Safety evaluation of neem (Azadirachta indica) derived pesticides

The neem tree, Azadirachta indica, provides many useful compounds that are used as pesticides and could be applied to protect stored seeds against insects. However in addition to possible beneficial health effects, such as blood sugar lowering properties, anti-parasitic, anti-inflammatory, anti-ulcer and hepatoprotective effects, also toxic effects are described. In this study we present a review of the toxicological data from human and animal studies with oral administration of different neem-based preparations. The non-aqueous extracts appear to be the most toxic neem-based products, with an estimated safe dose (ESD) of 0.002 and 12.5 microg/kg bw/day. Less toxic are the unprocessed materials seed oil and the aqueous extracts (ESD 0.26 and 0.3 mg/kg bw/day, 2 microl/kg bw/day respectively). Most of the pure compounds show a relatively low toxicity (ESD azadirachtin 15 mg/kg bw/day). For all preparations, reversible effect on reproduction of both male and female mammals seem to be the most important toxic effects upon sub-acute or chronic exposure. From the available data, safety assessments for the various neem-derived preparations were made and the outcomes are compared to the ingestion of residues on food treated with neem preparations as insecticides. This leads to the conclusion that, if applied with care, use of neem derived pesticides as an insecticide should not be discouraged.     

Radiosensitizing effects of neem (Azadirachta indica) oil.

Radiosensitization by neem oil was studied using Balbc/3T3 cells and SCID cells. Neem oil enhanced the radiosensitivity of the cells when applied both during and after x-irradiation under aerobic conditions. Neem oil completely inhibited the repair of sublethal damage and potentially lethal damage repair in Balbc/3T3 cells. The cytofluorimeter data show that neem oil treatment before and after x-irradiation reduced the G(2) + M phase, thus inhibiting the expression of the radiation induced arrest of cells in the G(2) phase of the cell cycle. However, SCIK cells (derived from the SCID mouse), deficient in DSB repair, treated with neem oil did not show any enhancement in the radiosensitivity. There was no effect of neem oil on SLD repair or its inhibition in SCIK cells. These results suggest that neem oil enhanced the radiosensitivity of cells by interacting with residual damage after x-irradiation, thereby converting the sublethal damage or potentially lethal damage into lethal damage, inhibiting the double-strand break repair or reducing the G(2) phase of the cell cycle.     

Molluscicidal activity of neem (Azadirachta indica A.Juss)

Molluscicidal property of Azadirachta indica A. Juss (neem) against the snails Lymnaea acuminata and Indoplanorbis exustus was studied. It was observed that the molluscicidal activity of the leaf, bark, cake, neem oil and the neem-based pesticides, achook and nimbecidine, was both time- and dose-dependent. The toxic effect of pure azadirachtin against both the snails was greater than the synthetic molluscicides.     

Anti-microbial activity of a new vaginal contraceptive NIM-76 from neem oil (Azadirachta indica)

Efficacy of NIM-76, a spermicidal fraction from neem oil, was investigated for its antimicrobial action against certain bacteria, fungi and Polio virus as compared to whole neem oil. The NIM-76 preparation showed stronger anti-microbial activity than the whole neem oil. It inhibited growth of various pathogens tested including Escherichia coli and Kleibsiella pneumoniae which were not affected by the whole neem oil. NIM-76 also exhibited antifungal activity against Candida albicans and antiviral activity against Polio virus replication in vero cell lines. It also protected mice from systemic candidiasis as revealed by enhanced % survival and reduced colony forming units of C. albicans in various tissues. This shows that NIM-76 has a potent broad spectrum anti-microbial activity.     

Isolation and characterization of an antimalarial agent of the neem tree Azadirachta indica

The isolation and structure elucidation of gedunin [1], the antimalarial agent of Azadirachta indica, are reported. Its 1H- and 13C-nmr spectra were assigned by using one- and two-dimensional nmr spectroscopy, especially homonuclear and heteronuclear COSY, nOe difference, and COLOC experiments.     

Cytotoxic and apoptosis-inducing activities of triterpene acids from Poria cocos

Six lanostane-type triterpene acids (1a-6a), isolated from Poria cocos , and their methyl ester (1b-6b) and hydroxy derivatives (1c-6c) were prepared. Upon evaluation of the cytotoxic activity of these compounds against leukemia (HL60), lung (A549), melanoma (CRL1579), ovary (NIH:OVCAR-3), breast (SK-BR-3), prostate (DU145), stomach (AZ521), and pancreas (PANC-1) cancer cell lines, 11 compounds (5a, 6a, 2b-5b, 1c, and 3c-6c) exhibited activity with single-digit micromolar IC(50) values against one or more cell lines. Poricotriol A (1c), a hydroxy derivative of poricoic acid A (1a), exhibited potent cytotoxicities against six cell lines with IC(50) values of 1.2-5.5 μM. Poricotriol A induced typical apoptotic cell death in HL60 and A549 cells on evaluation of the apoptosis-inducing activity by flow cytometric analysis. Western blot analysis in HL60 cells showed that poricotriol A activated caspases-3, -8, and -9, while increasing the ratio of Bax/Bcl-2. This suggested that poricotriol A induced apoptosis via both mitochondrial and death receptor pathways in HL60. On the other hand, poricotriol A did not activate caspases-3, -8, and -9, but induced translocation of apoptosis-inducing factor (AIF) from mitochondria and increased the ratio of Bax/Bcl-2 in A549. This suggested that poricotriol A induced apoptosis via the mitochondrial pathway mostly by translocation of AIF, independent from the caspase pathway in A549. Furthermore, poricotriol A was shown to possess high selective toxicity in lung cancer cells since it exhibited only weak cytotoxicity against a normal lung cell line (WI-38).     

Inhibitory potential of neem (Azadirachta indica Juss) leaves on dengue virus type-2 replication.

In the present study we report in vitro and in vivo inhibitory potential of crude aqueous extract of neem leaves and pure neem compound (Azadirachtin) on the replication of Dengue virus type-2. In vitro antiviral activity of aqueous neem leaves extract assessed in C(6/36) (cloned cells of larvae of Aedes albopictus) cells employing virus inhibition assay showed inhibition in dose dependent manner. The aqueous extract of neem leaves at its maximum non-toxic concentration of 1.897 mg/ml completely inhibited 100-10,000 TCID(50) of virus as indicated by the absence of cytopathic effects. The in vivo protection studies with neem leaves extract at its maximum non-toxic concentrations 120-30 mg/ml resulted in inhibition of the virus replication as confirmed by the absence of Dengue related clinical symptoms in suckling mice and absence of virus specific 511 bp amplicon in RT-PCR. The pure neem i.e. Azadirachtin did not reveal any inhibition on Dengue virus type-2 replication in both in vitro and in vivo systems.     

Early post implantation contraceptive effects of a purified fraction of neem (Azadirachta indica) seeds, given orally in rats: possible mechanisms involved

Neem seed and leaf extracts have immunomodulators that induce cellular immune reactions. These aspects of neem were exploited in earlier studies, where the oral administration of the neem seed extracts in rodents and primates could completely abrogate pregnancy at an early post implantation stage. Complete restoration of fertility was observed in the animals treated in the subsequent cycles. For the purpose of using neem as a long term contraceptive, an activity guided fractionation, followed by identification and characterization of the biologically active fraction from neem seeds was carried out. Sequentially extracted fractions of neem seeds were tested orally at an early post implantation stage in rats. The hexane extract of the neem seeds was found to be biologically active and was the precursor for the final active fraction. The active fraction, identified as a mixture of six components, could completely abrogate pregnancy in rodents up to a concentration of 10%. No apparent toxic effects could be seen following treatment with the fraction. The treatment with the active fraction caused a specific activation of T lymphocyte cells of CD8+ subtype as well as phagocytic cells followed by elevation in cytokines gamma-interferon and TNF. The results of the present study show that a pure active fraction of neem seeds could be obtained for the purpose of early post implantation contraception when given orally, and its mechanism of action seems to be by activating cell mediated immune reactions.     

Pretreatment with neem (Azadirachta indica) leaf preparation in Swiss mice diminishes leukopenia and enhances the antitumor activity of cyclophosphamide

Cancer chemotherapy is associated with several life threatening complications, including bone marrow suppression and leucopenia. To overcome this problem, colony stimulating factor (CSF), granulocyte colony stimulating factor (GCSF) and granulocyte macrophage colony stimulating factor (GMCSF), can be used, however, these therapeutics are expensive and have several disadvantages, including tumor growth promoting activities. This study attempted to use an immunostimulatory neem (Azadirachta indica) leaf preparation (NLP) to prevent the cyclophosphamide (CYP) induced reduction in the WBC count. Pretreatment of mice with NLP reduced the extent of leucopenia and neutropenia in normal and tumor bearing CYP treated mice. NLP pretreatment enhanced in vitro tumor cell cytotoxicity by peripheral blood mononuclear cells (PBMC) from CYP treated mice in either normal or tumor bearing conditions. Similarly, NLP pretreatment of mice enhanced the CYP mediated in vivo tumor growth inhibition and survivability of the host. Based on these observations, it is concluded that NLP would be an effective tool to reduce CYP-induced hematological complications.     

Cytoprotective and Anti‐secretory Effects of Azadiradione Isolated from the Seeds of Azadirachta indica (neem) on Gastric Ulcers in Rat Models

Azadirachta indica is well known medicinal plant mentioned in ancient herbal texts. It has been extensively used in Ayurvedic, Unani and Homoeopathic medicine and has become a luminary of modern medicine. As part of our drug discovery program we isolated azadiradione from the ethanolic extract of seeds of A. indica and evaluated for in‐vivo antiulcer activity in cold restraint induced gastric ulcer model, aspirin induced gastric ulcer model, alcohol induced gastric ulcers model and pyloric ligation induced ulcer model. Azadiradione exhibited potent antiulcer activity through the inhibition of H+ K+‐ATPase (proton pump) activity via its cytoprotective effect and also via its antisecretory effect. This combined effect has valuable potential in the future treatment of peptic ulceration. Copyright © 2015 John Wiley & Sons, Ltd.     

Antiproliferative effect on human cancer cell lines after treatment with nimbolide extracted from an edible part of the neem tree (Azadirachta indica)

Nimbolide, a triterpenoid extracted from the flowers of the neem tree (Azadirachta indica), was found to have antiproliferative activity against some cancer cell lines. Treatment of cells with 0.5-5.0 microm concentrations of nimbolide resulted in moderate to very strong growth inhibition in U937, HL-60, THP1 and B16 cell lines. Flow cytometric analysis of U937 cells showed that nimbolide treatment (1-2.5 microm) resulted in cell cycle disruption by decreasing the number of cells in G0/G1 phase, with initial increases in S and G2/M phases. Cells exposed to a higher dose of nimbolide for a longer period displayed a severely damaged DNA profile, resulting in a remarkable increase in the number of cells in the sub-G1 fraction, with a reciprocal decrease of cells in all phases. Quantification of the expression of phosphatidylserine in the outer cell membrane showed that doses of nimbolide higher than 0.4 microm exerted remarkable lethality, with over 60% of cells exhibiting apoptotic features after exposure to 1.2 microm nimbolide. The antiproliferative effect of nimbolide and its apoptosis-inducing property raise hope for its use in anticancer therapy by enhancing the effectiveness of cell cycle disruption.     

Chemopreventive potential of neem (Azadirachta indica) on 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis

The inhibitory effect of the aqueous extract of neem (Azadirachta indica A. Juss.) on 7,12-dimethylbenz[a]anthracene (DMBA) induced buccal pouch carcinogenesis was investigated in Syrian male hamsters. All hamsters painted on their buccal pouch with DMBA for 14 weeks developed squamous cell carcinoma. Administration of neem leaf extract effectively suppressed oral carcinogenesis initiated with DMBA as revealed by the reduced incidence of neoplasms. Lipid peroxidation, glutathione (GSH) content and the activities of glutathione peroxidase (GPx), glutathione S-transferase (GST) and gammaglutamyl transpeptidase (GGT) were used to biomonitor the chemopreventive potential of neem. Lipid peroxidation was found to be significantly decreased, whereas GSH, GPx, GST and GGT were elevated in the oral mucosa of tumour bearing animals. Our data suggest that neem may exert its chemopreventive effects in the oral mucosa by modulation of lipid peroxidation, antioxidants and detoxification systems.     

Screening Azadirachta indica and Pisum sativum for possible antimalarial activities

Solvent-free extracts obtained from the leaves of Azadirachta indica and Pisum sativum were screened for antimalarial action using Plasmodium berghei in mice. Four days of oral dosing with 500 mg/kg and 125 mg/kg of the methanol extract of A. indica showed a parasite suppression which was statistically significant although all test animals died after 5 days, just 1 day longer than the untreated control group. A 50 mg/kg oral dose of the aqueous extract of P. sativum was found to have significant prophylactic activity by producing a parasite suppression of 31.9%.     

Evaluation of mutagenic and antimutagenic activities of neem (Azadirachta indica) seed oil in the in vitro Ames Salmonella/microsome assay and in vivo mouse bone marrow micronucleus test

Aim of the study

The possible mutagenic and antimutagenic activity of neem oil (NO) and its DMSO extract (NDE) were, examined in the Ames Salmonella/microsome mutagenicity test and the mouse bone marrow micronucleus assay.

Materials and methods

Eight different strains of Salmonella typhimurium were, used to study the genotoxicity of neem oil both in the presence and absence of Aroclor-1254 induced rat liver homogenate (S9). Two-dose treatment protocol was, employed to study the cytogenetic activity in micronucleus assay. Similarly, the antimutagenic activity of neem oil and NDE was studied against mitomycin (MMC) and 7,12-dimethylbenz[a]anthracene (DMBA) in the above two test systems.

Results

Neem oil was non-mutagenic in all the eight tester strains of Salmonella typhimurium both in the presence and absence of S9 mix. In the present study, there was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in neem oil treated groups over the negative control (DMSO) group of animals, indicating the non-clastogenic activity of neem oil in the micronucleus test. Neem oil showed good antimutagenic activity against DMBA induced mutagenicity compared to its DMSO extract. However, neem oil showed comparatively less antimutagenicity against MMC in the Ames assay. In vivo anticlastogenic assays shows that neem oil exhibited better activity against DMBA induced clastogenicity.

Conclusion

These results indicate non-mutagenic activity of neem oil and significant antimutagenic activity of neem oil suggesting its pharmacological importance for the prevention of cancer.     

Concurrent administration of aqueous Azadirachta indica (neem) leaf extract with DOCA-salt prevents the development of hypertension and accompanying electrocardiogram changes in the rat

The effect of concurrent administration of Azadirachta indica leaf extract with DOCA-salt was investigated in the development of hypertension.Over 5-6 week old, inbred male Wistar rats with a starting weight of 190 g were given either: (1) twice weekly subcutaneous (s.c.) injections of vehicle (soyabean oil, 0.25 mL per animal) for the first 2 weeks, plus normal drinking water (controls); (2) twice weekly (s.c.) injections (weeks 1 and 2 only) of 15 mg/kg DOCA dissolved in vehicle, plus drinking water containing 1.0% NaCl and 0.03% KCl (DOCA-salt group); or (3) 20 mg/kg of aqueous neem extract daily, in addition to the DOCA-salt treatment (DOCA-salt-neem group). All groups (8-12 animals) received normal rat pellets ad libitum and their BP was measured weekly. Terminally, the animals were anaesthetized and ECGs recorded using s.c. pins in a lead II configuration.The mean arterial pressure was significantly lower (p < 0.05) in the control (97 +/- 3.7 mmHg) and DOCA-salt-neem (87 +/- 3.4 mm Hg) groups than in the DOCA-salt group (115 +/- 7.1 mm Hg). PR and RR intervals and the duration of the QRS complex were shorter (p < 0.05) in the DOCA-salt group than in the control and DOCA-salt-neem groups. Amplitude of the QRS complex was increased (p < 0.05) in the DOCA-salt group compared with both the DOCA-salt-neem and the control groups.Daily administration of 20 mg/kg neem-leaf extract concurrently with DOCA-salt for 5 weeks, prevents the development of hypertension and the accompanying alterations in the ECG patterns seen in DOCA-salt treated rats.     

Characterization of anti-complement compounds from Azadirachta indica

The crude aqueous extract of Azadirachta indica bark possesses an inhibitory activity on both classical (CP) and alternative pathway (AP) activation of human complement. Purification of the compounds with the guidance of the AP-inhibitory activity involved extraction with methanol, dialysis, ion-exchange procedures and gel-permeation chromatography. This sequence yielded two polymers, NB-I and NB-II, one a highly active compound with a relatively low molecular weight (NB-II) and the other a less active compound with a high molecular weight (NB-I). The polymers were characterized by using colour reactions, TLC, GLC and HPLC after hydrolysis and gel-permeation chromatography as peptidoglycans. The carbohydrate part consisted predominantly of glucose. Arabinose, galactose and mannose were present in minor amounts (NB-II) or only as traces (NB-I). Protein was present for 5.5% in NB-I and for 9.8% in NB-II.     

Two new triterpenoids from Azadirachta indica and their insecticidal activity

Two new triterpenoids, 22,23-dihydronimocinol (1) and desfurano-6alpha-hydroxyazadiradione (2), were isolated from a methanolic extract of the fresh leaves of Azadirachta indica (neem) along with a known meliacin, 7alpha-senecioyl-(7-deacetyl)-23-O-methylnimocinolide. The structures of 1 and 2 were elucidated through spectral and chemical studies. Compounds 1 and 2 showed mortality for fourth instar larvae of the mosquito (Anopheles stephensi), with LC(50) values of 60 and 43 ppm, respectively.     

苦楝皮中5个柠檬苦素化合物的细胞毒活性以及构效关系（英文）

目的：研究柠檬苦素化合物1-5的细胞毒活性以及阐明其构效关系。方法：从苦楝皮中分离获得化合物1-5,采用MTT法进行3种肿瘤细胞株的体外细胞毒活性实验。结果：化合物1-5均表现出潜在的抗肿瘤活性。尤其是化合物1对人肝癌BEL7402细胞的IC508.57μg·mL-1,对人肺癌H460细胞的IC506.29μg·mL-1,对人胃癌SGC-7901细胞的IC506.08μg·mL-1,较其他化合物表现出更强的细胞毒活性。结论：化合物1-5的构效关系研究显示16位羟基为柠檬苦素化合物具备细胞毒活性的重要基团。     

On the toxicology of Azadirachta indica leaves.

Brown Hisex chicks were fed diets containing 2% and 5% Azadirachta indica leaf from their 7th to 35th day of age. Thereafter, the chicks were fed control diet for 2 weeks. A depression in body weight gain and efficiency of feed utilization was observed in chicks fed A. indica leaf when compared with the control. The main clinicopathological changes were increases in lactic dehydrogenase, glutamic oxaloacetic transaminase and alkaline phosphatase activities and uric acid and bilirubin concentrations and decreases in the total protein levels in serum. Changes in the values of erythrocyte count, haemoglobin concentration, packed cell volume, mean corpuscular volume and mean corpuscular haemoglobin were remarkable and associated with yellow discoloration on the legs and combs and hepatonephropathy. Tissue recovery was incomplete 2 weeks after removal from the experimental diets.