SUMMARY OF Ph.D. THESIS

SUMMARY OF Ph.D. THESIS Stability evaluation of the lipophilic bioactive compounds from sea buckthorn (Hippophae rhamnoides) and apricot (Prunus armeniaca) during processing and their us as cosmetic ingredients with dermo-protective effect PhD student Elena-Andreea Pop Scientific coordinator Prof. univ. dr. Carmen Socaciu

CONTENTS INTRODUCTION 18 19 AIM AND OBJECTIVES THE CURRENT STATE OF KNOWLEGDE CHAPTER 1. Chapter 1. Sea buckthorn(hippophae rhamnoides)- Literature review 1.1. Morphological characterization of the plant 23 1.2. The composition and chemical characterization of sea buckthorn 23 1.3. Bioactive lipophilic compounds from sea buckthorn (carotenoids, tocopherols, 24 tocotrienols) 1.4. Effect of processing parameters on the lipophilic bioactive compounds from sea buckthorn 30 CHAPTER 2. Capitolul 2. Apricot (Prunus armeniaca)- Literature review 32 2.1 Morphological characterization of apricot 32 2.2 The composition and chemical characterization of apricot 32 2.3 Factors influencing the characteristics of apricots 34 2.4 Bioactive lipophilic compounds from apricot berries (carotenoids, tocopherols, 34 tocotrienols, fatty acids 2.5 Effect of processing parameters on the lipophilic bioactive compounds from apricot 35 CHAPTER 3. Methods for analysis of lipophilic bioactive compounds 36 3.1 Metode de analiza spectrometrică 36 3.1.1. Spectrometric analysis 36 3.1.2. FT-IR spectrometry 37 3.2. Separation and identification methods by high performance liquid chromatography 37 with mass spectrometric detection (HPLC-MS) 3.3. Analytical methods applied to identify specific bioactive compounds of plant (HPLC analysis) 20 23 PERSONAL CONTRIBUTIONS RESEARCH OBJECTIVES 41 CHAPTER 4. CHAPTER 4.1. Stability evaluation of the lipophilic bioactive compounds from sea buckthorn Stability evaluation of the lipophilic bioactive compounds (carotenoids) from frozen sea buckthorn 42 4.1.1. Introduction 42 4.1.2. Materials and methods 43 4.1.2.2 UV VIS spectroscopic fingerprint of extracts obtained from frozen sea buckthorn 43 berries 4.1.2.3. Determination of the carotenoids profile (including esters) of sea buckthorn using high performance liquid chromatography 44 4.1.2.4. Bio-statistical analysis 44 4.1.3. Results and discussion 44 4.1.3.1. UV VIS spectroscopic fingerprint of frozen sea buckthorn berries 44 4.1.3.2. Total carotenoids determined in frozen sea buckthorn 45 4.1.3.3. Identification and quantification of major pigments from frozen sea buckthorn 46 4.1.4. Conclusions 51 CHAPTER 4.2. Stability evaluation of the lipophilic bioactive compounds 52 2

(carotenoids) from dried sea bucktorn (Study 2) 4.2.1. Introduction 52 4.2.2. Materials and methods 52 4.2.3. Results and discussion 53 4.2.3.1. Total content of carotenoids content determined in dried sea buckthorn 53 4.2.3.2. Stability assessment of the major pigments samples identified in dried sea 54 buckthorn berries 4.2.4. Conclusions 57 CHAPTER 4.3. Stability evaluation of the lipophilic bioactive compounds (carotenoids) from sea buckthorn oil (Study 3) 4.3.1. Introduction 58 4.3.2. Materials and methods 59 4.3.3. Results and discussions 59 4.3.3.1. Identification and quantification of the major pigments from sea buckthorn oil 59 4.3.4. Conclusions 65 CHAPTER 5 CHAPTER 5.1. Stability evaluation of the lipophilic bioactive compounds apricots Stability evaluation of the lipophilic bioactive compounds (carotenoids) from frozen apricots (Study 4) 5.1.1. Introduction 66 5.1.2. Materials and methods 67 5.1.3. Results and discussions 68 5.1.3.1. Total carotenoid content determined in frozen apricots 68 5.1.3.2. Stability assessment of the lipophilic bioactive compounds (carotenoids) from 71 twelve apricot 5.1.4. Conclusions 74 CHAPTER 5.2. Stability evaluation of the lipophilic bioactive compounds (fatty acids) from frozen apricots varieties ( Study 5) 5.2.1. Introduction 75 5.2.2. Materials and methods 77 5.2.3. Results and discussions 78 81 CHAPTER 5.3. Stability evaluation of the lipophilic bioactive compounds (carotenoids) from dried apricots (Study 6) 5.3.1. Introduction 82 5.3.2. Materials and methods 83 5.3.3. Results and discussions 83 5.3.3.1. UV VIS spectroscopic fingerprint of dried apricots 83 5.3.3.2. Identification and quantification of the major pigments from dried apricots 85 5.3.4. Conclusions 87 CHAPTER 6. Carotenoids, tocopherols and antioxidant activity of lipophilic extracts from sea buckthorn berries (Hippophae rhamnoides), apricot pulp and apricot kernel (Prunus armeniaca) (Study 7) 6.1. Introduction 88 6.2. Materials and methods 88 6.2.1. Plant material 88 6.2.2. Separation of carotenoids by RP-HPLC 89 6.2.3. Separation of tocopherols by HPLC system with fluorescence detector 89 6.3. Results and discussions 90 6.3.1. Carotenoids, tocopherols and tocotrienols identified in sea buckthorn berries, apricot fruits and apricot kernels 6.3.2. Antioxidant capacity of lipophilic extracts from sea buckthorn berries, apricot fruits and apricot kernels 6.4. Conclusions 94 CHAPTER 7. Preparation and characterization of some cosmetic formulas 95 3 58 66 66 75 82 88 90 93

with dermo-protective effect (Study 8) 7.1. Introduction 95 7.2. Materials and methods 96 7.2.1. Preparation of cosmetic products (Anti-acne emulsion, Moisturizing day cream, Day cream for problematic skin, Sun screen, Anti-acne cream) 97 7.2.2. Sensorial tests 100 7.2.3. Microbiological tests 100 7.2.4. Assessment of the antioxidant capacity, using DPPH and TEAC methods. 102 7.2.5. Determination of bioactive components from the cosmetic formula using FT-IR spectroscopy 7.2.6. Bio-statistical analysis 103 7.3. Results and discussions 103 7.3.1. Results regarding the sensorial analysis 104 7.3.2. Results regarding the microbiological quality of the products 105 7.3.3. Results regarding the microbiological quality of the products 107 7.3.4. FT-IR fingerprints of the bioactive compunds identified in the cosmetic formulas 110 CONCLUSIONS 114 ORIGINALITY AND INNOVATIVE CONTRIBUTIONS OF THE THESIS 116 REFERENCES 117 103 4

THE PURPOSE AND OBJECTIVES OF THE THESIS The purpose of this scientific research was to deepen the approach and perfection of knowledge through the analysis of two fruits and herbs or sea buckthorn (Hippophae rhamnoides) and apricots (Prunus armeniaca). Comparative evaluation of the content of bioactive compounds of sea buckthorn and apricot was necessary to identify the main changes that occur during the process flow, identifying the factors that cause changes in the concentration of compounds and not least detecting the effect of certain processing parameters of biologically active substances. Most active decomposition principles is determined by enzymatic processes of oxidative decomposition under the action of oxidases or atmospheric oxygen and the process of hydrolysis to form inactive compounds. Fruits and vegetables are essential components of a healthy diet through their contribution in bioactive substances, including antioxidants. Sea buckthorn (Hippophae rhamnoides) is an important plant in the food and medical plan. Sea buckthorn fruits are used due to the high content of microelements, antioxidants, vitamins, in particular vitamin C, which is contained in the amount of ten times higher than citrus. The oil extracted from the pulp and seeds has regeneration, anti-inflammatory and antiulcerogenic effect, which are often linked to cytoprotective antioxidant activity. The fruits of apricot (Prunus armeniaca L.) are the most appreciated and consumed fruit throughout the world, both for their flavor and nutritional qualities. The high content of sugars, proteins, minerals, phytochemicals, vitamins, confer important biological properties such as the antioxidant, antimicrobial and anti-inflammatory. Bioactive compounds from sea buckthorn and apricots were analyzed using the method of alcohol extraction techniques, UV-VIS spectroscopy, FT-IR and HPLC-PDA/MS. All these compounds have a regenerative action on the metabolism. This is due to the high content of vitamins in fruits and other bioactive substances such as carotenoids, flavonoids, protein, essential fatty acids and trace elements. Due to their antioxidant, anti-inflammatory and antimicrobial properties both fruits gives immunity to the body and the combination of these two species offers spectacular results in dermatology. Currently, the public interest for natural products is evident due to the widely recognized benefits, wide accessibility and rare adverse effects. 1. Characterization of bioactive lipophilic compounds from sea buckthorn and apricots Making lipophilic extracts of sea buckthorn fruit, apricots and apricot kernels. Determination of total carotenoids of different varieties/cultivars of sea buckthorn and apricots. Determination of fatty acids in fruits of apricot and apricot kernels. Determination of tocopherols and tocotrienols in sea buckthorn fruits and apricots fruits. 2. Study the stability of lipophilic compounds during processing. Determination of lipophilic compounds from sea buckthorn fruit to thermal treatment (drying). Determination of light stability of carotenoids in sea buckthorn oil (controlled light exposure). Determination of light stability of carotenoids from fruits dried apricots (controlled light exposure). 5

3. Getting a product dermo-protective effect based on sea buckthorn oil and apricot kernel oil. Collection of sea buckthorn oil and apricot kernel oil. Getting creams according to the need of hydration and adaptation of each skin type. Thesis structure. The thesis is divided into two parts: the first part comprises bibliographic study on the scientific knowledge of the addressed areas, namely knowledge through analysis of two fruits and herbs, sea buckthorn (Hippophae rhamnoides) and apricots (Prunus armeniaca). The compounds of sea buckthorn and apricot bioactive investigated were analyzed using the alcohol extraction techniques, UV-VIS spectroscopy, FT-IR and HPLC-PDA/MS. The first part of the thesis is divided into three chapters: Chapters 1 and 2 include morphologic and physico-chemical characterization of sea buckthorn fruit and apricots, lipophilic fraction description and identification of factors that influence the characteristics of fruit. It has also shown the effect of processing parameters fot the lipophilic bioactive compounds from two fruits. Chapter 3 includes information about methods of analysis for the determination of organic compounds and to elucidate their structure (UV-VIS, FTIR, HPLC-PDA, HPLC-MS and GC). The second part of the thesis, totaling 4 experimental studies: Chapters 4.1, 4.2, 4.3 contains information about the evaluation of the stability of bioactive lipophilic (carotenoids) from the sea buckthorn subjected to the procedure of preservation by freezing, assess the stability of carotenoids in sea buckthorn subjected to thermal treatment (drying oven) and not least assess the stability of carotenoids in sea buckthorn oil, at room temperature and exposed to ambient light. Chapters 5.1, 5.2, 5.3 contains information about the evaluation of the stability of bioactive compounds (carotenoids, fatty acids) of different varieties of apricot frozen. The stability of carotenoids from dried apricots was also assessed. Chapter 6 is dedicated to determine the antioxidant capacity of lipophilic extracts of sea buckthorn berries (Hippophae rhamnoides), apricot kernels and apricot (Prunus armeniaca). Chapter 7 is dedicated to obtain and characterize cosmetic formulas containing ingredients with dermo-protective effect. 6

PERSONAL CONTRIBUTIONS CHAPTER 4. Evaluation of stability of lipophilic bioactive compounds (Carotenoids) from frozen sea buckthorn berries Chapter 4.1. Evaluation of stability of lipophilic bioactive compounds (Carotenoids) from frozen sea buckthorn berries Introduction The main issue addressed in this study refers to the experimental identification and characterization of carotenoids from fruits of sea buckthorn frozen. Materials and methods In order to extract the total carotenoid of the sea buckthorn fruit, there were used 20 grams of plant product to which was added 90 ml of a mixture of solvents (petroleum ether / methanol / ethyl acetate, 1: 1: 1, v / v / v). Spectophotometric characterization was carried out using the plant extract obtained by conventional solvent extraction. The chromatographic analysis to determine the profile of carotenoids was carried out using a Shimadzu LC20 AT with PDA detector SPD-M20A and a gradient formed from two solvents: Solvent A: methanol / tert-butyl methyl ether / water (v / v / v, 81: 15: 4) and solvent B: tert-butyl methyl ether / methanol / water (v / v / v; = 90: 7: 3). Results and discussions After analyzing UV-Vis extracts, it was found that while absorption spectra layout changes, a drop in absorbance in the visible (400-500 nm) and with an increase in absorbance in the 300-350 nm. This may indicate a degradation of carotenoids and forming cis isomers which are characterized by absorption bands in the ultraviolet range. After analyzing UV-VIS, assayed in total carotenoids in sea buckthorn carotenoids are expressed in mg/100 g. The results from this analysis show a variation of the total carotenoid content in the sea buckthorn fruits. These results demonstrate the influence while the process of preservation by freezing or low temperatures on the total carotenoid content of sea buckthorn fruit. By HPLC-PDA sea buckthorn fruits were many peaks, among which, zeaxanthin, β- cryptoxanthin with various fatty acids. To assess the stability of carotenoids in sea buckthorn fruits were frozen retrieve selected five major compounds in fruits cătină- β-carotene, lycopene, zeaxanthin di-palmitate and myristate zeaxanthin. The rate of degradation of the carotenoid varies depending on the chemical structure of the carotenoid compound, which can be seen from 7

the data quantity recorded for individual compounds. Following the assessment of the relative concentration of compounds majority retrieve the underbrush fruits analyzed, the higher the residual concentration was recorded for Di-zeaxanthin palmitate (71.19%), followed stubblezeaxanthin palmitate (69.01% ). Such stability is observed significantly higher zeaxanthin esters during storage compared to free zeaxanthin (54, 68%). Conclusion qualitative changes were revealed in the appearance of UV-Vis absorption spectra of the extracts of total carotenoid during storage, which indicates processes of isomerization/oxidative degradation of the pigments. Total carotenoids content in the underbrush gradually decreases during preservation by freezing, It is not statistically significant in the first three months, but significant after six months and after 12 months. The degradation rate of carotenoids varies is different depending on the chemical structure of carotenoids, such hydrocarbons are less stable than xanthophylls. The more stable forms have been shown to be zeaxanthin esters with saturated fatty acids, of which the rate of degradation was significantly lower than that of the free zeaxanthin. Chapter 4.2. Stability assessment lipophilic bioactive compounds (Carotenoids) of sea buckthorn subjected to thermal treatment (drying oven) Introduction The main issue addressed in this study refers to the experimental identification and characterization of carotenoids from dried sea buckthorn fruit. Materials and methods To assess the stability over time was carried out to 300.46 g dry fruit of sea buckthorn at 37 C for 14 days in an oven. Buckthorn fruits were reweighed periodically, finally yielding 68.082 g vegetable. For the extraction of carotenoids total fruit of sea-buckthorn made during storage, were used 3 grams of plant product to which was added 60 ml of a mixture of solvents (petroleum ether/methanol/ethyl acetate, 1: 1: 1 v/v/v). Determination of total carotenoids from dried sea buckthorn fruits was performed by UV-VIS spectrophotometric method, respectatăndu the protocol described above. To determine the profile of carotenoids (including esters) of sea buckthorn dried by means of high performance liquid chromatography (HPLC) was observed same protocol as previously. 8

Results and discussions The total carotenoids in seabuckthorn fruit dry carotenoid is expressed in mg / 100g. In this study it was found that there is a gradual degradation in terms of total carotenoid content in seabuckthorn fruit dry. Total carotenoid composition recorded variations in time, and the values obtained in this experiment were between 53.19 and includes 106, 49 mg / 100g. Changes in quantity or degradation of carotenoids, went with a very low speed if the monthly extractions track progress made, but nevertheless observed a highly significant difference between the control sample, the initial extraction performed on 2 June 2014 and the extraction carried out on October 14 2014. Drying process stops the growth of microorganisms and enzymes own fruit and vegetables, but studies have shown degradation of the active principles found in fruit composition, which influences food value of the finished product. At the same time produce a qualitative sea buckthorn fruit that you are suffering due to reduced volume and tissue shrinkage. Following chromatographic analysis HPLC-PDA, demonstrated degradation of β-carotene stronger than zeaxanthin and esters thereof. These observations are consistent with other studies showing increased stability of the xanthophylls and especially their esters, compared to hydrocarbons. It was determined the correlation between majority retrieve compounds in seabuckthorn fruit dry by the Pearson correlation coefficient. Correlations between hydrocarbons and xanthophylls were between -0.69 and 0.98. Lycopene linking with other compounds in fruits majority retrieve dry underbrush, it was found that there is a negative correlation coefficient, which shows an inverse correlation between them. Conclusion According to experimental data presented were obtained and quantified total carotenoids in sea buckthorn extracts subjected to thermal treatment (drying oven). The obtained results we can conclude that: total carotenoids content in the underbrush gradually decreases during preservation by drying, it is not statistically significant in the first three months, but significant in the next six months Compared with the original sample, the content of carotenoids six months after freezing was reduced to about 50% Following this heat treatment has been demonstrated that the drying and preservation of sea buckthorn fruit while this form leads to significant losses in terms of the total content of carotenoids. 9

Chapter 4.3. Stability assessment lipophilic bioactive compounds (Carotenoids) of sea buckthorn oil Introduction Sea buckthorn oil is a natural remedy that can be used for human consumption and also as a cosmetic skin care ingredient. Rich in various essential fatty acids of sea buckthorn oil has also higher palmitoleic acid content, which is a component of the natural skin lipids. Materials and methods This experimental study aimed at obtaining the first stage of sea buckthorn oil using methanol as solvent and chloroform. The resulting oil was stored at room temperature and ambient light in the transparent or brown vials. Major carotenoid concentration was evaluated monthly for 10 months by the same method HPLC / PDA described in the previous chapter. For determination of bioactive compounds from sea buckthorn oil, extraction was carried out initially, then the extracts obtained were analyzed by HPLC. Pigments extraction was performed from frozen sea buckthorn fruits, using methanol as solvent extraction and chloroform) (v/v/1:2). Results and discussions HPLC-PDA analysis revealed specific profile of the carotenoids in sea buckthorn oil, which includes free and esterified xanthophylls and carotenoid hydrocarbons. Like in the case of frozen and dried fruits were monitored during preserve five major compounds: β-carotene, lycopene, zeaxanthin and zeaxanthin esters (Di-palmitate and myristate-palmitate). Major compound found in samples in September 2014 when oil was exposed to white vial β-carotene, followed by Di-zeaxanthin palmitate. Qualitative and quantitative differences may be due to the consequences of oil extraction steps and not least changes are caused by the storage of samples under the influence of light and temperature over a long period. The content of sea buckthorn fruit oil varies from species to species, depending on the stage of harvest of the fruit, and not the least by the extraction method used. Compared with results for samples stored in transparent vial, the vial when exposed brown oil, Di-zeaxanthin palmitate was found in greater quantity, followed by β-carotene. Zeaxanthin and lycopene free were found in smaller quantities. There was a gradual change in the ratio of carotenoids, a decrease of carotenoids available and an increase in time of esterified forms. The major differences between samples exposed in the transparent vial and the vial brown exposed were observed in all compounds, identifying molecules greater stability of oil samples exposed brown vial. The major difference has occurred in lycopene, the compound that has changed during the typical processing procedures sea buckthorn oil, showing greater stability in oil samples exposed brown vial compared with the oil exposed in open vial. Due process of isomerization and oxidation of carotenoids, during the experimental study identified a qualitative 10

and quantitative change in the compound, so there was correlation coefficient negative if correlated with carotenoids zeaxanthin esters free. It is considered that xanthophylls esterification plant has physiological significance in that it helps to increase lipophilicity and chromoplasts assembly. Equally storing them in specialized structures (plastoglobule and fibrils) protects plants against fotooxidative degradation. Chapter 5.1. Identification of carotenoids of the apricots preservation by freezing Introduction In this experimental study was determined the total content of carotenoids in 12 varieties of apricots namely Harogem, Commodore, Sirena, Mamaia, Sulmona, Sulina, Red Baneasa HABU, Tudor, Olympus, Excelsior, Umberto. Materials and methods Analyses were performed on an HPLC system (Shimadzu, Milan, Italy) equipped with two pumps LC-20, a DGU-20A3 degasser, an autosampler SIL AC-20 and SPD-M20A photodiode. Data were processed with software version Labsolution. MS was used for the analyzes of the mass spectrometer (LC-MS-2020, Shimadzu) equipped with an APCI interface, both in positive ionization mode, and in the negative. The separations were carried out on a YMC C30 column - (250x4.6 mm, 5 mm); The mobile phase consisted of methanol: MTBE: water (83:15:2, v/v/v; eluent A) and methanol. Results and discussions The major amount of total carotenoids was estimated Harogem variety and the variety found in the majority compound was ß-carotene, which represents 57.4% of total carotenoids. ß- carotene is also composed mostly of the variety Olympus, where they found the rate of 44.2%. Along with ß-carotene were found in large quantities and ß-cryptoxanthin and other compounds, ß-zeacaroten. The latter compound was not detected in Tudor variety. At the same time, the amount of lycopene varied apricot varieties analyzed, the values were between 0,01-3.8%, the highest percentages being in the variety Sirena and in varieties Excelsior, Red Baneasa, Mamaia and Tudor, lycopene not it was detected. The biggest difference in terms of total carotenoid content was found in varieties Excelsior, Sirena, Sulina, variability in representing the predominant factor regarding carotenoids found. Concomitant use with photodiode detector and the detector MS has identified a large number of compounds, which is particularly important especially for the characterization fraction of β-cryptoxanthin esters. Since xanthophylls esters with the same spectral characteristics of free xanthophylls, they can not be identified solely on the basis of the absorption spectra. Moreover, 11

the absorption spectra esters are the same for all, regardless of the chemical nature of the fatty acid. Therefore HPLC/PDA/MS is the only method that allows the unequivocal identification of esters. Following this exploratory study, it was shown that apricot varieties show a major difference in their composition. The presence of carotenoids differs from one variety to the next and the information provided in this study are useful to identify varieties of interest and also in order to gain insight as to the variability of the composition from one variety to the other. Chapter 5.2. Identification of fatty acids of the varieties of apricot Introduction Lipids are basic components, structural role, being involved in all the vital processes in the body. Fatty acids are the cornerstones of lipid components entering into their composition. Isolation of free fatty acids from biological materials is a complex task and precautions to prevent or minimize the effects of hydrolysis enzymes. Materials and methods Analysis of the fatty acid profile of the extract of sea buckthorn fruits was carried out by gas chromatography coupled with mass spectrometry, a method that involves the initial fatty acids trans-esterification in methanol/sulfuric acid. Acid methyl esters (FAME) were separated and identified using a Perkin Elmer Clarus system T-600 GC-MS (PerkinElmer Inc., Shelton, CT, USA) equipped with a capillary column SUPELCOWAX 10 (Supelco Inc.). Identification of FAME was performed by comparing the retention times with those of known standards (FAME mix component, Supelco # 47885-U) and mass spectra results of the specific databases. The amount of fatty acid was expressed as a percentage of total fatty acids. Results and discussions Data from qualitative and quantitative analysis of the samples have values different from some studies in the literature (oleic 62.34% linoleic acid 30.33% of total fatty acids). This suggests that oil fatty acids from apricot kernels vary greatly, depending on the variety and often content is a parameter fatty acids which help to distinguish varieties. Content analysis of fatty acids of several varieties of apricots, by the method gas chromatography coupled with mass spectrometry (GC-MS) showed that fruit is an excellent source of fatty acids, including linoleic acid is found in a ratio of 43-46%. Variety Variety is an important factor that can affect the composition and content of bioactive compounds lipophilic. Quantification correct this analysis has important applications in nutrition, because the composition of fatty acids retrieve fruit and apricot kernels have a very important effect on health. The results of the experimental study has demonstrated that the varieties of apricot varieties have a different composition in terms of fatty acid composition. Also noted was the importance of 12

apricot kernels, which pri oil content, is a rich source of essential fatty acids. Chapter 5.3. Stability assessment lipophilic bioactive compounds (carotenoids) of apricots dehydrated Introduction The storage conditions may affect the structural integrity of the fruit. After heat treatment, will produce a series of changes, both in terms of physico-chemical stability of dried apricots and on the sensory profile. Thus, the isomerization of the carotenoid may be a useful marker for assessing the process of drying of apricots, at which point it can be seen the formation of the cis isomers, depending on the temperatures applied. Degradation and isomerization of carotenoids may reduce the nutritional value of fruits, therefore, this study is of real interest in improving the nutritional quality of the finished product. Materials and methods Fruits dried apricots were purchased commercially from a shop of organic products. These were kept at ambient temperature (25 C) in the dark. Samples were taken every month, and were extracted and analyzed in triplicate according to the procedure described in Methods. Total carotenoid extracts were dosed and then analyzed spectrophotometrically by high performance liquid chromatography with photodiode array detector. Results and discussions Total carotenoid analysis revealed less the total quantity of carotenoids, their concentration practically halved after 7 months, reaching 0.82 mg / 100 g after 12 months of storage. Considering the initial amount of carotenoids as 100%, only about 20% of the original can be found in samples after one year of storage. Just as in the case of sea buckthorn fruit analyzed in the previous step, there is a higher rate of degradation of hydrocarbons (β-carotene and its isomers), β-cryptoxanthin compared and its esters with saturated fatty acids. The degradation rate was most intense β-carotene registered if, while for cis residual concentrations are higher. A possible explanation is that β-carotene part of total trans isomerization is actually degraded by the various cis isomers. Although concentrations esters are small, they have shown increased stability β-cryptoxanthin and compared with β-carotene. Conclusions Dried apricots In total carotenoid content was 5.45 mg / 100 g. During keeping them suffered significant degradation, residual value is only 20% of the original after 12 months of storage at ambient temperature and darkness. β-cryptoxanthin esters have β-cryptoxanthin superior stability during preserve free and much higher than that of oil, which had the most intense degradation rate. 13

Chapter 6. Carotenoids, tocopherols and antioxidant activity of lipophilic extracts from sea buckthorn berries (Hippophae rhamnoides), apricot pulp and apricot kernel (Prunus armeniaca) Introduction Determination of antioxidant carotenoids and tocopherols activity is an area of interest due to multiple technical impediments caused by water insolubility of carotenoid and antioxidant that most methods used as radical generators hydrophilic substances. Materials and methods Sea buckthorn berries (Hippophae rhamnoides L., var. Mara),were collected from Sibiu and was selected for uniform size and color and stored at -20 C until analysis. The apricot (Prunus armeniaca) were collected from Amman, Jordan and stored at -20 C until analysis. The extraction was carried out starting from 20 g of freeze-dried fruit of sea-buckthorn, apricot and kernel apricot with a mixture of methanol/ethyl acetate/ petroleum ether (1: 1: 1, v/v/v) (Breithaupt and Schwack, 2000). After filtering the extract, the residue was re-extracted twice using same solvent mixture. The combined extract were partitioned in a separation funnel with water, diethyl ether and saturated saline solution. The upper organic phase was evaporated to dryness under vacuum, using a rotary evaporator. ABTS radical-scavenging activity for lipophilic fractions was determined according to Müller et al. (2011) with some modifications. In the present study, a distilled water solution of 5 mm aqueous solution ABTS (2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid) was filtered through manganese dioxide on a Whatman filter paper and then the excess of manganese dioxide was removed from the filtrate through PVDF syringe filter. Fresh ABTS + was prepared fresh each day by dilution with PBS (75 mm) in order to obtain an absorbance of 0.700 at 734 nm. The diluted sample (100 μl) was mixed with 600 μl for 30s, centrifuged and then the absorbance was read using a Biotek Synergy HT microplate reader. A standard Trolox curve (r2=0.997) was obtained using solutions with concentration between 3.125 μm and 350 μm. Antioxidant activity of the extract was expressed as μm Trolox Equivalent. Tocopherols and tocotrienols were separated using the same HPLC system but the detection was performed by a fluorescence detector RF20A operating at λ excitation = 290 nm and λ emission = 330 nm. A Lichrosorb Si60 column (250 x 4.6 mm; 5 μm) was used. The mobile phase consisted in hexane:2-propanol (99.5:0.5; v/v) operated in an isocratic mode with the flow 1 ml/min. Standard compounds α, γ and δ tocopherols and tocotrienols were provided from ChromaDex, USA. Calibration curves were performed with all standards in the concentration range 4-50 μg/ml. 14

Results and discussions In the variety analyzed (Mara) in the present study a total amount of 17.19±1.4 mg/100 fresh weight was found. It was previously reported that the content of carotenoids in some Romanian varieties of Hippophae rhamnoides ranged between 53.1 96.7 mg/100 dry weigh. Considering that the dry weight represents about 20 % of the berries, the total carotenoid content of this variety is high. As we used an unsaponified extract, the major compounds identified were zeaxanthin dipalmitate, other zeaxanthin esters, β-carotene, zeaxanthin, β-cryptoxanthin palmitate and lycopene. Other minor compounds were presents, most of them esters of zeaxanthin, β-cryptoxanthin and lutein with various fatty acids (retention time 70-120 min) which could not be unequivocally identified by PDA detector. Apricot fruits contain significantly lower amounts of carotenoids - 3.51±0.25 mg/100 fresh weight - and have a completely different profile of pigments. The all trans isomer of β-carotene is the main carotenoid, followed by different other of its geometric isomers. The tentative identification of cis isomers was based on the presence of the cis-peak which appears in the UV region of the absorption spectra of carotenoids (around 340 nm). Small amounts of β-cryptoxanthin and β-cryptoxanthin esters and unesterified lutein were also present. The HPLC-PDA analysis of apricot kernel extract showed a total carotenoid content 0.58±0.04 mg/100 fresh weight. Only one carotenoid could be identified the lycopene, the others being in to law amounts to allow a proper identification based only on the characteristics of UV- VIS spectrum. At our knowledge, there are no literature date regarding the presence or the content of carotenoids in apricot kernels. Apricot fruits are characterized by low amounts of tocopherols 1.43 mg/kg -compared to sea buckthorn berries. α-tocopherol was the major compound which accounts for almost 70 %, followed by α-tocotrienol. Small amounts of γ and δ tocopherols were also detected and, as in the case of sea buckthor berries, peaks corresponding to β-tocopherol have been observed but not properly identified. In contrast, apricot seeds had the highest amount of tocopherols and tocotrienols 131.6 mg/kg kernel, which represents 516 mg/kg oil. Chapter 7. Formulation and characterization of new cosmetics, containing dermoprotective extracts Introduction Herbal formulations have been used for many years, globally, not only as therapeutic, but also as prophylactic and health promoting agents. Sea buckthorn oil is a natural remedy which can be used both for human consumption and as a skin care product. 15

Materials and methods In order to test the quality of the creams obtained, several organoleptic and microbiological tests were created. Microbiological tests follow the detection of microorganisms on the surface under seeding on special plate counts in order to characterize the bactericide properties of certain substances both or for control in case of necessity. FT-IR spectra using attenuated total reflectance (ATR) and an internal reflection accessory made of composite zinc selenide (ZnSe) and diamond crystals were obtained on a Schimatzu IR Prestige-21 spectrometer. Each spectrum was registered from 4000 to 500 cm -1. Results and discussions Following the organoleptic testing of the five creams, was noticed the homogenous aspect, typical smell for each substance or oiled incorporated in the cosmetic formulation. The colour was also typical of the type of ingredient used. Following the tests made to assess the microbial load, it was noticed that the emulsion for acneic skin (P1), the anti-acne cream (P4) and the sunscreen (P5) do not contain pathological aerobic germs and micromycetes, Staphylococcus aureus or Escherichia Coli. In the case of the hydrating cream (P2) and of the cream for problematic skin (P3) the results proved to be unsuitable from the point of view of the number of aerobic, mesophilic germs, and there were identified colonies exceeding the maximum number allowed, and the creams are not suitable from a microbiological point of view. Conclusions The results of this monitoring showed that the presence of colonies in the cosmetic tests can be associated with the insufficient addition of curing agent and of the replacement of the bi-distilled water with rose water. According to this study it was noticed that the hydrating day formula shows extremely significant differences compared to the acneic skin emulsion and due to the method used, has the highest antioxidant activity. The results recorded by using the TEAC method confirm that there are significant statistical differences between the emulsion for acne prone skin and the anti-acne cream, between the emulsion for acne prone skin and the sunscreen. Therefore, it can be noticed that unlike the results previously obtained by using the DPPH method, the fact that the formulation for the acne prone skin (CA), which could not be analyzed, has in this case the highest antioxidant activity (0,17 μm Trolox/g sample), followed by the daily usage hydrating cream, the emulsion for acne prone skin and the day cream for problematic skin. The unsaturated compounds with an antioxidant potential, such as unsaturated fatty acids, carotenoids, tocopherols, characteristic to the 4th region, are found in significant quantities in the cream for problematic, mixed-oily skin, and in the sunscre 16

8. Originality and contributions Innovative thesis The original contributions of this thesis are: 8.1. Stability assessment lipophilic compounds from sea buckthorn fruits subject to freezing and heat treatment process (drying). 8.2. Evaluation of light stability of carotenoids in sea buckthorn oil (controlled light exposure). 8.3. Stability assessment carotenoids from fruits dried apricots. 8.4. Getting cosmetic formulas containing ingredients with dermo-protective effect 17