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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright   © 2021 The  Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                        Research Article 

Physicochemical, Druggable, ADMET Pharmacoinformatics and Therapeutic Potentials of Azadirachtin - a Prenol Lipid (Triterpenoid) from Seed Oil Extracts of Azadirachta indica A. Juss.

T. Loganathan¹, A. Barathinivas², C. Soorya³, S. Balamurugan⁴, T. G. Nagajothi⁵, S. Ramya³, R. Jayakumararaj³*

¹ Department of Plant Biology and Plant Biotechnology, LN Government College (Autonomous), Ponneri - 601204, TN, India

² Department of Zoology, Yadava College for Men, Tirupalai- 625 017, Madurai, TamilNadu, India 

³ Department of Botany, Government Arts College, Melur – 625106, Madurai, TamilNadu, India

⁴ Department of Mathematics, Government Arts College, Melur – 625106, Madurai, TamilNadu, India

⁵ Department of Botany, R D Government Arts College, Sivagangai – 630561, TamilNadu, India

INTRODUCTION

Azadirachta indica A. Juss commonly known as Neem or Margosa belongs to the family Meliaceae^1-3. Popular as Miracle tree it is a natural store-house of phyto-drugs since the dawn of civilization^4,5. This tree is one of the most versatile plant across the country and elsewhere known for its use in various Indigenous/ Traditional Systems of Medicine. A. indica has its origin from India and is commonly distributed in the South East Asian (SEA) Region (Bangladesh, Srilanka, Bhutan, Myanmar, Pakistan, and Nepal)⁶, however, it has been disseminated world over, in particular the tropical and sub-tropical regions⁷. 

Neem is a perennial, small to medium-sized (10 - 15 m) and fast-growing tree and grows well in locations with temperature to a maximum of 48-50 °C, the plant needs low annual rainfall (400 – 800 mm/annum). Furthermore, the plant grows well in poor/ degraded/ mined soils. However, growth is affected by low temperature (poor growth below 14 °C) and frosts. Being the storehouse/ repository of wide array of BASM, Neem tree remains the ideal target of interest for research. As most of the BASM are localised in the leaves and seeds, destruction of whole plant is not required for the isolation/ extraction of bioactive principles. Furthermore, being perennial, annual replenishment of leaves and seeds prevents whole-plant harvest. BASM of Neem contains high proportion of water-soluble substances that favours DIY extraction and application in folklore medicine. Moreover, majority of these metabolites are eco-friendly bioactive compounds that are biodegradable in nature, adhere to GRAS standards, therefore harmless to man and environment⁸. 

A. indica shows therapeutics potential in healthcare and management due to rich source of BASM^9-11. The most important active constituent is azadirachtin, while others include nimbolinin, nimbin, nimbidin, nimbidol, sodium nimbinate, gedunin, salannin, and quercetin. Leaves contain BASM such as nimbin, nimbanene, 6-desacetylnimbinene, nimbandiol, nimbolide, ascorbic acid, n-hexacosanol, 7-desacetyl-7-benzoylazadiradione, 7-desacetyl-7-benzoylgedunin, 17-hydroxyazadiradione, and nimbiol^12-14. Quercetin and β-sitosterol, polyphenolic flavonoids, obtained from fresh leaves have significant antibacterial and antifungal properties; while seeds are comparably rich in azadirachtin¹⁴. 

Since antiquity all parts of the plant, including root, stem, bark, leaves, fruits, and seeds are used to cure various ailments in humans and domestic animals therefore, Neem has been considered as a multi-purposes village dispensary^15-25. In fact, therapeutic applications attributed to Neem include abortive, analgesic, antibacterial, anticancer, antidiabetic, antifungal, anti-helminthic, anti-hyperglycemic, anti-inflammatory, antimalarial, antipyretic, antispasmodic, anti-spermatogenic, antiviral, diuretic, hyper-cholesteremic, immuno-modulatory, mouth-wash, contraception, dental plaque, head lice, heart disease, insect repellent, malaria, pesticide, psoriasis, skin diseases, wound healing, gastro-intestinal ailments^26-55. 

Neem is influenced by a myriad of factors, namely geographic area, climate, genetic variability, agronomic conditions, plant morphology and physiology, collection and storage of plant material which determines the therapeutic potential. Further, this variation affects the development processes as regulation of secondary metabolite synthesis is directly linked to gene expression. This boils down to the fact that growth of Neem plant and the biochemical composition of the active principle is significantly influenced by external parameters. Kaushik et al⁵⁶. and Tomar et al⁵⁷. independently, analysed trees from different regions of India and observed significant difference in the AZA content of seeds collected in different regions. Furthermore, Kaushik et al⁵⁶. evaluated the effect of climatic conditions in the AZA content of seeds and indicated that AZA values of samples from semi-arid regions with mild winters were different from values observed in hot sub-humid, hot arid and hot semi-arid with cold winter regions. Similarly, Zheng et al.⁵⁸ pointed out that season and ecosystem properties significantly affect neem seed oil yield and, in a less extent, AZA content. In fact, AZA quantity obtained in seed was significantly influenced by precipitation, with lower values observed in rainy season. Likewise, the procedure and time of collection of the plant material also influences AZA concentration in the seeds. In the case of seeds, AZA concentration is maximized when clean and healthy seeds are collected^59,60. 

Indeed, it has been reported that mechanical damage, insect infestation and fungal infection of seeds significantly affect quantity and quality of AZA content. Since its isolation for the first time in 1968, AZA has been the subject of intense research, particularly of biological, synthetic and structural studies⁶¹. Azadirachtin - limonoid group of compound is a bioactive secondary metabolite present in neem seeds^{12,13,26,27,60}. It is a highly oxidized tetranortriterpenoid that asserts a plethora of oxygen-bearing functional group which includes an enol-ether, acetal, hemiacetal, tetra-substituted epoxide structure with variety of carboxylic esters (Fig. 1). Increasing interest in AZA is mainly due to the unique biomolecular properties, including broad spectrum of activity even in trace amounts, no or low toxicity to mammals. Its complex structure makes its synthesis a daunting task. Biological activities attributed to AZA include application as a bioinsecticide, biopesticide, insect-pest repellent as it is non-toxicity to humans. Azadirachtin has been identified as potential inhibitor of SARS-CoV-2 main protease^62-64 and is expected to play a major role in the management of COVID-19. Furthermore, pharmacological characterization is expected to validate Azadirachtin as novel drug lead^65-68.

MATERIALS AND METHODS

Botanical Description:  Tree, up to 15 m tall. Branches glabrous; Leaves imparipinnate, pulvinus at the base; leaflets alternate to opposite, 2.5 - 7.0 cm long, 1.5 - 4.0 cm broad, ovate, subsessile, acuminate; Flowers white, sweet-scented; Sepals obovate, 1.5 mm long, puberulous, imbricate. Petals 6 mm long, obvoate to oblong, white, margin ciliate; Staminal tube 5 mm long, puberulous, 10-striate, 10-toothed; teeth 2-lobed; anthers oblong, basifixed; Ovary sub-globose; style linear 2.5 mm long; stigma trifid. Fruit: Drupe oblong, 1.3 - 2.0 cm long, greenish-yellow, Seed: 1-seeded. Plants were collected from the fields in the wild Palani Hills, Western Ghats, INDIA as described previously³³. 

GC-MS Analysis 

Neem Seed Oil Extracts of A. indica was obtained from the seed samples collected from the foothills of Alagar Hills, Alagarkovil Reserve Forest, Dindigul District, Tamil Nadu, India. Phyto-components were identified using GC–MS detection system as described previously, however with modification, whereby portion of the extract was analysed directly by headspace sampling. GC–MS analysis was accomplished using an Agilent 7890A GC system set up with 5975C VL MSD (Agilent Technologies, CA, USA). Capillary column used was DB-5MS (30×0.25 mm, film thickness of 0.25 μm; J&W Scientific, CA, USA). Temperature program was set as follows: initial temperature 50°C held for 1 min, 5°C per min to 100°C, 9°C per min to 200°C held for 7.89 min, and the total run time was 40 min. The flow rate of helium as a carrier gas was 0.811851 mL/ min. MS system was performed in electron ionization (EI) mode with Selected Ion Monitoring (SIM). The ion source temperature and quadruple temperature were set at 230°C and 150°C, respectively. Identification of phyto-components was performed by comparison of their retention times and mass with those of authentic standards spectra using computer searches in NIST 08.L and Wiley 7n.l libraries^3,33. 

ADMET Prediction 

PubChem database was applied to get the smiles structures of the natural compounds, and was further used for the ADMET prediction. The qualitative assessment of pharmacokinetics viz; absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of selected compounds were predicted computationally by using SwissADME and toxicity prediction using TOPKAT (Accelrys, Inc., USA). QikProp develops and employs QSAR/QSPR models using partial least squares, principal component analysis and multiple linear regression to predict physico-chemical significant descriptors^68-70.

RESULTS AND DISCUSSION 

Physicochemical Properties: The molecular weight of AZA was 720.72 (g/mol); the calculated LogP value was -0.20; LogD - 0.14; LogSw - -4.34. The total number of stereo-centers in the molecule was 16; the stereo-chemical complexity of the molecule was 0.457; the calculated Fsp3 value of AZA was 0.771; The overall calculated Topological polar surface area of AZA was 215.34(Å2). Likewise the calculated number of hydrogen bond donors in the molecule was 3; whereas the number of hydrogen bond acceptors was 16; the number of smallest set of smallest rings (SSSR) in the molecule analyzed was 2; the size of the biggest system ring in the molecule was 15; similarly, the total number of rotatable bonds in the molecule was 6; the number of rigid bonds was 38; the number of charged groups was 0; similarly the total charge of the compound was 0; the number of carbon atoms in the molecule was 35; whereas the number of heteroatoms in AZA was calculated as 16; the number of heavy atoms in the molecule was calculated as 51; the ratio between the number of non-carbon atoms and the number of carbon atoms in the compound was 0.46 (Fig. 2,3).

Druggability Properties: Lipinski's rule of 5 violations of the molecule was 2; Veber rule was Low for the molecule; similarly Egan rule for the molecule was also Low; the Oral PhysChem score (Traffic Lights) for the molecule was recorded as 5; GSK's 4/400 score for the molecule was Good; Pfizer's 3/75 score for the molecule was Good; Weighted quantitative estimate of drug-likeness (QEDw) score for the molecule was 0.164; Solubility Forecast Index was Good and the solubility score was 9441.49; 

ADMET Properties: Only when the ADME/Tox properties of a drug like compound are of high quality, and when the target has been validated, the compound could be developed into a pharma-drug. In silico drug-likeness evaluation of Azadirachtin for Human Intestinal Absorption (HIA+) value had a probability of 0.890; Blood Brain Barrier (BBB-) value for the molecule had a probability of 0.773; Caco-2 permeable (Caco2-) value for the molecule had a probability of 0.711 (Fig. 4); P-glycoprotein substrate (Substrate) value for the molecule had a probability of 0.835; P-glycoprotein inhibitor I (Inhibitor) value for the molecule had a probability of 0.672; P-glycoprotein inhibitor II (Non-inhibitor) value for the molecule had a probability of 0.534. CYP450 2C9 substrate (Non-substrate) value for the molecule had a probability of 0.857; CYP450 2D6 substrate (Non-substrate) - 0.872; CYP450 3A4 substrate (Substrate) - 0.714; CYP450 1A2 inhibitor (Non-inhibitor) - 0.887; CYP450 2C9 inhibitor (Non-inhibitor) - 0.845; CYP450 2D6 inhibitor (Non-inhibitor) - 0.944; CYP450 2C19 inhibitor (Non-inhibitor) - 0.833; CYP450 3A4 inhibitor (Non-inhibitor) - 0.770; CYP450 inhibitory promiscuity (Low CYP Inhibitory Promiscuity) - 0.886; Ames test (Non AMES toxic) - 0.756; Carcinogenicity (Non-carcinogens) - 0.946; Biodegradation (Not ready biodegradable) - 1.000; Rat acute toxicity (4.348 LD50, mol/kg) - PNA; hERG inhibition (predictor I) (Weak inhibitor) - 0.992; hERG inhibition (predictor II) (Non-inhibitor) - 0.569 respectively. Computational methods for analysing and estimating the toxicity of natural bioactive compounds are considered as useful tool for validation as it provides in-depth understanding of toxicogenomics. Therefore, determining the toxicity of BASM in-silico is warranted to identify their potential harmful effects on humans, animals, plants, besides the environment as in-vivo animal tests are constrained by time, ethical considerations, and financial burden. Data pertaining to the descriptors viz., Toxicity, Environmental toxicity, Tox21 pathway and Toxicophore Rules for Azadirachtin are summarized in Table 2. Furthermore, GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, enzyme inhibitor score for AZA were calculated as -0.71; -1.51; -1.46; -0.67; -0.35 and -0.71 respectively (Fig. 3). Swiss Target Prediction towards Macrophage migration inhibitory factor, Heat shock protein (HSP 90-alpha), Kappa Opioid receptor, Mu opioid receptor, Delta opioid receptor, Thrombin, Squalene synthetase, Glycogen synthase kinase-3 beta, Glycogen synthase kinase-3 alpha, Protein kinase C alpha, Apoptosis regulator Bcl-X, HMG-CoA reductase, Zinc finger protein GLI1, Proto-oncogene c-JUN, Vanilloid receptor for the compound has been provided in Table 4. Chemical and biological investigations on Azadirachta indica bioactive compounds indicates that the compound is safe for use as a drug molecule^3,72,72.

CONCLUSION

The present study is an example to insights into the broad scope of pharmacoinformatics to plant based natural product research with an emphasis on drug discovery. The study indicates that plant based natural products still possess an extraordinary challenge that has to be solved before taken for drug development. However, it is anticipated that as more quality data on natural product research, such as bio-activity, biomolecularinformatics, cheminformatics, toxicoinformatics integrated together with new algorithms and machine learning techniques to accelerate natural product based drug discovery. Furthermore, online databases serve as attractive sources for identifying novel natural product scaffolds with promising drug-like properties in NPs which is expected to accelerate the pace of Drug Discovery.  

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Table 1 Physicochemical, Medicinal Chemistry and ADMET properties of AZA

Table 2 Summary of Physicochemical, Druggability, ADMET of AZA 

The physicochemical properties were computed using FAF-Drugs4 (28961788) and RDKit open-source cheminformatics platform. The druggabiity scoring schemes were computed using FAF-Drugs4 (28961788) and FAF-QED (28961788) open-source cheminformatics platform. ADMET features were predicted using admetSAR (23092397) open-source tool.

Table 3 Molecular Properties and of Bioactivity Score of AZA

Table 4 Swiss Target Prediction