Characterization of bioactive natural products from edible, medicinal and toxic plants

Research performed during work on this thesis has focused on isolation, structure elucidation and biological activity of natural products. Seven plant species have been examined, namely Bromelia laciniosa Mart. ex Schult f., Encholirium spectabile Martius ex Schult. Schult. f., Metasequoia glyptostroboides Hu & Cheng, Narthecium ossifragum L. Huds., Neoglaziovia variegata Arruda da Camara Mez, Viscum album L. and Zamioculcas zamiifolia Lodd. Engl. Fifty-five compounds are reported throughout the thesis. Among them are twelve novel natural products. Artemia toxicity test, 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity and 15-lipoxygenase inhibition test have been performed on pure compounds and in one case on crude extracts. Four novel natural products and four known compounds are reported from European white-berry mistletoe (V. album L.). Two novel aminoalkaloids 4,5,4’- trihydroxy-3,3’-iminodibenzoic acid and 4,5,4’,5’-tetrahydroxy-3,3’- iminodibenzoic acid were isolated and identified from the leaves of V. album. This is the first time alkaloids that appear to be specific to this medicinal plant used for millennia are reported in the scientific literature. The two novel natural products 3-(3’-carbomethoxypropyl) gallic acid and 3-(3’-carbomethoxypropyl)- 7→3’’-protocatechoyl galloate make up a novel group of natural products. They are derived of the methyl ester of γ-hydroxybutyric acid (GHB) coupled to aromatic hydroxybenzoic acids including gallic acid. Radical scavenging activity for 3-(3’-carbomethoxypropyl) gallic acid (IC50 value 222 μM) were twentyfold reduced by substitution of the 3-hydroxyl of gallic acid with the methyl ester of gamma-hydroxybutyric acid compared with that of gallic acid ((IC50 value 27.7 μM). From dawn redwood (M. glyptostroboides) altogether eight compounds are reported including the novel natural product 6-carboxydihydroresveratrol 3-O-β- glucopyranoside. This dihydrostilbenoid exhibited both radical scavenging activity (IC50 value 164±39μM) and inhibition of 15-Lipoxygenase (IC50 value 246±30μM). Apigenin-6-C-(6’’-O-(3-hydroxy-3-methylglutaroyl)-β-glucopyranoside, a novel acylated C-glycosylflavone, and six other known natural products are reported from Z. zamiifolia. This ancient African medicinal plant has recently seen widespread use as an indoor ornamental plant. Z. zamiifolia has gained a reputation for being toxic. Despite this no lethality was observed even at the highest concentrations (1 mg/ml) when crude extracts of both leaves and petioles were applied in initial toxicological tests with Artemia salina. From the three species of the Bromeliaceae family, B. laciniosa (nine compounds), N. variegata (thirteen compounds) and E. spectabile (sixteen compounds), a total of twenty non-polar natural products are reported. Prior to this investigation very little information was available about the chemical composition of these three Bromeliaceae spp. Very long chain alkanes, fatty acids, tocopherols and triterpenoids are among the reported compounds. Twelve aromatic compounds are reported from the fruits of bog asphodel (N. ossifragum). Six novel natural products namely, (E)-4-(3-R-hydroxy-2,2- dimethylChroman-6-yl)but-3-en-3-one, (E)-4-(4-(((E)-4-hydroxy-3-methylbut- 2-en-1-yl)oxy)Phenyl)but-3-en-2-one, (2R,3S)-Naringenin(3→6’’)luteolin, (2R,3S)-Naringenin(3→6’’)chrysoeriol, liovil 4-O-β-glucopyranoside and (E)- 2,6-dimethoxycinnamic acid were reported for the first time. Previous to this investigation the rare natural product 4-hydroxy-3-(3-methylbut-2-enyl) benzaldehyde was only known from the pathogenic fungus Heterobasidion occidentale. This fungal metabolite was shown to be cytotoxic towards both normal rat kidney (NRK) (IC50 value 430 μM) and MOLM13 acute myeloid leukemia (AML) cells (IC50 value 68 μM). Naringenin(3→6’’)luteolin was the main aromatic compound, and was shown to be toxic to A. salina (LC50 value 130μM). This novel biflavonoid was cytotoxic for both NRK (IC50 value 230 μM) and MOLM13AML cells (IC50 value 115 μM).Forskningen utført i løpet av denne doktorgraden har fokusert på isolering, strukturoppklaring og biologisk aktivitet til naturprodukter. De syv planteartene Bromelia laciniosa Mart ex. Schult. f., Encholirium spectabile Martius ex Schult. Schult. f., Metasequoia glyptostroboides Hu & Cheng, Narthecium ossifragum (L.) Huds., Neoglaziovia variegata (Arruda da Camara) Mez, Viscum album L. and Zamioculcas zamiifolia (Lodd.) Engl har blitt undersøkt. Femtifem naturprodukter er rapportert i denne avhandlingen deriblant tolv hittil ukjente forbindelser. Rene forbindelser og i ett tilfelle et råekstrakt har blitt testet med Artemia toksistets test, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radikal sluk effekt og 15-lipoxygenase inhiberingstest. Fire nye og fire kjente forbindelser er rapportert fra europeisk hvitbæret misteltein (V. album L.). De to aminoalkaloidene 4,5,4’-trihydroksy-3,3’- iminodibenzosyre og 4,5,4’, 5’-tetraydroksy-3,3’-iminodibenzosyre er isolert og identifisert for første gang fra bladene til V. album. For første gang er det i den vitenskaplige litteraturen rapportert alkaloider som ser ut til å være spesifikke for denne medisinplanten som har vært brukt i årtusener. To nye naturstoffer basert på metyl esteren av gammahydroksysmørsyre (GHB) forbundet med aromatiske hydroksybenzosyrer inkludert gallussyre (3,4,5- trihydroksybenzosyre) er nå rapportert. De to nye forbindelsene 3-(3’- karbometoksypropyl) gallussyre og 3-(3’-karbometoksypropyl)- 7→3’’protocatechoyl galloat utgjør en ny gruppe av naturprodukter. Radikalslukeffekten av 3-(3’-carbometoksypropyl) gallussyre (IC50 verdi 222 μM) var redusert med en faktor på tjue i forhold til gallussyre uten substituering (IC50 verdi 27,7 μM). Totalt åtte forbindelser er rapportert fra urtreet M. glyptostroboides inkludert det nye naturproduktet 6-karboksydihydroresveratrol 3-O-β-glukopyranosid. Det ble påvist både radikal sluk effekt (IC50 verdi 164±39 μM) og inhibering av 15- lipoksygenase (IC50 verdi 246±30 μM) for denne nye dihydrostilbenoid forbindelsen. Apigenin-6-C-(6’’-O-(3-hydroksy-3-metylglutaroyl)-β-glukopyranosid, en ny acylert C-glykosylflavon, og seks andre naturprodukter er rapportert fra Z. zamiifolia. I det siste har denne gamle afrikanske medisinplanten fått ustrakt brukt som innendørs dekorasjonsplante. Z. zamiifolia har gjennom assosiasjon med andre planter i Araceae familien fått et rykte for å være giftig. Toksisitets test med Artemia salina viste imidlertid ingen dødelighet selv for de høyeste konsentrasjonene (1 mg/ml) for råekstrakt fra blader og stengel fra Z. zamiifolia. Totalt tjue upolare naturstoffer er rapportert fra de tre Bromeliaceae artene B. laciniosa (ni forbindelser), N. variegata (tretten forbindelser) og E. spectabile (seksten forbindelser). Svært lite informasjon var tilgjengelig om den kjemiske sammensetningen til disse tre artene før denne studien ble gjennomført. Fettsyrer, tokoferoler, svært langkjedede alkaner og triterpenoider er blant de rapporterte forbindelsene. Tolv aromatiske forbindelser er rapportert fra frukten til Rome (N. ossifragum) deriblant de seks nye naturproduktene (E)-4-(3-R-hydroksy-2,2-dimetylkroman- 6-yl)but-3-en-3-on, (E)-4-(4-(((E)-4-hydroksy-3-metylbut-2-en-1- yl)oksy)fenyl)but-3-en-2-on, (2R,3S)-Naringenin(3→6’’)luteolin, (2R,3S)- Naringenin(3→6’’)chrysoeriol, liovil 4-O-β-glukopyranosid og (E)-2,6- dimetoksykanelsyre. Før denne studien var det sjeldne naturproduktet 4- hydroksy-3-(3-metylbut-2-enyl) benzaldehyd kun kjent fra den patogene soppen Heterobasidion occidentale. Denne soppmetabolitten ble vist å være cytotoksisk for både normal rat kidney (NRK) (IC50 verdi 430 μM) og MOLM13 acute myeloid leukemia (AML) celler (IC50 verdi 68 μM). Naringenin(3→6’’)luteolin var den aromatiske hovedforbindelsen i fruktene av N. ossifragum og viste seg å være toksisk for A. salina (LC50 verdi 130 μM). Denne nye biflavonoiden var cytotoksisk for både NRK (IC50 verdi 230 μM) og MOLM13 AML (IC50 verdi 115 μM) celler

Novel GHB-derived natural products from European mistletoe (Viscum album)

Context: The European white-berry mistletoe [Viscum album L. (Loranthaceae)] is among the oldest known medicinal plants. At present the most important application of mistletoe extracts is in the treatment of cancer. However, natural products specific to mistletoe have rarely been encountered in the current literature. Objective: To discover novel natural products specific to European mistletoe. Materials and methods: European mistletoe was extracted with methanol, purified to partition against diethyl ether and further purified with XAD-7 column chromatography. Pure compounds were separated by Sephadex column chromatography and preparative HPLC. The structures of the novel compounds were established using a combination of several 2D NMR spectroscopic techniques and mass spectrometry. Results: A new type of natural product derived from the methyl ester of g-hydroxybutyric acid (GHB) coupled to hydroxybenzoic acids, namely 3-(3'-carbomethoxypropyl) gallic acid and 3-(3'-carbomethoxypropyl)-7→3''-protocatechoyl galloate were characterized from European white-berry mistletoe. Condensation of the 3-hydroxyl of gallic acid with the 4-hydroxyl of GHB significantly reduced the radical scavenging properties of the former compound. Discussion and conclusion: The characterized compounds define a novel group of natural products that may be of particular interest because it appears that the two new compounds are not closely related to any known natural product.acceptedVersio

Characterization of bioactive natural products from edible, medicinal and toxic plants

Research performed during work on this thesis has focused on isolation, structure elucidation and biological activity of natural products. Seven plant species have been examined, namely Bromelia laciniosa Mart. ex Schult f., Encholirium spectabile Martius ex Schult. Schult. f., Metasequoia glyptostroboides Hu & Cheng, Narthecium ossifragum L. Huds., Neoglaziovia variegata Arruda da Camara Mez, Viscum album L. and Zamioculcas zamiifolia Lodd. Engl. Fifty-five compounds are reported throughout the thesis. Among them are twelve novel natural products. Artemia toxicity test, 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity and 15-lipoxygenase inhibition test have been performed on pure compounds and in one case on crude extracts. Four novel natural products and four known compounds are reported from European white-berry mistletoe (V. album L.). Two novel aminoalkaloids 4,5,4’- trihydroxy-3,3’-iminodibenzoic acid and 4,5,4’,5’-tetrahydroxy-3,3’- iminodibenzoic acid were isolated and identified from the leaves of V. album. This is the first time alkaloids that appear to be specific to this medicinal plant used for millennia are reported in the scientific literature. The two novel natural products 3-(3’-carbomethoxypropyl) gallic acid and 3-(3’-carbomethoxypropyl)- 7→3’’-protocatechoyl galloate make up a novel group of natural products. They are derived of the methyl ester of γ-hydroxybutyric acid (GHB) coupled to aromatic hydroxybenzoic acids including gallic acid. Radical scavenging activity for 3-(3’-carbomethoxypropyl) gallic acid (IC50 value 222 μM) were twentyfold reduced by substitution of the 3-hydroxyl of gallic acid with the methyl ester of gamma-hydroxybutyric acid compared with that of gallic acid ((IC50 value 27.7 μM). From dawn redwood (M. glyptostroboides) altogether eight compounds are reported including the novel natural product 6-carboxydihydroresveratrol 3-O-β- glucopyranoside. This dihydrostilbenoid exhibited both radical scavenging activity (IC50 value 164±39μM) and inhibition of 15-Lipoxygenase (IC50 value 246±30μM). Apigenin-6-C-(6’’-O-(3-hydroxy-3-methylglutaroyl)-β-glucopyranoside, a novel acylated C-glycosylflavone, and six other known natural products are reported from Z. zamiifolia. This ancient African medicinal plant has recently seen widespread use as an indoor ornamental plant. Z. zamiifolia has gained a reputation for being toxic. Despite this no lethality was observed even at the highest concentrations (1 mg/ml) when crude extracts of both leaves and petioles were applied in initial toxicological tests with Artemia salina. From the three species of the Bromeliaceae family, B. laciniosa (nine compounds), N. variegata (thirteen compounds) and E. spectabile (sixteen compounds), a total of twenty non-polar natural products are reported. Prior to this investigation very little information was available about the chemical composition of these three Bromeliaceae spp. Very long chain alkanes, fatty acids, tocopherols and triterpenoids are among the reported compounds. Twelve aromatic compounds are reported from the fruits of bog asphodel (N. ossifragum). Six novel natural products namely, (E)-4-(3-R-hydroxy-2,2- dimethylChroman-6-yl)but-3-en-3-one, (E)-4-(4-(((E)-4-hydroxy-3-methylbut- 2-en-1-yl)oxy)Phenyl)but-3-en-2-one, (2R,3S)-Naringenin(3→6’’)luteolin, (2R,3S)-Naringenin(3→6’’)chrysoeriol, liovil 4-O-β-glucopyranoside and (E)- 2,6-dimethoxycinnamic acid were reported for the first time. Previous to this investigation the rare natural product 4-hydroxy-3-(3-methylbut-2-enyl) benzaldehyde was only known from the pathogenic fungus Heterobasidion occidentale. This fungal metabolite was shown to be cytotoxic towards both normal rat kidney (NRK) (IC50 value 430 μM) and MOLM13 acute myeloid leukemia (AML) cells (IC50 value 68 μM). Naringenin(3→6’’)luteolin was the main aromatic compound, and was shown to be toxic to A. salina (LC50 value 130μM). This novel biflavonoid was cytotoxic for both NRK (IC50 value 230 μM) and MOLM13AML cells (IC50 value 115 μM)

Growing with dinosaurs: natural products from the Cretaceous relict Metasequoia glyptostroboides Hu & Cheng - a molecular reservoir from the ancient world with potential in modern medicine

After the sensational rediscovery of living exemplars of the Cretaceous relict Metasequoia glyptostroboides—a tree previously known exclusively from fossils from various locations in the northern hemisphere, there has been an increasing interest in discovery of novel natural products from this unique plant source. This article includes the first complete compilation of natural products reported from M. glyptostroboides during the entire period in which the tree has been investigated (1954–2014) with main focus on the compounds specific to this plant source. Studies on the biological activity of pure compounds and extracts derived from M. glyptostroboides are reviewed for the first time. The unique potential of M. glyptostroboides as a source of bioactive constituents is founded on the fact that the tree seems to have survived unchanged since the Cretaceous era. Since then, its molecular defense system has resisted the attacks of millions of generations of pathogens. In line with this, some recent landmarks in Metasequoia paleobotany are covered. Initial spectral analysis of recently discovered intact 53 million year old wood and amber of Metasequoia strongly indicate that the tree has remained unchanged for millions of years at the molecular level

Novel GHB-derived natural products from European mistletoe (Viscum album)

Context: The European white-berry mistletoe [Viscum album L. (Loranthaceae)] is among the oldest known medicinal plants. At present the most important application of mistletoe extracts is in the treatment of cancer. However, natural products specific to mistletoe have rarely been encountered in the current literature. Objective: To discover novel natural products specific to European mistletoe. Materials and methods: European mistletoe was extracted with methanol, purified to partition against diethyl ether and further purified with XAD-7 column chromatography. Pure compounds were separated by Sephadex column chromatography and preparative HPLC. The structures of the novel compounds were established using a combination of several 2D NMR spectroscopic techniques and mass spectrometry. Results: A new type of natural product derived from the methyl ester of g-hydroxybutyric acid (GHB) coupled to hydroxybenzoic acids, namely 3-(3'-carbomethoxypropyl) gallic acid and 3-(3'-carbomethoxypropyl)-7→3''-protocatechoyl galloate were characterized from European white-berry mistletoe. Condensation of the 3-hydroxyl of gallic acid with the 4-hydroxyl of GHB significantly reduced the radical scavenging properties of the former compound. Discussion and conclusion: The characterized compounds define a novel group of natural products that may be of particular interest because it appears that the two new compounds are not closely related to any known natural product

Non-Polar Natural Products from Bromelia laciniosa, Neoglaziovia variegata and Encholirium spectabile (Bromeliaceae)

Extensive regional droughts are already a major problem on all inhabited continents and severe regional droughts are expected to become an increasing and extended problem in the future. Consequently, extended use of available drought resistant food plants should be encouraged. Bromelia laciniosa, Neoglaziovia variegata and Encholirium spectabile are excellent candidates in that respect because they are established drought resistant edible plants from the semi-arid Caatinga region. From a food safety perspective, increased utilization of these plants would necessitate detailed knowledge about their chemical constituents. However, their chemical compositions have previously not been determined. For the first time, the non-polar constituents of B. laciniosa, N. variegata and E. spectabile have been identified. This is the first thorough report on natural products from N. variegata, E. spectabile, and B. laciniosa. Altogether, 20 non-polar natural products were characterized. The identifications were based on hyphenated gas chromatography-high resolution mass spectrometry (GC-HRMS) and supported by 1D and 2D Nuclear Magnetic Resonance (NMR) plant metabolomics