A review of salivary composition changes induced by fasting and its impact on health

Nurul Fahilah Kamalul Aripin,N.Iayu Zahi,Moh Aizat Aul Rahim,Hashim Yaao,Parvz I.Haris,Zuaiah Haji A.Rahim*,Rauzah Hashim,*

a School of Chemical Engineering,College of Engineering,Universiti Teknologi MARA,Shah Alam 40450,Malaysia

b Centre for Fundamental and Frontier Sciences in Nanostructure Self-Assembly,Department of Chemistry,Faculty of Science,Universiti Malaya,Kuala Lumpur 50603,Malaysia

c Department of Oral and Craniofacial Sciences,Faculty of Dentistry,Universiti Malaya,Kuala Lumpur 50603,Malaysia

d UMSC Dental Specialist Clinic,UM Dentistry Tower,Universiti Malaya,Medical Centre,Kuala Lumpur 50603,Malaysia

e Leicester School of Allied Health Sciences,De Montfort University,Leicester LE1 9BH,United Kingdom

f Chancellery (Research and Innovation),Universiti Malaya,Kuala Lumpur 50603,Malaysia

Keywords:Saliva Fasting Nitrate Nitrite Nitric oxide

ABSTRACT Human saliva is an indispensable fluid that maintains a healthy oral cavity which otherwise can lead to oral diseases (dental caries and periodontitis).In addition,salivary metabolites and microbiome profile provide early detection of systemic diseases such as cancer and obesity.Salivary diagnostic has gained popularity due to its non-invasive sampling technique.Fasting (abstinence from food or drink or both) research for weight loss and improve health is common,but studies using fasting saliva are scarce.Some metabolites in fasting saliva have been reported with interesting results,which can be enhanced by considering different confounding factors.For example,fasting saliva contains higher salivary nitrite,which is related to nitric oxide(NO).NO is a vasodilator supporting the healthy function of endothelial cells and its deficiency is connected to many diseases.The timely supply of NO through exogenous and endogenous means is highlighted and the potential advantage of fasting salivary composition changes in relation to COVID-19 infection is speculated.This review aims to provide a general discussion on the salivary composition,properties,and functions of the whole saliva,including the health benefits of fasting.

1. Introduction

Since antiquity saliva has been used as a therapeutic medicine to cure diseases,from blindness,epileptic,fever,and pain,to cuts and bruises,and even to ward off evil spirits[1].Saliva was often mixed with other substances,such as mud or leaves (e.g.,Piperbetelleaves)[2],and applied by those with divine status,spiritualists,healers,or elders.Over 2 000 years ago,saliva was considered the spiritual fluid related to mind-brain function as documented in Huangdi Neijing,a canon of traditional Chinese medicine from the book of Han[3,4].In the Muslim tradition,saliva is used together with reciting verses from Al-Qur’an when treating illness[5].

About 1 000 years ago,the father of modern medicine,Avicenna (or Ibn Sina) demonstrated saliva to be the agent of contagious diseases like rabies and later tuberculosis[6].Perhaps this event reduced the reputation of saliva as medicine.As evidencebased medicine gains acceptance and the belief in mystical power diminishes,the therapeutic use of saliva is becoming less significant.However,it is still commonly practised in remote villages and within indigenous communities.In modern culture,saliva is considered unhygienic.Spitting (throwing out saliva excrement) habit is a bad manner and offensive.However,in the animal kingdom,for example,in cats,saliva recrement is used for cleansing and therapy,while poisonous snakes spit venomous saliva for defence.Thus,saliva is anthropologically and medicinally ambivalence.

Habitual spitting has been common in many cultures and in recent times,especially with respect to the COVID-19 pandemic,there has been greater effort being made in certain countries to discourage people from spitting in public areas including the introduction of fines as in India[7,8].Spitting has been used as a sign of aggression against others and during the COVID-19 pandemic.For example,in the United Kingdom,there has been incidences where spitting has been used as a weapon to target police officers[9].

Scientific research on saliva began as early as 1898 from the work of Chettenden and Menden on how alcohols influence saliva secretion[10].However,serious research on saliva was reported as early as the 1950s,focusing on the whole saliva,emphasising its potential antimicrobial activity and its role in microbial adhesion,mineralisation,taste,and lubrication[11].Later,saliva from the major glands was investigated individually for the isolation and biochemical characterisation of salivary components.

These days,saliva research is more complex,for example,the functional domains of salivary proteins have been mapped[12].There are many excellent books[11,13-16]and reviews[17-19]on saliva science describing its physiology,functions and compositions.From these studies,methods in salivary diagnostic have been developed to monitor oral diseases[17-20].Emerging biotechnologies (transcriptomic,proteomic and metabolomic) allow for an extensive range of salivabased diagnostics to reflect the state of human health.This allows salivary molecular analytes to determine the disease progression in cancer[21].Saliva analysis is playing an important role in public health research.For example,a recent analysis of saliva from electronic cigarette users has revealed that the salivary microbiome is altered and may increase periodontal risks[22].

Fig.1a (green bars) shows the growth in the scientific literature related to publications where the word saliva appears (last 22 years).As can be seen,there is a progressive increase in the number of publications that contain the word saliva with a marked increase in publications since 2019.The recent increase could partly be due to the COVID-19 related research with 888 publications where the word saliva appears along with COVID-19.Fig.1b1(green bars),shows the research areas related to these publications (top 15 subject areas).Dentistry and oral surgery have,by far,the largest number of publications followed by immunology,biochemistry,pharmacology,chemistry and microbiology.From Fig.1c1(green bars),most of the articles appear in journals related to dentistry and oral diseases.These figures also contain data from fasting saliva Fig.1b2and Fig.1c2(blue bars),which will be commented on later.

The increase in research using saliva is not surprising since the collection of saliva is non-invasive and easy,making it a potential biological fluid as a substitute for other body fluids like serum in clinical diagnosis[19,20].However,it is worth noting that research using saliva is lagging compared to those of urine and blood,as evidenced from the WoS published articles from 1970 to 2022 where there are 227 378 (urine),2 756 267 (blood),and only 54 529 (saliva).

To the authors’ best knowledge,the first study to show the potential of saliva for investigating the impact of fasting on salivary metabolites was reported by Rahim and Yaacob in 1991[23].Nearly two decades later,another group reported inflammatory parameters,physical performance,and metabolic changes associated with Ramadan fasting[24].There are only 11 articles in WoS between 1970 to 2022 containing the words “saliva”,“fasting” and “Ramadan”.When the search is conducted for “intermittent fasting” and“saliva”,only 2 results are found for the same period.This clearly demonstrates the lack of research on the use of saliva as a biofluid in fasting research.Interestingly,Rahim and Yaacob reported that one of the salivary components,nitrite (),increases significantly in fasting saliva[23].Sinceand nitrate ()[25,26]can be a source of nitric oxide (NO),a proven vasodilator agent[27],this may suggest the possible therapeutic value of saliva in fasting conditions (i.e.abstinence from food or drink or both).

Recently,a disposable electrode was developed for monitoring the salivary electrical conductivity.This is a portable sensing device to measure the dehydration level,in the diagnosis related to chronic kidney disease[28].In view of the growing use of saliva for diagnosis,especially for COVID-19 detection[27,29],we shall review the saliva science (including production,selected components,and major functions) for a general readership.

2. Production of saliva

2.1 Salivary glands: location and histological structure

Major salivary glands (parotid,submandibular and sublingual)open into the oral cavity via their respective ducts (Stensen’s,Wharton’s,and Bartholin) (Fig.2a) and secrete different types of secretion (parotid–mainly serous,submandibular–mixed serous and mucous and sublingual–mainly mucous).Minor glands (labial,buccal,glossopalatine,palatine and lingual (von Ebner’s gland)),their secretions are mainly mucous except for von Ebner’s.The percentage of the volume of secretion of the respective glands is shown in Fig.2a.

Fig.2 Salivary glands,(a) location,(b) duct structures,and (c) histology of part of the acinus.The pie chart in (a) represents the percentage of secretion volumes of the respective salivary gland.

The salivary glands are highly vascularised and innervated.Hence,biomolecules and ions in the blood circulation can infiltrate acini and ultimately secrete into the saliva[29]and the innervation assists in the control of the secretion.The salivary secretions play a crucial physiological role that includes mastication,lubrication,speech articulation,and cleansing[29-31].

2.2 Histology

The salivary glands are divided into lobules by connective tissue septa[31].Within each lobule,there are numerous secretory end pieces or acini.Each acinus is made up of acinar cells and it is located at the terminal part of the gland linked to the ductal system (Fig.2b).

The acinus is arranged concentrically around a central lumen that is in continuity with the proximal end of an intercalated duct.These ducts shape the lumina in the duct system and produce striated ducts which subsequently drain the saliva into the excretory ducts.The diameter of the tubules increases through intercalated,striated,and excretory ducts in the ductal system[31].

The acini can be categorised as serous or mucous or a mixture of serous and mucous with different characteristics of epithelial cells and secretory products[31-33].Serous acini are enclosed by a unique basement membrane that contains pear-shaped groups of epithelial cells while mucous acini have an uneven pattern and bigger size than the serous type.Mixed acini,usually found in the submandibular gland,are defined by the clustered mucous cells adjacent to the intercalated duct and are surrounded by a crescent-shaped arrangement of serous cells,called serous demilunes.This mixed type of acinar cells produces both mucous and serous secretions.A single layer of myoepithelial cells surrounds every acinus whose function is to contract,to expel saliva into the lumen during the secretion of this fluid (Fig.2c).

2.3 Mechanism of saliva production and control

The formation of saliva involves two-stages (1 and 2).In stage 1,the acinar cells produce the primary acinar fluid containing ions and the salivary proteins.In stage 2,the duct cells especially the striated ducts modify the ionic composition and protein secretion of the primary acinar fluid as they are secreted into the oral cavity.The primary acinar fluid is hypertonic or isotonic,whereas saliva in the oral cavity is hypotonic.Fig.3 shows the formation of primary acinar fluid.

Fig.3 Formation of primary acinar fluid.The labels numbered 1,2,3,and 4 are locations of ions transporters.Label 5 refers to the route for sodium ions directly into the lumen.Adapted from[34].

The autonomic nervous system (ANS) which is stimulated by smelling,tasting,and thinking of food and its aroma and food presence in the alimentary canal controls the salivary gland.The stimulation of the ANS nerve impulse (either parasympathetic or sympathetic) regulates both the volume and type of saliva (serous,mucous,and mixed) secreted.The parasympathetic nervous system,when stimulated,increases the salivary flow rates and enzymes.The sympathetic nervous system is mainly responsible for the secretion of proteins via exocytosis in acinar cells.Its stimulation will lead to a production of predominantly thicker mucous saliva,mainly contributed by the sublingual and partly the submandibular gland[14,35].

2.4 Types of saliva

Saliva can be categorised as a whole,individual gland,stimulated and unstimulated (resting).The whole (also called mixed) saliva also contains microorganisms and their products,shed oral epithelial cells,food particles,serum components,and inflammatory cells from the gingival crevice[19,36].The individual gland,for example,the parotids produce watery saliva loaded with enzymes,while the submandibular glands also contain the gelling material mucin,while the sublingual and minor glands secrete mucin rich viscous saliva with no enzymes.Unstimulated or resting whole saliva refers to the saliva produced in the absence of external factors[17,37].Different types of saliva possess unique salivary compositions that may have consequences on oral health both through their non-specific physico-chemical properties and specific effects[19,38].

2.5 Salivary flow rate

Many factors (direct stimulation of taste,olfactory receptor,and oral stimulation) controlling the salivary flow rate make its measurement difficult to quantify[41].The salivary flow rate during sleep is nearly zero.An average unstimulated salivary flow rate of 0.12–0.16 mL/min[42],while that of a hypofunction is less than 50% of this value[43].The average flow rate of UWS for 16 hours is 0.3 mL/min[44].Stimulated saliva contributed 80%–90% of saliva in volume and its flow rate is greater than 0.2 (minimum),0.7 (hypotonic),and 3(maximum) mL/min[44].During fasting the flow rate is low in the range of 0.08–0.1 mL/min[23,39,40](Table 1).

Table 1 The salivary flow rate in different conditions.

3. Saliva: compositions and functions

Whole saliva contains 99.5% of water and 0.5% of inorganic and organic components[11].The normal pH of UWS is between 5.7–6.2,while the SWS is as high as 8[13].Saliva contains inorganic components that include sodium,potassium,calcium,magnesium,chloride,fluoride,nitrite bicarbonate,thiocyanate,and phosphates,while the organic components include proteins,immunoglobulins,enzymes,mucins,and nitrogenous products,such as nitrite,nitrate,urea,and ammonia[13,19,23,45].Table 2 lists the concentrations of salivary components (UWS and SWS) taken from healthy subjects and whenever available,those of fasting saliva are included.These values have large errors reflecting the factors affecting these measurements.Thus,there is an urgent need for the standardisation of a) procedures for collection with respect to time of day and day of the week,b)storage and c) analysis of saliva samples.

Table 2 The concentration of salivary components (inorganic and organic) of UWS and SWS for non-fasting.For fasting conditions only UWS is considered.These values have been rounded to one or two significant figures,while the units remained as in the original references.

Each saliva component may serve a certain function or multifunction,or overlapping functions,while others,for example,salivary proteins and mucins can be ambivalent acting both for and against the host[46].The common functions of saliva are buffering,ion reservoir,cleansing/clearance,digestive,taste,lubrication,water balance,speech,healing,agglutination,and acquired pellicle formation.Bicarbonate,phosphates,and proteins are the buffering components of saliva.The ion reservoir in saliva,especially the calcium and phosphate components,provides an anti-solubility factor and modulates demineralisation and remineralisation[13].

When food substances are taken orally,the hypotonic saliva flowing in the oral cavity a) dissolves the food,enabling taste receptors in the gustatory buds to perceive tastes (bitter,salty,sweet,umami and sour)[19,31],and b) the mucins together with parotid proline-rich glycoprotein albumin complex provide coating andlubrication to form food bolus facilitating swallowing.Some of the salivary enzymes assist in the digestion of food and the removal of bacteria by agglutination[47,48].Saliva also has many protective functions including the antimicrobial action[17,19,49],and the following discussion selects a few functions that are related to diagnostic and therapeutic values.

3.1 The role of mucins

The slimy,stringy,and stretchy properties of saliva are attributed to its viscosity (the state of being viscous) and spinnbarkeit (the ability to draw a thread)[66].Both rheological properties are distinct and have a complex correlation.Two salivary mucins responsible for these properties are MUC5B (mw～1 MDa) and MUC7(mw～200–250 kDa) which contribute to the viscosity and the spinnbarkeit phenomenon,respectively.The salivary mucins are secreted from the minor salivary glands contributing 26% of the total protein content[67].Mucins are glycoproteins characterised by their dense coat of glycans,especially theO-glycan family.Mucins,the major component of mucus[68],form a gel and an extended rodlike structure transmembrane (Fig.4) and its extensional viscosity was measured recently using the flow-induced birefringence[69].Adding purified mucins to artificial saliva will increase its viscosity thus assists in alleviating the dry-mouth symptoms in patients[70].Therefore,detailed knowledge of saliva rheology may lead to the prevention and treatment of these oral diseases.

Fig.4 Schematic structure of salivary mucin: (a) the entire mucin domain (b) mucin monomer,(c) close-up of gel-forming mucins.Adapted from[64,65].

3.2 Antimicrobial properties of saliva

The oral cavity is one of the most heavily colonised parts of our body due to its warm,nutrient-rich,and moist environment which promotes the growth of diverse microflora.Saliva is an important biological fluid for its protective role against pathogens in the oral cavity.For example,immunoglobulin A (IgA),immunoglobulin G (IgG),and immunoglobulin M (IgM),play a crucial role in the immune function of mucous membranes[16].When two monomers of IgA form a dimer being linked by a secretory piece,it is known as secretory IgA (sIgA) (Fig.5a),which gives protection to the antibodies from being degraded in the oral fluids[13].Quantity-wise,the sIgA is higher compared to those of IgG and IgM and may have a role in combating cariogenic bacteria.

Fig.5 Antimicrobial activities of salivary components: (a) sIgA,(b) lysozyme,(c) lactoperoxidase and (d) lactoferrin.(e) The normal human salivary microbiome distribution,shown in the word cloud is taken from[72].Here,the major components are Streptococcus,Prevotella,Veillonella,Neisseria,and Haemophilus.A deviation from this normal distribution may be linked to certain diseases,including cancer,diabetes,and obesity.

Saliva contains a large variety of antimicrobial proteins,peptides,and enzymes.The major ones include lysozyme,lactoperoxidase,and lactoferrin which directly and indirectly inhibit the uncontrolled growth of bacteria.

Salivary lysozyme inhibits gram-positive bacterial growth by hydrolysing theβ-1,4-glycosidic bonds betweenN-acetylmuramic acid andN-acetyl-D-glucosamine in the polysaccharide layer of the bacterial cell wall (Fig.5b).Lysozyme is capable of aggregating bacteria like streptococci,hence promoting its clearance[19,47].

Salivary lactoperoxidase is produced by the parotid and submandibular salivary glands[71].In the oral cavity,the peroxidase catalyses the oxidation of thiocyanate (SCN?) by hydrogen peroxide(originates from bacterial metabolism) to produce hypothiocyanite(OSCN?) and hypothiocyanous acid (HOSCN) (Fig.5c).These by-products,in turn,affect bacterial metabolism (especially acid production) by oxidising the sulfhydryl groups of the enzymes involved in glycolysis and sugar transport[13,49].

Salivary lactoferrin is secreted by the serous acinar cells of major and minor salivary glands[47].It is a glycoprotein which is an ironbinding protein.It exerts its antimicrobial activity against bacteria with an iron requirement by depriving the bacteria of available iron in the surroundings and consequently inhibits their metabolic processes[49](Fig.5d).It also possesses another antimicrobial activity not related to its iron-binding ability.In this case,it binds to the lipopolysaccharide of the bacterial cell wall which results in the lysis of bacteria cells.

3.3 Salivary microbiota

Salivary microbiota refers to all the microorganisms (bacteria,viruses,and fungi) found in the oral cavity.Sometimes microbiota is used interchangeably with microbiome which is defined as a characteristic microbial community occupying a reasonably welldefined habitat having distinct physio-chemical properties[73,74].There are 700 different kinds of microorganisms existing in the human oral cavity[75].The salivary microbiome of different groups of people has been reported[72].A healthy individual has a general microbiota profile,which changes in an unhealthy individual.Compared to a healthy individual,the diseased individual has microbiota that contains some other “specialist” microorganisms with different metabolic functions and an increased virulence potential[76].Salivary microbiota is associated with a variety of oral diseases.Recent evidence also shows that the oral microbiota is closely related to human systemic diseases,such as diabetes,obesity,and cancer[77].For example,the salivary microbiota profiles of an obese person versus a healthy individual are different,i.e.,the periodontium of healthy people has less bacterial diversity and oral microbiota.Moreover,the oral microbiota of obese people has morePlasmodium,S.genus,andS.mutans,but the abundance ofHaemophilus,Corynebacterium,carbonophilic phage,andStaphylococcusare significantly decreased (Fig.5e).

4. Fasting saliva

In the modern health-conscious society,fasting is becoming fashionable to many people[78],even though fasting have been practised for religious reasons over aeon[79].While intermittent fasting has been shown to improve health,long-term fasting (continuous fasting over several days) can be harmful to health[80,81].Table 3 briefly describes the different types of fasting regimes for health reasons including some examples of fasting for religious reasons.

From the WoS data collected over 22 years,there is an increasing number of publications where the word saliva appears with fasting (Fig.1a,blue bars).This sudden increment in recent years,is understandably resulting from research related to saliva analysis during the COVID-19 pandemic[99].This data also shows the top 15 research areas where fasting and saliva appear together(Fig.1b2,blue bars).The highest number of publications is in chemistry followed by pharmacology,biochemistry,and dentistry.This could be explained by the fact that the fasting saliva composition analysis requires chemistry-based methods.Indeed,most of the publications are in journals related to chemistry and sensors (Fig.1c).

We shall now comment on the effect of fasting on the production of some saliva components as given in Table 2.There are reports on saliva composition under fasting conditions[23,40,50,53,60,88,100,101],but the data reported are relatively scarce and incomplete.Moreover,the fasting regimes differ from author to author.The commonly reported regimes are fasting without food and water for a given period[23,50,53,101],intermittent fasting[88],and fasting during the month of Ramadhan,i.e.RF[40,60,100].

In 1991,Rahim and Yaacob[23]reported on the content of saliva from fasting conditions.They analysed the concentrations of calcium,phosphate,protein,and nitrite in UWS collected at least after 6 h without food and water with those under standard control conditions.They found fasting saliva has a lower flow rate of 0.098 mL/min compared to that of the control by half;relatively unchanged concentrations of calcium and phosphate compared to the respective control,while the protein concentration was only slightly decreased.The concentration of nitrite under fasting conditions was 50%higher than that in control saliva (P<0.05)[23]. The significant increase in nitrite content could be due to the low salivary flow rate during fasting restricting time for bacterial activity to convert nitrate to nitrite.This finding is considered important since nitrite is a precursor of NO and will be discussed further in the nitrate/nitrite cycle (Section 5).

Research related to intermittent fasting is more directed toward weight loss rather than any linked to salivary function.Intermittent fasting includes alternate day fasting,fasting over a time period,and restricted daily scheduled feeding (Table 3).The health benefits of intermittent fasting and physiological mechanisms have been reported[102].However,cross-sectional studies on eating patterns associated with health outcomes and experiments on sample size are limited[102].The major physiological mechanisms such as,circadian biology,the gut microbiome,and modifiable lifestyle behaviours,such as diet,activity,and sleep are related to fasting regimens with human health[102].Thus,the scarcity of literature on intermittent fasting related to saliva provides ample scopes and opportunities for future research exploration (Section 6).

Ramadan fasting is a religious duty practised by the Muslims where they refrain themselves from eating and drinking between dawn and sunset according to the geographic location[103](Table 3).For many years,this religious fasting has been reported to provide many positive effects on human physiology and pathology including increased high-density lipoprotein cholesterol (HDL)[104]and reduced cortisol,body mass index,blood pressure,and triacylglycerols[105-108].Table 2 gives the salivary compositions of selected studies under Ramadan fasting (RF) conditions by several authors[40,60,100].

Other time restricted fasting studies such as by Illahi et al.[50]found that salivary pH increases,while Selviani et al.[53]found inorganic components like potassium and magnesium decrease.There were no significant changes in salivary sodium[53].Some of these results agree (within the error) with those of TRF reported by Rahim and Yaacob[23].The content of uric acid and aspartate aminotransferase(AST) decreases while alkaline phosphatase (ALP) increases and no significant change in sIgA[100].The decrease in salivary uric acid production concurs with the anti-inflammatory effect of the fasting regimen and one-year vegetarian diet in rheumatoid arthritis[109].Another study suggested that Ramadan fasting may strengthen cellular immunity despite decreasing humoral immunity,hence encouraging further research on serum IgG and IgM and salivary IgA concentrations to confirm these outcomes[60].

There is a lack of studies investigating the impact of fasting on the salivary microbiome. However,it was reported that overnight fasting did not induce major changes in the salivary microbiome[110].The study explored nocturnal fasting,and the findings may not be relevant to diurnal fasting,such as Ramadan fasting,due to impacts of the circadian rhythm and differences in mealtimes.Although the salivary microbiome has not been studied specifically during fasting,the impact of fasting (during Ramadan) has been investigated on the gut microbiome[111-113].In these studies,the gut microbiome was reported to alter during Ramadan fasting[111-113].It was suggested that upregulation of butyric acid-producing Lachnospiraceae may provide a mechanistic explanation for the health benefits of intermittent fasting[113].In another study (but not on Ramadan fasting),changes in the gut microbiome led to reduction in blood pressure and body weight in metabolic syndrome patients[114].A recent review article reported that alternate-day fasting,and TRF has positive impacts on intestinal microbiota which can bring health benefits[115].

5. Salivary ()/ ()/ NO and COVID-19

As discussed in the previous section,salivary antimicrobial constituents have many therapeutic values and may be used for diagnostic purposes for some illnesses.At one time,andwere thought to be the end-products of endogenous NO metabolism and have a negative effect on human health since these may be carcinogenic[117];but many studies later prove this to be inconclusive[118]. Moreover,the inert nitrate () and nitrite() are recycledin-vivoand shown to be an alternative source of NO apart from theL-arginine-NO-synthase pathway.

This section discusses the emerging importance and biological functions of the nitrate-nitrite-NO pathway,highlighting some therapeutic potentials and suggesting its clinical diagnostic applications,especially in relation to COVID-19[119].

5.1 Nitrate/nitrite cycle

In the human body,NO is generated within the nitrate/nitrite cycle as represented in Fig.6[119,120].The cycle involves the nitrate and nitrite sources exogenously derived from many vegetables such as celery,cress,lettuce,beetroot,and spinach.The dietarycan also be converted toby bacterial enzymes (nitrate reductase) in the oral cavity[121].This nitrite then enters the stomach whose environment is acidic (pH 1-3),where it is converted into NO via reduction by protonation[122].Any remainingandwill be absorbed by the intestine while the excess is excreted via the kidney.Some of the absorbedandenter the circulatory system and tissues[25,26,123]where under hypoxic conditions (usually during and after strenuous exercise) they get converted to NO.Furthermore,research performed over the past decade indicated that under physiological conditions,is recycled in blood and tissues to form NO and other bioactive nitrogen oxides[116,124,125].Thus,is now recognised as a reservoir of NO-related bioactivity that will be switched on when the enzymatic NO production catalysed by nitric oxide synthase is inefficient.The in the blood can also be taken up by the salivary glands and begins the whole cycle of NO regeneration (Fig.6).During physiological or pathological hypoxia,oxygen is lacking in the body,leading to the reduction of nitrite to NO.Consequently,this contributes to the physiological hypoxic signalling,vasodilation,modulation of cellular respiration,and the cellular response to ischaemic stress.

Fig.6 The cycle of dietary nitrate/nitrite in the production of NO[116].

5.2 Nitric oxide (NO) therapeutic

NO gas is a vasodilator agent which maintains the healthy function of endothelial cells that line the blood vessels.NO ensures these vessels dilate effectively to allow blood flow[26].NO has been used in the treatment of acute respiratory distress syndrome(ARDS)[126].The scientific and medical community has acknowledged NO as one of the most important molecules produced within the body.In 1998,the Nobel Prize in Physiology or Medicine was awarded to the discoverers of NO (Drs Louis J Ignarro,Robert Furchgott and Ferid Murad) for “Thesignaltransmissionbyagasthatisproduced byonecell,penetratesthroughmembranesandregulatesthefunction ofanothercell,representsanentirelynewprincipleforsignallingin biologicalsystems.”

Moreover,NO also acts as hypoxic signalling,host defence,cryoprotection and energetic mitochondrial respiration.Deficient in NO leads to endothelial dysfunction with a host of health problems from high blood pressure,pulmonary hypertension,stomach ulcer,stroke,myocardial infarction,liver and lung transplantation,sickle cell disease and subarachnoid artery aneurysm haemorrhage[119](Fig.7).Conventionally NO therapy is related to a timely provision increase in NO bioavailability.There are a few US FDA-approved products related to NO production such as nitro-glycerine in the treatment of acute angina[127],and inhaling NO for pulmonary hypertension in infants[128].Improved knowledge of the physiology and pharmacology of NO,may lead to better drugs for many contemporary diseases and medical problems[129].

Fig.7 Lifestyle change to improve NO bioavailability: (a) and from balance lifestyle,(b) mechanisms of NO production,(c) roles of NO,(d) deficiency of NO leading to (e) endothelial dysfunction related diseases.Adapted from [119].

5.3 Nitric oxide (NO) relationship to COVID-19 therapeutic and diagnostic: a perspective

Since the end of 2019,the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has plagued the world.It basically infects the respiratory tract and patients rapidly develop acute respiratory distress syndrome (ARDS)[130].This is followed by systemic viral spread and cytokine storm which damage severely the endothelial cells lining the blood vessels of the major organs such as the intestine,kidney,heart,and brain[131].In healthy endothelial cells,NO is naturally released and this regulates vascular tone and maintains its integrity by inhibiting platelet aggregation and coagulation[132].In severe COVID-19 infection (especially to patients with pre-existing endothelial dysfunction conditions,due to maybe aging,diabetes,hypertension,and obesity),persistent injury to endothelial cells decreases NO production,causes vascular integrity disruption,and increases systemic thromboembolic complications[133].SARS-CoV-2 mortality amongst male is higher than female since the former has less estrogen-mediated vascular endothelial protection[134].Thus,endothelial dysfunction is the common risk factor for the underlying pathology in COVID-19 patients[135].The healthy condition of the endothelial cells can be ensured by increasing the bioavailability of NO that may overcome the disease.

Fig.7 illustrates strategies to improve NO bioavailability through changing one’s lifestyle.This involves (a) regular exercise which can create the hypoxic condition favourable to NO production (b)consuming diet rich in nitrate and nitrite sources[136],and (c) fasting,which increases the level of salivary nitrite[23].Thus,a long-term preventive measure to decrease the severity of virulent effect of COVID-19 virus is possible with a better public health education.The public should be made aware of the role of NO as a vasodilator in combating the effect of COVID-19 infection.

There are many excellent reviews on NO therapeutics for COVID-19[129,137-139],hinging on the role of NO as a vasodilator to maintain normal vascular function and slow down the progression of inflammation and reduce endothelial function.The NO supplementation prevents cytokine storm,restores oxygen delivery,waste removal,and protects oxygen-sensitive organs like kidneys[139,140].

Section5.1 deliberated in detail the plausible conversion of the salivary nitrate and nitrite to NO.The,,and NO,(collectively referred to as NOx),play an important role in the cardiovascular and immune systems[141].It has been suggested that NOxmay serve as a predictor to track the health status of recovered COVID-19 patients.In fact,it is important to elucidate NO levels in post COVID-19 infected patients[141,142].Although those studies were conducted using serum,a similar investigation could be conducted using saliva.

Monitoring the levels of antibodies,cytokines,chemokines,and other bioanalytes can be used directly to detect viral pathogens.However,other strategies have been considered when using saliva diagnosis[143].For example,both the epithelial cells in the oral cavity which have an abundance expression of angiotensinconverting enzyme 2 (ACE2),and ACE2-positive cells in salivary glands are considered the target cells of SARS-CoV-2[144].Realtime quantitative analysis of the salivary proteins,glucose,urea,secretory IgA,cortisol,and phosphates have diagnostic potential for COVID-19 biomarkers discovery[145].In recent articles,it has been suggested that the temporal kinetics of IgG,IgA,and IgM in the saliva of COVID-19 patients were consistent with those observed in serum[146].Faustini et al.[147]have reported that the antibodies (IgA,IgG,and IgM) to the SARS-CoV-2 spike glycoprotein could be detected in serum and saliva.Thus,detectable salivary bioanalytes provide an early and accurate COVID-19 diagnosis which is comparable and more advantageous to those of serum since the saliva sampling process is non-invasive,inexpensive,and safe.

6. Discussion and future perspectives

6.1 Health benefit of fasting saliva

A previous study reported the antibacterial effects of saliva and suggested that this is due to nitrate and nitrite[148].The authors noted that fasting saliva contains approximately 0.2 mmol/L nitrate which suffices inhibition of theDesulfovibriospecies of bacteria they studied.In a treatise in 1756,Dr Nicholas Robinson (Member of the Royal College of Physicians,and Physician to Christ’s Hospital,London) mentioned the medicinal properties of fasting saliva for curing different health conditions when applied externally or given internally[149].One possible rationale to explain the observed medicinal properties of fasting saliva is related to the higher nitrate/nitrite concentration in fasting saliva[23].

Furthermore,one may hypothesise that lowerShigellaspp.seen during the month of Ramadan in patients attending a clinic could be due to antimicrobial activity from higher levels of salivary nitrate/nitrite exhibited by fasting.A study at a diarrheal hospital in Bangladesh monitored patients during Ramadan and non-Ramadan periods for infecting pathogens[150]. They detected a slight decrease inShigellaspp.during Ramadan periods.They attributed this reduction inShigellaspp.burden during Ramadan to differences in food preparation and hygienic practices[150]including more frequent hand and foot washing for special prayers as well as preparing foods closer to mealtimes,resulting in the consumption of fresh and hot foods. An additional possibility could be that fasting saliva may confer greater antibacterial effects that are not seen during the non-Ramadan period.

Interestingly,research from the UK reported that COVID-19 infection[151]and mortality[152]did not increase during the month of Ramadan.The reason for this could be that human saliva displays antiviral properties as previously reported[153,154].It is possible that fastinginduced salivary composition changes,such as increased nitrate/nitrite concentration[23],may enhance inhibition and clearance of COVID-19 from the body.A recent study discussed potential anti-COVID-19 properties of saliva[155]and future studies comparing anti-viral properties of fasting and non-fasting saliva may shed more light in this area.

6.2 Possible future work

The central role of oral health in the prevention of diseases including cancer,diabetes,cardiovascular disease and Alzheimer’s disease is being increasingly recognised[156].Intermittent fasting will undoubtedly impact oral health due to refraining from eating foods for certain periods of time and the mouth is being the first organ to receive food for processing.Benefits of intermittent fasting against various diseases have been reported including cancer[157],Alzheimer’s disease[158]and diabetes[159,160].Unfortunately,research using saliva for intermittent fasting is limited.For example,the impact of fasting on Alzheimer’s disease using saliva as a biofluid has not been well investigated.A search of WoS where the key words “saliva”,“fasting”and “Alzheimer’s” were combined led to only nine articles between 1970-2022.None of the articles specifically focused on the impact of fasting on Alzheimer’s disease.For diabetes,there were 188 articles but only a couple of these articles were specifically focused on investigating the impact of fasting per se on salivary metabolites.

Saliva is a very useful biofluid for research studies as its collection is non-invasive and easy to obtain from study participants.However,it has some limitations that need to be addressed.For example,there is controversy in the literature regarding if salivary lactoferrin levels can be used for the diagnosis of Alzheimer’s disease.One study suggested that there is a decrease in lactoferrin in Alzheimer’s disease patients[161],but it could not be replicated by another study[162].This has raised concerns regarding the use of saliva as a biomarker of disease.There are many factors that could alter the composition of saliva,and these should be considered before the collection of saliva for research studies.Following are examples of factors that could influence saliva composition:

1) Unstimulated saliva versus stimulated saliva

2) Saliva flow variation due to age differences

3) Medication use by study participants

4) Oral hygiene variation between study participants

5) Self-collection procedure could vary between study participants

6) Variations in the time of saliva collection

Therefore,there is a great need for harmonisation and standardisation of saliva collection procedures as well as storage conditions.In this context,it would be good to develop precise and reliable protocols through inter-laboratory validation of saliva collection,storage,and analysis.Such types of studies are necessary if the findings of scientific research based on saliva analysis are to be taken seriously by the scientific community.

Similarly,research using fasting saliva is difficult and challenging without a set of defined standard fasting procedures.Amongst factors to be considered include type and duration of fasting,geographical differences and individual variation associated with diurnal and circadian rhythms.With a standard protocol,improved methods,modern equipment,and applying the internet of things (IoT),scientists from all over the world can collaborate effectively.There are already huge volumes of data (e.g.,hospital,mobile phones etc.) that can be used to assess the impact of fasting on health and predict things in advance including allocation of resources-human and material.Artificial intelligence-assisted data analysis will then provide immediate health benefits (e.g.,a more accurate diagnostic and designer therapeutic),eventually contributing to the realisation of personalised molecular-based medicine.

7. Conclusion

Healing properties of saliva,reported since ancient times,are gaining attention especially when these are supported by modern scientific discoveries.The COVID-19 pandemic has led to a renewed interest in the role of saliva in health and disease.Here,we have reviewed the latest literature on the composition of saliva in relation to human wellness and diseases.

Interest in saliva is growing as its collection is non-invasive and that can be readily used for the diagnosis and monitoring of different diseases.Advances are being made in characterising the salivary microbiome and its role in health and disease.At the same time,a detailed analysis of salivary metabolites using an array of analytical techniques is providing valuable information regarding the mechanism of disease processes as well as the development of diagnostic tests.In recent years,there is great interest in intermittent fasting for health improvement and prevention of disease but not much research has been done using saliva.Here we have provided a detailed discussion of changes in the composition of saliva associated with fasting that can be used to not only explain some of the health benefits of fasting but can also be used for the prevention and treatment of diseases.However,the use of saliva for intermittent fasting research is still in its infancy even though fasting saliva has been used since ancient times for healing.

In the 1987 Predator movie,there was a scene where the intergalactic alien used its saliva to treat its injured body.Similarly in the future,human saliva may be used as a personal molecular medicine.When human starts to travel across space,this mode of carrying medicine using saliva may be advantageous over the conventional and cumbersome medicine tablets,bottles,and vials.

Conflict of interest

The authors dedare no conflict of interest.