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Phytomedicine

journal homepage: www.elsevier.com/locate/phymed

Original article

Effects ofMarantodes pumilum (Kacip Fatimah) on vaginal pH and expressionof vacoular ATPase and carbonic anhydrase in the vagina of sex-steroiddeficient female rats

Nelli Giribabu, Kamarulzaman Karim, Naguib Salleh⁎

Department of Physiology, Faculty of Medicine, University of Malaya, Kuala Lumpur 50603, Malaysia

A R T I C L E I N F O

Keywords:Marantodes pumilumVaginapH, V-ATPaseCarbonic anhydrases

A B S T R A C T

Background: In sex-steroid deficiency, increased in the pH of vaginal fluid is due to low estrogen levels.Hypothesis: Consumption of Marantodes pumilum leaves helps to ameliorate increased in vaginal fluid pH in sex-steroid deficient condition.Purpose: To investigate changes in vaginal fluid pH and expression of proteins that participate in pH changes i.evacoular (V)-ATPases and carbonic anhydrases (CA) in the vagina following M. pumilum leaves consumption.Methods: Ovariectomized adult female rats were treated orally withM. pumilum leaves extract (MPE) at 100, 250and 500mg/kg.b.w and estradiol at 0.2 µg/kg/b.w for 28 days. At the end of the treatment, vaginal fluid pH wasmeasured in anesthetised rats by using micropH probe. Following sacrificed, levels of V-ATPase and CA proteinsand mRNAs in the vagina were identified by Western blotting and real-time PCR, respectively. Protein dis-tribution was visualized by immunohistochemistry.Results: Administration of MPE causes the pH of vaginal fluid to decrease and expression and distribution ofvaginal V-ATPase A & B and CA II, III, IX, XII and XIII to increase.Conclusions: The decrease in vaginal fluid pH following MPE treatment suggested that this herb has potential tobe used to ameliorate vaginal fluid pH changes in sex-steroid deficient condition.

Introduction

Menopause is a state when changes in the pH of vaginal fluid be-come apparent, whereby pH increases (Panda et al., 2014). The in-creased in fluid pH (>4.5) predisposes vagina to infection. Besides,during menopause, low estrogen levels can cause the glycogen contentin the vagina to decrease and this results in lesser production of lacticacid by vaginal flora i.e Lactobacillus sp, ultimately causing vaginal fluidpH to increase (Mirmonsef et al., 2014). The pH of vaginal fluid de-pends upon balance between H+ and HCO3

− (Frobenius and Bogdan,2015; Shen et al., 2016). H+ secretion via V-ATPase, a multi-subunitprotein which utilizes energy from ATP to transport H+ across the cellinto the lumen could contribute to pH decrease (Karim et al., 2016a,2016b). Besides, increased H+ generation involving carbonic anhy-drase (CA) (Karim et al., 2016a) which exists in several isoforms in-cluding membrane-bound CAIV, CAIX, and CAXII and cytosolic CAI,CAII, CAIII, CAVII, and CAXIII could also lead to pH decrease. The

membrane-bound and cytosolic CA have been reported to be expressedin the female reproductive tract (Hynninen et al., 2004).

Marantodes pumilum is a herb popularly used by the women inSoutheast to involute the uterus post-delivery. Besides, it is also used tofacilitate childbirth, treat irregular menstrual cycle and painful mensesas well as help to promote sexual well-being of women (Chua et al.,2012). In Malaysia, this herb is known as Kacip Fatimah. All parts of theplant are consumed mainly in the form of decoction drink and studieshave found that M. pumilum leaves contains phenols, flavonoids and sa-ponins (Karimi and Jaafar, 2011; Karimi et al., 2016). In view of thereported pharmacological effects of this plant on the female re-productive system and the possibility that it might contain compoundsthat have estrogen-like activities, this study hypothesized that theleaves ofM. pumilum could be used to ameliorate the increase in vaginalfluid pH due to estrogen deficiency. Therefore, this study aims to in-vestigate changes in the vaginal fluid pH and the mechanisms under-lying the pH changes such as changes in expression of V-ATPase and CA

https://doi.org/10.1016/j.phymed.2018.05.018Received 1 June 2017; Received in revised form 27 April 2018; Accepted 28 May 2018

Abbreviations: AC, Adenylyl cyclase; BSA, Bovine serum albumin; CA, Carbonic anhydrases; DAB, 3,3′-Diaminobenzidine; FSH, Follicular stimulating hormone;hECE, Human cervical-vaginal epithelial; HRP, Horseradish peroxidase; PCR, Polymerase chain reaction; V-ATPase, Vacuolar-type H+ - ATPase⁎ Corresponding author.E-mail address: naguibsalleh@um.edu.my (N. Salleh).

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T

in the vagina of sex-steroid deficient animal model representing me-nopause.

Materials and methods

Plant and extraction of M. pumilum leaves

M. pumilum leaves were collected from Tapah district, Perak,Malaysia and were identified and authenticated by a senior botanistfrom botanical institute (Rimba Ilmu), University of Malaya, KualaLumpur, Malaysia. The herb was assigned a voucher specimen numberKLU 46767. M. pumilum leaves were air dried under shade. Then thedried leaves were crushed into powder. 500 g of the powder was ma-cerated in 1.5 l distilled. Maceration was followed by extraction at roomtemperature. The extraction solution obtained was filtered and thenlyophilized by using a freeze-dryer (Telstar, Barcelona, Spain). Theyield was a solid brown material (12.55% W/W) and was then kept in afreezer.

Identification of the bioactive compounds in M. pumilum leaves extract(MPE)

ReagentsThe solvent used in the present study like acetonitrile and methanol

were purchased from Fisher Scientific (HPLC grade; Fisher Scientific,Hampton, NH, USA). The standards gallic acid, rutin, naringin, myr-icetin, quercetin and kaempferol were procured from Sigma-Aldrich (St.Louis, MI, USA). All other chemicals were of analytical grade.

Chromatography and mass conditionsThe chromatographic analysis was performed by using a Flexar

FX15 ultra high-performance liquid chromatograph (UHPLC,PerkinElmer, Inc., Massachusetts, USA), a system coupled to AB Sciex3200 Q-Trap hybrid linear ion trap triple quadruple mass spectrometerequipped with turbo ion spray source. For tandem mass spectrometry(MS/MS) analysis, QTRAP 3200 mass spectrometer was used. Thismachine works based on conventional triple quadrupole ion path withproperties of ion-trap for the third quadrupole. Analyst Software (ver-sion 1.5.2) was used for data collection and processing. The conditionsfor ultra-performance liquid chromatography-tandem mass spectro-meter (UPLC-MS/MS) were listed in Table 1.

The target analytes were detected by using multiple reaction mon-itoring (MRM) negative mode. The ionization voltage was set to 4500 V,source temperature to 500 °C, nebulization and desolvation gas flow at40 psi. MRM dwell time was 50ms, pause between mass range was 5mswith entrance potential (EP) of −10 V. Data analysis, processing andinterpretation were carried out by using Analyst 1.5.2 (AB Sciex,Framingham, MA, USA) and further calculations and quantificationswere done by using Microsoft Excel 2010.

Preparation of standard solutionStock standard solutions were prepared by dissolving 1mg of gallic

acid, rutin, naringin, myricetin, quercetin and kaempferol in 10ml ofmethanol: water (4:1), making a working standards’ concentration of100 µg/ml. These standards were diluted with appropriate solvent toachieve 100 ppb, 200 ppb, 500 ppb, 1000 ppb and 2000 ppb con-centrations.

Sample preparationThe sample solution was prepared by dissolving 100mg MPE with

1ml of 80:20 methanol: water diluent. Then samples were filtered with0.45 um nylon syringe filter and 100 µl sample were further dilutedwith 900ml methanol: water diluent prior to injection into liquidchromatography (LC)-MSMS.

Calibration curves, limits of detection (LOD), and quantification (LOQ)A series of different concentrations of standard solutes were pre-

pared in order to establish calibration curves. Calibration curve wasobtained by using standard mixtures of analytes at the tested con-centration levels of 0.1, 0.2, 0.5, 1 and 2 ppm. The curve was drawn byplotting peak area (y) versus corresponding concentrations (x, ppm).Limit of detection (LOD) and limit of quantification (LOQ) were de-termined by using the following equations. LOD (3.3× Sy.x)/S; LOQ(10× Sy.x)/S (Sy.x) is standard deviation of residuals from line; S isslope). The results are listed in Table 2 & Fig 1. All calibration curvesindicated good linearity with correlation coefficients (r2) ranging from0.9989 to 0.9999 and are within the test ranges.

Animals

Female Sprague Dawley (SD) rats, aged 3 months with body weightbetween 180–210 g were housed in cages and acclimatized to stan-dardized lab conditions (temperature 23 ± 2 °C, humidity 50 ± 5%and 12 h light-dark cycles). The rats were fed with standard rodent foodpellet (Harlan Diet, Rossdoff, Germany) and had free access to tap waterad libitum. All experimental procedures were approved by InstitutionalAnimal Care and Use Ethics Committee, University of Malaya.

Ovariectomy, drug treatment, vaginal pH probing and vaginaltissue harvesting

Bilateral ovariectomy was performed to eliminate the effect of en-dogenous sex-steroids, following the procedures as previously described(Shahzad et al., 2014). Three weeks after ovariectomy, treatment wascommenced and rats (n=6 per group) were divided into the followinggroups:

Table 1Detailed UHPLC system and chromatographic conditions.

Liquid chromatography conditions

Pump type Flexar FX-15Column Phenomenex Synergy RP C18 column

(100A, 100mm x 3uM x 2.0mm)Mobile phase A: Water with 0.1% formic acid

B: Acetonitrile with 0.1% formic acidFlow rate 400 ul/minInjection volume 20 µlGradient Time (min) %A %B

0.1 95 53.0 5 955.0 5 955.1 95 57.0 95 5

Software Analyst 1.5.2 (AB Sciex, Framingham,MA, USA).

Mass spectrometerconditions

ESI voltage (Negative) −4500 VDesolvation gas 40 psiNebulization gas 40 psiSource temperature 500 °CDwell time for each MRM

transition50 ms

Table 2Quantification of table for standards.

Analytes r2 LOD ng/ml LOQ ng/ml

Gallic acid 0.9964 0.75 2.5Rutin 0.9993 0.15 0.5Naringin 0.9954 0.15 0.5Myricetin 0.9992 1.0 3.3Quercetin 0.9975 3.0 10.0Kaempferol 0.9983 0.6 2.0

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C-ovariectomized control (vehicle-treated)100MPE- treated with 100mg/kg/b.w. MPE250MPE- treated with 250mg/kg/b.w. MPE500MPE- treated with 500mg/kg/b.w. MPEE - positive control (treated with 0.2 µg/kg/b.w. estradiol)

MPE was dissolved in distilled water while estradiol was dissolvedin corn oil. Our preliminary studies showed that corn oil had no effecton vaginal fluid pH changes and the expression of proteins involved invaginal fluid acidification (data not shown). All drugs were given orallyby using oral gavage tube for 28 days. At the end of the treatment,animals were anesthetistized by using ketamine and xylazine anesthesiaand micropH probe (Hanna instrument, Singapore) was inserted intothe vagina of anesthetized rats to directly measure the pH. Followingthat, animals were sacrificed and vagina was harvested for biochemicaland molecular biological/ immunohistological analyses.

Quantification of RNA by using real-time PCR (qPCR)

One step Real Time PCR was performed to evaluate gene expressionwith application of TaqMan®RNA-to-CT 2-Step kit, following themethod as previously described (Loh et al., 2017). TaqMan® primerswere used and were pre-designed (Applied Biosystems, Foster City, CA,USA). The primers used were as follows: CAII (Rn00570700_m1), CAIII(Rn00695939_m1), CAIX (Rn01764733_m1), CAXII (Rn01418250_m1),CAXIII (Rn01493656_g1), V-ATPase A1 (Rn01763276_m1), V-ATPaseB1/2 (Rn01765558_m1), GAPDH (Rn0177576_g) and Hprt(Rn01527840_m1). All experiments were carried out in triplicates and

data were analysed according to comparative Ct (2−ΔΔCt) method.The relative quantity of target in each sample was determined by

comparing the normalized target quantity of the sample to averagenormalized quantity of the references. Then the amplified region ofcDNA was probed by using fluorescence-labelled probe. Validation ofthe assay was performed in silico by using whole rat genome and in-vitroby using whole rat cDNA (Applied Biosystems, Foster City, CA, USA).

Immunofluorescence staining to visualize protein distribution

Histology and immunofluorescence were performed following themethods as previously described (Giribabu et al., 2017). In brief, sec-tions were blocked with appropriate normal serum (Santa Cruz, CA,USA) prior to incubation with V-ATPase A1 (sc-28801), V-ATPase B1/2(sc-20943), CAII (sc-17244), CAIII (sc-50715), CAIX (sc-25600), CAXII(sc-25601) and CAXIII (sc-67334) at a dilution of 1:100 in PBS with1.5% normal blocking serum at room temperature for 1 h. After threetimes rinsing with PBS, sections were incubated with IgG–fluor-ochrome-conjugated secondary antibody (Santa Cruz, CA, USA), at adilution of 1:250 in PBS with 1.5% normal blocking serum at roomtemperature for 45min. The slides were rinsed three times with PBSand were mounted with Ultra Cruz mounting medium (Santa Cruz, CA,USA), counterstained with DAPI to visualize the nuclei.

Western blotting for quantification of protein expression level

Western blotting was performed according to the methods as pre-viously described (Shahzad et al., 2017). Once proteins were

Fig. 1. Calibration curve obtained with (A) gallic acid; (B) rutin; (C) naringin; (D) quercetin; (E) myricetin and (F) kaempferol standards by using UHPLC.

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transferred, membranes were exposed to the same primary antibodiesas above. β-actin primary antibody was used as loading control. Fol-lowing incubation with the primary antibodies, membranes were thenincubated with appropriate horseradish peroxidase (HRP) conjugatedsecondary antibodies.

Photos of the blots were captured by using gel documentationsystem and density of each band was determined by using Image Jsoftware (version 1.46j; National Institutes of Health, Bethesda, MD,USA). The ratio of target protein/β-actin was calculated and was con-sidered as the expression levels of the targets. The average ratio of theband was obtained from four different membranes representing fourdifferent animals per group of treatment.

Statistical analysis

Statistical differences were evaluated by student's t-test and one-way ANOVA. p value <0.05 was considered as significant. Tukeys’post-hoc statistical power analysis was performed and all values ob-tained were >0.85 which indicate adequate sample size.

Results

Bioactive compounds in MPE

A typical UHPLC chromatogram of all standard mixture was pre-sented in Fig. 1. The typical retention times of 0.98, 3.46, 3.52, 3.84,4.11 and 4.34min were observed for gallic acid, rutin, naringin, myr-icetin, quercetin and kaempferol, respectively (Fig. 2). These retentiontimes reflect expectations based on the chemical structures of thesecompounds.

By using LCMS-MS, gallic acid, rutin, myricetin, quercetin andkaempferol were identified in MPE. Their levels were quantified basedon ion transition and retention times (Table 3). The MS/MS spectra,fragmentation scheme of the five bioactive compounds are shown inFig. 3. In Fig. 3A, [M−H ]− ion in MS spectrum was observed at m/z168.98, and was comparable to the library spectrum of gallic acid ac-quired from green tea (MS spectrum=m/z 169.01). In Fig. 3B, 3C, 3Dand 3E, the compounds identified were rutin with m/z 609.15[M−H]−, myricetin with m/z 317.03 [M−H]−, quercetin with m/z301.11 [M−H]− and kaempferol with m/z 285.00 [M−H]− (ascompared to the respective library spectrum), respectively.

Table 4 shows fit values (Fit), reverse fit value (RevFit) and purity ofthe bioactive compounds from the library spectrum. The Fit providesinformation about similarity of the signals in the reference spectrumwith the unknown compound, whereas RevFit reflects similarity of thesignals in the registered spectrum with those in the reference spectrum.Purity gives information for combination of both Fit and RevFit valuesand measures the unmatched peaks between the registered spectra andthe library. For gallic acid, Fit value is 92.41, RevFit value is 95.53 andpurity is 92.04. For rutin, Fit value is 91.18, RevFit value is 89.15 andpurity is 86.01. For myricetin, Fit value is 82.97, RevFit value is 85.38and purity is 75.99. Meanwhile, quercetin has Fit value of 85.29, RevFitvalue of 77.52 and purity of 71.26 and for kaempferol, the Fit value is82.99, RevFit value is 75.1 and purity is 72.49.

Quantitative analysis of the bioactive compounds in MPE

Gallic acid (688.4 µg/g) was present in highest amount followed byquercetin (85.36 µg/g), rutin (37.7 µg/g), myricetin (8.8 µg/g),kaempferol (3.2 µg/g). No naringin peak was observed. MS spectra andMRM extracted ion chromatogram of six standards are shown in Fig. 4and Table 5, respectively.

Changes in vaginal fluid pH

Vaginal fluid pH of ovariectomised rats was found to be

approximately 5.78 ± 0.42 (Table 6), whereas following 28 daystreatment with 250, 500mg/kg/b.w. MPE and E, a decrease in vaginalfluid pH was observed (p<0.05).

Effects of MPE on expression and distribution of V-ATPase-A in the vaginaof OVX rats

In Fig. 5A, expression level of V-ATPase A protein was lowest in thevagina of ovariectomized control rats, whereas treatment with 100, 250and 500mg/kg/b.w. MPE and E resulted in higher expression of thisprotein isoform in the vagina. In the meantime, low distribution of V-ATPase A was observed in the vagina of ovariectomized control rats(Fig. 5B). Treatment with 100, 250 and 500mg/kg/b.w. MPE and Eresulted in a relatively higher distribution of this protein isoform.Consistent with the changes in protein expression, low level of V-AT-Pase A mRNA was observed in the vagina of ovariectomized controlrats, whereas treatment with 100, 250 500mg/kg/b.w. MPE and Eresulted in a significant increase in V-ATPase A mRNA levels (Fig. 5C).

Effects of MPE on expression and distribution of V-ATPase B in the vagina ofOVX rats

The levels of V-ATPase B in the vagina was lowest in ovariectomizedcontrol rats, whereas 28 days treatment with 100, 250 and 500mg/kg/b.w. MPE and E resulted in higher expression of this protein isoform(Fig. 6A). In the meantime, low distribution of V-ATPase B was ob-served in the vagina of ovariectomized control rats, whereas treatmentwith 100, 250 500mg/kg/b.w. MPE and E resulted in relatively higherV-ATPase B distribution (Fig. 6B). Similarly, low level of V-ATPase BmRNA was observed in the vagina of ovariectomized control rats, withtreatment with 100, 250 500mg/kg/b.w. MPE and E resulted in sig-nificantly higher V-ATPase B mRNA levels (Fig. 6C).

Effects of MPE on expression and distribution of CA isofenzymes in thevagina of OVX rats

CA IIExpression levels of CAII protein in the vagina of ovariectomized

control rats were lower than ovariectomized rats receiving 100, 250and 500mg/kg/b.w. MPE and E treatments (Fig. 7A) (p<0.05). In themeantime, low distribution of CAII protein was observed in the vaginaof ovariectomized control rats, whereas treatment with 100, 250500mg/kg/b.w. MPE and E resulted in a relatively higher CAII proteindistribution (Fig. 7B). Similarly, low level of CaII mRNA was observedin the vagina of ovariectomized control rats, with treatment with 100,250 500mg/kg/b.w. MPE and E resulted in significant increase in CaIImRNA levels (Fig. 7C).

CA IIIExpression levels of CAIII protein were lowest in the vagina of

ovariectomized control rats, whereas treatment with 100, 250 and500mg/kg/b.w. MPE and E resulted in expression level of this iso-enzyme to increase (Fig. 8A) (p<0.05). In the meantime, low dis-tribution of CAIII protein was observed in the vagina of ovariectomizedcontrol rats, whereas a relatively higher distribution was observedfollowing treatment with 100, 250 500mg/kg/b.w. MPE and E(Fig. 8B). Likewise, mRNA results confirmed that low CaIIImRNA levelswere observed in the vagina of control rats, whereas treatment with100, 250 500mg/kg/b.w. MPE and E resulted in higher CaIII mRNAlevels (p<0.05) (Fig. 8C).

CAIXExpression level of CAIX protein was low in the vagina of ovar-

iectomized control rats, whereas treatment with 100, 250 and 500mg/kg/b.w. MPE and E resulted in expression level of this protein to sig-nificantly decreased (Fig. 9A) (p<0.05). In the meantime, low dis-tribution of CAIX was observed in vagina of ovariectomized controlrats, whereas treatment with 100, 250 500mg/kg/b.w. MPE and E

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resulted in a relatively higher CAIX distribution (Fig. 9B). Similarly,mRNA results show that lower CaIX mRNA levels were observed in thevagina of OVX rats, whereas 28 days treatment with 100, 250 500mg/kg/b.w. MPE and E resulted in CaIX mRNA levels to increase (Fig. 9C).

CAXIICAXII protein expression levels were low in the vagina of ovar-

iectomized control rats, whereas treatment with 100, 250 and 500mg/kg/b.w. MPE and E caused the levels of this protein to significantlyincreased (Fig. 10A) (p<0.05). In the meantime, low distribution of

CAXII isoenzyme was observed in the vagina of ovariectomized controlrats, whereas treatment with 100, 250 500mg/kg/b.w. MPE and Eresulted in CAXII distribution to be relatively higher (Fig. 10B). Simi-larly, low level of CAXII mRNA was observed in the vagina of ovar-iectomized control rats, with treatment with 100, 250 500mg/kg/b.w.MPE and E resulted in significantly higher CAXII mRNA levels(Fig. 10C).

CA XIIIExpression level of CAXIII was lowest in the vagina of ovar-

iectomized control rats, whereas treatment with 100, 250 and 500mg/kg/b.w. MPE and E resulted in a significant increase in CAXIII proteinexpression levels (Fig. 11A) (p<0.05). In the meantime, low dis-tribution of CAXIII isoenzyme was observed in the vagina of ovar-iectomized control rats, whereas treatment with 100, 250 500mg/kg/b.w. MPE and E resulted in a relatively higher CAXIII distribution(Fig. 11B). Similarly, mRNA results shows low CaXIII mRNA levels invagina of ovariectomized control rats, whereas 28 days treatment with100, 250 500mg/kg/b.w. MPE and E resulted in higher CaXIII mRNAlevels (Fig. 11C).

Fig. 2. MRM extracted chromatogram of standards bioactive compounds mixture obtained by UHPLC-MS/MS in negative mode. (A) gallic acid; (B) rutin; (C)naringin; (D) quercetin; (E) myricetin and (F) kaempferol.

Table 3Retention time and main mass parameters of 6 constituents in MPE by UHPLCwith MRM analysis.

Peak RT (min) Analyte Molecularformula

Molecularweight (Da)

[M-H]−(m/z)

1 0.98 Gallic acid C7H6O5 170.12 168.982 3.46 Rutin C27H30O16 610.52 609.153 3.52 Myricetin C15H10O8 318.23 317.034 3.84 Quercetin C15H10O7 302.23 301.115 4.34 Kaempferol C15H10O6 286.23 285.00

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Discussion

The findings from this study indicated that treatment of sex-steroiddeficient ovariectomized rats with MPE restored the vaginal fluidacidity most probably due to an increase in the generation of H+ asreflected by increased levels of CA isoenzymes as well as could be due

to an increased extrusion of H+ as reflected by increased levels of V-ATPase isoforms in the vagina. This study showed that MPE extractcontains gallic acid, rutin, myricetin, quercetin and kaempferol, withgallic acid was present in the highest amount. Due to its quantity, gallicacid could be responsible for the observed pharmacological effects.However, the effect of other compounds could not be ruled out. As noprevious studies revealed the trophic effect of these compounds on thevagina, our study could be the first to identify their effects.

Maintaining the normal vaginal fluid acidity is important for va-ginal functions (Wagner and Levin, 1984). Estrogen has been shownessential in maintaining the acidity of vaginal fluid, in which its defi-ciency could cause vaginal fluid pH to increase (Gorodeski et al., 2005).After menopause, low estrogen levels have been found to increase therisk of vaginal and urinary tract infections (Raz and Stamm, 1993).Thus, restoring the estrogen levels near normal either via exogenouslyadministering estrogen or estrogen-like compounds are important to

Fig. 3. MS/MS Spectra of reference analytes with library matching (A) gallic acid (B) rutin (C) myricetin (D) quercetin (E) kaempferol of samples compared withstandards MSMS spectra.

Table 4The confirmation results of garlic acid; rutin; myricetin; quercetin andkaempferol in MPE, registered in the MRM mode.

S. No Name FIT RevFit Purity

1 Gallic acid 92.41 95.53 92.042 Rutin 91.18 89.15 86.013 Myricetin 82.97 85.38 75.994 Quercetin 85.29 77.52 71.265 Kaempferol 82.99 75.1 72.49

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Fig. 4. MRM extracted ion chromatogram of reference analytes (A) gallic acid (B) rutin (C) naringin (no peak) (D) myricetin (E) quercetin (F) kaempferol.

Table 5Quantitative analysis bioactive compounds in MPE.

S. No Name Concentration (µg/g)

1 Gallic acid 688.42 Rutin 37.73 Naringin No peak4 Myricetin 8.85 Quercetin 85.366 Kaempferol 3.2

Table 6Vaginal pH in MPE and E treated ovariectomisedrats.

Treatment Vaginal pH

C 5.78 ± 0.42MPE 100 5.43* ± 0.42MPE 250 5.17* ± 0.35MPE 500 4.68* ± 0.47E 4.53* ± 0.58

⁎ p<0.05 compared to C, Data were expressedas Mean ± SEM with n=6 per group.

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overcome complications related to low estrogen levels. We have shownthat oral treatment with MPE ameliorates the increase in vaginal fluidpH in sex-steroid deficient state, and this suggested that MPE could beused to overcome the abnormal vaginal milieu in E deficient condition.In view of the effect of MPE was almost similar to E, there is a possi-bility that the compounds present in MPE might possess estrogen likeeffects. Quercetin and kaempferol are the two known estrogen-likecompounds (Palma-Duran et al., 2015) which could produce effectssimilar to estrogen. Besides, rutin (Horcajada‐Molteni et al., 2000) andmyricetin (Aquila et al., 2013) which were also reported to possessestrogen-like activities might also be capable of producing the vaginaleffects resembles that of estrogen.

In this study, the mechanisms underlying the lesser increase in va-ginal fluid pH following MPE and E treatments have been proposed.MPE and E were found to up-regulate the expression of V-ATPase A & B

in the vagina. Furthermore, the findings from this study found that V-ATPase was expressed at the apical membrane of the vaginal epithe-lium, which suggest that this protein could be involved in H+ extrusioninto the vaginal lumen. Our findings were similar to the reported apicaldistribution of V-ATPase in human cervical-vaginal epithelial cells(hECE) (Gorodeski et al., 2005). In the meantime, our findings whichindicate V-ATPase expression in the vagina was increased under theinfluence of E was consistent with a report that shows estrogen depri-vation attenuates while estrogen treatment augments active H+ secre-tion into the vagina of post-menopausal women (Gorodeski, 2005).Therefore, up-regulation of V-ATPase A and B by MPE could be thelikely mechanisms that restore low vaginal fluid pH in sex-steroid de-ficient condition.

The other mechanisms that might also contribute towards the lesserincrease in vaginal fluid pH following E and MPE treatments are the up-

Fig. 5. (A) Representative immunofluorescence imagesshowing distribution of V-ATPase A in vagina. Green fluores-cence signals indicate V-ATPase A distribution site. (B)Representative Western blot V-ATPase A protein bands andratio of V-ATPase A/β-actin proteins in vagina. (C)Representative V-ATPase A mRNA levels in vagina. *p<0.05compared to C. Data were expressed as mean ± S.E.M from 6animals. Scale bar represents 50 µm. (For interpretation of thereferences to color in this figure legend, the reader is referredto the web version of this article.)

Fig. 6. (A) Representative immunofluorescence imagesshowing distribution of V-ATPase B in vagina. Red fluores-cence signals indicate V-ATPase B distribution site. (B)Representative Western blot V-ATPase B protein bands andratio of V-ATPase B/β-actin proteins in vagina. (C)Representative V-ATPase B mRNA levels in vagina. *p<0.05compared to C. Data were expressed as mean ± S.E.M from 6animals. Scale bar represents 50 µm. (For interpretation of thereferences to color in this figure legend, the reader is referredto the web version of this article.)

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regulation of CA isoenzymes i.e. CAII, CAIII, CAIX, CAXII and CAXIII,which are known to participate in H+ generation. To the best of ourknowledge, this study is the first to show that the levels of CA iso-enzymes in the vagina were increased under E influence. The im-portance of CA isoenzymes in general in luminal fluid acidification hasbeen described. CAII plays important role in the conversion of CO2 andH2O to H2CO3, which then dissociates into H+ and HCO3

−

(Boron, 2010). CAII has also been shown to interact with HCO3−

transporter including Cl−/HCO3− exchanger (Sowah and Casey, 2011).

These might affect the luminal fluid pH i.e. causing luminal fluid pH to

decrease. CAXII, which is expressed at the apical membrane of theepithelium could participate in the conversion of H+ and HCO3

− in thelumen into CO2 and H2O, where both diffuse back into the cell to ensurecontinuous H+ and HCO3 generation (Karim et al., 2016a). We haveshown that in vagina, CAXII expression is under the influence of E. Onthe other hand, in the uterus, expression of this isoenzyme is under theinfluence of progesterone (Karim et al., 2016a). Besides CAII and CAXII,other CA isoenzymes including CAIII, IX and XIII which are also ex-pressed in the vagina might also be involve in the acidification of va-ginal fluid. The ability of MPE to up-regulate expression of these CA

Fig. 7. (A) Representative immunofluorescence imagesshowing distribution of CAII in vagina. Green fluorescencesignals indicate CAII distribution site. (B) RepresentativeWestern blot CAII protein bands and ratio of CAII/β-actinproteins in vagina. (C) Representative CaII mRNA levels invagina. *p<0.05 compared to C. Data were expressed asmean ± S.E.M from 6 animals. Scale bar represents 50 µm.(For interpretation of the references to color in this figure le-gend, the reader is referred to the web version of this article.)

Fig. 8. (A) Representative immunofluorescence imagesshowing distribution of CAIII in vagina. Red fluorescencesignals indicate CAIII distribution site. (B) RepresentativeWestern blot CAIII protein bands and ratio of CAIII/β-actinproteins in vagina. (C) Representative CaIII mRNA levels invagina. *p<0.05 compared to C. Data were expressed asmean ± S.E.M from 6 animals. Scale bar represents 50 µm.(For interpretation of the references to color in this figure le-gend, the reader is referred to the web version of this article.)

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isoenzymes in the vagina suggested that H+ generation could be sti-mulated, leading to acidification of the vaginal fluid.

In conclusions, MPE effects in causing lesser increase in vaginalfluid pH could partly restore the vaginal acidity in sex-steroid deficientstate. MPE could exert its effect via enhancing both H+ generation aswell as H+ extrusion into the vaginal lumen. Although gallic acid is themain constituent in MPE, the involvement of rutin, myricetin, quercetinand kaempferol in mediating these effects are likely due to their

estrogen-like activities.

Acknowledgment

This study was supported by NKEA grant, Ministry of Agricultureand Agro-based Industries, Malaysia, grant number (GA014-2014).

Fig. 9. (A) Representative immunofluorescence imagesshowing distribution of CAIX in vagina. Green fluorescencesignals indicate CAIX distribution site. (B) RepresentativeWestern blot CAII protein bands and ratio of CAIX/β-actinproteins in vagina. (C) Representative CaIX mRNA levels invagina. *p<0.05 compared to C. Data were expressed asmean ± S.E.M from 6 animals. Scale bar represents 50 µm.(For interpretation of the references to color in this figure le-gend, the reader is referred to the web version of this article.)

Fig. 10. (A) Representative immunofluorescence imagesshowing distribution of CAXII in vagina. Red fluorescencesignals indicate CAXII distribution site. (B) RepresentativeWestern blot CAXII protein bands and ratio of CAXII/β-actinproteins in vagina. (C) Representative CaXII mRNA levels invagina. *p<0.05 compared to C. Data were expressed asmean ± S.E.M from 6 animals. Scale bar represents 50 µm.(For interpretation of the references to color in this figure le-gend, the reader is referred to the web version of this article.)

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Conflict of interest

We wish to confirm that there are no known conflicts of interestassociated with this publication and there has been no significant fi-nancial support for this work that could have influenced its outcome.

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Fig 11. (A) Representative immunofluorescence imagesshowing distribution of CAXIII in vagina. Green fluorescencesignals indicate CAXIII distribution site. (B) RepresentativeWestern blot CAXIII protein bands and ratio of CAXIII/β-actinproteins in vagina. (C) Representative CaXIII mRNA levels invagina. *p<0.05 compared to C. Data were expressed asmean ± S.E.M from 6 animals. Scale bar represents 50 µm.(For interpretation of the references to color in this figure le-gend, the reader is referred to the web version of this article.)

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