Introduction

The neem tree, botanically referred to as Azadirachta indica (A. Juss) is a fast growing hardy and evergreen tropical and sub-tropical plant belonging to the same family as mahogany, Meliaceae. They thrive in climates with annual rainfall of 400–800 mm and an extended dry season, even with poor soils (Schmutterer 1990; Ajayi 2002). It is believed to be native to the whole Indo-Pakistan sub-continent, especially southeast Asia (India, Burma/Myanmar, Sri Lanka, Thailand, Malaysia and Indonesia), from where it was introduced to Nigeria in 1928 through Ghana by a man named ‘Dogon Yaro’ and hence its local name in northern Nigeria (Sara and Folorunso 2002).

The neem tree gained prominence in northern Nigeria due to its adoption as a desertification control plant, and the subsequent planting of 14 million neem seedlings in 1978 by the Federal Government of Nigeria under the Arid Zone Afforestation project (AZAP). According to Sara and Folorunso (2002), the population of neem trees in the landscape of the dry lands of Nigeria was further boosted in 1986 when 70% of the 1 billion tree seedlings planted under the World Bank-Assisted Forestry II project was neem.

Azadirachta indica is also commonly found in other African countries like Ghana, Togo, Niger, Chad, Cameroon, Ethiopia, Sudan, Somalia, Kenya, Tanzania, Mozambique, Burkina Faso, and Cote’ Devoire.

In addition to its use in afforestation programs, authors from different countries have referred to it as “miracle tree”, “multipurpose crop”, “village dispensary” and “living pharmacy” because of its multiple uses. In fact, in its Asian countries of origin, every part of the neem tree has been extensively used in ayurveda, unani and homeopathic medicine as household remedy against various human ailments from antiquity, leading Biswas et al. (2002) to conclude that it is a cynosure of modern medicine. In this review, we critically evaluate the literature to provide evidence that neem tree is indeed a “multipurpose crop” or “living pharmacy” due to its multivariate pharmacological properties. For the purpose of this article, the name “neem” and A. indica A. Juss shall be used interchangeably and synonymously.

In this review, existing information on the biological and pharmacological activities attributed to different parts and extracts of the plant, A. indica commonly called neem, including antiplasmodial, antitrypanosomal, antioxidant, anticancer, antibacterial, antifungal, antiviral, nematicidal, antiulcer, spermicidal, anthelminthic, antidiabetic, anti-implantation, as well as immunomodulating, contraceptive, molluscidal, insecticidal, antifeedant, insect repellant and toxicological effects are critically evaluated. The review will attempt to update earlier information on the subject (Biswas et al. 2002; Brahmachari 2004; Subapriya and Nagini 2005). The literature considered are those abstracted on the Medline or CABS covering the period, 1965 to October, 2007. However, supplementary information released while the manuscript was undergoing review (Bhattacharyya et al. 2007; Girish and Shankara 2008) were also used to update the article. The keywords combination for the search was: A. indica or neem in combination with any of the desired biological or pharmacological activities. Additional information was obtained based on lead by other web sites on the internet, which are not reflected on the Medline or CABS. The review is structured into the different biological and pharmacological activities under consideration.

Anthelminthic properties

There are several reports on the anthelmintic property of A. indica in domestic and economic animals. In Kenya, Githiori et al. 2004 compared aqueous extracts of A. indica with seven other plants used traditionally for treatment of gastrointestinal helminthes, including, Haemoncus cortus in sheep. Their result showed no significant reduction in fecal egg count (FEC) and total worm counts (TWC) for any of the plants tested, including A. indica. In monogastric host–parasite model system (Githiori et al. 2003), it was shown earlier that A. indica had no significant effect on Heligmosomoides polygyrus infection. Similarly, Hordegen et al. (2003) did not observe any significant reduction in FEC and TWC of Trichostrongyloids in small ruminants when they were administered 3 mg/kg bw of an aqueous ethanol extract (70% v/v) of the seeds of A. indica. Although limited in number and experimental design, some of these reports appear to cast doubt on the widely believed anthelminthic effect of different extracts of different parts of A. indica in traditional ethno-veterinary and ethnomedical practices.

Antifertility effects of neem

Experiment in different animal models appears to suggest that different extracts of various parts of A. indica may have varied effects on fertility in male and female animals. Therefore, these effects will be discussed under two sub-headings, namely, effect on male fertility and effect on female fertility.

Effect on male fertility

Using Sander Cramer Test, Khillare and Shrivastav (2003) demonstrated that aqueous extracts of old and tender A. indica leaves immobilized and killed human spermatozoa within 20 s with minimum effective spermicidal concentrations of 2.91 ± 0.669 and 2.75 ± 0.754 mg/ml sperm, respectively. This spermicidal effect of leaf extracts of A. indica had earlier been reported in albino rats and mice (Aladakatti and Ahamed 1999; Aladakatti et al. 2001; Joshi et al. 1996; Choudhary et al. 1990; Deshpande et al. 1980). Similarly, other workers had reported on the male antifertility activity of neem seed oil in rats and mice (Upadhyay et al. 1993) as well as monkeys (Bardhan et al. 1991).

Studies on the mechanism by which A. indica may influence male fertility reveal that many processes may be at play. For example, ultrastructural studies by Ghodesawar et al. (2004) on Cauda epididymidal cell types revealed hypoandrogenic effect as characterized by enlarged nuclei of principal cells, reduced number of coated micropinocytotic vessels of the apical cytoplasm, missing microvilli and mitochondrial cristae, Golgi complex disruption, abounding lysosomal bodies in the cytoplasm, decreased chromatin content of nuclei, vacuolized cytoplasm and bulging nuclear membrane. These results appear to collaborate that of Kasturi et al. (2002), who observed changes such as intracellular spaces and vacuolization in Sertoli cells, diminished Lydig cells and cytoplasmic inclusions of testis, hence concluding that A. indica leaves extract might affect spermatogenesis through antispermatogenetic and antiandrogenic properties. Also, Ghosesawar et al. (2003) observed that A. indica extracts influences the physiological maturation of sperm through antiandrogenic effect as diminished levels of fructose and sperm parameters in vas deferens paralleled androgen deficiency.

Earlier, other workers had demonstrated that different A. indica extracts caused statistically significant decrease in serum testosterone (Parshad et al. 1994), decreased phosphatase activities as well as biochemical and histological regression in seminal vesicles and ventral prostrate, which are androgen dependent, again, suggesting antiandrogen property.

Some workers have argued that plants like A. indica that are reported to be used in traditional medicine for male fertility control, and may have indeed displayed some in vitro spermicidal activity under experimental conditions, should be treated with caution in the search for male fertility-control drugs (Farnsworth and Waller 1982). This is because such experiments may suffer from inadequacies like lack of adequate number of vehicle-treated controls, poor experimental design, improper solubility of extracts, variation in routes of administration, diversity in reproductive function and control among various laboratory species and problems in identifying plant names consistently. However, the effective experimental and practical deployment of A. indica extracts in male contraception in different parts of the world (Jensen 2002) suggest that the use of A. indica as male contraceptive has grown beyond such inadequacies.

Neem and female fertility

There are many reports in literature that tend to suggest that different parts of A. indica exert significant modulatory effects on female fertility. A single intrauterine application of 100 μl A. indica oil has been found to induce a pre-implantation block in fertility, which was reversible after 5–6 months (Upadhyay et al. 1990), suggesting its possible use as a long-term female contraceptive. Earlier workers (Prakash et al. 1988) had demonstrated that neem seed oil administered as a sub-cutaneous dose of 0.3 ml/rat exerted post-coital contraceptive effect, but with less possibility of eliciting less side effects than the steroidal contraceptives, since it does not posses estrogenic, antiestrogenic or progesteronal activity.

Similarly, use of purified neem seed extract had been found efficacious in the termination of pregnancy in both rodents and primates (Talwar et al. 1997a, b; Mukherjee et al. 1996; Mukherjee and Talwar 1996). These workers (Mukherjee et al. 1999) demonstrated that this abortive effect of neem seed and leaf extracts when administered orally seems to be propelled by activation of cell-mediated immune reactions. Other laboratories have reported the contraceptive efficacy of a vaginal preparation, NIM-76 from neem seed (Sairam et al. 2000), a polyherbal cream containing extracts of dried seeds of A. indica (Garg et al. 1993), and the immunocontraceptive effect of neem seed extract that has been attributed to a mixture of six components containing mono and di-unsaturated free fatty acids and their methyl esters (Garg et al. 1998).

Antiplasmodial property of Azadirachta indica

Antiplasmodial property is perhaps the earliest and most popular pharmacological activity of A. indica exploited by man in disease therapy. But some evidence in literature does not seem to completely support the efficacy of neem, at least extract of some parts, as antimalarial. Studies by Abatan and Makinde (1986) demonstrated that, although oral dosing with leaf extracts at 500 and 125 mg/kg bw of methanol extract of A. indica caused statistically significant parasite suppression in Plasmodium berghei-infested mice treated for 4 days; it did not protect them more than 1 day longer than the untreated group. Similarly, other workers (Omar et al. 2003) have demonstrated that purified extracts of A. indica and other members of the Meliaceae family containing gedunin showed significant in vitro, but poor in vivo antimalarial activity. However, Biswas et al. (2002), summarizing earlier work on the subject, noted that components of the alcoholic extracts of the leaves and seeds are effective against both chloroquine-resistant and chloroquine-sensitive strains of malaria parasite, while purified and fractions were shown to inhibit growth and development of asexual and sexual stages of drug-resistant strains of the human malarial parasite, Plasmodium falciparum. Thus, while others have demonstrated that some other plants may even be more antiplasmodial than neem (Ofulla et al. 1995), others have shown that some neem extracts are at best, effective in malaria prophylaxis (Abatan and Makinde 1986).

Antitrypanosomal activity

Some investigators have studied the efficacy of extracts of A. indica against different species of Trypanosomes, a protozoal organism responsible for incidence of sleeping sickness (trypanosomiasis) and nagana disease in man and animals. Using Microtiter Plate Bioassay and Radioactive Thymidine Incorporation Techniques, Yanes et al. (2004) have demonstrated that fractions of chloroformic extracts of A. indica leaves markedly inhibited growth of Trypanosoma cruzi epimastogotes with ultrastructural changes such as vacuolization, organelle degeneration and cell division disruptions.

Similarly, De Azambuja and Garcia (1992) have shown that a major phytochemical in neem, azadirachtin—a tetranortriterpenoid, when administered as a single dose through blood meal, blocked the development of T. cruzi and induced a permanent resistance of the vector against reinfection with T. cruzi.

Antioxidant, hepatoprotective and cancer chemopreventive potential

A number of studies have indicated that extracts and purified fractions of different parts of A. indica plant may possess significant antioxidant, hepatoprotective and cancer chemopreventive capacities. For instance, aqueous leaf extracts of A. indica protected against paracetamol-induced hepatic damage in rats (Chattopadhyay 2003; Yanpallewar et al. 2003), and boosted blood antioxidant status, suggesting a key role in preventing cancer development at extrahepatic sites (Arivazhagan et al. 2004). Treatments involving petroleum ether extracts of kernel and husk of neem alone, significantly protected animals against oxidative stress caused by Streptozocin (STZ) in heart and erythrocytes, but not against renal and hepatic toxicity in Streptozocin-diabetic animals (Gupta et al. 2004).

Other workers (Rao et al. 1998) have used reverse-phase HPLC to isolate the antioxidant principle from seed kernels of A. indica and showed the molecule to be a potent inhibitor of plant lipoxygenase.

In vivo studies have strongly suggested that extracts of A. indica possess significant anticancer activities. For example, Arivazhagan et al. (2000) demonstrated that leaf extract of neem, among other plants enhanced hepatic glutathione-dependent enzymes during N-methyl-N′-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in Wistar rats and protected against in vivo clastogenic effects of MNNG (Arivazhagan et al. 2003). The chemopreventive effect of ethanolic extract of neem leaf against MNNG-induced oxidative stress has also been reported (Subapriya et al. 2003); Subapriya and Nagini 2003). Similarly, other workers have demonstrated the high chemopreventive potential of neem leaf extract in murine carcinogenesis model system (Dasgupta et al. 2004) and against dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch carcinogenesis in model systems (Subapriya et al. 2004; Balasenthil et al. 1999), as well as in hepatocarcinogenesis (Hanachi et al. 2004). It also caused immune-mediated prophylactic growth inhibition of murine Ehrlich carcinoma and B16 melanoma (Baral and Chattopadhyay 2004).

Furthermore, azadirone, a limonoidal constituent of A. indica has been found to possess good in vivo antitumor activity in modified hollow fiber animal models (Nanduri et al. 2003), while prenylated flavones isolated from flowers of A. indica are potent antimutagenic compounds against heterocyclic compounds in Salmonella typhimurium TA98 assay systems (Nakahara et al. 2003). Prenylated flavones also inhibited 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced mammary gland carcinogenesis in female Sprague-Dawley rats, as well as aflatoxin B-induced hepatocarcinogenesis in Wistar rats (Tepsuwan et al. 2002).

Antibacterial and antiviral properties

Several reports have appeared in literature to suggest that different extracts of A. indica possess significant antibacterial, antiviral and antifungal properties. Using semiquantitative quadrant streaking method, Pai et al. (2004) demonstrated that mucoadhesive dental gel containing alcoholic neem extract significantly reduced (P < 0.05) the plague index and bacterial count in the oral cavity, while Wolinsky et al. (1996) showed that incubation of oral Streptococci with the neem stick extract resulted in a microscopically observable bacterial aggregation, indicating that neem stick extract can reduce the ability of some Streptococci to colonize tooth surfaces. Utilizing other techniques such as Ditch plate method, tube dilution technique and agar dilution methods, a strong antibacterial effect of neem extract has also been reported against Bacillus cereus, Escherichia coli, Salmonella infantis (Alzoreky and Nakahara 2003), Streptococcus mutans and Lactobacillus spp.(Vanka et al. 2001), Streptococcus faecalis (Almas 1999), Klebsiella pneumoniae (Sairam et al. 2000), Helicobacter pylori (Fabry et al. 1996) and a variety of pathogenic bacteria in India and East Africa (Fabry et al. 1998). Other studies have indicated that different neem extracts may also possess antibacterial activity against fish pathogenic bacteria such as Aeromonas hydrophila, Pseudomonas fluorescens, E. coli and Myxobacteria (Das et al. 1999a), as well as other organisms like Vibrio cholera, Mycobacterium tuberculosis and Mycobacterium pyogenes (Biswas et al. 2002),which are of public health importance.

Although limited in number, some studies have suggested that neem extract may possess significant capacity for antiviral activity. Parida et al. (2002) have demonstrated that azadirachtin obtained from neem inhibited Dengue virus type-2 replication as confirmed by the absence of Dengue-related clinical symptoms in sucking mice and absence of virus-specific 511 bp amplicon in Reverse Transcriptase–Polymerase Chain Reaction (RT–PCR). Similarly, the antiviral activity of neem leaf extract against group B Coxsackieviruses (Badam et al. 1999) and potato virus X (Verma 1974) have been reported. Biswas et al. (2002) suggested that the methanolic extract of neem leaves (NCL-11) is most effective in Coxsackie B-4 viruses as a virucidal agent, in addition to its influence at the early events of its replication.

Phytochemical studies have implicated limonoids such as mahmoodin and tetranortriterpenoids like azadirone, epoxyazadiradione, nimbin, gedunin, azadiradione, deacetylnimbin and 17-hydroxyazadiradione, as well as the protolimonoid, naheedin as phytochemicals responsible (Fig. 1; Table 1) for the reported antibacterial and other antimicrobial activities (Siddiqui et al. 1992; Govindachari et al. 2000; Biswas et al. 2002; Brahmachari 2004; Subapriya and Nagini 2005; Bhattacharyya et al. 2007; Girish and Shankara 2008).

Fig. 1
figure 1

Structure of some bioactive compounds in neem: GIa (14) and GIb are water soluble polysaccharides, while GIIa (15) and GIIIa (16) are other polysaccharides components (Biswas et al. 2002)

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Table 1 Some bioactive compounds found in neem (modified from Biswas et al. 2002; Girish and Shankara 2008)
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Antifungal properties

A number of in vitro and in vivo studies appear to confirm the fungistatic and fungicidal activity of neem extracts. These include fungicidal effect of neem extract against pineapple fruit-rotting fungus, Ceratocystis paradoxa (Damayanti et al. 1996); fungistatic and fungicidal activities against Aspergillus spp. and Candida spp. (Fabry et al. 1996; Sairam et al. 2000), Basidiobolus haptosporus and Basidiobolus ranarum (Nwosu and Okafor 1995) and Pestalotiopsis mangiferae, the causative agent for the serious leaf-spot disease of Magnifera indica (Rai 1996). Neem extract has also been found to possess fungistatic and fungicidal effects against plant pathogenic fungi like Fusarium oxysporum, Alternaria solani, Curvularia lunata, Helminthosporium spp. and Schlerotium rolfsii (Bhonde et al. 1999) and against dermatophytes such as Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton violaceum, Microsporum nanum, and Epidermophyton floccosum (Natarajan et al. 2002). Other workers have suggested that neem oil and leaf extracts significantly inhibit production of fungal toxic secondary metabolites, e.g. patulin produced by Penicillium expansum (Mossini et al. 2004).

Put together, these reports strongly suggest that neem products may be useful in the management and treatment of dermatophytic and fungal infections of animals and plants (Natarajan et al. 2002; Rai 1996; Sairam et al. 2000), as well as in the biological control of biodeterioration of stored agricultural products (Mossini et al. 2004; Damayanti et al. 1996).

Larvicidal, acaricidal and nematicidal effects

Larvicidal, acaricidal and nematicidal properties are the underlying bases for the use of neem products for control of agricultural pests. These effects have been reported for methanolic extracts of defatted neem seed kernels. In eggs, immature and adult stages of Hyalomma anatolicum excavatum at concentrations of 1.6, 3.2, 6.4 and 12.8%, Abdel-Shafy and Zayed (2002) observed a significant increase in the hatching rate during the first 7 days posttreatment, giving incompletely developed and dead larvae, and after 15 days, it resulted in hatching failure, induced a significant increase in mortality rates of newly hatched larvae, unfed larvae and unfed adults.

Similarly, azadirachtin extracted from neem seed and ethanolic extract of grounded seed blended into cow manure or administered orally to cattle, exhibited effects similar to those of insect growth regulators as they were found to be larvicidal against horn fly (Haemotobia irritans, L), stable fly (Stomoxys calcitrans) and house fly (Musca domestica; Miller and Chamberlain 1989). The larvicidal effect of A. indica and other plants on Aedes aedes aegyptica (Linnaeus) and Culex quinequefaciatus (Wandscheer et al. 2004; Monzon et al. 1994) third instar larvae of Drosophila melanogaster and fifth instar larvae of Manduca sexta (Mitchell et al. 1997) have been demonstrated.

There are also reports that aqueous extract of de-oiled neem seed kernel caused 100% mortality in the fourth instar larvae and pupae at the concentration of 100 ppm with no significant effect on the development period of Culex quinquefasciatus (Sagar and Sehgal 1996) and anopheline pupae (Rao et al. 1995).

Bioassay-guided fractionation and phytochemical studies revealed cardenolide (Al-Rajhy et al. 2003), azadirachtins A, B and H (Sharma et al. 2003) as well as salannin, nimbin and 6-desacetylnimbin as the larvicidal component of neem extracts, azadirachtins showing better efficacy (Mitchell et al. 1997). Furthermore, studies by Koul et al. (2003) showed 6β-hydroxygedunin isolated from A. indica A. Juss. to be the active agent against the gram pod borer, Helicoverpa armigera (Hubner), and Asian armyworm, Spodoptera litura (Fabricius; Lepidoptera: Noctuidae), alone and in combination with other limonoids, gedunin, salannin, nimbinene, and azadirachtin (Structures of some of these phytochemicals are presented in Fig. 1). However, work of Siddiqui et al. (2002) implicated two triterpenoids, 22,23-dihydronimocinol and desfurano-6α_-hydroxyazadiradione isolated from methanolic extract of the fresh leaves of A. indica as well as meliacin, 7α-senecioyl-(7-deacetyl)-23-O-methyl nimocinolide (which showed mortality for fourth instar larvae of the mosquito (Anopheles stephensi), with LC(50) values of 60 and 43 ppm, respectively), as other larvicidal agents in neem plant.

Insecticidal effect

Insecticidal effect of neem is closely related to the larvicidal, acaricidal and nematicidal properties, but this section is separated from the earlier for better comprehension.

It has been observed that ethanol extract of seeds of A. indica mixed with beans protected the grain against pest infestation during storage without affecting instrumental hardness (% hard to cook/mean gram force) and consumer acceptability (Dunkel et al. 1995). Neem seed oil and a fraction containing volatile components have been demonstrated to adversely affect the gonotrophic cycle and oviposition of female Anopheles stephensi and Anopheles culicifacies (Diptera: culicidae), causing irreversible impairment of vitellogenesis (Dhar et al. 1996). Similarly, a 1% azadirachtin formulation, commercially called “Neemazal,” has been found effective against roots sucking (Aphis fabae Hom.Apididae) and free-feeding pest insects (Heliothis armigera Lep. Noctuidae) in a concentration- and time-dependent manner (Hummel and Kleeberg 2002). Insecticidal activity of neem extracts have also been reported against Clavigralla scutellaris, Gryon fulviventre (Mitchell et al. 2004), cowpea bruchid, Callosobruchus maculates (Lale and Mustapha 2000) and female boll weevil (Showler et al. 2004).

Phytochemical and bioassay-guided fractionation suggest that the insecticidal properties of neem extracts and neem products are mediated by groups of compounds such as the tetranortriterpenoid, meliatetraolenone (Siddiqui et al. 2000), aromatics [2,6-bis-(1,1-dimethylethyl)-4-methyl phenol, 2-(phenylmethylene)-octanal, 1,2,4-trimethoxy-5-(1Z-propenyl)-benzene], benzopyranoids (3,4-dihydro-4,4,5,8-tetramethylcoumarin, 3,4-dihydro-4,4,7,8-tetramethylcoumarin-6-ol, 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethyl-cyclopenta[g]-2-benzopyran); one sesquiterpene methyl-3,7,11-trimethyl-2E,6E,10-dodecatrienoate); esters of fatty acids (methyl 14-methyl-pentadecanoate, ethyl hexadecanoate, ethyl 9Z-octadecenoate) and a monoterpene, 3,7-dimethyl-1-octen-7-ol (Siddiqui et al. 2004; Fig. 2).

Fig. 2
figure 2

Insecticidal Aromatics, benzopyranoids, sesquiterpene and monoterpene from fractions of the fruit coatings of Azadirachta indica (2,6-bis-(1,1)-dimethylethyl-4-methyl phenol (17); 3,4-dihydro-4,4,5,8-tetramethylcoumarin (18); 3,4-dihydro,4,4,7,8 tetramethyl-coumarin-6-ol (19); α-hexylcinnamaldehyde (20); methyl (2E,6E)-farnesoate (21); galoxolide (22); methyl 14-methylpentadecanoate (23); ethyl palmitate (24);ethyl oleate (25); dihydromyrcenol (26); _-asarone (27) (Compounds 1–16 were n-alkanes: structures not provided here; Siddiqui et al. 2004)

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Other insecticidal components of neem include volatile di-n-propyl disulfide from neem seeds (Koul 2004), 6-β-hydroxygedunin (Koul et al. 2003), nimbin and salannin (Simmonds et al. 2004) triterpenoids (22,23-dihydronimocinol and desfurano-6α-hydroxyazadiradione), meliacin (7α-senecioyl-(7-deacetyl)-23-O-methyl-nimocinolide) and meliacinol [24,25,26,27-tetranorapotirucalla-(apoeupha)-1α-trimethylacryloxy-21,23-6α,28-diepoxy-16-oxo-17-oxa-14,20,22-trien-3α,7α-diol; Siddiqui et al. 2000, 2002].

Insect repellant and antifeedant properties

Insects and other pests of medical, veterinary and agricultural importance may be controlled through repulsion, antifeedant effects or killing. Several investigations appear to suggest that different neem products may be useful in insect and vector control by one or more of these mechanisms. Different neem parts and products have found widespread use as mosquito repellants. For instance, smoke of leaves of A. indica was found to repel mosquito by up to 70% (Palsson and Jaenson 1999), while a number of reports have indicated that neem oil also may possess significant insect repellant activity.

Sharma and Ansari (1994) burnt kerosene containing 0.01–1.0% neem oil and found insignificant reduction in the biting of human volunteers and catches of mosquitoes resting on room walls in two Indian villages near Delhi. Similarly, 1–4% neem oil in coconut oil, when applied to the exposed body parts of humans has been demonstrated to provide complete protection for 12 h from the bites of all anopheline mosquito species (Mishra et al. 1995; Sharma et al. 1993). Other studies in literature indicate that neem oil at 5% in wood scraping balls prevented breeding of Anopheles stephensi and Aedes aegypi in overhead tanks for 45 days (Nagpal et al. 1995), and effectively controlled culicine mosquito vectors in rice field (Rao et al. 1995). However, potted live A. indica caused statistically insignificant repellency effect against Anopheles gambiae mosquito from human baits in semi-field experimental huts (Seyoum et al. 2002) and even in field trials (Das et al. 1999b). These data suggest that burning of leaves and oil of A. indica can reduce domestic exposure to mosquitoes, and hence may constitute a sustainable and readily applicable malaria vector control tool for incorporation into integrated vector management programs (Seyoum et al. 2002; Knols 2002) of relevant public health departments of many developing countries.

Molluscidal effects

Molluscidal activity refers to the ability of a substance to kill one or more members of the Snail family, through direct effect on the adult organism or any of the stages in the life cycle. Using criteria such as feed intake, cessation of crawling, mucus secretion, lack of response to mechanical stimuli, decomposition (Ebenso 2004) and even enzyme profiles (Rao et al. 2003), the molluscidal effect of A. indica products on several species and sub-species of snails has been evaluated.

At concentration of 350, 500 and 700 mg/kg bw extracts of neem seed oil showed no significant effect but those of the bark, roots and leaves produced mortality at 500 and 700 mg/kg bw after exposure of Limicolaria aurora, Jay and Achatina marginata for 48–72 h (Ebenso 2004). However, A. indica seed oil has been reported to significantly alter the activities of acetylcholinesterase, lactic dehydrogenase and acid/alkaline phosphatase in the nervous tissue of the harmful terrestrial snail, Achatina fulica (Rao et al. 2003) and showed synergistic molluscicidal effect in binary or tertiary combination with Cedrus deidara oil and/or piperonylbutoxide (Rao and Singh 2001) in Lymnae acuminata. Earlier have demonstrated the molluscicidal activity of the neem leaf, stem bark, seed cake, neem seed oil and the neem-based pesticides, “Achook” and “Nimbecidine”, in both time- and dose-dependent manner; with pure azadirachtin displaying greater activity than synthetic molluscicides.

Also in binary and tertiary combinations with other molluscicidal plant products like Lawsonia inermis, Embelia ribes fruit powder, Allium sativum, Linn, Zingiber officinale Rosc., Polianthes tuberose Linn and Annona squamosa, neem was shown to possess significant activity against Lymnaea acuminata and Indoplanorbis exustus (Singh and Singh 2001, 2004).

Antidiabetic action

The hypoglycemic and anti-hyperglycemic properties of A. indica plant parts have been evaluated using different animal models. In normal and streptozotocin-induced diabetic animals, aqueous leaf extracts of A. indica displayed anti-hyperglycemic effect, but showed little or no action on peripheral utilization of glucose and hepatic glycogen metabolism (Chattopadhyay 1996). The blood lowering effect of A. indica leaf extract, which was found to be either through significant blockage of the inhibitory effect of serotonin on insulin secretion mediated by glucose or peripheral utilization of glucose and glycogenolic effect due to epinephrine action blockage, was observed to be statistically higher than that of Catharanthus roseus, Gymnema sylvestre and Ocimum sanctum. Because A. indica extract exhibited antiperoxidative, hypoglycemic and cortisol lowering activities, Gholap and Kar (2004) suggested that it may potentially regulate corticosteroid-induced diabetes mellitus.

Also, in polyherbal preparation as an ayurvedic herbomineral formulation called “hyponidd”, oral administration of A. indica at 100 and 200 mg/kg bw for 45 days did not only cause decreased levels of glycosylated hemoglobin, plasma thiobarbituric acid reactive substances, hydroperoxides (which are part of the complications of diabetes), but also elevated the levels of endogenous antioxidants, especially, plasma reduced glutathione and vitamin C (Babu and Prince 2004) in streptozotocin-induced diabetic animals.

Moreover, in normal and alloxan-induced diabetic animal models, Khosla et al. (2000) demonstrated that pretreatment of rabbits with A. indica leaf extract or seed oil administration started 2 weeks prior to alloxan treatment, partially prevented the rise in blood glucose levels when compared to control diabetic animals. It also caused significant hypoglycemic effect comparable to that of glibenclamide after 4 weeks of administration. These studies have clearly demonstrated the hypoglycemic potential of A. indica in alloxan-induced diabetic animal model, either alone (Halim 2003; Kar et al. 2003), or in polyherbal combinations (Babu and Prince 2004).

Anti-inflammatory properties

Studies in some laboratories have indicated that certain fractions and extracts of neem may posses anti-inflammatory properties. Kaur et al. (2004) evaluated the anti-inflammatory effects of nimbin and nimbidin, which is a mixture of tetranortriterpenes that forms the major active principle of neem seed oil. Their results revealed that nimbin significantly inhibited some of the functions of macrophages and neutrophils relevant to the inflammatory response following both in vivo and in vitro exposure. They reported that oral administration of nimbidin at 5–25 mg/kg bw to rats for three consecutive days significantly inhibited the migration of macrophages to their peritoneal cavities in response to inflammatory stimuli and also inhibited phagocytosis and phorbol-12-myristate-13-acetate (PMA)-stimulated respiratory burst in these cells. Also, in vitro exposure of rat peritoneal macrophages to nimbidin also inhibited phagocytosis and PMA-stimulated respiratory burst in these cells as well as nitric oxide (NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-stimulated macrophages, while interleukin 1 (IL-1) was only weakly inhibited. Furthermore, Kaur et al. (2004) observed that nimbidin ameliorated NO inhibition via the induction of inducible NO synthase (iNOS) without any inhibition in its catalytic activity, and also attenuated degranulation in neutrophils were assessed in terms of release of beta-glucuronidase, myeloperoxidase and lysozyme. This demonstrated ability of nimbin and nimbidin to suppress the functions of macrophages and neutrophils relevant to inflammation, made these authors to conclude that they can be valuable in treating inflammation and inflammatory diseases.

Earlier, Chattopadhyay (1998) demonstrated that water soluble part of alcoholic extracts of A. indica leaves administered orally at a dose of 200 mg/kg bw exerted significant anti-inflammatory activity in Cotton Pellet Granuloma Assay (CPGA) in rats. Similarly, Jain and Basal (2003) studied Propionibacterium acnes, an anaerobic pathogen, that plays an important role in the pathogenesis of acne by inducing certain inflammatory mediators such as reactive oxygen species (ROS) and pro-inflammatory cytokines. The authors proved that A. indica extracts among other herbs showed anti-inflammatory activity by suppressing the capacity of P. acnes-induced ROS and pro-inflammatory cytokines, the two important inflammatory mediators in acne pathogenesis.

Anti-ulcerogenic effect

A number of workers have investigated the effect of neem extracts on gastric secretion and gastro-duodenal ulcer. In albino rats, Raji et al. (2004) found that stem bark extracts of A. indica significantly inhibited gastric ulceration induced by indomethacin (40 mg/kg bw) when administered orally at a dose of 100–800 mg/kg bw or intraperitoneally at a dose of 100–250 mg/kg bw. This action was found to be accompanied by a dose-dependent decrease in total gastric acidity, and when administered in combination with histamine (1 mg/kg bw) and cimetidine (0.12 mg/kg bw) in situ, A. indica at 250 mg/kg bw, significantly inhibited the basal and histamine-induced gastric acid secretion. This made them to conclude that the stem bark extract of A. indica contains antiulcer agents, which probably act via histamine receptor.

The anti-ulcerogenic effect of A. indica has indeed been demonstrated in clinical trials involving humans (Bandyopadhyay et al. 2004). In that trial, which involved administration of lyophilized aqueous extracts to Indian patients suffering from acid-related problems and gastroduodenal ulcers for 10 days at daily twice dose of 30 mg, it was observed that neem bark extract not only has therapeutic potential for controlling gastric hypersecretion and gastroesophageal and gastroduodenal ulcers, but can also heal the duodenal ulcers as monitored by Barium Meal X-ray and endoscopy. Using important blood parameters for organ toxicity, these investigators further established the relative safety of the extracts when administered at 30–60 mg/kg bw for 10 weeks.

In order to understand the mechanism by which neem extract brings about the anti-ulcerogenic effect, Chattopadhyay et al. (2004) compared its effects with standard antiulcer drugs using stress ulcer and pylorus-ligation models. Based on their observation that aqueous neem extract dose-dependently inhibited gastric lesions induced by restraint-cold stress, indomethacin and ethanol more effectively than ranitidine, but less effective than omeprazole; and also dose-dependently blocked pyloric ligation and mercaptomethylimidazole-induced acid secretion, H+-K+-ATPase activity, as well as protected against membrane damage caused by hydroxyl radical and stress-induced apoptotic DNA fragmentation, Chattopadhyay et al. (2004) concluded that neem leaf extracts offer antiulcer activity by blocking acid secretion through inhibition of H+-K+-ATPase and by preventing oxidative damage and apoptosis. The involvement of H+-K+-ATPase inhibition and scavenging of hydroxyl radical as mechanisms for the gastroprotective effect of aqueous neem bark extract have also been demonstrated by Bandyopadhyay et al. (2004).

Immunological properties

There are several reports in literature to suggest that neem products have significant modulating effect on the humoral and cell-mediated immune system. Upadhyay et al. (1992) studied the immunomodulatory effects of neem oil in mice following a single intraperitoneal dose. They observed that neem oil increased level of leukocytic cells, enhanced phagocytic activity of peritoneal macrophages and expression of MHC class II antigens, as well as induced production of gamma interferon. This led them to conclude that neem oil acts as a non-specific immunostimulant that selectively activates the cell-mediated immune mechanisms to elicit an enhanced response to subsequent mitogenic or antigenic challenge.

The immunopotentiating effect of neem leaves and stem bark extracts (Njiro and Kofi-Tsekpo 1999; Van der Nat et al. 1987; Labadie et al. 1989) have also been demonstrated in mice (Ray et al. 1996; Njiro and Kofi-Tsekpo 1999), broilers naturally infected with Infectious Bursal Disease (IBD) virus, as well as normal and stressed rats (Sen et al. 1992).

Other workers have exploited the immune-modulatory properties of neem for immunocontraception in rats and monkeys by using single intrauterine application of neem oil and neem seed hexane extracts (Garg et al. 1994, 1998). This opened the way for use of neem oil products as a reversible long-term contraceptive with little or no toxic effects in humans (Talwar et al. 1997a, b). This immunocontraceptive activity has been ascribed to a fraction of hexane seed extracts made of six components consisting of saturated, mono- and di-unsaturated free fatty acids and their methylesters (Garg et al. 1998).

Toxicological effects of Azadirachta indica products

The toxicological effects of various extracts from different parts of A. indica, as well as purified compounds and neem-based products, have been studied in a number of animal models ranging from rodents to primates, and utilizing animals such as rats, mice, chicken and monkeys.. (Table 2).

Table 2 Toxic effects of Azadirachta indica extracts and its products in animals of different species
Full size table

In a review on safety evaluation of neem-based pesticides, Boeke et al. (2004) noted that 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 μg/kg bw, while the unprocessed seed oil and the aqueous extracts with ESD of 0.26–0.3 mg/kg bw and 2 μl l/kg bw, respectively, were less toxic. Furthermore, they observed that most of the pure compounds show a relatively low toxicity (ESD azadirachtin 15 mg/kg bw), while 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. Hence, based on review of available data on safety assessments for the various neem-derived preparations, they concluded that, if applied with care, use of neem-derived products as insecticide should not be discouraged.

Toxicity in rodents

Acute toxicity studies of neem seed oil established the 24 h LD50 to be 14 ml/kg bw in rats and 24 ml/kg bw in rabbits. Prior to death, animals of both species and sexes exhibited comparable pharmacotoxicological symptoms, with lungs and central nervous system as the main target organ of toxicity (Gandhi et al. 1988). In another study (Kumar et al. 2002) revealed the radiosensitizing effects of neem seed oil in Balbc/3T3 cells and SCID cells by interacting with residual damage after X-ray irradiation thereby converting the sub-lethal damage or potentially lethal damage into lethal damage inhibiting double strand break repair or reducing the G (2) phase of the cell cycle.

However, when the debitterized neem oil was evaluated in a three generation study utilizing the WHO/FDA protocol, Chinnasamy et al. (1993) did not find any adverse effects on the reproductive parameters studied, and the mean organ weights, histopathological evaluations and mutagenicity test result were normal, suggesting that the toxic principle in neem seed oil may only be contained in the bitter and odoriferous components, but the possibility that aflatoxins contamination may also be involved must be considered.

The genotoxicity of neem and neem products has been reported in rodents. Oral administration of a soxhlated crude ethanolic extract of neem leaves to adult male mice for 6 weeks at a rate of 0.5, 1.0 and 2.0 g/kg bw, increased the incidences of structural changes and synaptic disturbances in meiotic chromosomes, and also caused disruption of meiosis, resulting in reduced sperm count, increased frequency of spermatozoa with abnormal head morphology and chromosome strand breakages or spindle disturbances (Awasthy 2001). Similar genotoxic effect of A. indica ethanolic leaf extract in rats had earlier been reported by the same workers (Awasthy et al. 1999).

Rahman and Siddiqui (2004) studied the biochemical effects of Vapacide—a neem-based pesticide, administered orally in coconut oil at doses of 80, 160 and 320 mg/kg bw for 45–90 days. They observed that exposure to Vapacide resulted in a significant but reversible increases in acid phosphatase, alkaline phosphatase in serum, kidney, lung and liver tissue (alkaline phosphatase only in liver), which was accompanied with a significant decrease of acid phosphatase in the liver of male and female rats after 45 and 90 days of moderate to high doses, with the lung tissues being the most susceptible and kidney the least susceptible. They attributed the decrease in liver acid phosphatase to the necrosis of cellular tissues, and suggested that these enzyme activities could be useful as biomarkers of exposure to Vepacide. Similar conclusions were reached when aspartate and alanine aminotranferases profiles were established in rats on sub-chronic doses of Vapacide (Rahman et al. 2001).

Toxicity in aquatic animals

In studies on aquatic organisms, a 100% mortality among three snail intermediate host species, Biomphlaria pfeifferi, Bulinus truncates and Lymnaaecea natalensis after 24 h exposure to freeze-dried aqueous or ethanolic extract of A. indica at 100 mg/l has been reported. Two aquatic crustaceans namely, Daphnia magna, Hyalella azteca and a dipteran, Chironomus riparius have also been found to be susceptible to the toxic effects of Margosan O—a neem seed kernel-based insecticide registered in the United states, at concentrations as low as 10 mg/l (Scott and Kaushik 1998). Other workers (El-Shazly and El-Sharnoubi 2000) have also reported varying toxic effects of NeemAzal—T/S, another neem-based insecticide on various developmental stages of different classes of aquatic organisms, including Bufo regularis (Amphibia), Aedes caspius (Insecta), Gambusia affinis (Poecilliidae), Cyclops sp. and Daphnia magna (Crustacea) at concentrations of 10–20 ppm, but other studies also reported NOEL values of 197.5 mg/l LC50:1000 mg/l for Daphnia magna. Similarly, the toxicity of azadirachtin—a component of neem, to some specie of fish—has been reported (Chandra and Khuda-Bukhsh 2004).

This selective toxicity of neem-based compounds toward these different classes of animals appears to be the singular most important basis for their varied use as insecticidal and molluscidal agents.

Toxicity in ruminants

The chronic and sub-chronic effects of neem and neem products have also been studied in ruminants. In a comprehensive study involving oral administration of aqueous leaf extract of A. indica at doses of 50 and 200 mg/kg bw for 8 weeks to goats and guinea pigs, Ali (1987) observed a progressive decrease in body weight, heart pulse and respiratory rates, weakness, inappetence, diarrhea and loss of condition. It was further observed that goats on higher doses of the plant leaves produced tremors and ataxia during the last few days of treatment, but no statistically significant hematological changes were observed after dosing the animals with A. indica leaves, although there was a tendency toward lowered erythrocyte counts, packed cell volume and hemoglobin concentration. The treatments also caused significant rises in the plasma activity of aspartate transferase, sorbitol dehydrogenase, and concentrations of cholesterol, urea, creatinine and potassium, but no significant changes in the plasma concentration of sodium, chloride or bilirubin were detected. They further reported areas of hemorrhagic erosions, flappy hearts and hydropericardium, while histopathologically; there was evidence of various degrees of hemorrhage, congestion and degeneration in the liver, kidney, lung, duodenum, brain and seminiferous tubules.

Toxicity in poultry

After feeding Brown Hisex chicks with 2 and 5% A. indica leaf supplemented diet for 28 days, Ibrahim et al. (1994) reported a depression in body weight gain and efficiency of feed utilization accompanied with clinicopatholological changes such as increases in activities/levels of lactic dehydrogenase, glutamic oxaloacetic transaminase, alkaline phosphatase, uric acid and billirubin concentrations with concomitant decreases in serum total protein. They also observed significant changes in the values of erythrocyte count, hemoglobin concentration, packed cell volume, mean corpuscular volume and mean corpuscular hemoglobin concentration associated with yellow discoloration of the legs and combs and hepatonephropathy.

In an earlier study on poultry by some of these workers (Ibrahim et al. 1992), a dose-dependent decrease in body weight gain and efficiency of feed utilization and hepatonephropathy accompanied by anemia and increases in lactate dehydrogenase, glutamic oxaloacetic transaminase, alkaline phosphatase, uric acid and bilirubin were observed in Brown Hisex chicks when fed diets contained 2, 5 and 10% ripe fruits of A. indica.

Toxicity in humans

Reports of toxicity of neem products in man are rather scarce in literature. However, some investigators have reported the allergizing potential of A. indica pollen among an Indian community (Boral et al. 2004). Two immunoglobulin reactive proteins have been isolated and characterized from A. indica pollen, and their allergenicity confirmed by skin prick test and Immunoblot Assay (Karmakar and Chatterjee 1994).

Structure and activity of major bioactive compounds found in Azadirachta indica

According to Girish and Shankara (2008), more than 135 compounds have been isolated from different parts of neem. These compounds are classified into two major groups—isoprenoids and others. The isoprenoids include diterpenoids and triterpenoids containing protomeliacins, limonoids, azadirone and its derivatives, genudin and its derivatives, vilarin type of compounds and secomeliacins such as nimbin, salannin and azadirachtin. The first compound studied was nimbin, but recently, the complete laboratory synthesis of azadirachtin, one of the major bioactive compounds in neem was accomplished in one of the longest, most involving and most expensive attempt at synthesizing any natural product in history, although synthesis of portions of the molecule had been reported since 1994 by Ley. The non-isoprenoids include proteins (amino acids) and carbohydrates (polysaccharides), sulphurous compounds, polyphenolics such as flavonoids and their glycosides, dihydrochalcone, coumarin and tannins, aliphatic compounds and phenolic acids. The structures of some of the bioactive compounds are presented in Figs. 1 and 2, while bioactivities of the few of these compounds that have been studied are presented in Table 1.

Conclusion

The multivariate biological and pharmacological activities attributable to different parts and extracts of the neem plant, A. indica A. Juss, enumerated in this review, appear to justify the reference to the plant as a “wonder plant”, “village dispensary” or “living pharmacy”. Most of the varied, but desirable biological and pharmacological activities of the plant extracts and purified bioactive compounds are observed at doses within the estimated safe doses (ESD). However, the observed differential toxicity in animals of different species, which ironically appears to be the singular most important basis for its divergent uses and applications, especially as biopesticide, must be taken into consideration when neem products are utilized by man.