Norhafizah Mohtarrudin

Antioxidant and anti-inflammatory activities contribute to the prophylactic effect of semi-purified fractions obtained from the crude methanol extract of Muntingia calabura leaves against gastric ulceration in rats

ETHNOPHARMACOLOGICAL RELEVANCE In traditional medicine, the leaves, flowers, barks and roots of Muntingia calabura L. (Muntingiaceae) have been employed as a treatment for various ailments including dyspepsia and to relieve pain caused by gastritis and peptic ulcer disease. The methanolic extract of Muntingia calabura leaves (MEMC) has been proven in the previous study to possess significant antiulcer activity. In this study, we attempted to determine the prophylactic effect of the fractions obtained from MEMC against ethanol-induced gastric lesion in rats and the involvement of antioxidants and anti-inflammatory mediators. MATERIALS AND METHODS The MEMC was fractionated with petroleum ether (PEF), ethyl acetate (EAF) and distilled water (AQF). These fractions were investigated for possible antiulcer property using ethanol-induced gastric ulcer rat model. The rats were administered orally once daily with 8% Tween 80 (control), 100mg/kg ranitidine, or the fractions, in the doses of 100, 250, and 500 mg/kg, for 7 days, followed by ulcer induction using absolute ethanol. The rats were euthanized; macroscopic and histological observations of the stomach were done. The ulcer area (UA) was determined and the percentage protection afforded by the fractions was calculated. The fractions were subjected to antioxidant studies including the superoxide and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, oxygen radical absorbance capacity (ORAC) and total phenolic content (TPC) assay. Involvement of nitric oxide (NO) and inflammatory mediators such as lipoxygenase (LOX) and xanthine oxidase (XO) were evaluated. Phytochemical screening and HPLC analysis of the fractions were also conducted. RESULTS Pre-treatment of PEF and EAF significantly (p<0.001) attenuated the gastric lesions as compared to the control group in a dose-dependent manner. On the other hand, 100 and 250 mg/kg of AQF significantly (p<0.001) prevented the ulcer formation but at the highest dose (500 mg/kg), AQF failed to significantly reduce the ulcer formation, showing a dose-independent antiulcerative effect of AQF. The histological evaluation supported the observed gastroprotective activity of PEF, EAF and AQF. All the fractions showed high superoxide and DPPH scavenging activity, meanwhile the EAF showed highest TPC followed by PEF and AQF. These fractions also significantly (p<0.05) inhibited the NO while maintaining the viability of the cells. EAF exhibited high inhibition towards both the LOX and XO enzymes, meanwhile PEF and AQF exerted high LOX inhibition but low XO inhibition. Phytochemical screening and HPLC profiling suggested the presence of flavonoid- and tannin based compounds in PEF and EAF. CONCLUSION It can be concluded that the prophylactic effect of the fractions on gastric ulceration in rats is associated with its high antioxidant activity and its ability to effectively inhibit the inflammation mediators. Presence of several flavonoids and gallic acid explains the effectiveness of the fractions in affording protection against gastric damages.

The anti-obesity effects of Lactobacillus casei strain Shirota versus Orlistat on high fat diet-induced obese rats

Background Obesity and overweight are major public health problems. Various factors, such as daily nutritional habits, physical inactivity, and genetic, are related to the prevalence of obesity. Recently, it was revealed that the gut microflora may also play an important role in weight management. Thus, this study aimed to determine the anti-obesity effects of Lactobacillus casei strain Shirota (LcS) compared with those of orlistat in an animal model fed a high-fat diet (HFD). Design Thirty-two male Sprague-Dawley rats were assigned to four groups fed various diets as follows: a standard diet group, HFD group, HFD supplemented with LcS (108109 colony-forming units (HFD-LcS) group, and HFD group treated with Orlistat (10 mg/kg body weight)). After 15 weeks, the weights of organs, body weight, body fat mass and serological biomarkers were measured. In addition, histological analysis of the liver and adipose tissue was performed. Results Body weight, body mass index, fat mass, leptin and glucose levels were lower, and high-density lipoprotein and adiponectin levels were higher in the HFD-LcS and HFD-orlistat groups than in the HFD group. In addition a significant difference in body fat mass was observed between HFD-LcS group with HFD-orlistat group (19.19±5.76 g vs. 30.19±7.98 g). Although the interleukin-6 level was significantly decreased in the HFD-LcS and HFD-orlistat groups compared with the HFD group, no significant change was observed in other inflammatory biomarkers. Conclusion The results of the present study show that LcS supplementation improves body weight management and the levels of some related biomarkers. In addition, LcS supplementation showed a better result in fat mass and alanine aminotransferase reduction than Orlistat. Further studies are needed to elucidate the anti-obesity effects of LcS, with a longer period of supplementation.

High Tibial Osteotomy in Combination With Chondrogenesis After Stem Cell Therapy: A Histologic Report of 8 Cases

PURPOSE To histologically evaluate the quality of articular cartilage regeneration from the medial compartment after arthroscopic subchondral drilling followed by postoperative intra-articular injections of autologous peripheral blood stem cells (PBSCs) and hyaluronic acid with concomitant medial open-wedge high tibial osteotomy (HTO) in patients with varus deformity of the knee joint. METHODS Eight patients with varus deformity of the knee joint underwent arthroscopic subchondral drilling of International Cartilage Repair Society (ICRS) grade 4 bone-on-bone lesions of the medial compartment with concomitant HTO. These patients were part of a larger pilot study in which 18 patients underwent the same procedure. PBSCs were harvested and cryopreserved preoperatively. At 1 week after surgery, 8 mL of PBSCs was mixed with 2 mL of hyaluronic acid and injected intra-articularly into the knee joint; this was repeated once a week for 5 consecutive weeks. Three additional intra-articular injections were administered weekly at intervals of 6, 12, and 18 months postoperatively. Informed consent was obtained at the time of hardware removal for opportunistic second-look arthroscopy and chondral biopsy. Biopsy specimens were stained with H&E, safranin O, and immunohistochemical staining for type I and II collagen. Specimens were graded using the 14 components of the ICRS Visual Assessment Scale II, and a total score was obtained. RESULTS Second-look arthroscopy showed satisfactory healing of the regenerated cartilage. Histologic analysis showed significant amounts of proteoglycan and type II collagen. The total ICRS Visual Assessment Scale II histologic scores comparing the regenerated articular cartilage (mean, 1,274) with normal articular cartilage (mean, 1,340) indicated that the repair cartilage score approached 95% of the normal articular cartilage score. There were no infections, delayed unions, or nonunions. CONCLUSIONS Chondrogenesis with stem cells in combination with medial open-wedge HTO for varus deformity correction of the knee joint regenerates cartilage that closely resembles the native articular cartilage. LEVEL OF EVIDENCE Level IV, therapeutic case series.

Hepatoprotective Activity of Methanolic Extract of Bauhinia purpurea Leaves against Paracetamol-Induced Hepatic Damage in Rats

In an attempt to further establish the pharmacological properties of Bauhinia purpurea (Fabaceae), hepatoprotective potential of methanol extract of B. purpurea leaves (MEBP) was investigated using the paracetamol- (PCM-) induced liver toxicity in rats. Five groups of rats (n = 6) were used and administered orally once daily with 10% DMSO (negative control), 200 mg/kg silymarin (positive control), or MEBP (50, 250, and 500 mg/kg) for 7 days, followed by the hepatotoxicity induction using paracetamol (PCM). The blood samples and livers were collected and subjected to biochemical and microscopical analysis. The extract was also subjected to antioxidant study using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay with the total phenolic content (TPC) also determined. From the histological observation, lymphocyte infiltration and marked necrosis were observed in PCM-treated groups (negative control), whereas maintenance of the normal hepatic structural was observed in group pretreated with silymarin and MEBP. Hepatotoxic rats pretreated with silymarin or MEBP exhibited significant decrease (P < 0.05) in ALT and AST enzyme level. Moreover, the extract also exhibited antioxidant activity and contained high TPC. In conclusion, MEBP exerts potential hepatoprotective activity that could be partly attributed to its antioxidant activity and high phenolic content and thus warrants further investigation.

Antiulcer activity of methanol extract of Melastoma malabathricum leaves in rats.

Objective: To determine the potential antiulcer activity of methanol extract of Melastoma malabathricum leaves (MEMM) using various established rat models. Materials and Methods: Ten groups of rats were used and orally administered 10% DMSO (negative control), 100 mg/kg ranitidine (positive control) or MEMM (50, 250 and 500 mg/kg) followed by gastric ulcer induction either using ethanol or indomethacin. The stomachs were collected and subjected to macroscopic and microscopic analyses. Results: MEMM exhibited significant (p < 0.05) antiulcer activity in the ethanol, but not in the indomethacin-induced gastric ulcer model. The percentage of antiulcer activity for 50–500 mg/kg MEMM ranged between 3 and 75%, respectively. The gross observations were supported by histological findings. MEMM also aggravated the indomethacin-induced gastric ulcer, leading to an increase in ulcer area formation and ulcer score. Conclusion: The M. malabathricum leaves showed antiulcer activity, which could be attributed to their antioxidant and anti-inflammatory activities. This requires further in-depth studies.

Antiulcer activity of Muntingia calabura leaves involves the modulation of endogenous nitric oxide and nonprotein sulfhydryl compounds

Abstract Context: Muntingia calabura L. (Muntingiaceae) is a native plant species of the American continent and is widely cultivated in warm areas in Asia, including Malaysia. The plant is traditionally used to relieve pain from gastric ulcers. Objective: This study was designed to determine the antiulcer activity of a methanol extract of M. calabura leaves (MEMC) and the possible mechanisms of action involved. Materials and methods: An acute toxicity study was conducted using a single oral dose of 2000 mg/kg MEMC. The antiulcer activity of MEMC was evaluated in absolute ethanol- and indomethacin-induced gastric ulcer rat models. MEMC was administered orally (dose range 25–500 mg/kg) to rats fasted for 24 h. The animals were pretreated with NG-nitro-l-arginine methyl esters (l-NAME) or N-ethylmaleimide (NEM) prior to MEMC treatment to assess the possible involvement of endogenous nitric oxide (NO) and nonprotein sulfhydryl (NP-SH) compounds in the gastroprotective effect of MEMC. Results: As the administered dose did not cause toxicity in the rats, the oral median lethal dose (LD50) of MEMC was >2000 mg/kg in rats. MEMC exerted significant (p < 0.001) gastroprotective activity in the ethanol- and indomethacin-induced ulcer models dose-dependently. Histological evaluation supported the observed antiulcer activity of MEMC. l-NAME and NEM pretreatment significantly (p < 0.05) reversed and abolished the gastroprotective effect of MEMC, respectively. Discussion and conclusion: The results obtained indicate that MEMC has significant antiulcer activity that might involve the participation of endogenous NO and NP-SH compounds. These findings provide new pharmacological information regarding the potential use of M. calabura.

Amelioration of paracetamol-induced hepatotoxicity in rat by the administration of methanol extract of Muntingia calabura L. leaves.

Muntingia calabura L. is a tropical plant species that belongs to the Elaeocarpaceae family. The present study is aimed at determining the hepatoprotective activity of methanol extract of M. calabura leaves (MEMC) using two models of liver injury in rats. Rats were divided into five groups (n = 6) and received 10% DMSO (negative control), 50 mg/kg N-acetylcysteine (NAC; positive control), or MEMC (50, 250, and 500 mg/kg) orally once daily for 7 days and on the 8th day were subjected to the hepatotoxic induction using paracetamol (PCM). The blood and liver tissues were collected and subjected to biochemical and microscopical analysis. The extract was also subjected to antioxidant study using the 2,2-diphenyl-1-picrylhydrazyl-(DPPH) and superoxide anion-radical scavenging assays. At the same time, oxygen radical antioxidant capacity (ORAC) and total phenolic content were also determined. From the histological observation, lymphocyte infiltration and marked necrosis were observed in PCM-treated groups (negative control), whereas maintenance of hepatic structure was observed in group pretreated with N-acetylcysteine and MEMC. Hepatotoxic rats pretreated with NAC or MEMC exhibited significant decrease (P < 0.05) in ALT and AST enzymes level. Moreover, the extract also exhibited good antioxidant activity. In conclusion, MEMC exerts potential hepatoprotective activity that could be partly attributed to its antioxidant activity and, thus warrants further investigations.

Hepatoprotective Activity of Methanol Extract of Melastoma malabathricum Leaf in Rats

The present study aimed to determine the hepatoprotective activity of a methanol extract of Melastoma malabathricum leaves (MEMM) using two established rat models. Ten groups of rats (n=6) were given a once-daily administration of 10% dimethyl sulfoxide (negative control), 200 mg/kg silymarin (positive control), or MEMM (50, 250, or 500 mg/kg) for 7 days followed by induction of hepatotoxicity either using paracetamol or carbon tetrachloride. Blood samples and livers were collected for biochemical and microscopic analysis. Based on the results obtained, MEMM exhibited a significant (p<0.05) hepatoprotective activity against both inducers, as indicated by an improvement in the liver function test. These observations were supported by the histologic findings. In conclusion, M. malabathricum leaves possessed hepatoprotective activity, which could be linked to their phytochemical constituents and antioxidant activity; this therefore requires further in-depth studies.

Annona muricata leaves extracts prevent DMBA/TPA-induced skin tumorigenesis via modulating antioxidants enzymes system in ICR mice

Annona muricata, locally known as soursop has been reported to exhibit antiproliferative activities against various cancer cell lines. In this current study, we have investigated the antitumor promotion of various fractions of Annona muricata leaves (AML); hexane (AMLH), dichloromethane (AMLD) and methanol (AMLM) fraction respectively on 7, 12-dimethylbenz[α]anthracene (DMBA) induced and 12-0-tetradecaboylphorbol-13-acetate (TPA) promoted skin tumorigenesis in mice via morphological assessment, biochemical analysis and histopathological evaluation. The results of the study revealed significant inhibition in tumor incidence, tumor burden and tumor volume in the groups received AMLH and AMLD, respectively, and suppressive effects in group received AMLM compared with carcinogen control group at week 21. Superoxide dismutase, catalase, and lipid peroxidation levels were returned to near normal by administration of AML to DMBA/TPA-induced mice. The above findings were supported by histopathological studies, in which the extensive epidermal hyperplasia in carcinogen control group was restored to normal in AML treated groups. Whilst, annonacin, a major annaonaceous acetogenin was found to be the highest in AMLH and AMLD. From the present study, it can be inferred that AML supressed DMBA/TPA-induced skin tumor and this antitumor-promoting activity may be linked to the antioxidant/free radical-scavenging constituents of the extract and annonacin contained in the extracts.

Semipurified Ethyl Acetate Partition of Methanolic Extract of Melastoma malabathricum Leaves Exerts Gastroprotective Activity Partly via Its Antioxidant-Antisecretory-Anti-Inflammatory Action and Synergistic Action of Several Flavonoid-Based Compounds

Recent study has demonstrated the gastroprotective activity of crude methanolic extract of M. malabathricum leaves. The present study evaluated the gastroprotective potential of semipurified extracts (partitions): petroleum ether, ethyl acetate (EAMM), and aqueous obtained from the methanolic extract followed by the elucidation of the gastroprotective mechanisms of the most effective partition. Using the ethanol-induced gastric ulcer assay, all partitions exerted significant gastroprotection, with EAMM being the most effective partition. EAMM significantly (i) reduced the volume and acidity (free and total) while increasing the pH of gastric juice and enhanced the gastric wall mucus secretion when assessed using the pylorus ligation assay, (ii) increased the enzymatic and nonenzymatic antioxidant activity of the stomach tissue, (iii) lost its gastroprotective activity following pretreatment with N-omega-nitro-L-arginine methyl ester (L-NAME; NO blocker) or carbenoxolone (CBXN; NP-SH blocker), (iv) exerted antioxidant activity against various in vitro oxidation assays, and (v) showed moderate in vitro anti-inflammatory activity via the LOX-modulated pathway. In conclusion, EAMM exerts a remarkable NO/NP-SH-dependent gastroprotective effect that is attributed to its antisecretory and antioxidant activities, ability to stimulate the gastric mucus production and endogenous antioxidant system, and synergistic action of several gastroprotective-induced flavonoids.