Vaginal microbiota contains several healthy microbes that remain in a mutually regulated relationship with the human body.¹ The body produces immunoglobulins (secretory IgA and IgG), mucins, neutrophil gelatinase-associated lipocalin, secretory leucocyte protease inhibitor and β-defensins, as well as other proteins possess antimicrobial action. Collectively, these substances create a strong initial defense against infection.² Lactobacilli spp. fortifies the defensive mechanistic pathway against the colonization of unhealthy microbes.³ The vaginal milieu becomes more prone to this kind of invasion because of continuous transformations in the vaginal microbiota during the menstrual cycle, menopause, and hormonal changes during the pregnancy.⁴ The DNA sequencing method describes different types of dominant Lactobacilli spp. in the vaginal microenvironment. These include four Lactobacillus spp. (L. gasseri, L. crispatus, L. iners, and L. jensenii) which are also known as Community State types (Type-I, II, III, and V) along with some anaerobic bacteria linked to bacterial vaginosis (BV) that can cause vaginal infections but remain dormant due to Lactobacilli acting as their defense.⁵ Lactobacilli spp. is associated with the production of L-isomers and D-isomers of lactic acid in the vagina. Lactobacillus iners produces L-isomer of lactic acid because it does not constitute the gene coding for hybridization of D-isomer.⁶ To regulate MMP-8 (matrix metalloproteinase) production, D-isomer modulates metalloproteinase-inducing activity. The MMP-8 is having the potential of degrading cervical plugs in vagina that prevent bacteria to reach to the upper part of genitals.⁷ The microbiota of vagina comprises immune-responsive specialized cells known as (natural killer cells, neutrophils, macrophages, cytotoxic cells and helper cells. It also contains specialized receptors known as NLRs and TLRs, which can detect invasive pathogens and initiate the release of interleukins and TNF-a. These immune responses are regulated by the MAPK signaling pathway.^8,9 The development of resistance by microorganisms in several antibiotic therapies sparked the interest of researchers in antimicrobial plant extracts and their secondary metabolites.¹⁰ Traditional studies reported a huge number of antimicrobial plants with their secondary metabolites, which act as antimicrobial molecules against bacteria, fungi, and disease-causing pathogens.¹¹ Plant secondary metabolites included alkaloids, saponins, monoterpenes, sesquiterpenes, and flavonoids, exhibit remarkable in-vitro activity against those pathogens that causes vaginitis.¹²

Vaginitis
Vaginitis reflects the changes in vaginal microbial community that is frequently correlated with several vaginal infections like as candidiasis, trichomoniasis, bacterial vaginosis, cytolytic vaginosis and aerobic vaginitis.¹³ Higher vaginal pH is directly associated with increased vulvovaginal infections due to the decrease in lactic acid concentration and the number of Lactobacilli spp. It results in a spectrum of symptoms such as irritation, foul odor, burning sensation, frequent urination, vaginal discharge, abdominal pain, and preterm delivery.¹⁴ Certain types of contraceptives, improper hygiene, dietary changes, lifestyle, and antibiotic resistance in the body have caused women to experience recurrent vaginitis episodes.¹⁵ The synchronous occurrence of these infections not only misleads the diagnosis, but it may also increase the probable risk of longer-term persistence of these microbes in the body, which may lead to overlapping of the symptoms and failure to identify an adequate treatment regimen.¹⁶ Major vaginal infections, including VVC vulvovaginal candidiasis, BV bacterial vaginosis, TV trichomoniasis, AV aerobic vaginitis and CV cytolytic vaginosis, with their associated changes in vaginal microflora, common causes, symptoms, and associated risk factors are listed in Table 1.^17,18

Table (1):
Causes, symptoms with associated risk factors of different types of vaginitis

Vulvovaginal Candidiasis (VVC)
Candidiasis is a commonly occuring inflammatory condition caused by Candida spp., which majorly affects about 70-75% women in their reproductive age.¹⁹ The clinical symptoms of candidiasis are characterized by itching and redness of the vulva and vagina, a burning sensation, clumpy white discharge, and a frequent urge for urination.²⁰ Onset of VVC is associated with many of the triggering factors, such as antibiotic resistance (after long term antibiotic therapy), oral contraceptives (which increase estrogen levels), hormonal replacement therapies, tight fitting clothes, multiple sex partners, and uncontrolled diabetes. Gene polymorphism is a predisposing factor for the occurrence of VVC, includes single nucleotide polymorphism (SNP in that gene, which codes for mannose binding lectin), dectin-1 receptor, IL-4, and variants of genes of IL-22 with enzymes of regulatory T cells.²¹ It is recently reported that out of 284 women, 78% had the history of one or more VVC previous episode, 65% had three VVC annual episodes, and 35% had four or more recurrence annual episodes.²²

Etiology and pathophysiology
Candida albicans is a main factor in the development of VVC. This fungus changes shape from a normal ‘Y’ yeast cell to a mycelial ‘H’ hyphae organism. This process is known as the dimorphic transition. Normally, its ‘Y’ form remains in commensalism with the host, while the ‘H’ form found in tissue’s samples collected from women, had VVC. When the ‘Y’ form does not remain in homeostasis with the host and its tolerance becomes defective for the body, it changes into ‘H’ form. Furthermore, the ‘H’ form makes a firmly attached biofilm to the vaginal epithelium. The clinical sign of VVC, vaginal discharge is the result of detached ‘H’ form, cell debris, particularly inflammatory cells, and vaginal fluid.^23,24 The ability of Candida albicans to infect other organisms is mostly linked to the Sap family of proteins (ten genes that code for ten proteins, Sap1-Sap10), which are expressed differently in different hosts. This Sap gene differential expression depends on the number of factors, like vaginal pH, the number of ‘Y’ forms present in the host, and growth stages and types of Candida albicans.²⁵ The natural defensive mechanism of the vaginal environment responds to eliminate the opportunistic pathogen. Cell activation or the secretion of immune mediators by vaginal epithelial cells triggers inflammation during infection.²⁶ Inflammasomes are the vaginal intracellular multiprotein complexes of NOD like receptors (complexed with ASC protein and procaspase-1 proenzyme) that translate the encountered signals, cause immune activation, and encase the delivery of β-defensin after pathogenic invasion. It results in production of cytokines or interleukins such as IL-1β, IL-18 and lymphocyte activation (T-1 and T-17), which are traced to anti-Candida activity.^27,28

Bacterial Vaginosis or Vaginosis (BV)
Vaginosis caused by specific bacteria, is one of the prevalent vaginal infections that are affecting about 60% of women at the ages of 16 to 45 years. It leads into an abrupt increase in the colonies of etiological microorganisms such as Gardnerella vaginalis, Mycoplasma hominis, Megasphaera phylotypes 1 and 2, Atopobium vaginae, Peptostreptococcus, Sneathia, Bacteroides spp., Prevotella spp. and bacterial vaginosis-associated bacteria also classified as (BVAB1, BVAB2 and BVAB3).²⁹ BV is commonly associated with several complications, like vaginal discharge with foul odor, pelvic inflammation, and infertility, susceptibility to STDs, cervicitis, miscarriages, preterm labour, postpartum infections, and fetal low birth weight. This inflammatory disease is the result of an imbalance in the vaginal microflora, increased vaginal pH, fewer Lactobacilli spp., frequent use of antibiotics, and an excessive number of anaerobic bacteria.³⁰ Amsel criteria are the standardized method used to evaluate the condition of BV microscopically. This criterion includes four characteristics, i.e. pH of vagina greater than 4.5, presence of uniform vaginal discharge (white or grey), presence of clue cells (epithelial cells that layered by bacteria), and whiff’s test (characterized by presence of the fishy odor, when discharge added to KOH solution) found positive. The Nugent’s score is an evaluating system used to assess the presence of Gram-positive morphotypes in numbers i.e. normal (0-3), intermediate (4-6) and (7-10) categorized as containing BV.^31,32 Other methods such as, targeted qPCR assays have been used to spot commonly present bacterial species in the discharge of BV patients using 16S rRNA sequencing. Further taxon-directed PCR assays and DNA hybridization studies have also been reported for sensitive detection of BVAB.^32,33

Etiology and pathophysiology
An excessive number of anaerobic bacteria replace the normally present Lactobacilli spp. in BV, resulting in a reduced level of lactic acid, antimicrobial hydrogen peroxide, and some organic acids.³⁴ BV is mostly asymptomatic until the vaginal discharge is found to have a fishy odor that is primarily caused by amine production by anaerobic bacteria. This odor is more common after intercourse (due to the presence of semen) and in menstruation (due to the presence of blood), which results in increased alkalinity of the vaginal flora.³⁵ In both symptomatic and asymptomatic conditions, BV increased the risk of HIV transmission, developing preterm birth, pelvic inflammatory disease and premature membrane rupture.³⁶ Gardnerella vaginalis is the widely described BV causative agent. It is the only bacteria that express virulence factors and the rest are virulent opportunistic pathogens in BV. It is likely associated with the ability to colonize the urogenital tract and form biofilms. It produces mucin-degrading enzymes, resulting in a decrease in the viscosity of vaginal fluid. Cytolysins are known as virulence factors that produced by Gardnerella vaginalis that leads into cell death in epithelial cells of vagina after trigger of protein kinase pathway. These cytolysins act as vaginolysins (pore forming toxins) that lyses RBC and vaginal epithelial cells. The presence of cytolysins increases the nutrient availability of Gardnerella vaginalis. It also produces some mucosal degrading factors like prolidase, putrescine, and sialidase, resulting in the exfoliation of vaginal membrane. The peptidases of Gardnerella vaginalis release amino acids and peptides in the vaginal environment, which stimulates bacterial growth and provides the nutrient facility for other BVABs.^37,38

Trichomoniasis (TV)
Trichomonas vaginalis causes Trichomoniasis (TV), a protozoan infection that primarily contributes to most sexually transmitted diseases such as STDs, HIV, vaginitis, and urethritis.³⁹ Trichomoniasis can be asymptomatic for several months, and it is mainly characterized by yellowish-green diffused malodorous thin vaginal discharge with a pH greater than 4.5 and irritation and itchiness in the vulva. It is majorly associated with many reproductive complications, like dysuria, UTIs, vulvar pruritis, painful intercourse, pelvic pain, underweight babies, preterm birth, infertility, and cervical cancer. TV is a white blood cell type flagellated motile protozoan that can exist in more than one cellular form. It can reside in the lumen of the urogenital tract of a woman for a longer time. It releases some cytotoxic proteins that destroy the vaginal and urethral epithelial linings. This parasite decreases Lactobacillus colonization, promotes the growth of anaerobic bacteria, and damages the vaginal epithelium, which predisposes women to other urogenital infections.^40,41

Etiology and pathophysiology
The trophozoite form of TV has unique patterns of protein phosphorylation and a specific signaling mechanism that only works in certain protozoan cell types and the right conditions.⁴¹ The binding of parasites to the host cell is adhesin mediated through TV lipoglycans (TVLG) (the surface molecule of parasites). In humans, galectin-1 is the receptor for TV. TVLG modulates the macrophages and inflammatory response of epithelial cells in the host body.⁴² Studies found 11 adhesion proteins on the surface of parasites that cause a high level of binding to epithelial cells. Trichomonas vaginalis phagocytoses epithelial cells, fungi, and bacteria in the human host to get nutrients. TV neutralizes host defense proteins by receptor mediated endocytosis or phagocytosis.⁴³ Phagocytosis or endocytosis of the vaginal microbiota induces dysbiosis (imbalance in microbial’s community) that resulted in other vaginal infections like BV due to activation of BVABs. The major cause of this type of imbalance is continuous use of antibiotics which actively trigger human microbe interaction. TV remains in symbiosis with Mycoplasma hominis and causes TV-Mycoplasma infections. These types of co-infections consume large amount of arginine that leads to cause reduction in nitric oxide produced by natural macrophages in vagina that ultimately interfere with defensive mechanism in human body.^44,45

Aerobic Vaginitis (AV)
Aerobic vaginitis is also called vaginal dysbiosis or desquamative inflammatory vaginitis and results from an increased number of bacteria such as Streptococcus (Group-B), Streptococcus viridans, Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella spp., Enterococcus faecalis, Pseudomonas spp., and Citrobacter spp. and remains asymptomatic in around 10-30% of the cases.⁴⁶ AV is associated with a reduced number or absence of Lactobacilli spp. in the vaginal microflora. Symptoms associated with AV included yellowish green putrid vaginal discharge with a foul smell. AV gives a negative Whiff test. Vaginal pH increased over 6, causing burning and inflammation in the vaginal mucosa with severe ulceration. AV is diagnosed by the increased number of parabasal cells, increased interleukins (IL-8, IL-6, IL-1β), and numerous leucocytes, which is most likely the condition in cervical neoplasias in women.⁴⁷

Etiology and pathophysiology
In AV, different types of pathogenic mechanisms are involved that trigger the release of inflammatory cytokines, i.e., interleukins and inhibitory factors (about three folds increase in comparison to BV).⁴⁸ Interleukins (IL-6 and IL-8) are linked with an increase in prostaglandins and their delivery. Interleukins activate the leucocytes, which results into thinning of vaginal mucosa and increases the desquamation of epithelial cells. Kallikrein relative peptidases increased in AV resulted in desquamation. Sialidases are hydrolytic enzymes that increase in AV and degrade IgA (the host’s defensive molecule). Another reason for AV or DIV is the lower level of oestrogen that might induce the development of DIV in lactating women and in menopause.^49-51

Cytolytic Vaginosis (CV)
Cytolytic vaginosis or Doderlein’s cytolysis or lactobacillus overgrowth syndrome is diagnosed by the presence of an increased number of Lactobacillus spp. that resulted in vaginal epithelial cell lysis.⁵² It is characterized by abundant white and frothy vaginal discharge, dyspareunia, vulvar dysuria, and pruritis. These symptoms are mostly occured in the luteal phase and during pregnancy. CV is also associated with the presence of false clue cells, in which increased Lactobacilli covered the vaginal epithelial cells. More likely CV has symptoms like BV except the pH of vagina (lower than 3.5), absence of Candida, Gardnerella, and Trichomonas and Whiff’s test results.⁵³

Etiology and pathophysiology
Hormones play a vital role in vaginal infections, including CV development. Due to lower pH and elevated glycogen degradation by an increased number of Lactobacillus spp. cytolysis in CV occurs. Lactobacilli produce isomers of lactic acid. An imbalance in the ratio of these two isomers leads to CV development causing an alteration in extracellular matrix (loproteinase) expression.⁵⁴ Patients who have previously received any antifungal treatment often experience CV mimicking the symptoms of VVC. CV is commonly associated with localized vulvodynia. Exposure of the vestibule to an increased number of Lactobacilli, low pH, and excessive amount of H₂O₂ often leads to vulvodynia. Researchers have reported over-acidification, an elevated level of l-lactic acid, and numerous fragmented bare nuclei with cytoplasm debris in a wet smear of the affected woman.^55,56

Monoterpenes: naturally present antimicrobial compounds
Terpene compounds (C₁₀H₆) are also classified as isoprenoid’s units and their derivatives. When it contains an excessive oxygen element, then it termed as terpenoids. They can exist as monoterpenoids, diterpenoids, triterpenoids, and sesquiterpenoids in nature. The antibacterial, anti-trichomonal and antifungal activity of tea tree oil (TTO) against Candida, Gardenella, and Trichomonas spp. have been widely reported due to the presence of monoterpenoids such as eucalyptol and terpinen-4-ol.⁵⁷ Thymol and carvacrol are the main extracted monoterpenoids from Thymus spp. that have been reported to have interesting anti-Candida activity in VVC.⁵⁸ Plants and their associated secondary metabolites such as geraniol, linalool, limonene, and menthol widely reported to possess remarkable antimicrobial activity against a broad spectrum of microbes.^59,60 In a murine model of VVC, nanoemulsions containing eucalyptol and limonene were used. The developed nanoemulsion showed superior anti-Candida activity than miconazole cream. In a further study, 16 essential oils were tested against 21 fungi isolates, of which the major monoterpenes citronellol, geraniol, thymol and carvacrol were the monoterpenoids that have been reported for their antifungal effect against all fungi isolates.⁶¹ Monoterpenes such as eugenol, carvacrol, thymol, p-cymene, a-pinene, terpinolene, eucalyptol, and a–terpineol against Gardenella spp. have shown therapeutic success up to 80% for BV.⁶² Monoterpenes are the phenolic compounds of plant’s essential oils. Phenolic monoterpenes contributed to intrinsic antimicrobial properties in vaginitis. Several monoterpenes and their role in the management of vaginitis with their mechanism of action in treatment of vaginal infections are represented in Figure.^63,64

Thymol
Thymol or 2-isopropyl-5-methylphenol or hydroxyl cymene is a monoterpene phenolic compound and an isomer with carvacrol, majorly present in extracted oils obtained from plants that belong to families Apiaceae, Scrophulariaceae, Lamiaceae, Ranunculaceae, and Verbenaceae.⁶⁵ Thymol (12-65%) is the major constituent obtained from the plant Thyme (Thymus vulgaris and other Thymus spp. of the Lamiaceae family) and many other plants. Thymol has been reported for different pharmacological activities like antiseptic, anti-inflammatory, antinociceptive, antioxidant, local anaesthetic, and most commonly for possessing antifungal and antibacterial properties.^66-70

Mechanism of action of thymol
Thymol has good antimicrobial action. The antibacterial action of thymol has been evaluated in various in-vitro studies that included Salmonella typhimurium, E. coli, Yersinia enterocolitica, Pseudomonas fluorescens, Proteus mirabilis, E. coli, Sarcina flava, Listeria innocua, Staphylococcus aureus, Micrococcus spp., and Bacillus licheniformis.^71,72 Its antibacterial activity mainly results from changes in the bacterial lipid bilayer and interactions with bacterial DNA. At very low concentrations, thymol can alter fatty acids composition (specially branched 14-methylhexadecanoic acid and 12- methyltetradecanoic acid) of lipid membrane in bacteria and at high concentration it can alter cell membrane integrity and reduces the viability of bacterial cells. The interaction with bacterial genomic DNA included the formation of a junction between bacterial DNA (minor groove) and thymol, which causes destabilization of secondary structures in DNA and results in DNA disaggregation.⁷³ Another study revealed that in bacterial cell membrane thymol causes uncontrolled intracellular material release, resulting in disturbances in the metabolic actions of bacteria. Thymol is also associated with a reduction in glutathione and nitric oxide production in bacteria.⁷⁴ In a microbial cell model, thymol distended DPPS (dipalmitoylphosphotidylserine) monolayers resulted in decreased surface elasticity and a change in the rheological properties of bacterial membrane films. Through this alteration in the cell membrane, thymol can easily permeate into the membrane, resulting in changes in membrane morphology, membrane structural order, membrane fluidity and lipid loading density.^75,76 An antifungal activity study of thymol against Candida spp. concluded that it inhibits the ergosterol synthesis in fungal cell membrane and leads into cell impairment. It acts as a chemo sensitizing agent with interfering agents of cell wall. After penetration it reaches to fungal DNA and interferes with DNA synthesis. Thymol also disrupts the oxidative stress response system in fungi when used in combination therapy.⁷⁷ A further study reported that thymol inhibits biofilm formation in Candida albicans by preventing further biofilm development or adherence. The morphological characteristics of thymol-treated biofilms change due to cell wall shrinkage and leakage in the intracellular matrix In Candida biofilms; thymol also inhibits sessile cells.⁷⁸ Thymol has superior anti-trichomonia activity.⁷⁹ The mode of action against Trichomonas spp. includes cell wall lysis and changes in the permeability. Thymol causes apoptosis in Trichomonas spp. cells and alters protozoan mitochondrial membrane permeability. It is also reported to interfere with Trichomonas metabolism by inhibiting enzyme dihydrofolate reductase, which leads to cell death.^80,81

Antimicrobial activity of thymol
A study in 1994 reported noteworthy activity of thymol against wide range of bacteria in an effective range, with the lowest minimum inhibitory concentration of 175 µg/mL for three different strains of Staphylococcus spp. and 125-245 µg/mL for E. coli.⁸² Sasso et al. identified the antibacterial activity of thymol on S. aureus and E. coli. In that study it was reported that the ½ MIC shows the highest inhibition for E. coli and 1/16 for Staphylococcus aureus on vaginal epithelial cells. For thymol, efficient inhibition for adhesion was reported between ½ and 1/32 MIC values.⁸³ Thymol containing vaginal cream has been reported to show a better response to vaginal and vulvar edema in comparison to the metronidazole-containing vaginal gel.⁸⁴ Antifungal activity of thymol in vaginal infections has been reported with highest MIC ≤0.0038% and 0.0078, <0.015%, v/v for three strains of Candida spp. Thymol effectively decreased the number of Candida albicans and its biofilm production in the dose range of 64-120 µg/mL. These studies concluded that thymol could suppress the transformation of Candida from its yeast to hyphae form.⁸⁵ In 2018, a study on five essential oils, including thymol, with seven E. coli spp. and twelve Candida spp., resulted in promising antibacterial and antimycotic activity with lower MIC values.⁸⁶ Cordoba et al., evaluated antifungal activity of thymol against four Candida spp., including other essential oils and concluded that thymol is an active essential oil at MIC values of 0.8-0.16 mg/L.⁸⁷ De Castro et al., reported antifungal activity of thymol with MICs range of 39-78 µg/mL for three strains of Candida spp. Researchers also reported synergistic antimicrobial activity with fluconazole and amphotericin B.⁸⁸ Thymol-loaded nanoparticles and nystatin combination have been found to have significant anti-Candida activity in comparison to the nystatin group with the best MIC of 0.158 µg/mL.⁸⁹ Metronidazole was not as effective against Trichomonas vaginalis as thymol, eugenol, and carvacrol extracts. Their hexane extract and methanol extract were more effective.⁹⁰ Researchers investigated the anti-trichomonal activity of pure thymol and two nanopreparations, namely nanoliposomes and nanoparticles of thymol, and compared them with metronidazole. The developed formulation resulted in the best activity with a lower inhibitory effect.⁹¹

Eugenol
Eugenol or 4-Allyl-2-Methoxyphenol is a hydroxyphenyl propene found in volatile oils extracted from a variety of plants, such as Eugenia cariophylata, Myristica fragrans, and Ocimum basilicum, of the family Myristicaceae, Lauraceae, Lamiaceae, and Myrtaceae.⁹² It is a mainly present in clove oil (45-90%), obtained from Syzygium aromaticum, Myrtaceae and widely used for flavouring in the food and cosmetic industry. Traditional medicine uses it for its many pharmacological activities such as antibacterial, anti-inflammatory, antifungal, anticancer, and antioxidant.⁹³ It has antibacterial, antifungal, and antitrichomonal properties, either alone or in combination.

Mechanism of action of eugenol
A free hydroxyl group present in eugenol is mainly responsible for its antimicrobial activity. The hydroxyl group is attached to bacterial cell proteins and prevents enzymatic action. In Gram-negative bacteria, eugenol penetrates the lipopolysaccharide of cell membrane and causes an alteration in cell structure.^94,95 Eugenol damages the bacterial cytoplasmic membrane, causing potassium ions to leak out and a significant loss of cellular substances, ultimately leading to cell death.⁹⁶ Eugenol mainly changes the portrayal of principal fatty acids and increases fatty acids with a low molecular weight.⁹⁷ In bacterial cells, eugenol causes cell cytotoxicity by creating reactive oxygen species (ROS). These ROS stop cell growth, damage cell membranes, and damage bacterial DNA, which ultimately causes cell death.⁹⁸ It also exhibits noticeable activity against bacterial enzymes like histidine carboxylase, protease, amylase, ATPase.⁹⁹ Eugenol causes diminish in production of pyocyanin, violacein elastase (some virulence factors in Pseudomonas aeruginosa) and biofilm formation. It also inhibits biofilm production by 65% the gene expression level; especially the QS synthase genes.¹⁰⁰ Eugenol changes the shape of the envelopes of bacteria and fungi due to presence of free hydroxyl group. It is highly effective in reducing colonies of Candida albicans and increasing the amount of damaged cells. Candida albicans envelope modification is relative to its pathogenicity, so it can cause compromises in host cell adhesion and some morphological transitions.¹⁰¹ Eugenol affects ion transportation in fungi. Eugenol reduces H⁺-ATPase activity in Candida albicans and inhibits H⁺ extrusion, leading to glucose stimulation. There is a high level of superoxide dismutase in Candida albicans that can cause an oxidative stress response. This leads to lipid peroxidation in the cytoplasmic membrane and then cell death.^102,103 Antitrichomonas activity was reported for eugenol in many of the recent studies.^104,105 The mechanism of action against Trichomonas vaginalis includes cell wall lysis and cell membrane permeability alteration. Metabolism interference is also one of the main causes for the cell disruption in Trichomonas spp.¹⁰⁶

Antimicrobial activity of eugenol
Eyambe et al. determined the antimicrobial activity of eugenol against a number of bacteria such as Staphylococcus aureus. The MBC value indicated that it could kill about 99% of bacteria at a minimum concentration. The MIC value was at 115 µg/mL and MBC value was found out at 230 µg/mL.¹⁰⁷ The activity of eugenol against Staphylococcus aureus, E. coli, and Candida spp. is well documented. The study estimated the MIC value of eugenol against E. coli to be 1.6 mg/L, for Staphylococcus aureus to be 128 mg/mL, and for Candida spp. to be 0.88 mg/mL. The study concluded that eugenol exhibited remarkable antimicrobial activity against the causative agents of vaginitis.¹⁰⁸ Saugella probiogel, which mainly contained eugenol as an active ingredient, has been tested on a group of 209 women who suffered from BV, VVC, and RVVC. Researchers discovered that eugenol interferes with the biofilms, leading to lower the number of bacterial and fungal pathogens. This resulted in a normal state for 80% of BV cases, 62.5% of VVC cases, and 100% of RVVC cases.¹⁰⁹ Banks and Taghread determined the antimicrobial activity of eugenol and extract of cloves against E. coli, Staphylococcus aureus (that were resistant to cefotaxime) and Candida spp. by disc diffusion sensitive method. It was found that both eugenol and non-polar clove extracts showed a significant inhibition of Staphylococcus aureus, E. coli, and Candida spp. with p ˂ 0.001.¹¹⁰ The antitrichomonal activity of eugenol showed that the extracts containing eugenol as an active compound cause growth inhibition of Trichomonas vaginalis with MIC range of 0.156 mg/mL.¹¹¹ Yassin et al., used three strains of Candida spp. and tested with ethyl acetate extract of clove containing eugenol 60% as an active ingredient. In the disc diffusion method, the zones of inhibition were 14.9, 20.9 and 30.7 for Candida glabrata, Candida albicans and Candida tropicalis.¹¹² In a study of 2022, it was found out that eugenol shows better antimicrobial effect than cinnamaldehyde against 18 Candida strains that was derived from vaginal strains cultures of infected women. MIC value for eugenol was 455.42 mg/L and MFC value was 690.09 mg/L and all the viable fungal cells were lost within 1 hour.¹¹³ In 2023, another study reported the anti-trichomonal vaginalis activity of eugenol. In this study the IC₅₀ value for eugenol was 1.21 mg/mL which was better than metronidazole.¹¹⁴

Eucalyptol (1, 8-Cineole)
Eucalyptol or (1,3,3-trimethyl-2- oxabicyclo[2.2.2]octane)  is a naturally produced monoterpene majorly obtained (70%) from leaves of Eucalyptus globulus as eucalyptus oil and other Eucalyptus spp. of the family Myrtaceae.¹¹⁵ Eucalyptol is widely reported to be used in the pharmaceutical, cosmetic and chemical industries. Its pharmacological activities are reported for anti-inflammatory, antiseptic, and antioxidant effects as well as having a strong antimicrobial activity in a broad spectrum.¹¹⁶ Numerous studies have documented its antimicrobial and antiprotozoal activity in vaginitis.

Mechanism of action of eucalyptol
Eucalyptol was reported to interfere with signaling in quorum sensing in bacteria. Quorum sensing is the technique of communication through chemical signals in microorganisms for promoting their growth, spread, and invasion. Biofilm formation is the result of quorum sensing, which protects the microbes from their external environment. Eucalyptol blocks those receptors that receive signals through different autoinducers.¹¹⁷ Eucalyptol changes the size and shapes of bacteria cells. In bacterial cells, it causes apoptosis in Staphylococcus aureus; when treated, it causes a nuclear chromatin condensation with necrosis in E. coli results into a reduction of nuclear chromatin and nucleoplasm.¹¹⁸ Eucalyptol makes Gram-negative bacteria more likely to have their cell walls damaged, which is a clinical benefit that can cover a wide range of antibacterial activity. Its activity is due to its hydrophobicity, which can synergistically increase the antimicrobial action of other agents.¹¹⁹ Eucalyptol affects cell membrane permeability, and it enhances the fluidity of the membrane, which results in membrane protein topology and lipid peroxidation. This inhibits bacterial cell respiration. It also increases the oxidative stress and ROS (reactive oxygen species) that inhibit the biological processes of bacterial cells.¹²⁰ Eucalyptol was reported to alter membrane fluidity and cell wall permeability of Candida albicans by modifying the properties and functions of membrane. Eucalyptol also caused distortion in the fungal hyphae wall, followed by cell membrane disruption. Eucalyptol and other agents can interfere with the activity of mitochondrial dehydrogenase, which is ultimately involved in fungal ATP biosynthesis. The effectiveness of eucalyptol on the H⁺-ATP_ase of fungal membrane interferes with intracellular pH and fungal cell nutrient uptake. It finally leads to the acidification of intracellular material and cell death. Eucalyptol acts as a potent antifungal for Candida albicans and biofilms due to metabolic interference in comparison to fluconazole.^121,122 Eucalyptol hydrophobicity and lipophilic nature allow it to enter the cell membrane of Trichomonas vaginalis. It increases cytoplasmic material permeability, which leads to impaired function in Trichomonas. Eucalyptol can interfere with the lipid composition leading to protein denaturation, cytoplasmic substance leakage, membrane disintegration, and cell death. Enzymatic inhibition and immune modulation are other modes of action of eucalyptol in protists.¹²³

Antimicrobial activity of eucalyptol
Trinh et al. found that eucalyptol and α-terpineol are effective at killing two vaginal bacteria: Candida albicans and Gardnerella vaginalis. The combination of eucalyptol and α-terpineol potently inhibits the growth of these microorganisms at MIC values (0.125 and 0.065% v/v) respectively.¹²⁴ In a study, eucalyptol lowered the number of Trichomonas vaginalis in a culture media at the effective concentration of 12.54 mg/mL after 48 hours.¹²⁵ Rosemary oil containing eucalyptol showed potent activity against Pseudomonas aeruginosa and E. coli, with MIC 50 mg/mL and 25 mg/mL, respectively. The antifungal activity for Candida reported in this same study, with MIC/MFC value of 1.26 mg/mL.¹²⁶ Bogavac et al., evaluated the antibacterial and antifungal activity of eucalyptus oil against two strains of of E. coli, Staphylococcus aureus and Candida albicans. The MIC values were reported to be 12.5-25, 6.255 and 6.254 µL/mL for E. coli, Staphylococcus aureus, Candida albicans, respectively. The study concluded that eucalyptol can be a possible potent antimicrobial agent in vaginal infections.¹²⁷ Zhou et al., demonstrated that eucalyptus oil containing eucalyptol as major active constituent having antimicrobial activity against Candida albicans, E. coli and Pseudomonas aeruginosa with MICs range from 5.5-40.2 µL/mL.¹²⁸ In a study of 2021, Sang-Youn et al., reported that eucalyptol possessed good antimicrobial and antioxidant effect against Candida albicans at MIC value 1.25 mg/mL.¹²⁹ In a recent study of 2020, it was demonstrated that eucalyptol is having an efficient anti-trichomonal activity against Trichomonas vaginalis at the concentration of 21 mg in a culture media that was found out to be similar as metronidazole.¹³⁰ In a further study of 2021, eucalyptol was tested against Gram-positive bacteria, methicillin resistant strains, Gram-negative bacteria, vancomycin resistant strains and Candida spp. It showed the inhibition in growth of these microbes in a very prominent range of MIC (0.25-8% v/v).¹³¹

R-Limonene
Limonene or 1-methyl-4-1-methyl phenyl cyclohexene is a naturally occurring monoterpene (R-Limonene or S-Limonene) containing two isoprene units with a lemon-like odour and is mainly found in citrus oil extracted from lemon, orange, and mandarin peels and in lemongrass oil, i.e., extracted from plants of the family Rutaceae.¹³² It is reported to possess different pharmacological activities like as antimicrobial, antioxidant, anticarcinogenic, antidiabetic and chemopreventive.^133-135 Limonene has been prominently reported to have strong antimicrobial activities in vaginal infections.

Mechanism of action of R-limonene
The antimicrobial activity and mechanism of action of R-limonene have been well documented. Limonene damages lipopolysaccharide, which is associated with barrel proteins. It also changes membrane permeability and inactivates the bacterial cell envelope.¹³⁶ At an optimum concentration, it was found that it leads to the formation of hydroxyl radicals (Fenton-mediated), which result in the oxidation of bacterial DNA and alter the membrane permeability.¹³⁷ Collapse in bacterial cell structure (Gram-positive and Gram-negative) was seen when treated with limonene-containing nanoemulsion, which further lead to cell lysis, cell deformation, exudation of cellular material and cell death.¹³⁸ The efficacy of antibacterial agents might differ for bacterial cells (Gram-positive and Gram-negative) because of differences in their targeted sites. Limonene also showed difference in its activity for both as Gram-negative bacterial cells are having efficient membrane homeostasis. Limonene is hydrophobic and Gram-negative bacterial cells are prone to develop a hydrophilic barrier to protect themselves, due to which these cells are lightly sensitive for limonene.^139,140 The antifungal activity of limonene included the overexpression of different genes that are associated with cell signaling pathway. It was observed that Candida cell deteriorated after treatment with limonene due to altered cell structure and accumulation of reactive oxygen species or ROS that ultimately leads to cell destruction.¹⁴¹ About 80% of the changes in membrane permeability observed in Candida isolates resulted from rupture of cell wall. Researchers found that limonene interferes with pectin methyl esterase and cellulase in fungus cells.¹⁴¹ Inhibition of biofilm formation by limonene is also reported in various studies against several microbes. Limonene was reported to have better anti-trichomonal activity by causing changes in the morphology of protists. After exposure to limonene, it was observed that it might cause ribosomal disappearance, organelle disintegration, and formation of vacuoles, endoplasmic reticulum dilation, and leakage in cytoplasmic material. It also causes damage to the cytoplasmic membrane and appearance of cytoplasmic vacuoles. Limonene developed holes and projections in cytoplasmic membrane, which finally caused cell death of Trichomonas vaginalis. The attachment capacity of protist was also interfered by the presence of limonene.^142,143

Antimicrobial activity of R-limonene
Vuuren and Viljoen assessed the antibacterial and antifungal efficiency of limonene and eucalyptol in a 1:1 ratio against wide range of bacteria and a fungus isolate. It was resulted into the MIC value range of 2-27 mg/mL for limonene enantiomers for Pseudomonas spp., Staphylococcus aureus and Cryptococcus neoformans.¹⁴⁴ Bassole et al., estimated that limonene and its epoxides possessed better antibacterial and antifungal activity against vaginal isolates such as E. coli, Pseudomonas spp., Staphylococcus aureus, and Candida albicans, with MIC range of 2.4-3.2%.¹⁴⁵ Essential oil containing lemongrass oil (active constituent limonene) shows a remarkable inhibitory effect against Streptococcus spp. with a mean value of 10.07 of the zone of inhibition after 48 hours. Limonene was found to be the second most active antibacterial against Streptococcus spp.^146,147 Zahi et al. determined the antimicrobial activity of limonene loaded in nanoemulsion (organogel-based) form against Staphylococcus aureus, Pseudomonas spp., and Candida albicans with a MIC range of 3.14-12.56 µg/mL. The microbial cell’s constituent release increased about two folds after the application of limonene organogel.¹³⁸ Gundel et al. estimated in vitro antifungal effect of limonene and eucalyptol in a murine model of vulvovaginal candidiasis. Nanoemulsion containing limonene and eucalyptol showed better antifungal activity in comparison to miconazole cream with p ˂ 0.05. The nanoemulsion showed better results due to the smaller size of the globules and larger surface area.^61,148 Another study of 2005 tested the activity (in vitro and in vivo) of limonene against different specimens of Candida spp. on vulvovaginal candidiasis model. It decreased cell viability due to treatment with limonene at EC 50% (444 ± 35 µM) after 8 hr. In vivo result was showing significant decrease in number of fungal cells at the concentration of 500 µM of limonene.¹³⁹ In another study, an anti-trichomonal activity was examined by using tea tree oil. It was found out that due to presence of limonene the number of protozoan Trichomonas vaginalis decreased at IC₅₀ 0.06 µL/mL.¹⁴⁹ Nanoemulsion containing limonene with resveratrol showed prominent inhibition of Staphylococcus aureus due to the increased skin permeation. Limonene showed synergistic antibacterial activity.¹⁵⁰

Alpha-Terpineol (α-Terpineol)
α–terpineol or 2-(4-methylcyclohex-3-en-1-yl)propan-2-ol is one of the isomers of terpineols (α, β, γ, and δ) naturally present as monoterpenoid alcohol in essential oils, mainly obtained from Mentha spp., Origanum spp., Artemisis spp., Thymus spp., Pinus spp., Salvia spp., Melaleuca spp., and Narcissus spp. in high fractions and in small fractions from many more plants. It is characterized by the floral fragrance and flavour.¹⁵¹ It has been widely known and documented to possess several biological properties as being anticarcinogenic, antioxidant, antimicrobial, anti-inflammatory, antiulcer, skin penetration enhancer, and anticonvulsant.^152,153

Mechanism of action of α-terpineol
Monoterpenes have been reported to cause hydrocarbon partitioning in microbial cell membranes and disrupt vital functions. It causes lysis of bacterial cell membrane and resulted in to extrusion of cytoplasmic matter with associated ions and respiration inhibition.¹⁵⁴ It disrupts microbial potassium homeostasis, eliminates propidium iodide, forms mesosomes, and modifies the glucose-dependent respiration mechanism in Staphylococcus aureus and Pseudomonas spp.¹⁵⁵ It also changes the structural functions of the bacterial cellular membrane. Alpha-terpineol resulted to cell shape irregularities, formation of edges in the bacterial nuclear part, condensation of cytoplasm, plasmolysis, and formation of vacuoles in E. coli bacterial cells.¹⁵⁶ In Candida spp. and Saccharomyces spp., it alters membrane permeability and inhibits cell respiration. It also affects the mitochondrial membrane of fungi by inhibiting medium acidification, which in turn interferes with the energy production of the fungi cells. Alpha-terpineol inhibits the germ tube formation; thereby obstructing the conversion of fungi into their mycelial form.¹⁵⁷ Treatment with a-terpineol also causes abnormal hyphae formation due to disruption in the fungi cell wall. It down regulates fungi metabolic pathway and disrupts energy formation.¹⁵⁸ Monoterpene extract contained a-terpineol, which is reported to block acetylcholinesterase in Trichomonas vaginalis. Due to acetylcholinesterase blockage, the level of acetylcholine increases, which increases the activation of nicotinic acetylcholine receptors, ultimately leading to muscular contraction and membrane depolarization.¹⁵⁹

Antimicrobial activity of Alpha-terpineol
Alpha-terpineol has been documented to have activity in vaginitis against Staphylococcus aureus and E. coli at MICs 0.065 and 0.252% v/v, respectively. In a similar study, anti-candida activity was also reported in the range of 0.064-0.2568% v/v.¹⁶⁰ Alpha-terpineol inhibited biofilms produce by Candida spp. after 2 h of exposure at 0.1256% v/v.¹⁵⁷ The activity of α-terpineol in bacterial vaginosis and vulvovaginal candidiasis has been reported against G. vaginalis and C. albicans at MIC 0.065% and 0.1254% v/v, respectively, by increasing cytokines (interleukins) expressions and NF-kB activation.¹²⁴ Alpha-terpineol has shown antifungal activity, along with other constituents.¹⁶¹ Alpha-terpineol showed prominent inhibitory activity against Candida albicans isolates that were resistant to fluconazole with MIC range of 300-3000 µg/mL.¹⁶² A study in 2023, reported to have antibacterial, anti-candida and anti-trichomonal activity of tea tree oil due to its main active constituent α-terpineol in a very effective way.¹⁶³ Alpha-terpineol is the majorly present in tea tree oil and possesses antibacterial and antifungal activities against ten bacterial isolates and Candida albicans isolates. The Rideal-Walker coefficient for α-terpineol was found to be 16, which was the highest among all the constituents of tea tree oil. The anti-inflammatory activity also possessed by α-terpineol in vaginal inflammation occurs in vaginal candidiasis. This study also demonstrated anti-trichomonal activity, concluding that -terpineol effectively combats Trichomonas vaginalis.^164,165 Alpha-terpineol is also effective against Aspergillus niger and effectively inhibits fungal mycelial growth and spore germination.¹⁶⁵ Alpha-terpineol has antibacterial activity against fifty Staphylococcus spp.¹⁶⁶

Geraniol
Geraniol or 3-7dimethylocta2-6-dien1-ol is a monoterpene found in a mixture of nerol and geraniol (cis and trans forms). It is mostly obtained from Rosa rubiginosa, Cymbopogon martini, Cymbopogon nardus, Pelargonium graveolens, and Monarda fistulosa. It has a sweet floral or rose-like fragrance, so it is widely used as a fragrance material.¹⁶⁷ It possesses various pharmacological activities, including antitumor, insecticidal, antioxidant, antimicrobial and anti-inflammatory properties. It has efficient antibacterial, antifungal, and antitrichomonal properties for vaginal infections.¹⁶⁸

Mechanism of action of geraniol
The antimicrobial activity of geraniol has been described against Staphylococcus aureus and E. coli. Geraniol induces DNA damage and increases osmotic stress in bacterial cells.¹⁶⁹ It disrupts the microbial cell membrane lipid structure, interacts with bacterial cellular components, and makes the bacterial cell more permeable to the outer compounds. It penetrates the cell interior and ultimately limits bacterial cell growth. It also disturbs the membrane efflux pumps in bacterial cells and reduces the level of ergosterol, leading to an alteration in ATP_ase activity in the membrane.^170,171 The activity of geraniol has been reported against a broad range of Candida and non-Candida spp. In fungal cells, it causes impaired iron homeostasis with mitochondrial dysfunction. It also alters the functioning of calcineurin signaling pathway in a fungal cell that is responsible for the antifungal activity of geraniol.¹⁷² In Candida albicans spp., membrane disruption is the major mechanism of action of geraniol that affects fungal cells. Geraniol changes the ATP_ase activity of the plasma membrane and alters intracellular pH. Geraniol damages DNA repair in Candida albicans. Geraniol is a potent inhibitor of hyphae formation in yeast cells. This inhibits biofilm formation.¹⁷³ In vaginal infections, geraniol has better anti-trichomonal activity. The treated cells of Trichomonas vaginalis showed morphological changes, formation of vacuoles, ribosomal disappearance, and dilation in the rough endoplasmic reticulum. After treatment with geraniol, the protozoan nucleus membrane damages and leads to leakage of cytoplasmic content, accumulation of chromatin in the cytoplasm, disintegrated organelles, and damaged cell membrane, which resulted into cell death.^174,175

Antimicrobial activity of geraniol
An in vitro study reported antifungal activity of geranium oil containing geraniol against Candida albicans, and it was found that vaginal washing with geranium oil inhibited fungal growth (IC₅₀ 25-26 mg/mL). The study concluded that geraniol is a potent antifungal agent in vaginal candidiasis.¹⁷⁶ Antimicrobial activity of geraniol has been accounted against a wide range of bacteria and fungi. It has remarkable bactericidal activity, with BA₅₀ value of 1.5 for E. coli. In its gaseous state, it has potent activity against Staphylococcus aureus.¹⁷⁷ Researchers have reported the activity of geraniol with carvacrol and fluconazole against seven different strains of Candida albicans. Geraniol showed a MIC value range 0.5-2 mg/mL for those tested strains. The synergism of geraniol has been reported with fluconazole against resistant fungal species.¹⁷⁷ A study involved antifungal activity of geraniol at the MIC₉₀ value was 16 µg/mL and it was recommended that geraniol as a potent anti-Candida agent.¹⁷⁸ Another study reported anti-trichomonal activity of geraniol against two isolates of Trichomonas vaginalis. Geraniol showed MLC values for TV1 and TV2 isolates as 45 mg/mL and 89.95 mg/mL, respectively. The IC₅₀ values were found out to be 22.5 mg/mL and 45 mg/mL, respectively.¹⁷⁴ The antimicrobial activity of rose oil containing geraniol as a major constituent against Staphylococcus aureus (methicillin-resistant) and mycelial growth of Candida reduced 99 percent of Staphylococcus aureus and inhibited the mycelial growth of Candida albicans within 1 h of geraniol treatment with an IC₅₀ value of 0.00045%.¹⁷⁹ Geraniol’s detailed antibacterial and antifungal activity with related mode of action in vaginitis have been well documented.¹⁷³ Antifungal activity of nanoemulsion containing geraniol with volatile oil and hydrogel containing essential oil with geraniol has been reported against five strains. The hydrogel containing geraniol showed about 64 times better anti-Candida activity.¹⁸⁰ Antibacterial activity of geraniol has been reported for a range of bacteria.¹⁸¹

Researchers have reported several monoterpenes, either individually or in combinations, for their intrinsic antimicrobial activities in vaginal infections, as well as their other pharmacological activities. This review reported a collective number of monoterpenes so that they can further be utilized in combinations to demonstrate their synergistic activities. These antimicrobial activities are against a broad range of micro-organisms, including fungi, bacteria (Gram-positive and Gram-negative bacteria) and other pathogens (Table 2).

Table (2):
Monoterpenes and their antimicrobial activity in vaginal infections (MIC/MBC)