Preparation of Antibacterial Lotion of Carex meyeriana Kunth

Mengnan Zhu

1,† a

, Jialin Song

and Hongli Zhou

1,* c

School of Chemistry and Pharmaceutical Engineering, Jilin Institute of Chemical Technology, Jilin, China

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

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

https://orcid.org/0000-0003-0915-1545

https://orcid.org/0000-0002-0878-2874

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

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)

C－PHMB content in sample solution (g/mL).

m－PHMB mass of the sample solution from the

standard curve (g).

V－Volume 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

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:

AX ×=

(2)

－Total number of bacterial colonies.

A － Total number of bacterial colonies on 5

nutrient agar plates.

K－Dilution 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:

=No－Nx (3)

－Killing log value.

No － Log value of average living bacteria

concentration in control group.

Nx－Log 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

/mL)

Dandelion

/mL)

Mentha

/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

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

4.02×10

3.80×10

3.87×10

3 2.89×10

3.05×10

2.95×10

2.96×10

4 2.61×10

2.95×10

2.86×10

2.81×10

5 3.02×10

3.20×10

2.63×1

2.95×10

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) .

REFERENCES

Aslani A, Zolfaghari B, F Davoodvandi. Design,

Formulation and Evaluation of an Oral Gel from

Punica Granatum Flower Extract for the Treatment of

Recurrent Aphthous Stomatitis[J]. Advanced

Pharmaceutical Bulletin, 2016, 6(3): 391.

Cheng X, Cheng Y, Zhang N, et al. Purification of

flavonoids from Carex meyeriana Kunth based on

AHP and RSM: Composition analysis, antioxidant,

and antimicrobial activity[J]. Industrial Crops and

Products, 2020, 157: 112900.

Gong D K, Zhang A, Luo H, et al. Polyhexamethylene

biguanide hydrochloride anchored polymeric elastic

fibers with robust antibacterial performance[J].

Journal of Applied Polymer Science.2021.

Hyun Kyu Seo, Ju Sun Kim, Sam Sik Kang. Labdane

Diterpenes and Flavonoids from Leonurus

japonicus[J]. Helvetica Chimica Acta, 2010, 93(10):

2045-2051.

Kim C S, Park S N, Ahn S J, et al. Antimicrobial effect of

sophoraflavanone G isolated from Sophora flavescens

against mutans streptococci[J]. Anaerobe, 2013,

19(Complete): 17-21.

Sato S, Takeo J, C A oyama, et al. Na+-glucose

cotransporter (SGLT) inhibitory flavonoids from the

roots of Sophora flavescens. [J]. Bioorganic &

Medicinal Chemistry, 2007, 15(10): 3445-3449.

Zou P, Tu P, Jiang Y. A simple and specific quantitative

method for determination of dictamnine in Dictamni

Cortex by 1H NMR spectroscopy[J]. Analytical

Methods, 2013, 5(4): 1062-1067.

Zhang J H, Zhu Y, Fan X H, et al. Efficacy-oriented

compatibility for component-based Chinese

medicine[J]. Acta Pharmacologica Sinica, 2015, 36(6):

654-658.

Zhang S T, Cheng G H, Lin J M, et al. Optimization of

Jatropha curcas for total flavonoids by response

surface methodology and antibacterial activity[J].

Academic Journals, 2011.

Zhengwu Jiangsupa/supsupb/sup, Bin Wangsupa/sup,

Hualin Chesupa/sup &, Liusupa/Sup B. Structural

Characterization and Bacteriostatic and Cytotoxicity to

3T3 Cells Study of Oligobiguanidine

(Polyhexamethylene Biguanidine Hydrochloride) and

its 3-Glycidoxypropyltrimethoxysilane Derivatives[J].

Journal of Macromolecular Science: Part A -

Chemistry, 2012, 49(11): 952-962.

Zhao W, Wolfender J L, Hostettmann K, et al. Antifungal

alkaloids and limonoid derivatives from Dictamnus

dasycarpus[J]. Phytochemistry, 1998, 47(1): 7-11.

Preparation of Antibacterial Lotion of Carex meyeriana Kunth

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