Secondary metabolite profiling and characterization of diterpenes and flavones from the methanolic extract of Andrographis paniculata using HPLC-LC-MS/MS

Background Andrographis paniculata is a well-known medicinal plant that contains various classes of bioactive secondary metabolites. It is widely used by the traditional medicinal healers for treatment of malaria and other diseases. There is an urgent need for screening of potent novel compounds from the methanol extract of A. paniculata. Earlier, we obtained appreciable in vitro anti-malarial activity (IC50-10.75 μg/ml) in the same plant. In current study, we developed novel analytical methods for rapid identification and characterization of diterpenes and flavones using chromatographic and spectroscopic techniques and identified major compounds that might possess anti-malarial activities. Results Based on the chromatographic and mass spectrometric features, we have identified a total of 74 compounds (25 compounds from positive ion mode; 49 compounds from negative ion mode). The mass spectrum data predicted andrographolide (15%) presence in the highest amount in both positive and negative ion modes. Based on the percentage purity, Andrographolide and skullcapflavone I was selected as representative class of diterpenes and flavones for fragmentation studies. Conclusions The result led to identification of Neoandrographolide, andrographolactone, 14-dehydroxy-11,12-didehydroandrographolide, skullcapflavone I, and 5-Hydroxy-2′,7,8-tri methoxy flavone from the methanolic extract of A. paniculata that is used in traditional medicine by tribal healers of Amarkantak region for treating malaria. These could be lead compounds for the development of novel anti-malarial drugs.

Pachypodostyflavone, a New 3-methoxy Flavone and Other Constituents with Antifilarial Activities from the Stem Bark of Duguetia staudtii

A new flavone derivative named pachypodostyflavone (1), along with 8 known compounds (2–9) and a mixture of β-sitosterol and stigmasterol were isolated from the stem bark of Duguetia staudtii (Annonaceae), based on a bioassay-guided fractionation. Their structures were determined using high-resolution mass spectrometry and NMR spectroscopic data, as well as by comparison with the literature values of their analogs. Selected isolated compounds were evaluated for their in vitro antifilaricidal activities on Onchocerca ochengi microfilariae and adult worms. Inhibition of motility was evaluated spectroscopically on microfilaria and adult male worms. Viability was determined on adult female worms by the MTT/ Formazan assay. Auranofin at 10 µM and 2% DMSO were used as positive and negative controls, respectively. Compounds 1 and 7 showed potent anti-onchocerca activities with 100% activity at 250 µg/mL on both O. ochengi adult male and female worms, while compound 5 displayed 100% activity at 30 µg/mL.

FORMULATION OF AN ANTI-BACTERIAL CREAM FROM PLANTOXALIS CORNICULATA ANDITS EVALUATION

Objective: Even in areas where modern medicine is available, the interest on herbal medicines and their utilization have been increasing rapidly in recent years. Plant-derived substances and herbal medicines have recently attracted the great interest towards their versatile application as medical plants are the rich source of bioactive compounds used in traditional and modern medicine. The present work is to formulate and evaluate the antibacterial cream of oxalis corniculata extract. Methods: The ethanolic extracts were prepared by using the maceration method. Results: The agrochemical potential of methanolic extract, n-hexane, chloroform, ethyl acetate,and n-butanol soluble sfractions showed excellent activites against Escherichia coli, Shigella dysenteriae, Salmonella typhi, and Bacillus subtilis. Similarly the crude n-hexane and chloroform fractions were also found to have significant activity against fungal strains including Fusarium solani, Aspergillus flexneri, and Aspergillus flavus. Conclusion: Oxalis corniculata is a common medicinal plant widely used against numerous infectious diseases. The two isolated compounds 5-hydroxy-6,7,8,4’-tetra methoxy flavone and 5,7,4’-trihydroxy-6,8-dimethoxyflavone were evaluated for antibacterial and antifungal activities. The results showed that latter compound was more active than that of the former.

Pharmacognostical, Phytochemical and Pharmacological profile of Colebrookea oppositifolia Smith

Colebrookea oppositifolia commonly known as ‘Bhaman’ is distributed throughout India from the Himalayas down to Deccan. The plant is used traditionally as such as dermatitis, dysentery, fever, headache, peptic ulcer, haemostatic, wounds, as anti-fertility agent, fungicide, and the roots of the plant has been most widely used for the treatment of epilepsy.. Medicinally, it has been proven to possess various pharmacological activities like treating corneal opacity or conjunctivitis, sore eyes due to its anti-inflammatory properties, cardioprotective, hepatoprotective, anti-inflammatory, antihelmintic, antifungal, antioxidant, antimicrobial, antinociceptive, cytotoxic activity, anticonvulsant, antiulcer, antimicrobial, anti-fertility, antipyretic and insecticide. Further, studies reveal the presence of various phytochemical constituents mainly flavone glycosides viz. chrysin, negletein, landenein; leaves contain 5,6,7- tri-methoxyflavone, 5,6,7,4'-tetramethoxyflavone, acteoside, and quercetin in the bark; root contains stearic, palmitic, oleic acids, triacontanol, flavone glycoside echioidin, 5,6,7-trimethoxyflavone and 4',5,6,7- tetra methoxy flavone; sugars and vitamins have also been isolated from this plant. These studies reveal that Colebrookea oppositifolia is a source of medicinally active compounds and have various pharmacological effects; hence, this drug encourage finding its new therapeutic uses. Keywords: Colebrookea oppositifolia, wound healing, anticonvulsant, Lamiaceae

Functional Group-Dependent Induction of Astrocytogenesis and Neurogenesis by Flavone Derivatives

Neural stem cells (NSCs) differentiate into multiple cell types, including neurons, astrocytes, and oligodendrocytes, and provide an excellent platform to screen drugs against neurodegenerative diseases. Flavonoids exert a wide range of biological functions on several cell types and affect the fate of NSCs. In the present study, we investigated whether the structure-activity relationships of flavone derivatives influence NSC differentiation. As previously reported, we observed that PD98059 (2′-amino-3′-methoxy-flavone), compound 2 (3′-methoxy-flavone) induced astrocytogenesis. In the present study, we showed that compound 3 (2′-hydroxy-3′-methoxy-flavone), containing a 3′-methoxy group, and a non-bulky group at C2′ and C4′, induced astrocytogenesis through JAK-STAT3 signaling pathway. However, compound 1 and 7–12 without the methoxy group did not show such effects. Interestingly, the compounds 4 (2′,3′-dimethoxyflavone), 5 (2′-N-phenylacetamido-3′-methoxy-flavone), and 6 (3′,4′-dimethoxyflavone) containing 3′-methoxy could not promote astrocytic differentiation, suggesting that both the methoxy groups at C3′ and non-bulky group at C2′ and C4′ are required for the induction of astrocytogenesis. Notably, compound 6 promoted neuronal differentiation, whereas its 4′-demethoxylated analog, compound 2, repressed neurogenesis, suggesting an essential role of the methoxy group at C4′ in neurogenesis. These findings revealed that subtle structural changes of flavone derivatives have pronounced effects on NSC differentiation and can guide to design and develop novel flavone chemicals targeting NSCs fate regulation.

Role of Some Food-Grade Synthesized Flavonoids on the Control of Ochratoxin A in Aspergillus carbonarius

Ochratoxin A (OTA) is a mycotoxin with a serious impact on human health. In Mediterranean countries, the black Aspergilli group, in particular Aspergillus carbonarius, causes the highest OTA contamination. Here we describe the synthesis of three polyphenolic flavonoids: 5-hydroxy-6,7-dimethoxy-flavone (MOS), 5,6-dihydroxy-7-methoxy-flavone (NEG), and 5,6 dihydroxy-flavone (DHF), as well as their effect on the prevention of OTA biosynthesis and lipoxygenase (LOX) activity in A. carbonarius cultured in a conducive liquid medium. The best control effect on OTA biosynthesis was achieved using NEG and DHF. In fungal cultures treated with these compounds at 5, 25, and 50 μg/mL, OTA biosynthesis significantly decreased throughout the 8-day experiment. NEG and DHF appear to have an inhibiting effect also on the activity of LOX, whereas MOS, which did not significantly inhibit OTA production, had no effect on LOX activity. The presence of free hydroxyls in catecholic position in the molecule appears to be a determining factor for significantly inhibiting OTA biosynthesis. However, the presence of a methoxy group in C-7 in NEG could slightly lower the molecule’s reactivity increasing OTA inhibition by this molecule at 5 μg/mL. Polyphenolic flavonoids present in edible plants may be easily synthesized and used to control OTA biosynthesis.

Turunan Senyawa Flavonoid dari Daun Macaranga involucrata (Roxb.) Baill dari Buton Tengah, Sulawesi Tenggara

Macaranga merupakan salah satu genus terbesar dari famili Euphorbiaceae yang terdiri dari 300 spesies dengan nama lokal mahang-mahangan. Tumbuhan Macaranga tersebar luas di wilayah Afrika dan Madagaskar di bagian barat hingga ke wilayah tropis Asia, Australia utara dan kepulauan Pasifik. Di Indonesia tumbuhan Macaranga tersebar di beberapa daerah yaitu daerah Papua, Maluku, Sulawesi, Kalimantan, Sumatera, Bangka, dan Jawa. Kajian fitokimia beberapa spesies Macaranga menunjukan adanya kelompok senyawa fenolik yaitu turunan flavonoid dan stilben, serta turunan terpenoid. Senyawa turunan fenolik tersebut memiliki keunikan dari struktur molekulnya, yaitu adanya subtituen tambahan dari metabolit terpenoid yaitu prenil (C5), geranil (C10), farnesil (C15), dan geranilgeranil (C20). Pada penelitian ini telah dilakukan isolasi metabolit sekunder dari daun M. involucrata (Roxb.) Baill dengan metode maserasi menggunakan pelarut aseton, kemudian dilanjutkan pemisahan dan pemurnian dengan menggunakan kromatografi cair vakum dan kromatografi radial untuk mendapatkan senyawa murni. Penentuan struktur dilakukan berdasarkan analisis data spektrum NMR 1D (1H-NMR dan13C-NMR), NMR 2D (NOESY, TOCSY, HSQC, dan HMBC), dan spektrum massa (MS). Berdasarkan metodologi tersebut, dua senyawa turunan flavon yaitu 5,7,4’-trihidroksi-3’(3-metilbut-2-enil)-3-metoksiflavon (1) dan makarangin (2), telah berhasil diisolasi dari tumbuhan ini. Berdasarkan hasil penelitian tersebut menunjukan daun M. involucrata (Roxb.) Baill yang berasal dari Kabupaten Buton Tengah, Sulawesi Tenggara menghasilkan senyawa fenolik turunan flavonoid. Kata kunci: Euphorbiaceae, Macaranga involucrata (Roxb.) Baill, flavon. Macaranga is one of the largest genera of the family Euphorbiaceae comprising 300 species with local name “mahang-mahangan”. Macaranga is widespread in the region of Africa and the west of Madagascar to the tropical regions of Asia, northern Australia, and the Pacific islands. In Indonesia Macaranga spread in several areas: Papua, Maluku, Sulawesi, Kalimantan, Sumatra, Bangka, and Java. Phytochemical studies showed the presence of several phenolic compounds such as flavonoids and stilbene derivatives. The phenolic compounds have a unique molecular structure with the addition of some substituents such as prenyl (C5), geranyl (C10), farnesyl (C15), and geranylgeranyl (C20). This research has been conducted on the isolation of secondary metabolites from the leaves of M. involucrata (Roxb.) Baill by maceration method using acetone, followed by separation and purification by using liquid vacuum chromatography and radial chromatography to obtain pure compounds. Determination of the structure is based on data analysis of 1D NMR spectrum (1H-NMR and 13C-NMR), 2D NMR (1H-1HCOSY, NOESY, TOCSY, HSQC, and HMBC), and mass spectra (MS). Based on this methodology, two flavone derivatives 5,7,4'-trihydroxy-3'(3-methylbut-2-enyl)-3-methoxy flavone (1) and macarangin (2), have been isolated from this plant. Based on these results showed that leaf of M. involucrata (Roxb.) Baill from Central Buton, Southeast Sulawesi produces phenolic compounds from flavonoid derivatives. Keywords: Euphorbiaceae, Macaranga involucrata (Roxb.) Baill, flavone.

LOCALIZATION AND ORIENTATION OF METHOXY FLAVONOIDS IN DPPC BILAYERS: EFFECT ON THEIR ANTI-PROLIFERATIVE ACTIVITY

Objective: Flavones and flavonols are an important class of naturally occurring flavonoids. They are well known for their pharmacological activity. This activity is associated with the ability of flavones and flavonols to influence membrane–dependent processes. In this paper, we have reported localization, orientation and interaction, of four synthesized flavone/flavonols with 1, 2–dipalmitoyl–sn–glycero–3–phosphocholine (DPPC) bilayers. These are compared with standard flavone; chrysin (CHY) and flavonol Quercetin (QUE).Methods: The molecules studied are 4ʹ–methoxy flavone (MF), 3ʹ,4ʹ–dimethoxyflavone (DMF), 4ʹ–methoxyflavonol (MF–ol) and 3ʹ,4ʹ–dimethoxyflavonol (DMF–ol). The techniques used are Differential Scanning Calorimetry (DSC) and multi–nuclear NMR.Results: Highest binding to lipid bilayers is shown by DMF, followed by QUE. Based on DSC studies it is seen, that maximum interaction of MF and DMF, takes place with the hydrophobic core of lipid bilayers. DMF–ol shows formation of a heterogeneous system at higher concentrations. The 1H NMR spectra of unilamellar vesicles of DPPC, incorporated with MF, DMF and MF–ol shows significant interaction of these compounds with the alkyl chain of the hydrophobic core. MF, DMF and MF–ol acquire parallel orientation in bilayers with the B–ring pointing towards hydrophobic core, while DMF–ol acquire mixed orientation. This may be ascribed to the presence of two methoxy and one hydroxyl group on the B–ring of DMF–ol which hinders its partitioning inside the hydrophobic core of lipid bilayer. Multi–lamellar vesicles (MLV) of DPPC incorporated with flavones, show maximum increase in Chemical Shift Anisotropy in 31P spectrum of DMF. This is followed by MF. DSC.Conclusion: NMR and binding studies indicate that DMF is partitioned deeply inside the hydrophobic core, while MF, MF–ol and DMF–ol are mostly located in the vicinity of sn–glycero region. Therefore, we conclude that DMF which penetrates deepest inside the hydrophobic core also shows the highest anti–proliferative activity against K562 and MCF–7 cancer cell lines. Its activity is also better than CHY.

FLAVONOIDS AND TRITERPENOIDS FROM CALLISTEMON CITRINUS AND THEIR INHIBITORY EFFECT ON NO PRODUCTION IN LPS-STIMULATED RAW264.7 MACROPHAGES

Phytochemical investigation of the leaves and stems of Callistemon citrinus (Curtis) Skeels led to the isolation of 12 flavonoid and triterpenoid compounds, including one new flavonoid, callistine A (1) and six known flavonoids 6,7- dimethyl-5,7-dihydroxy-4’-methoxy flavone (2), astragalin (3), quercetin (4), catechin (5), eucalyptin (6), and 8-demethyleucalyptin (7), along with 5 triterpenoids, 3-β-acetylmorolic acid (8), 3β-hydroxy-urs-11-en-13(28)-olide (9), betulinic acid (10), diospyrolide (11) and ursolic acid (12). Their chemical structures were determined from the spectroscopic evidences counting 1D- and 2D-NMR and HR-MS data. All the isolated compounds were examined for their anti-inflammatory activity against LPS-activated NO production in macrophage RAW264.7 cells. Among them, quercetin (4) and 3β-hydroxy-urs-11-en-13(28)-olide (9) showed potential inhibition activity in nitric oxide (NO) production in RAW264.7 cells exposed to LPS.