Bioactive compounds from Hypericum humifusum and Hypericum perfoliatum: inhibition potential of polyphenols with acetylcholinesterase and key enzymes linked to type-2 diabetes

Context: Natural products are reported to have a wide spectrum of pharmacological properties such as antimicrobial, anti-inflammatory and anti-cholinesterase. The genus Hypericum (Hypericaceae) is a source of a variety of molecules with different biological activities, notably hypericin and various phenolics.

Objectives: The goals of the present work were the determination of total phenolic and flavonoid content, hypericin and hyperforin concentration as well as the evaluation of biological of Hypericum humifusum L. (Hhu) and Hypericum perfoliatum L. (Hper).

Materials and methods: The various extracts of aerial parts were powdered, and then extracted with methanol. Antibacterial activity was performed according to minimum inhibitory concentration (MIC) and minimum bactericidal (MBC) methods against four Gram-positive bacteria, four Gram-negative bacteria and yeast.

Results: The results revealed that H. humifusum, bear the highest total phenolic and flavonoid content (48–113?mg GAE/g and 8–41?mg RE/g, respectively) as well as hypericin (60–90?mg/g) and hyperforin (8–30?mg/g) concentration. Both species showed significant antioxidant activity as revealed by DPPH, FRAP, ABTS, and metal chelating assays. H. humifusum exhibited a strong acetylcholinesterase (3.86–4.57?mg GALAEs/g), α-glucosidase (0.73–2.55?mmol ACEs/g) and α-amylase (3–8?mmol ACEs/g) inhibitory activity. The extract of H. humifusum exhibited strong antibacterial activity mainly against Staphylococcus epidermidis, Staphylococus aureus, and Enterococcus faecium (MIC values ranging from 200 to 250?μg/mL). The highest antifungal activity was showed for H. perfoliatum extract (MIC value = 250?μg/mL).

Conclusion: The data suggest that H. humifusum could be used as valuable new natural agents with functional properties for pharmacology industries.     

Anti-fibrotic effects of Cuscuta chinensis with in vitro hepatic stellate cells and a thioacetamide-induced experimental rat model

Context: Cuscuta chinensis Lam. (Convolvulaceae) has been used as a traditional herbal remedy for treating liver and kidney disorders.

Objective: Anti-fibrotic effects of C. chinensis extract (CCE) in cellular and experimental animal models were investigated.

Materials and methods: HSC-T6 cell viability, cell cycle and apoptosis were analysed using MTT assay, flow cytometry and Annexin V-FITC/PI staining techniques. Thioacetamide (TAA)-induced fibrosis model was established using Sprague Dawley rats (n?=?10). Control, TAA, CCE 10 (TAA with CCE 10?mg/kg), CCE 100 (TAA with CCE 100?mg/kg) and silymarin (TAA with silymarin 50?mg/kg). Fibrosis was induced by TAA (200?mg/kg, i.p.) twice per week for 13 weeks. CCE and silymarin were administered orally two times per week from the 7th to 13th week. Fibrotic related gene expression (α-SMA, Col1α1 and TGF-β1) was measured by RT-PCR. Serum biomarkers, glutathione (GSH) and hydroxyproline were estimated by spectrophotometer using commercial kits.

Results: CCE (0.05 and 0.1?mg/mL) and silymarin (0.05?mg/mL) treatment significantly (p?p?in vivo studies, CCE (10 and 100?mg/kg) treatment ameliorated the TAA-induced altered levels of serum biomarkers, fibrotic related gene expression, GSH, hydroxyproline significantly (p?Conclusions: CCE can be developed as a potential agent in the treatment of hepatofibrosis.     

Vasorelaxant mode of action of dichloromethane-soluble extract from Agastache mexicana and its main bioactive compounds

Context: Agastache mexicana (Kunth) Lint & Epling (Lamiaceae) is a plant used in Mexican traditional medicine for the treatment of hypertension, anxiety and so on.

Objective: To determine the vasorelaxant effect and functional mode of action of dichloromethane-soluble extract from A. mexicana (DEAm) and isolate the constituents responsible for the pharmacological activity.

Materials and methods: Extracts were prepared from the aerial parts of A. mexicana (225.6?g) by successive maceration with hexane, dichloromethane and methanol (three times for 72?h at room temperature), respectively. DEAm (0.01–1000?μg/mL), fractions (at 174.27?μg/mL), acacetin and ursolic acid (UA) (0.5–500?μM) were evaluated to determine their vasorelaxant effect on ex vivo rat aorta ring model. In vivo UA antihypertensive action was determined on spontaneously hypertensive rats.

Results and discussion: DEAm induced a significant vasorelaxant effect in concentration-dependent and endothelium-independent manners (EC_50?=_?174.276?±?5.98?μg/mL) by a calcium channel blockade and potassium channel opening. Bio-guided fractionation allowed to isolate acacetin (112?mg), UA (2.830?g), acacetin/oleanolic acid (OA) (M1) (155?mg) and acacetin/OA/UA (M2) (1.382?g) mixtures, which also showed significant vasodilation. UA significantly diminished diastolic (80?mmHg) and systolic blood pressure (120?mmHg), but heart rate was not modified.

Conclusion: DEAm produced significant vasorelaxant action by myogenic control cation. The presence of acacetin, OA and UA into the extract was substantial for the relaxant activity of DEAm. In vivo antihypertensive action of UA corroborates the use of A. mexicana as an antihypertensive agent on Mexican folk medicine.     

Protective effects of Ampelopsis brevipedunculata against in vitro hepatic stellate cells system and thioacetamide-induced liver fibrosis rat model

Context: Ampelopsis brevipedunculata Maxim (Vitaceae) is a traditional medicinal herb used for treating liver disorders.

Objective: The hepatoprotective effects of A. brevipedunculata ethanol extract (ABE) was investigated in experimental models of fibrosis.

Materials and methods: Hepatic stellate cells (HSCs) system in vitro and thioacetamide (TAA)-induced liver fibrosis rat model in vivo were used. Sprague–Dawley rats were divided into five groups of eight each (control, TAA, TAA with ABE 10?mg/kg, ABE 100?mg/kg and silymarin 50?mg/kg groups, respectively). Fibrosis was induced except to the control group by TAA (200?mg/kg, i.p.) twice per week for 13?weeks. ABE and silymarin was administered orally six times per week from the 7th week to the 13th week.

Results: In HSC-T6 cells, ABE (0.1?mg/mL) and silymarin (0.05?mg/mL) significantly (p?p?in vivo studies, ABE (10 and 100?mg/kg) treatment ameliorated the altered levels of serum biomarkers significantly (p?p?p?Conclusion: These results collectively indicate that ABE can potentially be developed as a therapeutic agent in the treatment of hepatic fibrosis.     

Effects of garlic polysaccharide on alcoholic liver fibrosis and intestinal microflora in mice

Context: Alcoholic liver fibrosis (ALF) is treatable and reversible consequence of liver disease. Intestinal microflora plays an important role in the progression of liver disease. Garlic (Allium sativum L. [Amaryllidaceae]) has been consumed as a traditional medicine to treat liver injury.

Objective: To investigate the effects of garlic polysaccharide (GP) on ALF and intestinal microflora in mice.

Materials and methods: KM mice were orally administered with alcohol (56%, 6?mL/kg) for 30?d to establish ALF model, and divided into four groups together with control group (water only). Hugan tablet (60?mg/kg) or GP (250 and 150?mg/kg) were given 5?h after each dose of alcohol. Biochemical markers in serum and liver homogenate were determined with kits. Alteration of intestinal microflora, and protein expressions of TGF-β1, TNF-α and decorin were detected.

Results: In GP-H group, ALT and AST decreased to 18.85?±?4.71?U/L and 40.84?±?7.89?U/L. MDA, TC, TG and LDL-C decreased to 2.32?±?0.86?mmol/mg, 0.21?±?0.12?mmol/L, 0.96?±?0.31?mmol/L and 0.084?±?0.027?mmol/L. SOD, GSH-Px and GSH increased to 118.32?±?16.32?U/mg, 523.72?±?64.20?U/mg and 0.56?±?0.05?mg/g. Ratios of TGF-β1 and TNF-α decreased to 0.608?±?0.170 and 1.057?±?0.058, decorin increased to 2.182?±?0.129. Lachnospiraceae and Lactobacillus increased, Facklamia and Firmicutes decreased with GP pretreatment.

Discussion and conclusions: Intestinal microflora provides novel insight into the mechanisms of GP that may be used to treat ALF and intestinal microflora dysbiosis.     

LC-MS identification and preparative HPLC isolation of Frankenia pulverulenta phenolics with antioxidant and neuroprotective capacities in PC12 cell line

Context: Frankenia pulverulenta L. (Frankeniaceae) is a medicinal species with carminative, analgesic and antiviral properties. However, phytochemical investigations, antioxidant and neuroprotective capacities of this plant remain unclear.

Objective: This work assesses the phenolic composition of F. pulverulenta shoot and root and evaluates their antioxidant and neuroprotective capacities.

Materials and methods: Successive fractionation of F. pulverulenta shoot and root using 6 solvents were used. Antioxidant capacity of these fractions was assessed through four in vitro tests (DPPH, ABTS, Fe-chelating activity and ORAC). Phenolic identification, purification as well as neuroprotective activity of ethyl acetate (EtOAc) fraction and purified molecules were assessed.

Results: Among the tested fractions, EtOAc shoot and root fractions possessed considerable phenolic contents (383 and 374?mg GAE/g E, respectively) because of their important ORAC (821 and 1054?mg of TE/g E), DPPH (586 and 750?mg of TE/g) and ABTS (1453 and 1319?mg of TE/g) results. Moreover, gallic acid, quercetin, quercetin galloyl glucoside, trigalloyl hexoside, procyanidin dimers and sulfated flavonoids were identified by LC-DAD-ESI-MS for the first time in this species. The relevant cytoprotective capacity (at 300?μg/mL) against β-amyloid peptide induced toxicity in PC12 cells of EtOAc fractions were corroborated with the chemical composition. In addition, purified molecules were tested for their ORAC and neuroprotective activity. Quercetin showed the best ORAC value (33.55?mmol TE/g polyphenols); nevertheless, procyanidin dimer exhibited an exceptionally efficient neuroprotective activity (100% of viability at 50?μg/mL).

Discussion and conclusions: These findings suggest that this halophyte is a promising source of antioxidant and neuroprotective molecules for pharmaceutical purposes.     

Oxidant effects and toxicity of Croton campestris in Drosophila melanogaster

Context: Croton campestris A.St.-Hil. (Euphorbiaceae) is a species native to Northeast Brazil used by traditional communities for the treatment of a variety of health problems. However, potential toxicological effects of this plant are unknown.

Objective: The potential toxicity of the hydroalcoholic extract of C. campestris leaves on Drosophila melanogaster insect model, additionally with phytochemical constitution and cellular mechanisms mediating the action of extract were analysed in this study.

Materials and methods: Constituents of the extract were evaluated by HPLC. In vitro antioxidant potential of extract was analysed by DPPH, ABTS and FRAP. Flies injected culture medium mixed with extract (0.1–50?mg/mL) for 72?h. After, ROS production was evaluated by DCF-DA oxidation. Phosphorylation of MAPK signalling pathway was investigated by Western blotting method. Activity of antioxidant enzymes was analysed in homogenates.

Results: Major components of the extract include quercetin (38.11?±?0.06?mg/g), caffeic acid (20.06?±?0.17?mg/g) and kaempferol (15.45?±?0.05?mg/g). Consumption of the extract impaired locomotor performance and induced fly death of flies (LC₅₀ of 26.51?mg/mL). Augmented ROS formation and SOD, CAT and GST activity were observed from 0.1?mg/mL. JNK and p38 kinases phosphorylation was modulated and Paraquat-induced toxicity was augmented by extract.

Discussion and conclusion: Our data show important toxicological effects of C. campestris leading to increased mortality and impaired locomotor performance accompanied by induction of cell stress markers in flies. The study draws attention to the indiscriminate use of plant extracts.     

Screening of in vitro antimicrobial activity of plants used in traditional Indonesian medicine

Context: In many regions of Indonesia, there are numerous traditional herbal preparations for treatment of infectious diseases. However, their antimicrobial potential has been poorly studied by modern laboratory methods.

Objective: This study investigates in vitro antimicrobial activity of 49 ethanol extracts from 37 plant species used in Indonesian traditional medicine for treatment against Candida albicans, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus.

Materials and methods: The plants were collected from the Biopharmaca collection garden, Bogor, Indonesia. The plant material was dried, finely grounded, extracted using ethanol, concentrated, and the dried residue was dissolved in 100% DMSO. Antimicrobial activity was determined in terms of a minimum inhibitory concentration (MIC) using a broth microdilution method in 96-well microplates.

Results: The extract of Orthosiphon aristatus (Blume) Miq. (Lamiaceae) leaf produced the strongest antimicrobial effect, inhibiting the growth of C. albicans (MIC 128?μg/mL), S. aureus (MIC 256?μg/mL), E. faecalis (MIC 256?μg/mL) and P. aeruginosa (MIC 256?μg/mL). The leaf extract of Woodfordia floribunda Salisb. (Lythraceae) also exhibited significant effect against C. albicans (MIC 128?μg/mL), S. aureus (MIC 256?μg/mL) and E. faecalis (MIC 256?μg/mL). Rotheca serrata (L.) Steane &; Mabb. (Lamiaceae) leaf extract inhibited the growth of S. aureus (MIC 256 µg/mL) and C. albicans (MIC 256 µg/mL).

Discussion and conclusions: The leaf extract of O. aristatus and W. floribunda exhibited a significant anti-candidal effect. Therefore, both of these plants can serve as prospective source materials for the development of new anti-candidal agents.     

Phytoextract of Indian mustard seeds acts by suppressing the generation of ROS against acetaminophen-induced hepatotoxicity in HepG2 cells

Abstract

Context: Indian mustard [Brassica juncea (L.) Czern. & Coss. (Brassicaceae)] is reported to possess diverse pharmacological properties. However, limited information is available concerning its hepatoprotective activity and mechanism of action.

Objective: To study the protective mechanism of mustard seed extract against acetaminophen (APAP) toxicity in a hepatocellular carcinoma (HepG2) cell line.

Materials and methods: Hepatotoxicity models were established using APAP (2.5–22.5?mM) based on the cytotoxicity profile. An antioxidant-rich fraction from mustard seeds was extracted and evaluated for its hepatoprotective potential. The mechanism of action was elucidated using various in vitro antioxidant assays, the detection of intracellular generation of reactive oxygen species (ROS), and cell cycle analysis. The phytoconstituents isolated via HPLC-DAD were also evaluated for hepatoprotective activity.

Results: Hydromethanolic seed extract exhibited hepatoprotective activity in post- and pre-treatment models of 20?mM APAP toxicity and restored the elevated levels of liver indices to normal values (p?<?0.05). Post-treatment suppressed the generation of ROS by 58.37% and pre-treatment effectively prevented the generation of ROS by 90.5%. The mechanism of ROS suppression was further supported by antioxidant activity (IC₅₀) data from DPPH (103.37?±?4.2?µg AAE/mg), FRAP (83.26?±?1.1?µg AAE/mg), ORAC (1115?µM GAE/ml), ABTS (83.05?µg GAE/ml), and superoxide (345.22?±?5.15?µg AAE/mg) scavenging assays and by the restoration of cell cycle alterations. HPLC-DAD analysis revealed the presence quercetin, vitamin E, and catechin, which exhibited hepatoprotective activity.

Discussion and conclusions: A phytoextract of mustard seeds acts by suppressing the generation of ROS in response to APAP toxicity.     

Essential oil composition and antinociceptive activity of Thymus capitatus

Context: The essential oil (EO) from Thymus capitatus Hoff. et Link. (Lamiaceae) has been traditionally used for its medicinal properties, such as anti-inflammatory, analgesic, antioxidant and antimicrobial properties.

Objective: Characterize the constituents from T. capitatus EO and further evaluate the antinociceptive activity by in vivo and in vitro procedures.

Materials and methods: Gas chromatography–mass spectrometry was used to identify and quantify the constituents of the T. capitatus EO. The antinociceptive activity was evaluated in vivo by the glutamate-induced nociception model in male Swiss mice (25?g), at doses of 3, 6 and 12?mg/kg, 1?h before evaluation of the licking time response (0–15?min). The mechanism of T. capitatus EO (1–500?μg/mL) on the isolated nerve excitability of Wistar rat (300?g) was assessed by the single sucrose technique.

Results and discussion: The EO of T. capitatus presented 33 components, mainly monoterpenes and sesquiterpenes, carvacrol (ca. 80%) was its major constituent. T. capitatus EO induced antinociception in orally treated mice (3, 6, and 12?mg/kg) reducing the licking time from control (100.3?±?11.9?s) to 84.8?±?12.2, 62.7.6?±?9.9, and 41.5?±?12.7?s, respectively (n?=?8; p?T. capitatus EO (500?μg/mL) decreased the compound action potential amplitude (V_CAP) of about 80.0?±?4.3% from control recordings (n?=?4; p?+?channels.

Conclusions: The present study demonstrated the antinociceptive activity of Thymus capitatus essential oil, which acts via peripheral nervous excitability blockade.     

In vivo antitumour potential of camel’s milk against hepatocellular carcinoma in rats and its improvement of cisplatin renal side effects

Context: Camel milk (CM) is recommended for liver disease patients in Egypt for a strong belief that it has a curative effect.

Objective: The effect of consumption of CM with or without chemotherapeutic drug cisplatin was evaluated on induced hepatocarcinogenesis in rats.

Materials and methods: Wistar male rats (56) were divided into eight groups (7 rats each). Group I was control. Hepatocarcinogenesis was initiated by a single dose of intraperitoneal injection of diethylnitrosamine (DENA) (200?mg/kg BW) and promoted by phenobarbitone (500?ppm) in drinking water in groups V, VI, VII and VIII. Treatment started from 28th till 38th week using CM (5?mL/day) and/or cisplatin (5?mg/kg/3?weeks) in groups II, III IV, VI, VII and VIII. Biochemical analysis, lipid peroxidation and superoxide dismutase (SOD) activity in liver tissue were performed. Histopathology of liver and kidney and immunohistochemistry of placental glutathione-S-transferase (P-GST) in liver were performed and analyzed using image analysis.

Results: Albumin concentration and SOD activity were 3.13?±?0.23 and 311.45?±?41.71 in group VII (DENA &; cisplatin), whereas they were 4.3?±?0.15 and 540.5?±?29.94 in group VII (DENA, CM and cisplatin). The mean area of altered hepatocellular foci and P-GST altered foci decreased in group VI (DENA and CM) (1049.6?±?174.78 and 829.1?±?261) and group VIII (cisplatin and CM) (1615.12?±?436 and 543.9?±?127) compared to group V (DENA only) (4173.74?±?510.7 and 3169.49?±?538.61). Cisplatin caused chronic interstitial nephritis, which was slightly alleviated in group VIII (CM and cisplatin).

Conclusions: CM had an antioxidant effect and together with cisplatin managed to decrease hepatocarcinogenesis.     

Antioxidant,anticholinesterase and antibacterial activities of Stachys guyoniana and Mentha aquatica

Context: Stachys guyoniana Noë ex. Batt. and Mentha aquatica L. are two Algerian Lamiaceae used in folk medicine.

Objective: To investigate their antioxidant, anticholinesterase and antibacterial activities.

Material and methods: n-Butanol (BESG), ethyl acetate (EESG) and chloroform (CESG) extracts of S. guyoniana and methanol (MEMA) and chloroform (CEMA) aerial part extracts of M. aquatica and methanol (MERMA) and acetone (AERMA) roots extracts of M. aquatica were evaluated for their antioxidant activity by the β-carotene-linoleic acid, DPPH^? and ABTS^?+?scavenging, CUPRAC and metal chelating assays. The anticholinesterase activity was tested against AChE and BChE. The antibacterial activity was assessed by MICs determination against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella heidelberg, Klebsiella pneumoniae, Enterobacter aerogenes and Morganella morganii strains.

Results: In the β-carotene test, the CESG (IC₅₀: 2.3?±?1.27?μg/mL) exhibited the highest activity. The BESG was the best scavenger of DPPH^? (IC₅₀: 2.91?±?0.14?μg/mL). In the ABTS test, AERMA was the most active (IC₅₀: 4.21?±?0.28?μg/mL). However, with the CUPRAC, the BESG exhibited the best activity (A_0.50: 0.15?±?0.05?μg/mL) and was active in metal chelating assay with 48% inhibition at 100?μg/mL. The BESG was the best AChE inhibitor (IC₅₀: 5.78?±?0.01?μg/mL) however, the AERMA showed the highest BChE inhibitory activity (IC₅₀: 19.23?±?1.42?μg/mL). The tested extracts exhibited a good antibacterial activity.

Conclusion: This study demonstrated good antioxidant, anticholinesterase and antibacterial potential of S. guyoniana and M. aquatica, which fits in well with their use in folk medicine.     

A mechanistic evaluation of the traditional uses of Nepeta ruderalis in gastrointestinal and airway disorders

Context: Nepeta ruderalis Buch.-Ham. (Lamiaceae), locally known as Badranj Boya, is an aromatic herb used traditionally as an antispasmodic, antidiarrhoeal, and anti-asthamatic remedy.

Objective: Aqueous methanolic extract of N. ruderalis was studied to investigate its traditional uses.

Materials and methods: Study was conducted from September 2015 to February 2016. In vitro spasmolytic and broncho-relaxant activity of crude extract of N. ruderalis (whole plant) was evaluated at 0.01–10?mg/mL final bath concentration in isolated rabbit jejunum and tracheal tissues, using PowerLab data acquisition system (Transonic Systems Inc., Ithaca, NY). In vivo antidiarrhoeal activity was evaluated in castor oil-induced diarrhoeal mice at the dose of 300 and 500?mg of crude extract orally.

Results: Crude extract of N. ruderalis completely relaxed spontaneously contracting, high K⁺ (80?mM) and carbachol (1?μM) induced contracted jejunum with an EC₅₀ value of 5.85 (5.45–6.27), 4.0 (3.80–4.23) and 2.86 (2.48–3.29), similar to verapamil. Nr.Cr relaxed high K⁺ and carbachol induced contractions, at 5 and 10?mg/mL with an EC₅₀ value of 2.37 (2.11–2.67) and 3.26 (2.9–3.67), respectively, and also displaced calcium concentration–response curves toward right at 0.1 and 0.3?mg/mL. Nr.Cr exhibited antidiarrhoeal protection at a dose of 300 and 500?mg/kg, similar to verapamil, whereas no acute toxicity signs were seen up to 5?g/kg in healthy mice.

Discussion and conclusion: Results suggest the presence of spasmolytic and broncho-relaxant effects in the crude extract of N. ruderalis, possibly mediated through calcium channel-blocking activity, providing the pharmacological basis for its traditional uses in gastrointestinal and airway disorders.     

Prophylactic and curative effect of rosemary leaves extract in a bleomycin model of pulmonary fibrosis

Context: Pulmonary fibrosis is a devastating disease without effective treatment. Rosemary is appreciated since ancient times for its medicinal properties, while biomolecules originated from the plant have an antioxidant and antifibrotic effect.

Objective: The effects of Rosmarinus officinalis L. (Lamiaceae) leaves extract (RO) on bleomycin-induced lung fibrosis were investigated.

Materials and methods: Male Wistar rats were given a single dose of bleomycin (BLM, 4?mg/kg, intratracheal), while RO (75?mg/kg, intraperitoneal) was administered 3 days later and continued for 4 weeks (BLM/RO1-curative group). Alternatively, RO was administered 2 weeks before BLM and continued 15 days thereafter (BLM/RO2-prophylactic group). Antioxidant activities of RO and lung tissues were studied by standard methods. Histological staining revealed lung architecture and collagen deposition. RO was characterized for its polyphenol content and by high-performance liquid chromatography.

Results: RO polyphenol content was 60.52?mg/g of dry weight, carnosic and rosmarinic acids being major components (6.886 and 2.351?mg/g). Antioxidant effect of RO (DPPH and FRAP assay) expressed as IC₅₀ values were 2.23?μg/mL and 0.074?μg/mL, respectively. In BLM/RO1 and BLM/RO2 lung architecture was less compromised compared to BLM, which was reflected in lower fibrosis score (2.33?±?0.33 and 1.8?±?0.32 vs 3.7?±?0.3). Malondialdehyde levels were attenuated (141% and 108% vs 258% of normal value). Catalase and glutathione-S-transferase activities were normalized (103% and 117% vs 59%, 85% and 69% vs 23%, respectively).

Discussion and conclusion: RO has a protective effect against BLM-induced oxidative stress and lung fibrosis due to its phenolic compounds.     

Production of eurycomanone from cell suspension culture of Eurycoma longifolia

Context: Eurycomanone is found in the Eurycoma longifolia Jack (Simaroubaceae) tree, exhibits significant antimalarial activity, improves spermatogenesis, suppresses expression of lung cancer cell tumour markers and regulates signalling pathways involved in proliferation, cell death and inflammation.

Objectives: Establishment of cell suspension culture of E. longifolia to determine the eurycomanone accumulation during cultures.

Materials and methods: Callus of E. longifolia was cultured in MS medium supplemented with 0.8% agar, 30/L sucrose, 1.25?mg/L NAA and 1?mg/L KIN for biomass production. Cell suspension culture was established by transferring friable calli to the same medium without agar. Eurycomanone content during cell culture was determined by HPLC with a C18 column, flow rate of 0.8?mL/min, run time of 17.5?min, detector wavelength of 254?nm. The stationary phase was silica gel and the mobile phase was acetonitric:H₂O. Roots of 5 year-old trees were used as the control.

Results: The cells from 3?g of inoculum increased in biomass with a maximum value of 16?g fresh weight (0.7?g dry weight) at 14th day of culture. The cell growth then decreased from day 14 to day 20. Eurycomanone was produced during culture from the beginning to 20th day, its highest content (1.7?mg/g dry weight) also obtained at 14th day (the control is 2.1?mg/g dry weight).

Discussion and conclusions: Cell suspension culture of E. longifolia is a suitable procedure to produce eurycomanone. The yield of eurycomanone biosynthesis in 14 days-old cells are relatively high, approximately 0.8 times the control.     

In vitro cultures of Bacopa monnieri and an analysis of selected groups of biologically active metabolites in their biomass

Context: Bacopa monnieri L. Pennell (Scrophulariaceae) is one of the most important plants in the system of Indian medicine (Ayurveda).

Objective: This paper studies the optimal growth of B. monnieri for effective accumulation of metabolites. Biomass growth of this plant could be accomplished in liquid cultures on Murashige & Skoog medium.

Materials and methods: Powdered shoots of in vitro cultures of B. monnieri were extracted by methanol for indole compounds, phenolic compounds and bacosides for RP-HPLC analysis. Fatty acid analysis was performed via gas chromatography. Anti-inflammatory effect of B. monnieri extracts was evaluated in the A549 cells. COX-2 and cPGES expression was analyzed using Western blots.

Results: l-Tryptophan and serotonin were found in biomass from in vitro cultures of B. monnieri on MS medium and in biomass from the MS mediums enriched with the different additions such as of 0.1?g/L magnesium sulphate, 0.1?g/L zinc hydroaspartate, 0.1?g/L l-tryptophan, 0.25?g/L serine, 0.5?g/L serine and 0.5?mg/L anthranilic acid. The content of l-tryptophan and serotonin compounds was significant in biomass from medium with the addition of 0.1?g/L zinc hydroaspartate (0.72?mg/g dry weight and 1.19, respectively). Phenolic compounds identified in biomass from the same variants of MS medium were chlorogenic acid (ranging from 0.20 to 0.70?mg/g dry weight), neochlorogenic acid (ranging from 0.11 to 0.40?mg/g dry weight) and caffeic acid (ranging from 0.01 to 0.04?mg/g dry weight). The main group of fatty acids in biomass was saturated fatty acids (53.4%). The predominant fatty acid was palmitic acid. A significant decrease of COX-2 and cPGES expression was observed in the A549 cells activated with LPS and treated with B. monnieri extracts.

Discussion and conclusions: As far as we know, this is the first analysis of indole compounds and phenolic acids in this plant. The multi-therapeutic effect of B. monnieri is expressed by the activity of bacosides. Information about the presence of indole and phenolic compounds, and fatty acids in this plant is limited, but the content of these compounds might participate in the physiological activity of B. monnieri.     

Protective activity of Hertia cheirifolia extracts against DNA damage,lipid peroxidation and protein oxidation

Context: Hertia cheirifolia L. (Asteraceae), a perennial shrub widely distributed in Northern Africa, is traditionally used to treat inflammatory disorders.

Objective: The protective effect of methanol (Met E) and aqueous (Aq E) extracts of Hertia cheirifolia against DNA, lipid and protein oxidation was investigated.

Materials and methods: Different concentrations (50–1000?μg/mL) of Hertia cheirifolia aerial part extracts were examined against DNA, lipid and protein oxidation induced by H₂O_2?+?UV, FeSO₄, and Fe³⁺/H₂O₂-ascorbic acid, respectively. The DPPH^?, metal ion chelating, reducing power and β-carotene bleaching tests were conducted.

Results: Both extracts were rich in polyphenols, flavonoids and tannins, and were able to scavenge DPPH^? with IC₅₀ values of 138 and 197?μg/mL, respectively. At 300?μg/mL, Aq E exerted stronger chelating effect (99%) than Met E (69%). However, Met E reducing power (IC_50?=_?61?μg/mL) was more than that of Aq E (IC_50?=_?193?μg/mL). Both extracts protected from β-carotene bleaching by 74% and 94%, respectively, and inhibited linoleic acid peroxidation. The inhibitory activity of Aq E extract (64%) was twice more than that of Met E (32%). Interestingly, both extracts protected DNA against the cleavage by about 96–98%. At 1?mg/mL, Met E and Aq E restored protein band intensity by 94–99%.

Conclusions: Hertia cheirifolia exhibits potent antioxidant activity and protects biomolecules against oxidative damage; hence, it may serve as potential source of natural antioxidant for pharmaceutical applications and food preservation. This is the first report on the protective activity of this plant against biomolecule oxidation.     

Polyphenol profile by UHPLC-MS/MS,anti-glycation,antioxidant and cytotoxic activities of several samples of propolis from the northeastern semi-arid region of Brazil

Context: Propolis has promising biological activities. Propolis samples from the Northeast of Bahia, Brazil – sample A from Ribeira do Pombal and B, from Tucano – were investigated, with new information regarding their biological activities.

Objective: This paper describes the chemical profile, antioxidant, anti-glycation and cytotoxic activities of these propolis samples.

Material and methods: Ethanol extracts of these propolis samples (EEP) and their fractions were analyzed to determine total phenolic content (TPC); antioxidant capacity through DPPH^?, FRAP and lipid peroxidation; anti-glycation activity, by an in vitro glucose (10?mg/mL) bovine serum albumine (1?mg/mL) assay, during 7?d; cytotoxic activity on cancer (SF295, HCT-116, OVCAR-8, MDA-MB435, MX-1, MCF7, HL60, JURKAT, MOLT-4, K562, PC3, DU145) and normal cell lines (V79) at 0.04–25?μg/mL concentrations, for 72?h. The determination of primary phenols by ultra high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and volatile organic compounds content by gas chromatography-mass spectrometry (GC-MS) were also performed.

Results: The EEP polar fractions exhibited up to 90% protection against lipid peroxidation. The IC₅₀ value for anti-glycation activity of EEP was between 16.5 and 19.2?μg/mL, close to aminoguanidine (IC₅₀?=?7.7?μg/mL). The use of UHPLC-MS/MS and GC-MS allowed the identification of 12 bioactive phenols in the EEP and 24 volatile compounds, all already reported.

Conclusions: The samples present good antioxidant/anti-glycation/cytotoxic activities and a plethora of biologically active compounds. These results suggest a potential role of propolis in targeting ageing and diseases associated with oxidative and carbonylic stress, aggregating value to them.     

Anti-inflammatory activity of standardized dichloromethane extract of Salvia connivens on macrophages stimulated by LPS

Context: A previous study demonstrated that the chloroform extract of Salvia connivens Epling (Lamiaceae) has anti-inflammatory activity.

Objective: Identification of the active components in the dicholorometane extract (DESC), and, standardization of the extract based in ursolic acid.

Material and methods: DESC was prepared by percolation with dichlromethane and after washed with hot hexane, its composition was determined by CG-MS and NMR, and standardized by HPLC. The anti-inflammatory activity was tested on acute TPA-induced mouse ear oedema at doses of 2.0?mg/ear. The cell viability of macrophages was evaluated by MTT method, and pro- and anti-inflammatory interleukin levels were measured using an ELISA kit.

Results: Ursolic acid, oleanolic acid, dihydroursolic acid and eupatorin were identified in DESC, which was standardized based on the ursolic acid concentration (126?mg/g). The anti-inflammatory activities of DESC, the acid mixture, and eupatorin (2?mg/ear) were 60.55, 57.20 and 56.40% inhibition, respectively, on TPA-induced ear oedema. The IC₅₀ of DESC on macrophages was 149.4?μg/mL. DESC (25?μg/mL) significantly reduced TNF-α (2.0-fold), IL-1β (2.2-fold) and IL-6 (2.0-fold) in macrophages stimulated with LPS and increased the production of IL-10 (1.9-fold).

Discussion: Inflammation is a basic response to injuries, and macrophages are involved in triggering inflammation. Macrophage cells exhibit a response to LPS, inducing inflammatory mediators, and DESC inhibits the biosynthesis of the pro-inflammatory and promote anti-inflammatory cytokines.

Conclusion: DESC has an anti-inflammatory effect; reduced the levels of IL-1β, Il-6 and TNF-α; and increases IL-10 in macrophages stimulated with LPS. Ursolic acid is a good phytochemical marker.     

Sempervivum davisii: phytochemical composition,antioxidant and lipase-inhibitory activities

Context: Sempervivum davisii Muirhead (Crassulaceae) is a traditional medicinal herb from Eastern Anatolia. To date the composition of phytochemicals and physiological properties of this herb were not subjected to any research.

Objective: This study identifies compounds in S. davisii hydrophilic extracts and evaluates their potential biological properties.

Materials and methods: Ethanol-based lyophilized extracts were obtained from aerial parts of plant (10?g of ground dry plant material in 200?mL of acidified aqueous ethanol, shaken for 2?h at 22?°C with supernatant collected and freeze-dried under vacuum). Phytochemical composition was investigated by liquid chromatography mass spectrometry (LC-MS/MS, phenolics) and gas chromatography mass spectrometry (GC-MS, volatiles). Phenolic compounds were quantified by high-performance liquid chromatography (HPLC) and the Folin–Ciocalteu assay. Subsequently, antioxidant capacity [ferric reducing antioxidant power (FRAP), oxygen radical absorbance capacity (ORAC) assays] and enzyme inhibitory properties (isolated porcine pancreatic lipase) of the extracts were determined.

Results: Polyphenolic compounds were the main constituents of lyophilized extracts, among which kaempferol glycosides and quercetin hexoside dominated. The extracts exhibited potent antioxidant (FRAP values of 1925.2–5973.3?μM Fe²⁺/g DW; ORAC values of 1858.5–4208.7?μM Trolox Eq./g DW) and moderate lipase inhibitory (IC₅₀: 11.6–2.96?mg/mL) activities. Volatile compounds (nonanal, dehydroxylinalool oxide isomers, 2-decenal, 2-undecenal, 2,6-di-tetr-butylphenol) were also found.

Conclusions: Phenolic compounds with the dominating kaempferol and quercetin derivatives are the sources of potent antioxidant properties of S. davisii hydrophilic extracts. The extracts exhibit moderate inhibitory properties towards isolated pancreatic lipase.