Preparation of Antibacterial Lotion of Carex meyeriana Kunth
Mengnan Zhu
1,† a
, Jialin Song
2b
and Hongli Zhou
1,* c
1
School of Chemistry and Pharmaceutical Engineering, Jilin Institute of Chemical Technology, Jilin, China
2
Bioinformatics Research Center, School of Life Science and Technology, Xian Jiaotong University, Xian, China
Keywords: Carex meyeriana Kunth, Lotion, Antibacterial, Safety.
Abstract: This paper is to prepare lotion of Carex meyeriana Kunth (CMK) with antibacterial effect. The lotion is
mainly composed of the following materials: CMK, Leonurus japonicas, Sophora flavescens, Dictamnus
dasycarpus and polyhexamethylene biguanide. Suspension quantitative germicidal test and toxicity test
were used to evaluate the antibacterial effect and safety of prepared lotion. Results showed that the lotion
exerts profound inhibitory effects on Escherichia coli, Staphylococcus aureus and Candida albicans, all the
inhibitory rates exceeded 99.9% after treatment for 20 min, 20 min and 30 min, respectively. After being
stored in a 40℃ incubator under strong light for 6 months, the antibacterial effects of prepared lotion were
not decreased significantly. Moreover, the lotion has no skin irritation and adverse reactions. Thus the lotion
reported in present investigation possesses better applicable potentials due to the excellent antibacterial
effects as well as better stability and safety properties.
1 INTRODUCTION
1
Lotion mainly includes plant medicine and synthetic
medicine. Traditional botanical drugs potentiate the
onset and rapid efficacy of synthetic drugs.
However, synthetic drugs are prone to generate
resistance and relapse. Therefore, antibacterial
products with Chinese herbal medicine as the main
component combined with synthetic drugs come into
being and have been widely used. As one of the
Northeast Three Treasures in China, Carex
meyeriana Kunth has a good bacteriostatic effect
(Cheng 2020), but its uses are currently limited to
the preparation ofmattresses and insoles. High value-
added development of Carex meyeriana Kunth is
urgent to be conducted. Leonurus japonicas
Houtt (LjH) is used in the treatment of
gynecologic disorders such as menorrhagia,
menostasia, and other irregular menstruation
disorders (Hyun 2010). Sophora flavescens (SF) is
used for external treatment of diseases such as red
and leucorrhea, Yin swelling and Yin itching,
eczema, wet sores, skin itching and trichomonas
vaginitis (Sato 2007). Dictamnus dasycarpus Turcz
a
https://orcid.org/0000-0003-0915-1545
b
https://orcid.org/0000-0002-0878-2874
c
https://orcid.org/0000-0002-5561-2587
(DdT) can dispel wind and relieve itching totreat
wind heating and dampness caused by rubella
andeczema (Zou 2013). From the theory of
compatibility of traditional Chinese medicine, the
combination of radix DdT and radix SF can greatly
increase the functions of detoxification, heat clearing
and dehumidification (Zhang 2015). In this study,
extracts of CMK, LjH, SF and DdT were synergized
with polyhexamethylene biguanide (PHMB)
toprepare the lotion with antibacterial potential.
Then, the stability and safety properties were further
evaluated.
2 MATERIALS AND METHODS
2.1 Materials
CMK were collected in October 2020 in Longtan
District, Jilin City, Jilin Province. LjH, SF and DdT
were purchased from Longtan pharmacy (Jilin,
China). PHMB was purchased from Shanghai Gaoju
Biotechnology Co, LTD. (Shanghai, China), lactic
acid was purchased from Tianjin Damao Chemical
Reagent Factory (Tianjin, China). All chemical
reagents are of 95% analytical pure. All the strains
used in the experiment were provided by China
910
Zhu, M., Song, J. and Zhou, H.
Preparation of Antibacterial Lotion of Carex meyeriana Kunth.
DOI: 10.5220/0011313000003443
In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 910-915
ISBN: 978-989-758-595-1
Copyright
c
2022 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
Industrial Microbiology Preservation and
Management Centre.
2.2 Methods
2.2.1 Formula Screening with Bacteriostatic
Test
Extracts of LjH, SF and DdT were prepared by hot-
water and ethanol extractions, and then the MIC of
the extracts against Escherichia coli (EC),
Staphylococcus aureus (SA) and Candida albicans
(CA) were determined by the doubled and half
dilution method.
CMK extract: CMK was cut into 2 - 3 cm and
refluxed with distilled water at a solid-liquid ratio of
1: 40 at 90℃ for 3h.
LjH and DdT extracts: LjH and DdT were
powdered and refluxed with distilled waterat a solid-
liquid ratio of 1: 12 at 90℃ for 1.5 h.
SF extract: SF was crushed and refluxed with
80% ethanol as the solvent by a solid-liquid ratio of
1:20 at 70℃ for 3h.
The lotion was prepared by 1: 1 of MIC of each
extract after the extracts were filtered, separately.
2.2.2 Product Appearance and
Determination of pH Value
Visual inspection of the product appearance
wasperformed under the conditions of ambient
temperature of 19℃ and relative humidity of 85%,
according to sanitary products standard GB15979-
2002.
The pH range of aqueous solution samples was
determined by pH test paper, and the pH meter was
corrected with the corresponding pH correction
liquid, and then the pH value of the sample was
determined. The pH value of organic samples was
measured by pH test paper.
2.2.3 Determination of PHMB Content
According to GB26367-2010, the absorbance was
measured at 545 nm, water was served as solvent.
The calculation formula of PHMB content is as
follows:
(1)
CPHMB content in sample solution (g/mL).
mPHMB mass of the sample solution from the
standard curve (g).
VVolume of sample solution (mL).
2.2.4 Stability Test
The accelerated test method was adopted, and the
samples were placed in a 40℃ constant temperature
box with strong light for 6 months. The inhibitory
effects of the samples on SA, EC and CA were
measured before and after storage to judge their
stabilities (Aslani 2016).
2.2.5 Toxicity Test
According to the disinfection technical specification
2002 edition, rabbits were selected for vaginal
mucosa irritation test. Rabbits were divided into the
infected and control groups (n = 3). The average
score of the vaginal mucosal stimulation of the
experimental group was calculated by the following
equation: add up all scores of three animals in the
experimental group, and divide by the total numbers
of observed animals. (number of animals × 3). The
scoring method for control group was the same as
above. The average score of the experimental group
was subtracted from the average score of the control
group to obtain the stimulation index. According to
the disinfection technical Specification 2002 edition,
guinea pigs were selected for skin allergy test. There
were 16 guinea pigs in the control group and the
experimental group, respectively. Take 0.5 mL of
sample and apply it to the animal hair removal area,
then negative control groupwas given tight subjects
stimulate processing, and the skin reaction was
observed and scored 24 h and 48 h after the
treatment. The animals with skin reaction (score≥1)
were divided by the number of experimental animals
in this group to obtain the sensitization rate (%).
2.2.6 Preparation of Bacterial Suspension
The bacterial stock solution used in the experiment
was configured with the corresponding inclined
surface culture medium, and the original bacteria
was dipped in the sterile operating table with the
inoculation stick, and the original bacteria was
evenly smeared on the surface of the inclined
surface culture medium by rotating the inoculation
stick, and then wrapped up and cultured for 24 h.
Take out the inclined culture medium, andann wash
the inclined plane with 10 mL of sterile water, then
make the bacterial suspension and pour it into the
sterile conical bottle (Zhang 2011).
2.2.7 Identification Test of Neutralizer
According to Disinfection Technical Specification
2002 edition of ministry of health, the neutralizer
1000V
m
×
=C
Preparation of Antibacterial Lotion of Carex meyeriana Kunth
911
identification test of antibacterial lotion was carried
out with TSB of 1% soap base, 6.5% tween 80 and
4.5% lecithin as neutralizer.
2.2.8 Microbial Index Detection
According to the hygienic standard for disposable
sanitary products GB-15979-2002, the ambient
temperature for inspection was fixed at 19℃ - 21℃.
Accurately weigh 10±1 g of the sample, add it into
200 mL of sterilized physiological saline, and mix
well to obtain a physiological saline sample solution.
Then, take the supernatant and count the colonies,
inoculate a total of 5 plates, add 1 mL of washing
liquid sample to each plate, and then pour 15 - 20
mL of melted nutrient agar medium cooled to about
45, and mixed well in each plate. After the agar
was solidified, turned the plate over and incubated at
35±2℃ for 48 h, and then counted the number of
colonies on the plate.
The formula calculating the total number of
bacterial colonies:
5
1
K
AX ×=
(2)
X
1
Total number of bacterial colonies.
A Total number of bacterial colonies on 5
nutrient agar plates.
KDilution degrees.
2.2.9 Bacteriostatic Test
According to Disinfection Technical Specification
2002 edition of ministry of health, quantitative
bacteriostatic test of bacteria and fungi for lotion
was carried out.
The formula killing log value:
K
L
=NoNx (3)
K
L
Killing log value.
No Log value of average living bacteria
concentration in control group.
NxLog value of live bacteria concentration in
experimental group.
3 RESULTS AND DISCUSSION
3.1 Screened Formula
The MIC of different plant extracts against different
strains were shown in Table 1.
Table 1: MIC of different plant extracts against different strains.
Stran LJH (mg/mL) SF (mg/mL)
DDT
(m
g
/mL)
Dandelion
(m
g
/mL)
Mentha
(m
g
/mL)
SA 24 5 5 50 /
EC / 8 6 / 32
CA / 5 10 / /
As shown in Table. 1, MIC of single dandelion
and peppermint was high, so LjH, SF and DdT were
choose to make antibacterial lotion.
3.2 Preparation of Antibacterial Lotion
Extracts of CMK, LjH and DdT, SF were mixed in
the ratio of 1: 1: 1, 95% PHMB was added to the
solution at a concentration of 0.8%, mixed well and
then 5%lactic acid solution was added, mixed well
and left for 48 h, the supernatant was taken, filtered
and centrifuged to obtain the antibacterial lotion.
3.3 Appearance and pH Value of
Lotion
The lotion was brown liquid, neat in appearance,
consistent with the inherent shape of the sanitary
product, without abnormal odor and foreign matter.
The pH value of the antibacterial lotion was 3.7 after
repeated determination for 3 times.
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
912
3.4 PHMB Content and Lotion
Stability
The content of PHMB was 0.2 ± 0.02%, which meet
the hygiene standards of guanidine disinfectants.
The bactericidal rate reached 99.9%, showing good
stability.
3.5 Vaginal Mucosa Irritation and Skin
Allergy
As shown in Table 2, the index of vaginal mucosa
stimulation to rabbits was 0.33, and the results
intensity test of vaginal mucosa stimulation was no
vaginal mucosa irritation. As shown in Table 3, the
sensitization rate of the sample was 0%, suggesting
none of skin allergy.
Table 2: Vaginal mucosa irritation rating table.
Group Number
Stimulus response score
Epithelial
tissue
Leukocyte
infiltration
Small vessels Edema Total
Infected
1 0 0 1 0 1
2 0 0 1 0 1
3 0 0 1 0 1
average score — — 0.33
contrast
4 0 0 0 0 0
5 0 0 0 0 0
6 0 0 0 0 0
average score — — 0
Stimulus index 0.33
Table 3: Skin allergy in guinea pigs.
Group Quantity
Induction
concentration
Stimulate
concentration
Time
(h)
Erythema response intensity
0 1 2 3 4
Test group 16 Sample apply Sample apply
24 h 16/16 0/16 0/16 0/16 0/16
48 h 16/16 0/16 0/16 0/16 0/16
Negative
control
16 Sample apply
24 h 16/16 0/16 0/16 0/16 0/16
48 h 16/16 0/16 0/16 0/16 0/16
Positive
control
16 0.60% 0.30%
24 h 0/16 15/16 1/16 0/16 0/16
48 h 0/16 12/16 1/16 0/16 0/16
continued table
Edema response intensity
Sensitized
animals
Sensitization rate
(%)
0 1 2 3
16/16 0/16 0/16 0/16 0 0
16/16 0/16 0/16 0/16 0 0
16/16 0/16 0/16 0/16 0 0
16/16 0/16 0/16 0/16 0 0
12/16 4/16 0/16 0/16 16 100
8/16 7/16 1/16 0/16 13 81.25
Preparation of Antibacterial Lotion of Carex meyeriana Kunth
913
3.6 Neutralizer Identification Test
Table 4 showed the experimental results of
neutralizer identification.
Table 4: Results of neutralizer identification test.
Concen
tration
group
Number of growing colonies(cfu/mL)
Average
(cfu/mL)
1 2 3
concentrate
1 0 0 0 0
2 3.06×10
4
4.02×10
4
3.80×10
4
3.87×10
4
3 2.89×10
7
3.05×10
7
2.95×10
7
2.96×10
7
4 2.61×10
7
2.95×10
7
2.86×10
7
2.81×10
7
5 3.02×10
7
3.20×10
7
2.63×1
7
2.95×10
7
Annotation: All the negative controls were sterile
The Table 4 showed that TSB neutralizer with
1% saponin, 6.5% Tween 80 and 4.5% lecithin could
neutralize the bactericidal components in
antibacterial lotion, and was suitable for the killing
test of fungi and bacteria to be carried out.
3.7 Microbial Index Test
The results of microbial indicators of the lotion were
in line with the requirements of the hygienic
standard for disposable sanitary products GB15979-
2002.
3.8 Bacteriostatic Test
The results in Table 5 showed that the killing
logarithms of the lotion against SA and EC for 20
min were greater than 5.00, and the killing logarithm
of CA treated for 10 min was greater than 4.00,
indicating that the lotion has killing effects on EC,
SA and CA.
Table 5: Killing effect of antibacterial lotion on EC, SA and CA.
Test
strains
Concentra
tion
Average colony
number logarithm
and range
Average killing logarithm and range at
different time (min)
0 1
EC stoste
7.07
(
7.03-7.11
)
>5.00 (>5.00) >5.00 (>5.00)
SA stoste
7.06
(
7.02-7.11
)
>5.00 (>5.00) >5.00 (>5.00)
CA stoste
6.26
(
6.20-6.31
)
2.47 (>4.00) >4.00 (>4.00)
PHMB is one of the commonly used antibacterial
agent, which exhibits low minimal inhibitory
concentration and rapid inactivating efficacy against
common microorganisms (Gong 2021). As an
antiseptic in the medical field for many years(Zheng
2012), CMK can inhibit EC, SA and CA (Cheng
2020). DdT can inhibit the growth of many
pathogenic bacteria in vitro (Zhao 1998). The
aqueous extract of SF has antimicrobial and anti-
inflammatory effects and has been widely used for
the treatment of skin problems (Kim 2013). Due to
the synergy of the above-mentioned extracts, the
experimental results showed that the combinatory
use of plant extracts and PHMB can inhibitEC, SA
and CA, significantly. The lotion prepared using
these components has good compatibility with skin,
no irritation and allergic reaction, long-action time
and low-recurrence rate, and improves the
shortcomings of the previous lotion.
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
914
4 CONCLUSIONS
This study innovatively used plant extract of CMK
for preparation of antibacterial lotion, expanded the
scope of its application, compound CMK lotion was
made from natural herbs CMK, LjH, SF, DdT
extract as the functional component. The lotion is a
brown liquid with no abnormal odour and a pH of
3.7. The lotion possesses good stability, and water
was used as solvent and PHMB with content of
0.2% and 0.5% lactic acid were added. The in vitro
antibacterial test showed that the lotion has
antibacterial activity against EC, SA and CA, and
the antibacterial effects can last for a long time, and
there were no irritation and adverse reactions to
vaginal mucosa. The results showed that the lotion
has promising market application potential and
development value.
ACKNOWLEDGEMENTS
This investigation was supported by the programs of
the Science and Technology Department of Jilin
province (Grant No. 20190304102YY) .
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