Natural matrices have historically been important sources of potential drug leads, with their use documented in traditional medicines as remedies, potions, and oils. Despite many bioactive natural products remaining unidentified, their potential continues to be recognized. The use of natural matrices in drug discovery faces significant challenges, such as variability in starting materials, characterization difficulties, and scaling issues for high-throughput screening of bioactivity. These challenges have slowed the development of the natural product-related pharmaceutical industry. Nonetheless, natural products from plants and animals persist in entering clinical trials or providing leads, especially for anticancer and antimicrobial agents. The exceptional structural diversity offered by natural matrices, compared to standard combinatorial chemistry, has renewed interest in revisiting natural products for drug discovery. In this work, two different natural matrices were selected in order to assess their potential bioactivity on three human renal cell lines, the non-tumor HK-2, and the tumor Caki-1 and RCC-Shaw: (i) the animal-derived natural matrix of dromedary urines, and (ii) the Fungi-derived natural matrix of Ganoderma adspersum extracts. Indeed, dromedary urinotherapy as a remedy against various illnesses is a widespread ethnomedical practice, well recognized in traditional dromedary countries, and several studies tried to unravel the therapeutic potential of this animal by-product. However, these studies often overlooked important factors, such as the measurement of urine osmolarity or the impact of concentration and/or composition of the serum used in cell cultures, which could deeply influence the in vitro tests results. The bioactivity of a set of seventeen dromedary urines, derived from animals of different sex and age, on the above-mentioned human renal cell lines. The possible influence of the samples’ osmolarity and the content of the fetal bovine serum (FBS) added to the cell culture medium were considered prior to bioactivity testing. Moreover, a preliminary characterization of the tested urine samples through Nuclear Magnetic Resonance (NMR) – based metabolomics analysis pointed out the presence of different metabolites between bioactive and inactive samples. On the other hand, Ganoderma adspersum is a type of medicinal fungus mainly distributed in the southern regions of Europe recently reported to have potential bioactive effects, including antibacterial, anticholinesterase, and antioxidant activities. In addition, a few works reported about its antiproliferative effects against cancer cells. Here, the antiproliferative activity of ethanolic extracts derived from Ganoderma adspersum was investigated. Both the ethanolic extracts obtained from the media after fermentation and of the mycelium were tested in vitro and spectroscopically analyzed. The study revealed that the mycelium ethanolic extract exhibited a significant reduction in the viability of tumor human renal cells Caki-1 and RCC-Shaw, up to 90% for RCC-Shaw cells after 72 hours of exposure. Conversely, no impact on the non-tumor human renal HK-2 cells was observed. Interestingly, the extract derived from the fermented medium had no significant influence on cell viability. Nuclear Magnetic Resonance (NMR) analysis revealed remarkable changes in the metabolic composition of fermented medium extract (not active) compared to that of mycelium extract (active), suggesting the role of specific metabolites in the activation of apoptosis observed on the cancer cells.
Le matrici naturali sono storicamente riconosciute come importanti fonti di potenziali farmaci: il loro utilizzo è infatti documentato dalla medicina tradizionale, sottoforma di rimedi, pozioni e oli. Nonostante molti prodotti naturali bioattivi rimangano tutt’ora non identificati, il loro potenziale continua ad essere riconosciuto e a destare l’interesse dei ricercatori. L'uso delle matrici naturali nel drug discovery deve tener conto delle problematiche associate alla natura stessa delle matrici, quali la variabilità biologica, le difficoltà nella caratterizzazione dei prodotti naturali e le complessità legata alla valutazione della loro bioattività: tali sfide hanno rallentato lo sviluppo dell'industria farmaceutica correlata ai prodotti naturali. Tuttavia, i prodotti naturali derivanti da piante e animali continuano a entrare nelle sperimentazioni cliniche o a fornire dei leads, soprattutto come agenti anticancro e antimicrobici. L'eccezionale diversità strutturale offerta dalle matrici naturali, rispetto alla chimica combinatoria standard, ha rinnovato l'interesse nel riconsiderare i prodotti naturali nel drug discovery. In questo lavoro, sono state selezionate due diverse matrici naturali per la valutazione in vitro della loro potenziale bioattività su tre linee cellulari renali umane, le non-tumorali HK-2 e due le tumorali Caki-1 e RCC-Shaw: (i) le urine di dromedario, come matrice di origine animale e (ii) gli estratti di Ganoderma adspersum, come matrice naturale di origine fungina. L’urinoterapia con le urine di dromedario come rimedio contro varie malattie è una pratica etnomedica diffusa nei paesi tradizionalmente associati ai dromedari, e diversi studi hanno cercato di svelare il potenziale terapeutico di questo sottoprodotto. Tuttavia, questi studi spesso trascurano fattori importanti, come l'osmolarità delle urine o l'impatto della concentrazione e/o composizione del siero utilizzato nelle colture cellulari, che potrebbero influenzare profondamente i risultati dei test in vitro. La bioattività di un set di diciassette urine di dromedario, provenienti da animali di sesso ed età diversi, è stata esaminata sulle linee cellulari renali umane sopra menzionate. Prima dei test di bioattività, sono stati considerati l'eventuale influenza dell'osmolarità dei campioni e il contenuto del siero fetale bovino (FBS) aggiunto al mezzo di coltura cellulare. Inoltre, una caratterizzazione preliminare dei campioni di urina testati attraverso analisi metabolomica basata sulla Risonanza Magnetica Nucleare (NMR) ha evidenziato la presenza di diversi metaboliti tra campioni bioattivi e inattivi. D'altra parte, il Ganoderma adspersum è un tipo di fungo medicinale principalmente distribuito nelle regioni meridionali dell'Europa, recentemente riconosciuto per avere potenziali effetti bioattivi, tra cui effetti antiproliferativi contro le cellule cancerose. In questo studio, è stata indagata l'attività antiproliferativa degli estratti etanolici derivati dal Ganoderma adspersum. Sia gli estratti etanolici ottenuti dal mezzo di fermentazione che dal micelio sono stati testati in vitro e analizzati spettroscopicamente. Lo studio ha rivelato che l'estratto etanolico del micelio ha mostrato una significativa riduzione della vitalità delle cellule tumorali renali umane. Al contrario, non è stato osservato alcun impatto sulle cellule renali umane non tumorali, mentre l'estratto derivato dal mezzo fermentato non ha avuto un'influenza significativa sulla vitalità cellulare. L'analisi tramite Risonanza Magnetica Nucleare (NMR) ha rivelato notevoli cambiamenti nella composizione metabolica dell'estratto del mezzo fermentato (non attivo) rispetto a quella dell'estratto del micelio (attivo), suggerendo il ruolo di specifici metaboliti nell'attivazione dell'apoptosi osservata nelle cellule cancerose.
Valutazione di composti bioattivi derivanti da matrici naturali per applicazioni in condizioni fisiologiche e patologiche
SGOBBA, MARIA NOEMI
2025
Abstract
Natural matrices have historically been important sources of potential drug leads, with their use documented in traditional medicines as remedies, potions, and oils. Despite many bioactive natural products remaining unidentified, their potential continues to be recognized. The use of natural matrices in drug discovery faces significant challenges, such as variability in starting materials, characterization difficulties, and scaling issues for high-throughput screening of bioactivity. These challenges have slowed the development of the natural product-related pharmaceutical industry. Nonetheless, natural products from plants and animals persist in entering clinical trials or providing leads, especially for anticancer and antimicrobial agents. The exceptional structural diversity offered by natural matrices, compared to standard combinatorial chemistry, has renewed interest in revisiting natural products for drug discovery. In this work, two different natural matrices were selected in order to assess their potential bioactivity on three human renal cell lines, the non-tumor HK-2, and the tumor Caki-1 and RCC-Shaw: (i) the animal-derived natural matrix of dromedary urines, and (ii) the Fungi-derived natural matrix of Ganoderma adspersum extracts. Indeed, dromedary urinotherapy as a remedy against various illnesses is a widespread ethnomedical practice, well recognized in traditional dromedary countries, and several studies tried to unravel the therapeutic potential of this animal by-product. However, these studies often overlooked important factors, such as the measurement of urine osmolarity or the impact of concentration and/or composition of the serum used in cell cultures, which could deeply influence the in vitro tests results. The bioactivity of a set of seventeen dromedary urines, derived from animals of different sex and age, on the above-mentioned human renal cell lines. The possible influence of the samples’ osmolarity and the content of the fetal bovine serum (FBS) added to the cell culture medium were considered prior to bioactivity testing. Moreover, a preliminary characterization of the tested urine samples through Nuclear Magnetic Resonance (NMR) – based metabolomics analysis pointed out the presence of different metabolites between bioactive and inactive samples. On the other hand, Ganoderma adspersum is a type of medicinal fungus mainly distributed in the southern regions of Europe recently reported to have potential bioactive effects, including antibacterial, anticholinesterase, and antioxidant activities. In addition, a few works reported about its antiproliferative effects against cancer cells. Here, the antiproliferative activity of ethanolic extracts derived from Ganoderma adspersum was investigated. Both the ethanolic extracts obtained from the media after fermentation and of the mycelium were tested in vitro and spectroscopically analyzed. The study revealed that the mycelium ethanolic extract exhibited a significant reduction in the viability of tumor human renal cells Caki-1 and RCC-Shaw, up to 90% for RCC-Shaw cells after 72 hours of exposure. Conversely, no impact on the non-tumor human renal HK-2 cells was observed. Interestingly, the extract derived from the fermented medium had no significant influence on cell viability. Nuclear Magnetic Resonance (NMR) analysis revealed remarkable changes in the metabolic composition of fermented medium extract (not active) compared to that of mycelium extract (active), suggesting the role of specific metabolites in the activation of apoptosis observed on the cancer cells.File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14242/217877
URN:NBN:IT:UNIBA-217877