Multi-Target Approach of Murraya koenigii Leaves in Treating Neurodegenerative Diseases

Neurodegenerative diseases (NDs) mainly affect neurons and gradually lead to a loss of normal motor and cognitive functions. Atypical protein homeostasis—misfolding, aggregations and accumulations, oxidative stress, inflammation, and apoptosis—are common features in most NDs. To date, due to the complex etiology and pathogenesis of NDs, no defined treatment is available. There has been increasing interest in plant extracts as potential alternative medicines as the presence of various active components may exert synergistic and multi-pharmacological effects. Murraya koenigii (Rutaceae) is utilized in Ayurvedic medicine for various ailments. Pharmacological studies evidenced its potential antioxidant, anti-inflammatory, anticancer, hepatoprotective, immunomodulatory, antimicrobial, and neuroprotective activities, among others. In line with our interest in exploring natural agents for the treatment of neurodegenerative diseases, this review presents an overview of literature concerning the mechanisms of action and the safety profile of significant bioactive components present in M. koenigii leaves to support further investigations into their neuroprotective therapeutic potential.


Introduction
Neurodegenerative diseases (NDs) are a broad term used to define a range of disorders (Alzheimer's disease, Parkinson's disease, dementia with Lewy body, frontal temporal dementia, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, prion disease, and many more) primarily affecting the neurons and leading, ultimately, to the progressive loss of normal motor functions and the decline in cognitive functions. The brain region affected, severity, and pace of neurodegeneration vary with the type of ND. According to a recent report, around 50 million people worldwide have such diseases [1]. The total number of Alzheimer's disease (AD) patients is estimated to be over 100 million by the year 2050 [2]. Similarly, the number of patients with Parkinson's disease (PD) is expected to double or more than double for populous countries such as China, India, and Indonesia [3].
The exact reasons for ND manifestation among some people and not for others are still not fully understood. Some NDs are suspected to occur due to genetic mutations; some are also linked to living conditions [4]. However, oxidative stress, atypical protein folding, inflammation, and apoptosis are common to the majority of NDs. Due to complex etiology and pathogenesis of NDs, to date, there are no defined treatments that can reverse the progressive degeneration of neurons and cure these diseases. Although research is progressing, drug development for NDs is slow. Over the past 30 years, only around

Phytochemicals of Murraya koenigii and Their Role in Neurodegenerative Diseases
This section describes the neuroprotective effects of M. koenigii leaf extracts and specified metabolites against their potential targets in NDs. The oral administration of the leaf aqueous extract (100, 200, 400 mg/kg body weight) to aluminum-treated rats was found to significantly restore antioxidant parameters (Catalase: CAT, Glutathione reduced: GSH, Lipid peroxidation: LPO), reversing them to normal through the neuroprotective properties of the extract [58]. The protective effect of M. koenigii on oxidative stress was most likely due to carbazole alkaloids, polyphenols, and flavonoids, which are known to have antioxidant properties [52,59,60]. Neuroprotective activity of the leaf aqueous extract (100,200, 400 mg/kg body weight) against paraquat (10 mg/kg body weight/i.p. per week for 4 weeks)-induced Parkinsonism in rats was also studied. The leaf extract-treated animals performed better in behavioral and locomotor activities in comparison to the untreated group [61]. The possible neuroprotective potentials of a methanolic leaf extract were also observed in a two-vessel occlusion (2VO) rat model of partial global cerebral ischemia. Cognitive functions were evaluated by the Morris water maze test and the viable neurons were evaluated in the hippocampal region. The study results indicated cognitive improvement in the group treated with M. koenigii leaves methanolic extract [62]. Diazepam-, scopolamine-and aging-induced amnesia behavioral models in rats were evaluated for memory enhancement after treatment with M. koenigii leaves (MKL). MKL (leaf powder mixed with wheat flour) were orally administered to various groups, and their behaviors were evaluated with the elevated plus-maze and Hebb-Williams maze trials. A significant dose-dependent improvement in the memory scores of young and aged rats was reported, and the significant reduction in amnesia could be due to the induction by scopolamine (0.4 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.) in the treatment groups [63]. In addition, the acetylcholine esterase (AChE) activity and the total cholesterol were reported to be decreased in the groups fed on the MKL diet, suggesting a role of AChE and cholesterol in inducing nootropic effects in the animals [64]. Further studies were conducted on animal models to better understand the mechanism of cognitive improvement. The 15-day treatment with M. koenigii alkaloid extract (MKA) caused a dose-dependent reduction in AChE levels in the brain of aged mice. It also prevented the brain from age-induced oxidative stress by decreasing lipid peroxidation and nitric oxide levels and increasing the level of antioxidant enzymes (superoxide dismutase: SOD, CAT, GSH) in the brain [65,66].
The neuroprotective potential of the carbazole alkaloid mahanimbine against lipopolysaccharides (LPS)-induced neuronal deficits in the SK-N-SH neuroblastoma cell line and its antioxidant potentials in the ICR mouse brain were evaluated. The pre-treatment of SK-N-SH cells with mahanimbine significantly mitigated the generation of LPS-induced reactive oxygen species (ROS) in vitro. In addition, mahanimbine also inhibited the β-secretase (IC 50 4 µg/mL), which was responsible for production of β amyloid (Aβ). In the in vivo study, mahanimbine supplementation re-equilibrated the levels of antioxidant markers (CAT, glutathione reductase: GR, malondialdehyde; MDA) in the brain in comparison to the untreated group [67]. Pre-treatment of LPS-challenged mice with mahanimbine enhanced the central cholinergic transmission by increasing acetylcholine (ACh) level through the AChE inhibition. Amyloidogenesis was also significantly decreased. Mahanimbine increased the levels of anti-inflammatory cytokines transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), and inhibited pro-inflammatory cytokines (IL-1 β and tumor necrosis factor-α: TNF-α). Decreased activity and expression of cyclooxygenase (COX-2) gene were observed with mahanimbine supplementation in the LPS-induced group. The overall findings supported the neuroprotective potential of mahanimbine against LPS-induced neuroinflammation [68]. The anti-AChE activity (IC 50 0.03 mg/mL) of mahanimbine was also established in vitro under acellular conditions [69]. A tricyclic sesquiterpene, isolongifolene (ILF), present in the essential oil of M. koenigii, was studied for its neuroprotective properties both in vitro [70] and in vivo [71]. ILF displayed effective scavenging activities in various antioxidant assays with EC 50 values of 77.34, 40.9, 16.27, 238.3, 25.01, 16.79, 1.311, 6.701, 0.418 µg/mL in the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, ABTS), hydroxyl radical, nitric oxide, hydrogen peroxide, super oxide radical scavenging, ferric reducing antioxidant power (FRAP), total radical-trapping antioxidant parameter (TRAP) and reducing power assays, respectively [72]. ILF attenuated the rotenone-induced mitochondrial dysfunction and cell apoptosis in SH-SY5Y human neuroblastoma cells. ILF treatment improved the rotenone-induced oxidative stress, apoptosis, and mitochondrial dysfunction in vitro. ILF mitigated rotenone induced apoptosis by downregulating the expression of BCL2-associated X protein (Bax), caspases-3, 6, 8, and 9, cytosolic cytochrome complex (cyt c), and increasing the expression of B-cell lymphoma family of proteins (Bcl-2), along with increased mitochondrial cyt c. Additionally, the regulation of the expression of phosphoinositide 3-kinase (p-PI3K), protein kinase B (p-AKT), and glycogen synthase kinase 3β (p-GSK3β) by ILF demonstrated its neuroprotective effects [70]. The PI3K/AKT/GSK3β signaling pathway is an important pathway for neuronal growth and function [73]. AKT regulated PI3K through the phosphorylation of GSK3β, which was identified to be associated with various vital cellular functions, such as the regulation of cell apoptosis and survival [74]. The active form of GSK-3β (p-GSK-3 Tyr216) was reported to be increased in postmortem striata of PD patients [75]. These events occur through mitochondrially mediated cell death pathways [76], which involve the activation and localization of Bcl-2 and mitochondrial complex I activity [77]. In a separate study [71], ILF improved the oxidative stress condition and movement impairment induced by rotenone in a rat PD model. The co-administration of ILF and rotenone significantly restored the levels of antioxidant enzymes (SOD, CAT, and glutathione peroxidase (GPx)) and decreased the level of LPO. In addition, ILF improved the motor dysfunction in muscles and catalepsy that were induced by rotenone. This suggested that ILF could protect the dopaminergic neurons through free radical scavenging activity [70,71].
A sesquiterpene alkene and atypical dietary cannabinoid, β-caryophyllene (BCP), is another essential bioactive compound in M. koenigii leaves [78]. It is a selective agonist of cannabinoid receptor 2 (CB2R), making it a favorable target for immune modulation, inflammation, and neuropathic pain conditions [79], and is involved in pathogenesis of cancer and NDs [80,81]. Through the CBR signaling pathways, the endocannabinoid system (ECS) controls stress linked to cognitive and emotional responses. The dysregulation of the ECS plays an important role in the pathogenesis and progression of various NDs, such as multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and Alzheimer's disease (AD) [82,83]. In the brain of MS patients, aggregates of CB1R-and CB2R-expressing cells were found in the active multiple sclerosis plaques [84]. Similarly, in case of ALS patients, the increased CB2R-positive microglia and macrophages were present in the areas of motor neuron damage [85]. In AD, the altered expressions of CB1R and CB2R were observed in the brain [86][87][88]. In addition, increased levels of endocannabinoid degradation enzymes namely, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), were found in human AD brains [87,89]. Hence, reversing ECS dysregulation through the activation of CB2R and the neuroprotection by BCP may influence the regulation of myelination and stimulating immune balance along with the inhibition of endocannabinoid degradation enzymes for their positive effects in NDs [90].
The effect of BCP on cognition was studied in a galactose (GAL) model of aging in mice. GAL induction lead to increased DNA oxidation in the prefrontal cortex and increased astrocyte numbers and interactions in the hippocampal region of the brain. Although the cognitive damage was not reversed by administering BCP, it was able to block the increases in the number of astrocytes and DNA oxidation in mice treated with GAL. Hence, the neuroprotective effects of BCP were demonstrated at the molecular and cellular level in the GAL model of aging [91]. The protective effect of BCP was also seen in isolated macrophages and lymphocytes and in vivo models of experimental autoimmune encephalomyelitis (EAE) [92]. The in vitro neuroprotective effect of BCP was reported against the LPS-induced oligodendrocyte toxicity, which was facilitated through the nuclear factor-erythroid 2-related factor 2 and heme oxygenase-1 (Nrf2/HO-1) antioxidant defense pathway and peroxisome proliferator-activated receptor gamma (PPAR-γ) signaling pathways through CB2R binding [93]. Similarly, BCP (48 mg/kg) alleviated the Alzheimer-like diseases by modulating CB2R and PPAR-γ signaling pathways in another study [94]. At 100 µM, BCP inhibited the expression of inducible nitric oxide synthetase (iNOS), interleukin (IL)-1β, IL-6, and COX2 in C6 microglial cells, and decreased the level of nitric oxide and prostaglandin E2. These studies show that BCP had good neuroprotective potential, which may be related to the modulation of inflammatory mediators [95] in association with high mobility group box 1 and toll-like receptor 4 (HMGB1/TLR4) signaling [96], reducing oxidative stress and modulating CB2R [97][98][99].
Spathulenol (5,10-cycloaromadendrane), a tricyclic sesquiterpenoid, is a component of essential oil from the leaves of M. koenigii [100]. Spathulenol exerted neuroprotective effects against 6-hydroxydopamine (6-OHDA), a synthetic neurotoxin, in SH-SY5Y human dopaminergic neuroblastoma cells. Spathulenol not only relieved the generated oxidative stress in the cells due to 6-OHDG, but the mitochondrial membrane integrity was maintained, highlighting its potentials as a promising therapy for treating NDs [101].
(+)-3-Carene, a bicyclic monoterpenoid from M. koenigii leaf [78], was reported to be a potent uncompetitive inhibitor of AChE (IC 50 0.2 mM) [102]. A molecular docking study envisaging ligand-target interactions of essential oil components with human AChE, led to seven hit compounds, which formed favorable bonds at the enzyme active site. Hence, the in vitro evaluation led to the discovery of two new active hits, namely caryophyllene oxide (IC 50 5.3 µM) and geranyl acetate (IC 50 244.5 µM) [103], as components of essential oil from M. koenigii leaf [26,78].
Linalool, a monoterpene alcohol, was also present in the essential oil of M. koenigii leaves. The improved cognitive-enhancing effects and anti-apoptotic activities in Aβ1-42-treated rats could mostly result from its antioxidant potential [104]. In another study, linalool displayed neuroprotective potential in male Wistar rats after acrylamide (50 mg/kg i.p.) treatment. Acrylamide was found in various food products during processing, which could present a potent neurotoxic effect in human and animals [105]. Linalool treatment (12.5 mg/kg i.p.) significantly reduced movement, which could be due to the acrylamide-induced abnormalities. Increased GSH levels in the rat brain could be regulated by linalool, indicating its role of antioxidant properties in neuroprotection [106]. In addition to improving cognitive functions, linalool displayed a neuroprotective effect through competitively inhibiting glutamate release and non-competitively blocking of N-methyl-D-aspartate (NMDA) receptors [107,108]. Moreover, it also decreased microgliosis and astrogliosis, reduced the formation of neurofibrillary tangles and β-amyloid plaques, and the levels of pro-inflammatory markers in the brain [109].
The neuroprotective effects of β-myrcene (MYR), a monoterpene present in the leaves of M. koenigii, were studied. MYR treatment protected the C57BL/J6 mice against oxidative stress, apoptosis, and histopathological damage induced by cerebral ischemia/reperfusion (I/R) [110].
The neuroprotective effect of nerolidol (NRD), a sesquiterpene alcohol, was discovered in the essential oil of M. koenigii leaves from the studies with a rotenone-induced model of PD [100]. NRD supplementation considerably improved the levels of oxidative stress markers (SOD, CAT, GSH, and MDA). NRD also inhibited the release of pro-inflammatory cytokines and inflammatory mediators. Moreover, NRD treatment prevented the rotenoneinduced activation of glial cells and the loss of dopaminergic neurons and nerve fibers, thus mitigating rotenone-induced dopaminergic neurodegeneration [111]. In another study, NRD significantly improved the locomotor activities and cognitive impairment, and reduced the AChE activity and reduction in oxidative stress [112]. Therefore, NRD displays its neuroprotective potential by antioxidant and anti-inflammatory action, which would be helpful in treating NDs. Table 1 summarized the mechanism of action of different extracts and bioactive components in ameliorating the symptoms of NDs. Structures of the biologically active natural products of M. koenigii against various NDs are shown in Figure 1. A summative representation of the mechanism of action of the M. koenigii extracts and natural products are shown in Figure 2.

Clinical Studies
Currently, no significant clinical trials have been conducted on curry leaves. A randomized controlled clinical trial was conducted to evaluate the efficacy of M. koenigii and chlorhexidine gluconate for the treatment of gingivitis [113]. In another study, curry leaf powder (5 g daily for 45 days) was given to volunteers to evaluate its effect on liver and renal functions, and no harmful effect was observed on the functions of both the liver and kidney [114]. An herbal medicine containing curry leaves along with pomegranate and turmeric was developed for the treatment of irritable bowel syndrome patients [115]. The effect of curry leaves was also investigated in hypertensive subjects [116]. However, despite numerous in vivo and in vitro studies exploring the neuroprotective potential of M. koenigii, the research was lacking on its preclinical and/or clinical efficacy. Therefore, there is an urgent need to conduct clinical trials to prove the neuroprotective capacity of M. koenigii.

Toxicity of Murraya koenigii Leaves
Curry leaves were rich in calcium, potassium, magnesium, and phosphorous, along with trace amounts of zinc, manganese, selenium, and iron. Additionally, lead, mercury, and cadmium were also present, but below the US FDA limits [117]. Various leaf samples of M. koenigii were collected from different regions, and their elemental analyses were performed by the instrumental neutron activation (INAA) and atomic absorption spectrophotometry (AAS). Mean elemental contents varied owing to different climate, soil, and other geo-environmental conditions. The changes in plants grown on polluted soils re-

Clinical Studies
Currently, no significant clinical trials have been conducted on curry leaves. A randomized controlled clinical trial was conducted to evaluate the efficacy of M. koenigii and chlorhexidine gluconate for the treatment of gingivitis [113]. In another study, curry leaf powder (5 g daily for 45 days) was given to volunteers to evaluate its effect on liver and renal functions, and no harmful effect was observed on the functions of both the liver and kidney [114]. An herbal medicine containing curry leaves along with pomegranate and turmeric was developed for the treatment of irritable bowel syndrome patients [115]. The effect of curry leaves was also investigated in hypertensive subjects [116]. However, despite numerous in vivo and in vitro studies exploring the neuroprotective potential of M. koenigii, the research was lacking on its preclinical and/or clinical efficacy. Therefore, there is an urgent need to conduct clinical trials to prove the neuroprotective capacity of M. koenigii.

Toxicity of Murraya koenigii Leaves
Curry leaves were rich in calcium, potassium, magnesium, and phosphorous, along with trace amounts of zinc, manganese, selenium, and iron. Additionally, lead, mercury, and cadmium were also present, but below the US FDA limits [117]. Various leaf samples of M. koenigii were collected from different regions, and their elemental analyses were performed by the instrumental neutron activation (INAA) and atomic absorption spectrophotometry (AAS). Mean elemental contents varied owing to different climate, soil, and other geoenvironmental conditions. The changes in plants grown on polluted soils revealed the higher levels of heavy metals, which posed a major health concern, where they could affect kidney, liver, and the central nervous system [118].
No signs of mortality or morbidity were seen in either male or female rats fed the ethanolic extract of M. koenigii leaf (300 and 500 mg/kg) for 28 days. In addition, no mortality was observed at higher doses (900 mg/kg), but congestion, hemorrhage, and lymphocyte infiltration were recorded. The study concluded that the safety consumption could be high as 500 mg/kg dosage without inducing any structural damage to the organs [118]. Similarly, no mortality or toxicity signs were observed in another toxicological study of Malaysian MKL. For MKL methanolic extract an LD 50 = 200 mg/kg/day was reported [119]. In addition, mice fed with the mahanine-enriched fraction (MEF) at different doses (5000 mg/kg BW single dose, 300-1500 mg/kg BW/day for 14 days, and 300 mg/kg BW for 180 days) did not exhibit significant toxicity, mortality, or behavioral changes [120]. Another study suggested that crude leaf powder and methanolic leaf extract was safe up to 9000 mg/kg in mice [121] However, methanolic leaf extract was found to be moderately toxic (LD 50 = 316.23 mg/kg body weight) to rats and to cause liver inflammation. However, this dose had no severe side effects on other organs [31].

Conclusions and Future Directions
Traditional medicinal plants have gained importance during the last few decades, and evidence has been presented for their therapeutic actions, allowing the traditional knowledge of plants to be streamlined with the modern system of medicine to achieve health benefits. This review presented substantial evidence in relation to the mechanistic aspects of M. koenigii leaves in ameliorating several neurodegenerative disorders. M. koenigii leaves are an integral part of Indian cuisine and are used for various ailments traditionally. Carbazole alkaloids are abundantly found in species of the Rutaceae family including M. koenigii (Curry leaves). Both the natural and synthetic derivatives of carbazole alkaloids revealed numerous pharmacological activities, including neuroprotection. The M. koenigii leaf extracts and their bioactive compounds, including carbazole alkaloids, sesquiterpenoids, and monoterpenoids, exhibited a multi-target approach by alleviating the oxidative stress, attenuating proinflammatory cytokines, inhibiting AChE and BACE 1, preventing/reducing Aβ protein aggregation, and improving cognitive dysfunction. Thus, M. koenigii leaves, their extracts, or purified compounds could offer a beneficial alterna-tive therapy to treat NDs through their multi-targeted neuroprotective properties and by improving cholinergic transmissions. Hence, the bioactive compounds from the leaves can serve as lead molecules in future drug discovery. Moreover, there have been no safety concerns associated with the use of M. koenigii leaves. Therefore, it is highly recommended to determine effective dose of leaves or their compounds in human for future clinical trials on NDs.