck Art. 7733, Darmstadt, Germany). Selected fractions were further rechromatographed on silica gel with a size of <0.063 nm (Merck Art. 7729, Darmstadt, Germany).
Dried and crushed herbs of S. coronata (620 g) was soaked three times in ethanol (EtOH) (2.85 L) for 24 h. The EtOH extract was evaporated and gave a residue-Extractum siccum (14 g)-which was used as the material for quantitative analysis and for the preparation of the standardized cream for application.

Extractionand Identification of Ecdysteroids
Dried herbs of S. coronata (330 g) were crushed and soaked in methanol (MeOH; Merck KGaA, Darmstadt, Germany) (1.32 L, three times). The MeOH extract was evaporated, and the residue was dissolved in distilled water (0.5 L). The aqueous extract was shaken with ethyl acetate (AcOEt; Merck KGaA, Darmstadt, Germany) (three times with 0.5 L). The AcOEt extract was evaporated, leaving 7.35 g of residue.
The compounds were separated by column chromatography on silica gel with a size of 0.063-200 nm (Merck Art. 7733, Darmstadt, Germany). Selected fractions were further rechromatographed on silica gel with a size of <0.063 nm (Merck Art. 7729, Darmstadt, Germany).
The AcOEt extract was chromatographed on silica gel with mixtures of n-hexane and Me 2 CO (ratio 2:1) as eluent (Merck KGaA, Darmstadt, Germany). Two collected fractions needed purifying and were rechromatographed on silica gel with a mixture of dichloromethane (CH 2 Cl 2 ; Merck KGaA, Darmstadt, Germany) and MeOH (ratio 5:1).
Dried and crushed herbs of S. coronata (620 g) was soaked three times in ethanol (EtOH) (2.85 L) for 24 h. The EtOH extract was evaporated and gave a residue-Extractum siccum (14 g)-which was used as the material for quantitative analysis and for the preparation of the standardized cream for application.

Ecdysteroids Characterization
For the characterization of compounds, 1 H NMR analysis was performed. Signals of vegetable ecdysteroids have been seen in the proton spectra of all three analysed compounds (Table S1): double bonds between C7-C8 (characteristic doublets from 5.80 for compounds 1 and 3 to 5.85 for compound 2).
The following signals are typical of compound 1 ( Figure S1): a 4.10 ppm signal on C11, which is from the proton on the hydroxyl group; and a 1.58 ppm signal on C25, which means that there is no OH group in this location in the ecdysone's aliphatic chain, resulting in typical signals of two methyl groups on C26 (0.916 ppm) and on C27 (0.920 ppm). The 2.38 ppm signal, on the other hand, indicating the presence of a proton on C5 in compound 3's spectrum, distinguishes it from compound 2 ( Figures S2 and S3).

Quantification of Ecdysteroids Using HPLC-DAD Analysis
Ajugasterone C (1) and polypodine B (2) were used for analysis after their purification and identification by spectral methods. The ecdysteroid 20-Hydroxyecdysone (3) (1 mg/mL of methanol) in the form of a Certified Reference Material was purchased from Merck (KGaA, Germany). Solvents used for chromatographic analysis were of HPLC grade (Sigma Aldrich Sp. z o.o., Poznan, Poland).
The dry ethanol extract of the Serratula coronata herb was dissolved in methanol, in a final concentration of 20 g/L.
Quantitative analysis was performed using a standard external method. Stock solutions of the analyzed compounds (1.0 g/L of methanol) were used to prepare the calibration curves. The peak area ratios of compounds 1-3 were determined for each calibration sample using a calibration curve from 300 to 1000 mg/L. The linear equation describing the relationship between ajugasterone C (1), polypodine B (2) and 20-hydroxyecdysone (3) concentrations and peak area ratios was determined by least-squares weighted regression methods.The validation of the method was performed in accordance with the Food and Drug Administration (FDA) guidelines [27].Three standard curves were prepared on threedifferent days, and the appropriate regression statistics were determined. The limit of detection (LOD) andthe limit of quantitation (LOQ) were calculated by the determination of the signal-to-noise ratio (S/N) (LOD = 3 × (S/N); LOQ = 10 × (S/N)). For the intra-day assay, six replicates at three different concentrations of quality control (QC) samples-low (300 mg/L), middle (500 mg/L) and high (1000 mg/L)-were analyzed. For the inter-day assay, six replicates of low, middle and high-quality control samples were analyzed daily on three consecutive days. The mean, standard deviation, relative standard deviation (RSD) and accuracy of the intra-day and inter-day experiments were calculated. The accuracy was calculated on the basis of the given formula (mean concentration found/concentration taken) × 100. Accuracy and precision data for intra and inter-day assays for analyzed samples are given in Table 2. Table 2. Intra-day and inter-day precision and accuracy of compounds 1-3 (quality control, QC; relative standard deviation, RSD).

Study Group and Antiseborrheic Activities
The study protocol was approved by the Bioethics Committee, based on Polish legislation and Good Clinical Practice at the Poznan University of Medical Sciences, Poznan, Poland (ethical approval number 1082/08). Written informed consent was obtained from all participants.
After dermatological consultation, the study included 36 patients: 17 women and 19 men aged 18-65. All of them were in a good general state of health. The patients were diagnosed with erythema and skin exfoliation and complained of itching. The changes in their skin were diagnosed as mild-to-moderate SD and were present on average for seven years. There were 64 diagnosed changes in total, located mostly (in 97%) on nasal alar creases, ears, central chest, anterior hairline.
The recipe for the cream was developed by the Department of Medicinal and Cosmetic Natural Products, Poznan University of Medical Sciences (Poznan, Poland). The active substance of the cream was previously described as a dry ethanol extract of the Serratula coronata herb containing three dominant ecdysteroids-ajugasterone C (1), polypodine B (2) and 20-hydroxyecdysone (3)-which constituted 2% of the cream. In this study, the examined ointments were prepared on the basis of a creamy Lekobaza ® Pharma Cosmetic base (Fagron, Kraków, Poland) with amphiphilic properties. Lekobaza is a commercially available, multi-component medium with a pH close to the skin condition [28].
Before the application of cream, all patients were subjected to skin allergy tests using patches with hypoallergenic and water-resistant chambers (IQ Ultimate™ by Chemotechnique Diagnostics) filled with a 25 µL aqueous extract from the Serratula coronata herb and 2 mg of the cream.
Each patient used 8 mg of the cream, which was applied directly to the changed skintwo times a week for six weeks. The effectiveness of the cream was evaluated by a dermatologist during each control visit-that is, after two, four, and six weeks of application-and documented in a Termoquolity index questionnaire. Patients in the study group did not use any other preparations for the skin changes and were asked not to ingest any oral medicines or food which could have affected the changed skin.
The effect of using the tested cream on changes in quality of life was determined using a Termoquality index survey (four-item itch questionnaire) [29]. An assessment of four pruritus features was made, which is the most troublesome symptom of SD. Patients (19 men and 17 women) scored on a scale of 1 to 5 the extent (location, duration), severity and frequency of pruritus and its effect on sleep difficulties.

Data Processing and Statistical Analysis
Test results were analyzed statistically using Statistica 12 software (StatSoftPoland, Cracow, Poland) by StatSoft. Statistical significance was α = 0.05. A result was deemed statistically significant if p < α. The Wilcoxon signed-rank test was used to compare the results before and after the application of cream.

Isolation of Dominant Phytoecdysones from the S. coronata Herb
As a result of our experiment, two fractions were isolated, one of which contained a chromatographically homogenous compound: ajugasterone C (1) (65.3 mg, m.p. 210-214 • C).

Thin Layer Chromatography (TLC) of the Isolated Compounds
After the visualization of the chromatograms of the AcOEt extract from the S. coronata herb, three principal spots appeared: one of them was purple and two were dark blue. Figure 1 shows a clear separation of the purple spot of compound 1, and Figure 2 shows a clear separation between the two dark blue spots of compounds 2 and 3. Interestingly, the TLC study showed the characteristic behaviour of polypodine B (2) on the chromatograms. This is a more polar compound than 20-hydroxyecdysone (3) (due to the additional hydroxyl group at C5) and should theoretically be placed below compound 3 on the chromatogram. However, this reasoning is valid only for a solvent system without methanol (Figure 1). When we used a developing phase containing methanol (Figure 2), compound 2 exhibited a higher running spot compared to compound 3. A possible explanation of the change in the polarity of polypodine B (2) is the presence of both the hydroxyl group at position 5 and carbonyl oxygen at C6 [30]. below compound 3 on the chromatogram. However, this reasoning is valid only for a solvent system without methanol (Figure 1). When we used a developing phase containing methanol (Figure 2), compound 2exhibited a higher running spot compared to compound 3. A possible explanation of the change in the polarity of polypodine B (2) is the presence of both the hydroxyl group at position 5 and carbonyl oxygen at C6 [30].
The limits of detection (LOD) for compounds 1-3 were 18, 4.5 and 15 μg/mL, respectively. The limits of quantification (LOQ) for ajugasterone C (1), polypodine B (2), 20-hydroxyecdysone (3) were 60, 15 and 50 μg/mL, respectively. below compound 3 on the chromatogram. However, this reasoning is valid only for a solvent system without methanol (Figure 1). When we used a developing phase containing methanol (Figure 2), compound 2exhibited a higher running spot compared to compound 3. A possible explanation of the change in the polarity of polypodine B (2) is the presence of both the hydroxyl group at position 5 and carbonyl oxygen at C6 [30].
The analysis of independent low, middle and high-quality control samples was used to perform the intra-day and inter-day assay. The intra-day RSD for compounds 1-3 ranged from 0.67% to 1.26% for ajugasterone C (1), from 0.60% to 0.95% for polypodine B (2) and from 0.51% to 1.10% for 20-hydroxyecdysone (3); additionally, an accuracy of 97% was achieved for compound 1, between 98% and 106% for compound 2 and 96% and 102% for compound 3 ( Table 2).
The validated method was applied to the determination of compounds 1-3 in plant material extract (Figure 3) after column chromatography separation. The obtained 22.89% concentration of phytoecdysteroids in the S. coronata herb extract of these compounds guarantees the possible use of the standardized extract as an effective ingredient in cosmetics and drug production with this unique group of natural compounds.
It should be noticed that isomers in rare cases are not identifiable by mass spectrometry (MS) because they produce identical spectra. In our work, the 1 H NMR analysis indicated the details of the structures of the three compounds from the plant extract. Two of the compounds (ajugasterone C and 20-hydroxyecdysone) are isomers; therefore, by MS, the substitution sites ofOH groups are indistinguishable. The consequence of this approach is the misinterpretation of ajugasterone C and 20-hydroxyecdysone as synonyms. Therefore, we decided to use NMR in our study as the best method for structural analysis.

Examination of 2% Phytoecdysteroids Cream for Seborrheic Dermatitis
During the six weeks of the application of the cream, the quality of the patients' life improved significantly, and in 97% of cases, following the use of the cream with phytoecdysteroids, the epidermis reverted to its physiological state as assessed by a dermatologist. Moreover, there was no report of any side effects of the cream. It has been shown that applying the cream with phytoecdysteroids resulted in a statistically significant alleviation of symptoms (p < 0.05), especially in terms of itching, pain or burning sensations in the affected areas in comparison to previous symptoms. The results obtained before and after the application of the cream are presented in Figure 4. The obtained 22.89% concentration of phytoecdysteroids in the S. coronata herb extract of these compounds guarantees the possible use of the standardized extract as an effective ingredient in cosmetics and drug production with this unique group of natural compounds.
It should be noticed that isomers in rare cases are not identifiable by mass spectrometry (MS) because they produce identical spectra. In our work, the 1 H NMR analysis indicated the details of the structures of the three compounds from the plant extract. Two of the compounds (ajugasterone C and 20-hydroxyecdysone) are isomers; therefore, by MS, the substitution sites ofOH groups are indistinguishable. The consequence of this approach is the misinterpretation of ajugasterone C and 20-hydroxyecdysone as synonyms. Therefore, we decided to use NMR in our study as the best method for structural analysis.

Examination of 2% Phytoecdysteroids Cream for Seborrheic Dermatitis
During the six weeks of the application of the cream, the quality of the patients' life improved significantly, and in 97% of cases, following the use of the cream with phytoecdysteroids, the epidermis reverted to its physiological state as assessed by a dermatologist. Moreover, there was no report of any side effects of the cream. It has been shown that applying the cream with phytoecdysteroids resulted in a statistically significant alleviation of symptoms (p < 0.05), especially in terms of itching, pain or burning sensations in the affected areas in comparison to previous symptoms. The results obtained before and after the application of the cream are presented in Figure 4. There are scientific reports which have suggested that phytoecdysteroids have antiinflammatory and antioxidant properties as well as a positive effect on metabolic diseases, infectious diseases and physical and mental weakness [3,8,11,12,[14][15][16][17][18]31]. Rosselli et al. have shown that ecdysones have cytotoxicity potential against rat C6 glioma cells [32]. However, further studies of the application of phytoecdysteroids in medicine are still needed.
The method used in this study could be adapted for the future evaluation of ajugasterone C (1), 20-hydroxyecdysone (2) and polypodine B (3) concentrations in patients' plasma during the application of the cream. The obtained concentration of ecdysteroids (approximately 23%)in the Serratula coronata herb extract enhances their potential use in cosmetics and drug production with this unique group of natural compounds.
The results obtained in this study, after six weeks of usage of phytoecdysteroid-containing cream, were satisfactory. The quality of patients' life improved considerably due to significant alleviation of symptoms (p < 0.05), especially in terms of itching, pain or burning sensations in the SD-affected areas. In 97% of the cases, following the use of the cream with phytoecdysteroids, the epidermis reverted to its physiological state. Moreover, there was no report of any side effects of the cream.
The precise molecular mechanisms describing the pharmacological activity of phytoecdysteroids in an unusual skin condition still remain unexplored. However, it is known that the action of phytoecdysteroids is associated with skin barrier function recovery. Data in the literature indicated that phytoecdysones might promote keratinocyte differentiation in vitro [33]. Thus, the positive effect of the application of phytoecdysteroid-containing cream in SD patients may be associated with the normalization of the keratinocytes differentiation process. It is postulated [34,35] that ecdysones may have a significant impact on the reduction of inflammation, probably due to their immunomodulatory function and the modulation of the level of the pro-inflammatory cytokine (e.g.,interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α)). Moreover, it has been reported that phytoecdysteroids improve skin quality by accelerating the healing of wounds and burns [36,37].Several studies [38] have claimed associations between Malassezia restricta lipase and SD. In light of this, ecdysteroids, especially 20-hydroxyecdysone, probably enhance antifungal immunity, resulting in a reduction of disease symptoms [39].
Therefore, it can be expected that the use of phytoecdysteroids might have a long-term impact on skin changes in patients with SD, and potential recurrences of the condition might be delayed. Cream with natural steroids may also be helpful for other skin disorders resulting in itching, as well There are scientific reports which have suggested that phytoecdysteroids have anti-inflammatory and antioxidant properties as well as a positive effect on metabolic diseases, infectious diseases and physical and mental weakness [3,8,11,12,[14][15][16][17][18]31]. Rosselli et al. have shown that ecdysones have cytotoxicity potential against rat C6 glioma cells [32]. However, further studies of the application of phytoecdysteroids in medicine are still needed.
The method used in this study could be adapted for the future evaluation of ajugasterone C (1), 20-hydroxyecdysone (2) and polypodine B (3) concentrations in patients' plasma during the application of the cream. The obtained concentration of ecdysteroids (approximately 23%)in the Serratula coronata herb extract enhances their potential use in cosmetics and drug production with this unique group of natural compounds.
The results obtained in this study, after six weeks of usage of phytoecdysteroid-containing cream, were satisfactory. The quality of patients' life improved considerably due to significant alleviation of symptoms (p < 0.05), especially in terms of itching, pain or burning sensations in the SD-affected areas. In 97% of the cases, following the use of the cream with phytoecdysteroids, the epidermis reverted to its physiological state. Moreover, there was no report of any side effects of the cream.
The precise molecular mechanisms describing the pharmacological activity of phytoecdysteroids in an unusual skin condition still remain unexplored. However, it is known that the action of phytoecdysteroids is associated with skin barrier function recovery. Data in the literature indicated that phytoecdysones might promote keratinocyte differentiation in vitro [33]. Thus, the positive effect of the application of phytoecdysteroid-containing cream in SD patients may be associated with the normalization of the keratinocytes differentiation process. It is postulated [34,35] that ecdysones may have a significant impact on the reduction of inflammation, probably due to their immunomodulatory function and the modulation of the level of the pro-inflammatory cytokine (e.g.,interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α)). Moreover, it has been reported that phytoecdysteroids improve skin quality by accelerating the healing of wounds and burns [36,37].Several studies [38] have claimed associations between Malassezia restricta lipase and SD. In light of this, ecdysteroids, especially 20-hydroxyecdysone, probably enhance antifungal immunity, resulting in a reduction of disease symptoms [39].
Therefore, it can be expected that the use of phytoecdysteroids might have a long-term impact on skin changes in patients with SD, and potential recurrences of the condition might be delayed. Cream with natural steroids may also be helpful for other skin disorders resulting in itching, as well as atopic dermatitis. No side effects were observed, which frequently occur during the application of a cream with synthetic steroids [23].
The results of our study give dermatologists, cosmetologists and patients hope for an efficient and safe skin care method. The positive results of applying the cream with a 2% content of ecdysteroids in seborrheic dermatitis are promising for the development of a new drug for skin diseases which cause pruritus.

Conclusions
The use of the S. coronata herb, with a high concentration of ecdysteroids-ajugasterone C (1), polypodine B(2) and 20-hydroxyecdysone (3)-and with bioactive properties, appears to be a promising strategy for the preparation of valuable pharmaceutical and cosmetic products. The positive results of applying the cream with a 2% standardized extract content of ecdysteroids in seborrheic dermatitis suggest its potential new application for skin diseases which cause pruritus. The simple but effective method for isolating active phytoecdysones presented in this paper maybe useful infuture study. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.