Effect of Elicitors and Precursors on Azadirachtin Production in Hairy Root Culture of Azadirachta indica

The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag⁺, Hg⁺², Co⁺², Cu⁺²) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l^?1 of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g^?1). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 %?v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g^?1) with respect to the control (3.3 mg g^?1). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l^?1 in 25 days) was found to be the highest with cholesterol (50 mg l^?1) addition as an indirect precursor in the medium.     

Strategies to Overcome Oxygen Transfer Limitations During Hairy Root Cultivation of Azadiracta indica for Enhanced Azadirachtin Production

The vast untapped potential of hairy root cultures as a stable source of biologically active chemicals has focused the attention of scientific community toward its commercial exploitation. However, the major bottleneck remains its successful scale-up. Due to branching, the roots form an interlocked matrix that exhibits resistance to oxygen transfer. Thus, present work was undertaken to develop cultivation strategies like optimization of inlet gas composition (in terms of % (v/v) O₂ in air), air-flow rate and addition of oxygen vectors in the medium, to curb the oxygen transfer limitations during hairy root cultivation of Azadirachta indica for in vitro azadirachtin (a biopesticide) production. It was found that increasing the oxygen fraction in the inlet air (in the range, 20–100% (v/v) O₂ in air) increased the azadirachtin productivity by approximately threefold, to a maximum of 4.42 mg/L per day (at 100% (v/v) O₂ in air) with respect to 1.68 mg/L per day in control (air with no oxygen supplementation). Similarly, increasing the air-flow rate (in the range, 0.3–2 vvm) also increased the azadirachtin productivity to a maximum of 1.84 mg/L per day at 0.8 vvm of air-flow rate. On the contrary, addition of oxygen vectors (in the range, 1–4% (v/v); hydrogen peroxide, toluene, Tween 80, kerosene, silicone oil, and n-hexadecane), decreased the azadirachtin productivity with respect to control (1.76 mg/L per day).     

In Vitro Azadirachtin Production by Hairy Root Cultivation of <Emphasis Type="Italic">Azadirachta indica</Emphasis> in Nutrient Mist Bioreactor

Azadirachtin, a well-known biopesticide is a secondary metabolite conventionally extracted from the seeds of Azadirachta indica. The present study involved in vitro azadirachtin production by developing hairy roots of A. indica via Agrobacterium rhizogenes-mediated transformation of A. indica explants. Liquid culture of hairy roots was established in shake flask to study the kinetics of growth and azadirachtin production. A biomass production of 13.3 g/L dry weight (specific growth rate of 0.7 day⁻¹) was obtained after 25 days of cultivation period with an azadirachtin yield of 3.3 mg/g root biomass. To overcome the mass transfer limitation in conventionally used liquid-phase reactors, batch cultivation of hairy roots was carried out in gas-phase reactors (nutrient spray and nutrient mist bioreactor) to investigate the possible scale-up of A. indica hairy root culture. The nano-size nutrient mist particles generated from the nozzle of the nutrient mist bioreactor could penetrate till the inner core of the inoculated root matrix, facilitating uniform growth during high-density cultivation of hairy roots. A biomass production of 9.8 g/L dry weight with azadirachtin accumulation of 2.8 mg/g biomass (27.4 mg/L) could be achieved in 25 days of batch cultivation period, which was equivalent to a volumetric productivity of 1.09 mg/L per day of azadirachtin.     

Enhanced Production of Artemisinin by Hairy Root Cultivation of Artemisia annua in a Modified Stirred Tank Reactor

Artemisinin is an important drug commonly used in the treatment of malaria as a combination therapy. It is primarily produced by a plant Artemisia annua, however, its supply from plant is significantly lower than its huge demand and therefore alternative in vitro production routes are sought. Hairy root cultivation could be one such alternative production protocol. Agrobacterium rhizogenes was used to induce hairy roots of A. annua. Statistical optimization of media was thereafter attempted to maximize the biomass/artemisinin production. The growth and product formation kinetics and the significant role of O₂ in hairy root propagation were established in optimized media. Mass cultivation of hairy roots was, thereafter, attempted in a modified 3-L Stirred Tank Bioreactor (Applikon Dependable Instruments, The Netherlands) using optimized culture conditions. The reactor was suitably modified to obtain profuse growth of hairy roots by segregating and protecting the growing roots from the agitator rotation in the reactor using a perforated Teflon disk. It was possible to produce 18 g biomass L^?1 (on dry weight basis) and 4.63 mg L^?1 of artemisinin in 28 days, which increased to 10.33 mg L^?1 by the addition of elicitor methyl jasmonate.     

Azadirachtin as a Biomarker Compound in HPTLC Assay of Seed and Seed Oil of Azadirachta indica A. Juss.

JPC – Journal of Planar Chromatography – Modern TLC -     

Chemical Constituents of the Flowers of Azadirachta indica

Studies on the chemical constituents of the flowers of Azadirachta indica have led to the isolation of two new flavanones, flowerine (=5‐hydroxy‐7,4′‐dimethoxy‐8‐(3‐methylbut‐2‐enyl)flavan‐4‐one; 1 ) and flowerone (=5,7,8,4′‐tetrahydroxy‐3′‐(3‐methylbut‐3‐enyl)flavan‐4‐one; 2 ), and two new triterpenoids, O‐methylazadironolide (=7α‐(acetoxy)‐23ξ‐methoxy‐21,23‐epoxy‐24,25,26,27‐tetranorapotirucalla‐1,14,20(22)‐trien‐3,21‐dione; 3 ) and diepoxyazadirol (=(20S,23S,24R)‐7‐α‐(acetoxy)‐25‐hydroxy‐21,24 : 23,24‐diepoxyapotirucalla‐1,14‐dien‐3‐one; 4 ) along with the known triterpenoid trichilenone acetate (=7α‐(acetoxy)‐14,15 : 21,23‐diepoxy‐24,25,26,27‐tetranorapotirucalla‐1,20,22‐trien‐3‐one; 5 ), two known flavanones, nimbaflavone (=5,7‐dihydroxy‐4′‐methoxy‐8,3′‐bis(3‐methylbut‐2‐enyl)‐flavan‐4‐one; 6 ) and 3′‐prenylnaringenin (=5,7,4′‐trihydroxy‐3′‐(3‐methylbut‐2‐enyl) flavan‐4‐one; 7 ), and 4‐(2‐hydroxyethyl)phenol ( 8 ). Their structures have been elucidated through spectral studies, including 2D‐NMR experiments, and chemical transformation. Compounds 5, 7 and 8 are heretofore unreported from any part of tree, while 6 has been isolated earlier from leaves.     

A new flavanoid from the flowers of Azadirachta indica

Studies on the chemical constituents of the flowers of Azadirachta indica have led to the isolation of one new flavanone named as azharone (5,7,4'-trihydroxy-3'-(3'-methyl-2',3'-epoxybutyl)flavan-4-one (3)) along with two known constituents azadirone (1), and isoazadironolide (2). Their structures have been elucidated through spectral studies including 2D-NMR (COSY-45, NOESY, J-resolved, HMQC, HMBC) experiments.     

Two New Degraded Triterpenoids and a Novel seco‐Norabietane Diterpene from the Root Bark of Azadirachta indica

Three new compounds, the degraded ring C‐seco‐tetranortriterpenoid nimbolicidin ( 1 ), the degraded hexanortriterpenoid nimbocin ( 2 ), and the seco‐norabietane diterpene nimbocinin ( 3 ), were isolated from the root bark of Azadirachta indica A.Juss . Compound 1 is O‐bearing both at C(28) and C(29), which has been hitherto unreported in tetranortriterpenoids; 2 represents the first hexanortriterpenoid with a truncated apotirucallane (or apoeuphane) skeleton; 3 is an unprecedented seco‐norabietane. Spectroscopic data and chemical transformations of these compounds provided their complete structures.     

A new tetracyclic triterpenoid from the leaves of Azadirachta indica

A new tetracyclic triterpenoid zeeshanol [25,26,27-trinor-apotirucalla-(apoeupha)-6alpha-, 21-dihydroxy, 7alpha-acetoxy, 1,14,22-tri-en-3, 16-dione] (1) along with a known constituent desfurano-6alpha-hydroxyazadiradione (2) have been isolated from the methanolic extract of the leaves of Azadirachta indica. The structure and the relative configurations of 1 were determined by the spectroscopic method (1H- and 13C-NMR, IR, and MS) and 2D-NMR experiments.     

Detection of phytoconstituents present in Azadirachta indica L. seeds extract by GC-MS analysis

Azadirachta indica L., commonly known as "Neem," belongs to the Meliaceae family. Traditionally, "Neem" has been used to cure diabetes, leprosy, and respiratory disorders. Azadirachta indica L. seed n-hexane extract was tested using GC-MS to determine the Phyto-components present. The analysis of A. indica L. seeds revealed the existence of Hexadecanoic acid, methyl ester (0.32%), n-Hexadecanoic acid (23.77%), 9-Octadecenoic acid (Z)- (0.07%), Methyl stearate (0.21%), Agaricic acid (0.06%), (E)-9-Octadecenoic acid ethyl ester (0.10%), 9-Octadecenoic acid (Z)-, methyl ester (0.48%), 6-Octadecenoic acid, (Z)- (64.25%), 9-Octadecenamide (0.06%), Oleic Acid (0.16%), Heneicosane (0.04%), Tetratetracontane (0.16%), Pentacosane (0.09%), Squalene (1.17%), Tetracontane (0.36%), γ-Tocopherol (0.16%), 2-[2-(6,6-Dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl]-6,6-dimethylbicyclo[3.1.1]hept-2-ene (0.44%), γ-Sitosterol (0.25%), Kryptogenin dioxime (1.53%.), 9-Hexadecenoic acid, 9-octadecenyl ester, (Z,Z)- (1.34%.), and Lupa-13(18),20(30)-dien-3-yl acetate (0.17%). The findings of this study provide a foundation for employing A. indica L. seeds as a herbal option for a variety of ailments. GC-MS analysis was used to determine the quality of these substances. In the pharmaceutical industry, GC-MS reports will be useful for identifying a wide range of phyto-bioconstituents in various plant extracts, polyherbal extracts, and the standardization of specific plant materials.     

Synthesis of natural products from the Indian neem tree Azadirachta indica

The synthesis of five natural products (3, 6, 7, 10, and 14), isolated from the Indian neem tree Azadirachta indica, is reported from a common intermediate (2). The judicious choice of transacetalization conditions allows efficient access to both the azadirachtinin (9 and 10) and the azadirachtin (3, 6, 7, and 14) skeletons.     

Tetracyclic triterpenoids from the leaves of Azadirachta indica and their insecticidal activities

A new tetranortriterpenoid, meliatetraolenone [24,25,26,27-tetranor-apotirucalla-(apoeupha)-6alpha-O-methyl, 7alpha-senecioyl(7-deacetyl)-11alpha,12alpha,21,23-tetrahydroxy-21,23-epoxy-2,14,20(22)-trien-1,16-dione] (1) was isolated from the methanolic extract of fresh leaves of Azadirachta indica along with the known compound odoratone (3) which was hitherto unreported from this source. Their structures have been elucidated by spectral studies including 2D NMR. The insecticidal activities of 1 as well as those of odoratone (3) are reported. 1 and odoratone both showed mortality on fourth instar larvae of mosquitoes (Anopheles stephensi) with LC(50) values of 16 and 154 ppm, respectively.     

铕对决明发根生长及有效成分生成的作用

以决明发根为材料,在培养基中附加０．００１～１０ｍｇ．Ｌ＾－１Ｅｕ＾３＋（ＥｕＣ１３）,测量发根的鲜重和干重,用高效液相色谱法测定游离蒽醌化合物含量。发有低剂量（０．００１,０．０１,１．０ｍｇ．Ｌ＾－１）Ｅｕ＾３＋抑制生长,０．００１ｍｇ．Ｌ＾－１Ｅｕ＾３＋抑制作用最强,干重比对照降低２２％;较低剂量（１０ｍｇ．Ｌ＾－１）Ｅｕ＾３＋处理的含量高于对照２６＾,高于Ｅｕ＾３＋１．０ｍｇ．Ｌ＾－１处理     

Determination of azadirachtin and fatty acid methyl esters of Azadirachta indica seeds by HPLC and GLC

A simple and economical method has been developed to estimate the azadirachtin content and fatty acid composition of neem kernels. Neem kernels are crushed and soaked overnight in ethanol. The extract obtained is analysed by HPLC after filtering through a 0.22 micro m membrane. The peaks are separated using acetonitrile-water (40:60) 1 mL min(-1) as the mobile phase on an RP-18 column and monitored at 214 nm. For the determination of fatty acid composition, the fatty acids are directly transmethylated in the kernel powder by heating with methanol-acetyl chloride-benzene (20:1:4, v/v) for 1 h in a water bath. The fatty acid methyl esters (FAMEs) obtained are extracted in hexane and analysed using GLC. The separation of the FAMEs is achieved using an RH-Wax column using temperature programming, 170-200 degrees C at 2 degrees min(-1). The peaks are detected using an FID. Both the methods do not require any clean up or defatting of seeds. This results in faster, easier and more economical sample preparation.     

Efficacy of the Aqueous Extract of Azadirachta indica Against the Marine Parasitic Leech and Its Phytochemical Profiling

Zeylanicobdella arugamensis (Hirudinea), a marine parasitic leech, not only resulted in the mortality of the host fish (Groupers) but also caused economic losses. The current study aimed to elucidate the antiparasitic efficacy of the aqueous extract of the Azadirachta indica leaves against Z. arugamensis and to profile the composition via LC-Q Exactive HF Orbitrap mass spectrometry. Different concentrations (25, 50 and 100 mg/mL) of A. indica extract were prepared and tested on the parasitic leeches. The total mortality of leeches was noticed with an exposure to the A. indica aqueous extract. The average times required for the aqueous extract at concentrations of 25, 50 and 100 mg/mL to kill the leeches were 42.65 ± 9.20, 11.69 ± 1.11 and 6.45 ± 0.45 min, respectively, in a dose-dependent manner. The Orbitrap mass spectrometry analysis indicated the presence of five flavonoids (myricetin 3-O-galactoside, trifolin, isorhamnetin, quercetin and kaempferol), four aromatics (4-methoxy benzaldehyde, scopoletin, indole-3-acrylic acid and 2,4-quinolinediol), three phenolics (p-coumaric acid, ferulic acid and phloretin) and two terpenoids (pulegone and caryophyllene oxide). Thus, our study indicates that A. indica aqueous extract is a good source of metabolites with the potential to act as a biocontrol agent against the marine parasitic leech in aquaculture.     

HPLC Analysis of Alizarin and Purpurin Produced by Rubia tinctorum L. Hairy Root Cultures

We have determined the quantities of the anthraquinones alizarin and purpurin, with especial regard to their effective antigenotoxic activity, in genetically transformed hairy root cultures of Rubia tinctorum L. Hairy roots were cultured on solid and in liquid Gamborg B5 and 1/2 NMS media in a shaking cabinet and in a bioreactor. Methanolic extracts of lyophilized hairy roots were hydrolysed, and then purified by solid phase extraction with good recovery. A new HPLC method was developed for the determination of alizarin and purpurin. The analysis was performed on a Luna C8 RP column using a 45:55 (v/v) mixture of acetonitrile:20 mM ammonium formate-formic acid buffer (pH 3.00) as eluent. Peaks were identified by addition of standards and by diode-array detection. External standardization allowed the determination of alizarin and purpurin with good sensitivity and reliability. The maximum purpurin content was observed in cultures cultivated on solid B5 medium (5.94 mg g⁻¹). The highest alizarin content was measured in cultures cultivated on solid 1/2 NMS medium (2.14 mg g⁻¹).

     

Purification of the seven tetranortriterpenoids in neem (Azadirachta indica) seed by counter-current chromatography sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography

Counter-current chromatography (CCC) sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography was used to isolate the seven bio-actives (azadirachtin A, azadirachtin B, azadirachtin H, desacetylnimbin, desacetylsalannin, nimbin and salannin) from the seed concentrate (NSC) of the neem tree (Azadirachta indica A. Juss). Reproducible, narrow polarity range, high purity fractions were obtained from repeated injections of the NSC (700 mg loadings/injection), on to a relatively small volume CCC coil (116 mL). The CCC biphasic solvent system chosen was hexane:butanol:methanol:water (1:0.9:1:0.9, v/v). A mass balance of injected material showed that 95+% were recovered.     

Natural control of bacteria affecting meat quality by a neem (Azadirachta indica A. Juss) cake extract

The antibacterial activity of an ethylacetate neem cake extract (NCE) against bacteria that affect meat quality, namely Campylobacter jejuni, Carnobacterium spp., Lactobacillus curvatus, Lactobacillus sakei and Leuconostoc sp., is reported. The antibacterial activity was detected using standardised disc diffusion and macrodilution methods. The bacterial growth inhibition zone ranged from 11.33 ± 0.58 to 22.67 ± 0.58 mm (100 μL NCE). There is significant difference between the growth inhibition zone of NCE and the control (ciprofloxacin 100 μg). The percent of bacterial growth reduction range was 79.75 ± 1.53 to 90.73 ± 1.53 (100 μg NCE) as compared with control (without NCE). NCE in different amounts counteracted the growth of all tested bacteria.     

Study on the Synthesis of ZnO Nanoparticles Using Azadirachta indica Extracts for the Fabrication of a Gas Sensor

This paper compared the effects of A. indica plant proteins over chemical methods in the morphology of zinc oxide nanoparticles (ZnO NPs) prepared by a co-precipitation method, and ethanol sensing performance of prepared thin films deposited over a fluorene-doped tin oxide (FTO) bind glass substrate using spray pyrolysis technique. The average crystallite sizes and diameters of the grain-sized cluster ZnO NPs were 25 and (701.79 ± 176.21) nm for an undoped sample and 20 and (489.99 ± 112.96) nm for A. india dye-doped sample. The fourier transform infrared spectroscopy (FTIR) analysis confirmed the formation of the Zn–O bond at 450 cm⁻¹, and also showed the presence of plant proteins due to A. indica dye extracts. ZnO NPs films exhibited good response (up to 51 and 72% for without and with A. indica dye-doped extracts, respectively) toward ethanol vapors with quick response-recovery characteristics at a temperature of 250 °C for undoped and 225 °C for A. indica dye-doped ZnO thin films. The interaction of A. indica dye extracts helps to decrease the operating temperature and increased the response and recovery rates of the sensor, which may be due to an increase in the specific surface area, resulting in adsorption of more oxygen and hence high response results.     

A new lactam 28-norlimonoid from the leaves of Azadirachta indica A. Juss. (Meliaceae)

From an EtOAc-soluble fraction of the leaves of Azadirachta indica, one new lactam 28-norlimonoid named nimbandiolactam-21 (1), together with 2 known limonoids (2 and 3) were isolated. Their relative structures were elucidated based on NMR spectroscopic analysis. Nimbandiolactone-23 (2) showed the most potent α-glucosidase inhibitory activity, with an IC₅₀ value of 38.7 μM. Compound 1 represents the first naturally occurring example of a 28-norlimonoid having the lactam moiety. The plausible biosynthetic pathway for the formation of lactam moiety in 1 was proposed.