Journal of Ethnopharmacology 164 (2015) 256–264

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Journal of Ethnopharmacology

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Review The genus in traditional Chinese medicine: An ethnomedical, phytochemical and pharmacological review

De-Gang Kong 1,a, Yu Zhao a,1, Guo-Hui Li b, Bang-Jiao Chen a, Xiao-Ning Wang a, Hong-Lei Zhou c, Hong-Xiang Lou a, Dong-Mei Ren a,n, Tao Shen a,n a Key Lab of Chemical Biology (MOE), School of Pharmaceutical Sciences, Shandong University, Jinan, PR b Department of Pharmacy, Jinan Maternity and Child Care Hospital, Jinan, PR China c School of Pharmaceutical Sciences, Shandong University of Traditional Chinese Medicine, Jinan, PR China article info abstract

Article history: Ethnopharmacological relevance: The genus Litsea, mainly distributed in the tropical and subtropical Received 19 October 2014 regions, has been used in traditional and indigenous Chinese medicines for the treatment of diarrhea, Received in revised form stomachache, dyspepsia, gastroenteritis, diabetes, edema, cold, arthritis, asthma, pain, traumatic injury, 5 February 2015 etc. for a long history. The present review aims to provide a comprehensive summary on the Accepted 8 February 2015 ethnomedical uses, phytochemistry, and pharmacology of the Litsea species used in traditional Chinese Available online 16 February 2015 medicine (TCM). Based on these data, evidences supporting their ethnopharmacological effectiveness are Keywords: illustrated, and opportunities for the future research and development as well as the therapeutic Litsea potential of this genus are analyzed to highlight the gaps in our knowledge that deserves further Phytochemistry investigation. Pharmacology Material and methods: Information on the Litsea species was collected via electronic search (using Ethnomedical uses Pubmed, SciFinder, Google Scholar, Web of Science and CNKI) and a library search for articles published in peer-reviewed journals. Furthermore, information was also obtained from some local books on ethnopharmacology. Results: Twenty of the genus Litsea are found to be important traditional medicines in China, and have a long medicinal application for diarrhea, stomachache, dyspepsia, gastroenteritis, diabetes, edema, cold, arthritis, asthma, pain, traumatic injury, etc. Over 200 ingredients have been identified from these 20 Litsea species used in TCM, and flavonoids, terpenoids and alkaloids are considered as the characteristic and bioactive constituents. The crude extracts and the isolated metabolites of these medicinal plants have exhibited some in vitro and in vivo pharmacological effects, including antimicro- bial, hepatoprotection, anti-inflammatory, antiasthmatic, immunomodulation, anti-diabetic, antichole- lithogenic, as well as function on central nervous system, etc. Conclusions: The extensive literature survey reveals Litsea species to be a group of important medicinal plants used for the ethnomedical treatment of gastrointestinal diseases, diabetes, inflammatory disorders, and microbial infection in TCM. Pharmacological investigations have supported the use of some Litsea species in the traditional medicines. In addition, further researches targeting individual ingredients responsible for the pharmacological effects, as well as their mechanisms of action are necessary. The outcome of these studies will further support the therapeutic potential of the genus Litsea, and provide convincing evidences to its future clinical applications in modern medicine. & 2015 Elsevier Ireland Ltd. All rights reserved.

Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CCAAT, cytidine–cytidine–adenosine–adenosine–thymidine; CHOP, CCAAT/enhancer binding protein homologous protein; CTGF, connective tissue growth factor; FFA, free fatty acid; GC, gas chromatography; GRP 78, glucose regulated protein 78; GSH-PX, glutathione peroxidase; γ-GT, γ-glutamyltransferase; HA, hyaluronic acid; ICR, Institute for Cancer Research; IL-1, interleukin-1; IL-1β, interleukin-1β;

LDL-C, low density lipoprotein-cholesterol; LN, laminin; MDA, malondialdehyde; MMP-9, matrix metalloproteinase-9; PGE2, prostaglandin E2; PIIINP, procollagen III N-terminal peptide; PPARα, peroxisome proliferator-activated receptor α; SOD, superoxide dismutase; TC, total cholesterol; TCM, traditional Chinese medicine; TFLC, total

flavonoids of Litsea coreana; TG, triglyceride; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor; TrACP, tartarate resistance phosphatase n Correspondence to: School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, Jinan 250012, PR China. Tel.: þ86 531 88382028; fax: þ86 531 88382548. E-mail addresses: [email protected] (D.-M. Ren), [email protected] (T. Shen). 1 These two authors contributed equally. http://dx.doi.org/10.1016/j.jep.2015.02.020 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved. D.-G. Kong et al. / Journal of Ethnopharmacology 164 (2015) 256–264 257

Contents

1. Introduction...... 257 2. Ethnomedical uses...... 257 3. Phytochemistry ...... 259 3.1. Flavonoids ...... 260 3.2. Terpenoids ...... 260 3.3. Alkaloids ...... 260 3.4. Butanolides and butenolactones ...... 260 3.5. Lignans ...... 260 3.6. Amides...... 260 3.7. Miscellaneous constituents ...... 260 4. Pharmacology ...... 260 4.1. Antimicrobial activity ...... 260 4.2. Hepatoprotection ...... 261 4.3. Anti-inflammatory activity ...... 261 4.4. Antiasthmatic activity ...... 261 4.5. Immunomodulation...... 261 4.6. Anti-diabetes ...... 262 4.7. Function on central nervous system...... 262 4.8. Anticholelithogenic activity...... 262 4.9. Miscellaneous bioactivities ...... 262 5. Toxicology...... 262 6. Conclusions ...... 262 Acknowledgments...... 263 Appendix A. Supplementary data ...... 263 References...... 263

1. Introduction 2007), and anti-diabetic properties (Sun et al., 2010), confirmed by experiments in vivo and in vitro. The genus Litsea, belonging to the family , contains Several reviews dealing with the phytochemistry and pharma- approximately 200 species, which are mainly distributed in cology of the genus Litsea have been reported. Two reviews focusing the tropical and subtropical regions around the world (Richter, 1981). on terpenoids and alkaloids from Litsea species and their biological It is documented that 74 plant species of this genus have been found activities have been published (Xie and Zhang, 1999; Zhao, 2006). in China, most of which are growing in the regions between 181 and Recently, Agrawal et al. (2011) comprehensively summarized the 341 north latitude of Southern and Southwest China, including Anhui, chemical ingredients isolated from the genus Litsea before the year Zhejiang, Fujian, Yunnan, Sichuan and Tibet provinces (Flora of China of 2009, and also briefly introduced their biological activities. Editorial Committee, 1994). In the aspect of ethnomedicine,20 plants Different from writing objectives of above literatures, the present in Litsea species have a long history of use in traditional and review focuses on the research progress targeting the Litsea species indigenous Chinese medicines (Xie and Yu, 1996). Fruit of the plant used in TCM, to provide a comprehensive summary on the is the commonly prescribed medicinal part for the treatment of ethnomedical uses, phytochemistry and pharmacology of these gastrointestinal diseases, pain, asthma, and traumatic injury. Mean- plants. Besides, correlations of ethnomedical uses, phytochemistry while, the , stems, velamina, roots and barks have also been and pharmacology have been discussed based on the research adopted to treat people suffering from stomachache, cold, pain, findings of these fields (Arora and Kaur, 2007; Mandal et al., arthritis, diarrhea, traumatic injury, etc.(Editorial Committee of 2000; Chen and Xu, 2013; Wang and Liu, 2010; Devib and Meera, Zhonghua Bencao National Traditional Chinese Herb Administration, 2010; Chen et al., 2004; Zhong et al., 2013; Zhang and Di, 2008; 1999; Xie and Yu, 1996; Xie and Liang, 1996). Zhou et al., 2007; Wang et al., 1999, 2009; Tu et al., 1985; Fang et al., A great deal of studies concerning the phytochemical and pharma- 2002; Qian et al., 1980; Yin et al., 2006; Lv et al., 2008). cological aspects of the genus Litsea have been carried out. More than 200 chemical ingredients, covering flavonoids, terpenoids, alkaloids, butanolides and butenolactones, lignans, amides, steroids, fatty acids, 2. Ethnomedical uses megastigmanes, etc., have hitherto been isolated from these 20 plants medicinally used in traditional Chinese medicine (TCM). Of these Common names, ethnomedical uses and medicinal parts of the chemical ingredients, flavonoids and terpenoids are regarded as the Litsea species used in TCM are listed in Table 1. On the basis of our two groups of bioactive substances that are responsible for the investigations, 20 Litsea species are used in a Chinese ethnomedi- observed pharmacological effects of Litsea species (Chen et al., 2004; cal system (Xie and Yu, 1996). Fruits, roots, leaves and barks of Tang et al., 2013; Wan et al., 2006; Wang and Liu, 2010; Wang et al., these plant species are adopted for the therapy of diseases in the 2009; Ye et al., 2006). Extracts of plants from this genus, as well as the different approaches including (i) pharmaceutics (e.g. decoction, purified molecules, demonstrate a wide spectrum of pharmacological pill, pulvis, etc.) of signal medicine, or compound preparations functions, involving in antifungal (Yang et al., 2010), antibacterial with other traditional Chinese medicines; (ii) drunk as tea; (iii) (Chen and Xu, 2013), antidiarrheal (Mandal et al., 2000), anti- eaten as spices. The Litsea plants in TCM are mainly used for the inflammatory (Tang et al., 2013), anti-arthritic (Zhou et al., 2010), therapies of diarrhea, stomachache, dyspepsia, gastroenteritis, anti-HIV (H. Zhang et al., 2003), anti-asthma (Yin et al., 2006), diabetes, edema, cold, arthritis, asthma, pain, traumatic injury, hepatoprotective (Wang et al., 2009), immunomodulatory (Hu et al., etc. (Editorial Committee of Zhonghua Bencao National Traditional 258 D.-G. Kong et al. / Journal of Ethnopharmacology 164 (2015) 256–264

Table 1 Medicinal plants of the genus Litsea used in TCM.

Species Common/vernacular Medicinal Ethnomedical uses References names parts

L. auriculata S.S. Bajiaoyang Fruits and Expelling parasite, treating teniasis and The Health Department of Zhejiang Province (1965) Chien and W.C. velamina enterobiasis Cheng Leaves Relieving pain, treating traumatic injury L. coreana var. sinensis Bopifeng, Barks Treating stomach distension Xie and Yu, (1996) (C.K. Allen) Yen C. HuakechaiBaopizhang Yang and P.H. Huang Roots Treating edema L. coreana var. Baicha, Hawk tea Leaves Treating stomach distension, diarrhea, Wang et al. (2010) and lanuginosa (Migo) lowering blood fat, and sunstroke Xiang and Lu (1998) Yen C. Yang and P. H. Huang L. cubeba (Lour.) Pers. Bichengqie, Shancangzishu, Fruits Relieving pain, promoting blood circulation, as Editorial Committee of Zhonghua Bencao National Changzimu, Manshanxiang, well as treating stomach distension, asthma, Traditional Chinese Herb Administration (1999), Xie Gangouzhang, emesia, diarrhea, turbid urine and traumatic and Yu (1996), and Xie and Liang (1996) Dongpizhuang injury Roots Treating cold, stomachache, headache, dermatophytosis and arthralgia Leaves Promoting blood circulation, treating mammitis, as well as externally used for hemostasis, sores furuncle, insect and snake bites L. euosma W.W. Sm. Bichengqie, Jiguxiang, Fruits Tonifying spleen, treating dyspepsia and sore Xie and Yu (1996) Daliwang, Quwenshu, Shuicang L. glutinosa (Lour.) C. Chuanshu, Shanjiaomu, Barks and Treating furuncle and traumatic injury Nanning City Institute of Traditional Chinese B. Rob. Chunguishu, Shangaozi, leaves Medicine (1960), and Editorial Committee of Changaomu, Yanggushu Zhonghua Bencao National Traditional Chinese Herb Administration (1999). Roots Treating diarrhea, traumatic injury, mumps, diabetes, acute or chronic gastritis and rheumatalgia L. glutinosa var. Houyezhang, Yegaoshu, Barks and Reducing swelling, and treating sore Xie and Yu (1996) brideliifolia Yuanweigao, Qingyegaomu roots (Hayata) Merr. L. hupehana Hemsl. Baichuncha, Laoyingcha Leaves Treating diarrhea Xie and Yu (1996) L. ichangensis Gamble Goujiangzishu, Fruits and Treating dyspepsia and diarrhea Xie and Yu (1996) Yehujiaozishu roots L. mollis Hemsl. Shanhujiao, Mujiangzi, Fruits Treating chronic eczema Xie and Yu (1996) Shancangzi, Yemujiangzi, Bichengqie Roots Treating traumatic injury L. monopetala (Roxb.) Jiashali, Jiashishu, Nagao, Leaves Treating fracture and dislocation Xie and Yu (1996) Pers. Muzhugao, Shanboluoshu L. moupinensis var. Bichengqie, Chengqiezi Fruits Relieving pain, treating stomachache and Editorial Committee of Zhonghua Bencao National szechuanica (C.K. emesia Traditional Chinese Herb Administration (1999) Allen) Yen C. Yang and P.H. Huang L. populifolia (Hemsl.) Laoyapi, Chengqiezi, Roots Treating dyspepsia, relieving pain, nausea and Xie and Yu (1996) Gamble Laoyapimimi, Muxiangzi emesia L. pungens Hemsl. Shanhujiao, Lajiangzi, Fruits and Strengthening spleen, treating dyspepsia, Xie and Yu (1996) Huanghuazi, Baidamu, leaves diarrhea, and sunstroke, as well as externally Muzhangzi used for sore Stems Treating stomach distension. Roots Relieving the pain, treating stomachache and arthralgia L. rotundifolia Hemsl. Yuanyechaipizhang Roots Treating rheumatic pain Xie and Yu (1996) L. rotundifolia var. Baichai, Xiangyezi, Roots and Relieving pain, treating rheumatic arthritis, Xie and Yu (1996) oblongifolia Shanhujiao, Guoshanxiang, barks traumatic injury, dysmenorrheal, stomachache (Nees) C.K. Allen Shanrougui and diarrhea L. rubescens Lecomte Shanhujiao, Mujiangzi, Fruits Treating enterogastritis, stomachache and The Tibet Autonomous Regional Revolutionary Hongmujiangzi, Muwuyu, dyspepsia Committees Health Bureau (1973) and Xie and Yu Yeqilazi (1996) Roots Treating traumatic injury and cold L. sericea (Wall. ex Mujiangzi, Bichengqie, Fruits Preventing corrosion, eliminating phlegm, and The Botany Institute of Jiangsu Province (1990) Nees) Hook. f. Shanhujiao, Tuguizhi improving digestion L. veitchiana Gamble Muxiangzi, Mujiangzi, Fruits Treating dyspepsia Editorial Committee of Zhonghua Bencao National Chengqiezi, Huashuye, Traditional Chinese Herb Administration (1999) Shanhujiao L. verticillata Hance Niulali, Diedalao, Gaoshu, Roots, Promoting blood circulation, relieving pain and Xie and Yu (1996) Jiawujia, Yingxiongjian leaves and treating traumatic injury barks D.-G. Kong et al. / Journal of Ethnopharmacology 164 (2015) 256–264 259

Chinese Herb Administration, 1999; The Health Department of Zhejiang Province, 1965; Xie and Yu, 1996; Xie and Liang, 1996). These ethnomedical uses of the Litsea species might originate from their antimicrobial, anti-inflammatory, anti-diabetic, and antiasth- matic activities as evaluated by the modern pharmacological investigations. As can be seen from Table 1, stomach diseases, traumatic injury, pain and diarrhea are the diseases most frequently treated by the Litsea species. Regarding the quantity of plants used for the therapy of each disease, it is concluded that 12 plants are adopted for the treatment of stomach diseases, seven plants for traumatic injury, seven plants for pain, and seven plants for diarrhea. The fruit of Litsea cubeba, named as ‘Bi-Cheng-Qie’(荜澄茄), is the most important and widely used medicine of this genus. It has been used for the treatment of cold and pain in TCM since the Tang Dynasty in 600 AD, which was recorded in the Chinese medical document Ben Cao Shi Yi (本草拾遗). Some prescriptions created by the ancient famous doctors are adopted for the treatment of stomach diseases and pain, such as ‘Bi-Cheng-Qie-Wan’ [pill recorded in Sheng Ji Zong Lu (圣济总录)], ‘Bi-Cheng-Qie-San’ [pulvis recorded in Bian Que Xin Shu (扁鹊心书)], ‘Bi-Cheng-Qie- Yin’ [decoction recorded in Pu Ji Fang (普济方)], etc. In the modern monograph of TCM, it is described as a medicine for treating traumatic injury, dermatophytosis, stomachache, toothache, etc. (Editorial Committee of Zhonghua Bencao National Traditional Chinese Herb Administration, 1999; Enshi in Hubei Province of Chinese Herbal Medicine Research Group, 1970; Jiangxi Province Health Bureau Revolutionary Committee, 1970; The Medicine Institute of Fujian Province, 1970). The tender leaves of Litsea coreana var. lanuginosa were initially acted as tea for drinking by the local residents of Dalou mountain of Guizhou province, and was named as ‘hawk-tea’ because hawk was accustomed to establish nest on the tree (Xiang and Lu, 1998). Since its special flavor and functions of treating stomach distension, fever, diarrhea and sunstroke, hawk-tea spread out in the southwest of China, such as Guizhou, Sichuan, Chongqing, etc. (Xu et al., 2012). Hawk- tea can be further processed using larvae of Aglossa dimidiata to furnish it with the favorable taste and beneficial functions on human health. Larvae of A. dimidiate are fed with hawk-tea to produce feces, and the feces are collected and named as ‘sandy- tea’ for drinking (Wang et al., 2010; Xiang and Lu, 1998). Sandy-tea bears similar functions on human health to hawk-tea. The fruits and roots of some Litsea plants are important and characteristic spices in south and southwest regions of China, exemplified by Guizhou, Hunan, Hubei, and Taiwan. The ethnomedical values and applications of these Litsea species are intimately known by the local residents, and have been recorded in the regional medical monographs, covering Hu Nan Yao Wu Zhi (湖南药物志), Gui Zhou Min Jian Yao Wu (贵州民间药物), Chong Qing Cao Yao (重庆草药), Dian Nan Ben Cao (滇南本草), etc. (Xie and Yu, 1996; Editorial Committee of Zhonghua Bencao National Traditional Chinese Herb Administration, 1999).

3. Phytochemistry

Agrawal et al. (2011) have reviewed the chemical ingredients isolated from the genus Litsea before the year 2009. With respect to these 20 medicinal plant species in China, more than 200 chemical ingredients, covering flavonoids, terpenoids, alkaloids, butanolides and butenolactones, lignans, amides, steroids, fatty Fig. 1. Chemical structures of typical constituents isolated from the plants of Litsea acids and megastigmanes, have been reported. Their structures species used in TCM. and resources have been comprehensively summarized, and given in (Fig. 1 and Supplementary data). Based on these results, we monoterpenes and sesquiterpenes, are both abundant and bioac- conclude that flavonoids and terpenoids, especially the tive constituents in this genus. 260 D.-G. Kong et al. / Journal of Ethnopharmacology 164 (2015) 256–264

3.1. Flavonoids were purified from methanolic extract of L. glutinosa (Agrawal et al., 2013). The traditionally used Litsea species are rich sources of flavo- noids. Currently, 26 flavonoids have been isolated from these 20 3.5. Lignans Litsea species (Table S1 and Fig. S1). Flavonoids mainly occur in the plants of L. coreana, Litsea glutinosa and L. cubeba, and are classified A total of 20 lignans have been discovered in four Litsea species, to be flavones, flavonols, flavanones, flavanonols, anthocyanidins, including L. cubeba, L. euosma, L. glutinosa and L. verticillata (Table chalcones, and flavan-3-ols. Flavones, flavonols, and flavanones S6 and Fig. S6). (þ)-Epiexcelsin (36) and (þ)-50-demethoxyepiex- mainly exist in the form of glycosides, consisted of glucose, celsin (37) were isolated from L. verticillata (Hoang et al., 2002). galactose and rhamnose. Flavonoids are regarded to be ingredients Recently, a series of lignans were obtained from L. glutinosa (Pan responsible for the therapeutic action of Litsea species. For instance, et al., 2010; Wang et al., 2011). the flavonoids (111) from L. coreana demonstrate anti-inflamma- tory, antioxidant and hepatoprotective activities (Chen et al., 2004; 3.6. Amides Tang et al., 2013; Wang et al., 2009; Ye et al., 2006). Some new flavonoids have been reported from these plants. Two new flava- Zhu and Yang (2007) firstly reported two amides N-feruloy- nocoumarins, isophyllocoumarin (8) and isoepiphyllocoumarin (9), ltyramine (38) and cis-N-feruloyl-3-methoxytyramine (39) from together with phyllocoumarin (10) and epiphyllocoumarin (11), L. cubeba. Subsequent investigations led to the isolation of five were isolated from the leaves of L. coreana. These four flavanocou- additional amides (4044) (Chen et al., 2010; Tanaka et al., 2009). marins possessed a similar catechin core and a pyran-2-one ring in Amides only occur in the plants of Litsea auriculata and L. cubeba each molecule (Tang et al., 2013). (Table S7 and Fig. S7).

3.2. Terpenoids 3.7. Miscellaneous constituents

There are approximately 60 terpenoids, covering monoterpenes Many other constituents have been obtained (Tables S8S11 and sesquiterpenes, isolated from these 20 Litsea species (Tables S2, and Figs. S8S11), such as phenyl esters (4547) (Agrawal et al., S3 and Figs. S2, S3). Monoterpenes mainly occur in volatile oil, and 2013; Xiao et al., 2006b), stilbenes (4850) (Sun and Guo, 2006), fi are identi ed by GC-based techniques. Many papers have described megastigamanes (Wang et al., 2011, 2012), etc. A water soluble the GC analysis of volatile oil from different Litsea species, including polysaccharide, arabinoxylan (51), was isolated from green leaves L. cubeba (Cheng and Cheng, 1983; Jiang et al., 2009; Yang et al., of L. glutinosa (Das et al., 2013). 2010; Zhan et al., 1985), Litsea euosma (Thang et al., 2006), L. glutinosa (Choudhury et al., 1996), Litsea mollis (Wang et al., 2002)andLitsea pungens (Jiang et al., 2009; Zhang et al., 1992). 4. Pharmacology There are 20 monoterpenes isolated from the essential oil of L. cubeba which have attracted the attention of researchers since their 4.1. Antimicrobial activity diverse biological activities, exemplified by antioxidative, antifungal, antiasthmatic, anti-anaphylactic properties, as well as function on The EtOH extract of the L. glutinosa leaves was evaluated for its the central nervous system (Chen et al., 2012; Chen, 2005; Gogoi et antibacterial effect in vitro against urinary tract infection causing al., 1997; Lu et al., 1988; Qian et al., 1980). Litsea verticillata is the pathogens, including Staphylococcus aureus, Pseudomonas aeruginosa, plant species containing the largest number of sesquiterpenes in Proteus mirabilis, Enterococcus faecalis and Escherichia coli using the this genus. So far, 31 sesquiterpenes, exemplified by 12–26, have disc diffusion assay method. The extract at the concentration of been isolated from L. verticillata (Hoang et al., 2002; H.J. Zhang et al., 250 μg/disc displayed good inhibition against these tested pathogens 2003; H. Zhang et al., 2003; Zhang et al., 2005). with the zones of inhibition ranging from 8.1 mm to 11.8 mm (Arora and Kaur, 2007). Mandal et al. (2000) have investigated antibacterial 3.3. Alkaloids effect of MeOH extract from the bark of L. glutinosa against 16 bacteria using the agar diffusion method. This MeOH extract inhibited both About 30 alkaloids have been isolated from these 20 Litsea Gram-positive and Gram-negative bacteria, with the zones of inhibi- species, covering aporphine, proaporphine, 1-benzylisoquinoline, tion in the range of 6.5–13.5 mm, which was comparable to the morphinane, phenanthrene, and dibenzopyrrocoline (Table S4 and positive control chloramphenicol. The EtOH extract of stem and bark Fig. S4). Alkaloids mainly exist in L. cubeba, and hitherto 23 alka- of Litsea populifolia demonstrated antibacterial activities against nine loids have been isolated from this species. In addition, L. glutinosa, pathogenic bacteria with the MIC values between 8 and 125 mg/mL. Litsea rotundifolia, L. rotundifolia var. oblongifolia and L. euosma are The antibacterial effect of L. populifolia was further confirmed by its reported to be the sources of alkaloids (Xiao et al., 2006a; Yan protective effect on the mice infected with S. aureus and E. coli in vivo et al., 2000; Yang et al., 2005). The aporphine alkaloids constitute (Chen and Xu, 2013). The essential oils from different parts of L. the biggest group of Litsea alkaloids, and 22 aporphine alkaloids cubeba, including roots, stems, leaves, flower buds, flowers, and fruits, have been found in this genus. A aporphine-type alkaloid, oxonan- were tested for their antibacterial activities against six bacteria using tenine (27), was recently isolated from L. cubeba (Yang et al., 2010). disc diffusion and micro-broth dilution assays. The zones of inhibition and MIC values for the tested strains were in the range of 10.1– 3.4. Butanolides and butenolactones 35.0 mm and 100–1000 mg/mL (Wang and Liu, 2010). These data supported the traditional use of Litsea species for the therapy of Fifteen butanolides and butenolactones have been found in three bacterial infection related diseases, such as diarrhea, gastritis and Litsea species (Table S5 and Fig. S5), including L. glutinosa (Agrawal turbid urine (Editorial Committee of Zhonghua Bencao National et al., 2013), L. rotundifolia var. oblongifolia (Zhao et al., 2005)and Traditional Chinese Herb Administration, 1999; Xie and Yu, 1996). L. verticillata (Zhang et al., 2005). Hydroxydihydrobovolide (28), The essential oils from the L. cubeba fruits or leaves have been 3-epilitsenolide D2 (29), 4-hydroxy-2-methylbut-2-enolide (30), lit- evaluated for their antifungal properties in vitro.TheL. cubeba leaves seabutenolide(31)wereisolatedfromtheleavesandtwigsof oil demonstrated potent inhibitory effects against eight skin patho- L. verticillata (Zhang et al., 2005). Recently, four butanolides (32–35) gens, including Crytococcus neoformans, Sporothrix schenckii, D.-G. Kong et al. / Journal of Ethnopharmacology 164 (2015) 256–264 261

Microsporum lanosum, Microsporum gypseum, Aspergillus niger, Asper- extract of L. glutinosa leaves inhibited edematous response with gillus flavus, Rhizopus nigricans,andChaetomium globosum,withMIC inhibition rates of 46%, 35%, and 43%, respectively, at a dose of values ranging 0.03–1.0 μg/mL (Wang et al., 1999). Tu et al. (1985) 500 mg/kg body weight in carrageenan, histamine and dextrin reported that 60% L. cubeba fruits oil emulsion completely inhibited induced rat paw edema assay. growth of nine fungi. The L. cubeba fruits oil was tested for antifungal TFLC, prepared from the diethyl ether partition of L. coreana effects against five Candida strains with MIC values of 14.2–76.2 μg/ leaves, has been evaluated for its anti-inflammatory properties (Chen mL in the broth microdilution method (Fang et al., 2002). The et al., 2004). Oral administration of TFLC at a dose of 100 mg/kg, antifungal effect in vivo of the L. cubeba fruit oil has been studied inhibited 17% xylene-induced ear edema response in rat. TFLC was using the mice infected with Candida albican. The result revealed that able to inhibit carrageenan-induced paw swelling and cotton ball- treatment with 40 mg/kg L. cubeba oil prolonged median survival induced granuloma in rat at doses of 133 and 266 mg/kg. A further time of infected mice and reduced the colony count in the kidney of study indicated that TFLC suppressed the expression of inflammatory mouse (Wan et al., 2006). markers and mediators, such as MDA, NO, PGE2 and TNF-α,and A series of compounds, including litseachromolaevane B (15-epi- inhibited protein levels of CHOP and GRP 78 in carrageenan-induced eudesm-4(15)-ene-1β,6β-diol (13), litseagermacrane (14), litseaverticil- rat paw edema model (Zhong et al., 2013). In adjuvant-induced lols A–H(16–23), isolitseane B (24), 1,2,3,4-tetrahydro-2,5-dimethyl-8- arthritis rat model, administration of TFLC, recovered secretory (1-methylethy)-l,2-naphthalenediol (25), oxyphyllenodiol B (26), verti- function of synoviocyte, suppressed IL-1β activity (Zhou et al., cillatol (15), hydroxydihydrobovolide (28), 3-epilitsenolide D2 (29), 2007), and inhibited the expression of protein MMP-9 (T.Y. Wang 4-hydroxy-2-methylbut-2-enolide (30), litseabutenolide (31), (þ)-epi- et al., 2007). These data supported that TFLC had a potential to be excelsin (36), and (þ)-50-demethoxyepiexcelsin (37), were isolated developed as an anti-inflammatory agent. from the leaves and twigs of L. verticillata, and subjected to the Roots of L. cubeba were used to cure humans suffering from evaluation for their anti-HIV effects. These compounds inhibited arthritis in traditional medicine (Liu, 1994; Zhang and Zhang, 1998). the replication of HIV in HOG.R5 cells with IC50 values from 2.0 to Oral administration of L. cubeba roots decoction relieved the swelling 34.5 μg/mL (Hoang et al., 2002; Zhang et al., 2001; H.J. Zhang et al., of ankle joint, and down-regulated TNF-α and IL-1β levels in collagen 2003; H. Zhang et al., 2003; Zhang et al., 2005). Of these, litseaverticillol induced rat rheumatoid arthritis model (Zhang and Di, 2008). B (17) is the most active compound (H. Zhang et al., 2003). These Four flavanocoumarins, isophyllocoumarin (8), isoepiphyllocou- findings provided potential leads for discovering new anti-HIV agents. marin (9), phyllocoumarin (10) and epiphyllocoumarin (11), were obtained from the leaves of L. coreana. Tang et al. (2013) have studied 4.2. Hepatoprotection their anti-inflammatory activity. These compounds (8–11) inhibited TNF-α and IL-1 production in lipopolysaccharides activated primary Total flavonoids of L. coreana (TFLC) mainly consist of seven mouse peritoneal macrophages at the doses of 0.2 and 0.02 mM. flavonoids, including kaempferol (1), kaempferol-3-O-β-D-galactoside (2), kaempferol-3-O-β-D-glucoside (3), quercetin-3-O-β-D-galactoside 4.4. Antiasthmatic activity (4), quercetin-3-O-β-D-glucoside (5), (2R,3S)-catechin (6) and (2R,3R)- epicatechin (7)(Wang et al., 2009; Ye et al., 2006). TFLC was evaluated Therapeutic effect of L. cubeba on asthma has been intimately for its prevention on steatohepatitis in rats fed with high fat diet known by local residents in many regions of China, and was (Wang et al., 2009). Oral administration of TFLC for 4 weeks dose- confirmed by guinea-pig models in vitro and in vivo. The essential dependently suppressed the levels of TG, TC, FFA and LDL-C, in serum oil of L. cubeba at a concentration of 90 μg/mL inhibited histamine and/or liver. Noteworthily, treatment with 400 mg/kg TFLC almost and/or acetylcholine induced guinea-pig tracheal smooth muscle blocked the formation of steatosis. Meanwhile, expression of PPARα, contraction in vitro (Qian et al., 1980). A further in vivo study which was a protein associated with increased SOD and decreased indicated that oral administration, intraperitoneal injection and MDA levels, up-regulated in TFLC-treated rat liver. Ni et al. (2006) inhalation of the essential oil of L. cubeba alleviated bronchial asthma reported that TFLC attenuated the increased level of MDA, and caused by histamine and/or acetylcholine in conscious guinea-pigs elevated SOD level in the fat emulsion-induced nonalcoholic steato- (Qian et al., 1980). Yin et al. (2006) have identified citral to be the hepatitis rat model. TFLC was also active in preventing alcoholic dominant component of the L. cubeba essential oil, and investigated steatohepatitis. After treatment with TFLC for 200 and 400 mg/kg, the its anti-asthma effect using two different guinea pig asthma models levels of ALT, AST, ALP, γ-GT, TG, TC, and MDA in serum and/or liver, induced by acetylcholine and ammonia, respectively. The results significantly reduced, while the activities of SOD and GSH-PX were demonstrated that citral was able to prolong incubation period and enhanced in EtOH and the fat emulsion-induced alcoholic steatohe- inhibit smooth muscle constriction induced by acetylcholine, as well patitis rat model (Y.Y. Wang et al., 2007). TFLC protected liver against as prolong the coughing incubation period and reduce coughing acute alcoholic hepatic injury in mice (Lv et al., 2010). In CCl4-induced frequency caused by ammonia. Above data displayed therapeutic hepatic fibrosis rat model, oral administration of TFLC at doses of 200 values of citral and the L. cubeba essential oil as anti-asthma agents. and 400 mg/kg, reduced the levels of ALT, AST, HA, LN, CIV and PIIINP, and inhibited mRNA expression of TGF-β1 and CTGF in liver tissue 4.5. Immunomodulation (Zhu et al., 2009). Cumulative evidences confirmed that TFLC exerted hepatopro- Arabinoxylan (51), a polysaccharide from the leaves of L. glutinosa tection against nonalcoholic and alcoholic steatohepatitis, hepatic stimulated the proliferations of splenocyte and thymocyte, and pro- fibrosis and acute alcoholic hepatic injury. The levels of TG, TC, FFA moted NO production in macrophages at tested concentration ranging and LDL-C in rat serum and/or liver significantly decreased by TFLC from 25 to 100 μg/mL (Das et al., 2013). Ni and Hong (2001) have treatment (Y.Y. Wang et al., 2007, 2009). All of these data suppor- studied immunomodulatory activity of polysaccharides from the roots ted the traditional use of L. coreana as a hypolipidemic drug in of L. pungens using cyclophosphamide-induced immunosuppressive southern China. mice models. Polysaccharides evidently enhanced immunomodulatory functions through increase of phagocytosis for macrophages, augment 4.3. Anti-inflammatory activity of the immune organ weights, and promotion of the lympho- cyte transformation rate. TFLC demonstrated similar function on Devib and Meera (2010) have investigated anti-inflammatory enhancement of immunity in cyclophosphamide-induced mice model activity of L. glutinosa using the rat paw edema model. The aqueous (Hu et al., 2007). Furthermore, productions of immunoglobulin M and 262 D.-G. Kong et al. / Journal of Ethnopharmacology 164 (2015) 256–264 immunoglobulin G, T lymphocyte cell CD4 þ and CD8þ ,aswellasthe 4.9. Miscellaneous bioactivities interleukin-2, a key modulator for T lymphocyte cell proliferation, were activated after oral administration with TFLC. L. glutinosa was evaluated for its osteoprotective effect using ovariectomized rat model (Parikh et al., 2009). ALP and TrACP have been identified to be biomarkers for osteoporosis. Oral adminis- 4.6. Anti-diabetes tration of food containing 5% the bark of L. glutinosa significantly inhibited ALP and TrACP in rat, and notably improved the quality The aqueous extract of L. coreana apparently reduced blood and micro-architecture of bone, which were benefit for bone glucose levels in adrenaline- and alloxan-induced rat diabetic models, remodeling. These results suggested L. glutinosa as a promising with the optimal doses of 100 and 300 mg/kg, respectively (Lv et al., phytomedicine for the therapy of osteoporosis. 2005). Later, Lv et al. (2008) investigated the hypoglycemic activity of The leaves of L. glutinosa were adopted for the treatment of TFLC, the active ingredients of L. coreana.TFLCdecreasedblood traumatic injury in traditional medicine (Editorial Committee of glucose levels in diabetic mice models induced by adrenaline and Zhonghua Bencao National Traditional Chinese Herb Administration, alloxan. Diabetic patients are commonly complicated with hyperli- 1999). Devib and Meera (2010) have evaluated its action on wound poidemia. Administration of TFLC evidently reduced the serum levels healing using excision and incision wound models in rat. Ointment ofTC,TG,andLDL-C,inthestreptozotocin-inducedratdiabeticmodel with 4% EtOH extract of L. glutinosa leaves significantly promoted (Lv et al., 2008; Sun et al., 2010), which was constituent with the wound healing by expediting wound contracting and increasing finding in hepatic injury (Lv et al., 2008; Y.Y. Wang et al., 2007, 2009). tensile strength. This effect was even comparable with that of the A further study using insulin resistance rat with hyperlipemia positive control 0.2% w/w nitrofurazone ointment. indicated that TFLC increased insulin sensitivity and improved the Using ceftriaxone sodium induced intestinal dysbiosis mice insulin resistance (Lv et al., 2009). model, the extracts of L. coreana were tested for their actions on ameliorating intestinal microbe dysbiosis (Wu et al., 2012). The results indicated that both aqueous and EtOH extracts were capable 4.7. Function on central nervous system of regulating and improving dysbiosis of intestinal flora. However, they bore different targets: the aqueous extract regulated the The essential oil of the L. cubeba fruits, used as spice in Taiwan, aerobes on the surface of intestinal canals, while the EtOH extract demonstrated neuropharmacological effect in ICR mice (Chen et al., coordinated the anaerobes in the depth of intestinal canal. Its 2012). Treatment with this essential oil at doses of 100, 300 and function on improvement of intestinal microbe dysbiosis might be 500 mg/kg extended pentobarbitone-induced mouse sleeping time by related with its traditional use for the therapy of diarrhea. 20%, 110%, and 159%, respectively compared with the control group. Citral, the dominant constituent of the L. cubeba oil, possessed Furthermore, oral administration of 500 mg/kg L. cubeba fruit oil antiarrhythmic function in vivo. Oral administration of 0.2 mL/kg citral significantly prolonged the reaction time of mice in the tail-flick test, relieved BaCl2- and aconitine-induced rat arrhythmia, and inhibited and its analgetic effect was comparable to that of the positive control CaCl2 and digoxin induced cardiac toxicity in rat (Cha et al., 1985). Hu acetaminophen at a dose of 90 mg/kg. This result is consistent with et al. (1988) have verified citral as a cardioprotective agent because of the use of L. cubeba fruits for relieving pain in traditional medicines its capacity of inhibiting platelet aggregation. This effect supported the (Editorial Committee of Zhonghua Bencao National Traditional Chinese traditional use of some Litsea plants on cardiovascular system. Herb Administration, 1999). TFLC has been evaluated for its neuropro- tective effect using a focal ischemia/reperfusion injury rat model. Oral administration of TFLC (25, 50 and 100 mg/kg) evidently alleviated 5. Toxicology cerebral ischemia-induced neurological deficits and reduced infarct volume. Specially, TFLC diminished the increased levels of nitrates plus The oil of L. cubeba has been evaluated for its acute toxicity nitrites, MDA and lactate dehydrogenase, and up-regulated the using mice by different research groups. Zhou et al. (1984) reduced levels of glutathione, superoxide dismutase and catalase reported that the 50% of lethal dose (LD50) for intragastric admin- activity in cerebral ischemia/reperfusion injury model. The potency istration was 3.25 mL/kg. Later, the LD50 for intraperitoneal injec- of TFLC at 100 mg/kg for intragastric administration was similar with tion was determined to be 381 mg/kg (Wan et al., 2006). The oil of that of positive control edaravone at 3 mg/kg for intraperitoneal L. cubeba is pungent for skin, leading to the skin inflammatory injection (Dong et al., 2013). These findings suggested that TFLC had response of guinea pig (Tu and Zhang, 1995). Intraperitoneal a potential on neuroprotection, which might be associated with its administration of the essential oil of L. glauca was poisonous to antioxidant activity (Ye et al., 2006). mice, with a LD50 value of 0. 62 mL/kg (Zhang et al., 1985).

4.8. Anticholelithogenic activity 6. Conclusions

The essential oil of L. cubeba fruits at the concentration of 10% The present review summarizes ethnomedical uses of the Litsea (w/w) demonstrated potent potency on dissolving both pigment species in TCM, and analyzes phytochemical and pharmacological calculus and cholesterol calculus in vitro, with the calculus dissolu- aspects of 20 medicinal plants from the genus Litsea in China. The tion ratios of 76% and 88%, respectively (Lu et al., 1988). Its antic- phytochemical results indicate a significant variety of structural types holelithogenic activity was further confirmed by experiment using of chemical constituents. Pharmacological studies indicated that these rcabbit implanted with gallstone in the gallbladder (Lu et al., 1989). plants and ingredients possessed various biological activities, especially Rabbits implanted with gallstone were treated with 2 mL/kg 10% in the areas of antimicrobial, hepatoprotection, anti-inflammatory, (w/w) the essential oil emulsion or 0.9% NaCl solution one time a antiasthmatic and anti-diabetes. To a certain extent, these data day for consecutive 14 days. It was observed that the dissolution validated the application of Litsea species in TCM, and provided the ratios were 73% for pigment calculus, and 69% for cholesterol evidences for the correlations between ethnomedical uses and bios- calculus, which were 7% and 2% in NaCl-treated group. These data cientific evaluations: ethnomedical uses for gastroenteritis, diarrhea indicated that the essential oil of L. cubeba fruits had a potential as and expelling parasite were related to antimicrobial effect; edema, anticholelithogenic agent. arthritis, enterogastritis, and mammitis to anti-inflammatory activity; D.-G. Kong et al. / Journal of Ethnopharmacology 164 (2015) 256–264 263 trauma and traumatic injury to antimicrobial and anti-inflammatory References activities. In addition, their traditional uses for the therapies of asthma, diabetes and pain have also been verified by bioassay in vivo.Regarding Agrawal, N., Choudhary, A.S., Sharma, M.C., Dobhal, M.P., 2011. Chemical constitu- the constituents contributed to therapeutic values, the findings are ents of plants from the genus Litsea. Chemistry & Biodiversity 8, 223–243. inadequate for survey. Significantly, TFLC has received more attention Agrawal, N., Pareek, D., Dobhal, S., Sharma, M.C., Joshi, Y.C., Dobhal, M.P., 2013. Butanolides from methanolic extract of Litsea glutinosa. Chemistry & Biodiver- since its biological functions on anti-inflammatory,anti-diabetic,neu- sity 10, 394–400. roprotective, and hepatoprotective aspects, and correspondingly is Arora, D.S., Kaur, G.J., 2007. Antibacterial activity of some Indian medicinal plants. – regarded to be substances responsibleforitsethnomedicalusesfor Journal of Natural Medicines 61, 313 317. fl Cha, Z.-L., Gao, J.-H., Zhan, J.-P., 1985. The experimental anti-arrhythmia effect of diabetes and in ammation-related diseases. The toxicity of Litsea citral. Zhongguo Yao Fang 5, 435–436. species is limited according to the reported data, and commonly Chen, C.-J., Tseng, Y.-H., Chu, F.-H., Wen, T.-Y., Cheng, W.-W., Chen, Y.-T., Tsao, N.-W., Wang, S.-Y., 2012. Neuropharmacological activities of fruit essential oil from possessed LD50 values higher than 300 mg/kg (or 0.6 mL/kg for Litsea cubeba Persoon. Journal of Wood Science 58, 538–543. essential oil) for mice in the acute toxic test. And comprehensive Chen, C., 2005. Study on the antioxidation of Litsea cubeba oil. Food Research and toxicological investigations of these plants are required in the future. Development 26, 155–158. Because of the limited distribution of the Litsea plants in the Chen, J., Zhu, C.-H., Xu, H.-Y., Ni, X., Yang, P.-M., 2010. Study on chemical constituents of the root of Litsea cubeba II chloroform portion and ethyl acetate Southern and Southwest China, the so far ethnomedical uses of these portion from methanol extract. Chinese Journal of Pharmaceuticals 41, plants are restricted, and have not been widely recognized by the 504–508. residents in the north region of China. Even the representative, ‘Bi- Chen, L., Cheng, W.-M., Hu, C.-M., Jin, Y., Li, R., Li, J., 2004. Study on anti- fl fl Cheng-Qie’, a traditional medicine record in Chinese Pharmacopoeia in ammatory effects of total avonoids of Litsea coreana Leve. var. Anhui Yi Ke Da Xue Xue Bao 39, 439–442. (2010 edition), is not extensively prescribed by the TCM doctors. Chen, Y.-Z., Xu, Z.-L., 2013. The antibacterial activity of the extract of Litsea Although great progresses on the phytochemistry and pharmacology populifolia bark. Asia-Pacific Traditional Medicine 9, 28–29. of the genus Litsea have been made, there are still some areas needed Cheng, M.C., Cheng, Y.S., 1983. Compositions of the essential oils of Litsea kostermanin Chang, Litsea gerciae Vidal and Litsea cubeba (Lour.) persoon. to be explored to gain a better understanding of this genus. (i) Most of Journal of the Chinese Chemical Society 30, 59–62. the pharmacological studies are conducted using crude and poorly Choudhury, S., Singh, R., Ghosh, A.C., Leclercq, P.A., 1996. Litsea glutinosa (Lour.) C.B. characterized extracts of Litsea species; however, the pharmacological Rob., a new source of essential oil from northeast . Journal of Essential Oil fi fi Research 8, 553–556. researches of identi ed compounds would be of particular signi - Das, D., Maiti, S., Maiti, T.K., Islam, S.S., 2013. A new arabinoxylan from green leaves cance. Hence, more bioactive components should be identified of Litsea glutinosa (Lauraeae): structural and biological studies. Carbohydrate using bioactivity-guided isolation strategies, and then their possible Polymers 92, 1243–1248. fl mechanisms of action targeting on ethnomedical uses need to be Devib, P., Meera, R., 2010. Study of antioxdant, antiin ammatory and woundhealing activity of extracts of Litsea glutinosa. Journal of Pharmaceutical Sciences and illustrated. (ii) Some traditional uses of the Litsea species have Research 2, 155–163. been validated by modern pharmacological investigations; however, Dong, S.-Y., Tong, X.-H., Li, J., Huang, C., Hu, C.-M., Jiao, H., Gu, Y.-C., 2013. Total fl plenty of ethnomedical applications were only confirmed by cell- avonoid of Litsea coreana leve exerts anti-oxidative effects and alleviates focal cerebral ischemia/reperfusion injury. Neural Regeneration Research 8, based bioassay in vitro, and thus further investigations in vivo using 3193–3202. laboratory animals are required to certify their therapeutic effects. For Editorial Committee of Zhonghua Bencao National Traditional Chinese Herb instance, seven Litsea species were adopted for the therapy of pain Administration, 1999. Zhonghua Bencao. Shanghai Scientific and Technical Publishing House, Shanghai. 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