The search for a direct and specific cure for COVID-19 has so far been unsuccessful. Consequently, the current approach to anti-COVID-19 strategies relies on repurposed drugs, e.g., remdesivir ; hydroxychloroquine ; dexamethasone ; and/or resort to traditional medicine (TM) [3,12,22,23,39,40].
These strategies yield some sort of palliative quelling of the symptoms, as the body’s innate and acquired immune surveillance mechanisms are deployed to gradually subdue the infection to obscurity in the mild case. As alluded to by Fara et al.  regarding resolution of the cytokine storm, “Initially, the localized response is meant to eliminate the trigger and involves protective mechanisms ….”. The vascular endothelium (VE) plays a pivotal and intricate role in all aspects of COVID-19, as will be reviewed herein.
Functions of the vascular endothelium (VE) derive from its unique structure
The VE is a continuous monolayer of cells (endothelial cells, ECs) physically delineating the blood with its circulating elements in the lumen (of every blood vessel) from the vascular smooth muscle layer of the wall of all blood vessels. Thus, VE seamlessly links them all from the largest arteries and veins, to the capillaries that connect the arterial and venous systems.
It is a highly dynamic organ system that engages in various far-reaching physiological homeostatic functions in which it serves as a signal transducer [13,14,19,35,36,60]. The VE is therefore not merely only a simple physical barrier, but should also be visualized as a bona fide endocrine organ being the source of various cellular signaling factors .
Another important implication here is that this VE single continuous monolayer of cells pervades through the entire body of the individual, connecting the blood, lungs, heart, liver, kidney, brain, and other metabolic niches. Consequently, the VE serves as the link connecting various cardiometabolic, pulmonary, septic, and renal diseases .
Indeed, its involvement in neuronal pathology has been implicated as well [18,38,47]. Any perturbation in the system would therefore spell danger, as in the development of activation, yielding reactive oxygen species (ROS), nitric oxide (NO), cytokines, acute phase proteins (e.g., C-reactive protein, CRP), and other oxidative stress determinants, leading to triggering the formation of an atherosclerotic plaque, for instance . The central and intricate involvement of the VE in many physiological and pathophysiological situations has been recently reviewed [47,50,60].
The quiescent, normal, or “healthy” endothelium
The quiescent endothelium is that in which the component ECs remain non-activated. Such is the normal “healthy” physiological state. In this state, the VE is a primary sensor of biomechanical stimuli which are transduced into biological responses. For instance, regular steady smooth laminar flow of blood, consistent with stable shear stress on the wall, i.e., against the endothelium itself, invokes the homeostatic physiological processes.
This signals the activation of constitutive enzymes, namely endothelial nitric oxide synthase (eNOS or NOSIII) and cyclooxygenase-1 (COX-1), leading to synthesis of the appropriate low levels of NO by the eNOS (NOSIII), and the prostanoid prostacyclin (prostaglandin I2; PGI2) from arachidonic acid by COX-1.
Both NO and prostacyclin are powerful vasodilators, and thus relax blood vessels. In addition, ECs also produce endothelin-1 and angiotensin II, which are potent vasoconstrictors, as well as other vasoactive factors like the prostanoid thromboxanes [19,27,35]. To ensure vascular tone, homeostasis is maintained by a fine balance of these vasodilators, vasoconstrictors and the other vasoactive factors in the normal quiescent state [19,35,36].
To exert its action, NO diffuses to the vascular smooth muscle cells where it stimulates soluble guanylate cyclase leading to enhanced cyclic guanosine monophosphate (cGMP) synthesis, causing relaxation. It also diffuses into the lumen, affecting the platelet and blood element functions; it prevents thrombosis and renders the blood more fluid by inhibiting platelet adhesion and aggregation . Prostacyclin, like NO, is also antithrombotic, and causes smooth muscle relaxation by activating adenylate cyclase to increase cyclic adenosine monophosphate (cAMP) production [35,36].
Endothelial dysfunction and the pathological state
Conversion of the quiescent state to the activated state of the endothelium creates “endothelial dysfunction”, which is a state of inflammation. It has been indeed referred to in various terms, e.g., as belonging to a “diseased vessel” . It is characterized by the usual hallmarks of inflammation, including up-regulation of the inducible nitric oxide synthase (NOSII; iNOS), and inducible cyclooxygenase-2 (COX-2), respectively and forming copious amounts of NO and prostanoids (e.g., prostacyclin, thromboxanes) from arachidonic acid in the vascular smooth muscle cells, associated with the “diseased” endothelium.
Some of these prostanoids are pro-inflammatory and drive disease pathogenesis, e.g., prostacyclin, though usually anti-inflammatory, paradoxically acts pro-inflammatory in rheumatoid arthritis . Production of pro-inflammatory cytokines, such as IL-6, TNFα, IL-1β, is increased; and upgrading of other innate and adaptive immune factors and processes, such as acute phase proteins like CRP [21,32,36,50,60] is enhanced as well, exacerbating oxidative stress.
Production of chemokines (e.g., IL-8), cytokines, and adhesion molecules, which recruit leukocytes and platelets, would cause inflammation in specific tissues for clearing intruding foreign particles and pathogens (viruses, bacteria, etc.). In the VE, this transformation from quiescence to diseased state could be triggered by cardiovascular risk factors . In such circumstances, the homeostatic balance that characterizes the healthy quiescent state (arising from the interplay of cytokines, chemokines and the other factors) is breached .
Acute and chronic inflammation
In acute and chronic inflammation caused by any of the triggers, e.g., microbial pathogens; Gram-negative lipopolysaccharide (LPS) or Gram-positive lipoteichoic acid (LTA); and dead cell debris, these reactions and products are exacerbated, leading to overwhelming excesses, and causing sepsis and, at a more aggravated state, septic shock [29,37,41,43,60]. The dysregulated oxidative stress in these circumstances responds to antioxidative and anti-inflammatory management approaches. This is the basis of the multiple effects of the antioxidant vitamin C in various pathological situations [2,6].
The manifestations of COVID-19 range from asymptomatic to mild, to severe ill-health conditions like respiratory failure, sepsis and subsequently to multi-organ dysfunction syndromes . The reason for this can be linked to the key involvement of the vascular endothelium . The viral pathogen SARS-CoV-2 is the trigger as it infects the VE for instance, and sets the entire inflammatory cascade into motion, creating endothelial dysfunction, with the oxidative stress and other manifestations described .
From early infection to the cytokine storm and severe COVID-19
Early in the infection, the respiratory tract is affected, yielding the early symptoms of mild COVID-19 . The triggered innate and adaptive immune responses would attempt to resolve the infection . Should this mitigation strategy not succeed, the result is acute respiratory distress syndrome.
Then, due to the interconnectedness of the vascular endothelium, the prevailing inflammatory response is cascaded far and wide through the vascular bed, and so, in addition, affects cardiovascular, renal, and other targets as well. The outcome of this is the exacerbated “endothelial dysfunction” consistent with severe COVID-19, caused by the created “Cytokine Storm” with the associated exaggerated thrombotic and other consequences . The cytokine storm is essentially the exaggerated mixture of pro-inflammatory cytokines (TNFα, IL-6, IL-1β) , chemokines (IL-8), and CRPs [16,21,, , ] .
Compromised health conditions as risk factors for serious COVID-19
A compromised health status involving cardiovascular, respiratory, renal, or other systems would be a determinant for the serious consequences (enhanced morbidity and mortality) of COVID-19 . So far in the pandemic, intervention strategies with chemical and natural product antioxidants, anti-inflammatory agents, and immunomodulators, aimed at ameliorating the cytokine storm or its production have been under investigation [6,40,42,45,52]. These efforts have yielded promising results and some treatments(e.g., with vitamin C) are in clinical trials .
It is therefore clear that the VE is a main focus of the unique pathological hallmarks of COVID-19 [24,33,59], which result from a complex blend of vascular dysfunction, dysregulated inflammation and thrombosis [59,60]. There is direct viral infection of the endothelium in different organs [1,47,59].
Also, pericyte cells, which have an exaggerated concentration of ACE2 (the receptor of SARS-CoV-2) are in proximity to the lung endothelial cells and, therefore, would exacerbate the endothelial cell injury [4,60] Normal function of the pericytes is maintenance of micro-vessel integrity. But on binding SARS-CoV-2, the consequences are grave.
This explains why pre-existing conditions that negatively impact vascular endothelial homeostasis cause severe COVID-19 . Therefore, preventing the access of SARS-CoV-2 to the vascular endothelial cells and pericytes and dislodging the bound virus from such sites have become reasonable strategies for avoiding the attendant problems, i.e., dysregulated inflammation.
COVID-19 and other disease severities caused by cytoadherence to vascular endothelium and other sites
Malaria, Gram-negative and Gram-positive infections, LPS (endotoxin) and LTA [9,10,37,55], and COVID-19, cause severe diseases involving the vascular endothelium and hyper-inflammation. The hallmarks of the disease in each case include a combination of various levels of exacerbated dysregulated cytokine production, NO and ROS release, oxidative stress, “cytokine storm”, thrombotic events and others.
Thus, in the Gram-negative bacterial and LPS triggered events, the outcome is endotoxin stress (sepsis), and septic shock. In the case of COVID-19, the critical severe outcomes have been linked to the cytokine storm, and some of the characteristics resemble sepsis-associated immune dysregulation . In these cases, effective intervention strategies have included the use of antioxidants, anti-inflammatory agents and immunomodulators [6,16,2].
Cytoadherence to vascular endothelium
Udeinya et al. [56,57] and others [31,34] have established that in the case of Plasmodium falciparum malaria infection, parasitized red blood cells (pRBCs) containing schizonts and trophozoites are preferentially sequestered by specific binding to the VE of the venules and capillaries through the parasite’s knobs. This specific event protects these malaria parasitic stages from the spleen, thereby saving them from immune destruction and clearance by the spleen. The malaria therefore persists, via this evasion strategy.
Cytoadherence also contributes prominently in the pathogenic mechanisms of other diseases like cancer metastasis [7,53], as well as bacterial  and viral infections . In the case of HIV, invasion of the target cell occurs when the viral surface envelope spike glycoprotein binds both CD4 and a seven-transmembrane coreceptor of the target lymphocyte. These interactions induce a conformational change in the spike protein resulting in the fusion event internalizing the HIV in the target cell .
Neem leaf acetone-water extract as potential mitigation strategy against COVID-19
Udeinya and associates  further discovered that the acetone-water extract of Azadirachta indica (Neem) dislodged the trapped pRBCs from the VE, which made it possible for these pRBCs to be conveyed to the spleen for immune killing and clearance from the system. The same Neem extract has also been shown to prevent the invasion of lymphocytes by HIV both in vitro and in vivo in humans. Thus, the Neem acetone-water extract displayed a broad-spectrum effect by inhibiting adhesion of malaria-infected pRBCs, adhesion of cancer cells, and invasion of human lymphocytes by HIV. In addition, it was reported that the extract had no observable toxicity among the cohort of individuals who received the experimental treatment at the University of Nigeria Teaching Hospital (tested in the limited clinical trials performed) .
On account of these findings, especially the fact that the pRBCs (just like the SARS-CoV-2 virus) bind to the VE, it is reasonable to hypothesize that the same acetone-water neem leaf extract would be effective in dislodging SARS-CoV-2 (the causative agent for COVID-19) from binding the cells. This would be a game changer in the fight against COVID-19 because the VE underpins the various pathologies and multi-organ involvements and disease severities. Part of the advantage is that, if the extract is effective as expected, it could be possible to administer it via a simple route; for instance, as a food or nutritional additive or an adjuvant to another drug, say remdesivir.
Indeed nature has provided the remedies for ailments and diseases for the benefit of humankind since ancient times. Azadirachta indica is one of the accredited and trusted sources of herbal therapy against a plethora of diseases and ill-health conditions since antiquity [15,25,26,61]. This acetone-water extract, considered in isolation, qualifies to be one of the numerous ways in which Neem phytochemicals (either as single applications, or in combination as cocktails with other components) safeguard humans against the onslaught of different pathogens.
Potential anti-covid-19 phytochemicals in Azadirachta indica (Neem) and other efficacious herbal resources
The desperate and worrisome absence of drugs for direct attack on the pathogenic agent SARS-CoV-2 has inspired and encouraged the exploitation of the properties of natural products against COVID-19 and its pathogenesis, symptoms and sequelae. Azadirachta indica (Neem), a prehistoric source for remedies against numerous ill-health conditions for various indigenous populations in Africa and other parts of the world presents, for advantage in this context, these age-old phytochemical agents endowed within its various parts. Excellent reviews on this topic regarding A. indica, and the other herbal and other resources abound [25,49,54,, ].
Rigorous clinical trials and phytochemical studies advocated
For this immediate moment therefore, it is recommended that appropriate rigorous clinical trials be done on the acetone-water extract, as currently prepared , administered to a large enough population of COVID-19 patients and non-infected controls. This is to unequivocally establish the appropriate dosage, and its efficacy, and empower its use as anti-COVID-19 remedy for people in Africa, India, and other zones endowed with abundant and renewable supplies of the Neem plant in their environments around the globe.
Thereafter, it would become necessary to further study the said acetone-water extract to isolate, identify and characterize the specific bioactive phytochemical components, as well as their levels present therein. This later phase of the work would inform the next step: formulating anti-COVID-19 remedies from the pure compounds.
Prerequisite in vitro confirmation that the Neem Extract Inhibits SARS-CoV-2 Binding on VECs:
As mentioned earlier (vide supra) in this paper, the Neem extract displayed in vitro broad-spectrum effects, and it: prevented malaria parasitized red blood cells (pRBCs) from adhesion to endothelial cells; cancer cell cytoadherence to endothelial cells, as well as cancer cell metastasis; human immunodeficiency virus (HIV) from binding to target lymphocytes.
Given this broad-spectrum effect, we hypothesized (vide supra) that the Neem extract will bind to the vascular endothelial cells (VECs) and prevent access of the spike glycoprotein of the SARS-CoV-2 to its main receptor, the VEC angiotensin converting enzyme 2 (ACE2). Also, as mentioned earlier, the inhibition of HIV is caused by the Neem extract preventing the viral surface spike glycoprotein from engaging the CD4 and a seven-transmembrane coreceptor of the target lymphocyte.
We present this hypothesis, considering (the speculation) that this could be one of the unique characteristics that have enabled the Neem to be such an effective remedy against diseases over the millennia of human existence on our planet , , , . However, to make assurance doubly sure, the plan being proposed herein is that the clinical trials will be preceded by in vitro confirmatory investigations involving SARS-CoV-2 (or simply SARS-CoV-2 spike glycoprotein) and the Neem leaf extract. The Neem leaf extract will be obtained as per the published procedure .
Udeinya and associates  had reported the in vitro antiretroviral activity of the Neem extract, and concluded that the mechanism of action may involve inhibition of cyto-adhesion. The work on viral interactions with the extract was performed  using HIV, and was patented . These authors reported that in the presence of the extract at 10 microgram/ml in vitro, 75% of the target lymphocytes were protected from HIV invasion . Adherence to, and replication in VECs have been highlighted as hallmark phenomena associated with various types of viral infections of humans and animals (e.g., SARS-CoV-2, Hantavirus, Influenza A, H5N1, H7N1, Ebola virus, Zikavirus, West Nile virus, Dengue virus, among others .
In a recent Case Report , it was concluded that circulating soluble ACE2 inhibited COVID-19. This is by binding of the soluble ACE2 to the VEC membrane-bound ACE2 receptors, thereby excluding SARS-CoV-2 from binding. In the said Case Report, the patient produced an overwhelming amount of soluble ACE2 as a defensive mitigation strategy against the SARS-CoV-2 infection and disease progression. This phenomenon has been reported for recombinant ACE2 administered to animal models of SARS-CoV-2 infection, which resolved the infection , . In addition, intravenously administered recombinant ACE2, ameliorated COVID-19 in seriously sick human patients .
Embarking on the proposed clinical trials with the Neem extract would therefore be predicated on the success of the Neem extract to inhibit the binding of SARS-CoV-2 (or simply its spike glycoprotein) to the membrane-bound ACE2 of the VECs.
Vascular-centric endothelial protective therapy
This simple proposed remedy is consistent with the “vascular-centric endothelial protective therapies” that have been advocated by Mangalmurti and collaborators , . However, if effective, the remedy proposed herein is indeed a superior approach given the fact that the Neem leaf extract presents the advantages of (i) being locally available to the target population; (ii) being easy to process; (iii) being inexpensive; and (iv) having no detectable toxicity at the appropriate low doses. These special attributes are in line with, and satisfy the conditions for applying natural products and resources as therapeutics to less privileged, resource-limited parts of the world [15,61].
The plant product or natural products show an important role in diseases prevention and treatment through the enhancement of antioxidant activity, inhibition of bacterial growth, and modulation of genetic pathways. The therapeutics role of number of plants in diseases management is still being enthusiastically researched due to their less side effect and affordable properties.
It has been accepted that drugs based on allopathy are expensive and also exhibit toxic effect on normal tissues and on various biological activities. It is a largely accepted fact that numerous pharmacologically active drugs are derived from natural resources including medicinal plants [1, 2].
Various religious documents such as Bible and Quran also supported the herbs role in health care and prevention. Islamic perspective also confirms the herbs role in diseases management and Prophet Mohammed (PBUH) recommended various plants/fruits in the diseases cure . Neem ingredients are applied in Ayurveda, Unani, Homeopathy, and modern medicine for the treatment of many infectious, metabolic, or cancer diseases [4, 5].
Different types of preparation based on plants or their constituents are very popular in many countries in diseases management. In this vista, neem (Azadirachta indica), a member of the Meliaceae family, commonly found in India, Pakistan, Bangladesh, and Nepal, has therapeutics implication in diseases cure and formulation based on the fact that neem is also used to treat various diseases. Azadirachta indica has complex of various constituents including nimbin, nimbidin, nimbolide, and limonoids and such types of ingredients play role in diseases management through modulation of various genetic pathways and other activities.
Quercetin and ß-sitosterol were first polyphenolic flavonoids purified from fresh leaves of neem and were known to have antifungal and antibacterial activities . Numerous biological and pharmacological activities have been reported including antibacterial , antifungal , and anti-inflammatory.
Earlier investigators have confirmed their role as anti-inflammatory, antiarthritic, antipyretic, hypoglycemic, antigastric ulcer, antifungal, antibacterial, and antitumour activities [9–12] and a review summarized the various therapeutics role of neem . This review summarizes the role of neem and its active ingredients in the diseases prevention and treatment through the modulation of various biological pathways.
- Botanical Description of Neem
Neem tree belongs to the family Meliaceae which is found in abundance in tropical and semitropical regions like India, Bangladesh, Pakistan, and Nepal. It is a fast-growing tree with 20–23 m tall and trunk is straight and has a diameter around 4-5 ft. The leaves are compound, imparipinnate, with each comprising 5–15 leaflets. Its fruits are green drupes which turn golden yellow on ripening in the months of June–August. Taxonomic position of Azadirachta indica (neem) is classified in Table 1 .
Taxonomic position of Azadirachtaindica (neem).
- Active Compounds of Azadirachta indica L. (Neem)
Azadirachta indica L. (neem) shows therapeutics role in health management due to rich source of various types of ingredients. The most important active constituent is azadirachtin and the others are nimbolinin, nimbin, nimbidin, nimbidol, sodium nimbinate, gedunin, salannin, and quercetin. Leaves contain ingredients such as nimbin, nimbanene, 6-desacetylnimbinene, nimbandiol, nimbolide, ascorbic acid, n-hexacosanol and amino acid, 7-desacetyl-7-benzoylazadiradione, 7-desacetyl-7-benzoylgedunin, 17-hydroxyazadiradione, and nimbiol [15–17]. Quercetin and ß-sitosterol, polyphenolic flavonoids, were purified from neem fresh leaves and were known to have antibacterial and antifungal properties  and seeds hold valuable constituents including gedunin and azadirachtin.
- Mechanism of Action of Active Compounds
Neem (Azadirachta indica), a member of the Meliaceae family, has therapeutics implication in the diseases prevention and treatment. But the exact molecular mechanism in the prevention of pathogenesis is not understood entirely. It is considered that Azadirachta indica shows therapeutic role due to the rich source of antioxidant and other valuable active compounds such as azadirachtin, nimbolinin, nimbin, nimbidin, nimbidol, salannin, and quercetin.
Possible mechanism of action of Azadirachta indica is presented as follows.
Neem (Azadirachta indica) plants parts shows antimicrobial role through inhibitory effect on microbial growth/potentiality of cell wall breakdown. Azadirachtin, a complex tetranortriterpenoid limonoid present in seeds, is the key constituent responsible for both antifeedant and toxic effects in insects . Results suggest that the ethanol extract of neem leaves showed in vitro antibacterial activity against both Staphylococcus aureus and MRSA with greatest zones of inhibition noted at 100% concentration .
Neem plays role as free radical scavenging properties due to rich source of antioxidant. Azadirachtin and nimbolide showed concentration-dependent antiradical scavenging activity and reductive potential in the following order: nimbolide > azadirachtin > ascorbate .
Neem ingredient shows effective role in the management of cancer through the regulation of cell signaling pathways. Neem modulates the activity of various tumour suppressor genes (e.g., p53, pTEN), angiogenesis (VEGF), transcription factors (e.g., NF-κB), and apoptosis (e.g., bcl2, bax).
Neem also plays role as anti-inflammatory via regulation of proinflammatory enzyme activities including cyclooxygenase (COX), and lipoxygenase (LOX) enzyme.
- Therapeutic Implications of Neem and Its Various Ingredients in Health Management
Active constitutes play role in the diseases cure via activation of antioxidative enzyme, rupture the cell wall of bacteria and play role as chemopreventive through the regulation of cellular pathways. Pharmacological activities of neem are discussed in detail (Figure 1).
5.1. Antioxidant Activity
Free radical or reactive oxygen species are one of the main culprits in the genesis of various diseases. However, neutralization of free radical activity is one of the important steps in the diseases prevention. Antioxidants stabilize/deactivate free radicals, often before they attack targets in biological cells  and also play role in the activation of antioxidative enzyme that plays role in the control of damage caused by free radicals/reactive oxygen species. Medicinal plants have been reported to have antioxidant activity . Plants fruits, seeds, oil, leaves, bark, and roots show an important role in diseases prevention due to the rich source of antioxidant.
Leaf and bark extracts of A. indica have been studied for their antioxidant activity and results of the study clearly indicated that all the tested leaf and bark extracts/fractions of neem grown in the foothills have significant antioxidant properties . Another important study was performed based on leaves, fruits, flowers, and stem bark extracts from the Siamese neem tree to assess the antioxidant activity and results suggest that extracts from leaf, flower, and stem bark have strong antioxidant potential .
A valuable study was carried out to evaluate in vitro antioxidant activity in different crude extracts of the leaves of Azadirachta indica (neem) and antioxidant capacity of different crude extracts was as follows: chloroform > butanol > ethyl acetate extract > hexane extract > methanol extract. Result of the current finding suggested that the chloroform crude extracts of neem could be used as a natural antioxidant .
Other results revealed that azadirachtin and nimbolide showed concentration-dependent antiradical scavenging activity and reductive potential in the following order: nimbolide > azadirachtin > ascorbate. Furthermore, administration of azadirachtin and nimbolide inhibited the development of DMBA-induced HBP carcinomas through prevention of procarcinogen activation and oxidative DNA damage and upregulation of antioxidant and carcinogen detoxification enzymes . Experimentation was made to evaluate the antioxidant activity of the flowers and seed oil of neem plant Azadirachta indica A. Juss. and results revealed that ethanolic extract of flowers and seed oil at 200 μg/mL produced the highest free radical scavenging activity with 64.17 ± 0.02% and 66.34 ± 0.06%, respectively .
The results of the study revealed that root bark extract exhibited higher free radical scavenging effect with 50% scavenging activity at 27.3 μg/mL and total antioxidant activity of this extract was found to be 0.58 mM of standard ascorbic acid . Other results of study concluded that tested leaf and bark extracts/fractions of neem grown in the foothills (subtropical region) have significant antioxidant properties .
Leaves, fruits, flowers, and stem bark extracts from the Siamese neem tree were evaluated for antioxidant and results of the study showed that leaf aqueous extract and flower and stem bark ethanol extracts showed higher free radical scavenging effect with 50% scavenging activity at 26.5, 27.9, and 30.6 microg/mL, respectively. Furthermore, total antioxidant activity of extracts was found to be 0.959, 0.988, and 1.064 mM of standard trolox, respectively .
5.2. Anticancerous Activity
Cancer is multifactorial disease and major health problem worldwide. The alteration of molecular/genetic pathways plays role in the development and progression of cancer. The treatment module based on allopathic is effective on one side but also shows adverse effect on the normal cell. Earlier studies reported that plants and their constituents show inhibitory effects on the growth of malignant cells via modulation of cellular proliferation, apoptosis, tumour suppressor gene, and various other molecular pathways . Neem contains flavanoids and various other ingredients that play an important role in inhibition of cancer development (Figure 2). Large number of epidemiological studies proposes that high flavonoid intake may be correlated with a decreased risk of cancer .
Neem oil holds various neem limonoids which prevents mutagenic effects of 7,12-dimethylbenz(a)anthracene . A study was performed to investigate the cytotoxic effects of nimbolide found in leaves and flowers on human choriocarcinoma (BeWo) cells and results showed that treatment with nimbolide resulted in dose- and time-dependent inhibition of growth of BeWo cells with IC50 values of 2.01 and 1.19 μM for 7 and 24 h, respectively . A study was made to assess the chemopreventive potential of the limonoids, azadirachtin, and nimbolide and results showed that azadirachtin and nimbolide inhibited the development of DMBA-induced HBP carcinomas through influencing multiple mechanisms such as prevention of procarcinogen activation and oxidative DNA damage, upregulation of antioxidant and carcinogen detoxification enzymes, and inhibition of tumour invasion and angiogenesis .
Azadirachta indica and their active compounds play pivotal role in the prevention of cancer development and progression. The exact molecular mechanism in this vista is not understood fully. Based on experimentation, it was considered that neem and its ingredients play role in the modulation of various cell signaling pathways. Azadirachta indica hold various ingredients and theses constituents activate the tumour suppressor genes and inactivate the activity of several genes involved in the cancer development and progression such as VEGF, NF-κB, and PI3K/Akt. Neem has been reported to be a good activator of tumour suppressor gene and inhibitor of VEGF and phosphoinositol PI3K/Akt pathways. It also activates apoptosis, suppression of NF-κB signaling, and cyclooxygenase pathway.
Neem and its constituents play role in the prevention of malignancies through the modulation of molecular pathways which are described below.
5.2.1. Effect of Neem and Its Constituents on Tumour Suppressor Genes
p53 is an important tumour suppressor gene and it plays role in the inhibition of the proliferation of abnormal cells, in that way inhibiting the development and progression of cancer. A study confirmed that ethanolic fraction of neem leaf (EFNL) treatment effectively upregulated the proapoptotic genes and proteins including p53, Bcl-2-associated X protein (Bax), Bcl-2-associated death promoter protein (Bad) caspases, phosphatase and tensin homolog gene (pTEN), and c-Jun N-terminal kinase (JNK) . A finding showed that ethanolic neem leaf extract enhanced the expression of proapoptotic genes, such as caspase-8 and caspase-3, and suppressed the expression of Bcl-2 and mutant p53 in the 7,12-dimethylbenz(a)anthracene-induced cancer cells [34, 35].
Nimbolide, a tetranortriterpenoid limonoid, is one of the important contributors to the cytotoxicity of neem extracts . Nimbolide downregulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and upregulated the proapoptotic proteins p53 and Bax .
pTEN activity is commonly lost via mutations, deletions, or promoter methylation silencing in various types of primary and metastatic cancers [38, 39]. Inactivation of pTEN has been noticed in various types of tumour. A study confirmed that ethanolic fraction of neem leaf treatment significantly increased the expression of pTEN, which could inhibit mammary tumourigenesis through its inhibitory effect on Akt .
5.2.2. Effect of Neem and Its Constituents on Apoptosis
bcl2 and bax play an important role in the regulation of apoptotic process. Any alteration in bcl2 and bax causes the development and progression of tumours . Altered expression of such genes has been noticed in many tumours. A study was performed to investigate the effect of extract in an in vivo 4T1 breast cancer model in mice and results confirmed that CN 250 and CN 500 groups had a higher incidence of apoptosis compared with the cancer controls . Another study reported that extract has been shown to cause cell death of prostate cancer cells (PC-3) via inducing apoptosis .
A study finding revealed that leaf extract downregulated Bcl-2 expression and upregulated Bim, caspase-8, and caspase-3 expression in the buccal pouch indicating that it has apoptosis inducing effects in the target organ  and study results confirmed that leaf extract induced a dose-dependent reduction in chronic lymphocytic leukemia (CLL) cell viability with significant apoptosis observed at 0.06% (w/v) by 24 h . Isolated compound and chief constituents from neem show a range of activities affecting multiple targets and also play role in the induction of apoptotic cell death in cancer [44, 45].
5.2.3. Effect of Neem and Its Constituents on Angiogenesis
Angiogenesis is complex process that supplies blood to the tissue and that is essential for growth and metastasis of tumour. Angiogenesis is regulated by activators as well as inhibitors. The development of antiangiogenic agents to block new blood vessel growth is crucial step in the inhibition/prevention of tumour growth. Medicinal plants and their ingredients play role in prevention of tumour growth due to their antiangiogenic activity.
An important study revealed that ethanolic fraction of neem leaf (EFNL) treatment effectively inhibited the expression of proangiogenic genes, vascular endothelial growth factor A, and angiopoietin, indicating the antiangiogenic potential of EFNL. Furthermore, inhibition of angiogenesis by ethanolic fraction of neem leaf (EFNL) could be a reason for reduction in mammary tumour volume and for blocked development of new tumours as observed in current studies . Another study was performed to evaluate the antiangiogenic activity of extract of leaves in human umbilical vein endothelial cells (HUVECs) and results showed treatment of HUVECs with EENL inhibited VEGF induced angiogenic response in vitro and in vivo and also EENL suppressed the in vitro proliferation, invasion, and migration of HUVECs . A study was made on zebra fish embryos via treatment of various concentrations of water soluble fractions of crude methanolic extract of neem root, imatinib (standard), and control and results of the study concluded that water soluble fractions of methanolic extract of neem root were found to have the ability to inhibit angiogenesis .
5.2.4. Effect of Neem on Oncogene
An oncogene is a mutated gene that plays significant role in the development and progression of tumours. Experiment was performed to investigate effect of leaf extract on c-Myc oncogene expression in 4T1 breast cancer BALB/c mice and results revealed that 500 mg/kg neem leaf extract (C500) group showed significant suppression of c-Myc oncogene expression as compared to the cancer control group .
5.2.5. Effect of Neem on PI3K/Akt Pathways
PI3K/Akt pathways show pivotal effect in the promotion of tumour. However, inhibition of PI3K/Akt pathways is one of the important steps towards regulation of tumour development. Effect of leaf extract on PI3K/Akt and apoptotic pathway in prostate cancer cell lines (PC-3 and LNCaP) was investigated and results suggested that effect of leaf extract induces apoptosis and inhibits cell proliferation through inhibiting PI3K/Akt pathway in both PC-3 and LNCaP cells .
Another study was performed to evaluate the molecular mechanisms involved in the induction of apoptosis and antiproliferative activity exerted by leaf extract on the human breast cancer cell lines and results confirmed that extract treated cells significantly decreased the protein expression such as IGF signaling molecules IGF-1R, Ras, Raf, p-Erk, p-Akt, and cyclin D1 .
Another study was carried out to evaluate the effects of nimbolide on apoptosis and insulin-like growth factor (IGF) signaling molecules in androgen-independent prostate cancer (PC-3) cells line and results of the study suggested that nimbolide acts as a potent anticancer agent by inducing apoptosis and inhibiting cell proliferation via PI3K/Akt pathway in PC-3 cells .
5.2.6. Effect of Neem on NF-κB Factor
The NF-κB transcription factor plays a major role in cancer and related diseases . However, the inhibition of NF-κB action is a vital step in the prevention of cancer development and progression. An important study was performed to investigate the efficacy of bioactive phytochemicals in inhibiting radiotherapy- (RT-) induced NF-κB activity, signaling, and NF-κB-dependent regulation of cell death and results showed that curcumin, leaf extract, and black raspberry extract (RSE) significantly inhibited both constitutive and RT-induced NF-κB  and other important study results demonstrate that nimbolide, a neem derived tetranortriterpenoid, concurrently abrogates canonical NF-κB and Wnt signaling and induces intrinsic apoptosis in human hepatocarcinoma (HepG2) cells .
- Effect of Neem as Anti-Inflammatory
Plants or their isolated derivatives are in the practice to treat/act as anti-inflammatory agents. A study result has confirmed that extract of A. indica leaves at a dose of 200 mg/kg, p.o., showed significant anti-inflammatory activity in cotton pellet granuloma assay in rats . Other study results revealed that neem leaf extract showed significant anti-inflammatory effect but it is less efficacious than that of dexamethasone  and study results suggest that nimbidin suppresses the functions of macrophages and neutrophils relevant to inflammation .
Earlier finding showed immunomodulator and anti-inflammatory effect of bark and leave extracts and antipyretic and anti-inflammatory activities of oil seeds [58, 59]. Experimentation was made to evaluate the analgesic activity of neem seed oil on albino rats and results of the study showed that neem seed oil showed significant analgesic effect in the dose of 1 and 2 mL/kg and oil has dose-dependent analgesic activity .
Another study was made to investigate the anti-inflammatory effect of neem seed oil (NSO) on albino rats using carrageenan-induced hind paw edema and results revealed that NSO showed increased inhibition of paw edema with the progressive increase in dose from 0.25 mL to 2 mL/kg body weight. At the dose of 2 mL/kg body weight, NSO showed maximum (53.14%) inhibition of edema at 4th hour of carrageenan injection .
Results of the study concluded that the treated animals with 100 mg kg−1 dose of carbon tetrachloride extract (CTCE) of Azadirachta indica fruit skin and isolated ingredient azadiradione showed significant antinociceptive and anti-inflammatory activities .
- Hepatoprotective Effect
Medicinal plants and their ingredients play a pivotal role as hepatoprotective without any adverse complications. A study was performed to investigate the hepatoprotective role of azadirachtin-A in carbon tetrachloride (CCl4) induced hepatotoxicity in rats and histology and ultrastructure results confirmed that pretreatment with azadirachtin-A dose-dependently reduced hepatocellular necrosis . Furthermore results of the study show that pretreatment with azadirachtin-A at the higher dose levels moderately restores the rat liver to normal .
Another study was carried out to evaluate the protective effect of active constituent of neem such as nimbolide against carbon tetrachloride (CCl4) induced liver toxicity in rats and results suggest that nimbolide possesses hepatoprotective effect against CCl4 induced liver damage with efficiency similar to that of silymarin standard  and another study finding revealed that leaf extract was found to have protection against paracetamol-induced liver necrosis in rats .
A study assesses the hepatoprotective activity of Azadirachta indica (AI) leaf extract on antitubercular drugs-induced hepatotoxicity and results confirmed aqueous leaf extract significantly prevented changes in the serum levels of bilirubin, protein, alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase and significantly prevented the histological changes as compared to the group receiving antitubercular drugs . Additionally, other results showed that ethanolic and aqueous leaf extracts of A. indica exhibited moderate activity over carbon tetrachloride treated animals . Hepatoprotective effect of methanolic and aqueous extracts of Azadirachta indica leaves was evaluated in rats and study result established that the plant has good potential to act as hepatoprotective agent .
An experiment was made to investigate the protective effect of neem extract on ethanol-induced gastric mucosal lesions in rats and results showed that pretreatment with neem extract showed protection against ethanol-induced gastric mucosal damage .
- Wound Healing Effect
Numerous plants/their constituents play an important role in the wound healing effect. A study was made to evaluate the wound healing activity of the extracts of leaves of A. indica and T. cordifolia using excision and incision wound models in Sprague Dawley rats and results revealed that extract of both plants significantly promoted the wound healing activity in both excision and incision wound models . Furthermore, in incision wound, tensile strength of the healing tissue of both plants treated groups was found to be significantly higher as compared to the control group . Other results showed that leave extracts of Azadirachta indica promote wound healing activity through increased inflammatory response and neovascularization .
- Antidiabetic Activity
A study was undertaken to evaluate the 70% alcoholic neem root bark extract (NRE) in diabetes and results showed that neem root bark extract showed statistically significant results in 800 mg/kg dose . Another experiment was performed to examine the pharmacological hypoglycemic action of Azadirachta indica in diabetic rats and results showed that in a glucose tolerance test with neem extract 250 mg/kg demonstrated glucose levels were significantly less as compared to the control group and Azadirachta indica significantly reduce glucose levels at 15th day in diabetic rats .
Studies using in vivo diabetic murine model, A. indica, and B. spectabilis chloroform, methanolic, and aqueous extracts were investigated and results showed that A. indica chloroform extract and B. spectabilis aqueous, methanolic extracts showed a good oral glucose tolerance and significantly reduced the intestinal glucosidase activity . Another important study suggested that leaves extracts of Azadirachta indica and Andrographis paniculata have significant antidiabetic activity and could be a potential source for treatment of diabetes mellitus .
- Antimicrobial Effect
Neem and its ingredients play role in the inhibition of growth of numerous microbes such as viruses, bacteria, and pathogenic fungi. The role of neem in the prevention of microbial growth is described individually as follows.
10.1. Antibacterial Activity
A study was performed to evaluate antimicrobial efficacy of herbal alternatives as endodontic irrigants and compared with the standard irrigant sodium hypochlorite and finding confirmed that leaf extracts and grape seed extracts showed zones of inhibition suggesting that they had antimicrobial properties . Furthermore, leaf extracts showed significantly greater zones of inhibition than 3% sodium hypochlorite .
The antibacterial activity of guava and neem extracts against 21 strains of foodborne pathogens was evaluated and result of the study suggested that guava and neem extracts possess compounds containing antibacterial properties that can potentially be useful to control foodborne pathogens and spoilage organisms .
Another experiment was made to evaluate the antibacterial activity of the bark, leaf, seed, and fruit extracts of Azadirachta indica (neem) on bacteria isolated from adult mouth and results revealed that bark and leaf extracts showed antibacterial activity against all the test bacteria used . Furthermore, seed and fruit extracts showed antibacterial activity only at higher concentrations .
10.2. Antiviral Activity
Results showed that neem bark (NBE) extract significantly blocked HSV-1 entry into cells at concentrations ranging from 50 to 100 μg/mL . Furthermore, blocking activity of NBE was noticed when the extract was preincubated with the virus but not with the target cells suggesting a direct anti-HSV-1 property of the neem bark .
Leaves extract of neem (Azadirachta indica A. Juss.) (NCL-11) has shown virucidal activity against coxsackievirus virus B-4 as suggested via virus inactivation and yield reduction assay besides interfering at an early event of its replication cycle .
10.3. Antifungal Activity
Experiment was made to evaluate the efficacy of various extracts of neem leaf on seed borne fungi Aspergillus and Rhizopus and results confirmed that growth of both the fungal species was significantly inhibited and controlled with both alcoholic and water extract. Furthermore, alcoholic extract of neem leaf was most effective as compared to aqueous extract for retarding the growth of both fungal species . Another finding showed the antimicrobial role of aqueous extracts of neem cake in the inhibition of spore germination against three sporulating fungi such as C. lunata, H. pennisetti, and C. gloeosporioides f. sp. mangiferae  and results of the study revealed that methanol and ethanol extract of Azadirachta indica showed growth inhibition against Aspergillus flavus, Alternaria solani, and Cladosporium .
Aqueous extracts of various parts of neem such as neem oil and its chief principles have antifungal activities and have been reported by earlier investigators [84–86]. A study was undertaken to examine the antifungal activity of Azadirachta indica L. against Alternaria solani Sorauer and results confirmed that ethyl acetate fraction was found most effective in retarding fungal growth with MIC of 0.19 mg and this fraction was also effective than fungicide (metalaxyl + mancozeb) as the fungicide has MIC of 0.78 mg .
10.4. Antimalarial Activity
Experiment was made to evaluate the antimalarial activity of extracts using Plasmodium berghei infected albino mice and results revealed that neem leaf and stem bark extracts reduced the level of parasitemia in infected mice by about 51–80% and 56–87%, respectively,  and other studies showed that azadirachtin and other limonoids available in neem extracts are active on malaria vectors [89–91].
Another finding based on crude acetone/water (50/50) extract of leaves (IRAB) was performed to evaluate the activity against the asexual and the sexual forms of the malaria parasite, Plasmodium falciparum, in vitro and results showed that, in separate 72-hour cultures of both asexual parasites and mature gametocytes treated with IRAB (0.5 microg/mL), parasite numbers were less than 50% of the numbers in control cultures, which had 8.0% and 8.5% parasitemia, respectively .
- Role of Neem in Dentistry
A study was made to assess the efficacy of neem based on mouth rinse regarding its antigingivitis effect and study confirmed that A. indica mouth rinse is equally effective in reducing periodontal indices as chlorhexidine .
Another study was carried out to evaluate the antimicrobial properties of organic extracts of neem against three bacterial strains causing dental caries and results showed that petroleum ether and chloroform extract showed strong antimicrobial activity against S. mutans. Chloroform extract showed strong activity against Streptococcus salivarius and third strain Fusobacterium nucleatum was highly sensitive to both ethanol and water extract . Earlier finding confirmed that dried chewing sticks of neem showed maximum antibacterial activity against S. mutans as compared to S. salivarius, S. mitis, and S. sanguis .
- Antinephrotoxicity Effect
An experiment was made to investigate the effects of methanolic leaves extract of Azadirachta indica (MLEN) on cisplatin- (CP-) induced nephrotoxicity and oxidative stress in rats and results confirmed that extract effectively rescues the kidney from CP-mediated oxidative damage . Furthermore, PCR results for caspase-3 and caspase-9 and Bax genes showed downregulation in MLEN treated groups .
- Neuroprotective Effects
A study was performed to investigate the neuroprotective effects of Azadirachta indica leaves against cisplatin- (CP-) induced neurotoxicity and results showed that morphological findings of neem before and after CP injection implied a well-preserved brain tissue. No changes, in biochemical parameters, were observed with neem treated groups .
- Immunomodulatory and Growth Promoting Effect
Experiment was performed to investigate growth promoting and immunomodulatory effects of neem leaves infusion on broiler chicks and results showed that neem infusion successfully improved antibody titre, growth performance, and gross return at the level of 50 mL/liter of fresh drinking water .
Another study investigated the effects of feeding of powdered dry leaves of A. indica (AI) on humoral and cell mediated immune responses, in broilers and results showed that AI (2 g/kg) treatment significantly enhanced the antibody titres against new castle disease virus (NCDV) antigen .
- Safety, Toxicities, and LD50 Values of Neem
The measurement of toxicities of natural compound is crucial before their application in health management. Various studies based on animal model and clinical trials confirmed the neem is safe at certain dose and on the other side neem and its ingredients showed toxic/adverse effect.
Several studies reported, in children, neem oil poisoning causing vomiting, hepatic toxicity, metabolic acidosis, and encephalopathy [100–102] and another study based on rat model showed that administration of leaf sap caused an antianxiety effect at low doses, whereas high doses did not show such types of effect . An important study based on rats model showed that azadirachtin did not show toxicity even at 5 g/kg bw . A study based on rabbit was performed to check the toxicological analysis and results of the study showed there was progressive increase in body weight in both the test and control animals, and during the entire duration of the administration of the neem extract, there was no observed sign of toxicity in both groups .
A study result showed that, in the acute toxicity test, the LD50 values of neem oil were found to be 31.95 g/kg . Another study was performed to evaluate the toxicity in chicken and finding showed that acute toxicity study of neem leaf aqueous extract revealed an intraperitoneal LD50 of 4800 mg/kg, and clinical signs were dose dependent .
A study reported that lethal median doses (LD50) recorded for neem leaf and stem bark extracts were 31.62 and 489.90 mg/kg body weight, respectively . The LD50 of water extract of A. indica leaves and seeds were 6.2, 9.4 mL kg−1, respectively . Lethal dose values were calculated with probit analysis and LD50 and LD90 values were found to be 8.4 and 169.8 µg/fly of neem extract, respectively . A test for acute oral toxicity in mice revealed that LD50 value of approximately 13 g/kg body weight .
- Clinical Studies Based on Neem
Various clinical trials based studies confirmed that herbal products or derivatives from the natural products play vital role in diseases prevention and treatment. A very few studies on active compounds such as nimbidin were made to check the efficacy in the health management. An important study was made based on human subjects to investigate the role of neem bark extract as antisecretory and antiulcer effects in human subjects. Administration of lyophilised powder of the extract for 10 days at the dose of 30 mg twice daily showed significant decrease (77%) of gastric acid secretion. The bark extract at the dose of 30–60 mg twice daily for 10 weeks almost completely healed the duodenal ulcers and one case of esophageal ulcer and one case of gastric ulcer healed completely when administrated at the dose of 30 mg twice daily for 6 weeks .
A double blind clinical drug trial study was performed to check the efficacy of drug made up of aqueous extract of neem leaves in 50 cases of uncomplicated psoriasis taking conventional coal tar regime and results revealed that patients taking drug in addition to coal tar had shown a quicker and better response in comparison to placebo group . A clinical study of six weeks was made to check the efficacy of neem extract dental gel with chlorhexidine gluconate (0.2% w/v) mouthwash as positive control and results of the study showed that the dental gel containing neem extract has significantly reduced the plaque index and bacterial count compared to that of the control group . A study showed that, in ulcer healing tests, nimbidin significantly enhanced the healing process in acetic acid induced chronic gastric lesions in albino rats and dogs .
reference link :https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791507/
reference link :https://www.sciencedirect.com/science/article/pii/S2468227622000928?via%3Dihub#sec0001