Comparative assessment of hepatoprotective properties of Artesunate and flavonoids from Artemisia annua on acetaminophen and carbon tetrachloride-induced cytotoxicity in primary mice hepatocytes.

Background: Artesunate (ART) is a semi-synthetized molecule from Artemisinin, an active compound isolated from the medicinal plant Artemisia annua, widely used for the treatment of malaria. Previous studies reported that ART may exert a dual effect on the liver. Accordingly, this study investigated the potential protective action of ART against Acetaminophen (APAP) and Carbon tetrachloride (CCl4)-induced hepatotoxicity in primary mice hepatocytes, in comparison to that of flavonoid extracted from A. annua (FAA). In addition, the antioxidant properties of FAA were also assessed. Methods: The antioxidant activities of FAA and Ascorbic acid (ASC) (0.01-100 µg/mL) were assessed through inhibition of lipid peroxidation, reduction of ferric and phosphomolydenum, and hydroxyl and DPPH radicals scavenging assays. The hepatoprotective effects of FAA and ART (0.1-100 µg/mL) were evaluated against APAP (11 mM) or CCl4 (4 mM) induced oxidative damage in primary mouse hepatocytes. Biochemical parameters associated with hepatotoxicity assessed include cell viability, cell membrane integrity, cellular glutathione, and antioxidant enzyme activities. Results: The obtained finding revealed FAA displayed a remarkable antioxidant activities as evidenced by the low IC50/EC50 values (3.85-19.32 µg/mL), comparable to that of ASC (3.26-18.04 µg/mL). When tested at 10 µg/mL, both FAA and ART significantly (p˂0.05) preserved cell viability, inhibited alanine aminotransferase leakage and lipid membrane peroxidation, and restored superoxide dismutase and catalase activities and glutathione content induced by APAP or CCl4 in a similar way as Silymarin. However, ART showed a significant (p˂0.05) cytotoxic effect on hepatocytes at 100 and 1000 µg/mL and did not confer obvious protection at 100 µg/mL. Conclusion: Overall, our data demonstrated that ART harms mice hepatocytes at high concentration while conferring relative protection against APAP and CCl4-hepatotoxicity at low concentration. In contrast, FAA effectively protects liver cells without cytotoxicity effect, event at 100 µg/mL. Accordingly, ART should be given to the patient only under a medical prescription.

Hydro-ethanolic extract of Khaya grandifoliola attenuates heavy metals-induced hepato-renal injury in rats by reducing oxidative stress and metals-bioaccumulation.

People living in developing countries are exposed to hepato-renal injuries induced by heavy metals like lead (Pb), cadmium (Cd), and mercury (Hg) since drinking water supplied is often polluted with a high concentration of those metals. Accordingly, it is necessary to search for antidotes against heavy metals poisoning. Hence, medicinal plants bearing anti-hepatotoxic properties represent a credible option; and such plant is Khaya grandifoliola. However, there is a paucity of knowledge regarding its protective effect on heavy metals-induced hepato-renal toxicity. Thus, this study was designed to assess the protective effect of the hydro-ethanolic stem bark extract of K. grandifoliola (HKG) against hepato-renal injuries induced by chronic consumption of drinking water containing high contents of Pb, Cd, and Hg; in addition to the investigation of the chemical antioxidant properties of HKG. For the antioxidant assays, HKG was tested as a potential inhibitor of lipid peroxidation, reducer of ferric and phosphomolybdenum, and scavenger of hydroxyl and 2,2-Diphenyl-Picryl-Hydrazyl radicals. Its protective effects were evaluated by daily co-treating rats with heavy metals solution (10 mL/kg b.w) containing 0.9, 0.58, and 1.13 ppm respectively for Pb, Cd and Hg and HKG (25 or 100 mg/kg b.w) for five consecutive months; and biochemical parameters associated to liver and kidneys functions, oxidative stress and metals bioaccumulation were assessed. HKG displayed a strong antioxidant capacity (IC50/EC50 range 3.95-17.17 µg/mL) correlated to its polyphenols content and comparable to that of Ascorbic acid. Serum levels of alkaline phosphatase, alanine/aspartate aminotransferase, and creatinine; renal and hepatic content of Cd and Pb, malondialdehyde and glutathione, activities of superoxide dismutase and catalase showed the protective effect of HKG, further evidenced by histopathological analysis. Taking together, these results demonstrated that HKG alleviates heavy metals-induced hepato-renal injuries in rats by reducing oxidative stress and metals-bioaccumulation.

Natural compounds flavonoids as modulators of inflammasomes in chronic diseases.

The use of dietary or medicinal plant based natural compounds to disease treatment has become a unique trend in clinical research. Flavonoids, a group of polyphenolic compounds have drawn significant attention due to their modulatory effects on inflammasomes associated with the initiation and progression of chronic disorders including metabolic, neurodegenerative diseases and cancer. In this article, the role of most commonly studied natural flavonoids with their disease-specific impact via inflammasomes as a potential molecular target has been described. Since the role of inflammation is evident in multiple diseases, flavonoids may serve as a promising tool in drug discovery for the intervention of chronic diseases by manipulating the status of inflammation via inflammasome targeting.

Protective Effects of Crude Plant Extracts against Aminochrome-induced toxicity in Human Astrocytoma Cells: Implications for Parkinson's Disease.

BACKGROUND/AIMS: Aminochrome, an endogenous compound formed during dopamine oxidation can induce neurotoxicity under certain aberrant conditions and induce Parkinson-like syndrome. Glutathione transferase M2 (GSTM2) activity of astrocytes by catalysing the conjugation of aminochrome with glutathione, can offer protection against aminochrome toxicity. Some medicinal toxicity through this plants may exert protective effect against aminochrome mechanism. METHODS: In the present study, extracts from plants native to Cameroon, such as Alchornea laxiflora (leaves), Dacryodes edulis (barks), Annona muricata (seeds), Annona senegalensis (barks) were evaluated for their protection against aminochrome-induced toxicity in human glioblastoma/ astrocytoma U373MG wild type and U373MGsiGT6 cells in which GSTM2 expression was 74% silenced. The cells were pre-incubated with the plant extracts for 2 hr before addition of aminochrome (75 µM) and measurement of cell death/viability by flow cytometry after 24 hr incubation. RESULTS: The extract of A. laxiflora (1 µg/ml), D. edulis (25 µg/ml), A. muricata (25 µg/ml) and A. senegalensis (25µg/ml) significantly decreased aminochrome-induced toxicity in U373siGST6 and U373MG cells. However, only A. laxiflora and A. muricata significantly increased the mitochondria membrane potential in U373siGST6 cells following aminochrome treatment. CONCLUSION: The results indicate that extracts of some Cameroon plants can provide protection against aminochrome-induced toxicity and mitochondria dysfunction in human glioblastoma/astrocytoma cells. Although further identification of active components of these extracts is needed, potential usefulness of these compounds in Parkinson's disease may be suggested.

Active chemical fractions of stem bark extract of Khaya grandifoliola C.DC and Entada africana Guill. et Perr. synergistically protect primary rat hepatocytes against paracetamol-induced damage.

BACKGROUND: Khaya grandifoliola (Meliaceae) and Entada africana (Fabaceae) are traditionally used in Bamun (a western tribe of Cameroon) traditional medicine for the treatment of liver related diseases. In this study, the synergistic hepatoprotective effect of respective active fractions of the plants were investigated against paracetamol-induced toxicity in primary cultures of rat hepatocytes. METHODS: Paracetamol conferred hepatocyte toxicity, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, alanine aminotransferase (ALT), superoxide dismutase (SOD), catalase (CAT) activities, malondialdehyde (MDA) and glutathione (GSH) content assays. The crude extracts were fractionated by flash chromatography and fractions were tested for hepato-(protective and curative) activities. The most active fractions of both plants were tested individually, and in combination based on their respective half effective concentration (EC50). RESULTS: The methylene chloride/methanol fractions of K. grandifoliola (75:25 v/v) (KgF25) and E. africana (90:10 v/v) (EaF10) were found to be the most hepato-protective with EC50 values of 10.30 ± 1.66 µg/ml and 13.47 ± 2.06 µg/ml respectively, comparable with that of silymarin (13.71 ± 3.87 µg/ml). These fractions and their combination significantly (P <0.05) improved cell viability, inhibited ALT leakage and MDA formation, and restored cellular CAT, SOD activities and GSH content. The combination was more effective in restoring biochemical parameters with coefficients of drugs interaction (CDI) less than 1. CONCLUSION: These findings demonstrate that the active fractions have synergistic action in the protection of rat hepatocytes against paracetamol-induced damage and suggest that their hepatoprotective properties may be maximized by using them in combination.

Khaya grandifoliola C.DC: a potential source of active ingredients against hepatitis C virus in vitro.

In this study, we examined the antiviral properties of Khaya grandifoliola C.DC (Meliaceae) on the hepatitis C virus (HCV) life cycle in vitro and identified some of the chemical constituents contained in the fraction with the most antiviral activity. Dried bark powder was extracted by maceration in a methylene chloride/methanol (MCM) system (50:50; v/v) and separated on silica gel by flash chromatography. Infection and replication rates in Huh-7 cells were investigated by luciferase reporter assay and indirect immunofluorescence assay using subgenomic replicons, HCV pseudotyped particles, and cell-culture-derived HCV (HCVcc), respectively. Cell viability was assessed by MTT assay, and cellular gene expression was analysed by qRT-PCR. The chemical composition of the fraction with the most antiviral activity was analysed by coupled gas chromatography and mass spectrometry (GC-MS). Five fractions of different polarities (F0-F100) were obtained from the MCM extract. One fraction (KgF25) showed the strongest antiviral effect on LucUbiNeoET replicons at nontoxic concentrations. Tested at 100 µg/mL, KgF25 had a high inhibitory effect on HCV replication, comparable to that of 0.01 µM daclatasvir or 1 µM telaprevir. This fraction also inhibited HCVcc infection by mostly targeting the entry step. KgF25 inhibited HCV entry in a pan-genotypic manner by directly inactivating free viral particles. Its antiviral effects were mediated by the transcriptional upregulation of the haem oxygenase-1 gene and interferon antiviral response. Three constituents, namely, benzene, 1,1'-(oxydiethylidene)bis (1), carbamic acid, (4-methylphenyl)-, 1-phenyl (2), and 6-phenyl, 4-(1'-oxyethylphenyl) hexene (3), were identified from the active fraction KgF25 by GC-MS. Khaya grandifoliola contains ingredients capable of acting on different steps of the HCV life cycle.

Hepatitis C in Cameroon: What is the progress from 2001 to 2016?

Chronic hepatitis C is a major public health problem in sub-Saharan countries and particularly in Cameroon where the prevalence rate is around 7.6% in the age group of 55-59 years. Recent investigations into this infection allowed defining a national seroprevalence, characterizing virological and biological profiles of infected patients and identifying medicinal plants of potential interest in hepatitis C therapy. However, in Cameroon, no existing report currently presents a good overview of hepatitis C research in relation to these parameters. This review seeks to discuss major findings published since 2001 that have significantly advanced our understanding of the epidemiology and treatment of hepatitis C in Cameroonian patients and highlight the major challenges that remain to overcome. We performed a systematic search in Pubmed and Google Scholar. Studies evaluating prevalence, treatment, coinfection, and genetic diversity of HCV infection in Cameroon were included. Studies suggest that HCV prevalence in Cameroon would be low (around 1.1%) with a lot of disparities according to regions and age of participants. Elders, pregnant women, blood donors, health care workers, patients on hemodialysis, and homozygous sickle cell patients have been identified as risk groups. Moreover, HCV/HBV coinfection was found more prevalent than HCV/HIV coinfection. Phylogenic studies reported circulation of three main genotypes such genotypes 1, 2, and 4 but little is known about antiviral candidates from the Cameroonian pharmacopeia. In conclusion, some epidemiological data prove that hepatitis C in Cameroon is well known but efforts are still necessary to prevent or control this infection.

Plant extracts from Cameroonian medicinal plants strongly inhibit hepatitis C virus infection in vitro.

According to some recent studies, Cameroon is one of the sub-Saharan African countries most affected by hepatitis C, with low access to the standard therapy based on the combination of pegylated interferon and ribavirin. A first ethnobotanical survey, conducted in the Western region of Cameroon, reported the use of several medicinal plants in traditional medicine for the healing of liver-related disorders. Crude organic extracts of five plants surveyed were prepared and their effect against hepatitis C virus (HCV) infection investigated. The HCV JFH1 strain cell culture system HCVcc was used. The antiviral activity was quantified by immunofluorescent labeling of HCV E1 envelope protein at 30 h post-infection in the presence of the plant extracts. Active compounds were then tested in time course infection experiments. Dose-response and cellular toxicity assays were also determined. Three extracts, methanol extracts from roots of Trichilia dregeana, stems of Detarium microcarpum and leaves of Phragmanthera capitata, showed anti-HCV activity, with half-maximal inhibitory concentration of 16.16, 1.42, and 13.17 µg/mL, respectively. Huh-7 cells were incubated with the extracts for 72 h and it appears that T. dregeana extract is not toxic up to 200 µg/mL, D. microcarpum up to 100 µg/mL and P. capitata up to 800 µg/mL. All the three extracts showed a strong inhibition of HCV entry and no effect on replication or secretion. Taken together, these results showed that extracts from Cameroonian medicinal plants are promising sources of anti-HCV agents.

Anticancer and antioxidant activities of methanol extracts and fractions of some Cameroonian medicinal plants.

OBJECTIVE: To obtain a scientific basis of the use of plant-derived preparations by many rural people in Cameroon, for their primary health care needs in the treatment of diseases such as cancer. METHODS: The antiproliferative effect of 11 plants methanol crude extracts on four cancer cells using sulforhodamine-B assay and their antioxidant activities using 1-diphenyl-2-picrylhydrazyl radical and nitric oxide radical scavenging ability were investigated. The Ekebergia senegalensis (E. senegalensis) and Protea elliotii (P. elliotii) extracts were selected based on their antioxidant and anticancer activities, and partition in hexane, dichloromethane, ethyl acetate, butanol and methanol was done. Each fraction was submitted to antioxidant and anticancer activities, and the effect of the dichloromethane fraction (the most antiproliferative fraction) on NCI-H460 cell cycle was determined by flow cytometry. RESULTS: The most antiproliferative substances were found for the extracts from E. senegalensis, P. elliotii, Terminalia macroptera and Vitellaria paradoxa. Whereas the most antioxidant substances were found for the extracts from Cissus populnea, E. senegalensis, P. elliotii, Terminalia macroptera, Vitellaria paradoxa, and Gardenia aqualla. Dichloromethane fraction of P. elliotii was found to be highly antiproliferative to NCI-H460 cancer cells and showed S phase arrest cell cycle progression. Ethyl acetate n-butanol and methanol fractions showed quite strong antioxidant activity for both E. senegalensis and P. elliotii, as compared to that of gallic acid. CONCLUSIONS: Overall, the antiproliferative and antioxidant activities of some of the extracts lend some support to their use in the traditional medicine of Adamawa Region, Cameroon to treat cancer.

Anti-hepatitis C virus activity of crude extract and fractions of Entada africana in genotype 1b replicon systems.

Entada africana (Ea) is a medicinal plant from the family of Fabaceae, used in Western and Central Africa regions to treat liver diseases. Antiviral properties of this plant were reported against Hepatitis B virus, while effects against Hepatitis C virus (HCV) remained unknown. This study reports for the first time, the effects of Ea crude extract and fractions on HCV replication. Furthermore, the effect of one Ea fraction on the transcriptional expression of two interferon-stimulated genes (ISGs) was also investigated. A methylene chloride-methanol (MCM) stem bark crude extract and different MCM fractions (EaF0, EaF5, EaF10, EaF25, and EaF100) were prepared and tested on LucUbiNeo-ET and Huh 5.15 cells lines used as genotype 1b (GT1b) replicon systems. The cells were incubated with crude extract and fractions at various concentrations. Then, the antiviral activity was assessed by luciferase reporter assay and the cell viability by MTT assay. Gene expression was also analyzed using quantitative real time RT-PCR. Results showed that the Ea crude extract dose-dependently inhibited HCV replication after 24 and 72 h of incubation. The MCM fraction (EaF10) exhibited the strongest anti-HCV properties with an IC50 = 0.453 ± 0.00117 mg/ml and no reduction of cell viability at antiviral concentrations. This fraction also significantly induced the expression of heme oxygenase-1 (HO-1) (5.36-fold), and 2'-5' oligoadenylate synthetase-3 (OAS-3) by 4.46-fold after 6 h and 2.31-fold after 24 h at the mRNA levels. Taken altogether, these results suggest that Ea may contain ingredients that indirectly regulate HCV replication.

Protective effect of Basella alba and Carpolobia alba extracts against maneb-induced male infertility.

CONTEXT: Male infertility is one of the leading causes of social frustration and marginalization, mainly in the developing world. It is attributed to many factors including exposure to agropesticides such as manganese ethylenebis (dithiocarbamate) (maneb), which is one of the most frequently used fungicides in Cameroon. Previous reports support efficiency of some medicinal plants commonly used in Cameroonian folk medicine for the treatment of this disorder. OBJECTIVE: The present study was aimed at assessing the protective effect of extracts from selected plant species, namely Basella alba L. (Basellaceae) (MEBa) and Carpolobia alba G. Don (Polygalaceae) (AECa), in alleviating the maneb-induced impairment of male reproductive function in Wistar albino rats. MATERIALS AND METHODS: The rats were treated with vehicle, plant extract (MEBa or AECa), maneb and maneb plus plant extract, respectively, and their fertility was assessed. Animals were thereafter sacrificed and organs (liver, kidneys and reproductive organs) were dissected out and weighed. Serum androgens together with alanine aminotransferase, liver glutathione and thiobarbituric acid reactive species (TBARS) were also measured. RESULTS AND DISCUSSION: From this study, both plant extracts stimulated testosterone and improved fertility. Administration of MEBa plus maneb prevented fertility reduction by maneb and minimized the inhibitory effect of maneb on testosterone levels. AECa also improved fertility of the maneb-exposed rats, though without restoring testosterone levels, and other investigated parameters remained unaffected by different treatments. CONCLUSION: These findings emphasized the beneficial effects of B. alba and C. alba extracts on male fertility, and suggest their protective effect against maneb-induced toxicity in male reproductive function.

Entada africana fraction CH2Cl2/MEOH 5% inhibits inducible nitric oxide synthase and pro-inflammatory cytokines gene expression induced by lipopolysaccharide in microglia.

BACKGROUND: Inflammatory response in the CNS mediated by microglia cells play an important role in host defense and is implicated in the pathology of neurodegenerative diseases. We investigated the capacity of Entada africana to protect microglia from inflammatory insults by exploring the effect of the CH2Cl2/MEOH 5% fraction (Ea5) on pro-inflammatory cytokines mRNA expression. Finally, we studied the effect of Ea5 on the inhibition of p38 MAPK Kinase. The results were compared to those obtained with Baicalin, a well reported anti-inflammatory flavonoid. METHODS: Barks from E. africana were harvested in 2010, in the west region of Cameroon. A crude extract was prepared using CH2Cl2/MEOH 1:1 V/V. The crude extract obtained was further fractionated by flash chromatography. A mouse microglia cell line (N9) was stimulated by LPS with or without different concentrations of Baicalin and Ea5. The release of NO was evaluated using the Griess method. The expression of pro-inflammatory cytokines mRNA (TNFα, IL-1ß, IL-6) and iNOS/NO were measured by RT- PCR. The inhibition of p38 MAPK Kinase was assessed using ELISA. RESULTS: We found that Ea5, as well as Baicalin inhibited LPS-induced NO production in a dose dependent manner. Ea5 was most active in term of NO inhibition (87.07%), in comparison to Baicalin (70.85%). The expression of TNFα, IL-1ß, IL-6 and iNOS was strongly suppressed by Ea5 in microglia. Ea5 also inhibited the activity of p38MAPK Kinase, up to 30% for the concentrations tested, whereas a prominent inhibition was obtained with Baicalin. CONCLUSION: These results suggest that E. africana may contain promising compounds useful for the treatment of diseases cause by over-activation of microglia such as Alzheimer disease and other neurological diseases.

A fraction of stem bark extract of Entada africana suppresses lipopolysaccharide-induced inflammation in RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Entada africana is a plant used in African traditional medicine for the treatment of stomachache, fever, liver related diseases, wound healing, cataract and dysentery. AIMS OF THE STUDY: This study aimed at evaluating the anti-inflammatory activity of fractions of the stem bark extract of the plant using lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages model. MATERIALS AND METHODS: The crude extract was prepared using the mixture CH2Cl2/MeOH (1:1, v/v) and fractionated by flash chromatography using solvents of increasing polarity to obtain five different fractions. The effects of the fractions on the cells viability were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and their inhibitory activity against LPS-induced nitric oxide (NO) production screened by Griess test. The most active fraction was further investigated for its effects on reactive oxygen species (ROS) production using flux cytometry, the expression of inducible nitric oxide synthase (iNOS), pro-and anti-inflammatory cytokines (IL1ß, TNFα, IL6, IL10 and IL13) by RT-PCR, and the activity of the enzyme p38 MAPK kinase by enzyme-linked immunosorbent assay (ELISA). RESULTS: The fractions presented no significant effect on the viability of macrophages at 100 µg/ml after 24h incubation. The CH2Cl2/MeOH 5% (Ea5) fraction was found to be the most potent in inhibiting NO production with a half inhibition concentration (IC50)=18.36 µg/ml, and showed the highest inhibition percentage (89.068%) in comparison with Baicalin (63.34%), an external standard at 50 µg/ml. Ea5, as well as Baicalin significantly (P<0.05) inhibited the expression of TNFα, IL6 and IL1ß mRNA, attenuated mRNA expression of inducible NO synthase in a concentration-dependent manner, stimulated the expression of anti-inflammatory cytokines (IL10 and IL13), and showed a 30% inhibition of the activity of p38 MAPK kinase. CONCLUSION: The results of the present study indicate that the fraction Ea5 of Entada africana possesses most potent in vitro anti-inflammatory activity and may contain compounds useful as a therapeutic agent in the treatment of inflammatory related diseases cause by over-activation of macrophages.

Effects of the methanol extract of Basella alba L (Basellaceae) on steroid production in Leydig cells.

In this study, Leydig cells were purified from 70 day-old Sprague Dawley male rats and incubated with 10 and 100 µg/mL of methanol extract of Basella alba (MEBa) for 4 hours followed by the evaluation of cell viability, steroid (testosterone and estradiol) production, and the level of aromatase mRNA. Results showed that MEBa did not affect Leydig cell viability. At the concentration of 10 µg/mL, MEBa significantly stimulated testosterone and estradiol production (p < 0.01 and p < 0.03, respectively), and enhanced aromatase mRNA level (p < 0.04). These observations suggest that MEBa directly stimulated testosterone, estradiol and aromatase mRNA levels in isolated Leydig cells.