Protective Potential of Methanol Extract of Drymaria cordata Willd. ex Schult (MEDC) on Letrozole-Induced Polycystic Ovary Syndrome Via Modulation of Apoptotic Markers, Sex Hormones and Antioxidant Status in Rat Model.

Polycystic ovary syndrome (PCOS) is a gynecological disorder among reproductive-aged women and a major cause of infertility. Different treatment options are being employed but with side effects. This has mandated alternative treatment options, especially complementary therapy. This study therefore investigated the possible protective effects of methanol extract of Drymaria cordata in Letrozole-induced PCOS. The plant is folklorically used in the treatment of diverse ailments including PCOS, fibroids, uterine/ovarian/breast tumors, and cancers. Forty-eight female Wistar rats were acclimatized and initially divided into two groups: group I(control group) and group II(PCOS group). PCOS was induced by the oral administration of letrozole/high-fat diet for 21 days. After the induction, the PCOS group was sub-divided into four groups (n = 4): group II (positive control with PCOS), group III (MET 2mg/kg), group IV (MEDC 200mg/kg), and group V (MEDC 400mg/kg). Rats were orally treated with MET and MEDC for six weeks after the PCOS induction. At the end of the experimental period, blood samples were collected, sera were separated, mitochondria were isolated, and the mPT, some apoptotic biomarkers, hormonal and lipid profiles, and oxidative stress markers were determined. Ovarian histological evaluation and GC-MS analysis of MEDC were carried out. There was no significant mPT pore opening in the PCOS (untreated) group. However, treatments with MEDC caused significant mPT pore opening, upraised caspase 9, caspase 3, and Bax, and decreased anti-apoptotic Bcl-2 levels. The MEDC treatments restored the hormonal and lipid profiles, increased the levels of GSH-Px and SOD and decreased TBARS. Histological examination revealed resolved ovarian cysts and improved follicular growth with MEDC treatments. Comparable results were observed for both MEDC and metformin. The GC-MS analysis revealed the presence of some major pharmacologically relevant compounds. These findings suggest that MEDC contains phytochemicals that can protect against letrozole-induced PCOS possibly by normalizing the impaired hormonal balance, restoring the lipid profile, and improving the antioxidant milieu of the system.

Mefloquine-curcumin combinations improve host mitochondrial respiration and decrease mitotoxic effects of mefloquine in Plasmodium berghei-infected mice.

Plasmodium infection is a health challenge. Although, antiplasmodial drugs kill the parasites, information on the effects of infection and drugs on the expression of some genes is limited. Malaria was induced in two different studies using NK65 (chloroquine-susceptible, study 1), and ANKA (chloroquine-resistant, study 2) strains of Plasmodium berghei in 30 male Swiss mice (n = 5) in each study. Mice orally received 10 mL/kg distilled water, (infected control), Mefloquine (MF) (10 mg/kg), MF and Curcumin (CM) (25 mg/kg), MF and CM (50 mg/kg), CM (25 mg/kg) and CM (50 mg/kg). Five mice (un-infected) were used as the control. After treatment, total Ribonucleic acid (RNA) was isolated from liver and erythrocytes while Deoxyribonucleic acid (DNA)-free RNA were converted to cDNA. Polymerase Chain Reaction (PCR) amplification was performed and relative expressions of FIKK12, AQP3, P38 MAPK, NADH oxidoreductase, and cytochrome oxidase expressions were determined. Markers of glycolysis, toxicity and antioxidants were determined using ELISA assays. While the expression of FIKK12 was blunted by MF in the susceptible study, co-treatment with curcumin (25 mg/kg) yielded the same results in the chloroquine-resistant study. Similar results were obtained on AQP3 in both studies. Curcumin decreased P38 MAPK in both studies. Plasmodium infection decreased NADH oxidoreductase and cytochrome oxidase but mefloquine-curcumin restored the expression of these genes. While glycolysis and toxicity were inhibited, antioxidant systems improved in the treated groups. Curcumin is needed for effective therapeutic efficacy and prevention of toxicity. Plasmodium infection and treatment modulate the expressions of some genes in the host. Curcumin combination with mefloquine modulates the expression of some genes in the host.

Mechanism of antimalarial action and mitigation of infection-mediated mitochondrial dysfunction by phyto-constituents of Andrographis paniculata ((Burm f.) Wall. ex Nees) in Plasmodium berghei-infected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Andrographis paniculata (AP) ((Burm f.) Wall. ex Nees) is a medicinal plant, documented for its folkloric use in the treatment of malaria. AIM: This study was designed to determine the potency of extract and fractions of A. paniculata (AP) as a curative, both for susceptible and resistant malaria and to also determine the plant's mechanism of action. This study was also designed to determine whether AP extract and its most potent fraction will mitigate infection-mediated mitochondrial dysfunction, and to assess the phytochemical constituents of the most potent fraction. MATERIALS AND METHODS: n-Hexane, dichloromethane, ethylacetate and methanol were used to partition the methanol extract of A. paniculata. Graded doses of these extract and fractions were used to treat mice infected with chloroquine-sensitive strain of P. berghei in a curative model. The most potent fraction was used to treat mice infected with resistant (ANKA strain) P. berghei. Inhibition of hemozoin formation, reversal of mitochondrial dysfunction and antiinflammatory potentials were determined. A combination of ultraperformance liquid chromatography-quadrupole time of flight-mass spectrometry and nuclear magnetic resonance spectroscopy were used for chemical analysis. RESULTS: Microscopy revealed that the dichloromethane fraction decreased the parasite burden the most, and inhibition of the hemozoin formation is one of its mechanisms of action. The dichloromethane fraction reversed parasite-induced mitochondrial pore opening in the host, enzyme-dependent ATP hydrolysis and peroxidation of host mitochondrial membrane phospholipids as well as its antiinflammatory potentials. The UPLC-qTOF-MS report and NMR fingerprints of the dichloromethane fraction of A. paniculata yielded fourteen compounds of which sibiricinone C was identified from the plant for the first time. CONCLUSION: Fractions of A. paniculata possess antiplasmodial effects with the dichloromethane fraction having the highest potency. The potent effect of this fraction may be attributed to the phytochemicals present because it contains terpenes implicated with antimalarial and antiinflammatory activities.

Reversal of mitochondrial permeability transition pore and pancreas degeneration by chloroform fraction of Ocimum gratissimum (L.) leaf extract in type 2 diabetic rat model.

Introduction: Unmanaged Diabetes Mellitus (DM) usually results to tissue wastage because of mitochondrial dysfunction. Adverse effects of some drugs used in the management of DM necessitates the search for alternative therapy from plant origin with less or no side effects. Ocimum gratissimum (L.) (OG) has been folklorically used in the management of DM. However, the mechanism used by this plant is not fully understood. This study was designed to investigate the effects of chloroform fraction of OG leaf (CFOG) in the reversal of tissue wastage in DM via inhibition of mitochondrial-mediated cell death in streptozotocin (STZ)-induced diabetic male Wistar rats. Methods: Air-dried OG leaves were extracted with methanol and partitioned successively between n-hexane, chloroform, ethylacetate and methanol to obtain their fractions while CFOG was further used because of its activity. Diabetes was induced in fifteen male Wistar rats, previously fed with high fat diet (28 days), via a single intraperitoneal administration of STZ (35 mg/kg). Diabetes was confirmed after 72 h. Another five fed rats were used as the normal control, treated with corn oil (group 1). The diabetic animals were grouped (n = 5) and treated for 28 days as follows: group 2 (diabetic control: DC) received corn oil (10 mL/kg), groups 3 and 4 were administered 400 mg/kg CFOG and 5 mg/kg glibenclamide, respectively. Body weight and Fasting Blood Glucose (FBG) were determined while Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) and beta cell (HOMA-ß), and pancreatic tissue regenerating potential by CFOG were assessed. Activity-guided purification and characterization of the most active principle in CFOG was done using chromatographic and NMR techniques. The animals were sacrificed after 28 days, blood samples were collected and serum was obtained. Liver mitochondria were isolated and mitochondrial permeability transition (mPT) was investigated by spectrophotometry. Results: CFOG reversed diabetic-induced mPT pore opening, inhibited ATPase activity and lipid peroxidation. CFOG reduced HOMA-IR but enhanced HOMA-ß and caused regeneration of pancreatic cells relative to DC. Lupanol was a major metabolite of CFOG. Discussion: Normoglycemic effect of CFOG, coupled with reversal of mPT, reduced HOMA-IR and improved HOMA-ß showed the probable antidiabetic mechanism and tissue regenerating potentials of OG.

Chloroform Fraction of Drymaria cordata Linn (CFDC) Suppresses Estradiol Benzoate- Induced Endometrial Hyperplasia.

BACKGROUND: The diagnosis of uterine dysfunction (endometrial hyperplasia) is on the rise. The available treatment is quite expensive and associated with some side effects. The therapeutic potential of natural products is now being explored, as they are easily available with little or no side effects. Drymaraia cordata is folklorically utilized in the treatment of diverse ailments including uterine fibroids. OBJECTIVES: This study aims to investigate the potential therapeutic effect of chloroform fraction of methanol extract of Drymaria cordata (CFDC) in estradiol benzoate (EB)-induced endometrial hyperplasia. METHODS: Thirty-six rats were randomly divided equally into six groups. These included control group, CFDC: (100 mg/kg), CFDC: (200 mg/kg), EB: (2 mg/kg), EB + CFDC (100 mg/kg), and EB + CFDC (200 mg/kg). Endometrial hyperplasia (EH) was induced by intraperitoneal injection of EB. The levels of estrogen (E2), progesterone (PG), Follicle stimulating hormone (FSH), Luteinizing hormone (LH), Malondialdehyde (MDA), Superoxide dismutase (SOD), and Glutathione peroxidase (GSH-Px) activities were determined using ELISA technique. The uterine histological assessment and immunohistochemical expression levels of estrogen receptor, Ki-67, cytochrome c, and caspase 3 were carried out. RESULTS: EH was severely expressed in the uterine section of EB-treated rats. However, CFDC administration improved the pathological features of the animal model. The sex hormones levels were increased in the EB-treated group, which were significantly reduced by CFDC. The antioxidant indices were also restored by CFDC. Immunoexpression levels of ERα and Ki-67 were downregulated while cytochrome c and caspase 3 were upregulated by CFDC. CONCLUSION: This study suggests that CFDC contains phytochemicals that can protect against EB-induced EH via modulation of hormonal signaling, apoptotic machinery, and oxidative indices.

In vitro effects of 2-methyl-3-propylbutane-1,4-diol purified from Alstonia boonei on erythrocyte membrane stabilization and mitochondrial membrane permeabilization.

A recent review on the ethnomedicinal, chemical, pharmacological, and toxicological properties of Alstonia boonei revealed the plant's potential in the treatment and management of a range of diseases. However, most of these pharmacological effects are only traceable to the crude form of the plant extract and not specific natural products. Phytochemical investigation of the methanol fraction of the methanol extract of the stem-bark of Alstonia boonei led to the isolation and identification of 2-methyl-3-propylbutane-1,4-diol. The structures were elucidated by the application of 1D-, and 2D-NMR spectroscopic analyses and by comparison with literature data. In this study, the membrane stabilizing activity, mitochondrial membrane permeability transition pore opening, cytochrome c release, mitochondrial ATPase activity, and prevention of mitochondrial lipid peroxidation activity of 2-methyl-3-propylbutane-1,4-diol (MPBD) isolated from A. boonei were determined. The results showed that MPBD significantly (p < .05) prevented peroxidation of mitochondrial membrane lipids and hemolysis using both the heat-induced and hypotonic solution-induced membrane stabilization assays. On the contrary, the compound caused large amplitude swelling of rat liver mitochondria in the absence of calcium, significant (p < .05) cytochrome c release and enhancement of mitochondrial ATPase activity in vitro. Our findings suggest that MPBD showed characteristic biological properties useful in modulating cell death.

Hexane fraction of Globimetula braunii induces mitochondria-mediated apoptosis in Plasmodium berghei-infected mice.

Background: Apoptosis is a common pathology in malaria and most antimalarial drugs induce apoptosis during chemotherapy. Globimetula braunii is an African mistletoe used for the treatment of malaria but its effect on mitochondria-mediated apoptosis is not known. Methods: Malarial infection was induced by the intraperitoneal injection of NK 65 strain Plasmodium berghei-infected erythrocytes into mice which were treated with graded doses (100-400 mg/kg) of methanol extract (ME), and fractions of n-hexane, dichloromethane, ethylacetate and methanol (HF, DF, EF and MF) for 9 days after the confirmation of parasitemia. Artequine (10 mg/kg) was used as control drug. The fraction with the highest antiplasmodial activity was used (same dose) to treat mice infected with chloroquine-resistant (ANKA) strain for 5 consecutive days after the confirmation of parasitemia. P-alaxin (10 mg/kg) was used as control drug. On the last day of the treatment, liver mitochondria were isolated and mitochondrial Permeability Transition (mPT) pore opening, mitochondrial F0F1 ATPase (mATPase) activity, lipid peroxidation (mLPO) and liver deoxyribonucleic acid (DNA) fragmentation were assessed spectrophotometrically. Caspases 3 and 9 were determined by Enzyme-Linked Immunosorbent Assay (ELISA) technique. Cytochrome c, P53, Bcl-2-associated X protein (Bax), and B-cell lymphoma-2 (Bcl2) were determined via immunohistochemistry. Phytochemical constituents of the crude methanol extract of Globimetula braunii were determined via the Gas Chromatography-Mass Spectrometry (GC-MS) analysis. Results: There was large amplitude mPT induction by malaria parasites, extract and fractions of Globimetula braunii. At 400 mg/kg, HF significantly (p < 0.01) downregulated mATPase activity, and mLPO in both (susceptible and resistant) models, caused DNA fragmentation (P < 0.0001), induced caspases activation, P53, bax and cytochrome c release but downregulated Bcl2 in both models. The GC-MS analysis of methanol extract of Globimetula braunii showed that α-amyrin is the most abundant phytochemical. Conclusion: The n-hexane fraction of Globimetula braunii induced mitochondrial-mediated apoptosis through the opening of the mitochondrial pore, fragmentation of genomic DNA, increase in the levels of P53, bax, caspase 3 and 9 activation and cytochrome c release with concomitant decrease in the level of Bcl2. α-Amyrin is a triterpene with apoptotic effects.

The roles of betulinic acid on circulating concentrations of creatine kinase and immunomodulation in mice infected with chloroquine-susceptible and resistant strains of Plasmodium berghei.

Complete malarial therapy depends largely on the immunological and inflammatory response of the host to the invading potentials of malarial parasite. In this study, we evaluated the roles of betulinic acid on immunological response, anti-inflammatory potentials, cardiac and skeletal muscle tissue damage in mice infected with chloroquine susceptible (NK 65) and resistant (ANKA) strains of Plasmodium berghei. Serum Interleukins 1ß and 6 (IL-1ß, IL-6), tumour necrosis factor alpha (TNF-α), immunoglobulins G and M (IgG and IgM), C-reactive protein (CRP) and creatine kinase (CK) were determined using ELISA technique. Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and gamma glutammyl transferase (GGT) were determined using ELISA technique. The results showed that betulinic acid decreased the levels of IL-1ß, IL-6, TNF-α and CRP relative to the infected control. The IgG and IgM levels significantly increased in both models while CK did not decrease significantly in both models although serum AST, ALT and GGT significantly decreased compared to the infected control. These results showed that betulinic acid possessed anti-inflammatory, immunomodulatory and remediating effects on tissue damage. Furthermore, the decrease in activity of CK brought about by betulinic acid is indicative of decrease in cardiac and skeletal muscle injury which is a major pathological concern in Plasmodium infection and treatment.

Terpene-rich fractions of Ficus mucoso (Welw) modulate lipopolysaccharide-induced inflammatory mediators and aberrant permeability of the inner mitochondrial membrane in murine animal model.

Ficus mucoso is traditionally used to treat bronchial infections. This study compared the efficacy of terpene-rich fractions of F. mucoso root bark on lipopolysaccharide(LPS)-induced inflammation, liver mitochondrial permeability transition (mPT), an index of mitochondrial health, and associated pathological alterations. Terpene-Rich Fractions of Dichloromethane (TRDF) and Ethylacetate Fractions of F. mucoso (TREF) were obtained according to standard procedures. To induce systemic inflammation, a single intraperitoneal injection of 1mgLPS/kgbw was given to mice. Spectrophotometric techniques were used to evaluate the effects of the oral administration of TRDF and TREF (3 days) on levels of pro-inflammatory mediators (TNF-α, IL-1ß, IL-6) using ELSA techniques as well as antioxidant indices in normal and LPS-treated mice. The mPT pore opening, mitochondrial ATPase activity and lipid peroxidation were monitored spectrophotometrically. Our results revealed that treatment with LPS caused significant elevation in serum cytokine levels while administration of 50 and 100 mg/kg TRDF and TREF significantly reduced elevated serum levels of cytokines (TNF-α, IL-1ß, IL-6) in LPS-challenged mice. In addition, activitities of superoxide dismutase, catalase and liver marker enzymes (ALT and AST) as well as levels of mitochondrial lipid peroxides were significantly reduced in mice treated with TRDF and TREF relative to LPS-fed mice. Furthermore, LPS caused induction of opening of the liver mPT pore which was significantly inhibited by TRDF at 100 and 200 mg/kg bw by 71% and 88%, respectively, but only at 100 mg/kg TREF. Furthermore, mitochondrial ATPase activity was inhibited largely by TRDF. UPLC-ESI-MS analysis revealed the presence of terpenoid derivatives and a few aromatic metabolites in TRDF. The terpene dominance of TRDF metabolites was further justified on the 1H NMR fingerprint. Overall, TRDF is more effective as a cocktail of anti-inflammatory compounds than TREF against LPS-induced acute systemic inflammation.

Antimalarial and Erythrocyte Membrane Stability Properties of Globimetula braunii (Engle Van Tiegh) Growing on Cocoa in Plasmodium berghei-Infected Mice.

INTRODUCTION: Resistant malaria is a fatal disease. Globimetula braunii (African Mistletoe) is traditionally used for malarial treatment but this fact has not been scientifically reported. METHODS: Plasmodium berghei (NK65)-infected male Swiss mice (20±2 g) were treated orally and once daily with 100, 200, and 400 mg/kg BW of methanol extract and its respective hexane, dichloromethane and ethyl acetate fractions for 9 days. P-alaxin was used as control drug P. berghei (ANKA)-infected mice were then treated with the most potent fraction for 5 days. Parasitemia and parasite clearance were determined by microscopy, while hematological parameters, heme, hemozoin, and mouse erythrocyte membrane stabilisation were assayed. The phytochemicals in the most potent fraction were identified using gas chromatography-mass spectrometry. RESULTS: Hexane fraction (HF)-treated mice (400 mg/kg BW) had the least mean parasite load (0.00 ± 0.00; 0.14 ± 0.05%) and highest clearance (100 ± 0.00; 75.50 ± 4.95%) compared with infected control (9.81 ± 0.09; 6.84 ± 0.09%) in susceptible and resistant models, respectively. Hexane fraction modulated hematological indices, minimised erythrocyte membrane damage in heat-induced (2.18 ± 0.94%) and hypotonic solution-induced (7.93 ± 0.93%) compared to artequin (5.05 ± 2.18; 6.38 ± 0.33%) and P-alaxin (67.45 ± 5.15; 56.78 ± 1.10%) in both models of membrane stabilisation, respectively. Hexane fraction (P<0.01) increased heme and decreased hemozoin contents. Friedelan-3-one was identified as the most abundant triterpene. CONCLUSION: The results indicated that G. braunii has anti-plasmodial properties and minimally dis-stabilised erythrocyte membrane. The major findings in this study are that n-hexane fraction of G. braunii possess excellent and moderate antiplasmodial activity against susceptible and resistant P. berghei, respectively. This was reflected via decreased parasite load, improved hematological parameters, increased heme and decreased hemozoin contents. Friedelan-3-one, a major constituent of the n-hexane fraction, may be responsible for this activity.

INFLUENCE OF ARTESUNATE COMBINATIVE THERAPY CO-ADMINISTRATION WITH RUTIN ON INFLAMMATORY CYTOKINES AND IMMUNOGLOBULINS IN PLASMODIUM BERGHEI-INFECTED MICE.

Some antimalarial drugs are immune-modulators that impact multiple pathways of innate immunity in malarial treatment. However, information on the immunomodulatory effects of artequine and rutin in the treatment of malaria remains elusive. Twenty-five Swiss mice (18 ± 2 g) were used for this study. Twenty were infected with Plasmodium berghei (NK65). Parasitemia was confirmed, and the animals were grouped (n = 5) as follows: Group A was not infected but treated orally with vehicle. Groups B to E were infected and treated (B) orally with vehicle (10 ml/kg), (C) with 10 mg/kg artequine, (D) with 10 mg/kg of artequine supplemented with 100 mg rutin/kg, and (D) with 10 mg/kg of artequine supplemented with 200 mg rutin/kg, for 7 days. Blood was collected for hematological, inflammatory cytokines, and immunoglobulins G and M assays. Post mitochondrial supernatant fraction was used for antioxidant assays. Rutin co-administration (200 mg/kg) significantly (P < 0.001) increased platelet and neutrophil counts (P < 0.01) but significantly (P < 0.01) decreased white blood cell count and lymphocyte relative to parasitized control. Also, it significantly (P < 0.05) decreased lipid peroxidation, xanthine oxidase, and superoxide dismutase activities but significantly (P < 0.05) increased reduced glutathione and glutathione S-transferase activity. Rutin co-administration also caused a significant (P < 0.001) increase in tumor necrosis factor-alpha, interleukin-6, and immunoglobulin M levels, while interleukin-1ß and immunoglobulin G decreased significantly (P < 0.001) compared with parasitized control. These results showed that rutin co-administration with artequine improved host antioxidant status and modulated the immune and inflammatory responses.

Anti-Inflammatory Potentials of the n-Hexane Fraction of Alstonia boonei Stem Bark in Lipopolysaccharide-Induced Inflammation in Wistar Rats.

BACKGROUND: Inflammation is a protective response of the host to infections and tissue damage and medicinal plants have been used to regulate inflammatory response. The phytochemical contents of the n-hexane fraction of Alstonia boonei and their anti-inflammatory potentials in lipopolysaccharide-induced inflammation were investigated in rat liver. MATERIALS AND METHODS: A quantity of 5 mg/kg lipopolysaccharide (LPS) was used to induce inflammation in twenty-five male Wistar rats, grouped (n = 5) and treated as follows: negative control (10 mL/kg saline), positive control (1 mg/kg ibuprofen); 50, 100 and 20 mg/kg of the n-hexane fraction of Alstonia boonei were administered to test groups. In another experiment, twenty rats (n = 5, without LPS) were administered the same doses of the n-hexane fraction of A. boonei and ibuprofen for seven days. At the end of the experiment, animals were sacrificed, serum was obtained from blood and liver mitochondria isolated in a refrigerated centrifuge. Mitochondrial permeability transition (mPT) pore opening and mitochondrial F0F1 ATPase (mATPase) were determined spectrophotometrically. Serum interleukins 1ß, 6 (IL-1ß, IL-6), tumour necrosis factor alpha (TNF-α), C-reactive protein (CRP) and creatine kinase (CK), gamma glutamyl transferase (GGT), aspartate and alanine aminotransferases (AST and ALT,) of the animals in which inflammation was induced using LPS but treated with graded doses of n-hexane fraction of A. boonei were determined using the ELISA technique. The phytochemical contents of the n-hexane fraction of A. boonei were determined using ultra performance liquid chromatography-tandem mass spectrometer (UHPLC-MS). RESULTS: Calcium induced mPT in 8 fold and LPS induced mPT 14 fold in the negative control while the n-hexane fraction reversed mPT in the treated groups (50, 100 and 200 mg/kg) to 2, 4, 4 folds, respectively. LPS treatment of the negative group enhanced F0F1 mATPase activity, increased CRP, TNF-α, IL-1ß, IL-6 levels as well as CK, AST, ALT and GGT activities. These values were significantly reduced by 100 and 200 mg/kg of the n-hexane fraction. UHPLC-MS analysis of the fraction revealed the presence of terpenoids, phenolics and sphingolipids. CONCLUSION: These results showed that bioactive phytochemicals present in the n-hexane fraction of A. boonei were not toxic, have an anti-inflammatory effect and could be used for the treatment of inflammatory diseases.

Studies on the mitochondrial, immunological and inflammatory effects of solvent fractions of Diospyros mespiliformis Hochst in Plasmodium berghei-infected mice.

The use of medicinal plants in the treatment of malaria is gaining global attention due to their efficacy and cost effectiveness. This study evaluated the bioactivity-guided antiplasmodial efficacy and immunomodulatory effects of solvent fractions of Diospyros mespiliformis in mice infected with a susceptible strain of Plasmodium berghei (NK 65). The crude methanol extract of the stem of D. mespiliformis (DM) was partitioned between n-hexane, dichloromethane, ethyl acetate and methanol. Male Swiss mice (20 ± 2 g) infected with P. berghei were grouped and treated with vehicle (10 mL/kg, control), Artemether lumefantrine (10 mg/kg), 100, 200 and 400 mg/kg of n-hexane, dichloromethane, ethyl acetate and methanol fractions of D. mespiliformis for seven days. Blood was obtained for heme and hemozoin contents while serum was obtained for inflammatory cytokines and immunoglobulins G and M assessments. Liver mitochondria were isolated for mitochondrial permeability transition (mPT), mitochondrial F1F0 ATPase (mATPase) and lipid peroxidation (mLPO) assays. The GC-MS was used to identify the compounds present in the most potent fraction. The dichloromethane fraction had the highest parasite clearance and improved hematological indices relative to the drug control. The heme values increased, while the hemozoin content significantly (P < 0.05) decreased compared with the drug control. The highest dose of HF and MF opened the mPT pore while the reversal effects of DF on mPT, mATPase and mLPO were dose-dependent. The levels of IgG, IgM and TNFα in the DF group were significantly higher than the drug control, while the IL-1ß and IL-6 values did not vary linearly with the dose. Lupeol and Stigmastan-3,5-diene were the most abundant phytochemicals in the DF. The outcome of this study showed that the DF has immunomodulatory effects in infected mice, reduced proliferation of the malaria parasite and thus protect liver cells.

ln vitro inhibition of beta-hematin formation and in vivo effects of Diospyros mespiliformis and Mondia whitei methanol extracts on chloroquine-susceptible Plasmodium berghei-induced malaria in mice.

Background and aim: This study was carried out to investigate antiplasmodial activities of Diospyros mespiliformis (DM) and Mondia whitei (MW) in Plasmodium berghei-infected mice. Materials and methods: Air-dried stem of DM and root of MW were soaked in methanol, decanted and concentrated to give extracts. Parts of these extracts were partitioned successively to give dichloromethane, ethylacetate and methanol fractions. Mice (18 ± 3 g) were infected with Plasmodium berghei-infected erythrocytes from a donor mouse and were treated with the extracts, while the drug control group received 10 mg/kg body weight of artesunate and the parasitized control received the vehicle (5% v/v DMSO). Percentage parasitemia and clearance were estimated from thin films of blood smear. Hematological parameters were determined using standard methods. Both extracts were also tested on in vitro inhibition of ß-hematin formation. Results discussion and conclusion: Results showed that DM had the least percentage parasitemia (0.67%) and highest percentage parasite clearance (84.7%) while the MW had 0.89% percentage parasitemia and 79.7% clearance at the highest dose used after the seventh day relative to untreated control. The cell free antiplasmodial activity of the fractions and extracts of both DM and MW revealed that DM significantly inhibited ß-hematin formation than MW. The packed cell volume, white blood cell count, Lymphocyte, Eosinophil, Monocyte and Neutrophil significantly increased in the treated groups compared with the control. The results showed that the DM had higher antiplasmodial activity.

Comparative antimalarial, toxicity and mito-protective effects of Diospyros mespiliformis Hochst. ex A. DC. and Mondia whitei (Hook.f.) Skeels on Plasmodium berghei infection in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Diospyros mespiliformis Hochst. ex A. DC. and Mondia whitei (Hook.f.) Skeels are traditionally used in Africa for the treatment of malaria. However, scientific evidence to substantiate this folkloric claim and their effects on liver mitochondria during malaria treatment have not been reported. AIM OF THE STUDY: This study investigated the efficacy of D. mespiliformis and M. whitei against chloroquine-sensitive and resistant strains of malarial parasites in mice. It also investigated the toxicity and protection against cellular organelles like mitochondria. MATERIALS AND METHODS: Male Swiss mice were infected with a chloroquine resistant (ANKA) strain of Plasmodium berghei and were treated via oral gavage with methanol extracts of D. mespiliformis and M. whitei reconstituted in diluted dimethylsulfoxide as vehicle (DMSO, 5% v/v) for five consecutive days. Percentage parasite load and clearance were assessed by microscopy. The infected control was treated with the vehicle. Hematological indices were assessed using standard procedures. Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were determined using assay kits. Hepatic mitochondria were isolated via centrifugation, and their permeability transition (mPT), ATPase (mATPase) activity and lipid peroxidation (mLPO) were determined spectroscopically. Liver tissue histology was carried out by standard laboratory procedures. Phytochemical analysis of both extracts were performed using LC-MS to identify the most prominent compounds from each of the extracts. RESULTS: After treatment on day 5, D. mespiliformis and M. whitei at 400 mg/kg decreased mean values for: percentage parasitemia (5.0 ± 1.0, 2.0 ± 0.2), increased Packed Cell Volume (PCV) (36.0 ± 1.4, 36.0 ± 0.0%) and platelets (2.0 ± 1.4, 2.0 ± 2.8 × 105mm3) relative to the untreated control (20.0 ± 5.2; 30.0 ± 0.0%; 1.4 ± 1.4 × 105 mm3, respectively). At the same dose, D. mespiliformis and M. whitei decreased ALT (8.0 ± 3.8, 24.2 ± 4.0U/L), AST (6.2 ± 0.8, 8.0 ± 0.9U/L) and ALP (56.0 ± 0.7, 51.0 ± 1.0U/L) activities compared to the infected control (77.0 ± 10.9U/L, 14.0 ± 0.7U/L and 76.0 ± 6.0U/L, respectively). Both D. mespiliformis and M. whitei reversed mPT opening, decreased mATPase enhancement and mLPO, relative to the control. Histopathology of the liver showed extensive hemorrhagic lesions and severe disseminated congestion in the infected control while both D. mespiliformis and M. whitei were well tolerated at the highest dose. The LC-MS analysis of D. mespiliformis showed the presence of betulinic acid, tocopherol and kaempferol with antimalarial and antioxidant properties while the M. whitei sample contained coumarin and chlorogenic acid that have antimalarial and hepato-protective properties. CONCLUSIONS: D.mespiliformis and M. whitei show antimalarial effects against resistant Plasmodium berghei infection, enhanced cell viability, mito-protection and are not toxic in mice.

Chemotherapeutic and prophylactic antimalarial drugs induce cell death through mitochondrial-mediated apoptosis in murine models.

Malaria is a parasitic disease that has defied many treatment plans. This study was carried out to investigate the host mitochondrial response to malarial infection and selected antimalarial chemotherapy using murine models. The effects of artesunate (ART) and proguanil (PRG) on mitochondrial Permeability Transition (mPT), mitochondrial ATPase (mATPase), level of malondialdehyde (MDA) and activities of antioxidant enzymes; catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), Xanthine oxidase (XO), glutathione S-transferase (GST) and reduced glutathione (GSH) were estimated in Plasmodium berghei-infected mice treated with ART and PRG. Besides, apoptotic markers, such as caspases 3, 9 and DNA fragmentation were estimated. Unparasitised (NORMAL) and parasitized but untreated (PU) animals were used as controls. The mPT pore opening fold of 9 (ART), 3 (PRG), and 4 (PU) were observed relative to calcium (23) for in vivo study. In vitro, graded concentrations (20, 40, 80 and 160 µg/mL) of ART gave mPT induction folds of 1, 21, 23 and 25, respectively, relative to calcium (9) while PRG did not have effect in the absence of calcium. In vivo, ART significantly (p < 0.001) enhanced mATPase activity than PRG. The PRG and ART increased the MDA levels in vivo. Oral administration of ART and PRG altered antioxidant enzymes status, Caspases 3 and 9 were significantly activated in PRG-treated groups; there was significant increase in DNA fragmentation in PU and PRG groups compared with the normal control. The results obtained showed that malaria parasite and antimalarial drugs cause mitochondrial-mediated apoptosis.

Antimalarial properties and preventive effects on mitochondrial dysfunction by extract and fractions of Phyllanthus amarus (Schum. and Thonn) in Plasmodium berghei-infected mice.

OBJECTIVES: Broad spectrum antimalarial drugs without deleterious effects on mitochondria are scarce. It is in this regard that we investigated the potency of methanol extract and solvent fractions of Phyllanthus amarus on chloroquine-susceptible and resistant strains of Plasmodium berghei, toxicity and its consequential effects on mitochondrial permeability transition (mPT) pore opening. METHODS: Malaria was induced in male Swiss mice with susceptible (NK 65) strain, divided into groups (n=5) and treated with 100, 200 and 400 mg/kg of methanol extract, n-hexane, dichloromethane, ethylacetate and methanol fractions daily for seven days. Percentage parasitemia and parasite clearance were determined microscopically. The two most potent fractions were tested on resistant (ANKA) strains. Heme and hemozoin contents were determined spectrophotometrically. The mPT, mitochondrial ATPase (mATPase) and lipid peroxidation (mLPO) were determined spectrophotometrically. Similar groups of animals were used for toxicity studies. RESULTS: Dichloromethane fraction (400 mg/kg) had the highest antimalarial curative effect via least parasitemia (0.49) and high clearance (96.63) compared with the negative control (10.08, 0.00, respectively), had the highest heme and least hemozoin contents (16.23; 0.03) compared with the negative control (8.2, 0.126, respectively). Malaria infection opened the mPT, caused significant increase in mLPO and enhanced mATPase; while dichloromethane fraction reversed these conditions. Serum ALT, AST, ALP, GGT, urea and creatinine of dichloromethane fraction-treated mice decreased relative to control. No significant lesion was noticed in liver and kidney tissue sections. CONCLUSIONS: Dichloromethane fraction of Phyllanthus amarus had the highest antimalarial activity with the highest mito-protective effect and it was well tolerated without toxic effects.

In vitro antiplasmodial activity and prophylactic potentials of extract and fractions of Trema orientalis (Linn.) stem bark.

BACKGROUND: Trema orientalis (T. orientalis Linn) has been used in the management of malaria in the western part of Nigeria and despite its application in ethnomedicine, there is dearth of scientific evidence to justify the acclaimed prophylactic antimalarial usage of the plant. The aim of this study is to assess the in vitro antiplasmodial cell-free assay and chemopreventive efficacy of the methanol extract of the stem bark of T. orientalis and its fractions as a prophylactic regimen for malaria prevention. Also, the antimicrobial activities of the extract and the fractions were investigated. METHOD: Vacuum liquid chromatography was used to obtain dichloromethane, ethylacetate and methanol fractions from the methanol extract of T. orientalis. The fractions were tested for their prophylactic and cell-free antimalarial activity using murine models and ß-hematin formation assay respectively. Disc diffusion method was used to determine the antibacterial activity of the extract and its fractions against both Gram-positive and Gram-negative bacteria. RESULTS: In the prophylactic experiment, dichloromethane (DCMF), methanol fraction (MF) and extract (ME) (in this order) showed significant chemopreventive effects against P. berghei invasion of the red blood cells when compared with both Sulfadoxine-Pyrimethamine (SP) and untreated controls. Results of the in vitro study showed that the DCMF had the highest effect in preventing the formation of ß-hematin when compared with other fractions. The DCMF also had the highest percentage inhibition of ß-hematin formation when compared with chloroquine. The extract and fractions showed a concentration dependent antibacterial activity. Methanol extract had a pronounced inhibitory effect on Enterobacter cloaca ATCC 13047 and Enterococcus faecalis ATCC 29212. Serratia mercescens ATCC 9986 and Pseudomonas aeruginosa ATCC 19582 were the most susceptible bacteria. CONCLUSION: The results obtained showed that both extract and fractions of T. orientalis possessed antiplasmodial and antimicrobial activity.

Protective effect of Aloe vera gel on the permeability transition pore in the inner membrane of rat liver mitochondria in vitro.

OBJECTIVE: Aloe vera is a perennial drought resisting, succulent plant belonging to the zanthorrhoeaceae family which historically has been used for a variety of medicinal purposes. This study seeks to determine the effect of varying concentrations of Aloe vera gel (50, 100, 150, 250, and 350 µg/ml) on mitochondrial permeability transition pore (MPTP) in rat liver mitochondria (RLM) (in vitro). METHODS: Fresh Aloe gel was prepared daily from the Aloe vera leaf and the effect of the gel on mitochondrial membrane permeability transition pore opening was estimated in vitro using the spectrophotometric method of Lapidus and Sokolove. RESULTS: Varying concentrations of Aloe vera gel (50, 100, 150, 250, and 350 µg/ml) induced (insignificantly at p < 0.05) the opening of the mitochondrial permeability transition pore in a concentration dependent manner in the absence of calcium (Δ540 nm as -0.020 ± 0.008, -0.021 ± 0.009, -0.031 ± 0.013, -0.031 ± 0.014, -0.034 ± 0.014 respectively) when compared with the control (-0.016 ± 0.009). In the presence of calcium, the various concentrations of Aloe vera gel further opened the MMPT pore with the highest effect noticed at 350 µg/ml concentration. CONCLUSIONS: These findings indicate that Aloe vera gel modulates the mitochondrial pore opening by further increasing the effect of calcium. This effect is needed in situations that requires tissue wastage such as in cancer treatment.