1.Porphyromonas gingivalis outer membrane vesicles activate Toll-like receptor 2 to promote osteoclast differentiation by carrying lipopolysaccharide
Jiekang ZOU ; Yumeng CAO ; Yi TIAN ; Xuan LI ; Ruixin WU ; Beimin TIAN ; Haihua SUN ; Faming CHEN ; Xiaotao HE
Chinese Journal of Stomatology 2024;59(3):237-246
		                        		
		                        			
		                        			Objective:To investigate the effects of Porphyromonas gingivalis derived outer membrane vesicles (Pg OMV) on osteoclast differentiation of macrophages and its underlying mechanisms. Methods:The morphology and the size distribution of Pg OMV were analyzed by transmission electron microscopy and nanoparticle tracing analysis, respectively. The osteoclast precursors were treated with 1, 3 and 10 mg/L Pg OMV (1, 3 and 10 mg/L OMV treatment group) or phosphate buffer solution (PBS)(control group). The formation of osteoclasts was analyzed by tartrate-resistant acid phosphase (TRAP) staining and F-actin staining and real-time quantitative PCR (RT-qPCR) were used to detect the expression of Fos and matrix metallopeptidase 9 (MMP9). Polymyxin B (PMB) was used to block lipopolysaccharide (LPS) and then Pg OMV was used to treat osteoclast precursor (PMB-OMV treatment group), and OMV treatment group was used as control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The effect of Pg OMV on the expression of Toll-like receptor (TLR) 2 and TLR4 in preosteoclasts was detected by Western blotting. The osteoclast precursors were pretreated with 10, 50, 100 and 200 μmol/L C29, an inhibitor of TLR2, and then treated with Pg OMV(OMV+10, 50, 100 and 200 μmol/L C29 treatment group) and OMV treatment group without C29 pretreatment was control. TRAP and F-actin staining were used to observe the formation of osteoclasts and actin rings. The osteoclast precursor cells were treated with OMV (OMV treatment group) and OMV incubated with PMB (PMB-OMV treatment group) and the expression of TLR2 in osteoclast precursor was detected by Western blotting.Results:Pg OMV showed classical vesicular structures, and the average particle size of Pg OMV were 179.2 nm. A large number of actin rings were observed in the 3 and 10 mg/L OMV treatment groups. The percentages of TRAP-positive osteoclast area in 3 mg/L OMV treatment group [(22.6±2.1)%] and 10 mg/L OMV treatment group [(32.0±2.3)%] were significantly increased compared with control group [(4.9±0.5)%] ( P<0.001). Compared with the control group (1.000±0.029), the mRNA relative expression of Fos in 3 mg/L OMV treatment group (1.491±0.114) and 10 mg/L OMV treatment group (1.726±0.254) was significantly increased ( P=0.013, P=0.001). Compared with the control group (1.007±0.148), the mRNA relative expression of MMP9 in the group of 10 mg/L OMV (2.232±0.097) was significantly increased ( P<0.001). Actin ring formation was less in PMB-OMV treatment groups than in OMV treatment groups. The proportion of TRAP-positive osteoclasts area [(14.8±3.8)%] in PMB-OMV treatment group was significantly lower than OMV treatment group [(31.5±6.7) %] ( P=0.004). The relative expression of TLR2 in OMV treatment group (1.359±0.134) was significantly higher than that in the control group (1.000±0.000) ( t=4.62, P=0.044). Compared with the OMV treatment group [(29.4±1.7)%], 50, 100 and 200 μmol/L C29 significantly decreased the formation of osteoclasts [(24.0±1.7)%, (18.5±2.1)%, (9.1±1.3) %] ( P=0.026, P<0.001, P<0.001). TLR2 protein expression in PMB-OMV group (0.780±0.046) was significantly lower than that in OMV group (1.000±0.000)( t=8.32, P=0.001). Conclusions:Pg OMV can promote osteoclast differentiation by carrying LPS, TLR2 plays an important role in Pg OMV mediated osteoclast differentiation.
		                        		
		                        		
		                        		
		                        	
2.Chinese expert consensus on the diagnosis and treatment of osteoporotic periarticular fracture of the shoulder in the elderly (version 2023)
Yan HU ; Dongliang WANG ; Xiao CHEN ; Zhongmin SHI ; Fengjin ZHOU ; Jianzheng ZHANG ; Yanxi CHEN ; Liehu CAO ; Sicheng WANG ; Jianfei WANG ; Hongliang WANG ; Yong FENG ; Zhimin YING ; Chengdong HU ; Qinglin HAN ; Ming LI ; Xiaotao CHEN ; Zhengrong GU ; Biaotong HUANG ; Liming XIONG ; Yunfei ZHANG ; Zhiwei WANG ; Baoqing YU ; Yong WANG ; Lei ZHANG ; Lei YANG ; Peijian TONG ; Ximing LIU ; Qiang ZHOU ; Feng NIU ; Weiguo YANG ; Wencai ZHANG ; Shijie CHEN ; Jinpeng JIA ; Qiang YANG ; Tao SHEN ; Bin YU ; Peng ZHANG ; Yong ZHANG ; Jun MIAO ; Kuo SUN ; Haodong LIN ; Yinxian YU ; Jinwu WANG ; Kun TAO ; Daqian WAN ; Lei WANG ; Xin MA ; Chengqing YI ; Hongjian LIU ; Kun ZHANG ; Guohui LIU ; Dianying ZHANG ; Zhiyong HOU ; Xisheng WENG ; Yingze ZHANG ; Jiacan SU
Chinese Journal of Trauma 2023;39(4):289-298
		                        		
		                        			
		                        			Periarticular fracture of the shoulder is a common type of fractures in the elderly. Postoperative adverse events such as internal fixation failure, humeral head ischemic necrosis and upper limb dysfunction occur frequently, which seriously endangers the exercise and health of the elderly. Compared with the fracture with normal bone mass, the osteoporotic periarticular fracture of the shoulder is complicated with slow healing and poor rehabilitation, so the clinical management becomes more difficult. At present, there is no targeted guideline or consensus for this type of fracture in China. In such context, experts from Youth Osteoporosis Group of Chinese Orthopedic Association, Orthopedic Expert Committee of Geriatrics Branch of Chinese Association of Gerontology and Geriatrics, Osteoporosis Group of Youth Committee of Chinese Association of Orthopedic Surgeons and Osteoporosis Committee of Shanghai Association of Chinese Integrative Medicine developed the Chinese expert consensus on the diagnosis and treatment of osteoporotic periarticular fracture of the shoulder in the elderly ( version 2023). Nine recommendations were put forward from the aspects of diagnosis, treatment strategies and rehabilitation of osteoporotic periarticular fracture of the shoulder, hoping to promote the standardized, systematic and personalized diagnosis and treatment concept and improve functional outcomes and quality of life in elderly patients with osteoporotic periarticular fracture of the shoulder.
		                        		
		                        		
		                        		
		                        	
3. Analysis on internal mechanism of zedoary turmeric in treatment of liver cancer based on pharmacodynamic substances and pharmacodynamic groups
Zeyu LI ; Erwei HAO ; Rui CAO ; Si LIN ; Linghui ZOU ; Tianyan HUANG ; Zhengcai DU ; Xiaotao HOU ; Jiagang DENG ; Zeyu LI ; Erwei HAO ; Rui CAO ; Si LIN ; Linghui ZOU ; Tianyan HUANG ; Zhengcai DU ; Xiaotao HOU ; Jiagang DENG ; Zeyu LI ; Erwei HAO ; Rui CAO ; Si LIN ; Linghui ZOU ; Tianyan HUANG ; Zhengcai DU ; Xiaotao HOU ; Jiagang DENG
Chinese Herbal Medicines 2022;14(4):479-493
		                        		
		                        			
		                        			 Zedoary tumeric (Curcumae Rhizoma, Ezhu in Chinese) has a long history of application and has great potential in the treatment of liver cancer. The antiliver cancer effect of zedoary tumeric depends on the combined action of multiple pharmacodynamic substances. In order to clarify the specific mechanism of zedoary tumeric against liver cancer, this paper first analyzes the mechanism of its single pharmacodynamic substance against liver cancer, and then verifies the joint anti liver cancer mechanism of its “pharmacodynamic group”. By searching the research on the antihepatoma effect of active components of zedoary tumeric in recent years, we found that pharmacodynamic substances, including curcumol, zedoarondiol, curcumenol, curzerenone, curdione, curcumin, germacrone, β-elemene, can act on multi-target and multi-channel to play an antihepatoma role. For example, curcumin can regulate miR, GLO1, CD133, VEGF, YAP, LIN28B, GPR81, HCAR-1, P53 and PI3K/Akt/mTOR, HSP70/TLR4 and NF-κB. Wnt/TGF/EMT, Nrf2/Keap1, JAK/STAT and other pathways play an antihepatoma role. Network pharmacological analysis showed that the core targets of the “pharmacodynamic group” for anti-life cancer are AKT1, EGFR, MAPK8, etc, and the core pathways are neuroactive live receiver interaction, nitrogen metabolism, HIF-1 signaling pathway, etc. At the same time, by comparing and analyzing the relationship between the specific mechanisms of pharmacodynamic substance and “pharmacodynamic group”, it is found that they have great reference significance in target, pathway, biological function, determination of core pharmacodynamic components, formation of core target protein interaction, in-depth research of single pharmacodynamic substance, increasing curative effect and so on. By analyzing the internal mechanism of zedoary tumeric pharmacodynamic substance and “pharmacodynamic group” in the treatment of liver cancer, this paper intends to provide some ideas and references for the deeper pharmacological research of zedoary tumeric and the relationship between pharmacodynamic substance and “pharmacodynamic group”. 
		                        		
		                        		
		                        		
		                        	
4.Meta-analysis of the correlation between growth and thyroid volume in children
Shuli AN ; Fan LI ; Xiaotao CAO ; Fangang MENG
Chinese Journal of Endemiology 2022;41(5):407-413
		                        		
		                        			
		                        			Objective:To systematically evaluate the relationship between growth and thyroid volume (Tvol).Methods:Chinese and English papers were searched in CNKI, Wanfang Data, VIP Database, PubMed, EMbase, Springerlink, Medline database, articles on the correlation between Tvol and growth in children published from the establishment of these databases to October 2021 were collected. Meta-analysis was conducted with R language 4.0. I2 and Q test were used for heterogeneity test. According to the heterogeneity test results, the fixed effect model or random effect model was selected, and sensitivity analysis was conducted. Egger's method was used to test publication bias. Results:A total of 17 papers were incorporated in this study, including 11 Chinese papers and 6 English papers. The correlation between body surface area (BSA) and Tvol was the strongest[estimated effect value ( COR) = 0.44, 95% confidence interval ( CI): 0.39 - 0.50], followed by weight ( COR = 0.39), height ( COR = 0.37) and age ( COR = 0.29). Sensitivity analysis showed that the estimated effects COR were all within 95% CI of the total effects after deleting any paper, which indicated that the results of Meta-analysis were reliable. Egger's method result showed P > 0.05, indicating that there was no obvious publication bias in this study. Conclusion:Age, height, weight and BSA all have effects on Tvol, among which BSA has the greatest effect.
		                        		
		                        		
		                        		
		                        	
5.Clinical guideline for spinal reconstruction of osteoporotic thoracolumbar fracture in elderly patients (version 2022)
Tao SUI ; Jian CHEN ; Zhenfei HUANG ; Zhiyi HU ; Weihua CAI ; Lipeng YU ; Xiaojian CAO ; Wei ZHOU ; Qingqing LI ; Jin FAN ; Qian WANG ; Pengyu TANG ; Shujie ZHAO ; Lin CHEN ; Zhiming CUI ; Wenyuan DING ; Shiqing FENG ; Xinmin FENG ; Yanzheng GAO ; Baorong HE ; Jianzhong HUO ; Haijun LI ; Jun LIU ; Fei LUO ; Chao MA ; Zhijun QIAO ; Qiang WANG ; Shouguo WANG ; Xiaotao WU ; Nanwei XU ; Jinglong YAN ; Zhaoming YE ; Feng YUAN ; Jishan YUAN ; Jie ZHAO ; Xiaozhong ZHOU ; Mengyuan WU ; Yongxin REN ; Guoyong YIN
Chinese Journal of Trauma 2022;38(12):1057-1066
		                        		
		                        			
		                        			Osteoporotic thoracolumbar fracture in the elderly will seriously reduce their quality of life and life expectancy. For osteoporotic thoracolumbar fracture in the elderly, spinal reconstruction is necessary, which should comprehensively consider factors such as the physical condition, fracture type, clinical characteristics and osteoporosis degree. While there lacks relevant clinical norms or guidelines on selection of spinal reconstruction strategies. In order to standardize the concept of spinal reconstruction for osteoporotic thoracolumbar fracture in the elderly, based on the principles of scientificity, practicality and progressiveness, the authors formulated the Clinical guideline for spinal reconstruction of osteoporotic thoracolumbar fracture in elderly patients ( version 2022), in which suggestions based on evidence of evidence-based medicine were put forward upon 10 important issues related to the fracture classification, non-operative treatment strategies and surgical treatment strategies in spinal reconstruction after osteoporosis thoracolumbar fracture in the elderly, hoping to provide a reference for clinical treatment.
		                        		
		                        		
		                        		
		                        	
6.Processing of Citri Reticulatae Pericarpium: A Review
Xiaoting ZHANG ; Ying ZHANG ; Daiying ZHANG ; Jiang MENG ; Yue SUN ; Shumei WANG ; Hui CAO
Chinese Journal of Experimental Traditional Medical Formulae 2022;28(17):267-274
		                        		
		                        			
		                        			Citri Reticulatae Pericarpium is a commonly used traditional Chinese medicine in China. By referring to herbal monographs and related research documents, and comparing all editions of Chinese Pharmacopoeia and local processing specifications, the processing history, modern processing specifications, study on chemical constituents and pharmacological effects before and after processing of Citri Reticulatae Pericarpium were summarized. It was found that the processing methods of Citri Reticulatae Pericarpium in the past dynasties were mainly cleansing, cutting and frying. The processing methods of Citri Reticulatae Pericarpium included in all editions of Chinese Pharmacopoeia were all cleansing and cutting. In modern local processing specifications, there were mainly processing methods such as frying, steaming and carbonizing. The components of Citri Reticulatae Pericarpium were mainly volatile oil and flavonoids. After processing, the total amount of volatile oil generally decreased, and the contents and compositions of volatile oil and flavonoids also changed. It may be the main reason for the difference of efficacy before and after processing. At present, the optimization of processing technology of Citri Reticulatae Pericarpium mainly focuses on steaming, processing with vinegar and baking. Pharmacological research on Citri Reticulatae Pericarpium mainly focuses on reducing blood lipid, relieving cough, relieving asthma and resolving phlegm, and inhibiting pulmonary fibrosis. The current research on the processing methods of Citri Reticulatae Pericarpium is not in-depth enough, the material basis of many processing methods has not been clarified, and the research on the efficacy before and after processing is not in-depth enough. Further research is needed to clarify the material basis and mechanism after the processing of Citri Reticulatae Pericarpium, so as to standardize the processing method and establish specific quality standards. 
		                        		
		                        		
		                        		
		                        	
7.Pharmacological Effect of Glycyrrizae Radix et Rhizoma Compatibility and Its Mechanism
Zeyu LI ; Erwei HAO ; Hui LI ; Jiayu WANG ; Xinxin LI ; Rui CAO ; Si LIN ; Huizhen QIN ; Jinling XIE ; Zhengcai DU ; Xiaotao HOU ; Jiagang DENG
Chinese Journal of Experimental Traditional Medical Formulae 2022;28(14):270-282
		                        		
		                        			
		                        			Glycyrrizae Radix et Rhizoma has high medicinal value and is widely used in compatibility. It is used most frequently in the compatibility of Chinese medicine prescriptions,and is known as ''Guolao''(national medicine) and "master of all medicines". The characteristic active ingredients are mainly liquitin,glycyrrizic acid,glycyrrizin,and licochalcone. In different compatibilities,based on traditional and modern pharmacological theories,the corresponding effect of Glycyrrizae Radix et Rhizoma are brought into play through different mechanisms. Based on the traditional pharmacology of Glycyrrizae Radix et Rhizoma for tonifying spleen,replenishing Qi,clearing heat,removing toxin,dispelling phlegm,relieving cough and pain,and harmonizing various medicines,this paper used herbal authentication to analyze its compatibility application and mechanism. It was found that Glycyrrizae Radix et Rhizoma played corresponding effect in compatibilities through "tonification","harmonization",and "regulation". For example,Glycyrrizae Radix et Rhizoma was combined with tonics including Ginseng Radix et Rhizoma and Atractylodis Macrocephalae Rhizoma to tonify the five Zang-organs through its strong tonifying effect,combined with Paeoniae Radix Alba and Aconiti Lateralis Radix Praeparata to relieve emergencies and pains through harmonizing medicine power and properties,and combined with Rhei Radix et Rhizoma and Natrii Sulfas to reduce medicine intensity through regulating medicine properties and body characteristics. The application law and mechanism of the modern pharmacological compatibility of Glycyrrizae Radix et Rhizoma were analyzed by data mining and network pharmacology. It was found that the modern clinical formula was often compatible with Glycyrrizae Radix et Rhizoma for anti-inflammation,cardiovascular and cerebrovascular protection,anti-virus,and anti-tumor,Ephedrae Herba and Scutellariae Radix for anti-inflammation,Bambusae Caulis in Taenias,Salviae Miltiorrhizae Radix et Rhizoma,and Aurantii Fructus Immaturus for cardiovascular and cerebrovascular protection,and Ophiopogonis Radix and Chuanxiong Rhizoma for nerves protection. Meanwhile,the important targets of the characteristic ingredients were protein kinase B1 (Akt1),interleukin-6 (IL-6),tumor necrosis factor (TNF),and epidermal growth factor receptor (EGFR). The important characteristic pathways such as tyrosine kinase inhibitor resistance pathway and cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal pathway played the role of cardiovascular and cerebrovascular protection,and proteoglycan pathway in cancer played a neuroprotective role. This study is expected to provide references for the rational compatibility and application of Glycyrrizae Radix et Rhizoma,as well as the compatibility application of Chinese medicine prescriptions. 
		                        		
		                        		
		                        		
		                        	
8.Cloning, expression, purification and identification of EgG1Y162-2 gene from Echinococcus granulosus
Huifang KONG ; Shangqi ZHAO ; Yanxia ZHOU ; Qiaoqiao GONG ; Yujiao LI ; Chunbao CAO ; Haimei MA ; Jianbing DING ; Xiaotao ZHOU
Chinese Journal of Endemiology 2021;40(8):635-639
		                        		
		                        			
		                        			Objective:To construct the pET30a-EgG1Y162-2 prokaryotic expression plasmid and induce the expression of EgG1Y162-2 protein, so as to provide a research basis for development of Echinococcus granulosus vaccine. Methods:Using Echinococcus granulosus cDNA as a template, the target gene of EgG1Y162-2 was synthesized by PCR, and after digestion with restriction enzymes EcoRⅠ and Hind Ⅲ, it was connected to the prokaryotic expression vector pET30a to construct the recombinant plasmid pET30a-EgG1Y162-2. The recombinant plasmid was transformed into competent cell BL21 (DE3) and induced by isopropyl β-D-thiogalactoside (IPTG) to express a large number of proteins. The recombinant protein was purified by affinity chromatography. The purification level was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the expression product was identified by Western blotting. Results:The recombinant plasmid pET30a-EgG1Y162-2 was successfully constructed. After inducting expression, the bacterial supernatant and the eluate were both at a relative molecular weight of about 15 × 10 3, and the protein antigen component eluted with 200 mmol/L imidazole was relatively pure. Western blotting results showed that the purified recombinant protein EgG1Y162-2 with His tag could be recognized by His monoclonal antibody. Conclusion:The pET30a-EgG1Y162-2 prokaryotic expression plasmid of Echinococcus granulosus is successfully constructed, and the recombinant protein of EgG1Y162-2 is induced to express, laying a foundation for further study on anti- Echinococcus granulosus vaccine.
		                        		
		                        		
		                        		
		                        	
9.Herbalogical study on Liangmianzhen (Zanthoxyli Radix).
Meng-Hua WU ; Zhi-Guo MA ; Ying ZHANG ; Li-Fang YE ; Hui CAO
China Journal of Chinese Materia Medica 2021;46(20):5436-5442
		                        		
		                        			
		                        			Liangmianzhen(Zanthoxyli Radix) has long been used as medicine. The current medicinal parts are different from those in the ancient. As recorded in the Chinese Pharmacopeia, the medicinal part is root. However, in ancient works, the medicinal parts include root, stem, leaf, and fruit. In an attempt to find the historical basis that stem is a reasonable medicinal part, the herbalogical study was carried out on this medicinal based on the formal names, synonyms, original plant, medicinal parts, habitat of the medicinal plant, producing area, processing and preparation methods, efficacy, and indications recorded in ancient Chinese materia medica and local gazetteers. The results showed that Liangmianzhen was firstly recorded as a medicinal in Shennong's Classic of Materia Medica with the formal name of "Manjiao". "Manjiao" was adopted from the Han Dynasty to the Qing Dynasty when it was changed to "Rudijinniu", the name originating from the folk in the south of the Five Ridges. Now, the formal name is "Liangmianzhen", which was firstly recorded in Wuxuan County Gazetteer in 1914 and then as a synonym in the Updated Records of Picking Herbs in the South of the Five Ridges. According to the formal names, synonyms, and the descriptions of the original plant, the medicinal plants of Liang-mianzhen have the characteristics of shrub-like young seedlings, vine adult seedlings, corymbiform thyrsus, stems with thorns, amphitropous golden-yellow roots with horn-like branches, and thorns on both sides of the leaves. Thus, "Manjiao", "Rudijinniu", and "Liangmianzhen" were from the same species of Zanthoxylum nitidum(Rutaceae), which was also verified based on the growth environment, habitat, processing and preparation methods, efficacy, and indications. In ancient times, the stem and root were the main medicinal parts and leaves and fruits were also used. However, in the Chinese Pharmacopeia, root is recorded as the only medicinal part, which is obviously inconsistent with the records in the ancient classics. In light of the limited medicinal resources for Liang-mianzhen, other medicinal parts of Z. nitidum is recommended. This study clarified the medicinal parts of Z. nitidum in history. It is recommended that the stem be added to the medicinal parts of Z. nitidum in the next edition of Chinese Pharmacopeia.
		                        		
		                        		
		                        		
		                        			China
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		                        			Drugs, Chinese Herbal
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		                        			Fruit
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		                        			Materia Medica
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		                        			Medicine, Chinese Traditional
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		                        			Plants, Medicinal
		                        			
		                        		
		                        	
10.Herbalogical study on olibanum(Ruxiang).
Zi-Han HUANG ; W U MENG-HUA ; Si-Min LUO ; Yu ZHOU ; Ying ZHANG ; M A ZHI-GUO ; Hui CAO
China Journal of Chinese Materia Medica 2020;45(21):5296-5303
		                        		
		                        			
		                        			As a representative foreign medicinal material, olibanum(Ruxiang) was imported to China since the Qin and Han Dynasties. Olibanum was first described as a medicinal by the name "Xunluxiang" in Miscellaneous Records of Famous Physicians(Ming Yi Bie Lu). This study investigated historical records on olibanum and conducted the herbalogical study. It was found that olibanum came from the resin mainly obtained from the bark of Pistacia lenticus before the Tang Dynasty. With the prosperity of the Maritime Silk Road, instead, the resin obtained from the bark of Boswellia carterii was mainly used as olibanum. In ancient time, the oleo-gum-resin secreted from the cut bark was collected in spring and summer, and the quality was judged based on transparency and shape. The processing methods of olibanum went through many evolutions, which changed from simple methods such as grinding and frying to complex methods such as levigating and grinding with wine, and now to frying and processing with vinegar. The usage of olibanum included alchemy, folk and religious incense, bathing, cosmetic and medicinal since ancient times. From the Song Dynasty, olibanum had been mainly used as medicinal because of its good effect to treat wounds. In traditional Chinese medicine, olibanum unblocks menstruation, relieves pain and reduces swelling and generated muscles. The medicinal efficacy of olibanum is not much different from ancient to modern. Only the efficacy of replenishing energy and promoting the movement of Qi was rarely mentioned in modern reference. In this article, the historical evolutions of olibanum about original plants, processing and medicinal efficacy were sorted out. The results could provide historical basis for the further development and clinical utilization of olibanum.
		                        		
		                        		
		                        		
		                        			China
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		                        			Drugs, Chinese Herbal
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		                        			Frankincense
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		                        			Medicine, Chinese Traditional
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		                        			Resins, Plant
		                        			
		                        		
		                        	
            
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