Protective and Ameliorative Effects of Hydroethanolic Extract of Piper nigrum (L.) Stem against Antiretroviral Therapy-Induced Hepatotoxicity and Dyslipidemia in Wistar Rats.

Antiretroviral therapy (ART) has revolutionized the lives of people living with HIV/AIDS by overall improving their quality of life and increasing life expectancy. However, ART-associated hepatotoxicity and metabolic disorders in HIV/AIDS patients are growing concerns to clinicians, especially due to the long-term use of the drugs. This study reported on the phytochemical and pharmacological profile of hydroethanolic extracts of Piper nigrum stem (PNS) and evaluated its protective effect against tenofovir/lamivudine/efavirenz (TLE)-induced hepatotoxicity and dyslipidemia in Wistar rats. Cytotoxic, antioxidant, and anti-inflammatory assays were performed on PNS. Thirty-six rats divided into 6 groups of 6 animals/group were administered: distilled water, 17 mg/kg TLE, 17 mg/kg TLE and 100 mg/kg silymarin, 17 mg/kg TLE, and Piper extract (200 mg/kg, 400 mg/kg, or 800 mg/kg) orally for 28 days. The body weight of animals was recorded every 7 days. On Day 29, the rats were sacrificed, and blood samples were collected for hematological and biochemical tests. Portions of the liver and kidneys were collected for histological evaluation, while liver homogenates were prepared from the rest to measure antioxidant enzymes. PNS possessed in vitro cytotoxic, antioxidant, and anti-inflammatory activities. A significant decrease (p < 0.05) in the body weight of rats treated with PNS was observed. A significant high platelet count (p < 0.05) was observed in the PNS800 mg/kg group. A considerable decrease in alkaline phosphatase and triglycerides was observed in the silymarin and PNS group compared to the TLE-only group. The findings also show a significant increase in catalase and glutathione in the TLE-only group compared to the normal group, while SOD decreased. Histological observations revealed normal hepatic and renal tissues in the silymarin, and PNS-treated groups compared to the normal control, while leucocyte infiltration was observed in the TLE-only group. These results suggest that PNS extract possessed antioxidant activity that alleviated TLE-induced toxicity. Further studies are necessary to understand the pharmacokinetic interactions between ART and PNS.

Moringa oleifera Seed at the Interface of Food and Medicine: Effect of Extracts on Some Reproductive Parameters, Hepatic and Renal Histology.

The lipid-rich Seed of Moringa oleifera has been promoted as an effective water clarifier. Aside its vital nutritional application as an emerging food additive, the seed has continued to gain a wider acceptance in various global ethnomedicines for managing several communicable and lifestyle diseases, howbeit, its potential toxic effect, particularly on fertility and pregnancy outcomes has remained uninvestigated; the effect of Moringa oleifera seed (MOSE) aqueous-methanol extracts on fertility and pregnancy outcome, was investigated in vivo using female Wistar rats that were divided into 50, 100, 300 and 500 mg per kilogram body weight. Group six was given Moringa oleifera seed treated water ad-libitum (ad-libitum group). Organs harvested for histological assessment included ovary, uterus, liver and kidney. In addition to HPLC fingerprint and a preliminary peptide detection, we determined the physico-chemical characteristics and mineral content of MOSE using standard methods. Data were analyzed with significance at p ≤ 0.05. There was no significant difference in the estrus cycle, mating index, gestation survival index, gestation index, fertility index and sex ratio among all groups. Gestation length was reduced in some groups. While the male pup birth weight was comparable among the different groups, female pups birth weights were significantly reduced in 50 and 100 mg groups. Anogenital distance indices of female pups in ad libitum group were significantly increased. Pathologies were observed in liver and kidneys of dams while kidneys of pups presented a dose dependent reduction in the number of glomeruli. There were no observed pathological changes in the ovary and uterus. This study showed for the first time in rodents, that the lipid-rich MOSE is unsafe to the kidney of rodents while the lipid-free MOSE appears to be safe at doses up to 300 mg/kg body weight. Findings from this study suggested that the female pups were masculinized. In conclusion, the lipid-rich seed extracts of MOSE appear to be unsafe during pregnancy, induce hepatic and renal toxicity while the lipid-free MOSE excludes inherent toxicity as the hydrophobic part has been linked to toxicity as observed in this study due to the developmental programming effect on female offspring in rodents.

Bioassay-guided isolation and identification of anti-ulcer ecdysteroids from the seeds of Sphenocentrum jollyanum Pierre (Menispermaceae).

Sphenocentrum jollyanum seeds (MeOH extract and n butanol fraction) exhibited urease inhibitory activity (IC50 40.0 ± 0.92, 28.6 ± 0.41). The Ethyl acetate (EtOAc) fraction gave significant antacid activity with an increase in the baseline pH value of 1.2 to 1.61 ± 0.00 and 1.53 ± 0.00 at 50 and 100 mg, respectively, compared to the antacid activity of sodium bicarbonate (1.53 ± 0.00, 1.47 ± 0.00). Five known ecdysteroid compounds isolated from S. jollyanum ethyl acetate and n butanol fractions are Pinnatasterone (1), Polypodine B (2), 20-hydroxyecdysone (3), 20, 26-dihydroxyecdysone, (4) and Atrotosterone A (5). The compounds' structures were determined using extensive 1D and 2D NMR experiments, and the molecular mass for each of the compounds was confirmed by FAB-MS. Compounds 1-5 were evaluated for their urease inhibitory and antacid activities. Fractions were active in comparison with the standard drug acetohydroxamic acid, and sodium bicarbonate, respectively. Compounds 2, 3 and 1 showed significant urease inhibitory activity (IC50 7.0 ± 0.56, 13.8 ± 0.49 and 14.1 ± 0.59), respectively. The activity of compounds 4 and 5 were moderate compared to that of acetohydroxamic acid (IC50 value 20.3 ± 0.43). Very few compounds have been isolated from this plant despite the numerous biological activities reported for it. The antacid and urease inhibitory activities of this plant and isolated compounds are described for the first time.

Effect-directed profiling and identification of bioactive metabolites from field, in vitro-grown and acclimatized Musa spp. accessions using high-performance thin-layer chromatography-mass spectrometry.

Bananas and plantains (Musa spp.) are used as nutritious foods, and at the same time, are a source of phytoconstituents for the pharmaceutical industry. As biological activities of especially the pulp and peel of Musa spp. have been documented, this study investigated the variation in the secondary metabolite profiles of the leaves from field, in vitro-grown and acclimatized accessions. The genetic fidelity of the diverse accessions was assessed using diversity array technology sequencing. It showed that the in vitro-grown accessions were true-to-type with the field samples. The antioxidant and anticholinesterase activities of the samples from different culture systems (field and in vitro) were evaluated by UV-spectrophotometry and compared to high-performance thin-layer chromatography-effect-directed analysis (HPTLC-EDA). The latter was applied for the first time for effect-directed profiling of the polar and medium polar sample components via different biochemical and biological assays. Compound zones showed acetyl-/butylrylcholinesterase inhibition (zones 1-4), α-/ß-glucosidase inhibition (zones 1 and 2) as well as antioxidative (zones 1-3) and antimicrobial (zone 4) activities. Structures were preliminary assigned by HPTLC-HRMS. The HPTLC was effective for bioactivity-guided characterization of the bioactive constituents in Musa spp. accessions. Accumulation of useful metabolites, especially compounds with antioxidant and anticholinesterase properties, was higher in samples from in vitro system. This validated the use of plant tissue culturing as an alternative method for large scale production of plant material and supply of bioactive constituents.

Isolation of flavonoids from Musa acuminata Colla (Simili radjah, ABB) and the in vitro inhibitory effects of its leaf and fruit fractions on free radicals, acetylcholinesterase, 15-lipoxygenase, and carbohydrate hydrolyzing enzymes.

Musa species are used traditionally for the management of many diseases. The study evaluated and compared anticholinesterase, anti-inflammatory, antioxidant, and antidiabetic activities of Musa acuminata (Simili radjah, ABB) fruits and leaves fractions and characterized the bioactive compounds using HPTLC-HRMS and NMR. Leaf fractions gave the higher biological activities than the fruit. Ethyl acetate fraction of the leaf had the highest total phenolic content (911.9 ± 1.7 mg GAE/g) and highest 2,2-diphenyl-1-picrylhydrazyl (DPPH· ) scavenging activity (IC50, 9.0 ± 0.4 µg/ml). It also gave the most effective inhibition of acetylcholinesterase (IC50, 404.4 ± 8.0 µg/ml) and α-glucosidase (IC50, 4.9 ± 1.6 µg/ml), but a moderate α-amylase inhibition (IC50, 444.3 ± 4.0 µg/ml). The anti-inflammatory activity of n-butanol (IC50, 34.1 ± 2.6 µg/ml) and ethyl acetate fractions (IC50 , 43.1 ± 11.3 µg/ml) of the leaf were higher than the positive control, quercetin (IC50 , 54.8 ± 17.1 µg/ml). Kaempferol-3-O-rutinoside and quercetin-3-O-rutinoside (rutin) were identified as the bioactive compounds with antioxidant and antidiabetic activities from the ethyl acetate fraction of M. acuminata leaf. PRACTICAL APPLICATIONS: All parts of Musa acuminata are known to be useful ethnomedicinally even as food. The leaves are mostly used to serve food and used for wrapping purposes. However, this study concluded that M. acuminata leaf is rich in bioactive flavonoids such as kaempferol-3-O-rutinoside and rutin, with relatively high antioxidative, antidiabetic, and anti-inflammatory activities. Therefore, aside the fact that the leaves can serve as potential drug leads for pharmaceutical industries, it can also be embraced in the food sector to produce supplements and/or nutraceuticals in the management of Alzheimer's, diabetes and other inflammatory diseases.

Antioxidant and acetylcholinesterase inhibitory activities of leaf extract and fractions of Albizia adianthifolia (Schumach) W.F. Wright.

BACKGROUND: This study was conducted to determine the antioxidant and acetylcholinesterase inhibitory activities of the leaf extract and different fractions of Albizia adianthifolia (Schumach.) W. Wright, and also to identify and quantify some phenolic compounds in the extract. METHODS: The antioxidant and acetylcholinesterase inhibitory activities of the methanol extract (CME), ethyl acetate (EAF), chloroform (CHF), and n-hexane (NHF) fractions of A. adianthifolia leaves were subjected to in vitro chemical analyses. RESULTS: Phytochemical screening revealed important classes of secondary metabolites. All extracts showed good antioxidant and acetylcholinesterase inhibitory activities. The EAF and CHF gave the highest total phenolic contents of 18.02±0.14 and 18.43±0.95 mg gallic acid equivalent per gram of extract, respectively. The polar extracts gave the highest activity in both assays with lower IC50 values. In the 2,2-diphenyl-1-picrylhydrazyl free-radical scavenging activity assay, the IC50 values of CME, EAF, CHF, and NHF were 77.74±0.84, 55.76±2.30, 58.47±0.72, and 232.24±2.70 µg/mL, respectively, while in the acetylcholinesterase inhibitory assay, the IC50 values of CME, EAF, CHF, and NHF were 11.80±0.88, 10.04±1.67, 17.44±1.74, and 124.38±1.51 µg/mL, respectively. CONCLUSIONS: The results of this study suggest that the leaf of A. adianthifolia could serve as a potential candidate for future search of antioxidant and acetylcholinesterase inhibitors for the management of neurodegenerative disorders such as Alzheimer's disease.

Antioxidant and antimicrobial activities of solvent fractions of Vernonia cinerea (L.) Less leaf extract.

BACKGROUND: Vernonia cinerea (L.) Less is used in folk medicine as a remedy for various diseases. OBJECTIVES: The present study reports antioxidant and antimicrobial activities of solvent fractions of Vernonia cinerea. METHODS: The antioxidant properties of solvent fractions of V. cinerea were evaluated by determining radicals scavenging activity, total flavonoid and phenolic contents measured with the 2,2-diphenyl-1-picryl hydrazyl (DPPH) test, the aluminum chloride and the Folin-ciocalteau methods, respectively. Antimicrobial activities were tested against human pathogenic microorganisms using agar diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each active extract were determined. RESULTS: The ethyl acetate fraction having the IC50 value of 6.50 µg/mL demonstrated comparable DPPH radical-scavenging activity with standard antioxidants, gallic acid and quercetin included in the study. All fractions displayed moderate antimicrobial potential against the tested pathogens with the zone of inhibition that ranged from 9.0 to 13.5 mm. The MIC (1.56 mg/mL) and MBC (3.13 mg/mL) indicated highest susceptibility of Candida albicans in all fractions. CONCLUSION: The results of this study showed that the solvent fractions of V. cinerea possess antioxidant and antimicrobial activities, hence justifying the folkloric use of the plant for the treatment of various ailments in traditional medicine.

Ethnobotanical survey of Rinorea dentata (Violaceae) used in South-Western Nigerian ethnomedicine and detection of cyclotides.

ETHNOPHARMACOLOGICAL RELEVANCE: People living in the tropical rain forest of South-Western Nigeria use Rinorea dentata (P. Beauv.) Kuntze (Violaceae) in ethno-veterinary medicine to facilitate parturition. There are no evidence-based pharmacological investigations for the uterotonic activity of this plant. AIMS OF STUDY: (i) Collection of data about the ethnopharmacological uses of R. dentata and evaluation of its uses and applications in health care; (ii) determining potential uterotonic effects in vitro, and (iii) chemical characterization of R. dentata, which is a member of the Violaceae family known to express circular cystine-knot peptides, called cyclotides. MATERIALS AND METHODS: The ethnopharmacological use of R. dentata in settlement camps within the area J4 of Omo forest has been investigated by semi-structured questionnaires and open interviews. Use index analysis has been performed by seven quantitative statistical models. Respondents' claim on the beneficial ethno-veterinary application of the plant to aid parturition has been investigated in vitro by myometrial contractility organ bath assays. The bioactive plant extract was screened by chemical derivatization and mass spectrometry-based peptidomics using reversed-phase HPLC fractionation and MALDI-TOF/TOF analysis. RESULTS: Based on the survey analysis, medicinal preparations of R. dentata have been used for anti-microbial and anti-malaria purpose in humans, and for aiding parturition in farm animals. The latter application was mentioned by one out of six respondents who claimed to use this plant for any medicinal purpose. The plant extract exhibited a weak uterotonic effect using organ bath studies. The plant contains cyclotides and the peptide riden A has been identified by de novo amino acid sequencing using mass spectrometry. CONCLUSION: Few dwellers around the settlement camps of the tropical forest of Omo (Nigeria) use R. dentata for various health problems in traditional veterinary and human medicine. The weak uterotonic effect of the cyclotide-rich extract is in agreement with the low use value index obtained for this plant. Cyclotides have been reported in the genus Rinorea confirming the ubiquitous expression of these stable bioactive plant peptides within the family of Violaceae.

A pharmacobotanical study of two medicinal species of Fabaceae.

OBJECTIVE: To carry out a pharmacobotanical study of Lonchocarpus cyanescens (Schum & Thonn) Benth (L. cyanescens) and Leptoderris micrantha Dunn (L. micrantha) which are two key medicinal plants from the family Fabaceae. METHODS: The epidermal peel was obtained by soaking the leaf in concentrated nitric acid (HNO3) in a petri dish. Both surfaces were carefully mounted on clean glass slides and dehydrated by ethyl alcohol, and stained with safaranin O for 2 min. Transverse sections of plant leaf were obtained by free hand sectioning. Phytochemical screening for various constituents was carried out on the powdered leaves. Other parameters such as, moisture content, ash value, acid insoluble ash, water-soluble ash, water and alcohol extractive values were obtained by standard techniques. RESULTS: THE DISTINCTIVE FEATURES OF THE SPECIES INCLUDE: the presence of stomata on both surfaces of L. cyanescens and the absence in L. micrantha. Presence of larger epidermal cells in both upper and lower surfaces of L. cyanescens [(35.25±1.64)×(31.25±2.36), (43.0±2.63)×(39.5±5.11)] respectively compared to L. micrantha. Glandular multicellular trichomes are present in L. micrantha but absent in L. cyanescens. Numerous trichomes surround the transverse section of the leaf of L. micrantha but absent in L. cyanescens. Preliminary phytochemical screening showed that both species contain secondary metabolites such as alkaloids, anthraquinones, cardiac glycosides, tannins, saponins, steroids and flavonoids. CONCLUSIONS: The microscopic and phytochemical data provided in this study are useful for the standardization of the medicinal plants.

Comparative micromorphological study of wild and micropropagated Dioscorea bulbifera Linn.

OBJECTIVE: To study the leaf epidermis of wild and micropropagated Dioscorea bulbifera Linn. (D. bulbifera) in order to document useful diagnostic features that may be employed for correct crude drug identification and to clear any taxonomic uncertainties in the micropropagated medicinal plant. METHODS: Growth responses of micropropagated D. bulbifera were observed on Murashige Skoog medium supplemented with 6-benzylamino purine (1.0 mg/L)+α-naphthaleneacetic acid (0.2 mg/L)+cysteine (20 mg/L) using nodal segments as explants. Leaves of the wild and micropropagated plants were studied microscopically. RESULTS: More than 80% shoot regeneration and formation of 10%-30% whitish-brown callus were observed within 3 weeks. The highest root proliferation was obtained from Murashige Skoog medium of 6-benzylamino purine (0.05 mg/L) and α-naphthaleneacetic acid (0.01 mg/L) with mean root length of (27.00±1.25) mm and elongated single shoot of mean length (38.00±11.09) mm. Leaf epidermal features that revealed similarities between the wild and micropropagated plants included amphistomatic condition, presence of mucilage, glandular unicellular trichome with multicellular head, polygonal cells with smooth walls, stomata type and shape. Slight variations included thick cuticular wall with closed stomata in wild plant compared to thin walled opened stomata in the in vitro plant. Opening of stomata accounted for larger average stomata sizes of (7.68±0.38) µm and (6.14±0.46) µm on the adaxial and abaxial surfaces, respectively of the micropropagated plant compared to the wild. CONCLUSIONS: The diagnostic features obtained in the study could serve as a basis for proper identification for quality control for standardization of the medicinal plant.

Antipsychotic property of solvent-partitioned fractions of Lonchocarpus cyanescens leaf extract in mice.

BACKGROUND: This study was carried out to evaluate the antipsychotic property of solvent-partitioned fractions of the leaf extract of Lonchocarpus cyanescens (LC), a reputable medicinal plant used in folk medicine for the treatment of mental illnesses in Nigeria. METHODS: The n-hexane fraction, ethyl acetate fraction (EAF), and aqueous ethanol fraction (AEF) of LC were tested for antipsychotic property based on the antagonism of stereotypy induced by apomorphine (APO). Antagonism of hyperactivity and lethality in aggregated mice induced by amphetamine (AMPH) were further employed for screening the antipsychotic effect of the fractions. EAF was tested for catalepsy utilising the horizontal plane paradigm. Thin-layer chromatography (TLC) was used to screen EAF for the presence of secondary metabolites. RESULTS: AEF (100-400 mg/kg) significantly (p<0.05) suppressed stereotypy induced by APO (2 mg/kg, intraperitoneal [IP]) in comparison with control, suggesting antipsychotic activity. However, EAF (200 mg/kg, IP) was most potent in inhibiting the stereotypic effect of APO. EAF was also the most active in antagonising AMPH-induced hyperactivity and in protecting against death caused by AMPH in grouped mice. However, in contrast to haloperidol, EAF did not produce cataleptic behaviour in the horizontal plane paradigm. The TLC analysis revealed that EAF contains several compounds, with some of them having Rf values similar to that of haloperidol, which suggests the presence of active substances with the same chemical structural identity. CONCLUSIONS: These findings suggest that EAF contains the major active constituent(s) mediating the antipsychotic property of LC and further support its use for the management of psychosis in traditional medicine.

Cyclotide discovery in Gentianales revisited--identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants.

Cyclotides are a unique class of ribosomally synthesized cysteine-rich miniproteins characterized by a head-to-tail cyclized backbone and three conserved disulfide-bonds in a knotted arrangement. Originally they were discovered in the coffee-family plant Oldenlandia affinis (Rubiaceae) and have since been identified in several species of the violet, cucurbit, pea, potato, and grass families. However, the identification of novel cyclotide-containing plant species still is a major challenge due to the lack of a rapid and accurate analytical workflow in particular for large sampling numbers. As a consequence, their phylogeny in the plant kingdom remains unclear. To gain further insight into the distribution and evolution of plant cyclotides, we analyzed ∼300 species of >40 different families, with special emphasis on plants from the order Gentianales. For this purpose, we have developed a refined screening methodology combining chemical analysis of plant extracts and bioinformatic analysis of transcript databases. Using mass spectrometry and transcriptome-mining, we identified nine novel cyclotide-containing species and their related cyclotide precursor genes in the tribe Palicoureeae. The characterization of novel peptide sequences underlines the high variability and plasticity of the cyclotide framework, and a comparison of novel precursor proteins from Carapichea ipecacuanha illustrated their typical cyclotide gene architectures. Phylogenetic analysis of their distribution within the Psychotria alliance revealed cyclotides to be restricted to Palicourea, Margaritopsis, Notopleura, Carapichea, Chassalia, and Geophila. In line with previous reports, our findings confirm cyclotides to be one of the largest peptide families within the plant kingdom and suggest that their total number may exceed tens of thousands.

Uterine contractility of plants used to facilitate childbirth in Nigerian ethnomedicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Pregnant women in Nigeria use plant preparations to facilitate childbirth and to reduce associated pain. The rationale for this is not known and requires pharmacological validation. AIM OF STUDY: Obtain primary information regarding the traditional use of plants and analyze their uterine contractility at cellular level. MATERIALS AND METHODS: Semi-structured, open interviews using questionnaires of traditional healthcare professionals and other informants triggered the collection and identification of medicinal plant species. The relative traditional importance of each medicinal plant was determined by its use-mention index. Extracts of these plants were analyzed for their uterotonic properties on an in vitro human uterine cell collagen model. RESULT: The plants Calotropis procera, Commelina africana, Duranta repens, Hyptis suaveolens, Ocimum gratissimum, Saba comorensis, Sclerocarya birrea, Sida corymbosa and Vernonia amygdalina were documented and characterized. Aqueous extracts from these nine plants induced significant sustained increases in human myometrial smooth muscle cell contractility, with varying efficiencies, depending upon time and dose of exposure. CONCLUSION: The folkloric use of several plant species during childbirth in Nigeria has been validated. Seven plants were for the first time characterized to have contractile properties on uterine myometrial cells. The results serve as ideal starting points in the search for safe, longer lasting, effective and tolerable uterotonic drug leads.

Antipsychotic property of aqueous and ethanolic extracts of Lonchocarpus cyanescens (Schumach and Thonn.) Benth. (Fabaceae) in rodents.

Lonchocarpus cyanescens (LC) is a medicinal plant commonly used in combination with other recipes in the treatment of psychotic disorders in traditional medicine. This study was designed to examine whether the aqueous and ethanolic extracts of LC possess antipsychotic property in rats. The antipsychotic effects of the extracts were assessed using the amphetamine animal model of psychosis in rats. The effect of the extracts on spontaneous motor activity was also studied in the open field test in mice. The extrapyramidal side effect of catalepsy was tested based on the ability of the extracts to alter the duration of akinesia in mice placed on a vertical wrapped string. Aqueous and ethanolic extracts of LC (25-400 mg/kg, i.p.) significantly (p < 0.05) suppressed stereotyped behaviour induced by amphetamine (10.0 mg/kg, i.p.) in rats, which suggest antipsychotic activity. The extracts (25-400 mg/kg, i.p.) further produced a significant (p < 0.05) reduction in spontaneous motor activity of the animals in the open field test. However, in contrast to chlorpromazine, a typical antipsychotic, the extracts did not induce cataleptic behaviour in the animals. Preliminary phytochemical screening showed the presence of alkaloids, anthraquinones, cardiac glycosides, cyanogenetic glycosides, flavonoids, saponins, steroids and tannins in the leaves of LC. The presence of these secondary metabolites was confirmed by thin-layer chromatography. Taken together, these findings suggest that the extracts possess phytochemically active constituents with antipsychotic property. Thus, this investigation provides evidence that may justify the ethnomedicinal applications of Lonchocarpus cyanescens as the major constituent of the recipe used for the management of psychosis in Nigeria.