The Indian Delicacy Nimbu Achar: Your Lemony Pathway to Brain Health

Lemon pickle has been made and consumed in India since times immemorial. Though the pickle is made in many different ways, using different ingredients, the basic pickle contains very few ingredients. These include lemons, red chillies, carom seeds and salt. All these ingredients have been found to have numerous health benefits, including promotive effects on the brain. These beneficial actions include anti-inflammatory, anti-oxidant, anti-dementia, anti-anxiety and anti-depressive effects. Several human studies have also ascertained that regular consumption of lemons and other citrus fruits improves cognition, mood and sleep patterns. Besides the direct effect of the lemon phytochemicals on the brain, lemon peel has also been found to boost the gut-brain axis via prebiotic effects


Introduction
Improved life expectancies have greatly increased the aging population, leading to a surge in metabolic disease burden, and a rising incidence of age-associated neurodegenerative diseases such as Alzheimer's Disease (AD). The pathology of AD comprises neurological dysfunction due to amyloid-β accumulation, tau hyperphosphorylation, oxidative stress, and neuroinflammation in the brain. In addition, lifestyle-related diseases such as dyslipidemia, diabetes mellitus, obesity, and cardio-vascular dysfunction increase the risk of developing dementia. This has brought to the fore the great need to develop new strategies to maintain brain health and prevent the onset of dementia in the older population.
Citrus fruits contain bioactives such as polyphenolic compounds which have shown potential in ameliorating the metabolic disease processes [1], and preventing cardiovascular disease [2]. Citrus fruits including lemons are abundant in polymethoxylated flavones (PMF)and flavanones such as nobiletin, hesperidin and narirutin. Preclinical studies reported that these compounds have neuroprotective effects in models of dementia such as AD, showing beneficial effects on cognition and related functions. These studies suggest that it would be worthwhile to include lemon and other citrus fruit in our daily diet. A simple and economical way to partake of all the benefits of lemons is by regular inclusion of lemon pickle in the meals.

The Indian Lemon Pickle
Lemon pickle is commonly made in most Indian homes, especially in rural areas. Though there are several recipes for the pickle, the Lemon juice contains significant amounts of the flavanones' hesperidin and eriocitrin, besides being quite rich in flavones, especially diosmin and di-C-glucosyl flavones. C. limon juice is also abundant in diosmetin 6,8-di-C-glucoside and contains some amount of apigenin di-C-glucoside [7].
Lemon peel essential oil contains: Limonene, sabinene, b-pinene, neral, borneol, linalool, cineole, geranial and myrcene [11]. All the flavonoid compounds were found to be more abundant in the peel than in the edible part and juice [12]. Flavonoid glycosides were present primarily in the peel of lemon fruit.

Bioavailability, Stability and Access to Brain
The flavonoids in lemon are reasonably stable to heat [9]. Bioavailability of the flavonoids is improved when these are ingested along with cereals. In a randomized study conducted by Egert et al, six healthy females (aged 22-28yrs) consumed 130 mg quercetin either in the form of quercetin-enriched cereal bars or quercetin powder filled capsules. Systemic availability in terms of plasma concentration-time curves was five times higher after quercetin-enriched cereal bar ingestion [13]. These findings suggest that carbohydrate composition of the food matrix is an important determinant of the total flavonoid absorption in the small intestine [14]. It is thus better to consume lemon pickle along with meals, rather than alone, to get maximum benefit.
To have an effect on the brain, the lemon phytochemicals must be able to cross the blood-brain barrier (BBB). Available evidence indicates that citrus flavonoids, namely hesperetin, naringenin, as well as their relevant metabolites, are able to reach the brain; though the extent to which citrus polyphenols cross the BBB remains to be fully determined [15][16][17][18][19]. Since PMF has been seen to be loaded onto chylomicron lipoproteins (in the intestines, after absorption), it is also possible for these flavonoids to reach the brain through the lymphatic route [20].

CNS Effects of Phytochemicals Present in Lemon
As given above, Lemons are a considerably rich source of bioactive compounds, particularly flavanones (such as hesperidin and narirutin), which are a sub-set of the flavonoid group. Preclinical studies have shown convincingly the neuroprotective effects of citrus flavonoids. Several animal studies have also demonstrated their anti-inflammatory and anti-oxidative properties. Newly emerging evidence indicates their actions upon blood-brain barrier function/integrity, and this may be the mechanism by which these neurological effects are mediated.
Besides animal and in-vitro studies, results from human studies, although limited in number, have demonstrated improvements in cognitive performance. The various actions of bio actives present in lemon fruit juice, pulp and peels are explained below.

Anti-oxidative Actions
The high metabolic activity of the brain leaves the brain susceptible to oxidative damage. Along with their ability to scavenge free radicles [21], citrus polyphenols have also demonstrated their ability to stimulate the endogenous antioxidant defense machinery. A number of studies have shown the activation of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione S-transferases (GST), glutathione reductase (GR) and glutathione peroxidase (GPx) by all the citrus polyphenols. Activation coincided with subsequent reduction of reactive oxygen species (ROS) and other oxidative markers [5].
Experimental studies have shown that upregulation of the transcription factor NRF2 occurs and is probably key to this polyphenol mediated anti-oxidative system through the activation of the antioxidant response element [22][23][24]. Hesperidin and hesperetin have also been shown to upregulate Haem-oxygenase (HO-1) and downregulate the superoxide radical generating enzyme Xanthine Oxidase (XO) respectively [25].

Anti-inflammatory Effects
Neuro-inflammatory modulation is inherent across all citrus flavonoids, and the immunomodulatory capabilities of citrus polyphenols within the brain have also been demonstrated [5]. The molecular targets which citrus polyphenols interact with appear to reduce pro-inflammatory cytokines IL-1β, IL-2, IL-6, IFN-γ, and TNF-α. This is likely mediated through the mitigation of hyperactive immune cells as is suggested by the reduction of NF-κB which governs chemokine and inflammatory mediator

Bioenergetic Actions
In experimental studies, Citrus flavonoids appear to ameliorate mitochondrial damage caused by exogenous factors which can predispose individuals to certain neurodegenerative conditions. The impact was consistent, resulting in an increase of mitochondrial respiratory chain complexes (I-IV) function [5]. Mitochondria function disturbance impacts mitochondrial enzyme bioenergetics, reducing ATP production, while simultaneously leading to substantial increases in ROS production. In addition to mitochondrial function, citrus flavonoids in general led to a reduction in acetylcholinesterase activity, accompanied by increased acetylcholine levels and cholinergic transmission [25,28-33].

Proteinopathy and Dementia
Lemon phytochemicals may have beneficial effect in Alzheimer's disease, with reports revealing improvements in Tau phosphorylation [16,34,27], and Aβ deposition [16,35,27]. A reduction of α-synuclein, the protein implicated in parkinopathy has also been described [36]. A report by Shagirtha in 2017, in which hesperetin was administered demonstrated restoration of brain proteolytic enzyme levels [37]. Studies on involvement of cathepsin D in proteinopathies, have linked citrus flavonoids to lysosomal degradation processes [38].
Despite the above preclinical evidence pointing to the beneficial effect of citrus flavonoids in models of Alzheimer's and Parkinson's diseases, etc. there remains very limited evaluation at the human level. In a retrospective cohort study by Zhang et al., 2017, the association between daily citrus intake and dementia incidence in 13,373 participants (age 65 years) was assessed over a 5.7year period. In general, an inverse dose-response relationship was noted between weekly citrus fruit intake and incident dementia [47].

Cognitive and Behavioral Effects
In animal studies, where cognitive and behavioral assessments were carried out, citrus flavonoid supplementation led to improvements in cognitive performance. These studies have demonstrated the neuroprotective properties associated with citrus flavonoids. Anxiolytic and anti-depressant actions are particularly prominent across the literature, suggesting modulatory effects on a fundamental anxiety and depression related process. Similarly, citrus polyphenol supplementation has been shown to improve deficits in learning and spatial memory, which may indicate protection of medial temporal lobe ( Figure 2). The latter lobe is particularly vulnerable in Alzheimer's like neurodegenerative diseases. Improvements in motor functions and locomotion were also apparent, even in most severe models such as middle cerebral artery occlusion [48], and traumatic brain injury [49]. In a human study conducted on healthy individuals in Norway, exploring the impact of different plant foods on cognitive performance in elderly, a very strong association was found between citrus fruits and cognition [50]. The tested individuals showed better episodic memory, executive function, perceptual speed, and visuospatial skills. A similar study by Kean et al.
(2015), on the chronic consumption of flavanone-rich orange juice in adults, showed an increase in global cognition. In addition, high flavanone intake significantly improved recall [51]. These effects were independent of mood and blood pressure which both remained unchanged. A clinical trial evaluated the anti-dementia effect of nobiletin-rich Citrus reticulata peel extract on AD patients taking donepezil [52]. The intervention group ingested extract three times daily for one year, after which cognitive function was again evaluated. The results showed that long-term intake of citrus peel extract suppressed cognitive decline in AD patients. Moreover, long-term citrus peel ingestion caused no apparent side effects. These results suggest that long-term intake of nobiletinrich citrus peel extract prevents AD progression [52]. The proposed mechanism by which Nobiletin-rich citrus peel extracts may improve cognitive function in the elderly and AD patients is by antioxidant and anti-inflammatory effects. Nobiletin may be involved in activating signaling pathways related to memory formation (i.e., the cAMP/PKA/CREB/BDNF pathway) and improving synaptic plasticity in the cortex and hippocampus [53-55].
Acute neurological responses to citrus flavonoids in healthy adults have also been investigated [56,57]. In a randomized trial, Alharbi et al. (2016) explored the cognitive benefits associated with Flavonoid rich orange juice (hesperidin, narirutin, and others); consumption of the juice led to higher non-significant global cognitive performance, as well as an increase in subjective alertness at 6 hours post consumption. Interestingly, this higher performance coincided with a peak in flavanone metabolites at 5-7 h [58].
Lamport et al. (2016) also assessed acute neurological response to a commercially available high flavanone beverage (hesperidin, naringin, narirutin, and caffeic acid) in healthy young adults [57]. They utilized an additional measure of cerebral blood flow (CBF). Participants underwent either cognitive testing 2 h post consumption or completed an fMRI assessment of CBF 2 and 5 h post consumption. High flavanone beverage intake significantly increased cerebral perfusion in the inferior frontal and middle right frontal gyrus in the right hemisphere at 2 h. Similarly, at 2 h, improvement in digit symbol substitution test (a measure of executive function) was seen, correlating with the increased regional perfusion of the inferior frontal gyrus, known to be involved in executive function [59]. Thus, both chronic and acute flavanone-rich beverage intake improved neurological function.
Depression is a common mental illness and complex mood disorder [60]. People with dementia often suffer from depression [61,62]. In a prospective NHS cohort study over 10 years, following 82,643 women with no previous diagnosis of depression, an inverse association between incident depression and citrus intake was found. It was observed that higher intakes of all flavonoid subclasses resulted in a significant reduction in incident depression risk [63]. Thus, high flavonoid intake may reduce the risk of depression, especially among older women [63]. The antidepressant effects of flavonoids are usually attributable to their antioxidant and anti-inflammatory actions, as also the inhibition of monoamine oxidases [64]. Brain-derived neurotrophic factor (BDNF), an important serological marker observed to be significantly lower in patients with major depressive disorder, is often increased in response to flavonoid consumption [65]. BDNF plays an important role in neuronal survival and growth, serves as a neurotransmitter modulator, and participates in neuronal plasticity, which is essential for learning and memory. It is widely expressed in the CNS, gut and other tissues. The anti-depressant effects of citrus flavonoids were recently re-tested by Park et al.
(2020), in a single blind, randomized control study examining the effects of daily flavonoid rich orange juice consumption on depressive symptoms and gut microbiota for an 8-week period in young adults. Compared with baseline, the results suggested potential improvement in BDNF and depression [66].

Effects on Vascular Function and Stroke Protection
Older individuals, who are at higher risk of dementia, can also suffer from stroke as a result of systemic hypertension. Stroke can then become a cause of dementia. Dietary interventions that reduce systemic blood pressure can be helpful in reducing incidence of stroke, and lemon pickle can be one such intervention.

Modulation of Circadian Rhythms
Circadian rhythms are bioactive rhythms inherent in many organ systems. These rhythms form the basic regulatory mechanism for several physiological functions

Effect of Citrus Components on Anxiety and Sleep
Kwangjai et al., in 2021 investigated C. reticulata essential oil (EO) inhalation on electrical brain waves and sleep parameters in the rat model. The rats had electrodes implanted on the skull over the frontal and parietal areas and were given the citrus EO inhalation while Electroencephalography (EEG) signals were recorded. EEG data analysis revealed that citrus EO effects are comparable to diazepam. C. reticulata EO inhalation was associated with reduced REM sleep latency and improved sleep-wake patterns. There was significantly increased total time and episode numbers of REM sleep [82]. Thus, it can be concluded that clinical applications of C. reticulata essential oil can be helpful to improve sleep quality in various neurodegenerative conditions.
In behavioral studies, oral administration of C. reticulata EO was observed to reduce anxiety in mice using light/dark box and marble-burying tests. The citrus EO also Ueda et al. in 2023 demonstrated specific effects of lemon essential oils on the human brain. Instant high brain activation was observed after lemon essential oil inhalation, exhibiting effects on memory processing, task performance, and cognitive function [88]. Another study suggested that lemon essential oil inhalation can reduce memory impairment induced by the administration of scopolamine [89]. Furthermore, inhalation of lemon essential oil can lead to positive effects on mood and emotions [90]. Citrus EOs contain a high concentration of terpenes that are known for their anti-inflammatory properties by inhibiting pro-inflammatory cytokines such as TNF-α, NF-κB, and IL-1β [91]. It has also been found that orange EO inhalation can induce mood-enhancing effects by decreasing oxyhemoglobin concentration in the brain's right prefrontal cortex [92].

Effect of Lemon Pulp and Peel on the Gut Microbiota: The Gut -Brain Axis
The gut microbes have an important part to play in human health, including brain health. The trillions of intestinal bacteria help in metabolizing and breaking down foodstuffs and complex carbohydrates such as dietary fibres. Some of the derivative metabolites have been found to act as neurochemicals and possess neuromodulatory properties. Short-chain fatty acids (SCFA) are a relatively well-characterized example of this [93] and are produced via fermentation of dietary fibres like pectin. Low levels of SCFA have been found to be associated with conditions such as depression, AD and Parkinson's disease [93]. The SCFA propionate has been recently reported to protect the BBB from oxidative stress via NRF2 (NFE2L2) signaling [94]. The influence of metabolites such as SCFA produced by our gut microbiome on the gut-brain axis has been extensively reviewed by Teratani et al., The gut-brain axis can be considered as a bidirectional system that encompasses both neuro-immune and neuro-endocrine communication as well as a direct neuronal affect (vagus nerve) [101], where each mode of transmission undergoes microbial modulation. The fact that beneficial shifts could be achieved by

Anti-inflammatory and Anti-oxidant Actions
The antioxidant activity of capsaicin was reported in the oxidation of methyl linoleate (ML) and also of soybean phosphatidylcholine liposomal membrane. This anti-oxidant activity was comparable to that of alpha-tocopherol which is one of the most important antioxidants in vivo [110]. The antioxidant property of capsaicin can even prevent cardiovascular diseases. Kim et al found capsaicin showed significant inhibition of the production of inflammatory molecule PGE2 in lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages. They also noted that Capsaicin inhibited the enzyme activity of COX-2 and the expression of the iNOS protein [111].

Metabolic activity
Capsaicin has also been found to reduce glucose intolerance and fasting glucose levels in experimental mice, demonstrating antihyperglycemic and antidiabetic activities [112]. Concentrationdependent reduction in LDL and cholesterol was observed when RC powder and standard diet were taken together, thus confirming the anti-atherogenic potential of red chilli powder. Red chilli consumption has been linked to reduced blood glucose in rabbit models [113]. Chilli pepper has demonstrated the potential to inhibit key enzymes of glucose metabolism such as α-glucosidase and α-amylase, which increase blood glucose by degrading carbohydrates [114].
Anti-Alzheimer disease. Capsicum annuum extracts have been found to be effective in suppressing major Alzheimer's associated enzymes such as butyrylcholinesterase, acetylcholinesterase, and β-secretase [115]. Capsaicin exhibits high free radical scavenging capacity [116], and also helps regulate the mitochondrial enzymes which are beneficial in minimizing the risk of lung carcinoma [117]. This finding holds significance for degenerative brain disorders, since increase of Mitochondrial Lipid peroxidase (LPO) levels are associated with damage to brain cells [118,119]. The carotenoids, lutein and β-carotene have demonstrated a positive effect on memory [120]. Additionally, red paprika contains capsanthin, which, alongwith b-carotene, protects against deteriorative diseases caused by the mechanisms of oxidative stress [121].

Contribution of Trachyspermum ammi towards Brain Health
Carom (Tachyspermum ammi) seeds, also called Carum copticum and ajwain seeds, have been shown to affect serum lipids and the cardio-vascular system in various beneficial ways. Considering the importance of metabolic and vascular disorders in causation of neuro-degenerative diseases, these effects are of significance. Javed et al in 2002 demonstrated the anti hyperlipidaemic potential of carom seeds; his study demonstrated that carom seed extract reduced total cholesterol, LDL-cholesterol, total lipids, and triglycerides in albino rabbits [122]. In a study conducted by Aftab and Usmanghani, C. copticum showed significant cardiovascular effects, reducing heart rate and blood pressure [123]. Gilani et al also in 2005 showed that T. ammi seed extract exhibited hypotensive activity and significantly reduced mean arterial blood pressure [124].
T. ammi seeds fixed oil contains palmitic acid, resin acids, petroselenic acid, linoleic acid, and oleic acid [125]. Extracts from Carum copticum seeds were found to affect kinnin, prostaglandin, lysozyme, and bradykinin synthesis, inhibiting inflammation in animal models [126]. Ajwain plant extracts exhibited the effects as a natural antioxidant on scavenging nitric oxide, hydroxyl, and superoxide radicals [127]. Soni and Parle in 2017 explored the positive effects of T. ammi seed powder on amnesia induced in mice. A 10-day administration led to an increase in brain The main phenolic compound in ajwain is thymol [132]. Thymol has demonstrated the potential to balance neurotransmitters and suppress proinflammatory cytokine expression in many types of depression models [133]. Thymol also has nociceptive actions, and this could be due to its action in increasing the spontaneous release of L-glutamate in substantia gelatinosa (SG) neurons by activating TRPA1 channels [134]. Rajput et al. in 2013 found that stimulation of GABA responses by thymol resulted in a significantly increased delay in the onset of convulsions, which indicates the potential application of Trachyspermum ammi in epileptic conditions [135]. Thymol inhibited cognitive impairments in scopolamine-treated rats, demonstrating its antioxidant, anticholineesterase, and antiinflammatory actions [131]. Additionally, thymol decreased H2O2-induced oxidative stress in the PC12 cell line (embryonic neural crest cells) [136]. These results exhibit therapeutic potential in the treatment of Alzheimer's Disease (AD). Thymol supplementation also improved endogenous antioxidants and specific PUFA and phosholipid proportions in the aging brain of rats [137]. Thymol enhanced acetyl choline (Ach) synaptic levels and nAchR responsiveness, demonstrating beneficial effects in cholinergic dysfunction, which is observed in a range of neurodegenerative and psychiatric disorders such as Alzheimer's and Parkinson's disease [138]. All these effects testify to the great therapeutic potential of T ammi in prevention and amelioration of degenerative brain disorders.

Conclusion
All the ingredients used in making the Indian lemon pickle have been found to have beneficial effects on the functional abilities of the brain. Lemons particularly contain bioactives that are effective in promoting better cognition, memory and mood. It may be noted that eating lemon pickle is probably one of the very few ways in which the bitter peel can be eaten. This is significant because the majority of lemon flavonoids are present in the peel. The protective effects of lemon are probably linked with the antioxidative and anti-inflammatory action of the phytochemicals contained. A great advantage of lemon phytochemicals is their safety profile [52,139-143].
The complex mixtures of phytochemicals present in lemon fruits, red pepper and carom seeds and their bioactive nuances likely exert greater benefit than one purified compound, accumulatively acting upon multiple targets, and producing synergistic effects. Given the multifactorial nature of neurodegenerative diseases, it can be hypothesized that this complex form offers greater efficacy. Eating lemon pickle regularly may be a safe and delicious way to ward off neurodegenerative diseases and the cognitive decline associated with old age. In any case, lemon pickle can prove a useful adjunct to therapeutic agents against various diseases such as AD.