Aqueous Azadirachta Indica Leaf Extract Research Articles

Altered presence of extra cellular matrix components in murine skin cancer: Modulation by Azadirachta indica leaf extract

Background and aimAlthough, the anticancer potential of Aqueous Azadirachta indica leaf extract (AAILE) has been robustly established against cutaneous squamous cell carcinoma (SCC) in mice, however, its ability in modulating tumor associated extra cellular matrix (ECM) is largely unknown. Therefore, the present study was conceived to explore changes in ECM during murine skin cancer and its chemoprevention by AAILE. Experimental procedureSkin tumors were induced using a two-stage model of carcinogenesis employing topical application of 7,12-Dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoyl phorbol-13-acetate (TPA) as carcinogen and promoter respectively. AAILE was administered orally to the animals. Male Laca mice were divided into four groups: control, AAILE, DMBA/TPA and AAILE + DMBA/TPA. ResultsThe tumors obtained in DMBA/TPA and AAILE + DMBA/TPA groups were histologically identified as SCC. Tumor induction in these groups was accompanied by raised serum carcinoembryonic antigen (CEA) levels when compared to control counterparts. Assessment of hydroxyproline levels and histochemical staining with sirius red and trichrome stain revealed an increase in collagen in tumors of DMBA/TPA group. An increase in glycosaminoglycans (GAGs) levels was also observed in DMBA/TPA group as made evident by biochemical studies and histochemical staining using mucicarmine and alcian blue-periodic acid schiff’s stain. Administration of AAILE to DMBA/TPA treated animals caused a decrease in collagen and GAG levels along with a decrease in serum CEA levels. ConclusionSkin tumors exhibited altered presence of ECM components which is indicative of a modified ECM. AAILE administration antagonised tumor associated ECM alterations which may be contributing to its chemopreventive activity as reported previously.

The effect of Azadirachta indica Leaf Extract on Early Stages of Chemically Induced Skin Cancer in Mice

ABSTRACTThe effect of Aqueous Azadirachta indica leaf extract (AAILE) administration during the early stages of chemically induced skin cancer in mice was assessed. 7,12-Dimethylbenz(a)anthracene was applied twice weekly for 2 weeks as a carcinogen followed by application of phorbol-12-myristate-13-acetate (twice weekly for 6 weeks) as a promoter on the skin of mice and AAILE was administered orally for 10 weeks. AAILE exhibited inhibitory effects in early stages as seen by decreased papilloma incidence and number. The decrease in cell proliferation in response to AAILE administration was apparent from the low epidermal thickness, epidermal cell count, and DNA/amide I ratio. The ameliorative effects on papilloma induction were associated with decreased expression of ornithine decarboxylase, ataxia telangiectasia mutated genes in skin, and serum lactate dehydrogenase activity.

NEUROPROTECTIVE POTENTIAL OF AZADIRACHTA INDICA LEAVES IN DIABETIC RATS

Objective: Azadirachta indica is a treasure of multiple pharmacological properties and presently leaves of this plant have been explored to evaluate the neuroprotective potential in diabetic rats.Methods: Male Sprague-Dawley rats were injected with single intra peritoneal dose of streptozotocin (60mg/ Kg body weight (BW.) to develop animal model of diabetes. Post twenty one days of streptozotocin induction, animals were treated with aqueous Azadirachta indica Leaf Extract (ALE, 600mg/Kg BW.) for seven consecutive days. Followed this, all animals were evaluated for the levels of blood glucose, lipid peroxidation (LPO), C Reactive Proteins (CRP), pro oxidant biomarkers and histological changes.Results: Streptozotocin treated rats exhibited elevated levels of blood glucose, LPO, CRP and altered pro oxidant biomarkers in comparison to control rats. Additionally, histological alterations/damage was evidenced as fragmentation, vacuolization, inflammation etc. However, ALE treatment to these rats significantly decreased blood glucose levels, LPO, CRP levels and restored pro-oxidants status. Light microscopic and ultra microscopic analysis also indicated less damage, tissue architectural changes in comparison to untreated diabetic rats. Further decrease in hyperalgesia and inflammation levels; along with protective and restorative changes following ALE treatment suggested the neuroprotective potential of Azadirachta indica leaves in diabetic rats.Conclusion: The oral administration of ALE to streptozotocin induced diabetic animals resulted in neuro-protection against degenerative oxidative stress associated with metabolic and histopathological damage in the brain.Key words: Azadirachta indica, Antioxidants, Hyperalgesia, Neuroprotection

Fourier Transform Infrared Spectroscopic Studies on Modulation of N-Nitrosodiethylamine-Induced Hepatocarcinogenesis by Azadirachta indica.

Fourier transform infrared spectroscopy was employed in the present study to obtain information about the molecular composition of hepatic tumor versus hepatic tissue. A hepatic cancer model was developed by administering N-nitrosodiethylamine (NDEA) to male Balb/c mice. The results revealed that NDEA-induced hepatic cancer tumor tissue had altered molecular composition compared with normal liver tissue. Compared with the normal tissue, the saturation level of membrane phospholipids was observed to be decreased in tumors along with an abnormal distribution of protein secondary structures. A significant decrease in glycogen and a significant increase in total nucleic acid content were also observed in tumor cells. The administration of aqueous Azadirachta indica leaf extract (AAILE) prior to NDEA treatment resulted in the normalization of saturation levels in phospholipids and total nucleic acid content and in the distribution of protein secondary structures in tumors. A significant increase in the amount of stored glycogen was observed in AAILE cotreated tumors compared with NDEA-induced tumors, which might indicate that AAILE cotreatment impeded the ability of tumor cells to consume glucose at a faster rate. The normalization of molecular composition upon AAILE cotreatment in hepatic tumors might indicate that AAILE hampered the process of evolution of tumors, which could be responsible for its observed chemopreventive action.

Azadirachta indica acts as a pro‐oxidant and modulates cell cycle associated proteins during DMBA/TPA induced skin carcinogenesis in mice

The present study was designed to determine the modulatory effect of aqueous Azadirachta indica leaf extract (AAILE) on cell cycle-associated proteins during two-stage skin carcinogenesis in mice. Considering the dual role of reactive oxygen species in cancer and its chemoprevention, the levels of lipid peroxidation (index of peroxidative damage) were also determined. Skin tumours were induced by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) as a carcinogen followed by the repetitive application of 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Skin tumours obtained in the DMBA/TPA group exhibited enhanced expression of proliferating cell nuclear antigen (PCNA, index of proliferation), p21 and cyclin D1, with no alterations in p53 expression in comparison to the control group. Tumours in AAILE + DMBA/TPA group exhibited low PCNA and cyclin D1 expression and enhanced expression of p53 and p21 in comparison to the DMBA/TPA group. The skin tumours obtained in the AAILE + DMBA/TPA group exhibited high lipid peroxidation levels in comparison to the tumours obtained in the DMBA/TPA group. The observations of the present study suggest that AAILE behaves as a pro-oxidant in the tumours, thereby rendering them susceptible to damage, which eventually culminates into its anti-neoplastic action. Also, cell cycle regulatory proteins may be modulated by AAILE and could affect the progression of cells through the cell cycle.

Histopathological, Enzymatic, and Molecular Alterations Induced By Cigarette Smoke Inhalation in the Pulmonary Tissue of Mice and Its Amelioration By Aqueous Azadirachta Indica Leaf Extract

The modulatory and protective effects of aqueous Azadirachta indica leaf extract (AAILE) had been investigated against cigarette smoke-induced pulmonary oxidative stress and risk of lung cancer. Male Balb/c mice were divided in 4 groups: control, cigarette smoke (CS), AAILE, and AAILE + CS. Animals in the CS group were exposed to cigarette smoke for a period of 10 weeks. AAILE oral administration at a dosage of 100 mg/kg body weight was started 2 weeks prior to CS exposure and continued until the termination of the experiment. The pulmonary tissue was analyzed for molecular changes and assessed for the risk of lung cancer and status of pulmonary detoxification enzymes. An increase in CH2/CH3 and decrease in PO2/amide II observed in Fourier transform infrared spectrometry correlated well with acute inflammation, infiltration, and lipid peroxidation levels observed in histopathological studies and malionaldehyde levels in mice exposed to CS. No change was observed in the amide I:amide II ratio. The subtle balance between carcinogen activation and detoxification was found to be derailed, and it was manifested as a 1.5-times increase in the risk of developing lung cancer in the CS-exposed group. However, these changes seemed to normalize upon AAILE supplementation.

Azadirachta indica Exerts Chemopreventive Action Against Murine Skin Cancer: Studies on Histopathological, Ultrastructural Changes and Modulation of NF-κB, AP-1, and STAT1

The present study reports the histopathological, ultrastructural changes and modulation of NF-kappaB, AP-1, and STAT 1 during skin carcinogenesis in LACA mice and its intervention with Azadirachta indica. Skin tumors were induced by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) (500 nmol/100 microl for 2 weeks) followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol/100 microl of acetone, twice weekly) as a promoter. Male LACA mice were divided into four groups: Control, DMBA/TPA, aqueous Azadirachta indica leaf extract (AAILE), and AAILE + DMBA/TPA. AAILE was administered orally at a dose level of 300 mg/kg body weight three times a week for 20 weeks. Topical application of DMBA/ TPA to the skin resulted in well-developed squamous cell carcinomas characterized by hyperproliferation, hyperkeratosis, and corrugation of the epidermis. Degenerative changes were observed in the tumors of AAILE + DMBA/TPA-treated animals. Scanning electron microscopy revealed surface disruptions and certain rounded structures on the skin tumors of DMBA/TPA-treated animals. Topographical changes were also observed in the tumors of AAILE + DMBA/TPA-treated animals, which resembled regions of degeneration. Tumors obtained in DMBA/TPA group were associated with enhanced expression of NF-kappaB and AP-1 when compared to the control counterparts. Inhibition in tumorigenesis in response to A. indica treatment was accompanied by an overexpression of STAT 1 and AP-1 and decrease in NF-kappaB expression. The results of the present study provide a basis for the chemopreventive potential of A. indica against murine skin carcinogenesis.

Chemopreventive activity of Azadirachta indica on two‐stage skin carcinogenesis in murine model

The present study reports the chemopreventive activity of aqueous Azadirachta indica leaf extract (AAILE) in a murine two-stage skin carcinogenesis model. Skin tumors were induced by topical application of 7,12-dimethylbenz(a)anthracene (DMBA) (500 nmol/100 µL for 2 weeks) followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) (1.7 nmol/100 µL of acetone, twice weekly) as a promoter. Male LACA mice were divided into four groups: control, DMBA/TPA, AAILE and AAILE + DMBA/TPA. AAILE was administered orally at a dose level of 300 mg/kg body weight thrice a week for 20 weeks. 100% tumor incidence was observed in the DMBA/TPA treated animals, whereas the AAILE + DMBA treated animals exhibited a tumor incidence of 58.3% only. A significant reduction in the mean tumor burden (54.5%) and mean tumor volume (45.6%) was observed in the mice that received AAILE along with DMBA/TPA. Topical application of DMBA/TPA to the skin resulted in well-developed carcinomas associated with decreased expression of pro-apoptotic protein such as caspase 3 and enhanced expression of antiapoptotic protein such as bcl-2 when compared with the control counterparts. However, adminstration of AAILE inhibited skin carcinogenesis with induction of pro-apoptotic proteins such as bax, caspase 3, caspase 9 and inhibition of antiapoptotic proteins such as bcl-2. These results suggest that the induction of apoptosis may be one of the mechanisms underlying the chemopreventive effects of A. indica.

Azadirachta indica modulates carcinogen biotransformation and reduced glutathione at peri‐initiation phase of benzo(a)pyrene induced murine forestomach tumorigenesis

The present study evaluated the effects of aqueous Azadirachta indica leaf extract (AAILE) on the activities of certain phase I (cytochrome P450, cytochrome b(5) and aryl hydrocarbon hydroxylase) as well as phase II (glutathione-S-transferase and UDP-glucuronosyl transferase) biotransformation enzymes; and reduced glutathione (GSH) (in forestomach and hepatic tissues) during/after intra-gastric instillations of B(a)P in murine forestomach tumorigenesis bioassay protocol. The activities of phase I biotransformation enzymes were found to increase, whereas a decrease in GSH content as well as glutathione-S-transferase was observed in mice receiving only B(a)P during as well as 2 weeks after B(a)P instillations. The activity of UDP-glucuronosyltransferase decreased during B(a)P instillations, whereas after the latter, the activity increased when compared with the control mice. However, in mice that received AAILE along with B(a)P instillations, a decrease in phase I enzymes was accompanied by an increase in phase II enzymes as well as GSH contents. Only AAILE treatment reduced the activities of phase I biotransformation enzymes and enhanced the GSH contents as well as the activities of phase II enzymes. Observations of the present study seem to be quite significant and (when taken together with our earlier findings) provides evidence for A. indica mediated modulation of the peri-initiation phase of the process of forestomach tumorigenesis.

Histochemical, Ultrastructural, and Biochemical Evidences for Azadirachta indica−Induced Apoptosis in Benzo(a)pyrene−Induced Murine Forestomach Tumors

The present study reports aqueous Azadirachta indica leaf extract (AAILE)-mediated induction of apoptosis in a murine forestomach tumorigenesis model. Histochemistry-based quantification of apoptosis revealed enhanced apoptotic index in the forestomach tumors of mice receiving AAILE along with benzo(a)pyrene (B(a)P). Transmission electron microscopy confirmed the presence of classical morphological features of apoptosis including chromatin condensation/marginalization, nuclear fragmentation, and formation of apoptotic bodies. Scanning electron microscopy showed surface modifications on the transformed squamous epithelial cells and certain mitotic cells among them over the forestomach tumors of mice receiving only B(a)P. In tumors of the mice receiving AAILE along with B (a)P, such mitotic cells were found to be absent; however, certain cells showing shrinkage and blebbings (characteristics of apoptosis) were observed. DNA fragmentation was observed to increase exclusively in the tumors of mice that received AAILE along with B(a)P. Lipid peroxidation (LPO) levels decreased in forestomach tissues of mice in all the groups studied when compared to control counterparts. However, levels of LPO were found to increase in the tumorous tissue of mice that received AAILE along with B(a)P when compared to mice receiving only B(a)P. Taken together, observations of the present study suggest that A. indica induces apoptosis in B(a)P-induced murine forestomach tumors.

Preventive effects of Azadirachta indica on benzo(a)pyrene‐DNA adduct formation in murine forestomach and hepatic tissues

In the present investigation, the effects of aqueous Azadirachta indica leaf extract (AAILE) on (3)H-benzo(a)pyrene-DNA [(3)H-B(a)P-DNA] adduct formation, the status of biotransformation enzymes and reduced glutathione (GSH) content were evaluated in the forestomach and liver of Balb/c mice. Two weeks of AAILE treatment reduced the (3)H-B(a)P-DNA adduct levels by 31.6% in forestomach tissue. Similarly, (3)H-B(a)P-DNA adduct levels were decreased by 34.7% in the liver of AAILE treated mice compared with their control counterparts. After AAILE treatment, the cytochrome P450 content decreased, whereas the GSH content increased significantly in the hepatic tissue. In the forestomach as well as in the liver, the cytochrome b5 content declined, whereas an increase in glutathione-S-transferase (GST) activity was observed in both tissues. These observations suggested that AAILE may have reduced the metabolic activation of (3)H-B(a)P with enhanced detoxification of its active metabolites, hence the observed decrease in the levels of (3)H-B(a)P-DNA adducts. These molecular and biochemical modulations observed at the initiation phase of carcinogenesis seems to be significant and could be correlated with the chemopreventive effects of A. indica against B(a)P induced forestomach tumorigenesis.

Chemomodulatory effects of Azadirachta indica on the hepatic status of skin tumor bearing mice

The liver plays an important role in the modulation of the process of carcinogenesis, as it is the primary site for the biotransformation of xenobiotics including carcinogens as well as anticancer drugs. The present study was designed to evaluate the biochemical alterations occurring in the liver of 7,12-dimethylbenz(a)anthracene (DMBA) induced skin tumor bearing male Balb/c mice and their modulation by aqueous Azadirachta indica leaf extract (AAILE). It was observed that skin tumor induction caused hepatic damage characterized by a decreased hepatosomatic index and significantly increased (p < 0.001) activities of the hepatic tissue injury marker enzymes, namely alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase. However, upon treatment with AAILE, the above-mentioned alterations, including the increased activities of hepatic tissue injury marker enzymes, were significantly reversed, which signified the hepato-protective efficacy of Azadirachta indica. Increased oxidative stress was also observed in the hepatic tissue of skin tumor bearing mice as revealed by a significant increase (p < 0.001) in lipid peroxidation levels and a decrease in reduced glutathione contents and activities of various antioxidant enzymes studied, namely glutathione-S-transferase, glutathione peroxidase and glutathione reductase. The AAILE treatment reduced oxidative stress by decreasing lipid peroxidation levels and enhancing the reduced glutathione contents and activities of various antioxidant enzymes. The activities of the xenobiotic biotransformation enzymes, namely cytochrome P450, cytochrome b5 and glutathione-S-transferase, were found to be decreased in the hepatic tissue of tumor bearing mice. Treatment with AAILE further caused a decrease in the activity of cytochrome P450 and cytochrome b5, whereas it up-regulated the activity of glutathione-S-transferase. The significance of these observations with respect to the progress of the process of carcinogenesis is explained in the present research article.

Inhibitory effects of Azadirachta indica on DMBA-induced skin carcinogenesis in Balb/c mice

Male Balb/c mice were divided into four groups on the basis of their respective treatments wherein mice of Group I served as controls. For induction of skin tumors, mice of Group II and IV were injected sub-cutaneously with 7,12-dimethylbenz(a)anthracene (DMBA). Mice of Group III and IV were administered aqueous Azadirachta indica leaf extract (AAILE) thrice a week throughout the experiment. After 14 weeks of the first DMBA injection, Group II and IV mice developed tumors. In the tumor-bearing mice that received AAILE (Group IV), a significant reduction in mean tumor burden and tumor volume was observed. The tumors were confirmed to be papillomas and interestingly, the extent of hyper-chromatia was observed to be much more in skin tumors of Group II mice vis a vis the mice receiving AAILE. An increase in the extent of lipid peroxidation was observed in tumorous tissue of Group IV when compared to that of Group II mice. Glutathione (GSH) content and the activities of GSH-based antioxidant enzymes viz. glutathione peroxidase (GPx) and glutathione reductase (GR) increased significantly in the skin tissues of all the groups of mice when compared to control counterparts. Catalase activity was found to decrease significantly in the skin of mice, which received AAILE treatment only (Group III). Activity of super-oxide dismutase (SOD) decreased significantly in all the tumorous tissues (Group II and IV mice). In light of the above observations, the role of AAILE in inhibition of DMBA-induced skin carcinogenesis is discussed in the present study.

Hematological and biochemical parameters in common carp, Cyprinus carpio, following herbal treatment for Aeromonas hydrophila infection

The common carp, Cyprinus carpio was injected 108 cfu/ml with a strain of the Gram-negative bacterium, Aeromonas hydrophila. After inoculation, the disease signs began on the 7th day as a haemorrhagic spot at the site of injection and the lesion subsequently progressed in size. After this period, the mortality of infected group was 10±5% daily; hence, they were dip treated with an aqueous Azadirachta indica leaf extract at 1 g/l for 10 min daily for 30 days until the lesions healed completely. The hematological and biochemical parameters of the infected and control fishes were monitored on the 10th, 20th and 30th day. The white blood cell (WBCs: 104 mm−3) counts significantly increased from 3.15±0.14 in control fish to 3.6±0.20 on the 10th day of treatment (P 0.05) on the 30th day. The red blood cell (RBC: 106 mm−3) counts also significantly decreased to 1.68±0.12 on the 10th day (P 0.05) values on the 30th day. In infected fishes the values continued to decrease significantly from 2.76±0.69 on the 10th day to 2.38±0.39 on the 30th day. Serum glucose, cholesterol and serum calcium levels were significantly lower in control fishes (119±16.93, 10.00±1.39 and 4.88±0.34) compared to treated fishes (77.63±15.57, 6.11±1.55 and 3.62±0.39) on the 10th day of treatment. In the infected fishes, the values continued to decrease significantly from the 10th day (64.6±11.97, 4.37±1.39 and 3.30±0.46) and by the 30th day reached 56.10±9.56, 4.31±0.91 and 2.88±0.52, respectively.