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Procedia Engineering 174 (2017) 410 – 415

13th Global Congress on Manufacturing and Management, GCMM 2016

Application of Atractylodes Macrocephala Koidz Extract in

Methicillin - Resistant Staphylococcus Aureus
Zhenguo Xu a, , Yuhua Cai a *, Gaofu Fan a, Xiushu Liu a, and Yin Daib
a
Department of Biotechnology Applications, Hefei vocational and technical college, Hefei 238000, PR China
b
Instiute of Animal Husbandry and Veterinary Science ,Anhui Academy of Agricultural Science, Hefei , China

Abstract

To study the effect of Atractylodes macrocephala Koidz extractive on MRSA. First we used hydrogen peroxide aqueous to
oxygenize the sample, and then added into ascorbic acid which had protected the oxidative product to extract effective consituent
of Actractylodes macrocephala Koidz, measuring its antibacterial activity with oxford cup method and the minimum inhibitory
concentration with double dilution method. Next, we divided mice with Otisis Media into six groups: high dose of Atractylodes
group, medium dose of Atractylodes group, low dose of Actractylodes group, Oxfoxacin Ear Drops group, Vancomycin group
and blank model group. The extracting method of oxidizing before preserving oxidative product leads to 9.5% of Actractylodes
macrocephala Koidz extractive, with a minimum inhibitory concentration of 0.781 mg/ml. Comparing with the blank model
group, the number of bacterial colonies had sharply declined after 1 day of medication. On the second day of medication
treatment, in the groups with Atractylodes in various concentration, an obvious decline of bacterial colonies were also observed.
Although the effect of Atractylodes had came slower than Oxfoxacin and Vancomycin, but it was proved to be effective to MRSA
within animal bodies. Besides, there were differences among various concentration of Atractylodes, higher concentration tended
to have better antibacterial function. The method of extracting Atractylodes in this research was easy and productive. Through
experiments in vivo and vitro, it proved that it could be independently used in MRSA, and we could speculate that the effect of
Atractylodes macrocephala Koidz extractive was concentration-based.
2016The
© 2017 TheAuthors.
Authors. Published
Published by by Elsevier
Elsevier Ltd. Ltd.
This is an open access article under the CC BY-NC-ND license
Peer-review under responsibility of the organizing committee of the 13th Global Congress on Manufacturing and Management.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of the 13th Global Congress on Manufacturing and Management
Keywords: Atractylodes macrocephala Koidz; Atractylodes macrocephala Koidz extractive; MRSA; Mice; Models.

1 Introduction
The British microbiologist Alexander Fleming found by chance a kind of green mold fungus that grown in the
culture dish for staphylococcus aureus in the 1920s, and then he found that its secretion can inhibit staphylococcus,
so he decided to name this antibacterial material as penicillin [1]. With the widespread application of antibiotics such

ѽ Corresponding author
E-mail address: cyh0551@163.com

1877-7058 © 2017 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of the 13th Global Congress on Manufacturing and Management
doi:10.1016/j.proeng.2017.01.160
 Zhenguo Xu et al. / Procedia Engineering 174 (2017) 410 – 415 411

as penicillin, the staphylococcus aureus has gradually developed resistance to antibiotics. In addition, since the
scientists isolated methicillin-resistant staphylococcus aureus (MRSA) for the first time in 1961[2], its clinical
detection rate has increased year by year, in particular, it may cause suppurative inflammation to human tissues and
organs and even lead to heart endometritis, sepsis and meningitis and other critically fatal diseases [3]. The sensitivity
of MRSA on glycopeptide vancomycin and teicoplanin has decreased [4,5] in recent years, so it is now in urgent need
to develop alternative antibiotics drugs. In recent decades, a large number of domestic and foreign scholars have
found that a variety of Chinese herbal medicine and Chinese herbal compound have different degrees of antibacterial
activity; besides, the traditional Chinese medicine has many advantages, such as not easy to produce drug resistance,
less side effects and broad antibacterial effect, not to mention that it can enhance the ability of the body to resist
pathogenic microorganisms after being absorbed by the body.
Atractylodes macrocephala Koidz (AMK) is the dried rhizome of atractylodes macrocephala that belongs to
Compositae , mainly produced in Zhejiang, and currently Anhui, Hebei, Fujian, Hunan and other provinces have the
plantation. It has warm nature, tastes sweet and bitter, and has the effect of invigorating spleen and supplementing Qi,
eliminating dampness and promoting urination, hidroschesis and miscarriage prevention, which is commonly used in
insufficiency of the spleen, abdominal distension and diarrhea, palpitations, edema, spontaneous perspiration,
threatened abortion and other symptoms. Anhui Province is rich in atractylodes macrocephala Koidz, that main
grows in Bozhou region of northern Anhui and Qimen, Shitai and Dongzhi of southern Anhui. Its active ingredients
contain the volatile component, lactones, glycosides, polysaccharides and amino acids, etc [6]. Studies at home and
abroad in recent years, have shown that atractylodes macrocephala Koidz has antibacterial, diuretic and anti-aging
effects; besides, it also plays a role on the nervous system, uterine smooth muscle and gastrointestinal motility [7].
However, the extract of atractylodes macrocephala Koidz was not used in anti-MRSA alone. The extraction method
in this study is used to obtain the antibacterial effect of atractylodes macrocephala Koidz extract on MRSA in vivo
and in vitro. It is expected that the extract of atractylodes macrocephala Koidz can be used to treat the disease caused
by MRSA.
2 Materials and Instruments
2.1 Materials
Medicinal materials: Atractylodes macrocephala Koidz planted in Bozhou, Anhui Province; Ofloxacin purchased
from Hefei People Pharmacy. Experimental strains: Hefei Aidikang Clinical Laboratory Co., Ltd provides the
Methicillin-Resistant Staphylococcus Aureus (MRSA) and confirms its resistance. Main reagents: double distilled
water, hydrogen peroxide water, 0.2% sodium pyrophosphate, 10% ascorbic acid and ethanol are all analytically
pure.
2.2 Instruments and equipment
Traditional Chinese medicine extraction and separation device (TS-NS-5), rotary evaporator (RE-52AA), hot air
circulation drying oven (CT-C-16), digital electronic constant temperature water bath (HH-4), one over ten-thousand
analytical balance (YP1002), UV lamp (UVA-340), clean bench (ZHJH-C111), constant temperature magnetic
stirrer (85-2), thermostat box (FYL-YS-50L), electric thermostat drying oven (DHG- 9140), automatic digital
display vertical pressure steam sterilization (LDZX-50KBS), Oxford Cup (antibiotic potency-specific), and so on.
2.3 Experimental animals
The 100 male mice of SPF grade was prepared, all of them weighed 18 ~ 22 g. The feeding temperature of
experimental animal was 22 ~ 25ć, and the relative humidity was 40% to 70%.
3 Method
3.1 The extraction, separation and purification of atractylodes macrocephala Koidz
(1) 60-mesh sieve of atractylodes macrocephala Koidz was taken, and rinsed with distilled water at 25ć
condition to obtain the solid shape after centrifuge. It was soaked for 8h after adding double-distilled water, and then
centrifuge was conducted again to dry the solid by temperature box, the temperature was kept at 40ć to obtain the
powder of atractylodes macrocephala Koidz;
(2) At room temperature, 3 times the volume of aqueous hydrogen peroxide solution and 0.2% sodium
pyrophosphate were added and stirred for 1 hour, and then the powder of atractylodes macrocephala Koidz was
filtered;
(3) The powder of atractylodes macrocephala Koidz obtained in step (2) was placed in a fume hood and irradiated
with an UV lamp for 10 successive times, each time lasted 2 minutes. The powder of atractylodes macrocephala
412 Zhenguo Xu et al. / Procedia Engineering 174 (2017) 410 – 415

Koidz after each irradiation was turned over;

(4) The powder obtained in the step (3) was dried in a vacuum oven at a temperature of 40 ° C by drying under
reduced pressure;
(5) 10% ascorbic acid was added into the powder of atractylodes macrocephala Koidz, followed by 2 times 40%
ethanol solution. At this time, it was assisted by ultrasonic and microwave for 30 minutes with the power of 300W.
(6) 6h of reflux extraction on 12 times 40% ethanol solution at 65ć was conducted, and then suction filtration
was performed to recover the filtrate;
(7) The extracting solution of atractylodes macrocephala Koidz was clarified by 0.2 μm micro-filtration
membranes, and then the separated solution was concentrated under reduced pressure and freeze drying to obtain an
extract of atractylodes macrocephala Koidz.
3.2 Determination of antibacterial activity
The antibacterial activity was determined by Oxford cup method. The experimental steps of Oxford cup method
was as follows: first, 0.1mL bacteria suspension was absorbed and evenly coated on the plate, and then four
sterilized Oxford cup was placed on the plate in equidistance, and then each Oxford cup was added with 100 μ L of
atractylodes macrocephala Koidz extract to cultivate for 37ć constant temperature. After 24 hours, the inhibition
situation was observed, and the diameter of the inhibition zone was measured with vernier caliper and recorded,
with the unit of mm. The experiment was repeated for 3 times. When the diameter of the inhibition zone ı 20, it
was extremely sensitivity; when 15mm İ the diameter of the inhibition zone <20 mm, it was high sensitivity;
when 10mm İ the diameter of the inhibition zone <15mm, it was mediate sensitivity; and when the diameter of
the inhibition zone <10mm, it was low sensitivity or invalidation.
3.3 Determination of the minimum inhibitory concentration
The MIC was determined by the double dilution method. Steps of the MIC determination method were as follows:
First, the atractylodes macrocephala Koidz extract was diluted to 100, 50, 25, 12.5, 6.25, 3.125, 1, 562, 0.781, 0.390
and 0.195 mg/ml respectively, and then 100 L of each concentration of ANK dilution was added in sequence to the
Oxford cup, to observe the growth at 37 ć cultivation after 24h. According to the plate colony counting method,
the bacteriostatic ability of the extract of atractylodes macrocephala Koidz needed to prepare the bacterial
suspension with the final concentration of about 0.5×107cfu/ml. The concentration of the extract at the highest
dilution where the colony was completely inhibited, i.e., without inhibition zone, was MIC.
3.4 Effect of MRSA on otitis media model
Preparation of bacterial solution: MRSA was diluted to 1.0 MCF/mL with sterile normal saline until the
logarithmic growth phase for use.
Animal model building: Under clean environment, 100 normal and healthy mice were selected and punctured
with a syringe needle in the anterior quadrant of the tympanic membrane in the mouse side and injected with 0.1mL
MRSA solution. After 2 days, the secretions from middle ear cavity were inoculated into LB solid culture medium
and cultivated at 37 ć, the results were observed after 24 h. The modeling was considered as successful if LB solid
culture medium was appeared with bacterial plaque.
Group administration of animal models: mice with successful modeling were divided into six groups, namely,
high-dose group, middle-dose group and low-dose group of the extract of atractylodes macrocephala Koidz, 5g/L
ofloxacin ear drops group, 100g/L vancomycin group and blank model group. The amount of atractylodes
macrocephala Koidz extract in the high-dose, middle-dose and low-dose groups was 8 times, 4 times and 2 times of
the MIC, respectively. Theadministration in the blank model group was sterile normal saline for ear dropping, it was
used three times a day, one drop each time, and lasted for 7 days.
Colony count: Before the daily administration of mice ear canal with cotton swab 75% ethanol disinfection, the
middle ear cavity of the mice was cleaned with a little saline. The cleaning solution was collected and diluted, and
then uniformly spread on the agar plate to cultivate at 37ćfor 24 h, and finally the MRSA was counted.
4 Results
4.1 Extraction, isolation and purification of atractylodes macrocephala Koidz
300g of atractylodes macrocephala Koidz were taken for crushing and cleaning, and then hydrogen peroxide
aqueous solution was added for stirring after the infusion, centrifugation and drying. In the following, the powder of
atractylodes macrocephala Koidz was filtered, dried and added with ascorbic acid, and ethanol solution of ultrasonic
microwave was used for extraction, and then reflux extraction and suction filtration were performed to recycle the
 Zhenguo Xu et al. / Procedia Engineering 174 (2017) 410 – 415 413

filtrate. The filtrate was performed with ceramic micro-filtration by 0.2μm micro-filtration membrane, and then
decompressed, concentrated and freeze-dried to obtain the extract of atractylodes macrocephala Koidz. The yield of
the obtained extract of atractylodes macrocephala Koidz was 9.50%.
4.2 Determination of antibacterial activity
The antibacterial activity of the extract of atractylodes macrocephala Koidz was determined by Oxford cup
method. The results showed that the extract of atractylodes macrocephala Koidz had antimicrobial activity against
methicillin-resistant staphylococcus aureus (see Table 1).
Tab.1 Antibacterial activity test results of atractylodes macrocephala Koidz extract

Bacterial Inhibition
No. Sensitive Type
Diameter˄mm˅
1 17.42±0.67 High sensitivity
2 17.92±0.55 High sensitivity
3 17.57±0.39 High sensitivity
Note: The concentration of bacteria solution was 50mg / ml.

4.3 Determination of the minimum inhibitory concentration

The minimal inhibitory concentration was determined by the double dilution method, and the concentration of the
extract at the highest dilution where the colony was completely inhibited, i.e., without inhibition zone, was MIC.
The MIC of the extract of atractylodes macrocephala Koidz obtained from the experiment was 0.781 mg/ml (see
Table 2) .
Tab.2 The minimum inhibitory concentration of atractylodes macrocephala Koidz extract (mg / ml)

Concentration of Atractylodes Inhibition Zone

No.
Macrocephala Koidz Extract˄mg/ml˅

1 100 +
2 50 +
3 25 +
4 12.5 +
5 6.25 +
6 3.125 +
7 1.562 +
8 0.781 +
9 0.390 -
10 0.195 -

Note: "+" indicates the presence of inhibition zone, "-" indicates there was no inhibition zone.

4.4 The role of MRSA in otitis media model

The 24 mice with successful modeling were taken and divided into 6 groups, and the experimental results were
shown in Table 3. It can be seen from the table that, compared with the blank model group, the number of colonies
in experimental groups was rapidly declined after 1 day of administration; the number of colonies also decreased
significantly on the second day of administration in atractylodes macrocephala Koidz extract groups with various
concentrations. Compared with ofloxacin and vancomycin, the effect initiating of atractylodes macrocephala Koidz
extract was slow, but it was effective for MRSA in animal experiments, and the effects of different concentrations of
atractylodes macrocephala Koidz extract on MRSA were also different: the higher the concentration, the higher the
bacteriostatic action. The results showed that although the effect initiating of atractylodes macrocephala Koidz
extract is slow compared with ofloxacin and vancomycin, it is effective in treating MRSA in animal experiments. At
the same time, it can also be seen from the table that, the control effects of atractylodes macrocephala Koidz extract
with different concentrations on MRSA are also different, the higher the concentration, the stronger the inhibitory
effect. Thus, it can be speculated that atractylodes macrocephala Koidz extract on MRSA antibacterial action is
concentration-dependent.
414 Zhenguo Xu et al. / Procedia Engineering 174 (2017) 410 – 415

Tab. 3 Effect of atractylodes macrocephala Koidz extract on MRSA-induced otitis model mice (unit: 103 cfu/ ml)
Group 0day 1day 2days 3days 4days 5days 6 days 7days
Blank model group 5087 4502 4302 3987 3479 2945 2000 992
AMK Extract (high) 5642 1400 200 98 67 39 21 5
AMK extract (middle) 5723 2000 312 157 111 68 30 20
AMK extract (low) 5502 2523 525 177 121 89 52 11
Ofloxacin 5253 300 68 56 15 9 7 1
Vancomycin 4998 380 113 33 20 10 5 2

5 Discussion
In this study, the method of oxidation and protection on oxidative products were adopted to increase the content
of atractylenolide in atractylodes macrocephala Koidz. The ethanol extract was used, instead of ethyl acetate and
petroleum ether or other organic solvent, to extract the effective components. On one hand, it could be made into
traditional Chinese medicine prescription to put into the market, which was cheap and had improved efficacy; on the
other hand, it could also be made into the Western dosage form, the price would be increased, but it would be Safer
and easy to take. Such treatment was able to realize the reasonable integration between traditional Chinese medicine
prescription and the Western dosage form, so it had great flexibility and strong adjustability. One thing needed to
mention: double distilled water soak could remove part of the polysaccharide and thus improving the purity of
atractylodes macrocephala Koidz extract; besides, atractylon in the atractylodes macrocephala Koidz could be
oxidized into atractylactone compounds by hydrogen peroxide solution to improve the anti-inflammatory activity of
atractylodes macrocephala Koidz extract. In addition, aiming at the shortcomings of hydrogen peroxide, such as
effumability, high temperature decomposition, and a large number of oxygen in the decomposition products, it was
suggested to add common food additive sodium pyrophosphate during the oxidation reaction, it played the role of
stabilizer in the reaction. After the oxidation, short-wave ultraviolet light was used to irradiate and decompose
excess hydrogen peroxide under atmospheric condition; meanwhile, the secondary oxidation could be conducted on
atractylodes macrocephala Koidz to improve the yield. Hydrogen peroxide solution also had the role of disinfection,
especially as a traditional Chinese medicine to be put into the market, because it could reduce the microbial breeding
produced from long-term placement.
During the extraction process, the natural antioxidant - ascorbic acid was able to protect atractylodes
macrocephala Koidz extract, and prevent it from further oxidation and degradation. Ascorbic acid was a water-
soluble vitamin, which was safe and reliable that could be directly added to the atractylodes macrocephala Koidz
extract without secondary treatment. In addition, it could also enhance the body’s resistance, and excessive
application could be discharged through the blood circulation, without affecting the health of human body.
The obtained atractylodes macrocephala Koidz extract plays a role in the in vitro and vivo effect of methicillin -
resistant staphylococcus aureus, and the details of the extracts were also optimized. The results showed that the
obtained extract with high activity can be used in MRSA, which had achieved good effect. This was consistent with
the reports in both domestic and abroad about the antibacterial activity of atractylodes macrocephala Koidz. Yan
Weilun et al. [8] studied that the water decoctum of atractylodes macrocephala Koidz had inhibiting effect on
epidermidium flocculans, star Nocardia, meningococcus, hemolytic streptococcus and bacillus subtilis, etc. Studies
of Li et al. [9] conducted on root chemistry of atractylodes macrocephala Koidz showed that its main active
constituents of sesquiterpenes and acetylenic compounds [10] had the antitumor and antibacterial effects [11,12]. Ruijun
W et al.[13]discovered that PAM of atractylodes macrocephala Koidz roots had the antibacterial activity. In addition,
the essential oil of rhizome of atractylodes macrocephala Koidz has many biological activities, including anti-
inflammatory[14], anti-carcinogenic[15] and anti-microbial [16]activities. The study of this paper found that, compared
with ofloxacin and vancomycin, although the effect initiating of atractylodes macrocephala Koidz was relatively
slow, it was still effective in vivo experiments on MRSA. It may be because the atractylodes macrocephala Koidz
stimulates the body Th1-type lymphocyte proliferation, produces antibodies and regulates immune response[17]. The
inhibitory effect of atractylodes macrocephala Koidz extract with different concentration on MRSA was different:
the higher the concentration, the stronger the bacteriostatic action. Therefore, it could be speculated that atractylodes
macrocephala Koidz extract on MRSA antibacterial action was concentration-dependent.
6 Conclusion
The method of extracting Atractylodes in this research was easy and productive. Through experiments in vivo and
vitro, it had proved that it could be independently used in MRSA, and we could speculate that the effect of
Atractylodes macrocephala Koidz extractive was concentration-based. However, the current research on atractylodes
 Zhenguo Xu et al. / Procedia Engineering 174 (2017) 410 – 415 415

macrocephala Koidz was mainly focused on its effects, such as antibiosis, diuresis, anti-aging, anti-spleen tumor and
so on. Moreover, many active ingredients to the pharmacological effects of atractylodes macrocephala Koidz were
still unclear, and the effect target and mechanism of these active components still need further study to be clarified.
Acknowledgements
The research was financially supported by grants from the National Natural Science Foundation of China (31302
044) and the Key projects of natural foundation research of universities in Anhui Province(KJ2016A613)and, the
Key projects of natural foundation research of universities in Anhui Province(KJ2015A406),and Anhui province
university outstanding youth talent support program key project˄gxyqZD2016547˅.
Zhenguo xu was mainly responsible for the test design and test development; Yuhua cai was responsible for the
collation and writing of this paper; Gaofu fan and Xiushu Liu were responsible for consulting the relevant literature;
Yin dai on the paper had put forward constructive suggestions and modifications. And the authors declare that they
have no conflictsof interest.

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