Biomedicine & Pharmacotherapy 88 (2017) 770–777

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Original article

Efficacy of vitamin B complex as an adjuvant therapy for the treatment

of complicated vulvovaginal candidiasis: An in vivo and in vitro study
Mei-Guo Suna , Ying Huangb , Yuan-Hong Xub , Yun-Xia Caoa,*
a
Department of Gynecology and Obstetrics, The First Affiliated Hospital of Anhui Medical University, Hefei 230031, PR China
b
Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei 230031, PR China

A R T I C L E I N F O A B S T R A C T

Article history: Objective: This study aimed to explore the efficacy of vitamin B complex as an adjuvant therapy for the
Received 5 October 2016 treatment of complicated vulvovaginal candidiasis (VVC) in vitro and in vivo.
Received in revised form 20 December 2016 Methods: One-hundred fifty-eight complicated VVC patients were randomly divided into group A
Accepted 1 January 2017
(treated with suppository + oral antifungal agents), group B (treated with suppository + vaginal cream),
and group C (treated with suppository + vaginal cream + oral vitamin B complex). A mouse model of VVC
Keywords: was established. Eighty VVC mice were randomly divided into 4 groups according to the dose of vitamin B
Complicated vulvovaginal candidiasis
complex (20 mice in each group): V1 group (injected with 150 mL normal salin), V2 group (injected with
Vitamin B complex
Adjuvant therapy
50 mL vitamin B complex solution + 100 mL normal saline), V3 group (injected with 100 mL vitamin B
In vivo complex solution + 50 mL normal saline), and V4 group (injected with 150 mL vitamin B complex
In vitro solution). After 4 weeks of treatment, the vaginal secretion was obtained for microscopic smear
examination. HE stainning was performed to observe histopathological changes of vaginal tissues. The
expressions of inflammatory factors were detected by ELISA. Meanwhile, VVC model of vaginal epithelial
cells was established. The effects of different concentrations of vitamin B complex on anti-fungal effect of
fluconazole were detected in vitro.
Results: After the treatment, complicated patients in the group C had significantly higher effective rates
than those in the group A and group B. After the intra-gastric administration, the microscopic smear
examination found that obvious pseudohypha in cluster with a lot of blastospores can be seen in the
vaginal secretions of mice in the V1 group under the microscope. There was significant difference
between mice treated with different dosages of vitamin B complex. The inflammatory response of mice in
the V1 group was significantly higher than those in other groups and the inflammation response reduced
with the increase of vitamin B complex dosage. The vitamin B complex elevated the curative effects of
fluconazole on VVC model of vaginal epithelial cells and significantly increased the anti-fungal effect of
fluconazole.
Conclusions: Our findings suggest that vitamin B complex could be an effective adjuvant therapy for
complicated VVC.
© 2017 Elsevier Masson SAS. All rights reserved.

1. Introduction VVC at least once in a lifetime, among which 5% women will

Vulvovaginal candidiasis (VVC) is a kind of opportunistic fungal
infections caused by Candida species, especially Monilia Albicans
[1]. VVC, the second major cause of vaginitis in women only after
bacterial vaginitis, is also known as fungal vaginitis or Candida
vaginitis [2]. About 75% premenopausal women are infected with
* Corresponding author at: Department of Gynecology and Obstetrics, The First
Affiliated Hospital of Anhui Medical University, No. 218, Jixi Road, Shushan District,
Hefei 230031, Anhui Province, PR China.
E-mail address: caoyunxia6@126.com (Y.-X. Cao).
develop into recurrent VVC, that is, 4 times a year or even more [3].
The clinical manifestations of VVC include frequent micturition,
dysuria, dyspareunia, leucorrhea, vulva and vaginal burning,
itching, etc. [4]. Pregnant women infected with VVC may also
end up with adverse pregnancy outcomes such as abortion,
premature delivery, premature rupture of membranes and
neonatal infections, leading to a huge physical and psychological
pain for a majority of patients [5]. Common drugs for the clinical
treatment of VVC usually are antifungal agents like nystatin and
fluconazole, miconazole, but the complexity of complicated VVC
contributes to different degrees of drug resistance in the clinical
course of treatment [6,7]. Therefore, the combined use of vitamins

http://dx.doi.org/10.1016/j.biopha.2017.01.001
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 M.-G. Sun et al. / Biomedicine & Pharmacotherapy 88 (2017) 770–777
and anti-fungal agents is proposed to improve the cure rate of VVC.
For example, vitamin B is necessary for the synthesis of specific
vaginal structural proteins, such as cytokeratin, and its deficiency
will lead to structural integrity loss and desquamation of vaginal
epithelium [8]. Hence, this paper proposed vitamin B complex as
an adjuvant therapy for complicated VVC.
Vitamin B is water-soluble and vitamin B complex mainly
contains vitamin B1, vitamin B2, vitamin B6, vitamin B12, and
calcium pantothenate [9]. As indispensable vitamins in human
body, vitamin B plays an important role in the metabolism of
carbohydrate, fat, and protein, and respiration [10,11]. It has been
reported that vitamin B complex combined with allicin can treat
acetic acid-induced toxic reaction in mice, making blood and
biochemical parameters return to normal state [12]. Vitamin B
metabolites can be used as therapeutic target agents for the
treatment of Plasmodium falciparum [13]. Besides, the supplement
of high dose of B vitamins to healthy population can improve their
psychological quality, ease their pressure, and effectively adjust
their cognitive ability under mental stress [14]. Intake of vitamin
B2 and B6 can reduce the risk of colorectal cancer in postmeno-
pausal women [15]. Additionally, vitamin B complex supplemen-
tation has a positive effect on the control of adult depression [16].
Furthermore, it has been demonstrated clinically that B vitamins
can slow down the development of diabetic nephropathy and
prevent vascular complications [17]. However, there are few
researches about the application of B vitamins in the treatment of
fungal infections. Therefore, this paper carried out in vivo and in
vitro study to investigate the efficacy of vitamin B complex as an
adjuvant therapy for complicated VVC.
2. Materials and methods
2.1. Ethical statement
All animals were raised and treated in accordance with the
Guide for the Care and Use of Laboratory Animals by National
Institutes of Health of the U.S.A. This study was approved by the
Ethics Committee of the First Affiliated Hospital of Anhui Medical
University, and informed consent was obtained from each patient,
or guardians prior to study.
2.2. Study subjects
The study included 158 patients with complicated VVC
diagnosed in the First Affiliated Hospital of Anhui Medical
University. The pathological tissue sections were identified after
separation and purification, showing positive results in germ tube
test and spore formation experiment. The diagnosis was strictly in
accordance with criteria of VVC [18]. The patients were 21–
50 years old (mean age of 35.39  4.37 years) with disease course of
15–127 d (mean duration of 46.45  17.87 d). The patients were
married and their major manifestations included different degrees
of burning pain of vulvar with bean curd residue-like secretions,
vaginal mucosal hyperemia, increasing leucorrhoea and edema,
white lumps attached to the inner side of labium minus and
swelling membrane. Pregnant patients with VVC, severe VVC and
recurrent VVC were all complicated types. Exclusion criteria were
as follows: (1) patients with simple VVC; (2) patients with liver
dysfunction; (3) patients with renal failure; (4) patients allergic to
drugs; (6) patients complicated with other types of vaginitis.
2.3. Grouping and treatment regimes
The 158 patients with complicated VVC were randomly divided
into three groups: the group A included 49 patients aging 24–
50 years old (mean age of 36.10  4.84 years) treated with local
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771
vaginal suppository for 1 week and oral antifungal agents for 4
weeks (1 tablet miconazole every night + 150 mg fluconazole once
for a week), with the disease course of 17–109 d (the mean
duration of 50.02  20.80 d); the group B enrolled 54 patients aging
21–48 years old (mean age of 35.04  4.58 years) treated with local
vaginal suppository for 1 week and vaginal cream for 4 weeks (1
tablet miconazole every night + compound ketoconazole ointment
every night), with the disease course of 18–127 d (the mean
duration of 45.52  19.05 d); the group C consisted of 55 patients
aging 25–47 years old (mean age of 35.11  4.06 years) treated with
local vaginal suppository for 1 week, vaginal cream for 4 weeks,
and oral vitamin B complex for 2 weeks (1 tablet miconazole every
night + compound ketoconazole ointment every night + 2 tablets
vitamin B complex twice a day), with the disease course of 15–
102 d (the mean duration of 47.35  17.37 d). During the period of
treatment, sexual life was prohibited.
2.4. Follow-up and efficacy evaluation
After treatment, patients would come back regularly to the
Department of Gynecology in the First Affiliated Hospital of Anhui
Medical University for outpatient or follow-up by telephone was
conducted. In order to facilitate follow-up and timely feedback,
phone numbers of follow-up worker will be given to patients so
that psychological counseling can be provided at any time.
Recovery meant that all symptoms and signs disappeared within
6 months after the treatment and the test result of Candida
albicans was negative. Marked effective meant that symptoms and
signs were significantly improved within 6 months after treatment
and the test result of Candida albicans was negative. Improvement
indicated that symptoms and signs were somewhat improved
within 6 months after treatment, but the test results of Candida
albicans were still positive. Ineffective meant that there was no
improvement in symptoms and signs with 6 months after
treatment and the test results of Candida albicans were still
positive [19].
2.5. Establishment of a mouse model of VVC
One hundred and one female mice of ICR strains (aging 8–10
weeks and weighing 22.31  3.26 g) were purchased from Better
Biotechnology Co., Ltd, (Nanjing, Jiangsu, China). The mice were fed
with standard diets for more than a week to reduce the stress
response caused by environmental change. Subcutaneous injection
of estradiol benzoate was conducted to cause fake estrous state of
mice (one injection in every two days with 0.1 mg each time).
Isolated and purified bacterial strain were inoculated into medium
containing 1% peptone and 0.1% glucose, and cultured in oscillating
water at 25  C for 16–18 h. At the same time, bacteria at the density
of 2.5  106/L were dissolved in 20 uL phosphate buffer saline (PBS)
bacterial suspension, indicating that spores of Candida albicans
were 5  104. On the 6th day after injection, the fluid of bacterial
was injected into absorbable hemostatic sponge and the sponge
was inverted into the vagina of mice. After the injection, each
mouse was maintained inverted for 5 min. On the 2nd, 4th 7th and
21st day after inoculation, 5 mice were selected for vaginal lavage
with 100 mL PBS stored in sterilized 1.5 mL Eppendorf (EP) tube.
Then the mice were killed by cervical dislocation and vaginal
tissues were obtained.
2.6. Identification of VVC mouse model
Lavage fluid was mixed thoroughly and lotion was (40 uL) added
to dilute 1 mL sterile saline. Sabouraud Dextrose Agar medium was
modified for inoculation and culture at 30  C for 72 h before CFU
counting (colony-forming unit, colonies/L). Gradient dilution of the
772 M.-G. Sun et al. / Biomedicine & Pharmacotherapy 88 (2017) 770–777
rest lavage liquid was performed according to the concentration;
then, the liquid was spread on Candida chromogenic medium. After
48 h culture at 28  C in incubator, Candida albicans were counted.
Candida albicans were bluish green, Candida glabrata purple, and
other Candida white. The growth of bacteria was inhibited.
2.7. Experimental grouping and treatment
About 21 d after successful establishment of mouse model (first
day for treatment), all mice received intragastric administration
with fluconazole 150 mg/time on the 1st, 4th, and 7th day, and later
once a week, with a total of 3 weeks. At the same time, injection of
vitamin B complex was performed as an adjuvant therapy. Vitamin
B complex injection (specification: 2 mL, 20 mg vitamin B1, 2 mg
riboflavin sodium phosphate (by vitamin B2), 2 mg vitamin B6,
50 mg nicotinamide and 1 mg sodium dextropantothenate) were
purchased from Shanghai Tongren Pharmaceutical Co., Ltd (China),
and the batch number was 070301, 060801. Mice were randomly
divided into 4 groups according to the dosage of vitamin B complex
(n = 20). Mice in V1 group were injected with 150 mL normal saline;
V2 group, 50 mL vitamin B complex solution + 100 mL normal
saline; V3 group, 100 mL vitamin B complex solution + 50 mL
normal saline; and V4 group, 150 mL vitamin B complex solution.
After 4 weeks of treatment, vaginal secretions of mice were
obtained for microscopic smear examination and the mice were
killed by cervical dislocation.
2.8. HE stainning
Vaginal tissues were fixed before paraffin embedding. Then
tissues were sliced into 4 mm, stained for 5 min with hematoxylin,
washed with tap water for 1 min, differentiated for 30 s (hydro-
chloric acid and ethanol), washed by warm water for 5 min (about
50  C), and finally placed into the eosin solution for 2 min before
regular dehydration and sealed with resinene. Under the micro-
scope, cytoplasm was pale red and the nuclei bluish black.
Apoptotic cells dispersed as individuals with nuclear fragmenta-
tion, and nuclear chromatin concentrated densely. Necrotic tissues
showed red substance without structure and not-stained nucleus.
2.9. Cell culture
Vaginal epithelial cells were obtained from patients enrolled in
this study who had received total hysterectomy because of
adenomyosis and hysteromyoma. Vaginal tissues with a size of
3 mm * 3 mm were washed 3 times in serum-free culture medium
of the D-Hanks solution, containing 100 U/mL streptomycin, 100 U/
mL penicillin and 50 mg/mL gentamicin, which were all purchased
from China National Pharmaceutical Group Corporation (Beijing,
China). Then, the cells were digested with 0.5 U/mL dispase at 4  C
overnight and 0.25% trypsase (Gibco Company, Grand Island, NY,
USA) at 37  C for 20 min, re-suspended in Dulbecco’s modified
Eagle's medium 12 (DMEM/F) (HyClone, Logan, Utah, USA) after
digestion and centrifuged at 1000g for 5 min, after which the
supernatant was aspirated. Afterwards, the cells were re-
suspended by keratinocyte serum-free medium (K-SFM) supple-
mented with 100 U/mL streptomycin, 100 U/mL penicillin and
0.25 mg/mL amphotericin B. After counting, cells were seeded into
culture medium at the density of 1.0 * 107/mL with 5% CO2 at 37  C.
Two or three days later, the medium was changed and cells were
passaged at the ratio of 1: 2.
2.10. Establishment of VVC vaginal epithelial cell model and grouping
The passaged vaginal epithelial cells (concentration was
1.0 * 108/mL) and Candida spore (concentration was 1.0 * 105/mL)
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were selected to construct the VVC cell model. The 24-well plates
were used and each well was added with 200 mL vaginal epithelial
cells and 50 mL Candida suspension. After a 24 h culture in
incubator, 50 mL fluconazole (the concentration of mother liquid
was 12 mg/mL) was added for further culture. Cells were assigned
into the V1 control (15 mL PBS), V2 (10 mL PBS + 5 mL vitamin B
complex), V3 (5 mL PBS + 10 mL vitamin B complex) and V4 (15 mL
vitamin B complex) groups. After 48 h, the supernatant was
collected and the levels of IL-1b, IL-6 and IL-8 were detected.
2.11. Enzyme-linked immunosorbent assay (ELISA)
Carbonate buffer solution of 50 mM (pH = 9) was used to dilute
the antibody into a concentration of 1–10 mg/mL protein. Then
0.1 mL of the diluted solution was added to the reaction hole of
polystyrene plate at 4  C overnight. On the next day, the solution in
the well was aspirated and the plate was washed with buffer
solution for 3 times (3 min/time). About 0.1 mL diluted sample was
added to the above reaction well before incubation for 1 h at 37  C
and washing (Same operations were conducted in blank control
well, positive control well and negative control well). In each
reaction well, 0.1 mL enzyme-labeled antibody that was diluted
accurately was added before incubation at 37  C for 0.5–1 h. Then,
the samples were washed. Afterwards, 0.1 mL newly-formulated
tetramethyl benzidine (TMB) substrate solution was added to the
reaction well before incubation at 37  C for 10–30 min. With the
zero setting in negative control well, each well was tested at the
wavelength of 450 nm with ELISA instrument. Standard results
were obtained according to standard solution and the concentra-
tion of each group was calculated.
2.12. Antifungal susceptibility test
The cultured Candida was seeded in shake flask culture for 24 h
(250 mL), containing 100 mL yeast extract-peptone, dextrose
(YEPD) under the condition of 30  C at 120 rpm/min. Roswell Park
Memorial Institute-1640 solution (RPMI) (Gibco Company, Grand
Island, NY, USA) was applied to dilute the Candida into 103 CFU/mL.
A total of 100 mL sample was seeded into the 96-well plate and the
Candida was divided into the V1 control (15 mL RPMI1640
solution), V2 (10 mL RPMI1640 solution + 5 mL vitamin B complex),
V3 (5 mL RPMI1640 solution + 10 mL vitamin B complex) and V4
groups (15 mL vitamin B complex). Different concentrations of
fluconazole were set and the final concentrations were as follows:
2.0 mg/mL, 1.8 mg/mL, 1.6 mg/mL, 1.4 mg/mL, 1.2 mg/mL, 1.0 mg/mL,
0.8 mg/mL, 0.6 mg/mL, 0.4 mg/mL, 0.2 mg/mL and 0 mg/mL. Three
replicates of each concentration were set up for the three groups.
After 24 h shake culture at 30  C in incubator, the optical density
(OD)630nm was detected using microplate reader (Multiskan MK3
type, Labsystems Diagnostics Ltd., Vantaa, Finland). The OD630nm
represented the growth state of Candida and minimum inhibitory
concentration (MIC)80 was analyzed. According to the National
Committee for Clinical Laboratory Standards M27-A2 document,
compared with the V1 control group (the concentration of
fluconazole was 0 mg/mL), the MIC80 value referred to the
concentration of fluconazole when the growth inhibition rate
was no less than 80%.
2.13. Statistical analysis
All the data were analyzed with SPSS 21.0 statistical analysis
(SPSS, Chicago, IL, USA). When assessing the curative effect,
recovery, marked effective and improvement were counted as
effective rate. Measurement data were presented by mean 
standard deviation (SD) (X  S). Differences between two groups
were analyzed using independent-sample t-test. Enumeration data
 M.-G. Sun et al. / Biomedicine & Pharmacotherapy 88 (2017) 770–777 773

Table 1
Comparisons of curative effects among three groups.

Group n Recovery (%) Marked effective (%) Improvement (%) Ineffective (%) Effective rate (%)
A 49 30 (61.22) 4 (8.16) 2 (4.08) 1 3(26.53) 73.47
[65_TD$IF] B 54 34 (62.96) 4 (7.41) 5 (9.26) 11 (20.37) 79.63
[6_TD$IF] C 55 40 (72.73)* 5 (9.09) 6 (10.91) 4 (7.27) 92.73*

Notes: group A, treated with local vaginal suppository for 1 week and oral antifungal agents for 4 weeks (1 tablet miconazole every night + 150 mg fluconazole once for a
week); group B, treated with local vaginal suppository for 1 week and vaginal cream for 4 weeks (1 tablet miconazole every night + compound ketoconazole ointment every
night); group C, treated with local vaginal suppository for 1 week, vaginal cream for 4 weeks, and oral vitamin B complex for 2 weeks (1 tablet miconazole every
night + compound ketoconazole ointment every night + 2 tablets vitamin B complex twice a day); *, P < 0.05 compared with group A.

were presented by percentage and the Kruskal-Waillis H test was
performed to compare the difference between two groups. P < 0.05
was considered statistically different.
3. Results
3.1. Comparisons of curative effects among three groups
By comparison, we found that there were significantly differ-
ences in curative effect and cure rate among VVC patients in the
three groups (all P < 0.05). According to Table 1, effective rate of
patients in the group C was 92.73%, which is significantly higher
than those in the A (73.47%) and B [67_TD$IF](79.63%) groups (all P < 0.05).
The effective rate was defined as the percentage of patients with
recovery, marked effective and improvement. Compared with the
A (61.22%) and group B (62.96%), the group C (72.73%) also
exhibited significantly higher recovery rate (all P < 0.05).
3.2. Successful establishment of VVC mouse model
were infected successfully and the infection rate was (100/105)
95.23%.
3.3. Comparisons of vaginal secretions among the V1, V2, V3 and V4
groups after intragastric administration
After 4 weeks of intragastric administration, microscopic smear
examination was performed to detect vaginal secretions of mice in
the 4 groups and the formation of hyphae and spores were also
observed. After a period of treatment, pseudohypha in cluster with
rich blastospores can be seen in the vaginal secretion of mice in the
V1 group under the microscope. In the V2 group, a lot of
pseudohypha with rich blastospores was visible but the pseudo-
hypha was not in cluster. In the V3 group, there were a few
pseudohypha and a small amount of blastospores in vaginal
secretion. In the V4 group, it was almost impossible to see
pseudohypha under the microscope, and blastospores were
occasionally observed (Fig. 2). These results suggested that
adjuvant therapy with vitamin B complex has a certain effect on
the treatment of complicated VVC.

Mice were observed for 21 d after inoculation with Candida
albicans and the number of fungal spores in their vagina was
recorded. Through microscopic smear examination of vaginal
lavage fluid, spores and false hyphae could be observed from the
second day, and genital swelling was also observed. On the fourth
day, the smear was the most intensive with pelotons and
symptoms such as severe infection, hyperemia and edema and
festering. Candida chromogenic medium was applied for the
identification and counting of Candida albicans (Fig. 1A). From the
2nd d of infection, high level of CFU (CFU/ml) in the vagina of mice
recorded (2.99  0.32), and the level peaked on the 4th d
(7.28  0.55). Then, it gradually decreased to 4.96  0.24 on the
7th d and to 3.13  0.19 on the 21st d (Fig. 1B). A hundred of mice
3.4. Comparisons of histopathological changes of vaginal tissues
among the V1, V2, V3 and V4 groups after staining
After staining, hyphae adhesion and invasion were observed in
vaginal mucosa tissues of mice. In the V1 group, vaginal tissues of
mice manifested severe hyperemia, edema, acute inflammatory
infiltration and many inflammatory cells can be seen in vaginal
mucosa and sub-mucosa. Compared with the V1 group, the V2
group showed significantly reduced lesions severity and decreased
degree of inflammatory infiltration, but there were obvious edema
and congestion. Mice in the V4 group had a milder edema than
those in the V3 group and no inflammatory infiltration was
observed (Fig. 3).

[(Fig._1)TD$IG]

Fig. 1. Successful establishment of VVC mouse model.

Notes: A: cultures of Candida albicans (40); B: CFU level in the vagina of mice after infection with Candida albicans; VVC, Vulvovaginal candidiasis; CFU, colony-forming unit.

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774 M.-G. Sun et al. / Biomedicine & Pharmacotherapy 88 (2017) 770–777
[(Fig._2)TD$IG]

Fig. 2. Comparisons of vaginal secretions among the V1, V2, V3 and V4 groups after intragastric administration.
Notes: A, V1 group; B, V2 group; C, V3 group; D, V4 group. V1 group, injected with 150 mL normal saline; V2 group, injected with 50 mL vitamin B complex solution + 100 mL
normal saline; V3 group, injected with 100 mL vitamin B complex solution + 50 mL normal saline; V4 group, injected with 150 mL vitamin B complex solution.

3.5. Comparisons of curative effects in VVC mouse model treated with With the increase of vitamin B complex, the inflammation was
four different regimens effectively alleviated (Table 3).

By comparison, significant difference of curative effects in VVC
mouse model was noted. Treated with fluconazole alone, the
effective rate was 60% and the recovery rate was 30%. With the
increase of vitamin B complex, the effective rate especially the
recovery rate significantly increased (all P < 0.05) (Table 2). The
effective rate in the V4 group was 90% which is significantly higher
than that of the V1 group (60%). In terms of the recovery rate, it was
significantly higher in the V4 group (65%) than that of the V1 group
(30%) (both P < 0.05). It was indicated that adjuvant therapy with
vitamin B complex has a certain effect on the treatment of VVC
mouse.
3.6. Comparisons of IL-1b, IL-6, and IL-8 levels in VVC mouse model
treated with four different regimens
3.7. Comparisons of IL-1b, IL-6, and IL-8 levels in VVC vaginal
epithelial cell model treated with four different regimens
ELISA was used to analyze the levels of inflammatory factors
including IL-1b, IL-6, and IL-8 in VVC vaginal epithelial cells. The
levels of IL-1b, IL-6, and IL-8 in the V1 group were
308.15  23.65 ng/L, 93.85  4.92 ng/L and 135.65  14.13 ng/L,
which were significantly higher than those in other groups (all
P < 0.05). No significant difference of IL-1b, IL-6, and IL-8 level was
observed between the V2 and V3 groups (P > 0.05). Compared with
the V2 and V3 groups, the V4 group showed significantly decreased
level of IL-1b, IL-6, and IL-8 (all P < 0.05). With the increase of
vitamin B complex, the inflammation was effectively alleviated
(Table 4).

ELISA was used to analyze the levels of inflammatory factors
including IL-1b, IL-6, and IL-8 in vaginal tissues of mice. The
expressions of IL-1b, IL-6, and IL-8 in mice of the V1 group were
308.15  23.65 ng/L, 93.85  4.92 ng/L and 135.65  14.13 ng/L,
which were significantly higher than those of mice in other
groups (all P < 0.05). There was no significant difference in IL-1b,
IL-6, and IL-8 levels between the V2 and the V3 groups (P > 0.05).
Compared with the V2 and V3 groups, the IL-1b, IL-6, and IL-8
levels in mice of the V4 group significantly decreased (P < 0.05).
3.8. Effects of vitamin B complex on MIC80 of fluconazole treated with
four different regimens
The antifungal susceptibility test was performed to detect how
vitamin B complex affects the effect of fluconazole on Candida in
vitro. As shown in Table 5, without vitamin B complex, the MIC80 of
fluconazole was 2.0 ug/mL. With the addition of vitamin B
complex, the MIC80 of fluconazole significantly decreased. When
the dosage of vitamin B complex reached 15 uL, the MIC80 of

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 M.-G. Sun et al. / Biomedicine & Pharmacotherapy 88 (2017) 770–777 775
[(Fig._3)TD$IG]

Fig. 3. Staining results of pathological vaginal tissues of mice (200).

Table 2
Comparisons of curative effects in VVC mouse model treated with four different regimens.

Group n Recovery Marked effective Improvement Ineffective Effective rate

(%) (%) (%) (%) (%)
V1 20 6 (30.00) 4 (20.00) 2 (10.00) 8 (40.00) (60.00)
V2 20 8 (40.00) 5 (25.00) 1 (5.00) 6 (30.00) (70.00)
V3 20 10 (50.00) 6 (30.00) 1 (5.00) 3 (15.00) (85.00)
V4 20 13 (65.00)* 4 (20.00) 2 (10.00) 1 (5.00) (90.00)*

Notes: *, compared with the V1 group, P < 0.05; V1 group, injected with 150 mL normal saline; V2 group, injected with 50 mL vitamin B complex solution + 100 mL normal
saline; V3 group, injected with 100 mL vitamin B complex solution + 50 mL normal saline; V4 group, injected with 150 mL vitamin B complex solution.

fluconazole was only 1.2 ug/mL. It was indicated that vitamin B 4. Discussion
complex enhanced the inhibitive ability of fluconazole on Candida
growth. In this study, we carried out a clinical experiment of
complicated VVC, established a reliable VVC mouse model with

Table 3
Comparisons of IL-1b, IL-6, and IL-8 levels in VVC mouse model treated with four different regimens (unit: ng/L).

Group Regimen IL-1b IL-6 IL-8

V1 150 mL NS 308.15  23.65 93.85  4.92 135.65  14.13
V2 VB 50 mL  NS 100 mL 199.65  21.43* 71.05  3.95* 96.55  16.27*
V3 VB 100 mL  NS 50 mL 205.31  22.18* 68.34  3.25* 89.09  13.05*
V4 VB 150 mL 118.16  21.52*# 55.49  2.41*# 66.08  12.92*#

Notes: *, P < 0.05 compared with the V1 group; #, P < 0.05 compared with the V2 and V3 groups; V1 group, injected with 150 mL normal saline; V2 group, injected with 50 mL
vitamin B complex solution + 100 mL normal saline; V3 group, injected with 100 mL vitamin B complex solution + 50 mL normal saline; V4 group, injected with 150 mL vitamin
B complex solution; NS, normal saline; VB, vitamin B complex.

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Table 4 The study also showed that, compared with those in the V2 and
Comparisons of IL-1b, IL-6, and IL-8 levels in VVC vaginal epithelial cell model
the V3 groups, the expressions of IL-1b, IL-6, IL-8 in mice of the V4
treated with four different regimens.
group were significantly lower, and the dosage increase of vitamin
Group Regimen IL-1b IL-6 IL-8 B complex can effectively reduce inflammation. Another related
V1 15 mL PBS 418.25  22.75 99.48  4.43 155.74  11.11 study confirmed that Candida albicans infection can induce pro-
V2 VB 5 mL  PBS 269.65  19.53* 86.18  4.05* 107.44  17.29* inflammatory reaction of the vaginal epithelial cells by inducing
10 mL
the expression of Toll-like receptors, IL-8 and TLR6 [29]. A study by
V3 VB 10 mL  PBS 223.34  21.28* 71.09  4.67* 96.01  13.52*
5 mL
Palma-Ramos et al. showed that Candida albicans could activate
V4 VB 15 mL 108.89  22.19*# 50.32  3.11*# 60.99  11.12*# the expression of IL-1b, and reducing its activity can affect the
down-expression of IL-1b [30]. Besides, IL-8 is a chemokine of
Notes: *, P < 0.05 compared with the V1 group; #, P < 0.05 compared with the V2
and V3 groups; V1 group, injected with 15 mL PBS; V2 group, injected with 10 mL neutrophils and can attract and stimulate white blood cells to
PBS + 5 mL vitamin B complex; V3 group, injected with 5 mL PBS + 10 mL vitamin B produce inflammatory response [31]. A research by Antonietta
complex; V4 group, injected with 15 mL vitamin B complex; PBS, phosphate buffer proved that content of IL-8 in oral epithelial cells increased
saline; VB, vitamin B complex.
significantly after Candida albicans treatment [32]. In addition, IL-6
has an extensive biological effect and can participate in the process
of inflammation by affecting various target cells [33]. B12 in vitamin
B complex can involve in the synthesis of nucleic acid in
the successful rate 94%, and detected the expressions of
inflammatory tissues to increase the content of nucleic acid
inflammatory factors IL-1, IL-6, and IL-8. These results finally
significantly, promoting the regeneration of damaged epithelium
demonstrated that routing medicine combined with vitamin B
cells and accelerating the cell repair of injury site [34]. According to
complex showed a higher efficacy than routine medicine only, and
the above mentioned researches, it can be speculated that with the
the animal experiments further confirmed the results. Besides, the
dose increase of water-soluble vitamin B complex, the concentra-
dosage of vitamin B complex was negatively correlated with the
tion of nucleic acid in inflammation site would increase
expressions of inflammatory factors.
accordingly, and the nucleic acid synthesis of normal cells and
Complicated VVC, as one of the most common vaginal
the repair of inflammatory sites would also be accelerated,
infections, has become a difficult and hot research in clinical
indicating the more obvious efficacy of adjunctive treatment. By
medicine because of its susceptibility to drug resistance, complex
the way, the dosage of vitamin B complex can also affect the
etiology and low cure rate [20,21]. Vitamin B contains eight water-
efficacy of gentamicin in the improvement of the liver toxicity
soluble vitamins and plays an important role in the metabolism of
[35,36]. This study demonstrated that the expressions of inflam-
the cell [22]. In this study, we found that there were significant
matory factors were down-regulated in a concentration-depen-
differences in the symptoms of mice depending on the use or not of
dent manner with the dosage increase of vitamin B complex, which
vitamin B complex as adjuvant therapy. Although the mechanism
was consistent with the results of previous studies.
of vitamin B complex fighting against pathogenic bacteria has not
In this study, both clinical experiments and in vivo experiments
been clarified, many different active ingredients in vitamin B
clarified that vitamin B complex as an adjunctive therapy has a
complex have a certain antibacterial effect. For example, vitamin
certain effect on the treatment of VVC, which provides a reference for
B1 plays an antimicrobial role by attaching to the cell membrane
clinical application of B vitamins. However, Samour et al. have
and inhibits cell proliferation and differentiation [23]. Vitamin B2
demonstrated that a suitable daily intake dose of vitamin B6, a part of
can inhibit the transmission of hyphae in the interstitial substance
vitamin B complex, is around 3.0 mg/kg of diet and excess dietary or
of host cells, and enhance the resistibility of host cells by producing
high doses of vitamin B6 may lead to ataxia, sensory neuropathy or
H2O2, regulating a series of defense related genes, and synthesizing
secondary photosensitization [37,38]. Thus, further study is needed
corpus callosum [24,25]. Besides, vitamin B2 might produce
clinically in terms of the dosage selection of vitamin B complex and
antibacterial function by regulating the biosynthesis of jasmonic
the adverse effect of adjuvant administration.
acid and corpus callosum [26,27]. Vitamin B12 usually can regulate
the repair mechanism of DNA and oxidative stress response,
Competing interests
protect DNA from damage by medicine, and enhance the
antibacterial effect of quinolones when used together [28]. In this
None.
study, both mice model experiment and clinical experiment
showed that adjuvant therapy with vitamin B complex can
Acknowledgments
effectively enhance the curative effect of VVC treatment, suggest-
ing that it may be related to the active ingredients in vitamin B
This study was supported by Annual Key Scientific Research
complex.
Projects in Anhui Province (No. 12070403064). We would like to
acknowledge the helpful comments on this paper received from
our reviewers.

Table 5 References
Effects of vitamin B complex on MIC80 of fluconazole treated with four different
regimens.
[1] E.E. Nash, B.M. Peters, E.A. Lilly, M.C. Noverr, P.L. Fidel Jr., A murine model of
Group Fluconazol candida glabrata vaginitis shows no evidence of an inflammatory
immunopathogenic response, PLoS One 11 (2016) e0147969.
Regimen MIC80 (ug/mL)
[2] F. Vicariotto, M. Del Piano, L. Mogna, G. Mogna, Effectiveness of the association
V1 2 of 2 probiotic strains formulated in a slow release vaginal product, in women
V2 1.6 affected by vulvovaginal candidiasis: a pilot study, J. Clin. Gastroenterol. 46
V3 1.4 (Suppl) (2012) S73–S80.
V4 1.2* [3] C. Iavazzo, I.D. Gkegkes, I.M. Zarkada, M.E. Falagas, Boric acid for recurrent
vulvovaginal candidiasis: the clinical evidence, J. Womens Health (Larchmt.)
Notes: *, P < 0.05 compared with the V1 group; V1 group, injected with 15 mL PBS; 20 (2011) 1245–1255.
V2 group, injected with 10 mL PBS + 5 mL vitamin B complex; V3 group, injected [4] M. Ilkit, A.B. Guzel, The epidemiology, pathogenesis, and diagnosis of
with 5 mL PBS + 10 mL vitamin B complex; V4 group, injected with 15 mL vitamin B vulvovaginal candidosis: a mycological perspective, Crit. Rev. Microbiol. 37
complex. (2011) 250–261.

Downloaded for Mahasiswa KTI UKDW (mhsktiukdw01@gmail.com) at Universitas Kristen Duta Wacana from ClinicalKey.com by Elsevier on July 31, 2017.
For personal use only. No other uses without permission. Copyright ©2017. Elsevier Inc. All rights reserved.
 M.-G. Sun et al. / Biomedicine & Pharmacotherapy 88 (2017) 770–777
[5] T.J. Aguin, J.D. Sobel, Vulvovaginal candidiasis in pregnancy, Curr. Infect. Dis.
Rep. 17 (2015) 462.
[6] S. Fan, X. Liu, Y. Liang, Miconazole nitrate vaginal suppository 1,200 mg versus
oral fluconazole 150 mg in treating severe vulvovaginal candidiasis, Gynecol.
Obstet. Invest. 80 (2015) 113–118.
[7] J.D. Sobel, Factors involved in patient choice of oral or vaginal treatment for
vulvovaginal candidiasis, Patient Preference Adherence 8 (2013) 31–34.
[8] M. Peacocke, E. Djurkinak, S. Thys-Jacobs, Treatment of desquamative
inflammatory vaginitis with vitamin D: a case report, Cutis 81 (2008) 75–78.
[9] C.M. Kopruszinski, R.C. Reis, E. Bressan, P.W. Reeh, J.G. Chichorro, Vitamin B
complex attenuated heat hyperalgesia following infraorbital nerve
constriction in rats and reduced capsaicin in vivo and in vitro effects, Eur. J.
Pharmacol. 762 (2015) 326–332.
[10] L. Kjer-Nielsen, O. Patel, A.J. Corbett, J. Le Nours, B. Meehan, L. Liu, et al., MR1
presents microbial vitamin B metabolites to MAIT cells, Nature 491 (2012)
717–723.
[11] O. Patel, L. Kjer-Nielsen, J. Le Nours, S.B. Eckle, R. Birkinshaw, T. Beddoe, et al.,
Recognition of vitamin B metabolites by mucosal-associated invariant T cells,
Nat. Commun. 4 (2013) 2142.
[12] H.N. Mustafa, A.M. Hussein, Does allicin combined with vitamin B-complex
have superior potentials than alpha-tocopherol alone in ameliorating lead
acetate-induced Purkinje cell alterations in rats? An immunohistochemical
and ultrastructural study, Folia Morphol. (Warsz.) [68_TD$IF]75 (2016) 76–86.
[13] I.B. Muller, J.E. Hyde, C. Wrenger, Vitamin B metabolism in plasmodium
falciparum as a source of drug targets, Trends Parasitol. 26 (2010) 35–43.
[14] D.O. Kennedy, R. Veasey, A. Watson, F. Dodd, E. Jones, S. Maggini, et al., Effects
of high-dose B vitamin complex with vitamin C and minerals on subjective
mood and performance in healthy males, Psychopharmacology (Berl.) 211
(2010) 55–68.
[15] S. Zschabitz, T.Y. Cheng, M.L. Neuhouser, Y. Zheng, R.M. Ray, J.W. Miller, et al., B
vitamin intakes and incidence of colorectal cancer: results from the Women’s
Health Initiative Observational Study cohort, Am. J. Clin. Nutr. 97 (2013) 332–
343.
[16] J.E. Lewis, E. Tiozzo, A.B. Melillo, S. Leonard, L. Chen, A. Mendez, et al., The effect
of methylated vitamin B complex on depressive and anxiety symptoms and
quality of life in adults with depression, ISRN Psychiatry 2013 (2013) 621453.
[17] A.A. House, M. Eliasziw, D.C. Cattran, D.N. Churchill, M.J. Oliver, A. Fine, et al.,
Effect of B-vitamin therapy on progression of diabetic nephropathy: a
randomized controlled trial, JAMA 303 (2010) 1603–1609.
[18] E. Hong, S. Dixit, P.L. Fidel, J. Bradford, G. Fischer, Vulvovaginal candidiasis as a
chronic disease: diagnostic criteria and definition, J. Low. Genit. Tract Dis. 18
(2014) 31–38.
[19] J.E. Swain, T.B. Pool, ART failure: oocyte contributions to unsuccessful
fertilization, Hum. Reprod. Update 14 (2008) 431–446.
[20] S.R. Fan, X.P. Liu, In vitro fluconazole and nystatin susceptibility and clinical
outcome in complicated vulvovaginal candidosis, Mycoses 54 (2011) 501–505.
[21] X.P. Liu, S.R. Fan, F.Y. Bai, J. Li, Q.P. Liao, Antifungal susceptibility and genotypes
of Candida albicans strains from patients with vulvovaginal candidiasis,
Mycoses 52 (2009) 24–28.
[22] A.D. Raval, D. Thakker, A.N. Rangoonwala, D. Gor, R. Walia, Vitamin B and its
derivatives for diabetic kidney disease, Cochrane Database Syst. Rev. 1 (2015)
CD009403.
Downloaded for Mahasiswa KTI UKDW (mhsktiukdw01@gmail.com) at Universitas Kristen Duta Wacana from ClinicalKey.com by Elsevier on July 31, 2017.
For personal use only. No other uses without permission. Copyright ©2017. Elsevier Inc. All rights reserved.
777
[23] J.S. Cho, Y.C. Seo, T.B. Yim, H.Y. Lee, Effect of nanoencapsulated vitamin B1
derivative on inhibition of both mycelial growth and spore germination of
fusarium oxysporum f. sp. raphani, Int. J. Mol. Sci. 14 (2013) 4283–4297.
[24] E. Haggi, N. Blasich, L. Gutierrez, G. Vazquez, S. Criado, S. Miskoski, et al., On the
generation and quenching of reactive-oxygen-species by aqueous vitamin B2
and serotonin under visible-light irradiation, J. Photochem. Photobiol. B 113
(2012) 22–28.
[25] I. Matsumoto, Y. Ohta, R. Waseda, M. Tamura, M. Oda, G. Watanabe, Vitamin B2
as a tracer for intraoperative pulmonary sentinel node navigation surgery,
Anticancer Res. 30 (2010) 4109–4114.
[26] P. Taheri, S. Tarighi, A survey on basal resistance and riboflavin-induced
defense responses of sugar beet against Rhizoctonia solani, J. Plant Physiol. 168
(2011) 1114–1122.
[27] I. Hassan, S. Chibber, A.A. Khan, I. Naseem, Riboflavin ameliorates cisplatin
induced toxicities under photoillumination, PLoS One 7 (2012) e36273.
[28] M.M. Masadeh, K.H. Alzoubi, S.I. Al-azzam, Flouroquinolones-induced
antibacterial activity atteneuation by pretreatment with vitamin B12, Int. J.
Pharmacol. 11 (2004) 67–71.
[29] R.D. Wagner, S.J. Johnson, Probiotic lactobacillus and estrogen effects on
vaginal epithelial gene expression responses to Candida albicans, J. Biomed.
Sci. 19 (2012) 58.
[30] A. Palma-Ramos, G. Casillas-Pétriz, Laura Estela Castrillón-Rivera, et al.,
Activation and IL-[69_TD$IF]1é secretion of human peripheral phagocytes infected with [70_TD$IF]
Actinomadura madurae, Nocardia asteroides and Candida albicans, [71_TD$IF]Asian Pac. J.
Trop. Med. [72_TD$IF]639 (2016) 962–967.
[31] B.M. Tavares-Murta, A.D. de Resende, F.Q. Cunha, E.F. Murta, Local profile of
cytokines and nitric oxide in patients with bacterial vaginosis and cervical
intraepithelial neoplasia, Eur. J. Obstet. Gynecol. Reprod. Biol. 138 (2008) 93–
99.
[32] A. Rizzo, A. Losacco, C.R. Carratelli, Lactobacillus crispatus modulates epithelial
cell defense against Candida albicans through toll-like receptors 2 and 4,
interleukin 8 and human beta-defensins 2 and 3, Immunol. Lett. 156 (2013)
102–109.
[33] W.A. Neveu, J.B. Allard, O. Dienz, M.J. Wargo, G. Ciliberto, L.A. Whittaker, et al.,
IL-6 is required for airway mucus production induced by inhaled fungal
allergens, J. Immunol. 183 (2009) 1732–1738.
[34] I. Altun, E.B. Kurutas, Vitamin B complex and vitamin B12 levels after
peripheral nerve injury, Neural Regen. Res. 11 (2016) 842–845.
[35] N.A. Abdulmajeed, H.S. Alnahdi, N.O. Ayas, A.M. Mohamed, Amelioration of
cardiotoxic impacts of diclofenac sodium by vitamin B complex, Eur. Rev. Med.
Pharmacol. Sci. 19 (2015) 671–681.
[36] S.O. Bello, A. Chika, Dose-dependent amelioration of gentamicin-induced
nephrotoxicity in adult Swiss albino rats by vitamin B-complex – a preliminary
study, Trop. J. Pharm. Res. 2 (2009) 111–116.
[37] J. Samour, J. Perlman, J. Kinne, V. Baskar, U. Wernery, G. Dorrestein, Vitamin B6
(Pyridoxine hydrochloride) toxicosis in falcons, J. Zoo Wildl. Med. 47 (2016)
601–608.
[38] H. Hellmann, S. Mooney, Vitamin B6: a molecule for human health? Molecules
15 (2010) 442–459.