Isolation and characterization of plant bioactive compounds and assessment of their use in veterinary science

The research activity of the PhD project in Veterinary Science was aimed at the isolation and structural characterization of secondary metabolites from medicinal plants and the evaluation of their use in veterinary science. The selected plants are species used in folk medicine or in the veterinary medicine, particularly plants used as antiparasitic and antimicrobial The chemical-biological investigation was carried out on the following plants: • Psiadia punctulata (DC.) Vatke of the Asteraceae family it is found in tropical arid areas from the grasslands and shrubs in arid pastures to the edge of the forest, South-Eastern Africa, and Middle East, some plants of this family are widely used as antimicrobial; • Hypoestes forskaolii (Vahl) Roem. & Schult, herbaceous plant belonging to the Acanthaceae family, widely distributed throughout the southern region of Saudi Arabia, some plants of this family are widely used as a natural anthelmintic; • Trichilia maynasiana C.DC belonging to the family of the Meliaceae is a tree widely distributed in tropical areas of South Africa used in folk medicine to treat various anti-inflammatory diseases; • Vernonia nigritiana Oliv. & Hiern. (Asteraceae) is an herbaceous plant or shrub widely distributed in West Africa, the leaves and other parts of the plant are traditionally used against skin inflammations, rheumatism, fever, headache and impairment. In veterinary medicine many plants of Vernonia genus are used for bacterial infections and intestinal worms. The isolation of secondary metabolites was achieved by chromatographic techniques such as column chromatography, thin layer chromatography, HPLC (High Pressure Liquid Chromatography). The structural characterization of all the isolates was performed by spectroscopic techniques, nuclear magnetic resonance experiments monodimensional (1H-NMR, 13C-NMR) and two-dimensional (COSY, HSQC, HMBC, ROESY), and mass spectrometry. The investigation of Psiadia punctulata led to the isolation of 30 compounds including diterpenes and flavones, of which 13 were new natural compounds. The antimicrobial activity of the dicloromethan extract was tested against Gram-positive bacteria (Staphylococcus aureus), Gram-negative (Pseudomonas aeruginosa) and dimorphic fungus Candida albicans. The results of this study highlighted that the dicloromethan extract showed antibacterial activity against S. aureus (MIC50 = 100 ug/mL), whereas no activity was detected against Gram-negative bacteria. Moreover, the extract showed antifungal activity against Candida albicans (MIC2 = 50 μg/mL and MIC0 = 130 μg/mL). The nine fractions A-I obtained after a silica gel column separation were tested for their antimicrobial activity at doses ranged from 20 to 100 μg/ml. The A,B and I fractions were found to be inactive on all species, instead six fractions (C-H) showed a remarkable activity. All the compounds isolated from active fractions were tested for their antimicrobial activity. Compound 3, 3',4',5,7-tetramethoxyflavone, at a concentration of 40 g/mL was able to inhibit by 50% the production of biofilm of the S. aureus and 90% for Candida albigans. The phytochemical investigation of H. forskaolii leaves led to the isolation of 13 compounds belonging to the class of diterpenes fusicoccane and isopimarane, among these 4 of fusicoccane diterpenes are new natural compounds. The n-hexane, chloroform, chloroform: methanol 9:1 and methanolic extracts of Hypoestes forskaolii were tested fot in vitro anthelmintic activity via Egg hacth Assay on different species of gastrointestinal netamathodes that naturally parasitize small ruminants. Polar extracts have not been able to show significant ovicidal activity (less than 50% of hatching), while the n-hexane extract at a concentration of 1 mg/ml showed moderate inhibition of 30% hatching. All the isolated compounds were also tested in vitro for the antiproliferative activity on HeLa and Jurkat U973 cell lines, all compounds did not significantly affect growth and cell viability. The phytochemical investigation of Trichilia maynasiana led to the isolation of nine compounds of which two are new natural compounds. These compounds belonging to the class of tirucallane and secoguaiane terpenes. The phytochemical study of V. nigritiana led to the isolation of seven secondary metabolites including two sesquiterpene lactones, never described before

Chemical Profiling of Astragalus membranaceus Roots (Fish.) Bunge Herbal Preparation and Evaluation of Its Bioactivity

Astragalus membranaceus (Fish.) Bunge is a perennial herb distributed in the northern part of China, and its roots, namely, Hang qi, are included as a natural ingredient in dietary supplement formulations commonly used to treat different disorders such as respiratory infections, diabetes, and heart failure. The availability of a simple method for the determination of the quality of Astragalus herbal preparations could be a challenging issue for commercial purposes. In this study, a liquid chromatography–mass spectrometry (LC–MS)/MS based approach was used to characterize specialized metabolite recovery of 3 commercial hydroalcoholic extracts of A. membranaceus (AMG1, AMG2, AMG3) in addition to a hydroalcoholic extract of A. membranaceus root (AST). The hypoglycemic effect, cholinesterase inhibition, and antioxidant activities were also evaluated. Thirty-one compounds, of which 19 polyphenols and 12 saponins, were identified. The extracts were also quantified by using a sensitive and selective Q-Trap system for their content in flavonoids and astragalosides, selecting astragaloside I and IV as chemical markers. From our results, AMG3 preparation (Axtragyl) was the most abundant in terms of both specialized classes of metabolites, showing a fingerprint similar to that of AST. Interestingly, tested enzyme inhibition ability of flavonoids, daidzein (11) and formononetin (19), reported a higher α-glucosidase inhibition in comparison with that of acarbose used as positive control. The in silico study clarified the interactions among the molecules and the importance of having a free hydroxy group. Moreover, Axtragyl was able to exert protective effects in Caco-2 cells treated with hydrogen peroxide, confirming its ability as a potential protective agent in intestinal injury

A Novel Tirucallane-type Triterpene and Sesquiterpene from Trichilia maynasiana

One new tirucallane-type triterpene 3β, 24-dihydroxytirucallan-7,25-diene, 24-sulfate (1), one new sesquiterpene 7-epi-10-hydroxychabrol-1(2)-en-4,5-dione A (2), together with three known tirucallanes, and four aromadendranes were isolated from the leaves of Trichilia maynasiana C. DC.. Their structures were determined by means of NMR spectroscopy, mass spectrometric analysis, and chemical methods

In Vitro anthelminthic efficacy of hypoestes forskaolii (Vahl) R.Br (Acanthaceae) extracts on gastrointestinal nematodes of sheep

The anthelminthic efficacy of the crude extracts of Hypoestes forskaolii (Vahl) R.Br (Acanthaceae) against gastrointestinal nematodes (GIN) in sheep was investigated using the in vitro egg hatch inhibition assay. Faecal samples were collected from sheep with naturally occurring infection of GIN (Trichostrongylus spp., Chabertia ovina, Cooperia spp., Haemonchus contortus and Teladorsagia spp.). Crude leaf extracts of H. forskaolii was obtained using increasing polarity solvents: n-hexane, chloroform, chloroform:methanol 9:1, methanol. Thiabendazole (0.2 µg/mL and 0.5 µg/mL) was used as a positive control and untreated GIN eggs in deionised water served as the negative control. All the extracts exhibited a weak ovicidal activity against GIN (less than 50% of egg hatch). Noteworthy, the n-hexane extract showed a percentage of inhibition of egg hatching greater than other extracts inhibiting the 30.8% at the concentration of 1 mg/mL showing a dose-dependent effect on nematode eggs hatching. Further studies are needed to investigate the effects of extracts used and to evaluate the ovicidal effects of other extracts of H. forskaolii

Phytochemical study and antioxidant activity of Calligonum azel and C. comosum

One new phenolic compound (1) together with nine known derivatives were isolated from the aerial part apolar extracts of Calligonum azel Maire and Calligonum comosum L'Hér (Polygonaceae).Their structures were established on the basis of 1D and 2D NMR spectroscopy, as well as ESI-MS analysis. The anti-inflammatory and antioxidant potential of pure compounds was evaluated in J774A.1 murine macrophages and Jurkat cells. Among tested molecules, 4-ethoxy-1,2-benzendiol, tamgermanetin, and α,β-diamino-4-hydroxybenzene butanoic acid exerted the more interesting activity

Antiprotozoal activity of diterpenoids isolated from Zhumeria majdae - absolute configuration by circular dichroism

Zhumeria majdae, a unique species of the Zhumeria genus, is an endemic Iranian plant in the Lamiaceae family. Phytochemical investigation and biological activity of this plant are rarely reported. The current study aimed to find new antiprotozoal compounds from the roots of Z. majdae and to determine the absolute configuration of isolated compounds by circular dichroism. The extraction process from roots and aerial parts of Z. majdae was carried out by hexane, ethyl acetate and methanol followed by testing their antiprotozoal effects against Leishmania donovani, Trypanosoma brucei rhodesiense, T. cruzi, and Plasmodium falciparum, respectively. Structure elucidation was done using 1D and 2D NMR spectroscopy and HREIMS spectrometry. In addition, experimental and theoretical circular dichroism spectroscopy was used to establish absolute configuration. In comparison with aerial parts, the hexane extract from roots showed superior activity against T. b. rhodesiense, L. donovani and P. falciparum with IC50 values of 5.4, 1.6 and 2.1 μg/ml, respectively. From eight abietane-type diterpenoids identified in roots, six were reported for the first time in the genus Zhumeria. 11,14-dihydroxy-8,11,13-abietatrien-7-one (6) exhibited a promising biological activity against P. falciparum (IC50 8.65 μM), with a selectivity index (SI) of 4.6, and lanugon Q (8) showed an IC50 value of 0.13 μM and SI of 15.4 against T. b. rhodesiense. Altogether, according to the results, of 8 isolated compounds, dihydroxy-8,11,13-abietatrien-7-one (6) and lanugon Q (8) exhibited a promising activity against T. b. rhodesiense and P. falciparum. In conclusion, these compounds could be potential candidates for further analysis and may serve as lead compounds for the synthesis of antiprotozoal agents

Fusicoccane Diterpenes from Hypoestes forsskaolii as Heat Shock Protein 90 (Hsp90) Modulators

Ten new (1-10) and six known (11-16) fusicoccane diterpenes were isolated from the roots of Hypoestes forsskaolii. The structural characterization of 1-10 was performed by spectroscopic analysis, including 1D and 2D NMR, ECD, and HRESIMS experiments. From a perspective of obtaining potential Hsp90α inhibitors, the isolates were screened by surface plasmon resonance measurements and their cytotoxic activity was assayed using Jurkat and HeLa cancer cells. Compound 6, 18-hydroxyhypoestenone, was shown to be the most active compound against Hsp90, and its interactions were studied also by biochemical and cellular assays and by molecular docking

Antioxidant Potential of Herbal Preparations and Components from Galactites elegans (All.) Nyman ex Soldano

Galactites is a genus of flowering plants belonging to Asteraceae family. This genus is mainly represented by the Galactites elegans (All.) Nyman ex Soldano, the milky thistle, a plant of Mediterranean origin. Galactites elegans is consumed as a monofloral boar thistle honey. Chromatography separation of CHCl3 and n-BuOH extracts of aerial parts of G. elegans led to isolation of 18 pure compounds. Their structures were elucidated by 1D-and 2D-NMR spectroscopy and confirmed by mass spectrometry analysis. Sinapic aldehyde, abietin, chlorogenic acid, neochlorogenic acid, 8 alpha-ahydroxypinoresinol, 9 alpha-hydroxypinoresinol, pinoresinol, 4-ketopinoresinol, nortrachelogenin, and erythro-guaiacylglycerol-beta-O-4'-dihydroconiferyl alcohol were isolated from CHCl3 extract, while luteolin 4'-O-glucuronide, naringenin-7-O-neohcsperidoside, kaempferol-3-O-alpha-L-rhamnopyranosyl-(1 -> 6)-beta-D-glucopyranoside, apigenin-7-O-alpha-L-rhamnopyranosyl-(1 -> 6)-beta-D-glucopyranoside, quercitrin, quercetin-3-O-alpha-L-rhamnopyranosyl-(1 -> 6)-beta-D-glucopyranoside, ciwujiatone, and nortrachelogenin-4,4'-di-O-beta-D-glucopyranoside were obtained from n-BuOH extract. The majority of isolated compounds displayed a significant antioxidant potential in vitro test (DPPH). The ability of compounds to reduce the level of peroxides in control and BHP-treated Jurkat cells was studied. The lignan derivatives were also able to reduce at 50 mu M the basal level of peroxides in Jurkat cells as well as counteract peroxide increase induced by BLIP treatment. Particularly 8 alpha-hydroxypinoresinol was the most active showing 70\% of peroxide level inhibition

Phytochemistry of compounds isolated from the leaf-surface extract of Psiadia punctulata (DC.) Vatke growing in Saudi Arabia

The surface extract of an accession of Psiadia punctulata (DC.) Vatke (Asteraceae) growing in Saudi Arabia was investigated for its phytochemical composition. A bio-guided investigation of the extract led to the isolation of thirteen ent-kaurane and trachylobane diterpenes and seventeen compounds previously described, including nine flavonoids and eight diterpenes. Three flavonoids and one ent-kaurane diterpene showed antimicrobial activity with MIC100 values ranging from 25 to 150 μg/ml. The extract showed antibacterial activity against Staphylococcus aureus (MIC100 = 180 μg/ml) and antifungal activity against Candida albicans (MIC0 = 130 μg/ml). The isolated 3',4',5,7-tetramethoxyflavone, at a concentration of 40 μg/ml, displayed the ability to reduce biofilm formation of S. aureus and C. albicans by 50% and 90% respectively