Effect of Azadirachta Indica Extract On Hepatocarcinogenesis-Induced Rats

The effects of 5% A. indica aqueous extract (AI), or more commonly as Neem, on hepatocarcinogenesis induced Spraque-Dawley male rats were investigated. Hepatocarcinogenesis was induced in rats by employing a two carcinogen system: an intraperitoneal injection of 200 mg/kg diethyl nitrosamine (DEN) as initiator; followed by 0.02% of 2-acetylaminofluorene (AAF) in rat chow for two weeks to promote carcinogenesis. The rats were then left for two weeks to allow hepatic preneoplastic lesions to occur. The plant extract was prepared in 5% w/v in distilled water. Fresh leaves were collected, blended and mixed with distilled water. Twenty male rats Spraque-Dawley weighmg 150-250g were acclimatized for 1 week before use. The rats were divided into four groups of five rats each. iii The groups were: DENIAAF-iduced rats (C), DENIAAF-iduced rats treated with 5% A. indica (CAI), normal control rats (N) and normal rats treated with 5% A. indica extract group (NAI). The rats in group N and NAI were not induced with cancer however were injected once intraperitoneally with corn oil and act as normal control. The plant extract was fed to CAI and NAI groups to study its effects on both cancer and normal groups, respectively. In this study several parameters were evaluated as means of determining the effects of A1 on DENIAAF-induced hepatocarcinogenesis in rats. Body and liver weight profiles, foremost, hepatic lesions were scored in rats induced with DENIAAF cacinogens especially in the portal and lobular regions of the liver sections examined for histology. Loss of normal cell organization was also observed once the hepatocarcinogenesis was induced. In addition to histological observations, the TUNEL Assay, liver antioxidant enzyme Glutathione S transferase (GS-T), Glutathione Peroxidase (GPx) in the serum and liver, tumor marker alpha-fetoprotein (AFP) in serum and molecular detection of AFP and albumin genes expressions were conducted. The observation of the lesion scoring have shown significant difference (p<0.05) between DENIAAF and normal control groups (N, NAI). Histologically there were sigruficant changes in the lesion scoring of the liver in portal and lobular region in DENIAAF induced group (C) compared to the DENJAAF treated with A.indica (CAI). TUNEL assay supported that there was more numbers of apoptotic cells in the liver of (CAI) group compared to(C) group. The liver enzymes (GST & GPx) activity was measured and the result for both glutathione Stransferase and glutathione peroxidase were sigruficantly (p<0.05) higher in the (C) compared to the other groups (CAI, N, NAI). This result revealed that A. indica extract could reduce the activity of liver and serum GPx and GST enzymes of rats during hepatocarcinogenesis. However, the results of body and liver weight profiles showed that the CAI group was not significantly different (pN.05) from N, C and NAI groups. Alpha fetoprotein (AFP), a notable liver tumor marker, level was measured. The DENIAAF induced group (C) showed the highest increase in AFP levels while in CAI group illustrated significant (pc0.05) decrease in AFP level. There was no significant (pM.05) difference between N, NAI and CAI group. Mokdar detection of gene expression was done by RT-PCR for a-fetoprotein and albumin specific genes. However, the expression of the AFP gene was observed only in DEN/AAF induced group (C). Albumin gene expression was observed in all the study groups C, N, NAI and CAI proving the hepatic nature of the studied tissue and used as a housekeeping control gene in the RT-PCR experiments. As a conclusion, A. indica (Neem) has revealed a chemopreventive capability by regressing the hepatacarcinogenesis induced by DENJAAF carcinogens. This capability can be seen from the modulating effects of the plant in the biological indicators used in this study which can encourage the researchers to consider the A. indica (Neem) for further on mechanism and toxicology study

Investigations of antioxidant and antibacterial activities of Typhonium flagelliforme (Lodd.) Blume leaves.

The antioxidant and antibacterial activity of different extracts from of Typhonium flagelliforme (L.) Blume leave (family: Araceae) commonly called 'Rodent Tuber' was assessed towards different antioxidant models as well as in selected bacteria. None of the extracts showed significant activity against the selected strains. The only exception is hexane extract (2.0±0.15 mm diameter) against Pseudomonas aeruginosa. The positive control, Streptomycin had shown zone of inhibition of 20±1.5, 20±1.3, 23±1.5 and 23±1.0 mm in Methicillin Resistant Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella choleraesuis and Bacillus subtilis, respectively. All the extracts were subjected to screening for their possible antioxidant activity. Two complementary test systems, namely DPPH free radical scavenging and total phenolic compounds, were used for the analysis. The results showed that the inhibitory activity of Dichloromethane (60.7±3.2%) and Methanol (60.1±2.3%) extracts were comparatively commendable inhibition capacity when compared to the positive control BHT (95.3±1.3%). The total phenolic content of Methanol extracts (5.69±0.15 GAE mg g-1 extract) was superior to all other extracts, followed by dichloromethane and ethyl acetate. Considering all the results collectively T. flagelliforme appears to be a promising plant demonstrating antioxidant activity that requires further investigation

Oncolysis of breast, liver and leukemia cancer cells using ethyl acetate and methanol extracts of goniothalamus umbrosus.

This current study, aims to investigate the ethyl acetate and methanol extracts of Goniothalamus umbrosus for their anticancer effects on several human cancer cells namely, the MCF-7 breast cancer, HT-29 colon cancer and CEM-ss leukemia cell lines using a 3 days MTT (3-(4 and 5-dimethylthiazol-2-yl)-2 and 5-diphenyltetrazolium bromide) assay. Morphological changes and probable mode of cancer cell death induced by bioactive G. umbrosus extract were examined. DNA laddering assay was performed to assess endo-nucleosomal fragmentation. The MTT assay results revealed that only the ethyl acetate extract has anticancer effects on human breast cancer cells (MCF-7). Half maximal Inhibitory Concentration (IC50) of the ethyl acetate extract was found to be 24.5±0.12 μg mL-1. Both inverted and fluorescence microscopic studies demonstrated that treated MCF-7 breast cancer cells using IC50 of the extract displayed a number of typical morphological changes. Appearance of membrane blebs, DNA condensation and fragmentation are significant signs of apoptosis, were observed. The above findings suggested that the ethyl acetate extract of Goniothalamus umbrosus has potential therapeutic effect towards human breast cancer cells that requires further investigations in future

In vivo anti-tumor effects of Azadirachta indica in rat liver cancer.

The aim of the current study is to determine the effects of A. indica aqueous extract on Diethyl Nitrosamine (DEN) and 2-Acetylaminofluorene (AAF) induced-hepatocarcinogenesis on Spraque-Dawley rats. The plant, A. indica, extract was prepared into 5% w/v in distilled water. Spraque-Dawley male rats were divided into 3 groups of 7 rats each. The groups were: DEN/AAF-induced rats (C), DEN/AAF-induced rats treated with 5% A. indica (CAI) and normal control group (N). In situ detection of DNA fragmentation, TUNEL assay, was used to investigate the apoptogenic properties of A. indica. RT-PCR was used to amplify AFP mRNA. TUNEL assay supported that there was more numbers of apoptotic cells in the liver of (CAI) group compared with (C) group. AFP gene was suppressed by the supplementation of A. indica to DEN/AAF rats (CAI). A. indica (Neem) has revealed a chemopreventive capability by regressing the hepatocarcinogenesis induced by DEN/AAF carcinogens. This capability can be seen from the modulating effects of the plant in the biological indicators used in this study

Potential chemoprevention of diethylnitrosamine-initiated and 2-acetylaminofluorene-promoted hepatocarcinogenesis by zerumbone from the rhizomes of the subtropical ginger (Zingiber zerumbet)

Zerumbone (ZER), a monosesquiterpene found in the subtropical ginger (Zingiber zerumbet Smith), possesses antiproliferative properties to several cancer cells lines, including the cervical, skin and colon cancers. In this study, the antitumourigenic effects of ZER were assessed in rats induced to develop liver cancer with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and dietary 2-acetylaminofluorene (AAF) (0.02%). The rats also received intraperitoneal ZER injections at 15, 30 or 60 mg/kg body wt. twice a week for 11 weeks, beginning week four post-DEN injection. The hepatocytes of positive control (DEN/AAF) rats were smaller with larger hyperchromatic nuclei than normal, showing cytoplasmic granulation and intracytoplasmic violaceous material, which were characteristics of hepatocarcinogenesis. Histopathological evaluations showed that ZER protects the rat liver from the carcinogenic effects of DEN and AAF. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (AP) and alpha-fetoprotein (AFP) were significantly lower (P < 0.05) in ZER-treated than untreated rats with liver cancer. The liver malondialdehyde (MDA) concentrations significantly (P < 0.05) increased in the untreated DEN/AAF rats indicating hepatic lipid peroxidation. There was also significant (P < 0.05) reduction in the hepatic tissue glutathione (GSH) concentrations. The liver sections of untreated DEN/AAF rats also showed abundant proliferating cell nuclear antigen (PCNA), while in ZER-treated rats the expression of this antigen was significantly (P < 0.05) lowered. By the TUNEL assay, there were significantly (P < 0.05) higher numbers of apoptotic cells in DEN/AAF rats treated with ZER than those untreated. Zerumbone treatment had also increased Bax and decreased Bcl-2 protein expression in the livers of DEN/AAF rats, which suggested increased apoptosis. Even after 11 weeks of ZER treatment, there was no evidence of abnormality in the liver of normal rats. This study suggests that ZER reduces oxidative stress, inhibits proliferation, induces mitochondria-regulated apoptosis, thus minimising DEN/AAF-induced carcinogenesis in rat liver. Therefore, ZER has great potential in the treatment of liver cancers

In vivo and in vitro genotoxic effects of zerumbone.

Zerumbone (ZER) is derived from Zingiber zerumbet smith from the Zingiberaceae family. It has been shown to have anti-cancer and apoptosis-inducing properties against various human tumour cells. The aim of our study was to assess the genotoxic effects of ZER in cultured human peripheral blood lymphocytes, Chinese Hamster Ovary (CHO) cells and rat bone marrow polychromatic erythrocytes (PCEs) using micronucleus test (MN). All in vitro treatments were carried out in the absence of any exogenous metabolic activation system. Mitomycin C (MMC) was used as a positive control for in vitro treatments, while cisplatin was used as a positive micronucleus inducer in rat bone marrow PCEs. ZER at high concentrations induced an apparent signifi cant increase in the frequency of micronuclei in vivo (1000 mg/kg b.w) and in vitro (40 and 80 μM) compared to concurrent control values. Our in vivo and in vitro cytogenotoxicity studies suggest that high doses of ZER may be genotoxic and cytotoxic

Erectogenic Effects of Clerodendron capitatum

Clerodendron capitatum (Willd) (family: verbenaceae) is locally named as Gung and used traditionally to treat erectile dysfunction. Therefore, the current study was designed to investigate the erectogenic properties of C. capitatum. The relaxation effect of this plant was tested on phenylephrine precontracted rabbit corpus cavernosum smooth muscle (CCSM). The effects of C. capitatum were also examined on isolated Guinea pig atria alone, in the presence of calcium chloride (Ca2+ channel blocker), atropine (cholinergic blocker), and glibenclamide (ATP-sensitive K+ channel blocker). These effects were confirmed on isolated rabbit aortic strips. The extract, when tested colorimetrically for its inhibitory activities on phosphordiesterase-5 (PDE-5) in vitro towards p-nitrophenyl phenyl phosphate (PNPPP), was observed to induce significant dose-dependent inhibition of PDE-5, with an ID50 of 0.161 mg/ml (<.05). In conclusion, our results suggest that C. capitatum possesses a relaxant effect on CCSM, which is attributable to the inhibition of PDE-5, but not mediated by the release calcium, activation of adrenergic or cholinergic receptors, or the activation of potassium channels

Phytochemical profiling of Costus (Saussurea lappa Clarke) root essential oil, and its antimicrobial and toxicological effects

Purpose: To carry out gas chromatography-mass spectrometry (GC-MS) analysis of the phytochemical content of the root essential oil of Saussurea lappa Clarke Asteraceae (Costus, SLEO), and to evaluate its physicochemical, antimicrobial and cytoxic properties. Methods: The oil was extracted from the plant’s roots by steam distillation using a Clevenger system. Various physicochemical parameters for the oil including refractive index, color, acid value, saponification number, ester and peroxide values were measured. Flavonoid content was assessed using thin layer chromatography (TLC). Thermoscientific trace ultra gas chromatograph equipped with a Thermoscientific capillary TR-5MS column was utilized to determine the volatile components of SLEO. Antimicrobial activity of SLEO was performed against various Gram (+ve) and Gram (-ve) microorganisms, viz, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans, while cytotoxic effect was monitored using Artemia salina (brine shrimp) lethality assay. Results: Essential oil yield was good (3 %). Concentration-dependent antimicrobial effects were observed on all test microorganisms and no marked difference in lethality levels was observed among the tested SLEO concentrations on brine shrimp (p &lt; 0.05). The main component of SLEO was costunolide or eudesma-5,11(13)-dien-8,12-olide (52.01 %). Conclusion: The results indicate the promising therapeutic properties of S. lappa. However, further phytochemical and biological investigations are required to establish the mechanism of action and toxicological the extract

Combination of zerumbone and cisplatin to treat cervical intraepithelial neoplasia in female BALB/c mice.

Recent in vitro and in vivo studies have demonstrated that zerumbone (ZER) possesses anticancer properties. The main objective of this study was to examine the effectiveness of the combination of ZER and cisplatin (CIS) to treat cervical intraepithelial neoplasia (CIN) in vivo. Microculture tetrazolium assay and immunohistochemistry of proliferating cellular nuclear antigen were used to study the antitumor effect of ZER.Prenatally exposed female BALB/c micewere used as a model. The progenies with CIN were injected peritoneally with isotonic sodium chloride solution (positive control), CIS, ZER,and a combination of both compounds. All treated and untreated mice were humanely killed, and serum and cervix were obtained for interleukin 6 analysis and histopathologic studies using hematoxylin and eosin staining, respectively. Zerumbone has revealed an antitumor effect on human cervical cancer cells and downregulates immunoexpression of proliferating cellular nuclear antigen (P < 0.05). In vivo study indicates that ZER at 16 mg/kg and CIS at 10 mg/kg have a regressing effect on CIN. The combination of ZER and CIS also showed similar effectiveness in regressing CIN. Our results indicate that the combination of ZER and CIS has modulated the serum level of interleukin 6 when compared with that in mice treated with isotonic sodium chloride solution (P < 0.05). The effectiveness of combining ZER and CIS could be further explored as a new therapeutic intervention of early precancerous stages of carcinogenesis before the invasive stage begins

Typhonium flagelliforme induces apoptosis in CEMss cells via activation of caspase-9, PARP cleavage and cytochrome c release: Its activation coupled with G0/G1 phase cell cycle arrest.

Ethnopharmacological relevance: The plant Typhonium flagelliforme (TF), commonly known as ‘rodent tuber’ in Malaysia, is often used as traditional remedy for cancer, including leukemia. Aim of the study:Wehad previously identified morphologically that the linoleic acid rich fraction (DCM/F7) from the tubers of this plant induces selective anti-proliferative effects and apoptosis in CEMss cells. In this present study, we subjected the same DCM/F7 fraction to cell based activity analyses in order to determine the possible mechanism of cell death in leukemic CEMss cells in vitro. Materials and methods: Extraction of Typhonium flagelliforme tuber has done and fractionation has been done by vacuum liquid column chromatography. The anti-proliferative activity was assayed using MTT and the apoptosis detection was done by Annexin V and DNA laddering assay. Colorimetric caspase assay and immunoblot analysis were employed to detect the expression of protein associated with cell death. Cell cycle analysis was done using flow cytometry. Results: We found that the cancer inhibitory effect of the DCM/F7 fraction in CEMss cells was 3±0.08g/ml (IC50). An early apoptotic induction in CEMss cells was observed by Annexin V assay, which showed a clear dose-dependent DNA fragmentation being observed in gel electrophoresis at 10 and 20g/ml. The DCM/F7 fraction at 3g/ml significantly arrested CEMss cells at G0/G1 phase (p < 0.05). A constant but increasing pattern-related Sub-G0/G1 index was observed between 12 and 72 h treatment. In relation to this, we further investigated the biochemical events leading to cell death and found that the DCM/F7 fraction increased the cellular levels of caspase-3 and -9 on treated cells. Our results indicated that cytochrome c from mitochondria into the cytosol increased gradually as the DCM/F7 concentration increases, which later lead to the subsequent cleavage of PARP in to 85 kDa fragments. On the contrary, Bcl-2 protein was found to decrease concomitantly during treatment. Conclusions: Collectively, results presented in this study demonstrated that the DCM/F7 fraction inhibited the proliferation of leukemia cells, leading to the programmed cell death, which was confirmed to be through the mitochondrial pathway