









































Influence of puerarin, paeoniflorin, and menthol on structure and barrier function of tight junctions in MDCK and MDCK-MDR1 Cells


CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2024 | Vol 7 | Issue 1 

 

    ISSN : 2693 6356 

2024 | Vol 1 | Issue 1 

  
 

 

 

 

Influence of puerarin, paeoniflorin, and 

menthol on structure and barrier function of 

tight junctions in MDCK and MDCK-MDR 

1 Cells 

Lin Zhang, Shouying Duao*, Yang Lui **, Chang Lqiu, Huichao, Bing 
Yang, Bai, Pengyue 

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China 

 

 

 

Introduction 
 

Medications designed to target certain areas of the 

brain either do not reach those areas or do not reach 

enough concentrations once they do. The blood-

brain barrier (BBB) governs and controls the 

molecular permeation between the brain's 

periphery and interior, and when the levels of drugs 

administered to the brain are inadequate, it is often 

due to the barrier function of the brain's capillary 

endothelial cells.1e4 One of the best cell models to 

mimic the BBB in vitro is MadineDarby canine 

kidney epithelial (MDCK) cells or MDCK cells 

transfected with the human multidrug resistance 1 

(MDR1) gene (MDCK-MDR1).5, 6 The BBB is a 

barrier system that keeps the brain's internal 

environment steady and supports appropriate brain 

processes. It consists of a single layer of brain 

capillary endothelial cells joined by tight junctions 

(TJs). When it comes to molecules that go via the 

paracellular route, TJs are crucial for molecular 

permeability of the BBB. In addition to defining 

cell polarity by separating the apical and 

basolateral cell surface regions, they also serve as 

a barrier and fence.Upon examination under 

electron microscopy, transmembrane junctions 

(TMJs)—which include integral membrane 

proteins—are seen as a region of tightly connected 

 

Abstract—  

To find out how the drugs cross the blood-brain barrier (BBB), this study examined how puerarin, paeoniflorin, and 

menthol affected the structure and function of tight junctions (TJs) in MDCK and MDCK-multi-drug resistance 1 

(MDR1) cells.  

The cells were first treated with puerarin, paeoniflorin, and menthol. Then, they were stained with occludin, claudin-

1, and F-actin using immunohistochemistry. Next, laser-scanning confocal microscopy was used to examine the cells. 

An epithelial voltage voltmeter was used to assess transepithelial electrical resistance (TEER), and ImageJ software 

was used to analyse the average optical density (AOD) of the protein immunofluorescence pictures. 

The results showed that tight junction proteins treated with puerarin and paeoniflorin were visible under confocal 

microscopy, but menthol reduced their expression. Similarly, the menthol group's AOD value was downregulated, 

while the control group's value was not different from the AOD values of cells treated with either puerarin or 

paeoniflorin, or both. After three hours, the TEER of cells that were not exposed to menthol were comparable to those 

of the control group, but treatment with  

 

With a p-value less than.05, menthol considerably reduced the TEER score. And menthol's TEER-lowering effects 

on MDCK cells were noticed before those on MDCK-MDR1 cells.  

Ultimately, it seems that menthol, in contrast to puerarin and paeoniflorin, has the potential to decrease the barrier 

function of TJs, which in turn enhances paracellular transport and drug penetration of the blood-

brain barrier. 

 



membranes at the apex of neighbouring cells that 

face the lumen.A variety of transient junction 

proteins, such as occludin, claudins, zona 

occuldens protein 1 (ZO-1), and F-actin, are 

expressed by MDCK and MDCK-MDR1 cells 

because their transient junction structures are 

similar to those of brain capillary endothelial cells. 

Through their hydrophobic barrier function and 

intramembranous fence, TJs limit medication 

delivery to underlying tissues and reduce drug 

permeability of the blood-brain barrier (BBB). 

Some medications may change the structure and 

characteristics of transmembrane junctions (TMJs) 

by acting on TJ proteins to make them more 

permeable to the brain or to other drugs, or both. 

This is because TJ tightness is dictated by the 

protein composition of the webs of the related 

proteinaceous filaments (called strands). In order to 

create medications that target the brain and 

effectively cure disorders, it is crucial to research 

how chemicals affect TJ proteins and how they 

may cross the blood-brain barrier (BBB).  

An essential aspect of the folkloric therapeutic 

practice is the prescription of tongqiaosanyu, a 

traditional medicinal formula from China. Kudzu 

root (Pueraria lobata), white peony root (Paeonia 

albiflora), and mint (Mentha hap-localyx Briq.) are 

among the plants that make up the mix. 

Tongqiaosanyu is a treatment for stroke in China. 

The primary active ingredients have been 

determined to be menthol, pseudoniflorin, and 

pierin by pharmacodynamic screening. “Puerarin 

(8-[beta-D-glucopyranosyl] “-7-hydroxy-3-[4- 

hydroxyphenyl]”Traditional Chinese Medicine 

(TCM) has made extensive use of -4H-1-

benzopyran-4-one (C21H20O9), the main 

isoflavone glycoside extracted from kudzu root, for 

the treatment of ischemic stroke and cardiovascular 

diseases.17 with e19 5-Beta-[{Benzoyloxy} 

methyl] paeoniflorin5,4-dioxacyclobuta[cd] 

tetrahydro-5-hydroxy-2-methyl-2,5-methano-1H-

[2H] pentalen-1alphaThe monoterpene glucoside 

iso-lated from white peony root is known as -yl-

beta-D-glucopyranoside (C23H28O11). It 

enhances glucose uptake and possesses 

neuroprotective, anti-inflammatory, anti-allergy, 

and antihyperglycemic properties.20 The chemical 

formula for menthol is C10H20O, and it is a 

primary 

component of peppermint oil, which increases 

blood-brain barrier (BBB) permeation and has 

excitatory effects on the central nervous system 

(CNS).21 Analytical methodologies for the 

primary active constituents were developed in our 

prior examination of Tongqiaosanyu. Furthermore, 

we investigated the in vivo pharmacokinetic 

behaviour of Tongqiaosanyu, its compatibility with 

other medications, and several methods of 

administration. The processes by which puerarin 

crosses the blood-brain barrier (BBB) have been 

documented, as have its cytotoxicity and transport 

in MDCK and MDCK-MDR1 cells. Nevertheless, 

it is still not known how these chemicals 

paracellularly cross the blood-brain barrier (BBB), 

particularly how they affect protein structure and 

barrier function. Hence, we aimed to find out how 

this formula TJs worked and what part each 

chemical played in transport by simulating the 

BBB using MDCK and MDCK-MDR1 cells. 

Researchers looked at how these medications 

entered the brain and how certain medicines 

improved their penetration.  

 

Materials and methods 
 

Materials 

 
Puerarin, paeoniflorin, and menthol were purchased 

from the National Institute for Food and Drug Control 

(Beijing, China). Polyester (PET) cell culture inserts and 

12-well plates (12-mm diameter, 0.4-mm pore size) 

were obtained from Corning Life Sciences (Corning, NY, 

USA). Rabbit anti- occludin antibody (ab31721) was 

obtained from Abcam Shanghai (Shanghai, China). 

Mouse anti-claudin-1 antibody (2H10D10) was purchased 

from Invitrogen (Camarillo, CA, USA). Anti-rabbit IgG -

tetramethylrhodamineisothiocyanate (TRITC) conjugate 

was purchased from ZSG-BIO (Beijing, China). Anti-

mouse-fluoresceinisothiocyanate (FITC) con- jugated 

antibody was purchased from Kangwei Century 

Biotechnology (Beijing, China). Acti-stain 488 phalloidin 

staining for F-actin was obtained from Cytoskeleton 

(Den- ver, CO, USA). 

 

Cell culture 

 
MDCK and MDCK-MDR1 cells were generously provided 

by Dr. Zeng (Zhejiang University, China). Both cell lines 

were cultured with Dulbecco’s modified Eagle’s media 

(DMEM) supplemented with 10% heat-inactivated fetal 

bovine serum (FBS, Gibco, Fremont, CA, USA), 100 U/mL 

penicillin, and 0.1 mg/mL streptomycin in a humidified 

atmosphere of 

5% CO2 at 37◦C. The medium was replaced with fresh 

me- dium every other day until the cells reached 
approximately 

90% confluence. 

 

Grouping and drug administration 

 



CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2024 | Vol 7 | Issue 1 

 

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2024 | Vol 1 | Issue 1 

  
 

In a previous study,22 the cytotoxicity of puerarin, 

paeoni- florin, and menthol in MDCK and MDCK-MDR1 

cells using 3- (4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetr-azolium bro- mide (MTT) assay. Results 

showed puerarin, paeoniflorin, and menthol were not 

cytotoxic at concentration ranges of 

0e0.3 mg/mL, 0e0.4 mg/mL, and 0e0.04 mg/mL, respec- 

tively. Furthermore, puerarin plus paeoniflorin (1:0.4 w/v, 

PP), puerarin plus menthol (1:0.5 w/v, PM), and puerarin 

plus paeoniflorin plus menthol (1:0.4:0.5, w/w/w, PPM) 

groups exhibited no cytotoxicity at concentration ranges of 

0e0.2 mg/mL (paeoniflorin), 0e0.05 mg/mL (menthol), and 

0e0.04 mg/mL (menthol), respectively. Based on the re- 

sults of MTT assay, this study included the following six 

treatment groups: puerarin, paeoniflorin, and menthol 

(0.1 mg/mL, 0.04 mg/mL, and 0.05 mg/mL, respectively), 

as well as the PP group (0.1 mg/mL puerarin and 0.04 mg/ 

mL paeoniflorin, respectively), PM group (0.1 mg/mL 

puerarin and 0.05 mg/mL menthol, respectively), and PPM 

group (0.1 mg/mL puerarin, 0.04 mg/mL paeoniflorin, and 

0.05 mg/mL menthol, respectively) groups. 

 

Immunofluorescence microscopy 

For immunocytochemical analysis, the cells were seeded on 

15 mm coated glass coverslips. When cell fusion attained 

approximately 80e90%, the regular media were replaced 

with media containing the various compounds (puerarin, 

paeoniflorin, or menthol at 0.1 mg/mL, 0.04 mg/mL and 

the previously described drug combinations was measured 

at 30, 60, 90,120,150, and 180 min. TEER was measured 

immediately following drug treatment (0 min) and was set 

to 100%, with all the other values calculated and expressed 

relative to this value. Finally, TEER at each time point was 

compared to that of the control group and statistically 

analyzed. 

 

Data analysis and statistics 

 
Immunofluorescence images were analyzed semi- 

quantitatively using ImageJ software based on the 

following equations: 

IntDen 
Z 

Area 
(1)

 

where IntDen is the integrated optical density (IOD) of 

the image, Area is the region of fluorescence in the 

image, and AOD is the average optical density. 

Percentage AOD (%) of the TJ proteins was calculated 

using the following equation: 

AODsample 

0.05 mg/mL, respectively) and the cells were further 

cultured for 3 h, and then fixed with cold 4% para-

elative AOD (%)Z 

formaldehyde for 30 min. After washing thrice for 

5 min each with phosphate-buffered saline (PBS), the cov- 

erslips were incubated for 1 h with goat serum as the 

blocking buffer. Then, after rinsing with PBS thrice, some 

coverslips were incubated with polyclonal anti-occludin or 

monoclonal anti-claudin-1primary antibodies (1:100, each) 

at 4◦Covernight followed by incubation with TRITC- 

conjugated anti-rabbit IgG or FITC-conjugated anti-mouse 

IgG. Other cells were only stained with Acti-stain 488 
phalloidin stain (1:150) at 25◦C for 30 min. Cell nuclei were 

counterstained with 4', 6-diamidino-2-phenylindole (DAPI, 
Beijing Solarbio Science and Technology, Beijing, China). 

The cells were visualized using an inverted fluorescence 

microscope equipped with appropriate filters (Olympus, 

Tokyo, Japan). Images were acquired using a laser scanning 

confocal microscope and accompanying analysis software. 

Fluorescence intensity of the images was measured using 

ImageJ software (National Institutes of Health, Bethesda, 

MD, USA). 

 

Measurement of transepithelial electrical 

resistance 

 
To investigate changes in the TJs barrier function of the 

MDCK and MDCK-MDR1 cells, transepithelial electrical 

resistance (TEER) of the monolayers was detected using the 

EVOM epithelial voltohmmeter (EMD Millipore, Billerica, 

MA, USA). Both cell types were cultured separately 

on0.4 mm pore size filters of 12 mm Transwellsand grown to 

confluence. Then, 0.5 mL and 1.5 mL of drug solution and 

Hank’s basic salt solution (HBSS) were added to the apical 

(A) side and basolateral (B) side, respectively to simulate 

A / B transport while in a parallel experiment, B / A 

transport was simulated by reversing the order of the drug 

solution and HBSS. TEER of untreated or puerarin-, paeo- 

niflorin-, and menthol-treated cells or those treated with 

where AODsample and AODcontrol are the AODs of the sample 

and control, respectively. 

Percentage TEER was calculated using the following 

equation: 

Relative TEER (%)Z
 TEERt  

× 100 (3) 
TEERt0 

where TEERt is the TEER value at different times and 

TEERt0 is the initial TEER value before the experiment 

was performed. 

Results are presented as means (SD) of 3 separate 

par- allel experiments. Data were analyzed using a one-

way analysis of variance (ANOVA), followed by Dunnett’s 

test to determine the difference between multiple 

groups compared to the control group using SPSS 

version 17.0 

software. A P < .05 was considered statistically significant. 

 

Results 
 
Effect of compounds on expression of TJ proteins 

 
To determine the effects of the compounds on TJ 

proteins of the MDCK and MDCK-MDR1 cells, we 

immunohis- tochemically analyzed occludin, claudin-1, 

and F-actin using laser scanning confocal microscopy 

(Fig. 1). All three TJ proteins showed positive staining 

in both cell types and occludin stained red, claudin-

1and F-actin stained green, and the nuclei stained blue. 

Both cell types had similar TJ barrier function systems. 

AOD 



After treatment with puerarin and paeoniflorin, the 

visual morphology of occludin, claudin-1, and F-actin in 

both kinds of cells was not difference from that of the 

untreated cells. However, incubation with menthol, 

decreased the TJ proteins in both MDCK and MDCK-

MDR1-cells and the morphology was 

hardly observed (Fig. 1A, B, respectively).

 

Fig. 1 Effect of puerarin, paeoniflorin, and menthol on 
tight junction (TJ) proteins in (A) MadineDarby canine 

kidney epithelial (MDCK) and (B) MDCK-multi-drug 

resistance 1 (MDR1) cells (×600). (A) Immunocytochemistry 

for occluding, claudin-1, and F-actin in MDCK cells with 

puerarin or paeoniflorin showed similar fluorescent 

morphology with the control group. All three proteins were 

deceased in MDCK cells by menthol. (B) Fluorescence 
intensity in MDCK-MDR1 cells with different compounds was 

similar to that in MDCK cells. Scale bar corresponds to 1.0 

mm. 

Changes in AOD of TJ proteins after treatment with 

different compounds 

 
Fluorescence intensity of the immunofluorescent images 

was semi-quantitatively analyzed. AOD values of each 

protein were calculated using ImageJ software and statis- 

tical analysis was conducted (Fig. 2). The control, puerarin, 

and paeoniflorin groups showed similar approximate rela- 

tive AOD values in the MDCK and MDCK-MDR1 cells, while 

the menthol group was significantly different compared to 



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the control group (P < .05). 

 

Changes in TEER of cells treated with various 

compounds 

 
To determine the effects of the compounds on the function 

of the TJ barrier, we measured the TEER values of MDCK 

and MDCK-MDR1 cells after exposure to the various 

 

Fig. 2 Change in relative average optical density (AOD) of 

occludin, claudin-1, and F-actin in (A) MadineDarby canine 

kidney (MDCK) epithelial and (B) MDCK-multi-drug resistance 1 

(MDR1) cells after treatment with different compounds. (A) 

Relative AOD values of tight junction (TJ) proteins in MDCK 

cells treated with puerarin or paeoniflorin showed no signifi- 

cant difference compared to the control group while AOD 

values of the menthol group were significantly reduced. (B) 

Relative AOD values in MDCK-MDR1 cells treated with different 

compounds were the same as in MDCK cells. Only menthol 

reduced AOD of all three proteins. Data presented as mean (SD) 

(n Z 3). )P < .05 compared to control group. 

compounds for up to 3 h. TEER values of each 
group treated with the compounds for different 
times were statistically compared with those of 
the control group (Fig. 3). MDCK cells (Fig. 3A) 
treated with puerarin, paeoniflorin, and PP 
exhibited similar changes in TEER compared to 
the control group in the A / B and B / A 
transport processes. At 
90 min, TEER of the menthol, PM, and PPM groups 
decreased significantly compared with that of the 

control group (P < .05). In MDCK-MDR1 cells (Fig. 3B), 

groups not treated with menthol exhibited a steady 

TEER change similar to the control group and menthol-
treated groups showed a significant decrease at 120 

min(P < .05)in both the A / B and B / A transport 

processes compared to control. 

Discussion 

Use of both in vivo and in vitro methods has led to the 

elucidation of certain aspects of the drug transport 

process across the BBB. In vivo studies of the BBB 

provide valuable observational information and direct 

analysis of drug permeation processes such as drug 

distribution in each brain region. However, there are 

some limitations and shortcomings to this method 

including the laborious nature of the experiments, 

individual differences, and compli- cated analytical 

methods. Accordingly, development of in vitro models 

is highly desired to facilitate the clarifica- tion of the 

BBB permeability mechanisms. Such desired models 

mainly include those that would predict the asso- ciated 

BBB processes using computer simulation technol- ogy, 

parallel artificial membrane permeability assay 

(PAMPA), and in vitro cell culture techniques.6 Cell 

culture is currently the favored tool for simultaneously 

obtaining complex and comprehensive information of 

passive and active transport processes. Generally, there 

are two types of cell culture models that are used to 

simulate the BBB. One consists of the “real BBB model,” 

which is based on primary cultures of brain capillary 

endothelial cells or sin- gle cell lines that may be 

complemented or co-cultured with 

astrocytes/pericytes.23,24 The other type is the “sur- 

rogate BBB model,” which uses similar epithelial cells 

such as MDCK, MDCK-MDR1,and human colon carcinoma 

cell lines (Caco-2).5,25,26 Among these, MDCK and MDCK-

MDR1 have been recognized as the ideal models for in 

vitro simulation of BBB drug penetration.6,27e29 They 

have several advan- tages including (1) stable source 

with no batch-to-batch variability, (2) convenient for 

routine experimental appli- cation and thus reduce 

labor intensity, (3) adequately and closely mimic 

relevant in vivo properties of the BBB, (4) ensure 

adequate monolayer integrity and display a proper TEER 

value under conventional experimental conditions, and 

(5) show superior P-glycoprotein (P-gp) expression and 

activity, particularly in MDCK-MDR1 cells transfected 

with the human MDR1 gene, which is a very important 

efflux transporter in the BBB.30e34 Therefore, we chose 

MDCK and MDR1-MDCK cells to establish our in vitro BBB 

model. 

Drugs cross the blood brain barrier by two main 

methods: transcellular and paracellular transport. In 

addi- tion, numerous elements impede penetration of 

drugs and substances from the extra-brain region to the 

brain area, including the BBB morphologic 

characteristics (such as 



 

Fig. 3  Change in transepithelial electrical resistance (TEER) in (a, b) MadineDarby canine kidney (MDCK) epithelial and (c, d) MDCK 

multi-drug resistance 1 (MDCK-MDR1) cells after treatment with different compounds. (a, b) TEER of MDCK cells gradually decreased 

following treatment with compounds alone or in combination with menthol. (c, d) TEER in menthol-treated MDCK-MDR1 cells similarly 

declined. Data presented as mean (SD) (n Z 3). PP: puerarin and paeoniflorin; PM: puerarin and menthol; PPM: puerarin, paeoniflorin 

and menthol groups. 

 

existence of TJs), enzymes in the cytosolor on the extra- 

cellular membrane, and P-glycoprotein (P-gp). For 

hydro- philous compounds, paracellular transport is a 

crucial pathway for penetrating the brain and exerting a 

thera- peutic effect. The physiologic function of the TJ 

is insep- arable from paracellular transport and its barrier 

and fence functions are regulated by various factors.35e37 

Using on the simulated BBB MDCK and MDCK-MDR1 cell 

model, we focused on studying the mechanism of the 

main active constituents in the Tongqiaosanyu 

prescription to deter- mine their effects on the structure 

and barrier function of TJs. 

The TJ structure is composed of various types of 

proteins and occludin is regarded as the primary 

identifying mole- cule of the TJ strands. It is a tetraspan 

membrane protein of approximately 60-kD, with a short 

intracellular curve, two extracellular annuli, and N- and 

C-terminals in the cytoplasmic domain.38 Assembly of TJs 

and concomitant increase in TEER have been 

demonstrated to be associated with tyrosine 

phosphorylation of occludin, which is a phe- nomenon 

commonly observed after recovery from adeno- sine 

triphosphate (ATP) depletion or during calcium 

repletion.39,40 Claudins have different sequences from 

occluding and are 18- to 27-kDtetraspan proteins with 

two extracellular annuli, a short N-terminus, and a C-

terminal in the cytoplasmic domain.41 Claudin-1 is also 

considered an important strand or even the back bone of 

TJs.42 Recent studies have shown that claudin-1 is the 

decisive compo- nent of TJs and possesses cell adhesion 

activity.43 It can directly affect the regulation of 

paracellular transport and the selectivity of solute size. 

F-actin, which is one of the peripheral membrane 

proteins of TJs, is related to organ- elle movement, 

protoplasmic streaming, and intercellular 

junctionregulation.7,44e47 Importantly, the F-actin belt 

can control TJ function and limit the material intake.48 

Although we did not investigate all TJ protein types 

in MDCK and MDCK-MDR1 cells, the proteins we chose 

were excellent representations. We individually 

analyzed the immunofluorescence staining of occludin, 

claudin-1, and F- actin in MDCK and MDCK-MDR1 cells 

after treatment with puerarin, paeoniflorin, or 

menthol. The puerarin and paeoniflorin group did not 

affect the staining properties of TJ proteins. Only 

menthol inhibited the expression and reduced the 

fluorescence intensity (AOD value) of all three 

kinds of TJ proteins. This characteristic of menthol indi- 

cated that it directly disrupted the structure and integrity 

of TJs to open the BBB barrier. These results combined with 

previous reports led us to infer that disruption of the 

configuration and integrity of TJ by menthol might be 



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2024 | Vol 1 | Issue 1 

  
 

caused by phosphorylation of TJ components, activation of 

protein kinases, or calcium depletion.49e52 

TEER measurement has been widely applied in evalu- 

ating the integrity of the monolayer and studying the 

permeability of transport processes. Changes in TEER 

values have been directly linked to the function of the 

paracellular occluding barrier53,54 TEER is caused by cell- 

substrate contact, and if the distance of the cell- substrate 

is short, a high TEER value is observed. Based on this 

physiologic characteristic, paracellular resistance and the 

average cell-substrate distance can be estimated by the 

TEER. Paracellular resistance consists of the serial 

resistance of the TJs as well as lateral intercellular resis- 

tence.55 Therefore, to establish the relationship between 

the change in TEER and the state of TJ barrier function, we 

used EVOM instrumentation to determine the TEER of two 

kinds of cells at different time points after treatment with 

the compounds. The results were consistent with those of 

immunocytochemistry, and revealed that TEER value 

gradually declined following treatment of cells with 

menthol, and values of the other groups were relatively 

stable during the test period. Therefore, this experiment 

showed that menthol is capable of weakening the BBB and 

enhancing paracellular transport. The mechanism of the 

decrease in TEER also involves Ca2+ influx and the Ca2+ 
chelator EGTA blocked cofilin dephosphorylation. Further- 

more, the alteration may have been caused by activity 
variation of intrinsic membrane proteins.56 For example, 

removal and addition of Ca2+ to the filter where MDCK cell 

layers form a monolayer may reversibly lead to their TJs 
opening and resealing, and the cell-substrate separation 

may correspondingly increase or decrease.57 In addition, 

we were surprised to find that reduction in TEER of the 

MDCK cells occurred earlier than it did in MDCK-MDR1 cells, 

which suggests that the TJ barrier in the MDCK-MDR1 was 

stronger than it was in MDCK, and menthol might have a 

rapid onset of action in MDCK cells. 

Our previous research in rats and mice on the pharma- 

cokinetics of puerarin in Tongqiaosanyu prescription used 

different methods of administration, including caudal vein 

injection as well as nasal and oral administration. The study 

employed reversed-phase high-performance liquid 

chromatography(RP-HPLC) to determine the concentration 

of the puerarin in blood and brain tissue samples, which 

were collected at different time points. Pharmacokinetics 

in rats showed that AUC0eN following nasal administration 

was 376.56 93.93 mg/min/L and absolute bioavailability 

of puerarin was 47.78% by nasal administration, which was 

significantly higher than oral administration. These results 

were the basis for our choosing nasal administration of 

Tongqiaosanyu prescription. Plasma and brain pharmaco- 

kinetics in mice indicated that the brain targeting coeffi- 

cient (Re) was 132.25% following intranasal administration 

and the brain drug targeting index (DTI) was 2.70, both of 

which were significantly higher than Re and DTI obtained 

following injection administration. These results showed 

that intranasal administration enhanced the medicine’s 

penetration and absorption into the brain.58 Furthermore, 

we used the abdominal skin of the American bullfrog Rana 

catesbeiana as an in vitro model to study the effect of 

different concentrations of puerarin and the effect of 

different proportions of menthol on the apparent perme- 

ability coefficient (Papp) using the Franz diffusion cell sys- 

tem and HPLC. The results showed there was no significant 

difference in the effects of different concentrations of 

puerarin on Papp, indicating that the permeation of puerarin 

is via passive diffusion. Furthermore, when the mass con- 

centration of menthol was 5 mg/L, there was a significant 

difference compared with the puerarin group, which 

proved that menthol enhanced the permeation of puerarin 

at a certain concentrationrange.59 

The main limitation of this study is that we only 

explored a single drug dose. Future studies will further 

investigate the dose-effect relationship. Therefore, the 

mechanism underlying the effect of drugs on the TJ and 

transport through the BBB needs further elucidation. 

 

Conclusion 

In conclusion, this preliminary investigation 

demonstrated the mechanism of puerarin, paeoniflorin, 

and menthol on TJ proteins and TEER. Compared with 

puerarin and paeo- niflorin, menthol in Tongqiaosanyu 

prescription affected the TJ structure and function in 

MDCK and MDCK-MDR1 cells, which were used as in vitro 

BBB models. Menthol inhibited occludin, claudin-1, and 

F-actin, which suggests that it may be a potential drug 

for enhancing the transport of other drugs though the 

BBB and, thereby, improving their permeation in the 

brain. Moreover, menthol downregulated the TEER, 

which verified the relationship between TJ and TEER 

value, and contributed to opening the barrier to in- 

crease paracellular transport. This study was carried out 

to advance our understanding of the effect of puerarin, 

paeoniflorin, and menthol on the BBB and provide 

insight into how Tongqiaosanyu affects TJ structure and 

function. The study also helps explain the interaction of 

drugs in a multicomponent herbal prescription. 

 

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