CONTENTS INTRODUCTION THE PURPOSE AND OBJECTIVES OF THE PHD THESIS THE CURRENT STATE OF KNOWLEDGE...

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2 1.1.Definition and history Capsules clasification. Types of matrices and materials that are being encapsulated Capsules clasification Matrices that are utilized for microencapsulation Techniques for obtaining the microcapsules Spray drying technique ( atomization or Spray Drying) Fluid bed coating technique Emulsions Ionotropic gelification technique Microcapsules characterization methods Microcapsules dimensions Microcapsules morphology Microcapsules mechanical strenght Mechanisms and methods for delivery of actice principles Release mechanism of active principles Factors that influence the release of active principles Applications of microencapsulation technique The physiology an pathology of the prostate Prostate physiology Prostate pathology Medicinal herbs, fruits and seeds that can influence the prostate metabolic disorders Bioactive substances that play a role in protection of prostate cells Description of some medicinal herbs, seeds and fruits containing bioactive priciples that play a role in prostate protection White seabuckthorn (Hippophae rhamnoides) Nettle (Urtica dioica) Green tea (Camellia sinensis) Tomato (Solanum lycopersicum) Whilow (Epilobium parviflorum) Pumpkin (Curcubita maxima) Sun flower (Helianthus annus) Yeast (Saccharomyces cerevisiae) Methods for spectrometric analysis UV-VIS spectrometry FT-IR spectrometry Separation and identification methods based on liquid chromatography with mass spectrometric detection (LC-MS) Analytical methods applied to identify specific bioactive compounds of plant

3 Introduction Materials and Methods Plant ingredients used for obtaing PROMEN Extraction of bioactive compounds in plants and PROMEN UV-VIS analysis Poliphenols content (Metoda Folin-Ciocalteu) Extraction efficiency FT-IR analysis HPLC-DAD and LC ESI(+)QTOF-MS analysis Biostatistical data analysis Results and discussion UV-Vis spectroscopy fingerprint extracts, and efficiency extraction Polyphenol content-ingredient and product PROMEN FT-IR analysis ingredients and product PROMEN HPLC-DAD analysis of ingredients compared with the product PROMEN LC-(ESI+) QTOF MS analysis of ingredients compared with the product PROMEN Conclusion Introduction Materials and Methods Extraction of bioactive compounds of ingredients UV-VIS analysis Dosage of polyphenol content Calculation of total carotenoids and extraction efficiency FT-IR analysis of the ingredients Method for the preparation of microspheres and microcapsules Determination of the viscosity of the emulsion obtained The process of obtaining microspheres and microcapsules Results and discussion UV-VIS analysis of chloroformic extracts, extraction efficiency and calculation of total carotenoids UV-VIS analysis of methanolic extracts, extraction efficiency and calculation of total polyphenols FT-IR analysis of the ingredients Determination of the viscosity of the emulsion obtained The morphology of the microspheres and microcapsules Conclusion Introduction Materials and Methods

4 Extraction of bioactive compounds of microcapsules / microspheres Dosage polyphenol content of methanol extracts (METODA FOLIN - CIOCALTEU) Extraction efficiency Total carotenoids content FT-IR analysis Testing the microcapsules in simulated gastric and intestinal juice Biostatistical data analysis Results and discussion The efficiency of extraction and concentration of total carotenoids in microcapsules and microspheres Methanol extraction efficiency and total polyphenols content FT-IR analysis Testing the stability of microcapsules and microspheres in the simulated gastric and intestinal juice Conclusion Introduction Materials and Methods The process of obtaining microcapsules / microspheres with chitosan coating Extraction of bioactive compounds microcapsules / microspheres UV-Vis analysis of the methanol and chloroform extracts of microcapsules / microspheres The release rate and various compounds as solvents - comparisons between microcapsules with coating and without coating of chitosan Results and discussion The chloroformic extracts spectroscopic fingerprint The chloroformic extracts spectroscopic fingerprint Conclusion

5 The purpose of the conducted research is to characterize and identify the bioactive components of some medicinal herbs, fruits and lyophilized yeast to obtain an original formula for a dietary supplement (PROMEN), and also a microencapsulated formula with benefiting potential in prostate diseases prevention. For this study seven medicinal herbs and fruits have been utilised: nettle ( ), green tea ( ), willow herb ( ), tomatoes ( ), the white sea buckthorn ( ), pumpkin seeds ( ), sunflower seeds ( ), and lyophilized yeast. The bioactive compounds from these herbs have been analysed through UV-Vis, FTIR spectrometry and HPLC-DAD and LC-QTOF-MS chromatography techniques, with photo-diode detection (DAD) and mass spectrometry (MS). 1. The spectrometric and chromatographic extraction and characterization of bioactive compounds from the herbs used as ingredients in the PROMEN product, namely the polyphenolic and carotenoid compounds. 2. Obtaining the PROMEN product and characterizing its composition in comparison with ingredient herbs. 3. Applying microencapsulation technologies on alginate with or without chitosan coating matrices, in order to obtain microspheres and microcapsules which incorporate carotenoid and polyphenol bioactive compounds, with controlled release, as generic nutraceutical products. 4. Morphological, spectrometric (UV -Vis and FTIR) characterization of microcapsules and microspheres obtained. 5. Testing the stability of microspheres and microcapsules in the simulated gastric and intestinal environment. 6. Determining the bioactive compounds release rate from microspheres and microcapsules, during a timeframe and in various solvents, based on the type of microcapsules and the chitosan coating. The thesis is structured in two parts, the first part contains literature data referring to microencapsulating techniques, physiological and pathological functions of the prostate, but also medicinal herbs, fruits and seeds which affect metabolic derangements at the prostate level, and also analysing methods of vegetal originated bioactive compounds (3 chapters). The second part is focused on personal contributions made, including 4 experimental studies which include methods and materials used, results, and resulting conclusions. : includes a short description of the microencapsulating technologies, a classification of the capsules and the types of encapsulating matrices, the microcapsules obtaining techniques and a brief description of the most utilised techniques in the food industry, the characterization and evaluation methods of the active principles release of microcapsules, and also the applications of microencapsulation. includes the description of the physiological and pathological functions of the prostate, and also medicinal herbs, fruits and seeds which affect metabolic derangements at the prostate level. This chapter also includes a short description of the plants, fruits and seeds which have a protective role on the prostate. The medicinal and scented herbs which contain bioactive compounds important for 5

6 the protection of prostate are: the white sea-buckthorn, nettle, green tea, yeast, tomatoes, willow herb, pumpkin and sun flower seeds. includes a short description of the main vegetal bioactive compounds analysing techniques. Vegetal products include a complex of biochemical compounds, from secondary and intermediary metabolites, compounds with small molecular masses, to polymeric macromolecules (proteins, policarbohydrates, and complex lipids). The analysis of these elements involves the utilisation of several extraction techniques, separation, and measurement (UV-VIS, FT-IR and LC-MS). of the thesis represents the personal contribution and sums up 4 concluding studies: shows the results obtained through methanolic extracts analysis of seven herbs and the PROMEN product using several separation, measurement techniques (UV-VIS, FT-IR and LC-MS). has been dedicated to obtaining microcapsules/microspheres from white sea-buckthorn juice, tomatoes, and pumpkin oil and the UV-VIS, FT-IR analysis of the ingredients. objectives were to test the microcapsules in the simulated gastric and intestinal environment and the UV-VIS analysis of the obtained extracts. has been dedicated to determining the rate of release of the bioactive compounds from the microspheres and microcapsules, during a timeframe and in different solvents, based on the microcapsules type and the chitosan coating. of this study was to obtain methanolic extracts from each ingredient herb and the realisation of an innovative formula from the mixture of herbs with prostate protection driven functions, called PROMEN. The utilised ingredients have been: nettle, green tea, willow herb, tomatoes, white sea-buckthorn, pumpkin seeds, sunflower seeds, lyophilized yeast and PROMEN powder. The extraction of the bioactive compounds has been made in acidulated methanol, and the samples have been analysed using UV-VIS, FT-IR and LC-ESI(+)-Q- TOF-MS spectrometry. The statistical analysis has been made through PCA (Principal Component Analysis). Following the UV-VIS analysis of all samples it can be observed that green tea, pumpkin seeds, sunflower seeds, and yeast show 3 absorption zones namely at 280, 330 and 400 nm while PROMEN, tomatoes, willow herb, and the white sea-buckthorn only show 2 absorption regions at 280 and 400 nm. Based on UV-VIS prints the extraction factor has been calculated leading to the observation that in general the phenolic acids have been much better extracted than the flavonoids. The willow herb has had the best extraction degree showing a 10 times higher one than the nettle. Due to the high concentration of nonpolar compounds of the pumpkin seeds, sunflower 6

7 seeds, white sea-buckthorn, and tomatoes, the low concentration of phenolic acids, flavonoids, and quinones can be explained. Following the total polyphenols calculus and based on the herbs composition report from the PROMEN product, it should theoretically have shown 80, 56 mg GAE/100 ml extract instead of 76,611. This concludes that through the processing of the product approximately 5% of the total of phenolic compounds have been lost. The IR spectres analysis on the print specific domain ((1000 and 1500 cm-1) highlights three absorption zones, as following: 1 st zone: specific to mono-, oligoglucidic compounds; 2 nd zone: corresponding to the vibrations of the CO stretch carbonyl groups; and 3 rd zone: specific to CO vibrations (amide). Through LC-ESI(+)-Q-TOF-MS analysis over 21 compounds have been identified in the PROMEN product, some of them being: juglone, Resveratrol, Quercetin, epigallocatechin, Gallocatechin, Biochanin A, Isorhamnetin 3-O-glucoside 7-Orhamnoside, Quercetin 3-O-galactoside 7-O-rhamnoside, Campferol 3,7-O-diglucoside, and acid p Coumaroylquinic. 1. Based on UV-Vis prints, the extraction factors of the phenolic acids, flavonoids, and quinones have been calculated and it has been observed that phenolic acids have had a better extraction efficiency than the flavonoids. 2. Based on the FTIR prints the discrimination was possible between the PROMEN product and its ingredients. According to PCA analysis of the FTIR spectres, the samples were mainly connected to the strips in cm-1 zone , which mainly contributed to the spectre grouping. Troupes in the zone cm-1 were also important in the classifying of the samples. The data shown in this study have indicated that FTIR spectroscopy is a suitable comparative fingerprinting technique and also to evaluate the extraction efficiency of medicinal herbs and dietary supplements. 3. Based on the HPLC-DAD chromatographic analysis of the extracts with 280nm and 330 nm detection, a number of 3-7 major signals of phenolic acids and flavonoids were highlighted by comparing herbs extracts and the PROMEN product with pure standards of phenolic acids and flavonoids. The most important identified compounds are ferulic acids, ellagic acid, myricetin and trans-cinnamic acid (identified at 280 nm), and rutin, quercetin, ferulic acid and myricetin (identified at 330nm). 4. Based on LC - QTOF MS- analysis 21 compounds have been separated and identified, specific to the final product PROMEN. The results of this study have been published in the following works: 1.CSERNATONI FLORINA, CARMEN SOCACIU, RALUCA MARIA POP, FLORICUTA RANGA, FLORINA BUNGHEZ, FLORINA ROMANCIUC, 2013, Comparative Fingerprint of Aromatic Herbs and Yeast Alcoholic Extracts used as Ingredients for Promen, a Prostate Preventive Nutraceutical, Bulletin USAMV Food Science and Technology 70(1), CSERNATONI FLORINA, CARMEN SOCACIU, RALUCA MARIA POP, FLORINELA FETEA, FLORINA BUNGHEZ, 2013, Application of FTIR Spectroscopy for Fingerprinting Bioactive Molecules in a Nutraceutical PROMEN, comparatively with Plant ingredients, Bulletin USAMV Food Science and Technology 70(1),

8 3.CSERNATONI FLORINA, ANCA BACIU, RALUCA POP, FLORINA ROMANCIUC, CARMEN SOCACIU, 2014, Characterization of seven medicinal plants included in an original formula Promen, to prevent prostate diseases, Hop and Medicinal Plant, No. 1-2, CSERNATONI FLORINA, CARMEN SOCACIU, RALUCA MARIA POP, FLORINA ROMANCIUC, FLORINA BUNGHEZ, 2014, HPLC PDA and HPLC-ESI(+)QTOF-MS fingerprints of polyphenols in a nutraceutical product (Promen) comparatively with plant ingredients, Romanian Journal of Biochemistry 51, of this study has been to characterize tomato juice and white sea-buckthorn juice combined with pumpkin oil (through spectrometric methods) and to obtain microspheres and microcapsules with or without coating, through ionotropic gelation technique. Two types of extractions have been made for the determination of the bioactive compounds (methanolic and chloroformed) after which the obtained extracts have been analysed through UV-VIS and FT-IR spectrometry. Based on UV-VIS fingerprints the extraction factors have been calculated, total polyphenols (methanolic extracts) and the total carotenoid content ( chloroformed extracts). In order to obtain emulsions the juice samples have been weighted and then placed into a Berzelius glass on top of a magnetic stirrer and warmed up to 30 0 C, then the alginate has been added and it was left to stirr for an hour and a half until the sodium alginate was completely dissolved. Then the pumpkin oil has been added for the microspheres matrices, the sample has been ultraturraxed for 5 minutes until an emulsion was obtained, and for the microcapsules the oil represents the nucleus of the microcapsule. Then the hardening bath has been prepared from distilled water adding 2%CaCl2, and for a part of the microcapsules obtained from the samples 1C and 2C a 0.1% chitosan coating has been made added in the hardening bath, and the samples were noted as 1C* and 2C*. The viscosity determination has been achieved using a Fungibal viscometer utilising R2 geometry expressed in cp, and for obtaining microcapsules and microspheres a Buchi B 395 PRO microencapsulator has been utilised. The UV-VIS analysis of the chloroformed extracts allowed the identification of 4 compound classes: lipids, phenolic acids, flavonoids and carotenoids, and based on the extraction factor it has been observed that the 5% addition of pumpkin oil significantly increases the lipids quantity of the samples. By evaluating the total content of carotenoids through UV-Vis spectrometry, the fact that tomato juice and white sea-buckthorn juice are rich sources of carotenoids is highlighted. The UV-Vis analysis of the methanolic extracts allowed the identification of 4 classes of compounds: lipids, phenolic acids, flavonoids and quinones, and from the total polyphenols calculus it can be observed that the white sea-buckthorn juice is an extremely rich source of phenolic acids by having a 6 times higher concentration than the tomato juice. By determining the viscosity it could be observed that the emulsions which contain tomato juice (1A, 2A) have a lower viscosity than the ones that contain white 8

9 sea-buckthorn juice (1B, 2B), and the 5% addition of pumpkin oil significantly increases the viscosity of the samples. After the morphological evaluation of the microcapsules it could be observed that all the microspheres types 1A, 1B, 1C have spherical shapes with dimensions between 750 and 800 μm of the of the shelling and dimensions of um of the nucleus represented by the pumpkin seed. In the case of 2A sample the nucleus is well represented and delimited, and in the case of the sample 2B it cannot be observed due to the high quantity of pigments in the shelling. 1. Based on the UV-Vis chloroformed extracts 4 classes of compounds have been identified (lipids, phenolic acids, flavonoids and carotenoids) and the efficiency of their extraction has been calculated, and also the total content of carotenoids, which has proven that the best extraction was found on the individual samples (tomato juice and white sea -buckthorn juice) unlke the mixed samples. 2. The UV-VIS spectrometric analysis of the methanolic extracts allowed the identification of 4 compound classes: lipids, phenolic acids, flavonoids and quinones and following the calculus of the efficiency of the extraction it can be observed that the lipids and the polyphenols have had the best extraction performance. 3. Through the FT-IR analysis of the of the chloroformed extracts 4 absorption zones have been identified on the cm-1 interval, and in the case of methanolic extracts 3 extraction zones have been identified on the cm-1 interval. 4. By determining the viscosity of the emulsions which contain tomato juice (1A, 2A), it could be observed that they had a lower viscosity than the ones that contain sea buckthorn juice (1B, 2B). 5. The form and morphology of the microcapsules and microspheres has also been characterised through optical microscopy. All types of microspheres/microcapsules have spherical form and normal dimensions. The results have been published in the following works: CSERNATONI FLORINA, RALUCA POP, FLORINA ROMANCIUC, OANA POP, FLORINELA FETEA, FLORICUŢA RANGA, RAMONA BIANCA MOŞ, CARMEN SOCACIU, 2015, Preparation and Comparative Characterization of Alginate-Made Microcapsules and Microspheres Containing Tomato, Seabuckthorn Juices and Pumpkin Oil, Bulletin USAMV Food Science and Technology 72(1), of this study has been to evaluate the bioactive compounds incorporated in microspheres and microcapsules and to test their stability in the simulated gastric and intestinal environment. In order to determine the bioactive compounds from every type of microspheres/ microcapsules 2 types of extractions were made (methanolic one and chloroformed one), followed by analysis of the obtained extracts through UV-VIS and FT-IR spectrometry techniques. Based on UV-VIS fingerprints the following were calculated: the extraction factors, total polyphenols (methanolic extracts), and the total content of carotenoids (chloroformed extracts). 9

10 The simulated gastric liquid has been prepared through the adding of pepsin 3g/L (P7000, 1:10,000) into concentration sterile sodium chloride (0.5%, w/v) and by adjusting the ph to 2.0 with concentrated HCl or NaOH. The simulated intestinal liquid has been prepared by adding pancreatin 1g/L USP (P -1500) and bile salts 4,5% into concentration sterile sodium chloride (0.5%, w/v) and by adjusting the ph to 8.0 with concentrated HCl or NaOH Samples of 2 g microcapsules (1A, 2A, 1B, 2B, 1C, 2C, 1C*, 2C*) have been placed into 20 ml of simulated gastric environment and incubated at 37 0 C and easily stirred for 30 minutes, followed by the transfer of the microcapsules into 20 ml of simulated intestinal environment and incubated at 37 0 C and again easily stirred for 2 hours. Following the calculus of the chloroformed extraction factor of the compounds, it could be observed that the phenolic acids and carotenoids have had the greatest extraction factor, and the flavonoids have had the lowest. After determining the total carotenoids it could be observed that the tomatoes are very rich sources of carotenoids and have had an extraction factor of 90% compared to the raw material. The total calculus of polyphenols from the microcapsules has shown that by performing a similar extraction to the raw materials one, it was possible to evaluate over 50% of the polyphenols total while the rest was found in the remaining pellet after the centrifugation of the samples. In order to compare and highlight the FTIR spectroscopic fingerprint differences of the extracts, the method Principal component analysis (PCA) fig 1 - has been used, based on the intensity of the FT-IR registered signals of the chloroformed and methanolic extracts. PCA-Scores for raw materials and PCA-Scores for raw materials and chloroform extracted microcapsules methanol microcapsules Following the PCA analysis of the chloroformed extracts 3 separation zones have been identified, groups formed of: 1B, 1A, 2A and 1C, 2B, 2C situated in the inferior and superior side of the PC2 axis. The 3 rd group represented by P2, P4 and P6 is situated in the right end of the PC1 axis and diametrically opposed to the other 2 groups. The wavelengths which have influenced the samples are: cm -1 and also 2920 cm-1 which corresponds to C-H vibration strips, specific to CH3 and CH2 of lipids, characteristic to the first 2 groups and corresponding to the cm -1 interval with major signals at: 1743cm -1 and 1745cm -1, which corresponds to vibrations strips N-H (amino acids), aldehydes, ketones and esters and also fat acids and glycerides, characteristic to the 3 rd group. The wavelengths of the methanolic extracts which influences the grouping of the samples (P2, P6, P4) situated along the PC1 axis, on the left side there are the wavelengths between cm-1 with major signals at: 1055, 1072, 1074 and 1076cm -1, which correspond to C-O vibrations strips of mono-, oligo- and carbohydrates. And to continue, 3 more groups forming could be identified, oppositely 10

11 situated to the first identified group, as follows: the group represented by the sample 2B situated on the superior side of the PC2 and PC1 axis, at the middle of the right quadrant; the group represented by samples 1B, 1A and 2A situated almost to the centre of the plot, and the groups represented by the samples 1C-2C situated almost diametrically opposed to the first identified group (P2P6P4) along the PC1 axis, on the right side. The distribution of the 3 groups along the 2 PC1 and PC2 axis has been mostly influenced by the wavelengths between cm -1 characteristic to 2B group, which corresponds to N-H vibrations strips (amino acids), aldehydes, ketones and esters and also fat acids and glycerides, and cm -1 with major signals at 3311, 3332, 3356, 3358, 3383, 3385cm -1, which corresponds to O-H group vibration strips (of water, alcohol or phenolic compounds) characteristic to 1C, 2C group. Following the microcapsules stability evaluation in the gastric and intestinal simulated environment it can be observed that the microcapsules/microspheres do not disintegrate in the simulated gastric environment, they only release a part of the compounds, while in the intestinal environment they disintegrate. The UV-VIS spectrometric analysis of the extracts in the simulated gastric environment has allowed the identification of 2 compounds classes: lipids and phenolic acids, and in the case of intestinal environment 3 classed of compounds have been identified: phenolic acids, flavonoids, and carotenoids. 1. The total calculus of carotenoids has shown that both the sea buckthorn and the tomatoes are rich sources of carotenoids. 2. Following the total calculus of polyphenols it could be observed that the sea buckthorn juice is an extremely rich source of phenolic compounds, having a 6 times higher concentration as opposed to the tomato juice. 3. The FTIR fingerprint of the chloroformed extracts has allowed the highlighting of 4 absorption zones on the interval cm -1, and in the case of methanolic extracts 3 absorption zones on the interval cm The stability testing of the microcapsules in the gastric and intestinal simulated environment has proven that microcapsules/microspheres do not disintegrate in the simulated gastric environment at acid ph and also a part of the bioactive compounds are released; while in the intestinal environment, at neutral ph, after 2 hours the microcapsules disintegrate completely with the exception of the tomato microcapsules. Accepted article ongoing publishing: CSERNATONI FLORINA, RALUCA MARIA POP, FLORINA ROMACIUC, FLORINELA FETEA, OANA POP, CARMEN SOCACIU,2015, Sea buckthorn juice, tomato juice and pumpkin oil microcapsules/ microspheres with health benefit on prostate disease obtaining process, characterization and testing properties, Romanian Biotechnological Letters of this study was to determine the release rate of the bioactive compounds from microspheres and microcapsules in time and different solvents, according to the microcapsules type and chitosan coating. 11

12 In order to obtain the emulsions the juice samples have been weighted and then placed into a Berzelius glass on top of a magnetic stirrer and warmed up to 30 0 C, then the alginate has been added and left to stir fir an hour and a half until the sodium alginate was completely dissolved. For the microspheres matrices the pumpkin oil was subsequent added, then the sample was ultraturraxed for 5 minutes until and emulsion was obtained, and for the microcapsules the oil represents the nucleus of the microcapsule. Then the hardening bath was prepared from distilled water to which 2%CaCl2 was added, and for a part of the microcapsules obtained from the samples 1C and 2C there was also a chitosan coating made 0.1% added to the hardening bath, the samples being noted as 1C* and 2C*. The release rate of the compounds was made in three different types of solvents: acidulated methanol, 7, 5 ph methanol, and chloroform. These were incubated at 37 0 C and easily stirred for 1h, 2h, 4h and 6h. For the incubation and stirring processes a Heidolph Unimax Inkubator 1000 was used. After incubation the samples have been filtered and analysed with a spectrophotometer UV-Vis JASCO V530. Following the evaluation of the release rate of the compounds in time and different solvents, it can be observe that in the case of the extraction in acidulated methanol and chloroform there are significant differences of the release rate of the compounds in time, and also between the samples with or without chitosan coating. In the case of the extraction with ph=7,5 methanol there are no significant differences of the extraction in time, only regarding the release rate of the compounds from the samples with or without chitosan. It can also be observed that the extraction rate from the samples without chitosan coating is higher than in the case of the samples with chitosan coating, which proves that the chitosan coating can be used for obtaining products with controlled release. 1. Microcapsules and microspheres have been obtained through the microencapsulation technique in alginate followed by chitosan coating 2. For the evaluation of the bioactive compounds from the two types of microcapsules/microspheres two types of extractions have been performed (in methanol and chloroform). 3. The UV-VIS spectrometric analysis of the methanol extracts has allowed the identification of 4 compound classes: lipids, phenolic acids, flavonoids and quinones, and following the extraction efficiency calculus it can be observed that lipids and polyphenols have had the best extraction performance compared to flavonoids and quinones. 4. The extraction rate from the samples without chitosan coating is higher than in the case of the samples with chitosan coating, which proves that chitosan coating can be used for obtaining products with controlled release 12

13 The original and innovative contributions of this Doctorate Thesis can be synthetized through the following significant results: 1. The application of innovative techniques for obtaining microspheres and microcapsules with or without coating by utilising natural matrices (alginates and chitosan) which incorporate and stabilise the bioactive compounds that bring benefits to our health. 2. The realisation of an original PROMEN formula which includes ingredient herbs with controlled functionality to prevent prostate cancer 3. The utilisation of high performance analytical techniques such as liquid chromatography coupled with mass spectrometry, UV-Vis and FTIR spectrometry, in order to fingerprint, qualitatively and quantitatively determine the share of the bioactive compounds from the herbs extracts and microcapsules/microspheres. 4. The harnessing of the indigenous herbs (sea buckthorn, tomato, pumpkin oil) through innovative technological solutions in order to obtain microcapsules and microspheres with controlled release, with important applications in alimentation, and prevention of metabolic disorders. 13