Summary:
In recent decades, there has been an ever-increasing consumer preference for products derived from Medicinal Aromatic Plants (MAPs), which is consistent with their widespread use in the pharmaceutical, cosmetic and food supplement industry. However, during the processes of MAPs for the development of final products, the production of unavoidably large amounts of by-products and/or wastes has been recorded, which, due to the absence of alternative solutions, they are discarded to the environment, causing serious ecological problems. It is therefore important to make an evaluation of the raw materials to investigate the possibility of their utilization for the development of added value products.
In this thesis, which is a continuation of the master thesis entitled "Production and evaluation of essential oils and their by-products through distillation from cultivated plants of the Prefecture of Kozani", a systematic effort is made to highlight the value of the by-products that come from the processing of well-known commercial MAPs. Based on the results of the above-mentioned master thesis, it was clear that the by-products of the hydrodistillation of the MAPs are characterized by a rich phenolic content (>80mg GAE/g extr.) and a high antioxidant activity (>70% DPPH Inhibition at 200μg/ml), which made the need for their further study imperative. Thus, the subject of this work was the study of superior and inferior quality products of cultivated MAPs, as well as by-products of their processing, regarding their chemical content and their biological properties.
More specifically, within the framework of the EXANDAS project, the object of which was the utilization of MAP by-products for the development of high added value products, the supply of raw materials (superior and inferior quality) and by-products (aqueous extracts from hydrodistillation, non-marketable plant material, plant residues from steam distillation) of commercial MAP (Rosa damascena, Lavandula angustifolia, Hyssopus officinalis, Ocimum basilicum and Chamaemelum nobile). Accordingly, products and by-products of the sorting process of Sideritis raeseri, Matricaria recutita, Origanum vulgare subspp. hirtum, Origanum dictamnus, Thymus vulgaris and Thymbra capitata, kindly provided from the company 'Bagatzounis & Sons SA' from Kozani and the Cooperative 'Krites-Rhizotomi' from Chania, were studied. Finally, the company 'ANASSA' supplied us with different quality leaves of the species Aloysia citrodora, as well as residual aerial parts (Chapter 1, Part B).
The study of the high-quality raw materials was carried out to evaluate the quality of the selected MAPs and compare the produced extracts with the corresponding ones resulting from the by-products of their processing. The essential oils were obtained by classical hydrodistillation and microwave-assisted hydrodistillation, while the extracts were prepared by the techniques of decoction and ultrasonic assisted maceration. Accordingly, the processing of raw material of secondary quality was carried out by various techniques, such as classical hydrodistillation, maceration (classical and assisted by ultrasound and microwaves), liquid-liquid extraction and adsorption resin technology. All the preparations were evaluated in terms of their phytochemical content with simple and coupled chromatographic and spectroscopic techniques (HPTLC: High Performance Thin Layer Chromatography, HPLC: High Performance Liquid Chromatography, LC-MS: Liquid Chromatography – Mass Spectrometry, GC- MS: Gas Chromatography – Mass Spectrometry, NMR: Nuclear Magnetic Resonance), and (b) colorimetric methods (determination of total phenolic content, determination of total flavonoid content). Also, the majority of the extracts and enriched fractions thereof were evaluated for their biological properties, such as the free radical scavenging activity (DPPH, ABTS), but also the inhibitory effect against the enzymes tyrosinase, elastase and collagenase.
Considering the results of the study, as well as the yields of the applied processes and the stability of the quality of the raw materials, we concluded on the possibility of utilizing all the examined secondary quality products and the by-products of the selected MAPs. More specifically, in the case of the species Rosa damascena (Chapter 2, Part B), the aqueous extract resulting from the hydrodistillation process for the production of rose oil, as well as its phenolic fraction, proved to have a rich phenolic (>200 mg GAE/g extr.), and flavonoid content (>100 mg QUE/g extr.) and a remarkable antioxidant activity (IC50 24.8μg/ml). The analysis of several samples of the products of the industrial processing of rose petals (rose oil, 'absolute' and 'concrete') highlighted the consistently good quality of the raw material. Also, the yields of the by-product treatment processes were high and quite repeatable, which made the need to develop a methodology for its exploitation imperative. In order to investigate the possibility of exploiting the aqueous extract of the hydrodistillation of rose petals on an industrial scale, it was decided to check the stability of the quantitative and qualitative composition of the raw material and its products. Thus, samples were collected from the same retort on different days of production, samples from the same retort on the same day of production and samples from different retorts. The collected aqueous extracts were treated with XAD-4 adsorbent resin and countercurrent extraction to obtain the beneficial phenolic fraction. The study of the initial samples and their preparations, in terms of their chemical composition and biological properties, highlighted a satisfactory stability in the yields of the processes and in the composition of the final products of the processing, which shows that the utilization of the by-product is possible. Afterwards, a pilot scale development of the enriched extract in phenols was carried out with two techniques (resin treatment and liquid-liquid extraction), and it was shown that the industrial utilization of the aqueous by-product of the hydrodistillation of rose oil is possible, either by using adsorbent resins or by the application of liquid-liquid extraction, for the production of an extract with a high phenolic content, high flavonoid content and remarkable antioxidant properties. However, the huge amounts of aqueous by-product produced daily, the difficulty of maintaining it for a long time and the high cost of transporting it to processing units are necessary to be considered for the selection of the most suitable process. The preliminary techno-economic analysis, which took place in this study, showed that from a cost point of view it is feasible and profitable to exploit the aqueous by-product of the distillation of rose oil, which until today is discarded as waste in the environment. The preliminary market research carried out by the company 'GALEN-N Ltd', which has a significant number of cooperating companies worldwide, showed that there is a significant number of companies in the market that are interested in utilizing this extract as a raw material for the development of cosmeceuticals and nutritional supplements with antiaging and antioxidant properties.
On the other hand, the study of the products and by-products (plant residues) of the industrial steam distillation of the plants Lavandula angustifolia (Lavender), Hyssopus officinalis (Hyssop), Ocimum basilicum (Basil) and Chamoemelum nobile (Chamomile) (Chapter 3, Part B) showed that (a) the raw material of the studied AMPs was characterized by acceptable quality and stable composition, as was shown by the analysis of volatile components of a series of essential oil samples, (b) the by-products of the steam distillation of lavender and hyssop present a great potential for their exploitation for the production of high added value products, due to the high phenolic content, the remarkable free radical scavenging activity and the standardized composition of the produced preparations. On the contrary, in the other cases, the large variation in the yields of the processes and the composition of the extracts raised doubts about the possibility of their industrial exploitation.
In addition to the by-products of the distillation of MAPs for the production of essential oils, by-products of the sorting process of the marketable parts of selected MAPs were studied in this thesis, regarding the species Sideritis raeseri, Matricaria recutita, Origanum vulgare subspp. hirtum, Origanum dictamnus, Thymus vulgaris and Thymbra capitata (Chapter 4, Part B). The goal was to investigate the possibility of utilizing the by-products of the process of the above-mentioned AMP, through the comparison of their chemical composition and antioxidant activity with the corresponding marketable plant raw materials. Initially, the essential oils were produced by hydrodistillation process, and a comparative study of their chemical composition obtained. Subsequently, the aqueous extracts resulting from the above-mentioned process, the fractions enriched in phenols obtained after treatment with resins, and the hydroalcoholic extracts of the examined plant raw materials prepared by microwave extraction as well as with the maceration technique and the use of ultrasound, were studied. The phytochemical profile of the aforementioned preparations was evaluated, as well as their phenol and flavonoid content, but also their antioxidant activity and specifically their free radical scavenging activity ability. The results of the tests showed that the by-products of the examined MAPs are valuable natural raw materials for the development of final products and the isolation of high added value natural products, as they show great similarities with the corresponding first-quality plant materials. However, it is necessary to investigate the quality of the raw materials and the preparations thereof on a pilot scale, while the stability of the composition and the properties of the exploited by-products will be also a determining factor.
The fifth chapter of the second part describes the study of the by-product of the process of receiving the stigma of Crocus sativus (saffron), which is the commercially exploitable part of the crocus flowers. The tepals of the flower, which are the flowers’ main part (> 95%), are still discarded in the fields. According to numerous scientific studies, the tepals of the plant are rich in flavonoids with remarkable biological properties. Due to the remarkable annual production of tepals, during the collection period of the saffron stigmas in the area of Kozani, their utilization for the preparation of extracts with significant antioxidant and probably antiaging properties, was an attractive alternative for their management. In the present thesis, dry and frozen tepals were studied and the results of the experimental processes of receiving flavonoids on a laboratory scale are presented for the extracts obtained through microwave (MicroWave assisted Extraction - MWE) and ultrasound extraction (UltraSound assisted Extraction-USE) for the selection of the appropriate method for the preparation of the enriched extract on a pilot scale. The chemical content and the free radical scavenging activity were evaluated. In order to remove the contained sugars and to obtain extracts enriched in bioactive flavonoids, the aqueous extracts of frozen tepals, were processed with XAD-4 resin to yield enriched extracts in phenolic content (>70-150 mg GAE/g extr.). The study showed that the frozen tepals of the Crocus sativus are a valuable raw material, from which it is possible to obtain an extract rich in phenolic derivatives and especially in flavonoids and anthocyanins. However, further investigation on the qualitative and quantitative composition of both the raw material and the produced extract is required. Also, a techno-economic analysis is required to investigate the utilization of the enriched extract based on its production cost and commercial value.
The sixth chapter (Part B) includes the study of lower quality products from the sorting process of the leaves of Aloysia citrodora. During the sorting process of lemon verbena plant material at the 'ANASSA' company, apart from the by-product from stems and other discarded aerial parts, a significant amount of plant raw material is characterized as secondary quality (inferior quality) and it remains unexploited. In the present work, a comparative study was carried out between the plant material of the superior and inferior quality as well as the by-product obtained during the leaf screening process. Initially, decoctions from superior and inferior quality leaves were studied, and the results showed similar composition and similar antioxidant properties for both materials. Then, the inferior quality plant were extracted by the microwave maceration method in various experiments by varying the parameters of extraction time and plant/solvent ratio. The resulting aqueous extracts were also treated with resins to obtain enriched fractions. All the extracts obtained from the inferior quality material, especially the preparations obtained from the adsorbent resin treatment, showed a very high phenolic content (200-400 mg GAE/g extr.). On the other hand, the extracts derived from the by-product showed a lower phenolic content (50-150 mg GAE/g extr.). and a limited content compared to the preparations derived from the inferior quality raw material, which is attributed to the high content of woody parts with poor phenolic content. Apart from phenolic derivatives, Aloysia citrodora contains phenylpropanoid glycosides that are of particular pharmacological interest. Thus, the inferior quality plant material was studied for the preparation of an extract with a high content of acteoside and remarkable antioxidant properties. In search of optimal conditions with the accelerated extraction (ASE) technique (or pressure extraction (PLE)) to obtain an extract with a high phenolic content, rich in flavonoids, significant content of acteoside (a phenylpropanoid with remarkable biological properties) and remarkable free radical scavenging activity, the DOE (Design of Experiments) methodology was applied, and 30 duplicate extractions were performed. Based on the results, the values of the parameters (temperature, static extraction time, water-alcoholic solvent mixture composition, number of extraction cycles) that give the best responses (extraction yield (48.5%), total phenolic content (186.2mg GAE/g extr.), total flavonoid content (99.1mg QUE/g extr.), antioxidant activity (99.8% DPPH Inhibition at 50μg/ml & 79.6% ABTS Inhibition at 50μg/ml) and acteoside content (36.1%)}.
In conclusion, from the above study it happens that the by-products, which are produced in very large quantities during the processing of MAPs, constitute a huge pool of valuable metabolites with important biological properties (antioxidant, antimicrobial, etc.) and their utilization is deemed imperative for the development of high added value natural products instead of being treated as waste.
Keywords:
Medicinal aromatic plants, hydrodistillation, steam distillation, essential oil, hydrosol, extraction, sugars, enriched phenolic fraction, phenolic content, flavonoid content, antioxidant activity, plant by-products, plant residues, high-added value product, Rosa damascena, Lavandula angustifolia, Hyssopus officinalis, Ocimum basilicum, Chamaemelum nobile), Sideritis raeseri, Matricaria recutita, Origanum vulgare, Origanum dictamnus, Thymus vulgaris, Thymbra capitata, Crocus sativus, Aloysia citrodora
