The effect of Calendula officinalis versus metronidazole on bacterial vaginosis in women: A double-blind randomized controlled trial

Abstract

Bacterial vaginosis (BV) is a common disorder among women of reproductive age. This study aimed to compare the effect of a Calendula officinalis extract-based cream and metronidazole on BV among women of reproductive age. In this study, 80 women of reproductive age with BV were randomly assigned to the C. officinalis (n = 40) or metronidazole (n = 40). Diagnosis of BV was confirmed when at least 3 of the 4 Amsel criteria were met (pH> 4.5, whitish grey or thin homogeneous discharge, release of a fishy odor on adding 10% KOH, and detection of clue cells on microscopic examination). For each group, either a methanol extract of C. officinalis or metronidazole vaginal cream (5 g) was used for 1 week intravaginally, and all signs and symptoms were assessed 1 week after treatment completion. Before the intervention, the two groups did not differ significantly with regard to vaginal burning, odor, dysuria, and dyspareunia, but itching was significantly more common in the C. officinalis group than in the metronidazole group (22.5% vs. 2.5%, P = 0.01). One week after the intervention, all women in both groups were free of symptoms, including vaginal itching and burning sensation, odor, dysuria, and dyspareunia. None of the women in either group suffered any side effects from C. officinalis or metronidazole. C. officinalis was effective for the treatment of BV in women of reproductive age, without any side effects. This herb could be recommended for women of reproductive age who uncomfortable with the potential side effects of synthetic drugs.

Full text

15
© 2018 Journal of Advanced Pharmaceutical Technology & Research | Published by Wolters Kluwer ‑ Medknow
The eect of Calendula ocinalis versus metronidazole
on bacterial vaginosis in women: A double‑blind
randomized controlled trial
Abstract
Bacterial vaginosis (BV) is a common disorder among women of reproductive age.
This study aimed to compare the effect of a Calendula officinalis extract‑based cream
and metronidazole on BV among women of reproductive age. In this study, 80 women
of reproductive age with BV were randomly assigned to the C. officinalis (n = 40) or
metronidazole (n = 40). Diagnosis of BV was confirmed when at least 3 of the 4 Amsel
criteria were met (pH >4.5, whitish grey or thin homogeneous discharge, release of a
fishy odor on adding 10% KOH, and detection of clue cells on microscopic examination).
For each group, either a methanol extract of C. officinalis or metronidazole vaginal
cream (5 g) was used for 1 week intravaginally, and all signs and symptoms were
assessed 1 week after treatment completion. Before the intervention, the two groups did
not differ significantly with regard to vaginal burning, odor, dysuria, and dyspareunia,
but itching was significantly more common in the C. officinalis group than in the
metronidazole group (22.5% vs. 2.5%, P = 0.01). One week after the intervention, all
women in both groups were free of symptoms, including vaginal itching and burning
sensation, odor, dysuria, and dyspareunia. None of the women in either group suffered
any side effects from C. officinalis or metronidazole. C. officinalis was effective for the
treatment of BV in women of reproductive age, without any side effects. This herb
could be recommended for women of reproductive age who uncomfortable with the
potential side effects of synthetic drugs.
Key words: Bacterial, Calendula officinalis, metronidazole, vaginitis
Zahra Pazhohideh,
Solmaz Mohammadi1,
Nosrat Bahrami2, Faraz Mojab3,
Parvin Abedi4, Elham Maraghi5
Department of Midwifery, Faculty
of Medical Sciences, Shushtar,
1Department of Midwifery, Ahvaz
Jundishapur University of Medical
Sciences, 4Department of Midwifery,
Menopause Andropause Research
Center, Ahvaz Jundishapur University
of Medical Sciences, 5Department of
Biostatistics and Epidemiology, School
of Public Health, Ahvaz Jundishapur
University of Medical Sciences, Ahvaz,
2Faculty of Nursing and Midwifery,
Dezful University of Medical Sciences,
Dezful, 3Pharmaceutical Sciences
Research Center, Shahid Beheshti
University of Medical Sciences,
Tehran, Iran
J. Adv. Pharm. Technol. Res.
Access this article online
Quick Response Code:
Website:
www.japtr.org
DOI:
10.4103/japtr.JAPTR_305_17
INTRODUCTION
Bacterial vaginosis (BV) is a common disorder among
reproductive-aged women, and its underlying cause is
primarily a change in vaginal ora.[1] BV is characterized
by the suppression of the normal vaginal ora (hydrogen
peroxide-producing Lactobacillus) and an increase in
facultative and anaerobic bacteria.[2] The main cause of BV is
unclear; however, it is more commonly seen with anaerobic
bacteria than with aerobic bacteria.[3]
The prevalence of BV was reported to be 29.2% in a large
study in the United States, which corresponds to 21.4 million
women.[4] One study in Iran showed that the prevalence of
BV among reproductive-aged women was 16.2%[5] while
another study in Iran found a prevalence of 15.6% among
these women.[6]
Address for correspondence:
Dr. Parvin Abedi,
13th East Kianpars Ave, 1st Maroon St., No: 46, Ahvaz, Iran.
E‑mail: parvinabedi@ymail.com This is an open access article distributed under the terms of the Creative
Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows
others to remix, tweak, and build upon the work non‑commercially, as long as
the author is credited and the new creations are licensed under the identical
terms.
For reprints contact: reprints@medknow.com
How to cite this article: Pazhohideh Z, Mohammadi S, Bahrami N,
Mojab F, Abedi P, Maraghi E. The eect of Calendula ocinalis versus
metronidazole on bacterial vaginosis in women: A double-blind
randomized controlled trial. J Adv Pharm Technol Res 2017;9:15-9.
original articlE
[Downloaded free from http://www.japtr.org on Wednesday, January 17, 2018, IP: 151.244.66.7]

Pazhohideh, et al.: Calendula ocinalis and bacterial vaginosis
16 Journal of Advanced Pharmaceutical Technology & Research | Volume 9 | Issue 1 | January‑March 2018
Because of the unpleasant odor associated with BV,
most women experience embarrassment and feel dirty
and believe that the smell can be detected by others.
In addition, women with BV have low self-esteem and
lower sexual satisfaction.[7] BV can be treated with a
variety of antimicrobial agents, such as ampicillin,
penicillin, and metronidazole. However, metronidazole
in the form of tablets, gel, or suppositories used for
7 days shows beer results.[8] The use of antimicrobial
agents has been associated with drug resistance and in
some patients, recurrence of BV.[9] Furthermore, using
metronidazole during pregnancy and while breastfeeding
may pose some risks to the fetus and infant.[10] Therefore,
researchers are looking for alternative treatment for BV.
One alternative is the use of herbal medicine, which is
associated with fewer complications and which could
preserve the natural flora of the body.[11] Calendula
ocinalis L (pot marigold) is a herbaceous plant from
the Asteraceae family that contains phytochemicals such
as avonoids, saponins, carotenoids, triterpenoids, and
tannins, which possess antibacterial activity.[12] Bissa and
Bohra studied the antibacterial potential of the leaves,
roots, and owers of C. ocinalis and found good activity
against Escherichia coli, Salmonella, Klebsiella pneumoniae,
Enterobacter aerogenes, and Agrobacterium tumefaciens.[13]
The anti-inammatory, antifungal, and antioxidant eects
of C. ocinalis have been demonstrated, but there is a
lack of studies regarding its eect on BV. Therefore,
the aim of this study was to compare the eect of C.
ocinalis and metronidazole vaginal cream on BV among
reproductive-aged women.
MATERIALS AND METHODS
This was a double-blind randomized controlled trial
for which 80 nonpregnant reproductive-aged women
in Dezful, Iran, were recruited. The study setting
was a public health center in Dezful, Iran (Health
center no: 3). The study protocol was approved by the
Ethics Commiee of Ahvaz Jundishapur University of
Medical Sciences (Ref no. IR.AJUMS.REC.1394.302).
In addition, the protocol was registered in the Iranian
Registry for Randomized Controlled Trials (Ref no.
IRCT2015082921414N3). Written informed consent
was obtained from all women before data collection.
This study was started in early May 2016 and ended in
late July 2016. The inclusion criteria were women aged
18–45 years who were married and had BV. Women who
complained of other types of vaginitis or were pregnant
were excluded from the study.
Sample size
The required sample size with regard to recovery after
using metronidazole and C. ocinalis (70 and 80%),[14] with
a maximum acceptable error of 0.3 either side and a 20%
arition rate, was calculated to be 40 for each group.
Herbal extract
The fresh owers of C. ocinalis were purchased from an
Iranian herbal market, and they were converted to a powder
at the Pharmacy School of Shahid Beheshti University
of Medical Sciences. The C. ocinalis vaginal cream was
made using the stages of extraction and formulation. The
rst and second stages of extraction were as follow: the
minimum inhibitory concentration (MIC) was determined
and the vaginal cream was prepared. All drug preparation
processes were performed in the Bacteriology Department
of Shahid Beheshti University of Medical Sciences, Tehran,
Iran. The mean MIC (0.07 µmol/ml) was considered against
particular bacterial strains in this study. The direct method,
in which the concentration of the extract was determined
and the drug then added, was used to add the plant extract
to the culture. Considering the antibacterial potency of the
methanol extract (MIC = 0.079 µmol/ml) compared to that of
the ethanol extract (MIC = 0.125 µmol/ml) and considering
the lower cost of methanol, the methanol extract was
chosen for the formulation. Composition of base cream was
cetostearyl alcohol, petroleum jelly, glycerin, mineral oil,
preservative, and antioxidant. After preparation, 80 tubes of
cream were made in two stages with the necessary testing
done in each stage.
Randomization
A total of 80 women with BV were randomized into the
C. ocinalis and metronidazole groups. Permuted block
randomization with a block size of 4 (using a random
permutation table) was used. The metronidazole and C.
ocinalis labels were encoded as A or B by a person who
was not aware of the purpose of the study. The C. ocinalis
and metronidazole formulations were similar in appearance
and consistency. The person who distributed metronidazole
and C. ocinalis was not aware of the purpose of study.
Intervention
A detailed history was taken from all patients who were
seen at the health centers and a physical examination was
performed. Vaginal pH was measured using pH strips.
A Pap smear was then performed on all women who gave
consent. Clinical signs and symptoms such as edema,
inflammation, and changes in vaginal discharge were
recorded for each participant. Next, a sample of vaginal
discharge was obtained to look for clue cells and perform a
whi test using 10% KOH. Diagnosis of BV was conrmed
when at least 3 of the 4 Amsel criteria were met (pH >4.5,
whitish gray or thin homogenous discharge, release of a
shy odor on adding 10% KOH, and presence of clue cells
in microscopy). Women who were diagnosed with BV
were randomly treated with metronidazole or C. ocinalis
coded as A or B. All women in both groups received advice
regarding avoiding intercourse, using coon underwear,
changing underwear regularly, keeping the perineum dry,
and instruction on washing the applicator before starting
treatment and after each use. Each woman in both groups
[Downloaded free from http://www.japtr.org on Wednesday, January 17, 2018, IP: 151.244.66.7]

Pazhohideh, et al.: Calendula ocinalis and bacterial vaginosis
17
Journal of Advanced Pharmaceutical Technology & Research | Volume 9 | Issue 1 | January‑March 2018
received a tube of cream containing 50 g C. ocinalis or
placebo, and women were instructed to use one applicator
of vaginal cream (5 g) before sleep for 1 week. The eect
of treatment was assessed 1 week after completion of the
treatment through an interview, observation, and laboratory
testing of vaginal discharge. If a patient did not aend the
follow-up, one of the investigators called her, and if she still
did not aend, she was considered a dropout. The vaginal
discharge samples were assessed using a microscope and
appropriate tests. All data were entered in the medical
records of the patients. All women were asked to report
any side eects of the treatment.
Statistical analysis
All data were entered in Chicago, Illinois: SPSS Inc., USA.
The normal distribution of data was evaluated using the
Shapiro–Wilk test. The independent t-test or Mann–Whitney
test was used for comparing continuous data between the
two groups while the Chi-square test was used for categorical
data. P < 0.05 was considered statistically signicant.
RESULTS
None of the participants withdrew from the study [Figure 1].
Table 1 shows the sociodemographic characteristics of the
participants of the two groups. The median age of the
women in the metronidazole and C. ocinalis groups was
31 and 32.38 years, respectively. Almost all women (99.9%)
in both groups used one method of contraception. The
method that most women used was withdrawal (used by
35% of women in each group). Women did not show any
signicant dierence in age, marriage age, body mass index,
parity, amount of coitus per month, job, history of BV, and
method of contraception.
Before the intervention, most women in both groups had
symptoms such as vaginal burning, itching, and discharge;
odor; and dysuria, with dysuria being the most common
in both groups. Itching was signicantly more common
in the C. ocinalis group than in the metronidazole group
(22.5% vs. 2.5%, P = 0.01). The two groups did not show
any signicant dierence with regard to other symptoms
before intervention. One week after intervention, all women
in both groups were free of symptoms [Table 2]. None of
the women in either group suered any side eects from
C. ocinalis or metronidazole.
DISCUSSION
This study aimed to compare the eect of C. ocinalis and
metronidazole on BV in reproductive-aged women. Our
results showed that both C. ocinalis and metronidazole
could eliminate all symptoms of BV in women. In support
of our ndings, Roopashree et al. showed that an aqueous
extract of C. ocinalis had an antibacterial eect against both
Gram-positive and Gram-negative strains.[15] In addition,
C. officinalis has shown that antifungal activity was as
eective as clotrimazole in women with a Candida albicans
infection.[16] In the present study, we used fresh owers
of C. ocinalis. Other studies have shown that all types of
extracts (aqueous, alcoholic, chloroform, and petroleum
ether) of the leaves and owers of C. ocinalis were more
active than extracts of the roots of this plant.[13]
In a study, Tedeschi et al. found that vaginal gel comprising
isoavones, Lactobacillus sporogenes, and C. ocinalis could
signicantly reduce the signs and symptoms of vaginal
dystrophy (itching, burning, vulvovaginal erythema, and
vaginal dryness) compared to placebo in postmenopausal
women.[17] Palmieri et al. found that a mixture containing
Saliva, Melaleuca, and Calendula plants had a very good
eect on the vaginal surface of women with damage to their
vaginal mucosa. Furthermore, they found that Calendula
could enhance the body’s defense against external agents
and exert anti-inflammatory, antioxidant, and wound
healing effects.[18] The results of the aforementioned
studies are in line with ours. In the present study, we used
a methanol extract of C. ocinalis, and other studies have
shown that the methanol extract of C. ocinalis had beer
antimicrobial activity than that of the ethanol extract.[19] Our
study showed that women in both groups did not suer
any side eects from metronidazole or C. ocinalis. Other
studies have also demonstrated that the methanol extract
of C. ocinalis has very few side eects on the skin, such
as skin irritation, with no sensitization or phototoxicity.[20]
Figure 1: Flowchart of recruitment and retention and participants
in the study
[Downloaded free from http://www.japtr.org on Wednesday, January 17, 2018, IP: 151.244.66.7]

Pazhohideh, et al.: Calendula ocinalis and bacterial vaginosis
18 Journal of Advanced Pharmaceutical Technology & Research | Volume 9 | Issue 1 | January‑March 2018
Table 1: Demographic characteristics of participants in two groups of metronidazole and Calendula
ofcinalis
Variables Median (IQR); Range or n (%) P
Calendula officinalis (n=40) Metronidazole (n=40)
Age (year) 32.38 (26.2‑37.5); 29 31 (28‑36); 22 0.63
Marriage duration (year) 9.5 (5.25‑15); 32 11 (5.2‑17.7); 26 0.96
Body mass index (kg/m2)27.6 (24.3‑31.1); 26 26.7 (24.1‑29.4); 17 0.29
Gravida 2.03 (1‑2.75); 8 2 (1‑3); 6 0.28
Para 1 (1‑2); 8 2 (1‑3); 5 0.09
Number of children 1 (1‑2); 5 2 (1‑3); 5 0.06
Number of coitus per month 10 (8‑13.5); 20 10 (7.25‑14); 20 0.92
Job
Housewife 37 (92.5) 39 (97.5) 1
Employee 3 (7.5) 1 (2.5)
Mode of delivery
Normal vaginal delivery 15 (37.5) 26 (65) 0.71
Cesarean 25 (62.5) 14 (37.8)
History of bacterial vaginosis
Yes 22 (55) 29 (72.5) 0.16
No 18 (45) 11 (27.5)
Method of contraception
Oral contraception pills 2 (5.7) 4 (10.8) 0.73
Depot medroxyprogesterone acetate 3 (8.6) 0
Condom 6 (17.1) 7 (18.9)
Tubal ligation 3 (8.6) 3 (8.1)
Withdrawal 14 (37.8) 14 (40)
Vasectomy 1 (2.9) 1 (2.7)
Intrauterine device 6 (17.1) 8 (21.6)
No contraception 5 (12.5) 3 (7.5)
The
P
values are based on the results of Mann‑Whitney U‑test or Pearson’s Chi‑square test. IQR: Interquartile range (25th‑75th percentiles)
Table 2: Clinical signs and symptoms before and after treatment in two groups of Calendula
ofcinalis and metronidazole
Variables Calendula officinalis (n=40) Metronidazole (n=40) P value before intervention
Before, n (%) After, n (%) Before, n (%) After, n (%)
Burning
Yes 13 (32.5) 018 (45) 0 0.35
No 27 (67.5) 40 (100) 22 (55) 40 (100)
Itching
Yes 9 (22.5) 01 (2.5) 0 0.01
No 31 (77.5) 40 (100) 39 (97.5) 40 (100)
Vaginal discharge
Yes 11 (27.5) 024 (60) 0 0.06
No 29 (72.5) 40 (100) 16 (40) 40 (100)
Bad odor
Yes 21 (52.5) 016 (40) 0 0.37
No 19 (47.5) 40 (100) 24 (60) 40 (100)
Dysuria
Yes 39 (97.5) 040 (100) 0 1
No 1 (2.5) 40 (100) 040 (100)
Dyspareunia
Yes 0 0 3 (7.5) 0 0.24
No 40 (100) 27 (92.5)
pH* 4.7±0.25 (4.5‑5.5) 4.7±0.23 (3.5‑4.5) 4.9±0.28 (4.5‑5.5) 4.1±0.28 (3.5‑4.5) 0.58
*The values are expressed as mean±SD (range). The
P
values are based on the results of Fisher’s exact test or independent
t
‑test. SD: Standard deviation
[Downloaded free from http://www.japtr.org on Wednesday, January 17, 2018, IP: 151.244.66.7]

Pazhohideh, et al.: Calendula ocinalis and bacterial vaginosis
19
Journal of Advanced Pharmaceutical Technology & Research | Volume 9 | Issue 1 | January‑March 2018
Strengths and limitations of study
Although other studies have shown that C. ocinalis is
eective against vaginal candidiasis,[15] to the best of our
knowledge, this is the first time that C. officinalis was
compared with metronidazole for the treatment of BV. We
relied on participant responses regarding how the vaginal
cream was used and the presence of symptoms. These
answers might have been aected by recall bias.
CONCLUSION
C. officinalis is effective for the treatment of BV in
reproductive-aged women without any side eects. The
use of this herb could be recommended in women who are
uncomfortable with the potential side eects of conventional
pharmaceuticals.
Acknowledgment
We would like to thank Ahvaz Jundishapur University of
Medical Sciences for nancial support of this study. Thanks
also extended to women who participated in this study.
Financial support and sponsorship
This study nancially supported by Ahvaz Jundishapur
University of Medical Sciences.
Conicts of interest
There are no conicts of interest.
REFERENCES
1. Mårdh PA. The denition and epidemiology of bacterial vaginosis.
Rev Fr Gynecol Obstet 1993;88:195-7.
2. Fredricks DN, Fiedler TL, Marrazzo JM. Molecular identication
of bacteria associated with bacterial vaginosis. N Engl J Med
2005;353:1899-911.
3. Hillier SL. The complexity of microbial diversity in bacterial
vaginosis. N Engl J Med 2005;353:1886-7.
4. Koumans EH, Sternberg M, Bruce C, McQuillan G, Kendrick J,
Suon M, et al. The prevalence of bacterial vaginosis in the United
States, 2001-2004; associations with symptoms, sexual behaviors,
and reproductive health. Sex Transm Dis 2007;34:864-9.
5. Bahram A, Hamid B, Zohre T. Prevalence of bacterial vaginosis
and impact of genital hygiene practices in non-pregnant women
in Zanjan, Iran. Oman Med J 2009;24:288-93.
6. Bafghi AF, Hoseizadeh A, Naghshi Jouzsheri M, Mohseni P.
Frequency and etiology of vaginitis in women refereed to health
centers in Yazd city. JCHR 2014;3:163-7.
7. Bilardi JE, Walker S, Temple-Smith M, McNair R, Mooney-Somers J,
Bellhouse C, et al. The burden of bacterial vaginosis: Women’s
experience of the physical, emotional, sexual and social impact of
living with recurrent bacterial vaginosis. PLoS One 2013;8:e74378.
8. Sobel JD, Ferris D, Schwebke J, Nyirjesy P, Wiesenfeld HC, Peipert J,
et al. Suppressive antibacterial therapy with 0.75% metronidazole
vaginal gel to prevent recurrent bacterial vaginosis. Am J Obstet
Gynecol 2006;194:1283-9.
9. Beigi RH, Austin MN, Meyn LA, Krohn MA, Hillier SL.
Antimicrobial resistance associated with the treatment of bacterial
vaginosis. Am J Obstet Gynecol 2004;191:1124-9.
10. Briggs GC, Freeman RK, Yaffe SJ. Drugs in Pregnancy and
Lactation. 9th ed. Philadelphia, PA: Lippinco Williams & Wilkins;
2011.
11. Guaschino S, De Seta F, Sartore A, Ricci G, De Santo D,
Piccoli M, et al. Ecacy of maintenance therapy with topical
boric acid in comparison with oral itraconazole in the treatment
of recurrent vulvovaginal candidiasis. Am J Obstet Gynecol
2001;184:598-602.
12. Radioza SA, Iurchak LD. Antimicrobial activity of Calendula L.
plants. Mikrobiol Z 2007;69:21-5.
13. Bissa S, Bohra A. Antibacterial potential of pot marigold.
J Microbiol Antimicrob 2011;3:51-4.
14. Foladi Z, Afshari P, Gharibi T, Dabagh M. Comparison of eective
clotrimazole cream with thyme herbal vaginal cream in treat
candidia vaginitis. South Med J 2010;12:214-24.
15. Roopashree TS, Dang R, Shobha Rani RH, Narendra C. Antibacterial
activity of antipsoriatic herbs: Cassia tora, Momordica charantia and
Calendula ocinalis. Int J Appl Res Nat Prod 2008;1:20-8.
16. Saffari E, Mohammad-Alizadeh-Charandabi S, Adibpour M,
Mirghafourvand M, Javadzadeh Y. Comparing the effects of
Calendula officinalis and clotrimazole on vaginal candidiasis:
A randomized controlled trial. Women Health 2017;57:1145-60.
17. Tedeschi C, Benvenuti C; Research Group EG. Comparison of
vaginal gel isoavones versus no topical treatment in vaginal
dystrophy: Results of a preliminary prospective study. Gynecol
Endocrinol 2012;28:652-4.
18. Palmieri B, Vadala M, Fornabaio N, Laurino CA. New vaginal gel
for femalegenital wellness and disease prevention: Rationale of
a multi-herbal derived treatment on a high-tech pharmaceutical
perspective. AJPCT 2016;4:1-18.
19. Efstratiou E, Hussain AI, Nigam PS, Moore JE, Ayub MA, Rao JR,
et al. Antimicrobial activity of Calendula ocinalis petal extracts
against fungi, as well as gram-negative and gram-positive clinical
pathogens. Complement Ther Clin Pract 2012;18:173-6.
20. Final report on the safety assessment of Calendula ocinalis extract
and Calendula ocinalis. Int J Toxicol 2001;20 Suppl 2:13-20.
[Downloaded free from http://www.japtr.org on Wednesday, January 17, 2018, IP: 151.244.66.7]