Author’s Accepted Manuscript

Therapeutic effects of
traditionalChinesemedicineNiubeixiaohe in mouse
tuberculosis models

Yan Liang, Xiaomei Wang, Jinying Song, Lan

Wang, Dan Chen, Yourong Yang, Xuejuan Bai, Jie
Wang, Yingchang Shi, Shibing Chen, Jun Liu,
Cunwei Yang, Huafeng Luo, Guangling Liu, www.elsevier.com/locate/jep

Xueqiong Wu

PII: S0378-8741(16)31998-5
DOI: http://dx.doi.org/10.1016/j.jep.2016.11.037
Reference: JEP10581
To appear in: Journal of Ethnopharmacology
Received date: 4 March 2016
Revised date: 28 September 2016
Accepted date: 19 November 2016
Cite this article as: Yan Liang, Xiaomei Wang, Jinying Song, Lan Wang, Dan
Chen, Yourong Yang, Xuejuan Bai, Jie Wang, Yingchang Shi, Shibing Chen, Jun
Liu, Cunwei Yang, Huafeng Luo, Guangling Liu and Xueqiong Wu, Therapeutic
effects of traditionalChinesemedicineNiubeixiaohe in mouse tuberculosis models,
Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2016.11.037
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 Therapeutic effects of traditionalChinesemedicineNiubeixiaohe in mouse tuberculosis models

Yan Liang1,,Xiaomei Wang1,, Jinying Song1, Lan Wang1, Dan Chen1, Yourong Yang1, Xuejuan Bai1, Jie

Wang1, Yingchang Shi1, Shibing Chen1, Jun Liu2, Cunwei Yang2, Huafeng Luo2, Guangling Liu1, Xueqiong

Wu1,*

Yan Liang: amy5919@sina.com; Xiaomei Wang: wangxiaomei_1a@163.com; Jingying Song:

xiangfeidexin_20@sina.com; Lan Wang: wanglan922@163.com; Dan Chen: lxrjchd@163.com; Yourong

Yang: yangyourong@163.com; Xue-juanBai: baixuejuan2003@163.com; Jie Wang:2312262968@qq.com;

Yingchang Shi: Shiyingchang@sina.com ； Shibing Chen:chenshibing70@163.com; Jun Liu:

liujunalla@163.com; Cunwei Yang: qfy@21cn.net; HuafengLuo: luohuafeng@outlook.com; Guangling Liu:

liu.guangling@163.com; Xueqiong Wu: wu-xueqiong@263.net

1
Army Tuberculosis Prevention and Control Key Laboratory, Beijing Key Laboratory of New Techniques of

Tuberculosis Diagnosis and Treatment, Institute of Tuberculosis Research, the 309th Hospital of Chinese

PLA, Beijing 100091, China.

2
Guangdong Qifang Pharmaceutical co., LTD, Guangzhou 510075, China.

Corresponding author. Mailing address: Army Tuberculosis Prevention and Control Key Laboratory,

Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment, Institute for

Tuberculosis Research, the 309th Hospital of Chinese PLA, Beijing 100091, China. Fax: (008610)80115555

ext. 768415. E-mail: wu-xueqiong@263.net.


These authors contributed equally to this study.

Abstract

Ethnopharmacological relevance—The traditional Chinese medicine Niubeixiaohe (NBXH) is an

effective anti-tuberculosis prescription, which is made up of Bulbus Fritillariae Cirrhosae, Rhizoma

Bletillae, Radix Platycodonis, Fructus Arctii, Herba Houttuyniae and Glutinous rice. In this study, NBXH

powder (I) and three kinds of NBXH extracts (II, III, and IV) were prepared. The water decoction of NBXH
1
 had been used to treat TB in clinic sixteen years suggested that it was effective to treat TB.

Aim of the study—This study evaluated the effects of different processing products of NBXH on

mouse TB model in vivo and provide a new Chinese medicine for the clinical treatment of TB.

Materials and methods—In this study, 120 female BALB/c mice infected with Mycobacterium

tuberculosis H37Rv, were treated with distilled water, M. vaccae vaccine, the low, middle and high doses of

NBXH I, the low, middle and high doses of NBXH II, the low, middle and high doses of NBXH III, the low,

middle and high doses of NBXH IV for 12 weeks, respectively.

Results—The body weights of mice in all NBXH groups were higher than that in the water group. The

weight indexes of the spleens in M. vaccae group, the middle dose of NBXH II group, the low dose of

NBXH IV group and in the high dose of NBXH IV group were significantly lower than that in the water

group(P<0.05). Compared with the water group, the spleen colony counts in the low dose of NBXH I group,

the high dose of NBXH II group, the low dose of NBXH III group and the high dose of NBXH IV group

reduced by 0.43, 0.46, 0.73, 0.58 logs (P<0.05), respectively. But the lung colony counts had no significant

difference between each group. Pulmonary general pathology and histopathology displayed that the lung

lesions in treatment groups were improved at certain degree, especially in the low dose of NBXH IIIand IV

groups, in which their areas of the lesions were less than50%, and the half normal lung structure in half of

the mice could be observed.

Conclusion— Powder and three extracts of traditional Chinese medicine NBXH all had anti-tuberculosis

therapeutic effects on mouse tuberculosis model, and this study provided a base for the further development of

Chinese patent medicine NBXH. Also, this is the first report on comprehensive experimental research of

NBXH extracts coming from six kinds of traditional Chinese medicine.

Keywords: Traditional Chinese medicine; Niubeixiaohe (NBXH); Tuberculosis; Mycobacterium

tuberculosis

1. Introduction

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (M.tb) and which is a global health issue.

The new TB cases increased more than 6 million, and nearly 1.5 million people died from TB in 2014

(WHO, 2015). At present, the annual incidence of TB was about 1.3 million in China (Technical Guidance

2
 Group of the Fifth National TB Epidemiological Survey, 2012). The current TB treatment uses mainly the

combination chemotherapy, but the severe liver and kidney injuries were produced in some TB patients by

long-term chemotherapy. The rapid increase of multidrug-resistant M.tb strains and extensively

drug-resistant M.tb strains made the TB treatment into a difficult situation without available anti-TB drugs.

The integrated treatment of traditional Chinese medicine and Western medicine has a complementary

advantage on TB, in which the strong bactericidal efficacy of Western medicine plays a leading role, and

traditional Chinese medicine has the characteristics of multi-components, multi-path way, multi-targets and

multi-effect and effect synergistically on target network in the human body, tonic righting, enhances the

immune function, improves the poisoning symptoms and the curative effect of TB patient, and plays a

comprehensive treatment effect. The traditional Chinese medicine Niubeixiaohe (NBXH) (Chen SB, 2007)

is anti-TB prescription, which is made up of Bulbus Fritillariae Cirrhosae, Rhizoma Bletillae, Herba

Houttuyniae, Radix Platycodonis, Fructus Arctii and Glutinous rice. Bulbus Fritillariae Cirrhosae matched

Rhizoma Bletillae are the monarch drug of NBXH, Herba Houttuyniae, Radix Platycodonis and Fructus

Arctii are the minister drug of NBXH, Glutinous rice is the assistant drug of NBXH. The water decoction of

NBXH had been used to effectively treat TB in clinic for sixteen years (Chen D, 2015). The clinical research

proved that NBXH could nourish Yin, moisten lung, soften hard masses, eliminate node, remove the

necrotic tissue, promote granulation plaster, repair the pulmonary lobe and relieve the TB symptoms, for

example low fever, emaciation, chest pain, cough, hemoptysis. However, this traditional medicine decoction

has many disadvantages, for example its taking is not convenient, its effect is not stable, its quality is

difficult to be controlled, etc. Therefore, we concentrated effective components of NBXH after different

physical and chemical extraction and separation processes. This study evaluated the effects of different

processing products of NBXH on mouse TB model to lay the foundation for the development of a new

anti-TB Chinese medicine preparation.

2. Materials and methods

2.1. The preparation of NBXH powder and extracts

The herbal slices of Bulbus Fritillariae Cirrhosae, Rhizoma Bletillae, Radix Platycodonis, Fructus Arctii,
Herba Houttuyniae (Zhongyong Pharmacy Company Limited, Sichuan, China) and Glutinous rice (Qinghe
Rice Company Limited, Chendu, China) were purchased from No.155 chain store of Kangzheng Pharmacy
Company Limited, Neijiang, China. The herbal slices were identified by Professor Qin XU. The voucher
specimens were deposited Zhongyong Pharmacy Company Limited in Sichuan, China. The list of plant used in
this study is shown in Table 1.
3
 NBXH consist of 4 Bulbus Fritillariae Cirrhosae, 8 Rhizoma Bletillae, 4 Herba Houttuyniae, 4 Radix

Platycodonis, 4 Fructus Arctii, 5.5 Glutinous rice (ratio of weight) (The 309th Hospital of Chinese PLA and

Guangdong Qifang Pharmaceutical co., LTD., 2015). The powder of NBXH raw material was prepared and

used as control (named as NBXH I). NBXH raw materials were extracted, separated by the treatment

processes of water and/or ethanol, and then concentrated as three kinds of NBXH extracts by Guangdong

Qifang Pharmaceutical co., LTD, Guangzhou 510075, China. The details of NBXH extraction process was

showed in the patent No.201510142873.0 (The 309th Hospital of Chinese PLA and Guangdong Qifang

Pharmaceutical co., LTD., 2015).The aqueous and ethanol extract of NBXH was named as NBXHⅡ,

aqueous extract was named as NBXH III, and the ethanol extract was named as NBXH IV.

2.2. Mice

One hundred and twenty specific pathogen-free female BALB/c mice with 6-8 weeks of age were

purchased from the Academy of Military Medicine and Science, Beijing, China, maintained under barrier

conditions in an animal room in the 309th Hospital of Chinese PLA, Beijing, China, and fed a sterile

commercial mouse diet (Beijing KeAoXieLi Company Limited, China).

2.3. M.tb strain

M.tb H37Rv was provided by Chinese Academy for Food and Drug Control.M.tbH37Rv was grown on

LowensteinJensen medium for 4weeks. Bacterial colonies were collected and homogenized in 0.05% Tween

80 saline, and then stored at -20℃. After thawing, colony forming units (CFUs) were enumerated for viable

bacteria by plating serial dilutions on Lowenstein-Jensen medium.

2.4. The preparation of mouse TB model

One hundred and twenty female BALB/c mice 6-8 weeks of age were infected intravenously by tail

vein with 5×105 colony-forming units (CFUs) of M.tb.

2.5. Treatment of mouse TB model

One hundred and twenty M.tb-infected mice were randomly divided into the following fifteen groups

and treated at 1 week after infection: (1) Water group: each mouse was given 0.5 ml distilled water by

intragastric administration as a negative control; (2)Vaccae group: each mouse was injected intramuscularly

22.5 g M. vaccae vaccine for three times at two weeks intervals (Longcome Biological Pharmacy, Anhui,

China) as a positive control; (3) Low dose of NBXH I group: each mouse was given 3.3 mg NBXH I

suspended in 0.5 ml distilled water by intragastric administration; (4) Middle dose of NBXH I group: each

4
mouse was given 6.7 mg NBXH I suspended in 0.5 ml distilled water by intragastric administration; (5)

High dose of NBXHⅠgroup: each mouse was given 13.3 mg NBXH I suspended in 0.5 ml distilled water

by intragastric administration; (6) Low dose of NBXH II group: each mouse was given 2 mg NBXH II

suspended in 0.5 ml distilled water by intragastric administration; (7) Middle dose of NBXH II group: each

mouse was given 4 mg NBXH II suspended in 0.5 ml distilled water by intragastric administration; (8) High

dose of NBXH II group: each mouse was given 8 mg NBXH II in 0.5 ml distilled water suspended by

intragastric administration; (9) Low dose of NBXH III group: each mouse was given 1.67 mg NBXH III

suspended in 0.5 ml distilled water by intragastric administration; (10) Middle dose of NBXH III group:

each mouse was given 3.35 mg NBXH III suspended in 0.5 ml distilled water by intragastric administration;

(11) High dose of NBXH III group: each mouse was given 6.67 mg NBXH III suspended in 0.5 ml distilled

water by intragastric administration; (12) Low dose of NBXH IV group: each mouse was given 0.75 mg

NBXH IV suspended in 0.5 ml distilled water by intragastric administration; (13) Middle dose of NBXH IV

group: each mouse was given 1.5 mg NBXH IV suspended in 0.5 ml distilled water by intragastric

administration; (14) High dose of NBXHIV group: each mouse was given 3 mg NBXH IV suspended in 0.5

ml distilled water by intragastric administration; (15) TB model group: The mice were killed at 1 week after

infection to observe the development of mouse TB model. The dosages of NBXH given in mice were

determined according to effective dose in adult and the weight conversion. 0.5ml of NBXH was given by

intragastric administration from Monday to Friday (except Saturday and Sunday) and continuous 12 weeks.

2.6. Weight indexes of the lungs, livers and spleens

The body weights of mice were observed and recorded weekly. The growth of body weight each mouse

was calculated from difference in body weight of each mouse at 12th week of treatment and that of before

treatment. The lungs, livers and spleens of the mice in the fourteen groups were weighed by electronic scale,

then the indexes of lungs, spleens and livers of the mice were calculated. Weight index of organ = weight of

organ (g)/ body weight of mouse (g) ×100%.

2.7. Bacterium counts

Mice of TB model group were killed at 1 week after infection, and the other mice were killed at 13

weeks after infection with M.tb H37Rv. The left lungs and spleens of the mice were taken and homogenized

in saline. The tissue suspension was serially diluted 10-fold, and 0.1 ml of each dilution was plated in

duplicate onto Lowenstein-Jensen plates and incubated for at 37℃4 weeks. M.tb colonies on each plate

5
were enumerated and the results were expressed as CFUs per organ.

2.8. Lung histopathological examination

The right lungs of the mice were fixed in 10% buffered formalin and paraffin-embedded. The

paraffin-embedded tissue sections were prepared and stained with hematoxylin and eosin, and then were

analyzed by a certified pathologist.

2.9. Statistical analyses

Data were expressed as means and standard deviations. Statistical significance of differences between

groups was calculated using One way ANOVA followed by Dunnett’s t test, and a P-value of <0.05 was

considered to be statistically significant.

3. Results

3.1. Mouse survival rate

One mouse died in water group, the low dose of NBXH I group and the middle dose of NBXH I group.

The survival rates of these three groups were 90%. Three mice died in the middle dose of NBXH IV group,

the survival rates of this group was 70%. Mice in other treatment groups were all 100% alive.

3.2. Body weights

The body weights of the mice each group increased at different degree after treatment.Body weights of

the mice in vaccae group and the high dose of NBXH IV group were significantly higher than those in the

water group (P<0.05) (Figure 1).

3.3. Weight indexes of the lungs, livers and spleens

Except for the middle dose of NBXH I group, the weight indexes of the lungs in vaccae group and

others NBXH groups were lower than that in the water group, but there were no significant difference

(P>0.05). Except for the middle dose of NBXH III group, the weight indexes of the livers in vaccae group

and the others NBXH groups were lower than that in the water group, but only that in the low dose of

NBXH I group were significantly lower than those in the water group (P<0.05).The weight indexes of the

spleens in vaccae group, the middle dose of NBXH II group, the low and high doses of NBXH IV group

were significantly lower than that in the water group (P<0.05) (Figure 4).

3.4. Bacterial numbers in the lungs and spleens

The numbers of live bacteria were determined in lungs and spleens of mice at 1 week and 13 weeks

after infection with M.tb H37Rv. The lung CFUs of TB model group at 1 week after infection were 5.93

6
log10. The lung CFUs from water group, vaccae group, the low, middle and high doses of NBXHI groups,

the low, middle, and high doses of NBXH II groups, the low, middle, and high dose of NBXH III groups, the

low, middle and high doses of NBXH IV groups at 13weeks after infection (namely at 12 weeks after

treatment) were 6.53±0.14, 6.33±0.19, 6.37±0.15, 6.49±0.13, 6.42±0.18, 6.52±0.13, 6.38±0.28, 6.31

±0.26, 6.43±0.13, 6.35±0.42, 6.45±0.32, 6.35±0.26, 6.12±0.18 and 6.37±0.31 log10 respectively, and

the spleen CFUs each group mentioned above were 5.99±0.11, 5.62±0.23, 5.56±0.97, 5.77±0.31, 5.61±0.34,

5.75±0.25, 5.69±0.20, 5.53±0.36, 5.26±0.30, 5.82±0.29, 5.64±0.30, 5.78±0.48, 5.63±0.17 and 5.41±0.16

log10, respectively (Figure 3). Compared with the water group, the splenetic bacterial loads in the low dose

of NBXH I group, the high dose of NBXH II group, the low dose of NBXH III group and the high dose of

NBXH IV group reduced by 0.43, 0.46, 0.73, 0.58 logs (P<0.05).

3.5. Histopathological changes

The lung sections from TB model group at 1 week after infection showed extensive lung lesions. The

lung histopathological changes from the water group were severe and extensive, the percentage of

pathological area was larger than 85%. Compared with the water group, the lung histopathological changes

from the other treatment groups relieved at different degrees. The lung lesion from the low dose of NBXH II

group was lighter than that from the water group, the percentage of pathological area was more than 73%.

The lung lesions from the low, middle and high doses of NBXHI groups, the middle and high doses of

NBXH II groups, the middle dose of NBXH IV groups were lighter and limited as from vaccae group, their

percentages of pathological areas were 50-67%. The lung lesions from the low dose, middle and high doses

of NBXH III groups, the low and high doses of NBXH IV groups were slight than those from vaccae group,

their percentages of pathological areas were less than 50%, and the alveolar wall structures were clear and

had normal structures in half the areas. Representative histopathology each group is presented in Figure 4.

4. Discussion

Traditional Chinese Medicine could inhibit the growth of M.tb, enhance immunity of the body, relieve

the tuberculosis poisoning symptoms, alleviate the side effects of anti-TB drugs, decrease the resistance of

chemotherapy drugs (Hao and Yang, 2003; Kuang et al., 2001; Wang et al., 2011). Traditional Chinese

Medicine combined with chemotherapy drugs could promote negative conversion rate of the sputum M.tb,

cavity closure rate, lesion absorption, shorten the course of treatment and improve the efficiency against TB

(Gao et al., 2006; Li, 2010；Zhang and Gao, 2012). In order to realize the modernization of traditional

7
Chinese medicine, the traditional Chinese medicine was combined with modern science and technology to

clarify the pharmacodynamic components and effective mechanism of traditional Chinese medicine.

Therefore, we prepared different extracts of NBXH and evaluated the effects of NBXH on mouse TB model

in vivo.

In this study, the lung CFUs and histopathalogical changes of mice at 1 week after M.tb infection all

suggested that the mouse TB model was successfully developed. Mice infected M.tb appeared generally

anorexia, less activity, and loss of body weight, which is in accordance with the research from Li et al., their

results showed that the body weights of mice increased at early stage of infection, and decreased at 9 weeks

after infection (Li et al., 2007). Yangshixinlaoning capsules made up of Radix Rtemonae, Paris Polyphylla,

Astragalus and Radix Ophiopgonis could increase the body weights of mice at different degrees (Wang et al.,

2007). Our results showed that NBXH could also increase the body weights of mice, especially the high

dose of NBXH IV significantly increased the body weights of mice, which suggested NBXH could improve

the general status of TB mice. This result also suggests that NBXHs were safe, which is in accordance with

the research from Chen D. Dr. Chen in our research group had evaluated the acute toxicity and the general

pharmacology of NBXH aqueous extracts (NBXH III) and ethanol extracts (NBXH IV), which showed they

had no acute toxicity reaction and no obvious side effects on neurological system and the functions of liver

and kidney(Chen D., 2014).

Weight Index of organ is the ratio of the organ weight and body weight. It can reflect the severity

degree of organ lesion (Feng and Wang, 2011), the larger the index means the heavier the lesion. The weight

indexes of livers, spleens, lungs and kidneys obviously increased after mice infected intravenously with

M.tb (Li and Lu, 2003). Our results showed that NBXH could decrease organ weight indexes at different

degrees, especially the weight indexes of the livers in the low dose of NBXH I group, and weight indexes of

the spleens in the middle dose of NBXH II group, in the low dose and the high dose of NBXH IV group

were significantly lower than those in the water group (P<0.05). The lung histopathalogical examination

also exhibited that NBXH decreased the range of lung lesion at different degree, which is consistent with the

reports of Ou-Yang, their results showed Yuehua capsules (composed of RhizomaBletillae, Bulbus

Fritillariae Cirrhosae, prepared rhizome of Rehmannia, Radix Rehmanniae, Dwarf Lilyturf, Radix

Stemonae, Radix Notoginseng, Donkey Hide gelatin, Mulberry Leaf, Chrysanthemum Morifolium, etc.

(Ou-Yanget al., 2006) could extensively reduce the area of TB nodule. These results suggested that NBXH

8
could reduce the organ lesions.

Organ bacterial loads are one of the important indicators to evaluate curative effects on animal TB

experiments (Lowrie et al., 1999). The less the organ colony count suggests that the stronger the bactericidal

effect of drug and the better the treatment effect is. TB mainly occurs in the lungs. But NBXH in this study

could not inhibit and kill M.tb in the lungs each group, which is because in general the bactericidal effect of

traditional Chinese medicine is not as strong as that of western medicine, and mainly plays the role of

comprehensive regulation (Wu and Liang, 2016).Injecting M.tb through the tail vein could develop mouse

TB model of systemic infection, including extra-pulmonary TB (Zeng et al., 2006). NBXH could decrease

the splenic bacterial loads in different degree, suggesting that the NBXH has better antibacterial and

sterilization effects in extra-pulmonary organs. Our team’s further study showed that NBXH had effects of

immunomodulation and anti-inflammation by inhibiting c-Jun N-terminal kinase signal pathway using gene

expression chips and ingenuity pathway analysis (Ling et al., 2016), but its effects mechanism still needs

further verification and research.

5. Conclusion

The present study demonstrated that powder and three extracts of NBXH from six kinds of traditional

Chinese medicine all had anti-tuberculosis effects on mouse tuberculosis model, in which the mouse body

weights increased, organ indexes decreased, areas of pulmonary lesions lessened and splenetic bacterial

loads significantly reduced. This study provided a base for the further development of Chinese patent

medicine NBXH. At present, we are studying active chemical components and elucidate the mechanism of

action of NBXH.

Acknowledgment

This study was supported by the grant from Beijing Science and Technology Research Project

Foundation of China (No.Z141100002214002).

Author Contributions

Conceived and designed the experiments: XW GL. Performed the experiments: YL XWJS LWDC JW

YC SC JL HL. Analyzed the data: YL XW. Contributed reagents/materials/analysis tools: YYXB JZ YY CY.

Wrote the paper: YL XW XZ.

Competing interests

The authors have no competing interests to disclose.

9
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Table 1 The list of plant used in this study

Family Genus and Species Authoritie English Collectio Voucher Plant part Collection pl
s name* name n time specimen ace

11
Liliaceae Fritillaria cirrhosa Fritillaria Bulbus 2010.11. fc-20101108-0 bulb Songpan,
(D.Don) e Fritillaria 08 01 Sichuan
Cirrhosae e
Bulbus Cirrhosae
Orchidaceae Bletilla striata Bletillae Rhizoma 2011.09. bs-20110920-0 tuber Chengdu,
(Thunb.) Reichb.f. Rhizoma Bletillae 20 01 Sichuan

Campanulac Platycodongrandiflo Platycodo Radix 2011.12. pd-20111211-0 root Haozhou,

eae rum (Jacq.) A.DC nis Radix Platycodo 11 01 Anhui
nis
Composite Arctium lappaL. Arctii Fructus 2010.10. al-201005-001 fruits Northeast
Fructus Arctii 05

Saururaceae Houttuynia cordata Houttuyni Herba 2010.09. hc-2010.0902- stems and Chengdu,
(Thunb.) ae Herba Houttuyni 02 001 leaves Sichuan
ae

Gramineae Oryza sativa L.var. no record Glutinous 2012.08. no record polished ker Heilongjiang
Glutinosa Matsum rice 25 nel
(Batch.
No)

Note: * The authorities names of traditional Chinese medicine were from the 2015-ed Pharmacopeia of the
People's Republic of China.

12
Figure 1 Growth of body weights of mice each group at 12 weeks after treatment than before treatment.

0.06 0.08
Weight indexes of the spleens

0.04
Weight indexes of the lungs

Weight indexes of the livers

0.05
0.07
0.03
0.04
0.06
0.03 0.02

0.05
0.02
0.01

0.01 0.04
r
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dl se va r

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ed

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13
A B C
A A A
Figure 2 Weight indexes of the lungs (A), livers (B) and spleens (C)at 12 weeks after treatment with M.tb

H37Rv.

7.0
7.0

6.5
Log10 CFU ( Lung )

6.5
Log10 CFU（ Spleen）

6.0

5.5
6.0

5.0

5.5 4.5
er e Ⅰ Ⅰ Ⅰ Ⅱ Ⅱ Ⅱ Ⅲ Ⅲ Ⅲ Ⅳ Ⅳ Ⅳ
at cca XH XH XH XH XH XH H dl se NB Ⅲ

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ed

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.
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e

e
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h
ig

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M M M M
M

A B
A A
Figure 3 The numbers of live bacteria in the lungs (A) and spleens (B) at 12 weeks after treatment with

M.tbH37Rv.

14
Figure 4 This figure lists the representative photomicrographs of lung tissue obtained from the mouse TB

model at 1 week (a) (H＆E, 20) and the other groups at 13 weeks (H＆E, 40) after infection with M.tb

H37Rv. (b) Distilled water group; (c) M. vaccae group; (d) Low dose of NBXH I group; (e) Middle dose of

NBXH I group; (f)High dose of NBXH I group; (g) Low dose of NBXH II group; (h) Middle dose of

NBXH II group; (I) High dose of NBXHII group; (j) Low dose of NBXH III group; (k) Middle dose of

NBXH III group; (l) High dose of NBXH III group; (m) Low dose of NBXH IV group; (n) Middle dose of

NBXH IV group; (o) High dose of NBXH IV group.

15