Citrus limon (L.) Osbeck Fruit Peel Extract Attenuates Carbon Tetrachloride-Induced Hepatocarcinogenesis in Sprague-Dawley Rats.

Background: Traditional herbal medicine practitioners in the Ashanti region of Ghana use the fruit peels of Citrus limon (L.) Osbeck (C. limon) in preventive and curative treatment of many cancers including liver cancer. This ethnobotanical claim remains to be verified scientifically. Aim of the Study. This study investigated prophylactic hepatoprotective and anti-HCC effects of C. limon peel extract (LPE) in CCl4/olive oil-induced HCC-like rats. Materials and Methods: After preparation of LPE, it was subjected to phytochemical screening using standard phytochemical methods. A total of 30 healthy adult male Sprague-Dawley rats (weighing 150-200 g) were randomly assigned into six groups of 5 rats each. Rats in the control group received olive oil (5 mL/kg ip) twice weekly for 16 weeks. Rats in the model group received CCl4/olive oil (2 mL/kg, ip) twice weekly for 16 weeks. Rats in capecitabine (10 mg/kg po) and LPE (50, 100, and 200 mg/kg po) groups received CCl4/olive oil (2 mL/kg, i.p) in the morning and their respective treatments in the afternoon twice a week for 16 weeks. Rats in all groups had free access to food and water ad libitum. Body weight and survival rates were monitored. Rats were sacrificed under deep anesthesia, blood was collected, and liver and other organs were isolated. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), prothrombin time, bilirubin, C-reactive protein (CRP), alpha- (α-) fetoprotein (AFP), and liver histology were assessed. Results: Alkaloids, tannins, flavonoids, terpenoids, and saponins were detected in LPE. Model rats demonstrated increased serum levels of AFP, CRP, ALP, GGT, ALT, and AST, prothrombin time, total bilirubin, direct bilirubin, blood lymphocyte, and monocyte counts, but decreased serum albumin and total protein compared to control rats. Unlike the control, model rats demonstrated fat accumulation in periportal and centrilobular hepatocytes and neoplastic transformation. Semiquantitation of periodic acid Schiff- (PAS-) stained liver sections showed decreased glycogen storage in hepatocytes of model rats compared to control rats. Compared to the model, LPE treatment protected against CCl4-induced hepatocarcinogenesis, which was evidenced by decreased AFP, CRP, liver enzymes, total and direct bilirubin, prothrombin time, and blood lymphocyte and monocyte counts; attenuation of fat accumulation; and increased glycogen storage, albumin, and total protein. Conclusion: LPE abates CCl4-induced hepatocarcinogenesis by attenuating liver inflammation and improving metabolic, biosynthetic, and detoxification functions of the liver. The prophylactic hepatoprotective and anti-hepatocarcinogenic effects of LPE are attributable to its phytochemical composition raising hopes of finding potential anticancer bioactive compounds from C. limon fruit peels.

Antiplasmodial, Antitrypanosomal, and Cytotoxic Effects of Anthonotha macrophylla, Annickia polycarpa, Tieghemella heckelii, and Antrocaryon micraster Extracts.

Malaria and trypanosomiasis are protozoan diseases which pose a devastating challenge to human health and productivity especially, in Africa where their respective vectors (female Anopheles mosquito and tsetse fly) abound. Various medicinal plants are used to treat these parasitic diseases. However, the scientific basis of their use and toxicological profiles have not been assessed. We have, therefore, evaluated the antiplasmodial, antitrypanosomal, and cytotoxic activities of four African medicinal plant extracts namely, Anthonotha macrophylla leaf (AML), Annickia polycarpa leaf (APLE), Tieghemella heckelii stem bark (THBE), and Antrocaryon micraster stem bark (AMSBE) extracts in vitro against P. falciparum (W2mef laboratory strain), T. brucei (GUTat 3.1 strain), and mammalian RAW 264.7 macrophage cell line, respectively. The most active antiplasmodial extract was AML (IC50 = 5.0 ± 0.08 µg/mL with SI of 21.9). THBE also, produced the most effective antitrypanosomal activity (IC50 = 11.0 ± 0.09 µg/mL and SI of 10.2) among the extracts. In addition, none of the extracts produced toxic effect in the RAW 264.7 macrophage cell line except APLE which was moderately cytotoxic and also produced the least SI in both antitrypanosomal and antiplasmodial assays. These results suggest that AML and THBE could offer safe and alternative therapy for malaria and trypanosomiasis. This is the first study to report the antitrypanosomal and in vitro antiplasmodial activities of these four plants/plant parts. The cytotoxicity of the plant parts used is also being reported for the first time except for the T. heckelii stem bark.

Anti-inflammatory activity and mechanism of action of ethanolic leaf extract of Morinda lucida Benth.

BACKGROUND AND AIM: Most developing countries resort to medicinal plants for treating diseases, but few of these have scientific backing for their use. The aim of the study was to validate traditional use of Morinda lucida leaves in treating inflammation and determine the mechanism of action. EXPERIMENTAL PROCEDURE: Effect of hydroethanolic leaf extract of M. lucida (HEML) on localized inflammation was evaluated using rat paw edema presented by sub-planter injections of λ-carrageenan, histamine or serotonin in separate experiments. Systemic inflammation was evaluated by lipopolysaccharide (LPS)-induced hyperthermia. Antioxidant activity of HEML was also evaluated using the free-radical scavenging assay. RESULTS AND CONCLUSION: No mortalities were recorded in acute toxicity assay after administering 5000 mg/kg HEML to rats. It showed very good activity against localized and systemic inflammation in inverse dose-dependent manner and caused reduction in nitric oxide and prostaglandin E-2 levels by affecting expression of inducible nitric oxide synthase, but not cyclooxygenases-2 in LPS-activated RAW 264.7 murine macrophages. HEML reduced pro-inflammatory cytokines interleukin (IL)-1ß and tumor necrotic factor, but elevated levels of anti-inflammatory cytokine IL-10 in vitro. HEML contains saponins, reducing sugars, polyphenols and flavonoids and showed antioxidant activity with EC50 = 0.6415 ± 0.0027 mg/ml. In conclusion, this study provides evidence that HEML possesses anti-inflammatory activity, possibly through modulation of production of early/late phase inflammation mediators.

Intervention of standardized ethanol leaf extract of Annickia polycarpa, (DC.) Setten and Maas ex I.M. Turner. (Annonaceae), in Plasmodium berghei infested mice produced anti-malaria action and normalized gross hematological indices.

ETHNOPHARMACOLOGICAL RELEVANCE: Malaria is a global public health burden due to large number of annual infections and casualties caused by its hematological complications. The bark of Annickia polycarpa is an effective anti-malaria agent in African traditional medicine. However, there is no standardization parameters for A. polycarpa. The anti-malaria properties of its leaf are also not known. AIM OF THE STUDY: To standardize the ethanol leaf extract of A. polycarpa (APLE) and investigate its anti-malaria properties and the effect of its treatment on hematological indices in Plasmodium berghei infected mice in the Rane's test. MATERIALS AND METHODS: Malaria was induced by inoculating female ICR mice with 1.0 × 107P. berghei-infected RBCs in 0.2 mL (i.p.) of blood. Treatment was commenced 3 days later with APLE 50, 200, 400 mg/kg p.o., Quinine 30 mg/kg i.m. (Standard drug) or sterile water (Negative control) once daily per group for 4 successive days. Anti-malarial activity and gross malaria indices such as hyperparasitemia, mean change in body weight and mean survival time (MST) were determined for each group. Changes in white blood cells (WBCs), red blood cells (RBCs), platelets (PLT) counts, hemoglobin (HGB) concentration, hematocrit (HCT) and mean corpuscular volume (MCV) were also measured in the healthy mice before infection as baseline and on day 3 and 8 after inoculation using complete blood count. Standardization was achieved by UHPLC-MS chemical fingerprint analysis and quantitative phytochemical tests. RESULTS: APLE, standardized to its total alkaloids, phenolics and saponin contents, produced significant (P < 0.05) dose-dependent clearance of mean hyperparasitemia of 22.78 ± 0.93% with the minimum parasitemia level of 2.01 ± 0.25% achieved at 400 mg/kg p.o. on day 8. Quinine 30 mg/kg i.m. achieved a minimum parasitemia level of 6.15 ± 0.92%. Moreover, APLE (50-400 mg/kg p.o.) evoked very significant anti-malaria activity of 89.22-95.50%. Anti-malaria activity of Quinine 30 mg/kg i.m. was 86.22%. APLE also inverse dose-dependently promotes weight gain with the effect being significant (P < 0.05) at 50 mg/kg p.o. Moreover, APLE dose-dependently increased the MST of malaria infested mice with 100% survival at 400 mg/kg p.o. Quinine 30 mg/kg i.m. also produce 100% survival rate but did not promote (P > 0.05) weight gain. Hematological studies revealed the development of leukocytopenia, erythrocytosis, microcytic anemia and thrombocytopenia in the malaria infected mice which were reverted with the treatment of APLE 50-400 mg/kg p.o. or Quinine 30 mg/kg i.m. but persisted in the negative control. The UHPLC-MS fingerprint analysis of APLE led to identification of one oxoaporphine and two aporphine alkaloids (1-3). Alkaloids 1 and 3 are being reported in this plant for the first time. CONCLUSION: These results indicate that APLE possessed significant anti-malaria, immunomodulatory, erythropoietic and hematinic actions against malaria infection. APLE also has the ability to revoke deleterious physiological alteration produced by malaria and hence, promote clinical cure. These properties of APLE are due to its constituents especially, aporphine and oxoaporphine alkaloids.

New anti-trypanosomal active tetracyclic iridoid isolated from Morinda lucida Benth.

Human African trypanosomiasis (HAT), commonly known as sleeping sickness has remained a serious health problem in many African countries with thousands of new infected cases annually. Chemotherapy, which is the main form of control against HAT has been characterized lately by the viewpoints of toxicity and drug resistance issues. Recently, there have been a lot of emphases on the use of medicinal plants world-wide. Morinda lucida Benth. is one of the most popular medicinal plants widely distributed in Africa and several groups have reported on its anti-protozoa activities. In this study, we have isolated one novel tetracyclic iridoid, named as molucidin, from the CHCl3 fraction of the M. lucida leaves by bioassay-guided fractionation and purification. Molucidin was structurally elucidated by (1)H and (13)C NMR including HMQC, HMBC, H-H COSY and NOESY resulting in tetracyclic iridoid skeleton, and its absolute configuration was determined. We have further demonstrated that molucidin presented a strong anti-trypanosomal activity, indicating an IC50 value of 1.27 µM. The cytotoxicity study using human normal and cancer cell lines indicated that molucidin exhibited selectivity index (SI) against two normal fibroblasts greater than 4.73. Furthermore, structure-activity relationship (SAR) study was undertaken with molucidin and oregonin, which is identical to anti-trypanosomal active components of Alnus japonica. Overlapping analysis of the lowest energy conformation of molucidin with oregonin suggested a certain similarities of aromatic rings of both oregonin and molucidin. These results contribute to the future drug design studies for HAT.