Dietary Supplements and the Gut–Brain Axis: A Focus on Lemon, Glycerin, and Their Combinations

Abstract

Dietary supplements are products taken orally, and they contain an ingredient intended to augment the diet. Many studies demonstrate clear alterations in microbe abundances and the production of microbiota-derived metabolites, such as short-chain fatty acids, following dietary changes. This review comprehensively explores the possible interactions among gut microbiota, lemon extracts, glycerin, and their mixture products. Lemon extracts/components are associated with a vast array of health benefits, including anti-inflammation, antioxidant, anti-atherosclerotic, and anti-diabetic effects. They are also associated with increased memory and decreased depression. Glycerin can reduce serum free fatty acids and mimic caloric restriction; its metabolites can function as a broad-spectrum antimicrobial. Additionally, glycerin has a dehydrating effect on the central nervous system and can reduce focal cerebral edema and improve performance by expanding plasma volume. However, it may also have side effects, such as hyperglycemia. Therefore, combined consumption of lemon extracts and glycerin may, in part, mitigate each other’s side effects while exerting their benefits. There is growing evidence that both lemon components and glycerin are metabolized by the gut microbiota and may modulate the intestinal microbiome composition. Therefore, gut microbiome alterations are also explored as an important mechanism in the gut–brain axis regulating various effects of these dietary supplements and their application in various noncommunicable neurological disorders.

1. Introduction

Many studies have investigated dietary supplements for promoting good health. The Dietary Supplement Health and Education Act (DSHEA) of 1994 defines a dietary supplement as a “product taken by mouth that contains a dietary ingredient intended to supplement the diet”. These dietary ingredients may consist of vitamins, minerals, herbs, amino acids, botanicals, enzymes, organ tissues (e.g., glandulars), and metabolites. It may also be a dietary substance for use by humans to supplement the diet by increasing the total dietary intake, or a concentrate, metabolite, constituent, extract, and a combination of any ingredients mentioned above. Furthermore, dietary supplements are products intended for ingestion and are not to be used as a conventional food or as a sole item in a meal or the diet. Dietary supplements can take various forms, including tablets, capsules, soft gels, liquids, powders, and bars.

This review discusses the evidence reported on the involvement of gut microbiome (GMB) and the gut–brain axis in mediating the health benefits of dietary supplements, specifically lemon extracts/components, glycerin, and their mixture products. Cross-disciplinary approaches, including those in neuroscience, toxicology, physiology, and immunology, have been used to critically assess their contributions to the gut–brain axis, beneficial or detrimental. For this review, various databases, including Google Scholar and PubMed, were searched using terms such as dietary supplements, gut–brain axis, lemon, glycerin/glycerol, inflammation, neurotoxicity, short-chain fatty acids, and microbiome/microbiota.

2. Dietary Supplements and Their Relationship with the Gut–Brain Axis

Despite representing slightly more than 2% of adult body weight, the human brain accounts for 20% of the body’s total energy requirement. Excitatory (glutamatergic) neurons consume 80–85% of the brain’s energy, with inhibitory neurons and other cells accounting for the remainder [1]. Glucose (and glycogen) reserves within the brain can supply its energy needs for only a few minutes. Impaired brain glucose metabolism compromises transmembrane ion transport, vesicle recycling, and synaptic signaling, leading to hyperexcitability, excitatory–inhibitory imbalance, functional impairment of cortical networks, disrupted glutamatergic transmission, abnormal astrocyte and oligodendrocyte function, and neuroinflammation [1]. Evidence has been accumulating that impaired brain energetics are involved in the cause and progression of neurodegenerative disorders, and a broad range of ‘brain energy rescue’ strategies by improving, preserving, and/or restoring brain energy status have been explored. These strategies include mitochondrial function promotion, utilization of alternative fuel sources such as ketone bodies (ketones), and hormonal interventions to increase insulin sensitivity and brain glucose metabolism. Additional strategies, such as complementary lifestyle and dietary approaches, have become increasingly popular because they make use of the body’s natural healing abilities and do not interfere with other forms of treatments and medications. In this context, we have discussed the role of various dietary supplements, including lemon extracts, glycerin, and their combinations, on the gut–brain axis in the following sections.

The gut is the host for a wide range of microbial organisms, including viruses, bacteria, and fungi, that have a critical role in gastrointestinal (GI) health and digestion. It is estimated that there are more gut bacteria than the number of cells in an adult human [2]. The gut microbiota plays a crucial role in the host’s health, and there are many factors that affect the diversity and composition of the gut microbiota [3,4]. These include age, genetics, environment [5], and more transient factors such as sleep quality [6]. Diet is another important factor. Certain diets can promote growth of specific bacterial strains. This can in turn lead to an increase in inflammation if the diet is heavier in sugar, fat, and sodium, or a decrease in inflammation if the diet is higher in components such as dietary fibers [7]. Additionally, many food particles can be broken down by the gut microbiota to produce metabolites, such as short-chain fatty acids (SCFAs) and neurotransmitters, e.g., serotonin [8,9], which may lead to changes in health, including glycemic control [8], immunity [7], and mental health [4]. SCFAs act as ligands for G protein-coupled receptors, including GPR109A, GPR43, GPR41, and G protein-coupled hydroxycarboxylic acid receptors (such as HCAR2). They are key fuels for intestinal cells and can also modulate the immune system, stimulate the release of hormones such as glucagon-like peptide 1 from the gut, and inhibit histone deacetylases [10].

Dietary supplements often exert their effects through modulating the GMB. Dysbiosis, the destabilization of the normal GMB, is difficult to treat because medications that can kill the pathogenic bacteria also kill some bacteria that may be necessary to maintain a healthy microbiome. The most promising treatment for dysbiosis is currently fecal transplant. However, a safety alert has been put on the use of fecal transplants by the USDA in June 2019, after two immunocompromised patients developed extended-spectrum beta-lactamase-producing Escherichia coli, leading to the death of one patient [11]. There are additional limitations of fecal transplants. As of 2024, they are only approved as a treatment for recurrent Clostridium difficile colitis. For patients who do decide to undergo fecal transplants, the procedure may not provide the results they are hoping for: only 50.5% to 52.8% were successful after a single transplant, and there was an overall failure rate of 12.4% [12]. Furthermore, probiotic pills may also not be the most efficient way since it has been reported that 50% of patients do not follow prescription regimes given by medical practitioners [13]. There is also a concern of what percentage of the bacteria in pills can survive the packaging process and, if they make it to the digestive system, how many can colonize the gut and produce a notable effect. One solution is to make a dietary supplement that tastes good and that can be used as preventative treatment as well as a self-care treat. Potential dietary supplements that could meet these criteria are lemon extracts, glycerin, and their mixture products. For example, lemon cough syrups are already used for sore throat relief, lemon-glycerin swab sticks for oral lubrication, and the liquid dietary supplement LemonGlycerin for relieving vertigo and supporting gut and brain health [14]. These supplements can act as prebiotics, a substrate that is selectively utilized by host microorganisms to confer a health benefit as defined by the International Scientific Association for Probiotics and Prebiotics.

There have been many studies focused on the role of GMB on mental illnesses. The gut–brain axis consists of bidirectional communication between the central and the enteric nervous system, linking emotional and cognitive centers of the brain with peripheral intestinal functions [15]. The enteric nervous system (ENS), found in the gut, includes neurons that allow communication between microbes and the body [16]. Studies in animals have shown that an impaired gut–brain axis is associated with various neurological diseases including autism spectrum disorder, depression, Alzheimer’s disease, Parkinson’s disease, and Multiple Sclerosis [17,18,19]. Through the gut–brain axis, the gut microbiota communicate with the central nervous system (CNS), specifically the brain, to modulate emotions and behavior; and reciprocally, the brain can modulate the intestinal functions and the activities of other peripheral organs through the excretion of neurotransmitters such as dopamine and serotonin. Within the axis, the GMB affects brain health through the host’s immune response and generating nutrients and modulating overall energy homeostasis, as well as through neurotransmitters released during digestion [20]. The major pathway for gut–brain interactions is the vagus nerve, which extends from the brainstem to the gut [21].

3. Lemon Components and Their Relationship with the Gut–Brain Axis

Lemon, the species C. limon, belongs to Citrus, the genus Citrus L. of the family Rutaceae, and the subfamily Aurantioideae. According to the U.S. Department of Agriculture’s Economic Research Service, the production of lemons was 1.12 million tons in 2022–23 and has been annually increasing at a rate of over 5%. One third of these lemons undergo processing, and the other two thirds have gone to the fresh market. There are three main types of lemons—acidic, rough, and sweet. In 1622, the British explorer Sir Richard Hawkins recorded that sour lemons (Citrus limon) were effective in preventing scurvy, a disease resulting from a lack of vitamin C. In 1747, James Lind, the father of British naval medicine, carried out one of the first controlled clinical trials recorded in medical science and demonstrated that lemon could be used to counteract scurvy. In the 1940s, the extract of lemon was used as a detoxifying diet to improve insulin levels and is still being used today for such purpose [22]. Lemons can be processed into many products, including juice, oils, and dried peel. Fresh lemons are juiced using mechanical juicers or extraction machines, and the juice is strained to remove pulp and seeds and clarified by centrifugation or filtration following treatment with enzymes and coagulants. Lemon oil can be extracted from lemon peels using cold pressing, and lemon essential oil can be further isolated by freezing the lemon oil. The outer zest or peel of the lemon can be dried and ground into a fine powder to make products such as candied lemon peel, lemon pepper, lemon marmalade, and gremolata.

3.1. Nutrients and Bioactive Components of Lemon

The compositions of nutrients and bioactive components of lemon extracts are complex, including carotenoids, phenolic compounds (mainly flavonoids), terpenes, limonoids, vitamins, essential oils, minerals, dietary fiber, and many other compounds of nutritional and nutraceutical values. Flavonoids are the secondary plant metabolites. More than sixty individual flavonoids have been identified in Citrus sp., and most of them can be classified into three groups: flavanones, flavones and polymethoxyflavones (PMFs), and flavonols [22]. In addition, other phenolic compounds (phenolic acids, etc.) are also present in Citrus species. There are at least four different types of flavanones (naringin, naringenin, hesperidin, and hesperetin) and seven PMFs across the citrus genus that may have a combination of health promoting effects, including antioxidant, anti-inflammation, anticancer, anti-atherosclerotic, and/or anti-diabetic effects. Specifically in lemons, the structures of several representative bioactive components are shown in Figure 1, including: (1) Eriocitrin, a flavanone; (2) Nobiletin, a PMF; (3) Pectin, also known as pectic polysaccharides, the major dietary fiber present in lemon peel; (4) d-Limonene, the major component (45–75%) of the essential oils from lemon; and (5) Citric acid, the most abundant organic acid in lemon, representing as much as 8% in dry weight. In addition to dietary sources, citric acid (2-hydroxy-1,2,3-propane-tricarboxylic acid) can be synthesized from acetyl-CoA and oxaloacetate inside the mitochondria and, thus, be found in all animal tissues for energy generation. It can also be anti-inflammatory and reduce lipid peroxidation [23].

3.2. Neurological Effects of Lemon Extracts/Components and Their Relationship with the Gut-Brain Axis

Preclinical studies have demonstrated the neuroprotective potential of citrus flavonoids and have highlighted both the well-established (anti-inflammatory and anti-oxidative properties) and newly emerging (influences upon blood–brain barrier function/integrity) mechanistic actions by which these neurological effects are mediated [24]. Studies in animals have also shown that supplementing the diet with citrus fruits improves learning and memory, particularly spatial memory. The flavonoid nobiletin can reduce memory impairment and diminish Aβ pathology in a transgenic mouse model of Alzheimer’s disease [25]. The possible mechanisms include functioning as N-methyl-D-aspartate (NMDA) receptor agonists and upregulating the expression of NMDA receptors, which are essential for memory and learning as well as increasing the expression of neprilysin, an enzyme for degrading Aβ. Nobiletin has a high permeability and high lipophilic nature, and it is readily absorbed [26]. In addition, nobiletin can decrease the symptoms of Parkinson’s disease by enhancing the release of dopamine in the striatum and hippocampal CA1 region [26]. Furthermore, nobiletin can elicit an antidepressant-like response after five weeks of administration to patients who are diagnosed with chronic unpredictable mild stress-induced depression. This is likely due to upregulation of brain-derived neurotrophic factor to activate tropomyosin receptor kinase B (TrkB) signaling pathways that are important for not only regulating long term potentiation required in learning, but also decreasing the inflammation that causes the chronic unpredictable mild stress-induced depression [27]. Some studies have shown that eriocitrin may also have neuroprotective effects in the brain. In rats, eriocitrin treatment reduced cerebral infarct volume, cerebral water content, and cerebral indexes [28].

Other components in lemon that are associated with neuroprotection, e.g., decreased symptoms of depression, include pectin oligosaccharides [29], hesperidin, and naringin, all of which possess anti-inflammatory properties [30]. Studies have also suggested that lemon IntegroPectin, a product of lemon obtained via hydrodynamic cavitation of lemon in water, has significant neuroprotective properties by reducing oxidative stress [31]. Lemon essential oil, rich in limonoids and terpenes, is additionally known for its high levels of antioxidants and for its antitumor effect. The antioxidants in lemon essential oils act as free radical scavengers in a concentration or dose-dependent manner [32]. Lemon fragrance is known for its stimulatory and excitatory properties as well as for stress-alleviating effects. It can also modulate mood, cognition, and emotion, and their associated changes in brain waves, heart and respiratory rate, and blood pressure. It has been shown that the lemon oil vapor causes antidepressant-like and anti-stress effects via modulating the serotonin and dopamine activities in mice [33]. The present evidence also suggests that inhalation of lemon essential oils can increase the level of alertness by integrating cortical regions involved in olfaction and emotion processing [34].

The lemon fruit contains many compounds that can modulate GMB and produce health benefits (Table 1). Human clinical studies have shown that fibers and flavonoids, which are abundant in citrus fruits, can have prebiotic effects on the intestinal microbiota by stimulating growth of beneficial bacteria and inhibiting growth of pathogenic bacteria [35]. Through the fiber pectin that feeds healthy gut bacteria, lemons can promote an anti-inflammatory response in the colon [36,37,38]. Pectin cannot be metabolized by mammalian cells in the GI tract but is fermented by the gut microbiota in the colon. Studies have shown that the fibers in lemons stimulate the growth of Bifidobacterium and Lactobacillus [39,40], which are useful in preventing the progression of neurodegenerative diseases [41]. These beneficial bacteria can reduce the abundance of pathogens or mucin-degrading bacteria. Neurotransmitters can also be produced by the gut microbiota through the metabolism of indigestible and soluble fiber pectin [42]. The lemon flavonoids, including eriocitrin, hesperidin and naringin, can also influence the intestinal microbiota [43]. Eriocitrin is metabolized first to eriodictyol (the aglycone) by GMB, then to homoeriodictyol and hesperetin through methoxylation, and finally to 3,4-dihydroxycinnamic acid and phloroglucinol [22]. Lemons can produce naturally occurring exosome-like nanoparticles that have remarkable effects on both the host and gut microbiota. For example, lemon-derived exosome-like nanoparticles selectively increase Lactobacilli abundance in the small intestine of mice by increasing their resistance to bile [44]. Bile is one of the most challenging obstacles for bacteria in the gut to overcome as it kills bacteria and controls their growth by damaging bacterial DNA and the cell membrane.

Gut microbiota can ferment pectin because of the presence of bacterial species that carry polysaccharide utilization loci and produce carbohydrate-active enzymes. These enzymes can break down pectin molecules and allow pectin to be used as a carbon source that subsequently ends in the production of SCFAs [45]. The key pectin-degrading bacteria has been identified to be a Bacteroides species (e.g., Bacteroides thetaiotamicron). Furthermore, species belonging to the Lachnospiraceae family also contain pectin-degrading enzymes, such as hydrolases, lyases, and esterases; pig studies have shown increases in this family following dehydrated citrus pulp supplementation [46]. Some other microbes that are associated with pectin fermentation are Faecalbacterium prausnitzii and Eubacterium eligens. The breakdown products can be used by surrounding bacteria as growth substrates and increase their abundance [47]. The pectin enzyme hydrolysate from citrus fruits can significantly increase levels of Bifidobacterium bifidum and Lactobacillus acidophilus [39]. As discussed above, B. bifidum is a known probiotic that has a wide array of beneficial effects, including neuroprotection [41]. L. acidophilus is another well-known probiotic; it can prevent pathogenic bacterial infections such as C. difficile and candidiasis by producing lactic acid and hydrogen peroxide to create an acidic environment that is harmful to pathogens [48].

SCFAs include butyrate, propionate, and acetate. They are metabolic products derived from the fermentation of dietary fibers under anaerobic conditions. Treatments with Citrus extracts increase the production of butyrate and acetate in fecal samples from volunteers with metabolic syndrome, which have been linked to a decrease in intestinal inflammation [49]. An earlier in vitro study by this group using fecal samples of healthy volunteers reported increased acetate and reduced butyrate levels following treatment with Citrus fruit extract that contained 88.2% hesperidin and 6.5% naringin [50]. Lemon powder rich in dietary fibers also produces an anti-inflammation response in the intestines by protecting tight junction barriers and suppressing the inflammatory reaction in colitic mice, and it has been suggested that this may also be a result of increased levels of acetate [51]. These increases in SCFAs may enhance the gastrointestinal immune barrier, which consists of a mucus layer, a cellular layer of epithelial cells, and the lamina propria containing immune cells. However, rats fed a high-calorie diet treated with lemon fermented with Lactobacillus OPC1 exhibited reduced lipid metabolism and decreased content of acetate [52]. Further, treatment of male non-obese diabetic (NOD) mice with lemon extracts did not affect acetate production [14]. The inconsistencies in the production of SCFAs among the different studies may be related to the variations in GMB composition, characteristics of pectins, fermentation substrates, or the influence of the host. Indeed, it has been suggested that the production of SCFAs is strongly influenced by the rhamnogalacturonan I structures or the degree of methyl-esterification of pectins [47,53,54,55].

Table 1. Summary of gut microbiome studies on lemon components.

Lemon Components	Model/Diseases	Model/Population	Exposure Window/Period	Dose/Concentration	Routes of Administration	Diet/Medium	Effects	Reference
Lemon extract	Type 1 diabetes	Adult male NOD mice	6 months	Not known	Oral gavage	The 5053-PicoLab® Rodent Diet 20	Decrease in the Verrucomicrobia, Cyanobacteria and TM7 phyla, the RF39 and YS2 orders, F16 and Clostridiaceae families, and the muciniphila species; and an increase in the rc4-4 genus; predicted to decrease both propanoate metabolism and glycolysis/gluconeogenesis	[14]
Pectin-derived oligosaccharides	Continuous colon model (TIM-2)	Elderly Donors/GMB	3 days	6.5 g per day	In vitro	Standard ileal efflux medium (SIEM)	Increases in beneficial species such as Faecalibacterium prausnitzii and alpha diversity and SCFAs, but not butyrate	[29]
Pectin (galacturonide oligosaccharides DP4 and DP5)	In vitro cell-based assays	Eubacterium eligens and Faecalibacterium prausnitzii strains	24 h	0.2%	In vitro	M2GSC medium	Promotes the production of the anti-inflammatory cytokine IL-10	[36]
Extracts from citrus fiber	Tolerance to different pH values and bile salts	L. paracasei, L. fermentum, L. rhamnosus and B. animalis subsp. paracasei	0–24 h	10 g/L	In vitro	MRS broth	Showed great prebiotic activities	[37]
Citrus Pectin Enzyme Hydrolysate	Probiotics	B. bifidum and L. acidophilus cultures	6, 12, 24, 48 h	1%, 2%, 4%	Supplemented into growth media	Glucose-free MRS broth	Higher populations of B. bifidum and L. acidophilus	[39]
Eriocitrin	Metabolism	Six-week-old male ICR mice	2 weeks	100 mg/kg	Oral gavage	Standard chow	Altered the beta diversity; the probiotics such as Lachnospiraceae_UCG_006 were enriched, and the production of butyrate, valerate and hexanoate were increased	[43]
Lemon-derived exosome-like nanoparticles	Toleration to bile	C57BL/6J mice	Not given	Not given	Oral gavage	Not given	Lactobacillus rhamnosus GG was increased while the S24-7 was decreased	[44]
Citrus extract rich in citrus flavonoids	In vitro model of the colon (TIM-2)	Fecal samples with metabolic syndrome	3 days	500 mg/day	In vitro	Standard ileal efflux medium (SIEM)	Increased production of butyrate, acetate, and valerate	[49]
Citrus extract rich in citrus flavonoids	Volunteers with metabolic syndrome	Fecal samples	12 weeks	500 mg/day	In vivo/Oral	Habitual diet without foods high in citrus flavonoids	Increased SCFAs with significantly more butyrate. A trend towards a reduction in intestinal inflammation (calprotectin)	[49]
Citrus Fruit Extract (88.2% hesperidin and 6.5% naringin)	In vitro model of the colon (TIM-2)	Fecal samples of healthy volunteers	3 days	250 or 350 mg/day	In vitro	Standard ileal efflux medium (SIEM)	Increased Roseburia, Eubacterium ramulus and Bacteroides eggerthii. Increased acetate while reduced butyrate	[50]
Citrus limon peel (LP) powder rich in dietary fibers	Dextran sulfate sodium (DSS)-induced colitis	7 weeks old male BALB/c mice	16 days	5% by diet weight	Oral/diet	AIN-93G-based diet	LP powder increased levels of acetate and n-butyrate, reduced colitis, restored normal colon length and reduced intestinal damage	[51]
Lemon fermented with Lactobacillus OPC1	Obesity	Male Wistar rats	9 weeks	2.89 g/kg	Oral gavage	High calorie diet	Reduced the ratio of Firmicutes/Bacteroidetes and increased the abundance of Firmicutes Clostridia, decreased content of acetic acid and propionic acid.	[52]
Four types of pectins	Fermentation patterns	Male Wistar rats	7 weeks	3% by diet weight	Oral/diet	The control diet RMH-B ± pectins	Low methyl esterified citrus pectin increased the production of total SCFAs, propionate and butyrate	[53]
Nine structurally diverse pectins	TIM-2 colon model	Fecal samples of healthy volunteers	24, 48, 56, and 72 h	7.5 g pectin per day	In vitro fermentation	Simulated ileal efflux medium (SIEM)	Cumulative production of the total short chain fatty acids and propionate was largest in fermentations of the high methoxyl pectins	[54]
Rhamnogalacturonan-I (RG-I)-enriched pectin	Gut microbiota	C57BL/6J male mice	9 weeks	100 mg/kg	Oral gavage	Standard chow diet	Increased the abundance of prebiotics such as Bifidobacterium spp., Lactobacillus spp., and increased SCFA producers including species in Ruminococcaceae family	[55]

Table 1. Summary of gut microbiome studies on lemon components.

Lemon Components	Model/Diseases	Model/Population	Exposure Window/Period	Dose/Concentration	Routes of Administration	Diet/Medium	Effects	Reference
Lemon extract	Type 1 diabetes	Adult male NOD mice	6 months	Not known	Oral gavage	The 5053-PicoLab® Rodent Diet 20	Decrease in the Verrucomicrobia, Cyanobacteria and TM7 phyla, the RF39 and YS2 orders, F16 and Clostridiaceae families, and the muciniphila species; and an increase in the rc4-4 genus; predicted to decrease both propanoate metabolism and glycolysis/gluconeogenesis	[14]
Pectin-derived oligosaccharides	Continuous colon model (TIM-2)	Elderly Donors/GMB	3 days	6.5 g per day	In vitro	Standard ileal efflux medium (SIEM)	Increases in beneficial species such as Faecalibacterium prausnitzii and alpha diversity and SCFAs, but not butyrate	[29]
Pectin (galacturonide oligosaccharides DP4 and DP5)	In vitro cell-based assays	Eubacterium eligens and Faecalibacterium prausnitzii strains	24 h	0.2%	In vitro	M2GSC medium	Promotes the production of the anti-inflammatory cytokine IL-10	[36]
Extracts from citrus fiber	Tolerance to different pH values and bile salts	L. paracasei, L. fermentum, L. rhamnosus and B. animalis subsp. paracasei	0–24 h	10 g/L	In vitro	MRS broth	Showed great prebiotic activities	[37]
Citrus Pectin Enzyme Hydrolysate	Probiotics	B. bifidum and L. acidophilus cultures	6, 12, 24, 48 h	1%, 2%, 4%	Supplemented into growth media	Glucose-free MRS broth	Higher populations of B. bifidum and L. acidophilus	[39]
Eriocitrin	Metabolism	Six-week-old male ICR mice	2 weeks	100 mg/kg	Oral gavage	Standard chow	Altered the beta diversity; the probiotics such as Lachnospiraceae_UCG_006 were enriched, and the production of butyrate, valerate and hexanoate were increased	[43]
Lemon-derived exosome-like nanoparticles	Toleration to bile	C57BL/6J mice	Not given	Not given	Oral gavage	Not given	Lactobacillus rhamnosus GG was increased while the S24-7 was decreased	[44]
Citrus extract rich in citrus flavonoids	In vitro model of the colon (TIM-2)	Fecal samples with metabolic syndrome	3 days	500 mg/day	In vitro	Standard ileal efflux medium (SIEM)	Increased production of butyrate, acetate, and valerate	[49]
Citrus extract rich in citrus flavonoids	Volunteers with metabolic syndrome	Fecal samples	12 weeks	500 mg/day	In vivo/Oral	Habitual diet without foods high in citrus flavonoids	Increased SCFAs with significantly more butyrate. A trend towards a reduction in intestinal inflammation (calprotectin)	[49]
Citrus Fruit Extract (88.2% hesperidin and 6.5% naringin)	In vitro model of the colon (TIM-2)	Fecal samples of healthy volunteers	3 days	250 or 350 mg/day	In vitro	Standard ileal efflux medium (SIEM)	Increased Roseburia, Eubacterium ramulus and Bacteroides eggerthii. Increased acetate while reduced butyrate	[50]
Citrus limon peel (LP) powder rich in dietary fibers	Dextran sulfate sodium (DSS)-induced colitis	7 weeks old male BALB/c mice	16 days	5% by diet weight	Oral/diet	AIN-93G-based diet	LP powder increased levels of acetate and n-butyrate, reduced colitis, restored normal colon length and reduced intestinal damage	[51]
Lemon fermented with Lactobacillus OPC1	Obesity	Male Wistar rats	9 weeks	2.89 g/kg	Oral gavage	High calorie diet	Reduced the ratio of Firmicutes/Bacteroidetes and increased the abundance of Firmicutes Clostridia, decreased content of acetic acid and propionic acid.	[52]
Four types of pectins	Fermentation patterns	Male Wistar rats	7 weeks	3% by diet weight	Oral/diet	The control diet RMH-B ± pectins	Low methyl esterified citrus pectin increased the production of total SCFAs, propionate and butyrate	[53]
Nine structurally diverse pectins	TIM-2 colon model	Fecal samples of healthy volunteers	24, 48, 56, and 72 h	7.5 g pectin per day	In vitro fermentation	Simulated ileal efflux medium (SIEM)	Cumulative production of the total short chain fatty acids and propionate was largest in fermentations of the high methoxyl pectins	[54]
Rhamnogalacturonan-I (RG-I)-enriched pectin	Gut microbiota	C57BL/6J male mice	9 weeks	100 mg/kg	Oral gavage	Standard chow diet	Increased the abundance of prebiotics such as Bifidobacterium spp., Lactobacillus spp., and increased SCFA producers including species in Ruminococcaceae family	[55]

3.3. Other Effects of Lemon Extracts/Components and Their Side Effects

Lemons have many other health benefits. For example, hexane extracts from lemon peels have antidiarrheal functions, and the possible mechanism of action is through anti-secretory and anti-motility properties through the β adrenergic system [56]. The components in lemon, e.g., citric acid and flavonoids, may contribute to the postprandial hyperglycemia-suppressing action as well as the increases in insulin sensitivity [30,35]. With a high content of vitamin C and soluble fibers, low glycemic index, and other beneficial characteristics, the American Diabetes Association lists lemons as a diabetic superfood to lower blood glucose levels and reduce insulin resistance. In rats, oral administration of naringenin, a flavonoid in citrus peels, improved ketoacidosis and symptoms associated with type 1 diabetes by reducing chronic inflammation as well as oxidative damage in proteins and lipids that lead to insulin resistance [57,58]. Citrus extracts have also been reported to decrease weight [59]. Angiotensin converting enzyme is a biomarker for weight loss, and inhibition of the enzyme improves insulin sensitivity. Lemon extracts could also decrease the activity of angiotensin converting enzyme in mice to improve insulin sensitivity and increase breakdown of lipids in adipocytes [60]. Additional studies have shown that lemon polyphenols promote weight loss, probably through a combination of different factors, including increased acetate production, fat oxidation, and suppressed appetite [50]. The soluble fiber pectin in the fruit helps people feel full for a longer time [61]. Citric acid and vitamin C assist in the absorption of iron, which helps to prevent iron deficiency and reduce iron deficiency-based anemia by increasing hemoglobin concentration [62]. Citric acid can also increase urine output and reduce calcium oxalate crystal formation to decrease kidney stones [63]. The flavonoids in citrus fruits may also help lower the risk of ischemic stroke in women [64].

Lemons can also have side effects. Due to its acidic properties, lemon consumption may cause tooth decay and stomach irritation that can lead to nausea, bloating, and acid reflux, as well as exacerbate canker sores and peptic ulcers. It may also lead to dehydration due to its diuretic properties. People consuming lemons may also experience migraines because of the presence of amino acid tyramine and have worsened heartburn symptoms.

4. Glycerin and Its Relationship with the Gut–Brain Axis

Glycerol (1,2,3-propanetriol; Formula: C3H8O3, CAS No. 56-81-5) is a sugar alcohol or a polyol used in foods, soaps, medicines, and skin products. It can be derived from animal products, plants, or petroleum. Glycerol occurs naturally in plants, fermented foods, and beverages, and it can be produced from the hydrolysis of fats and oils. In 1779, Swedish chemist K. W. Scheele produced the first man-made glycerol from olive oil. It became economically and industrially significant in the late 1800s when it was first used to make dynamites. Glycerol is marketed as glycerin, which consists of 95% glycerol or more. Many commercially manufactured glycerin products contain different amounts of glycerol and other impurities, including salt, water, and other organic compounds. The U.S. Food and Drug Administration (FDA) has considered glycerin to be safe for human intake. Vegetable glycerin is made by heating triglyceride-rich vegetable fats, typically soybean, coconut, or palm oils, under pressure or together with a strong alkali, such as lye. Vegetable glycerin is a common ingredient in pharmaceutical drugs, including heart medication, suppositories, cough remedies and anesthetics. Additionally, vegetable glycerin can be found in toothpaste, as it helps prevent the toothpaste from drying out or hardening in the tube. In the food industry, it is often added to foods to help oil and water-based ingredients mix and to sweeten or moisten the final product. Glycerin can act as a cryoprotective agent and is used to prevent ice crystals from forming in frozen foods such as low-fat frozen yogurt, ice cream, and other desserts. Glycerin is a common food additive used in processed meats, dairy products, sweetened beverages, and confectionaries [65]. Its sweet taste and smooth texture make it a sugar substitute and a preservative to prolong product shelf life. Glycerin can also be useful in numerous industrial, therapeutic, and diagnostic applications.

4.1. Glycerol Metabolism

Glycerol is present in many species, including unicellular protists and mammals. It can be endogenously produced from glucose, proteins, pyruvate, triacylglycerols, and other glycerolipid metabolic pathways [66]. Glycerol can also be obtained exogenously through food and dietary supplements. The normal endogenous fasting serum concentration of glycerol in humans is about 46 µg/mL. Glycerol has long been known to play fundamental roles in several vital physiological processes of both prokaryotes and eukaryotes and is an important intermediate of energy metabolism. Glycerol can serve as a source of energy, yielding 4.32 calories/g when oxidized to carbon dioxide and water and contributing about 10% of total respiratory CO₂. Approximately 80% glycerol is metabolized by the liver, while 10–20% occurs in the kidney [67]. Some utilization of glycerol has been demonstrated in the nerve tissues, thyroid, lung, pancreas, mammary gland, lymphatic tissues, white adipose tissues, leukocytes, spermatozoa, granuloma tissues, and diaphragm. Glycerol kinase, which converts glycerol to glycerol-3-phosphate, has also been found in small amounts in intestinal mucosa. The slow metabolic clearance of glycerol from the blood contributes to the sustained hydration potential of this compound [68].

Glycerol is a normal intermediary metabolite via two major pathways: approximately 70–90% is oxidized by glycerol-3-phosphate dehydrogenase to dihydroxyacetone phosphate after phosphorylation by glycerol kinase, which then enters the Embden–Meyerhof pathway at the level of glyceraldehyde-3-phosphate. The Embden–Meyerhof pathway allows the metabolic use of glucose to generate ATP, NADH, and several biosynthetic precursors such as 3-phosphoglycerate or pyruvate. The remaining 10–30% combines with free fatty acids to form triglycerides. Glycerol can be metabolized to glucose via gluconeogenesis or lactate via glycolysis. Under normal health and diet conditions, gluconeogenesis from glycerol accounts for less than 5% of total glucose; however, more than 20% of such production is derived from glycerol metabolism after 62–86 h of starvation. In prolonged fasting, glycerol can be used as the only source for gluconeogenesis, since glycogen reserves are depleted within two fasting days. Because glycerol is a substrate for gluconeogenesis, it has been employed satisfactorily as a partial dietary substitute for carbohydrates over an extended period of time. Glycerol consumption can mimic caloric restriction by shifting metabolism away from glycolysis and towards oxidative phosphorylation [69].

When glycerol is consumed orally, it is rapidly absorbed primarily in the small intestine via passive diffusion and an active transport mechanism, e.g., sodium-dependent carrier-mediated transport. A small amount can also be absorbed in the stomach. Once it is in circulation through the blood, glycerol can provide an alternative source of energy to the brain [70,71]. Glycerol can also be metabolized directly by the brain and enhance lipogenesis. Although glycerol concentrations are very rapidly balanced in most tissues, glycerol permeability is considerably reduced in the brain, cerebrospinal fluid (CSF), and the aqueous humor of eyes. Glycerol decreases the sodium and water content of mammalian tissues, especially that of cerebral white matter. In the brain and CSF, glycerol can contribute to oxidative metabolism, increase blood flow to ischemic areas, decrease serum free fatty acids, and increase synthesis of glycerides [72]. Aquaporins (AQPs) are a family of homologous water channels; tissues, including skin, intestines, skeletal muscle, and lungs, express aquaporin channels that enable glycerol transport across cell membranes. Among the AQPs identified in humans, AQP3, 7, 9, and 11 are subcategorized as aquaglyceroporins, which permeabilize glycerol as well as water [73,74,75,76].

4.2. Neurological Effects of Glycerin and Its Relationship with the Gut-Brain Axis

Glycerin is a potent osmotic dehydrating agent that may directly affect brain metabolism. It reduces serum free fatty acids that can cause coma, increased intracranial pressure, cerebral edema, and mitochondrial swelling and dysfunction in experimental animals [67]. This effect of glycerin has led it to be used therapeutically for the treatment of increased intracranial pressure, as it lowers pressure in the brain and eyes. This reduction in serum free fatty acids can also increase blood flow to ischemic areas to treat acute stroke [67]. It can be especially beneficial to patients with recent cerebral infarction by reducing focal cerebral edema [77]. Cerebral edema is the leading cause of death in type 1 diabetic children presenting with diabetic ketoacidosis [78]. Moreover, people with meningitis, encephalitis, CNS trauma, and stroke are given glycerol intravenously to help lower the pressure inside the brain. Glycerin is also used to treat glaucoma and other eye conditions where there is increased pressure on the eyes. The consumption of glycerin can reduce body weight [79]. It is speculated that glycerin helps to control body weight by modifying the chemical balance maintained by the CNS. Under anaerobic conditions (Table 2), glycerin is metabolized into 1,3-propanediol by human GMB in the genera Lactobacillas, Kelbiella, Enterobacter, Citrobacter, and Clustridioum [80]. 1,3-propanediol can inhibit CNS expression of AQP4, a water channel protein that plays a critical role in pathologies such as brain edema [81]. The increase in 1,3-propanediol is likely the reason for increased Lactobacillus-mediated reuterin production following glycerin supplementation [82]. In the gut, 1,3-popanediol can be further metabolized by Lactobacillus reuteri into reuterin, a broad-spectrum antimicrobial that inhibits microbial growth, including Clostridium difficile [83]. This is important because antibiotic resistant Clostridium difficile contributes to 29,000 deaths each year [84], and as the number of people who take antidepressants increases, so does the number of people more susceptible to developing a Clostridium difficile infection [85]. Reuterin is also particularly effective against E. coli, but it is not harmful to beneficial bacteria like Lactobacillus-Enterococcus [82,86].

Glycerin is a suitable substrate for microbial fermentation, which may lead to more SCFA production (Table 2). Glycerin has been seen to increase acetate but not propionate, malonate, and butyrate in male NOD mice after 6 months of oral treatment [14]. Using in vitro batch incubations of fecal samples from 10 individuals, it was demonstrated that glycerin addition (140 mM) significantly affects the metabolism and composition of the microbial community and yields proportionally higher levels of acetate [80]. Increased acetate levels were also observed in infants who took glycerin-containing vitamin D liquid formulations at 3 months of age [87]. The genus Blautia is commonly found in the gut and associated with human well-being, and it can convert glycerin to acetate [88]. Acetate was produced when bovine rumen fluid was fermented anaerobically with glycerin [89]. Acetate is the main SCFA distributed throughout the body, with a blood concentration between 50 and 100 µM, while blood butyrate is usually <5 µM. Acetate renders the gut environment unfavorable to pathogenic/detrimental bacteria [90]. Long-term acetate deficiency due to vancomycin-induced reduction in acetate-producing bacteria, including Alistipes, Blautia, Ruminclostridium_9, and Roseburia, resulted in learning and memory impairments in streptozotocin-induced type 1 diabetic mice [91]. In the brain, acetate is taken up by astrocytes, where it is used to produce energy and acetylcholine and participates in the recycling of gamma-aminobutyric acid (GABA) and glutamate [92]. In rat models of various neurological diseases, acetate supplementation increases intermediary short chain acetyl-CoA metabolism and attenuates neuroinflammation [93,94,95]. Acetate also stimulates myelin deposition and reduces the tremor phenotype in a rat model of Canavan’s disease [96], a group of genetic diseases that cause progressive damage to nerve cells and white matter in the brain [97]. It should be noted that at high concentration, however, glycerin may reduce acetate production [88,98].

Table 2. Summary of gut microbiome studies on glycerol.

Products	Model/Diseases	Model/Population	Exposure Window/Time	Dose/Concentration	Routes of Administration	Diet/Medium	Effects	Reference
Vegetable glycerin	Type 1 diabetes	Adult male NOD mice	6 months	Not known	Oral gavage	The 5053-PicoLab® Rodent Diet 20	Acetate was increased in the fecal Samples; decreased Peptostreptococcaceae, Turicibacter and Coprobacillus genera, and the prausnitzii species	[14]
Glycerol	In vitro batch incubations	Fecal samples from 10 individuals	3 weeks	140 mM	Supplemented into growth media	Standard culture medium	Altered GMB metabolism and composition. Higher levels of acetate and 1,3-propanediol, and more Lactobacillus-Enterococcus in fast conversion samples	[80]
Glycerol	In vitro colonic fermentation model	Adult human immobilized fecal microbiota	12–24 h	100 mM	Supplemented into growth media	Standard culture medium	Glycerol increased numbers of Lactobacillus-Enterococcus group and decreased Escherichia coli. In combination with L. Reuteri, glycerol decreased E. coli populations	[82]
Glycerol/ Human-derived L. reuteri	Clostridium difficile infection	Human fecal microbial communities	8–72 h	2%–10% glycerol	Supplemented into growth media	MRS medium; brain heart infusion (BHI) medium	Codelivery of L. reuteri with glycerol was effective against C. difficile colonization	[83]
Glycerol	PolyFermS chicken cecal microbiota models	Immobilized cecal microbiota from broiler chickens	6–8 days	50 and 100 mM	Supplemented into growth media	Custom nutritive medium	Increases in butyrate production, reduction in Enterobacteriaceae, and 1,3-propanediol accumulation	[86]
Glycerin-containing vitamin D liquid formulations	Fecal microbiota and their metabolites	Infants at 3 months of age	3 months	Not known	Oral	Infant supplementations were administered to mothers	Fecal 1,2-propanediol and glycerol concentrations were negatively correlated. Positive correlations between fecal 1,2-PD, Bifidobacteriaceae, Lactobacillaceae, Enterobacteriaceae and acetate levels were observed.	[87]
Glycerol	Metabolic potential	B. schinkii, the genus Blautia	15–30 h	20 mM	Supplemented into growth media	CO2/KHCO3-buffered complex medium	Producing acetate and ethanol	[88]
Glycerol		Bovine rumen fluid	24 h	25 mM	Supplemented into growth media	Fermentation medium	Acetate, propionate, butyrate, valerate, and caproate concentrations, in decreasing order, all increased with incubation time	[89]
Glycerol	In vitro fermentation	Pig fecal inoculum	24 and 36 h	10%	Supplemented into growth media	Normal or modified medium	Increased the abundances of Firmicutes, Anaerovibrio, unclassified_f_Selenomonadaceae, and decreased that of Proteobacteria; decreased the acetate production and increased butyrate	[98]

Table 2. Summary of gut microbiome studies on glycerol.

Products	Model/Diseases	Model/Population	Exposure Window/Time	Dose/Concentration	Routes of Administration	Diet/Medium	Effects	Reference
Vegetable glycerin	Type 1 diabetes	Adult male NOD mice	6 months	Not known	Oral gavage	The 5053-PicoLab® Rodent Diet 20	Acetate was increased in the fecal Samples; decreased Peptostreptococcaceae, Turicibacter and Coprobacillus genera, and the prausnitzii species	[14]
Glycerol	In vitro batch incubations	Fecal samples from 10 individuals	3 weeks	140 mM	Supplemented into growth media	Standard culture medium	Altered GMB metabolism and composition. Higher levels of acetate and 1,3-propanediol, and more Lactobacillus-Enterococcus in fast conversion samples	[80]
Glycerol	In vitro colonic fermentation model	Adult human immobilized fecal microbiota	12–24 h	100 mM	Supplemented into growth media	Standard culture medium	Glycerol increased numbers of Lactobacillus-Enterococcus group and decreased Escherichia coli. In combination with L. Reuteri, glycerol decreased E. coli populations	[82]
Glycerol/ Human-derived L. reuteri	Clostridium difficile infection	Human fecal microbial communities	8–72 h	2%–10% glycerol	Supplemented into growth media	MRS medium; brain heart infusion (BHI) medium	Codelivery of L. reuteri with glycerol was effective against C. difficile colonization	[83]
Glycerol	PolyFermS chicken cecal microbiota models	Immobilized cecal microbiota from broiler chickens	6–8 days	50 and 100 mM	Supplemented into growth media	Custom nutritive medium	Increases in butyrate production, reduction in Enterobacteriaceae, and 1,3-propanediol accumulation	[86]
Glycerin-containing vitamin D liquid formulations	Fecal microbiota and their metabolites	Infants at 3 months of age	3 months	Not known	Oral	Infant supplementations were administered to mothers	Fecal 1,2-propanediol and glycerol concentrations were negatively correlated. Positive correlations between fecal 1,2-PD, Bifidobacteriaceae, Lactobacillaceae, Enterobacteriaceae and acetate levels were observed.	[87]
Glycerol	Metabolic potential	B. schinkii, the genus Blautia	15–30 h	20 mM	Supplemented into growth media	CO2/KHCO3-buffered complex medium	Producing acetate and ethanol	[88]
Glycerol		Bovine rumen fluid	24 h	25 mM	Supplemented into growth media	Fermentation medium	Acetate, propionate, butyrate, valerate, and caproate concentrations, in decreasing order, all increased with incubation time	[89]
Glycerol	In vitro fermentation	Pig fecal inoculum	24 and 36 h	10%	Supplemented into growth media	Normal or modified medium	Increased the abundances of Firmicutes, Anaerovibrio, unclassified_f_Selenomonadaceae, and decreased that of Proteobacteria; decreased the acetate production and increased butyrate	[98]

4.3. Other Effects of Glycerin and Side Effects

Glycerin has many other health benefits. It helps retain moisture in the skin [99], increases its hysteresis (creep phenomenon) and distensibility [100], and protects skin against irritants, inflammation, and microbes [99,101,102]. Glycerin can improve bowel movements and aid the passage of stool to relieve constipation and strengthen the gut, and it also improves hydration and cardiovascular functions [103,104]. In sports, the ingestion of glycerin in combination with excess fluid can lead to increased plasma osmolality, reduced urine volume, and expanded plasma volume [70]. The expanded plasma volume can improve performance by increasing maximal cardiac output and muscle perfusion [105]. Glycerin supplementation has several other beneficial effects, including lifespan extension, improved stress resistance, and enhanced locomotory and mitochondria activity in older age classes of Brachionus manjavacas rotifers [69].

It has been known for a long time that glycerin may cause headaches, dizziness, nausea, vomiting, and excessive thirst in some people when ingested directly and in large amounts [106]. The effect of glycerin on kidneys, e.g., a reversible decline in renal function, has also been recognized for some time [70]. Since glycerol is incorporated into the Embden–Meyerhof pathway, serum glucose concentration may be increased to cause hyperglycemia. Additional adverse reactions of glycerin in people who receive intravenous injection of glycerol include intravascular hemolysis, hemoglobinuria, renal damage, hyperglycemia, and hyperosmolality. Furthermore, rare cases of allergic reactions to glycerol have been reported [107].

5. The Combination of Lemon and Glycerin and Its Relationship with the Gut–Brain Axis

Lemons are a popular fruit that people use in small quantities to add flavor to baked goods, sauces, salad dressings, marinades, drinks, and desserts, but are rarely consumed alone due to their intensely sour taste. Glycerin ingestion has been associated with some side effects, including a transient hyperglycemic effect [108]. However, there are reasons to believe that the combination of glycerin with lemon extracts could mitigate the potential side effects elicited by glycerin consumption while gaining advantage of their mutual benefits (Figure 2). As discussed in previous sections, prior research about lemon and glycerin shows improved heart health, enhanced weight loss, increased absorption of iron, and boosted immune responses and, furthermore, they can modulate the gut–brain axis [109,110,111,112,113]. In studies of male NOD mice that were dosed daily with the combination of lemon and glycerin, it was found that mice treated with the combination had a better glucose control (Figure 2). Thus, it is hypothesized that the combination of glycerin with lemon extracts may synergistically promote neurological well-being, locally in the brain, through increased energy and metabolites, and distally, through an improved gut microbiome and gut–brain axis. To support this hypothesis, apparent health advantages in using lemon extracts and glycerin in combination have been confirmed in animal studies [14]. A consistent finding was an increased glucose tolerance and/or a decreased insulin resistance following the combined treatment (Figure 2). Impaired glucose tolerance and increased insulin resistance are common features of neurodegenerative diseases and several neurological disorders, including diabetic neuropathy, Alzheimer’s disease, Myotonic dystrophy, Crow–Fukase syndrome, Wolfram syndrome, Friedreich ataxia, and Spinal muscular atrophy of the Kennedy–Alter–Sung [1,114,115].

Glycerin treatment reduced the level of leucine; however, such effect was not observed following the combined treatment of lemon extract and glycerin [14]. This is important as the GMB-mediated branched-chain amino acid metabolism is responsible for metabolic syndromes [116], and disturbance of leucine metabolism leads to the development of metabolic diseases associated with neuropathological symptoms. The branched-chain amino acids, particularly leucine that is obtained from the diet, play an important role in regulating glutamate metabolism by serving as important donors of amino groups for the purpose of glutamate synthesis in the brain [117]. Leucine is the most effective amino acid to activate the mTOR (mammalian target of rapamycin) pathway, and mTOR-controlled signaling pathways regulate many physiological functions of the nervous system, including neuronal development, synaptic plasticity, memory storage, and cognition [117]. Leucine also prevented lipopolysaccharide-induced depression-like behavior in mice [118], and insufficient leucine levels exacerbate maternal separation-induced cognitive impairment in rats [119]. Additionally, when leucine is ingested with glucose, it stimulates insulin secretion and lowers blood glucose levels [120]. This is consistent with the increased glucose tolerance and/or a decreased insulin resistance following the combined treatment of lemon and glycerin in NOD mice [14]. Leucine may also help with weight loss in several ways, e.g., by regulating appetite [121].

Acetate is the main SCFA distributed throughout the body, and it was significantly increased in fecal samples following the combined treatment in NOD mice [14]. This finding could have important implications in some populations such as patients with Parkinson’s disease, who were reported to have significantly lower concentrations of fecal acetate [122]. In overweight and obese mice, acetate has been shown to increase fat oxidation when produced in the distal gut and stimulate secretion of appetite-suppressive neuropeptides [123]. As discussed in Section 4.2, acetate from the gut is an essential bacteria-derived molecule driving metabolic pathways and functions of microglia during health and perturbation [124]. Acetate ameliorated surgery-induced cognitive deficits of aged mice and inhibited the activation of IBA-1, a marker of microglial activity. Acetate also reduced expression of inflammatory proteins (tumor necrosis factor-α, interleukin-1β, and interleukin-6), oxidative stress factors (NADPH oxidase 2, inducible nitric oxide synthase, and reactive oxygen species), and signaling molecules (nuclear factor kappa B and mitogen-activated protein kinase) in the hippocampus [125]. Acetate can be produced by most of the enteric bacteria, such as Prevotella spp., which were increased following the combined treatment for one month in male NOD mice [14]. It has been observed that the fecal microbiota transplant of healthy human microbiota increased abundances of Bifidobacteria and Prevotella, and improved the behavioral symptoms in autistic patients [126,127]. Furthermore, Prevotella copri transplantation promoted neurorehabilitation in a mouse model of traumatic brain injury [128].

Pseudomonadota (synonym Proteobacteria) is the most diverse phylum of Gram-negative bacteria, comprising several known human pathogens. A core aspect of Proteobacteria-related diseases is inflammation [129]. It has been reported that children with autism spectrum disorder have neuroinflammation and higher levels of Proteobacteria in their gut microbiota than healthy controls [130]. Proteobacteria was significantly decreased in mice treated with the combination of lemon and glycerin for six months [14]. Some species of Cyanobacteria, also known as blue-green algae, may be present in small numbers in the human GI tract and produce neurotoxins that could be linked to neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis [131,132]. Cyanobacteria were significantly decreased following treatment with either the lemon or the combination of lemon extract and glycerin for six months when compared to the vehicle control in NOD mice [14]. In a prospective investigation into the association between the gut microbiome composition and cognitive performance among healthy young adults, it was found that the “Ruminococcaceae- and Coriobacteriaceae-dominant community” was positively associated with the fluid intelligence score, which measures the ability to think abstractly, reason quickly, and problem solve independently of any previously acquired knowledge [133]. Interestingly, Ruminococcus spp. and Coriobacteriaceae families were increased following the combined treatment in male NOD mice for one and six months, respectively [14]. Coriobacteriaceae perform significant tasks in the gut, including metabolizing bile salts and steroids and activating dietary polyphenols, and they are lower in abundance among patients with Parkinson’s disease when compared with the healthy controls [134].

Therapeutic strategies for countering neurodegenerative disorders of aging include improving, preserving, or rescuing brain energetics [1]. The brain requires a continuous supply of ATP, and the energy is mostly produced from glucose by oxidative phosphorylation in mitochondria, complemented by aerobic glycolysis in the cytoplasm. Glycolysis is the pathway by which glucose degrades into lactate. When glucose levels are limited, ketone bodies generated in the liver and lactate derived from exercising skeletal muscle can also become important energy substrates for the brain. The glycolysis pathway was predicted to decrease following treatment with either lemon or glycerin in the 6-month adult male NOD study, and this negative effect on energy metabolism was no longer present following the combined treatment for 6 months, although not for 1 month [14]. Taken together, there are possible advantages in using lemon extracts and glycerin in combination in terms of preserving brain energetics, improving the gut–brain axis, decreasing insulin resistance or increasing glucose tolerance, and decreasing body weight.

6. Conclusion and Future Directions

In conclusion, the intake of dietary supplements (lemon, glycerin, and their combination, etc.) can exert beneficial effects on the host in various areas, such as neurological health, glucose homeostasis, and energetics, via many routes such as the gut microbiota and metabolites. While the dietary supplements in studies may be able to have a direct effect on their targets, such as glycerin acting as a fuel for GABAergic neurons when injected intracerebrally [135], they often exert more noteworthy effects via the gut microbiota as a prebiotic, by selecting for commensal bacteria, or by acting as the source materials for beneficial bacteria to metabolize them into metabolites such as SCFAs. Therefore, prebiotics should be mindfully incorporated according to the desired outcome, with the understanding that this may impact multiple functions that would affect one’s health.

The globally rising prevalence of neurological diseases as well as obesity and diabetes pandemics and their close association with GMB suggest a need to explore the modulation of the gut–brain axis as a potential treatment and preventive measure. These studies should be carried out with a global perspective encompassing high-income countries and low- and middle-income countries. Although many studies have been conducted on the components of lemon and glycerin and how they affect the organism, the full spectrum of exact mechanisms remains elusive. Much could be inferred but must be further verified. For example, the flavonoid nobiletin reduces Aβ pathology, but research has yet to determine the pathways for this function. Further research is also needed to study the effects of lemon fermented products on lipid metabolism. Based on the interactions of lemon extracts with the body, it has been suggested that lemon extracts decrease the activity of angiotensin-converting enzyme, and this must be further investigated. Additional research is also needed to understand the effects of serum fatty acids on diseases and how glycerin impacts these.

Very little research has been conducted on the combined effects of lemon extracts/components and glycerin. The previously discussed interactions between the combined treatment of lemon extracts and glycerin and the gut–brain axis should be further investigated for their application in various noncommunicable neurological disorders. These include neurodegenerative and neuromuscular diseases such as Alzheimer’s and Parkinson’s disease. The mechanisms and pathways of interactions between the ENS, CNS, and GMB are logical targets for investigation with this dietary supplement in more in vivo studies and human trials. More studies are needed to identify the “good” bacteria and “bad” pathogens in GMB that initiate and/or exacerbate these diseases. Its components, e.g., lemon, have already provided some insights on the gut–brain axis and have functioned as a noninvasive and inexpensive manner to control blood glucose levels. Further understanding of the interactions between these networks could be key in slowing neurodegenerative diseases, reducing body weight and appetite, as well as in preventing diabetes.