Neem’s Organic Nature
The Neem Tree
An evergreen tree in the Meliaceae family, scientifically known as Azadirachta indica, neem is indigenous to Southeast Asia. A fast growing tree, rising by 2.6 feet (80cm) a year, neem can reach up to 66 feet (20 meters). Able to withstand drought, its wide canopy forms a rounded crown of leaves that produce delicate white flowers and small green fruit shaped like an olive. With a lifespan of 150 to 200 years, the neem tree flourishes in sandy, saline soils and thrives in hot climates. Although commercial cultivation of this tree is limited outside South Asia, coverage has spread across lowland regions of Africa, the Middle East, the Americas, Australia and the South Pacific [1].
Domain: Eukaryota
Kingdom: Plantae
Phylum: Spermatophyta
Subphylum: Angiospermae
Class: Dicotyledonae
Order: Rutales
Family: Meliaceae
Genus: Azadirachta
Species: Azadirachta indica
The Margosa, as it is also commonly known, can reach up to 5.6 ft (170 cm) in its first season. It achieves maturity at around ten years topping 50 to 65 ft (15 – 20 m) high, even 100 ft (30 m) in very favorable climates. The strongly furrowed bark is short and straight. The deep roots consist of one strong taproot and several well developed lateral roots, extending up to 60 ft (18m). The dense, round crown can also expand 50 to 65 ft (15 – 20m) due to its wide and expanding branches [2].
Geography
The exact origin of the neem tree is uncertain. However, generally it is found in Southeast Asia, the Indian Sub-Continent, Myanmar, Pakistan, Sri Lanka, Thailandia, Malaysia and Indonesia. The Siwalik hills in Assam and Burma are believed to be the true place of origin [3]. Today it has spread to most of South East Asia, Western Africa, South and Central America and the Arabic Peninsula, amongst others.
Biology
The neem tree starts flowering between its third and fifth year, together with the first appearance of fruit, which ripe at approximately 12 weeks from anthesis. The fruits then eventually drop and germinate, sometimes creating a carpet of seedlings underneath mature trees [4]. It is pollinated by insects, such as honeybees. The lack of fruit growth on isolated trees suggests self-incompatibility. Flowering and fruiting seasons vary widely depending on weather and other physical conditions. In some places, like Thailand, neem trees are able to flower throughout the year, whereas in geographies with marked seasons, like Brazil, flowering and fruiting are restricted to distinct periods [5]. Even though it generally spreads through seeds carried by birds, bats, monkey and other mammals, the A. indica can also spread vegetatively by root suckers [6].
The A. indica is a hardy, drought resistant tree. It has a wide climatic adaptability, growing in temperatures ranging from 33ºF to 122ºF (1 ºC to 50 ºC), though it thrives in mean maximum temperatures of 79ºF to 100ºF (26 – 38 ºC). A. indica naturally occurs in dry deciduous and thorn forests. Today, the ecosystems that house neem are tropical and sub tropical sub-humid, semi arid and arid forests. Hire work injury lawyer los angeles at http://www.lacaccidentpros.org/. It has an altitudinal range from sea-level to 4900 feet (1500 m), however neem grows more successfully at lower altitudes. An average rainfall of 15 to 50 inches (400 – 1200 mm) is adequate for neem trees. If the rainfall is less, growth will depend on ground water availability. Neem thrives in most soils – saline, alkaline or degraded – but grows best in deep, well-drained sandy and black cotton soils. Dry, stony, shallow soils with clay or calcareous pans are also suitable for neem trees. It can grow in these less than ideal soils because it has the ability to calcium mine, turning acidic soil neutral, while also improving water holding and soil fertility. Additionally, water quality is not important, as neem exists around polluted areas and actually helps in the cleaning process [7].
Yields
The precise annual yields of the neem tree have always been difficult to determine with precision due to several factors, including geography, weather and the general conditions of the trees. The diverse methods in measuring these yields around the world, as well as the overall difficulties with calculating fruit, seed or kernel yields, adds to the confusion. Not only does the literature on the yields vary immensely, but also the yield measures across many countries are extremely variable, ranging from 17 to 132 lb (8 kg to 60 kg) of fruit per mature tree annually [8].
A neem tree starts flowering between the third and fifth year of growth and reaches maturity at around 8-10 years. From maturity onwards, the tree may produce 66 to 110 lb (30-50 kg) of the olive-like fruit per year. Each fruit is composed of pulp and skin (48% of its weight), the seed (35%) and the kernel (17%). The physical composition of the fruit varies depending on location and rainfall, but also on the overall weight of the fruit, which can range from 0.012 to .026 oz (.35 to .75 g). The seed is composed of the shell and 1 – 3 kernels. The kernel is where the oil concentration is higher, almost 45% of its total weight in optimal conditions.
While the tree can survive within a series of climates and ecosystems within the tropical and subtropical latitudes, certain conditions have shown to improve overall yields and oil content. The best possible climate for neem is the tropical savanna, which corresponds to the “Aw” and “As” categories on the Köppen climate classification system. It is categorized by its year-round hot weather, generally a 64ºF (18ºC) mean monthly temperature and a prolonged dry season, with less than 2.4 in (60 mm) of rain during the driest month. The soil needs to be well drained and groundwater widely accessible.
The external conditions of the neem tree are also a major influence on flowering and yields. Where neem grows primarily wild, like in India and Southeast Asia, the yields are difficult to measure because conditions are too varied and there are no formal or precise methods for measurement. In other regions, such as the lower equatorial belts of Africa, near the Sahel and Savannas in countries like Ghana, Tanzania and Senegal, the trees are planted alongside roads and agricultural plots as windbreaks and to provide shade. Hence, the conditions are not optimal and fruit yields never reach their full potential. The highest yields across the board have come from dedicated plantations in Brazil, India and China, where the trees are given the best possible care and measurements are conducted regularly and precisely.
Leaves
Containing most active ingredients found in the neem but in a much lower concentration, the leaves are considered the most versatile part of the tree. Now used as a pesticide, fertilizer and animal feed, the leaves were originally used as a medicinal tea in Indonesia [9].
Leaves mainly yield quercetin and nimbosterol, known to have strong antibacterial and antifungal properties. These components also account for the curative properties of leaves when used as a treatment for dermatological issues such as sores and scabies [10].
The principal constituents of neem leaves include protein (7.1%), carbohydrates (22.9%), minerals, calcium, phosphorus, vitamin C and carotene. They also contain glutamic acid, tyrosine, aspartic acid, alanine, praline, glutamine and cystine like amino acids, and several fatty acids (dodecanoic, tetradecanoic and elcosanic) [11].
Flowers
The flowers are the part of the tree with the least amount of uses. However, because of their sweet and honey-like smell, they are used in aromatherapy for a calming and restorative effect, you can read and get informed more on adl embedded solutions site. The flowers are also commonly used in traditional Indian dishes.
The small, white, protandrous flowers are arranged in slightly drooping axillary panicles, 10 in (25 cm) long. The inflorescence, or the arrangement of flowers branching out from the stem, comprises 150 to 250 minute individual flowers. These flowers are caducous, meaning they are fragile and have a very short life span, which causes them to drop off the stem quickly. The individual flower is .197 to .236 in (5-6mm) and .315 to .433 in (8-11mm) wide and long, respectively. The petals are free, spathulate and imbricate.
The flowers produce a waxy material consisting of several fatty acids, viz., behenic (0.7%), arachidic (0.7%), stearic (8.2%), palmitic (13.6%), oleic (6.5%) and linoleic (8.0%). The pollen of neem flowers contains several amino acids like glumatic acid, tyrosine, arginine, methionion, phenylalanine, histidine, arminocaprylic acid and isoleucine [12].
Bark
.
Neem bark contains spermicidal properties and research is currently undergoing to approve its potential use as a sexual contraceptive for both women and men [13].
The trunk bark contains nimbin (0.04%), nimbinin (0.001%), nimbidin (0.4%), nimbosterol (0.03%), essential oil (0.02%), tannins (6.0%), a bitter principle margosine and 6-desacetyl nimbinene. The bark contains anti-inflammatory polysaccharides consisting of glucose, arabinose and fructose. The bark also yields an antitumor polysaccharide. Besides polysaccharides, several diterpenoids such as nimbinone, nimbolicin, margocin, nimbidiol and nimbione have been isolated in the bark from the stem as well as the root [14].
.
Gum
.
The tree exudes a gum, which on hydrolysis yields L-arabinose, L-fucose, D-galactose and D-glucoronic acid. The older tree exudes a sap containing free sugars (glucose, fructose, mannose and xylose), amino acids (alanine, aminobutyric acid, arginine, asparagines, aspartic acid, glycine, norvaline and praline) and organic acids (citric, malonic, succinic and fumaric) [15]. The sap is reported to be useful in the treatment of general weakness, skin diseases and oral hygeine [16].
.
.
Fruit
.
Generally taking the form of an ellipsoidal glabrous drupe, neem fruit is similar in shape and texture to an olive. When ripe, the fruit turns from light green to yellowish and almost light brown. The fruit measures between .55 in and 1.1 in (14 – 28 mm) long and .39 to .59 in (10 – 15 mm) wide. Consisting of a thin smooth skin (exocarp) and a bitter-sweet, yellowish-white pulp (mesocarp) enclosing a seed (endocarp), neem fruit is used across the pharmaceutical, cosmetic, agricultural and veterinary industries.
.
.
Seed and Kernel
The seed is composed of a shell and one (occasionally two) kernels. Each kernel is ovoid and apex pointed, normally darker than and approximately half the weight of the seed.
The kernel is very important both because of its high lipid content as well as the occurrence of a large number of biologically active constituents, including azadirachtin, gedunin and nimbin in considerable quantities.
Neem kernels are 45% oil, which is composed of oleic acid (50-60%), palmitic acid (13-15%), stearic acid (14-19%), linoleic acid (8-16%) and arachidic acid (1-3%). It is a brownish yellow, non-drying oil with an acrid taste. The quality of the oil differs with each method of processing [17].
.
Neem DNA
The neem tree is a rich source of limonoids, including complex tetranortriterpenoids such as azadirachtin, salanin and nimbin, that offer incredible and diverse application. Nevertheless, neither the molecular aspects of these terpenoids nor the biochemical pathways of these metabolites have been fully studied.
Nimbin was the first of more than 150 bioactive chemical compounds isolated from several neem tissues. The molecular structure of the major tetranortriterpenoid, azadirachtin, required more than 20 years of research for its dilucidation. Because the chemical synthesis of azadirachtin is not viable for commercial production, the interest to understand the in-vivo biosynthesis of its pathway has increased.
To provide a much needed model for metabolic pathways of this bioactive compound, as well as to identify each individual gene found within neem, next generation sequencing is needed. The molecular phylogenetic analysis has confirmed its taxonomic classification by grouping the A. Indica with the citrus (rutaceae) family, more specifically with the Citrus sinensis, the orange.
Using Ilumina and 454 sequencing reads resulted in 267 Mb, from the 44,000 plus genes predicted, 32,278 were actually expressed, of which around 32% are repetitive DNA elements. To quantify azadirachtin, salanin, and nimbin, ultrahigh performance liquid chromatography-mass spectrometry-selected reaction monitoring (UHPLC-MS/SRM) method was used, which can detect picogram (pg) levels of metabolites in neem. The results show that the mature seed always has the greatest concentration of metabolites: azadirachtin 11,046 pg/μg, salanin 5,235 pg/μg and nimbin 3,607 pg/μg.
Almost 70% of the genome has been assembled, based on estimates of the neem genome size by Ohri, Bhargava & Chatterjee (2004). Of the 30,000 genes supported by expression evidences, more than 13,000 of them were expressed in all the tissue while nearly 3,000 were tissue specific.
These studies added transcriptomic, metabolites, and genomic data to identify biochemical pathways of neem limonoids in the future. Science and entrepreneurship will take us into the future, where neem organic pesticides and fertilizers will be available and affordable to change the very nature of modern agriculture.
Neem’s Biologically Active Compounds
Scientists around the world, fascinated by neem’s varied attributes and applications, have spent the last several decades isolating the plant’s most varied and interesting chemical compounds. Neem boasts a complex chemical structure, with the seeds containing hundreds of biologically active compounds of which azadirachtin, nimbin, nimbidin and nimbolides are major molecules [18]. Many of these compounds have antifeedancy, ovicidal activity, fecundity suppression in addition to insect growth regulation and repellence against insects [19].
The most exciting chemical characteristic of neem is its composition of liminoids – a class of phytochemicals, found in citrus fruit and certain other plants, proven to have various medicinal, therapeutic and insecticide effects. These include:
Azadirachtin
Renowned for its robust insecticidal properties against 500 species of insects, institutions such as the Environmental Protection Agency, the US Department of Agriculture and the European Union have endorsed the extraordinary compound for agricultural use [20].
Biodegradable and non-toxic to mammals, azadarichtin disrupts the growth cycle of insects and deters them from feeding on plants. A naturally occurring substance that belongs to an organic molecule class called tetranortrriterpenoids, azadirachtin is a chemical that is similar in structure to the insect hormone ‘ecdysones.’ These hormones control metamorphoses in insects and are disrupted when exposed to azadirachtin (neem’s most active ingredient). By affecting the hormonal balance of insects, azadirachtin reduces their rate of reproduction and inhibits normal feeding behaviors. Since neem-based products must be ingested to take effect, only those that feed on plant tissues are exposed, thereby eliminating any risk towards pollinators and other natural enemies.
The extraction of azadirachtin from neem is a crucial process in ensuring the highest quality of the bio-insecticide. Azadirachta Indica oil and azadirachtin can be acquired through solvent dilution, aqueous extraction or cold pressing. These extraction methods produce various oil strengths, and are priced accordingly. The azadirachtin concentration in neem seeds ranges from 0.1%-0.9% [21], and with 30-60 grams of the compound needed to combat key pests per hectare, per hectare costs can reach up to $60. Conventional pesticides can be nearly double the price at $108 per hectare [22]. Solvent extraction, the most expensive method, produces the purest type of azadirachtin by separating all other chemicals found in neem. Aqueous extraction, despite obtaining the weakest concentration, is the cheapest option and is often used in developing regions of the world where resources are scarce [23].
Neem seeds that have gone thorough the oil extraction process do not go to waste. The residue, known as ‘cake’, contains vital nutrients necessary for plant development. When returned to the soil, the cake nourishes both plants and crops. Improving the soil’s organic content while protecting plant roots from pests, diseases and nematodes, the cake strengthens soil texture and increases water retention [24]. Packed with nutritional value, neem cake has more nitrogen, phosphorous, potassium, calcium and magnesium than ordinary farmyard manure. It is used to fertilize rice, cotton, and sugarcane, while also protects a variety of other crops, including coconut trees [25].
Nano-biotechnology is the latest frontier for botanical insecticides such as azadirachtin, offering the ability to provide a controlled release of the chemical compound at the point of action. It can also help to minimize undesirable toxic effects on non-target organisms, as well as improve the stability of its physical and chemical properties, while preventing the degradation of the active agent by microorganisms [26].
The reason for the extensive research into azadirachtin over the past several decades is simple: an increasing population that will require more food, crop yields at risk of decline and rapidly developing resistance to conventional pesticides will require environmentally sustainable methods for agricultural development in future. The increasing concern of regulatory authorities towards chemical-based fertilizers will also convince both farmers and consumers to opt for safer practices. There is no question, agriculture’s future is organic.
Gedunin
Gedunin, another tetranoterpenoid obtained from the neem tree, has been used as a natural remedy across Asia for centuries [27]. Over the past decade, Georgia Regents University Cancer Center discovered that the chemical compound showed promising effectiveness in disabling cancer cells [28].
Cancer cells work by ‘hijacking’ molecular chaperones, which are responsible for protecting the proteins that take care of normal cellular functions. These molecular chaperones are then tricked into keeping the cancerous cells alive.
Recent cancer drug development has focused on the molecular chaperone Hsp90. Up to now, the clinical efficacy of Hsp90 inhibitors has been poor, but neem-extracted gedunin attacks a ‘co-chaperone’ or helper protein of Hsp90 called p23. Gedunin works by binding directly to p23, which results in the Hsp90 becoming inactive, and causing the cancer cell to die [29].
Gedunin would therefore also have applications across hormone-dependent cancers such as breast, prostate and endometrial.
Nimbin
Nimbin, another chemical compound isolated from neem, is considered as responsible for much of the plant’s biological activities. The compound is reported to have anti-inflammatory, antipyretic, antifungal, antihistaminic and antiseptic properties [30].
Salannin
Salannin is a limonoid of the tetranortriterpenoid type found to occur only in the different types of neem trees [31]. The biological effects of salannin include insect antifeedant or feeding deterrency activity against the house fly, striped cucumber beetle, spotted cucumber beetle, Egyptian cotton leafworm, spiny bollworm, locust and others. [32].
Although the insect antifeedant activity of salannin is well documented, little is known about its structure-bioactivity relationship.
Neem Oil
Extracted and pressed from the kernels inside the seeds, the oil is recognized and valued as a safe and effective bio-pesticide for organic farming. The oil is also widely studied for its medicinal properties, while being used in a variety of cosmetic products such as creams, soaps and shampoos.
Neem oil varies in color, depending on the health of the seeds and how the oil is extracted. Neem oil can be golden yellow, yellowish brown, reddish brown, dark brown, greenish brown or bright red. Neem oil is composed mainly of triglycerides and contains many triterpenoid compounds, which are responsible for the bitter taste. The oil is hydrophobic in nature; in order to emulsify it in water for application purposes, the product must be formulated with appropriate surfactants [33].
Azadirachtin is the most well known and studied triterpenoid in neem oil. Nimbin is another triterpenoid which has been credited with some of neem oil’s properties as an antiseptic, antifungal, antipyretic and antihistamine [34].
Neem Oil Extraction
The extraction process will depend on the desired use of the neem oil, however there are four main types of extraction [35]:
Cold Pressing – The fruits are collected and the kernels separated. The leftover seeds are then woven, dried and fed into stainless steel presses to extract the oil. At no point in the extraction process can the temperature rise above 120ºF for the oil to be considered cold pressed. By using this method the oil retains all of its flavor, aroma and nutritional value.
Steam Pressure Extraction – Neem seeds, after the drying process, are fed into a steam boiler. The seeds are then swollen and the increasing pressure in the broiler drives the oil out from the seed without any pressing.
Solvent Extraction – Solvents such as hexane, acetone and methanol are mixed in with the seeds after they are woven and dried. The resulting oil is stored in a silo where pure neem oil is derived from the crude oil.
Aqueous Extraction – The simplest technique and most used is the extraction in water. It consists of crushing or grinding the seed or neem leaves, submerging them in water, straining them into thin fabric and collecting the extract. This extract can be used in spray for the control of pests without additional modification.
A by-product obtained by the cold-pressing of neem tree fruits and kernels, neem cake acts as an organic fertilizer with pesticidal properties [36].
Uses of Neem Oil
Neem oil is used for medicinal, agricultural and cosmetic purposes, and has been a feature of community life in South East Asia for centuries. The most frequent use of neem oil is in the treatment of skin diseases, inflammations and fevers [37]. The oil is also known for its effectiveness as an insecticide and insect repellent [38].
Traditional Ayurvedic medicine uses of neem include the treatment of acne, fever, leprosy, malaria, ophthalmia and tuberculosis. Various folk remedies for neem include use as an anthelmintic, antifeedant, diuretic, emmenagogue, contraceptive, febrifuge, parasiticide and insecticide. It has been used in traditional medicine for the treatment of tetanus, urticaria, eczema, scrofula and erysipelas [39].
Formulations made from neem oil also find wide usage as a biopesticide for organic farming, as it repels a wide variety of pests including the mealy bug, beet armyworm, aphids, the cabbage worm, thrips, whiteflies, mites, fungus gnats, beetles, moth larvae, mushroom flies, leafminers, caterpillars, locust, nematodes and the Japanese beetle [40]. Neem oil is not known to be harmful to mammals, birds, earthworms or beneficial insects such as butterflies, honeybees and ladybugs (ladybirds) if it is not concentrated directly into their area of habitat or on their food source [41]. Neem oil is used as a mite repellent for bees, sometimes sprayed directly on the hive. It can be used as a household pesticide for ant, bedbug, cockroach, housefly, sand fly, snail, termite and mosquitoes both as repellent and larvicide. Neem oil also controls black spot, powdery mildew, anthracnose and rust fungi [42].
Neem Cake
Neem cake contains NPK in organic form, which is conducive for plant growth. As a completely organic and botanical product, neem cake contains 100% natural NPK content and other micro nutrients such as Nitrogen (2.0% to 5.0%), Phosphorus (0.5% to 1.0%), Potassium (1.0% to 2.0%), Calcium (0.5% to 3.0%), Magnesium (0.3% to 1.0%), Sulphur (0.2% to 3.0%), Zinc (15 ppm to 60 ppm), Copper (4 ppm to 20 ppm), Iron (500 ppm to 1200 ppm) and Manganese (20 ppm to 60 ppm). The cake is also rich in both sulphur compounds and liminoids [43].
The Organic Content of Neem Cake
Use as an Organic Fertilizer
Neem cake improves the organic content in the soil by reducing alkaline levels and producing organic acids during decomposition. Being completely natural, neem is compatible with soil microbes, improves rhizosphere microflora and ensures stronger texture, higher water holding capacity and aeration in the soil for better root development [44]. Neem cake also protects the plant roots from nematodes, soil grubs and white ants [45].
Neem makes the soil more fertile due to an ingredient that blocks soil bacteria from converting nitrogenous compounds into nitrogen gas. As a nitrification inhibitor, neem prolongs the availability of nitrogen to both short-duration and long duration crops [46].
Applying neem along with nitrogenous fertilizers can also slow down the conversion process of nitrogenous compounds in ammonia, nitrates and nitrites, improving the overall efficiency of the soil [47].
Neem cake is applied by mixing it with the soil on and around the roots of the plans. Currently, the different types of commercial neem cake on the market are roughly identified as oiled and deoiled cake, but several other differences can be detected.
Quantity of Neem Cake Necessary According to Crop
Neem Cake as an Organic Pesticide
Neem cake is effective in the management of insects and pests. The bitter principles of the soil and cake have been reported to act as an antifeedant, attractant, repellent, insecticide, nematicide, growth disruptor and antimicrobial [48].
[1] Puri, H.S. Neem: The Divine Tree (2005) [2] HDRA – The Organic Organization. The Neem Tree (2015) [3] Akhila, A., and K. Rani. "Chemistry of the neem tree (Azadirachta indica A. Juss e/Progress in the Chemistry of Organic Natural Products. Springer Vienna (1999) [4] Girish, K., and S. Shankara Bhat. Neem - A Green Treasure. Electronic journal of Biology 4.3 (2008) [5] Koul, Opender. "Neem: A Global Perspective." Neem: Today and in the New Millennium. (2004) [6] Tomar, Alka, and K. K. Singh. Neem: An Introduction (2009) [7] Neem International Network Seed Source, United Nations Food and Agriculture Organization. [8] National Research Council. Neem: a Tree for Solving Global Problems. The Minerva Group (2002) [9] Schmutterer, Heinrich. Properties and Potential of Natural Pesticides from the Neem Tree, Azadirachta Indica. Annual Review of Entomology 35.1 (1990) [10] Subapriya, R., and S. Nagini. Medicinal properties of neem leaves: a review. Current Medicinal Chemistry-Anti-Cancer Agents 5.2 (2005) [11] Govindachari, T. R. Chemical and Biological Investigations on Azadirachta Indica (1992) [12] Kupradinun, Piengchai, et al. Toxicity Testing of Flowers of Neem tree (Azadirachta indica A. Juss)." Thail J Vet Med 40.1 (2010) [13] Livestrong, Neem Bark Benefits (2015) [14] Singh, Kamal Kishor. Neem, A Treatise. IK International (2009) [15] Neem Foundation (2016) [16] Mukherjee, S., and H. C. Srivastava. "The structure of neem gum." Journal of the American Chemical Society 77.2 (1955) [17] Biswas, Kausik, et al. Biological Activities and Medicinal Properties of Neem (Azadirachta indica (2002) [18] Neem Foundation (2016) [19] Biswas, Kausik, et al. Biological Activities and Medicinal Properties of Neem. (2002) [20] Schmutterer, Heinrich. "Properties and potential of natural pesticides from the neem tree, Azadirachta indica." Annual review of entomology 35.1 (1990) [21] Nisbet, Alasdair J. "Azadirachtin from the neem tree Azadirachta indica: its action against insects." Anais da Sociedade Entomológica do Brazil 29.4 (2000) [22] Warthen Jr, J. D., et al. "Estimation of Azadirachtin Content in Neem Extracts and Formulations." Journal of Liquid Chromatography 7.3 (1984) [23] The Primal Group (2015) [24] Morgan, E. David. "Azadirachtin, a scientific gold mine." Bioorganic & Medicinal Chemistry 17.12 (2009) [25] Rao, P. Udayasekhara. Chemical Composition and Biological Evaluation of Debitterized and Defatted Neem (Azadirachta indica) Seed Kernel Cake. Journal of the American Oil Chemists’ Society (1987) [26] Prasad, C. Devakumar, and Y. S. Shivay. Significance in Increasing Fertilizer Nitrogen Efficiency. Neem Research and Development (1993) [27] University of Sao Paolo Department of Environmental Engineering. Application of Nanotechnology for the Encapsulation of Botanical insecticides for Sustainable Agriculture (2014) [28] Biswas, Kausik, et al. "Biological activities and medicinal properties of neem (Azadirachta indica). (2002) [29] Kamath, Siddharth G., et al. "Gedunin, a novel natural substance, inhibits ovarian cancer cell proliferation." International Journal of Gynecological Cancer 19.9 (2009) [30] Memorial Sloane Kettering Cancer Center. Integrative Medicine – Neem (2014) [31] Koul, Opender, Murray B. Isman, and C. M. Ketkar. Properties and Uses of Neem, Azadirachta Indica." Canadian Journal of Botany 68.1 (1990). [32] Johnson, Shaun, and E. David Morgan. Supercritical Fluid Extraction of Oil and Triterpenoids from Neem Seeds." Phytochemical Analysis 8.5 (1997) [33] Mitchell, M. J., et al. "Effects of the Neem tree Compounds Azadirachtin, Salannin and Nimbin. Archives of Insect Biochemistry and Physiology 35.1‐2 (1997) [34] Biswas, Kausik, et al. Biological Activities and Medicinal Properties of Neem (2002) [35] Isman, Murray B., et al. Insecticidal and Antifeedant Bioactivities of Neem Oils and their Relationship to Azadirachtin Content. Journal of Agricultural and Food Chemistry (1990) [36] Liauw, Maria Yuliana, et al. Extraction of Neem Oil (Azadirachta Indica A. Juss) Using n-hexane and ethanol: Studies of oil Quality, Kinetic and Thermodynamic." ARPN Journal of Engineering and Applied Sciences 3.3 (2008) [37] Rao, P. Udayasekhara. "Chemical Composition and Biological Evaluation of Debitterized and Defatted Neem (Azadirachta indica) Seed Kernel Cake. Journal of the American Oil Chemists’ Society 64.9 (1987) [38] Roy, Amit, and Shailendra Saraf. Limonoids: Overview of Significant Bioactive Triterpenes Distributed in Plants Kingdom." Biological and Pharmaceutical Bulletin 29.2 (2006) [39] Stark, John D., and James F. Walter. Neem Oil and Neem Oil Components Affect the Efficacy of Commercial Neem Insecticides. Journal of Agricultural and Food Chemistry 43.2 (1995). [40] Brahmachari, Goutam. Neem: An Omnipotent Plant - A Retrospection."Chembiochem 5.4 (2004) [41] Immaraju, John A. The Commercial Use of Azadirachtin and its Integration into Viable Pest Control Programmes." Pesticide Science 54.3 (1998). [42] Raizada, R. B., et al. Azadirachtin, A Neem Biopesticide: Subchronic Toxicity Assessment. Food and chemical toxicology 39.5 (2001). [43] Hirose, Edson, et al. Effect of Biofertilizers and Neem Oil on the Entomopathogenic Fungi Beauveria Bassiana (Bals.) Vuill. and Metarhizium Anisopliae (Metsch.) Sorok." Brazilian Archives of Biology and Technology 44.4 (2001). [44] Rao, P. Udayasekhara. Chemical Composition and Biological Evaluation of Debitterized and Defatted Neem (Azadirachta indica) Seed Kernel Cake. Journal of the American Oil Chemists’ Society 64.9 (1987). [45] Gopal, Murali, et al. "Impact of Azadirachtin, an Insecticidal Allelochemical from Neem on Soil Microflora, Enzyme and Respiratory Activities." Bioresource Technology 98.16 (2007) [46] Saxena, R. C., H. D. Justo, and P. B. Epino. "Evaluation and Utilization of Neem Cake Against Homoptera: Delphacidae. Journal of economic entomology 77.2 (1984). [47] Sahrawat, K. L., and B. S. Parmar. Alcohol Extract of Neem (Azadirachta indica L.) Seed as Nitrification Inhibitor." Journal of the Indian Society of Soil Science 23.1 (1975). [48] Radwanski, S. A., and G. E. Wickens. "Vegetative Fallows and Potential Value of the Neem Tree (Azadirachta indica) in the Tropics." Economic Botany 35.4 (1981).