Leonurus japonicus Houtt.: ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine.

Journal of Ethnopharmacology 152 (2014) 14–32

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Review

Leonurus japonicus Houtt.: Ethnopharmacology, phytochemistry and pharmacology of an important traditional Chinese medicine Xiaofei Shang a, Hu Pan a,n, Xuezhi Wang a, Hua He b, Maoxing Li c,n a Key Lab of New Animal Drug Project, Gansu Province, Key Laboratory of Veterinary Pharmaceutics Discovery, Ministry of Agriculture, Lanzhou Institute of Animal Science and Veterinary Pharmaceutics, Chinese Academy of Agricultural Sciences, Lanzhou 730050, PR China b Department of Pharmacy, Wenzhou Medical University, Wenzhou 325035, PR China c Department of Pharmacy, Lanzhou General Hospital of PLA, Key Laboratory of the Prevention and Treatment for Injury in Plateau of PLA, Lanzhou 730050, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 22 September 2013 Received in revised form 30 December 2013 Accepted 30 December 2013 Available online 8 January 2014

Ethnopharmacological relevance: Leonurus japonicus Houtt. (Labiatae), commonly called Chinese mother), is an herbaceous flowering plant native to Asia. For thousands of years in China, the aerial wort ( part of Leonurus japonicus has been used to treat menoxenia, dysmenorrhea, amenorrhea, lochia, edema of the body, oliguresis, sores, ulcerations and other diseases in women. Now, Leonurus japonicus is listed in the Pharmacopoeia of the People0 s Republic of China. The present paper reviewed the ethnopharmacology, phytochemistry, biological actions and toxicology of Leonurus japonicus. Materials and methods: Information on Leonurus japonicus was gathered via the Internet (using Elsevier, ACS, Medline Plus, CNKI, VIP, Web of Science, Google Scholar and Baidu Scholar) and libraries. Results: Approximately 140 chemical compounds have been isolated from Leonurus japonicus, and the major components have been determined to be alkaloids, diterpenes and flavones. Among these active compounds, the effects of leonurine and stachydrine have been widely investigated. The primary active components in Leonurus japonicus possess wide pharmacological actions, such as effects on the uterus as well as cardioprotective, anti-oxidative, neuroprotective and anti-cancer activities. Conclusions: Modern pharmacological studies have demonstrated that Leonurus japonicus has marked bioactivities, especially on the uterus and as a cardioprotective agent. These activities are related to its traditional use and provide prospects for the development of novel drugs, therapeutics and health care products for women. However, the toxicity of Leonurus japonicus will require further study, and the nomenclature for Leonurus japonicus will require additional clarification. Crown Copyright & 2014 Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: Leonurus japonicus Women0 s diseases Leonurine Stachydrine Effects on the uterus Cardioprotective activity

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Botany and ethnopharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Botany . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Ethnopharmacology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

15 15 15 16

Abbreviations: ABTS, 2, 20 -Azino-bis (3-Ethylbenzothiazoline-6-Sulfonic Acid); ACh E, Acetyl Cholinesterase; ALT, Alanine Transarninase; AMI, acute myocardial infarction; ANP, Atrial Natriuretic Peptide; AP-1, Activator Protein 1; AST, Aspartate Amino Transferase; ATPase, Adenosine triphosphatase; bFGF, Basic Fibroblast Growth Factor; BNP, Brain Natriuretic Peptide; BPH, benign prostatic hyperplasia; BUN, Blood Urea Nitrogen; CAT, Catalase; CCl4, carbon tetrachloride; CK, Casein Kinase; CPK, creatine phosphate kinase; DAD, Diode-Array Detection; DNA, Deoxyribonucleic Acid; 7 dp/dtmax, Left Ventricular Pressure Maximum Rate of Change; ECM, extracellular matrix; EGF, Epidermal Growth Factor; ER, estrogen receptor; ESI, Electronic Spray Ion; ET, Endothelin; GnRH, Gonadotropin Releasing Hormone; GPx, glutathione peroxidase; GSH, glutathione; HPLC, High Performance Liquid Chromatography; HPTLC, High Performance Thin Layer Chromatography; IC50, 50% Maximal Inhibitory Concentration; IGF-1, Insulin-like Growth Factor-1; LD50, Median Lethal Dose; LDH, Lactate Dehydrogenase; LN, Laminin; LPS, Lipopolysaccharide; LVSP, left ventricular systolic pressure; MBC, minimum bactericidal concentration; MCAO, middle cerebral artery occlusion; MDA, malondialdehyde; MHC, Myosin Heavy Chain; MIC, minimum inhibitory concentration; MPO, Myeloperoxidase; MS, Mass Spectrometer; MSMC, myometrial smooth muscle cells; MTT, Methyl Thiazolyl Tetrazolium; NGF, Nerve Growth Factor; NO, Nitric Oxide; ONOO , Peroxynitrite; PGE2, Prostaglandin E2; ROS, reactive oxygen species; SOD, superoxide dismutase; T, Testosterone; TCM, traditional Chinese medicine; TEAC, Trolox Equivalent Antioxidant Capacity; TGF-β1, Transforming growth factor-β1; TIMP-1, Tissue-Inhibitor of Metalloproteinase-1; TLC, Thin Layer Chromatography; TNF-α, tumor necrosis factor-α; UCP4, Uncoupling Protein 4; UPLC, Ultra Performance Liquid Chromatography n Corresponding authors. Tel.: þ 86 931 2115261; fax: þ 86 931 2115191. E-mail address: [email protected] (X. Shang). 0378-8741/$ - see front matter Crown Copyright & 2014 Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2013.12.052

X. Shang et al. / Journal of Ethnopharmacology 152 (2014) 14–32

15

3.

Phytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.1. Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.2. Diterpenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 3.3. Flavones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.4. Spirocyclic nortriterpenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.5. Sesquiterpene glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.6. Megastigma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.7. Phenylethanoid glycosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.8. Nonapeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.9. Essential oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.10. Others. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 4. Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 4.1. Effects of chemical compounds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 4.1.1. Leonurine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 4.1.2. Stachydrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.2. Effects of crude extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.2.1. Effects on the uterus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.2.2. Cardioprotective activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4.2.3. Anti-oxidative activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.2.4. Anti-cancer activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.2.5. Neuroprotective activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.2.6. Analgesic and anti-inflammatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.2.7. Anthelminthic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 4.2.8. Antibacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 4.2.9. Others. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 5. Analysis of active constituents and quality control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 6. Toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

1. Introduction Leonurus japonicus Houtt. (Labiatae), commonly called Chinese motherwort, is an herbaceous flowering plant native to several regions in Asia, including China, Korea, Japan and Cambodia. It has escaped cultivation to become naturalized in other parts of the world, including South and North America as well as Europe and Africa (http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl? 407691). For thousands of years in China, the aerial part of Leonurus japonicus has been primarily used to treat menoxenia, dysmenorrhea, amenorrhea, lochia, edema of body, oliguresis, sores, ulcerations and other diseases in women and was thus named “Yi Mu Cao” (Chinese: ), meaning literally “beneficial herb for mothers.” Since 1990, Leonurus japonicus has been listed in the Pharmacopoeia of the People0 s Republic of China, and more than 300 prescriptions containing Leonurus japonicus have been used to treat various diseases in China, especially those specific to women (Cai, 2005). Modern pharmacological studies show that the active components in Leonurus japonicus possess wide pharmacological actions, such as effects on the uterus as well as cardioprotective, anti-oxidative, anti-cancer, analgesic, antiinflammatory, neuroprotective and antibacterial actions. Most of these actions are consistent with those for Leonurus japonicus in traditional medicine. Because of the marked effects on women0 s health and diseases, researchers began focusing attention on Leonurus japonicus, extensively studying its chemical components. Alkaloids, diterpenes, flavones, spirocyclic nortriterpenoids, phenylethanoid glycosides, sesquiterpene glycosides, essential oils and other compounds were the main components isolated and studied. Among them, stachydrine has been the most studied and shows the best bioactivity. According to the Pharmacopoeia of China, stachydrine is now used as the official indicator to monitor the quality of the herb and of

the preparations with Leonurus japonicus (Committee for the pharmacopoeia of P.R. China, 2010). In this review, we examine the advances in the ethnopharmacology, phytochemistry, pharmacology and toxicology of Leonurus japonicus as well the increasing data that support the utilization of Leonurus japonicus as a novel drug. Considering that Leonurus japonicus has many synonyms (http://www.theplantlist.org), we use Leonurus japonicus as the name of the plant throughout this review, except in those instances where we indicate a synonym has been used.

2. Botany and ethnopharmacology 2.1. Botany The Leonurus japonicus plants are either annual or biennial, growing from taproots with dense and fibrous rootlets. The stems

OO N+ H 3C

CH 3

Stachydrine Fig. 1. The flower of Leonurus japonicus and chemical structure of stachydrine.

16


Table 1 The preparations, in which Leonurus japonicus was the main compositions, listed in Chinese Pharmacopoeia and approved by governmenta. Preparation name

Main compositions

Usage

Yi Mu Cao Gao

Herb Leonuri

Promoting blood circulation to restore menstrual flow. Treating menoxenia induced by blood stasis and few menstruations Promoting blood circulation to restore menstrual flow. Treating menoxenia induced by blood stasis and few menstruations Uterotonic. Regulating menstruation and hemostasis

Yi Mu Cao Kou Herb Leonuri Fu Ye Yi Mu Cao Zhu Herb Leonuri She Ye Yi Mu Cao Pian Herb Leonuri Yi Mu Wan

Herb Leonuri, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix Aucklandiae

Ba Zhen Yi Mu Wan

Herb Leonuri, Radix Paeoniae Alba, Rhizoma Atractylodis Macroce phalae, Rhizoma Chuanxiong, Radix Angelicae Sinensis, Radix Codonopsis Pilosulae, Poria, Radix Glycyrrhizae, Radix Rehmanniae Herb Leonuri, Radix Angelicae Sinensis, Rhizoma Chuanxiong, Radix Paeoniae Alba, Radix Rehmanniae, Radix Aucklandiae

Fu Fang Yi Mu Cao Gao Jia Wei Yi Mu Cao Gao Fu Fang Yi Mu Cao Liu Qin Gao Fu Kang Ning Pian

Herb Leonuri, Radix Angelicae Sinensis, Radix Rehmanniae, Radix Paeoniae Alba, Rhizoma Chuanxiong Herb Leonuri, Radix Rehmanniae, Radix Angelicae Sinensis

Kang Gong Yan Pian Tong Jing Wan

Herb Leonuri, Herba Callicarpa, Radix Linderae

Herb Leonuri, Radix Paeoniae Alba, Rhizoma Cyperi, Radix Angelicae Sinensis, Radix Notoginseng, Folium Artemisiae Argyi, Radix ophiopogonis, Radix Codonopsis Pilosulae

Herb Leonuri, Radix Angelicae Sinensis, Radix Paeoniae Alba, Rhizoma Chuanxiong, Radix Rehmanniae, Rhizoma Cyperi, Fructus Pericarpium, Fructus Crataegi, Rhizoma Corydalis, Rhizoma Zingiberis, Cortex Cinnamomi, Radix Salviae Miltiorrhizae, Fructus Leonuri, Flos Carthami, Faeces Trogopterorl, Radix Aucklandiae Kun Ning Ke Li Herb Leonuri, Radix Angelicae Sinensis, Radix Paeoniae Alba, Radix Salviae Miltiorrhizae, Radix Curcumae, Radix Achyranthis Bidentatae, Fructus Aurantii, Herba Schizonepetae, Rhizoma Zingiberis, Radix Rubiae, Radix Aucklandiae Jia Wei Ba Zhen Herb Leonuri, Radix Ginseng, Poria, Rhizoma Atractylodis Macroce phalae, Yi Mu Gao Radix Angelicae Sinensis, Radix Paeoniae Alba, Rhizoma Chuanxiong, Radix Rehmanniae, Flos Carthami, Radix Salviae Miltiorrhizae, Herba Lycopi, Radix Glycyrrhizae, etc. Fu Fang Yi Mu Herb Leonuri, Radix Rehmanniae, Radix Angelicae Sinensis, Radix Paeoniae Yang Shen Alba, Rhizoma Chuanxiong, Fructus Corni, Mudanpi, Radix Morindae Officinalis, Kou Fu Ye Herba Epimedii, Radix Salviae Miltiorrhizae, Radix Ginseng, Rhizoma Anemarrhenae, etc. Xian Yi Mu Cao Fresh Herb Leonuri Jiao Nang Qian Lie Tai Jiao Herb Leonuri, Herba Polygonum, Flos Carthami, Bee pollen, Rhizoma Nang Anemarrhenae, Cortex phellodendri a

Promoting blood circulation to restore menstrual flow. Treating the few menstruations Regulating menstruation and nourishing blood. Treating Qi regurgitating and blood stasis, menstrual abdominal pain, leucorrhea, sore waist and tired, dizzy and tinnitus induced by cold in blood and blood-deficiency Invigorating vital energy and regulating menstruction. Treating Qi and blood deficiency of women, physically weak and few menstruations Regulating menstruation and nourishing blood. Treating menoxenia, abdominal pain in the period of menorrhea induced by stagnation of Qi and blood Regulating menstruation and nourishing blood. Treating menoxenia and few menstruations Regulating menstruation and nourishing blood, removing blood stasis. Treating menoxenia, insufficiency of uterus resetting after childbirth, lochia Nourishing blood and regulating vital energy, promoting blood circulation to restore menstrual flow. Treating menoxenia induced blood-deficiency few menstructions, abdominal pain in menstrual period Expelling dampness. Treating leucorrhea Warming meridians and activating blood circulation, regulating menstruation and analgesia. Treating dysmenorrheal, menoxeni induced by blood stasis., etc.

Activating blood circulation and promoting Qi, hemostasis andregulating menstruction. Treating menorrhagia and menostaxis induced by Qi-stagnancy and blood stasis Invigorating vital energy and regulating menstruction, removing blood stasis and regulating menstruation. Treating insufficiency of vital energy and blood, menoxenia, menstruction retrocession, etc. Nourishing liver and kidney, nourishing blood and regulating vital energy. Treating burn sensation of five centers, soreness and weakness of waist and knees, dizzy, amnesia and insomnia in female change of life Uterotonic, regulating menstruction. Treating menoxenia, postpartum metrorrhagia, uterus meromorphosis Clearing away heat evil and promoting dieresis, activating blood circulation and eliminating stagnation. Treating chronic prostatitis

Cited from ‘Chinese Pharmacopoeia’ and the Website: http://www.sda.gov.cn.

are upright, growing to a height from 30 to 120 cm and are approximately 5 mm in diameter. The main stem, filled with marrow, and the 4 prismatic stems are covered in fine hair-like filaments. The flowers are sessile and are produced in verticillasters. The calyx is tubular-campanulate shaped and 6–8 mm long with broad triangular-shaped teeth. The corolla is white or reddish to purplish-red. The flowering period is from June to September, and the plants bloom from July to October (Fig. 1) (http://frps. eflora.cn, Cai, 2005). In China, Leonurus japonicus mainly grows along the roadsides, hillsides and in fields in most regions with an altitude of 0–3000 m. Different regions have provided various names for Leonurus japonicus, such as Kuncao or Yimuhao (in the Jilin and Liaoning provinces), Yema or Tianzhima (Jiangsu), Sanjiaohuma (Zhejiang), Qinghao (Sichuan province), Qiancengta (Qinghai) and Tougucao (Yunnan province). In addition, Leonurus artemisia (Lour.) S.Y. Hu, Leonurus heterophyllus Sweet and Leonurus sibiricus auct. pl. and Stachys artemisia Lour. were thought to be synonyms

of this species. Now, Leonurus japonicus has become distributed in other regions, including Cambodia, Japan, Korea, Laos, Malaysia, Myanmar, Thailand, and Vietnam as well as in some countries of Africa, North America and South America (http://frps.eflora.cn). Because of its beauty and long flowering period, Leonurus japonicus also has been used as an ornamental plant in some regions. 2.2. Ethnopharmacology Due to its marked effect in treating blood diseases and diseases stemming from multiparity, Leonurus japonicus played an important role in traditional Chinese medicine (TCM) and was considered for thousands of years a superior medicine for treating women0 s diseases. In Sheng Nong Ben Cao Jing, the oldest classical medicinal book, Leonurus japonicus was listed as “Top grade.” In Ben Cao Gang Mu, the most famous medicinal book in China, Leonurus japonicus was thought to be innocuous and useful for the treatment of vaginal bleeding during pregnancy, dystocia, retained


17

Table 2 The compounds isolated from Leonurus japonicus. (The structure of main compounds illustrated in Fig. 2.) No. Compounds Alkaloids 1 Leonuridine 2

Leonurine

3 4

Leonurinine Stachydrine

Diterpenes 5 Ballatenol 6 Leosibirin 7 Isoleosibirin 8 Leosibiricin 9 Galeopsin 10 Hispanolone

Synonym

References

Leonurus heterophyllus Leonurus Artemisia Leonurus sibiricus Leonurus artemisia Leonurus heterophyllus

Luo et al. (1985)

Savona et al. (1982) Savona et al. (1982) Savona et al. (1982) Savona et al. (1982) Narukawa et al. (2014) Hon et al. (1993)

11

Leoheterin

12

Prehispanolone

13

Preleoheterin

14

Leopersin G

15 16 17

15,16-Epoxy-3α-hydroxyllabda-8, 13(16), 14-trien-7-one Leojaponin 13-Epi-preleoheterin

Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus japonicus Leonurus heterophyllus Leonurus japonicus Leonurus heterophyllus Leonurus japonicus Leonurus japonicus Leonurus japonicus

18

Iso-preleoheterin

Leonurus japonicus

19 20

15,16-Epoxy-3α,6β,9α-trihydroxylabda-13(16),14-dien-7-one Heteronone A

21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Sibiricinone A Sibiricinone B Sibiricinone C Sibiricinone D Sibiricinone E 15-Epi-sibircinone D 15-Epi-sibircinone E Leosibrinone A Leosibirinone B Preleosibirone A 13-Epi-preleosibirone A Isopreleosibirone A 15-Epi-leosibirone B 3α-Acetoxyleoheteronone A 3α-Acetoxyleoheteronone C 3α-Acetoxyleoheteronone E 3α-Acetoxy-15-epileoheteronone E 7β-Hydroxy-3α-acetyl-9α,13; 15,16-bicyclicepoxy-15α-ethoxylabdane-6-ketone

39

7β-Hydroxy-3α-acetyl-9α,13; 15,16-diepoxy-15α-methoxylabdane-6-ketone

40

6β,13-Dihydroxy-15,16-epoxy-15α-ethoxy-8,9-enelabdane-6-ketone

41

3α,7β-Hydroxy-9α,13; 15,16-bicyclicepoxy-15α-ethoxylabdane-6-ketone

42

3α,7β-Hydroxy-9α,13; 15,16-bicyclicepoxy-15β-ethoxylabdane-6-ketone

43

6β,9α-Dihydroxy-3β-acetyl-15,16-cyclicepoxy-14,15;13,16-dienelabdane-7-ketone

44

9α, 13R; 15,16-Diepoxy-labdane-14-en-7-one

45 46

3α-Acetoxy-15-O-methylleopersin C Leoheteronin F

47

Leoheteronin A

48

Leoheteronin D

Leonurus japonicus Leonurus heterophyllus Leonurus japonicus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus japonicus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus sibiricus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus japonicus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus

Yeung et al. (1997) Hayashi (1962) Yeung et al. (1997) Luo et al. (1985)

Hon et al. (1993) Ming et al. (1991) Xu et al. (1992) Hon et al. (1993) Xu et al. (1992) Hung et al. (2011) Khan et al. (2012) Moon (2010) Romero-González et al. (2006) Romero-González et al. (2006) Moon (2010) Fuchino et al. (2013) Zhang and Peng (2004) Cai (2005) Boalino et al. (2004) Boalino et al. (2004) Boalino et al. (2004) Boalino et al. (2004) Boalino et al. (2004) Boalino et al. (2004) Boalino et al. (2004) Seo et al. (2010) Moon et al. (2010) Wu et al. (2011) Wu et al. (2011) Wu et al., (2011) Wu et al. (2011) Moon et al. (2010) Moon et al. (2010) Moon et al. (2010) Moon et al. (2010) Gong (2011) Gong (2011) Gong (2011) Gong (2011) Gong (2011) Gong (2011) Hon et al. (1991) Seo et al. (2010) Hung et al. (2011) Hung et al. (2011) Hung et al. (2011)

18


Table 2 (continued ) No. Compounds

Synonym

49 50 51 52 53 54 55 56 57 58 59 60 61 62

Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus

[þ ]-Hispanone [þ ]-6β-Hydroxy-15,16-epoxylabda-8,13(16),14-trien-7-one [ ]-6,9:15,16-Diepoxy-9α-hydroxy-8,9-seco-13(16),14-labdaiene-7-one [ ]-8ξ-Acetoxy-15,16-epoxy-8,9-seco-13(16),14-labdadiene-7,9-dione 6β,9α-Dihydroxy-15,16-epoxy-13(16),14-labdadien-7-one 15,16-Epoxy-8,17-dinor-9-oxo-7,9-seco-13(16),14-labdadien-7-oic acid 6,9:15,16-Diepoxy-6-hydroxy-6,7-seco-13(16),14-labdadien-7-oic acid 6β,15ξ-Dihydroxy-7-oxo-8,13-labdadien-15,16-olide LS-1a LS-2a Leonotinin Leonotin Dubiin Nepetaefuran

Flavones 63 Rutin

64 65 66 67

Wogonin 5,7,30 ,40 ,50 -Pentamethoxy-flavone Apigenin-7-O-β-D-glucopyranoside Tiliroside

68 69 70 71 72 73 74 75 76 77 78

Quercetin Isoquercetin (Quercetin-3-O-D-glucoside) Quercetin-3-neohesperidoside Quercetin-3-O-rutinoside Kaempferol-3-O-β-D-glucopyranoside Kaempferol-3-O-β-D-galactopyranoside Kaempferol-3-O-β-D-robinobinoside Kaempferol-3-neohesperidoside Kaempferol-3-O-β-D-glucopyranoside-7-O-α-L-rhamnoside Kaempferol-7-O-α-L-rhamnoside Genkwanin

79 80 81 82 83 84 85 86 87 88 89 90 91

Quercetin-3-O-robinobioside Isoquercitrin Hyperoside Apigenin 2‴-Syringylrutin Nicotiflorin Cosmosiin Quercetin 3-O-[(3-O-syringoyl-α-L-rhamnopyranosyl)-(1-6)-β-D-glucopyranoside] Leonurusoide A Leonurusoide B Leonurusoide C Leonurusoide D Leonurusoide E

Spirocyclic nortriterpenoids 92 Leonurusoleanolide A

References sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus sibiricus

Leonurus heterophyllus Leonurus japonicus Leonurus japonicus Leonurus japonicus Leonurus japonicus Leonurus japonicus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus Leonurus

japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus sibiricus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus japonicus

Narukawa et al. (2014) Narukawa et al. (2014) Narukawa et al. (2014) Narukawa et al. (2014) Narukawa et al. (2014) Narukawa et al. (2014) Narukawa et al. (2014) Narukawa et al. (2014) Satoh et al. (2003) Satoh et al. (2003) Satoh et al. (2003) Satoh et al. (2003) Satoh et al. (2003) Satoh et al. (2003) Zhang et al. (2009) Cai (2005) Cai (2005) Cai (2005) Cai (2005) Cong et al. (2003) Seo et al. (2010) Tao et al. (2009) Seo et al. (2010) Deng et al. (2013) Deng et al. (2013) Deng et al. (2013) Deng et al. (2013) Deng et al. (2013) Deng et al. (2013) Tao et al. (2009) Tao et al. (2009) Boalino et al. (2004) Seo et al. (2010) Cong et al. (2009) Cong et al. (2009) Cong et al. (2009) Cong et al. (2009) Seo et al. (2010) Seo et al. (2010) Seo et al. (2010) Chang et al., (2010) Zhang et al. (2013) Zhang et al. (2013) Zhang et al. (2013) Zhang et al. (2013) Zhang et al. (2013)

Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus japonicus Leonurus japonicus

Liu et al. (2012)

Sesquiterpene glycosides 98 7α(H)-eudesmane-4,11 (12)-diene-3-one-2β-hydroxy-13-β-D-glucopyranoside 99 ( )-(1Sn,2Sn,3Rn)-3-Ethoxycupar-5-ene-1,2-diol 100 ( )-(1Sn,4Sn,9Sn)-1,9-Epoxybisabola-2,10-diene-4-ol 101 Arteannuin B

Leonurus Leonurus Leonurus Leonurus

japonicus japonicus japonicus japonicus

Li et al. (2012) Xiong et al. (2013b) Xiong et al. (2013b) Xiong et al. (2013b)

Megastigma 102 9-Hydroxy-megastigma-4,7-dien-3-one-9-O-glucopyranoside 103 Staphylionoside E 104 Megastigmane 105 Citroside A

Leonurus Leonurus Leonurus Leonurus

japonicus japonicus japonicus japonicus

Li et al. (2012) Li et al. (2012) Cong et al. (2003) Li et al. (2012)

93

Leonurusoleanolide B

94

Leonurusoleanolide C

95

Leonurusoleanolide D

96 97

Leonujaponin A Phlomistetraol B

Phenylethanoid glycosides 106 Lavansulifolioside, 107 Leonoside E

Leonurus heterophyllus Leonurus japonicus

Liu et al. (2012) Liu et al. (2012) Liu et al. (2012) Zheng et al. (2012) Zheng et al. (2012)

Zhang et al. (2009) Li et al. (2012)


19

Table 2 (continued ) No. Compounds

Synonym

References

108 Leonoside F 109 Verbascoside 110 2-(3,4-dihydroxyphenethy)-O-α-arabinopyranosyl-(1-2)-α-L-rhamnopyranosyl-(1-3)-6-O-β-Dglucopyranoside 111 Cistanoside E

Leonurus japonicus Leonurus japonicus Leonurus japonicus

Li et al. (2012) Li et al. (2012) Cai (2005) Li et al. (2012) Li et al. (2012)

112 Lavandulifolioside

Leonurus japonicus

113 Isolavandulifolioside

Leonurus japonicus

Nonapeptides 114 Cycloleonuripeptide A 115 Cycloleonuripeptide B 116 Cycloleonuripeptide C 117 Cycloleonuripeptide D 118 Cycloleonuripeptide E 119 Cycloleonuripeptide F Others 120 Benzoic acid

121 Salicylic acid 122 Syringic acid

123 Adenosine

124 Stigmasterol

125 2,6-Dimethyl-2E,7-octadiene-1,6-diol 126 β-Sitosterol 127 Daucosterol 128 129 130 131 132 133

(2S,5S)-2-Hydroxy-2,6,10,10-tetramethyl-1-oxaspiro[4.5]dec-6-en-8-one 3-Oxo-α-ionone ( þ)-Dehydrovomifoliol ( þ)-3-Hydroxy-β-ionone Chamigrenal β-Sitosterol glucopyranoside

134 Aurantiamide acetate 135 Auraptenol 136 137 138 139 140 a

Heterolignan Chlorogenic Caffeic Ferulic Cichoric acid

Leonurus japonicus

Cai (2005) Li et al. (2012) Cai (2005)

Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus

Morita et al. (1997)

Leonurus heterophyllus Leonurus japonicus Leonurus heterophyllus Leonurus heterophyllus Leonurus japonicus Leonurus heterophyllus Leonurus japonicus Leonurus heterophyllus Leonurus japonicus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus japonicus Leonurus japonicus Leonurus japonicus Leonurus japonicus Leonurus japonicus Leonurus heterophyllus Leonurus heterophyllus Leonurus heterophyllus Leonurus japonicus Leonurus japonicus Leonurus japonicus Leonurus japonicus Leonurus japonicus

Zhang et al. (2009)

Morita et al. (1997) Morita et al. (1997) Morita et al. (1997) Morita et al. (2006) Morita et al. (2006)

Cai (2005) Zhang et al. (2009) Zhang et al. (2009) Cong et al. (2003) Zhang et al. (2009) Cai (2005) Zhang et al. (2009) Cai (2005) Cong (2004) Cong (2004) Cong (2004) Xiong et al. (2013b) Xiong et al. (2013b) Xiong et al. (2013b) Xiong et al. (2013b) Xiong et al. (2013b) Liu et al. (2012) Liu et al. (2012) Liu et al. (2012) Cai (2005) Kuchta et al. Kuchta et al. Kuchta et al. Kuchta et al.

(2012a) (2012a) (2012a) (2012a)

The structures of two compounds were explained in Fig. 2.

fetal membranes, bruising, metrorrhagia, metrostaxis, hemuresis and other diseases. Since its listing in Ben Cao Gang Mu, Leonurus japonicus has been recorded in virtually all classical medicinal books in China (Cai, 2005). The aerial part of Leonurus japonicus has been listed in the Pharmacopoeia of the People0 s Republic of China since 1990. It is now listed in China as one of the 50 fundamental herbs used in traditional Chinese medicine. More than 15 preparations, in which Leonurus japonicus was the main and active component, were listed in the Chinese Pharmacopoeia and approved by the State Administration of Traditional Chinese Medicine of the People0 s

Republic of China, such as “Yi Mu Cao Gao” and “Ba Zhen Yi Mu Wan”, which were widely used as therapies in women to promote blood circulation and restore menstrual flow, induce dieresis and reduce edema, and clear the heat-evil and expel superficial evils (Table 1) (Committee for the pharmacopoeia of P.R. China, 2010, http://www.satcm.gov.cn). Chen et al. (2010a) reviewed the records of women who had received Chinese herbal medicine therapies for menopausal symptoms at the Traditional Medicine Center of Veterans General Hospital in Taipei between January 2003 and December 2006. Their results showed that the most frequently prescribed single Chinese herb was Yi-Mu-Tsao

20


(Leonurus japonicus). However, at the same time, it was forbidden in the clinics of China to treat diseases in pregnant women because of the potential effect on the uterus. Leonurus japonicus has also been employed in health care products with other medicines or food to improve the body and prevent illness in the women of China. For example, after decocting 1 h, the herb Leonuri (30–60 g) with rhizoma Corydalis Yanhusuo (20 g) and two eggs could be used to prevent and treat menalgia. Another concoction with the herb Leonuri, rhizoma Zingberis Recens, fructus Jujubae and brown sugar could be used to treat postpartum abdominal pain, etc. In addition, Leonurus japonicus has an excellent effect in cosmetology and halts the progression of caducity. In the Tang dynasty, Zetian Wu, the only woman emperor in the history of China, commonly used a cosmetic made from Leonurus japonicus to maintain her skin and to provide rejuvenation. Currently, the plant is used in cosmetics and other beauty products, such as facial masks or ointments, as well as in food products along with other materials to activate blood circulation and to treat skin diseases (Deng, 2010).

15,16-epoxy-8,17-dinor-9-oxo-7,9-seco-13(16),14-labdadien-7-oic acid (54), 6,9:15,16-diepoxy-6-hydroxy-6,7-seco-13(16),14-labdadien-7-oic acid (55), [þ ]-6β-hydroxy-15,16-epoxylabda-8,13(16),

3. Phytochemistry Currently, approximately 140 chemical components have been isolated and identified from Leonurus japonicus, including alkaloids, diterpenes, flavones, phenylethanoid glycosides, and essential oils (Table 2). The chemical structures of the primary compounds are shown in Fig. 2. 3.1. Alkaloids Although alkaloids are the main chemical compounds and active components in Leonurus japonicus, to date, only four alkaloids have been identified and studied: leonuridine (1), leonurine (2), leonurinine (3) and stachydrine (4). Of these, leonurine (2) and stachydrine (4) are the most studied, have the best bioactivities and have been used to monitor the quality of the herb and preparations of Leonurus japonicus in the Pharmacopoeia of China. 3.2. Diterpenes Since 1982, four diterpene compounds (ballatenol (5), leosibirin (6), isoleosibirin (7) and leosibiricin (8)) have been isolated from Leonurus sibiricus (Savona et al., 1982). Currently, more than 50 diterpene compounds have been isolated and identified from Leonurus japonicus, and all are labdane diterpenoids. These compounds can be sorted to five types based on the pentacyclic ring on C12: a furan ring, dihydrofuran ring, lactone ring, α, β-undersaturated lactone ring and tetrahydrofuran ring. Many of these chemical compounds have shown marked bioactivities in vivo or in vitro. In 2004, seven new labdane diterpenes, sibiricinones A-E (21–25) and 15-epi-sibiricinones D (26) and E (27), were isolated from the aerial part of Leonurus sibiricus (Boalino et al., 2004). Then, in 2010, six new bis-spirolabdane-type diterpenoids were isolated from the aerial part of Leonurus sibiricus: leosibrinone A (28), leosibrinone B (29), 3α-acetoxyleoheteronone A (34), 3αacetoxyleoheteronone C (35), 3α-acetoxyleoheteronone E (36) and 3α-acetoxy-15-epileoheteronone E (37) (Moon et al., 2010). Khan et al. (2012) isolated and identified 15,16-epoxy-3α-hydroxylabda8, 13(16), 14-trien-7-one (15) from Leonurus japonicus. Narukawa et al. (2014) isolated twelve diterpenoid compounds from the acetone extract of the aerial part of Leonurus sibiricus, and studied the estrogen sulfotransferase inhibitory activity of these compounds. Their results demonstrated that the IC50 of

Fig. 2. The chemical structure of the main compounds from Leonurus japonicas.


14-trien-7-one (50), [þ ]-hispanone (49) and [ ]-6,9:15,16-diepoxy-9α-hydroxy-8,9-seco-13(16),14-labdadiene-7-one (51) were 2.9, 23.2, 31.3, 19.8 and 40.5 mm, respectively. Satoh et al. (2003) studied and isolated six furanoditerpene-lactones from Leonurus sibiricus: LS-1 (57), LS-2 (58), leonotinin (59), leonotin (60), dubiin (61) and nepetaefuran (62). Their results demonstrated that these compounds exhibited moderate cytotoxic activity (IC50 ¼50– 60 μg/ml) against leukemia cells (L1210) in tissue culture. 3.3. Flavones To date, approximately 30 flavones from Leonurus japonicus have been isolated and identified. Cai (2005) isolated four compounds: rutin (63), wogonin (64), 5,7,30 ,40 ,50 -pentamethoxy-flavone (65) and apigenin-7-O-β-D-glucopyranoside (66). Then, Deng et al. (2013) isolated and identified quercetin-3-neohesperidoside (70), quercetin-3-O-rutinoside (71), kaempferol-3-O-β-D-glucopyranoside (72), kaempferol-3-O-β-D-galactopyranoside (73), kaempferol-3-O-β-D-robinobinoside (74) and kaempferol-3neohesperidoside (75). Cong et al. (2009) isolated eight chemical constituents from Leonurus heterophyllus and studied their antitumor activity against the leukemia K562 cell line. Their results using an MTT viability assay indicated that the IC50 for quercetin3-O-robinobioside (79), rutin (63), isoquercitrin (80), hyperoside (81), quercetin (68), apigenin (82) and benzoic acid (120) against K562 cells was 17.29, 21.56, 21.67, 24.12, 6.3, 10.4 and 116.5 mg/l, respectively, whereas the IC50 for the positive control drug adriamycin was 1.48 mg/l. Zhang et al. (2013) isolated five new syringyl acylated flavonol glycosides from the aerial part of Leonurus japonicus, leonurusoides A (87), B (88), C (89), D (90) and E (91), and suggested that these compounds inhibited triglyceride accumulation in free fatty acid-induced HepG2 cells.

3.4. Spirocyclic nortriterpenoids In 2012, four new spirocyclic nortriterpenoids, leonurusoleanolide A (92), B (93), C (94) and D (95), were isolated from the methanol extract of the fruit of Leonurus heterophyllus, and they were found to exist as equilibrium mixtures of trans and cis isomers. The results of pharmacological tests demonstrated that a mixture of 92 and 93 and a mixture of 94 and 95 at concentrations of 1, 10, and 30 μM promoted neurite outgrowth in NGFtreated PC12 cells (Liu et al., 2012). Meanwhile, two new C-28 nortriterpenoids leonujaponin A (96) and phlomistetraol B (97) were isolated and identified from the fruit of Leonurus japonicus (Zheng et al., 2012).

3.5. Sesquiterpene glycosides In 2012, a new sesquiterpene glycoside from the aerial part of Leonurus japonicus was isolated, namely, 7α (H)-eudesmane4, 11 (12)-diene-3-one-2β-hydroxy-13-β-D-glucopyranoside (98) (Li et al., 2012). At the same time, ( )-(1Sn, 2Sn, 3Rn)-3-ethoxycupar-5-ene-1, 2-diol (99), ( )-(1Sn, 4Sn, 9Sn)-1, 9-epoxybisabola-2, 10-diene-4-ol (100) and arteannuin B (101) were isolated from Leonurus japonicus (Xiong et al., 2013b).

3.6. Megastigma To date, four megastigma compounds have been isolated from Leonurus japonicus: 9-hydroxy-megastigma-4, 7-dien-3-one-9-Oglucopyranoside (102), staphylionoside E (103), megastigmane (104) and citroside A (105) (Cong et al., 2003; Li et al., 2012).

21

3.7. Phenylethanoid glycosides Li et al. (2012) isolated two new phenylethanoid glycosides from the aerial part of Leonurus japonicus, leonoside E (107) and leonoside F (108), These compounds exhibited potent hepatoprotective activity against D-galactosamine-induced toxicity in HL7702 cells at a concentration of 1 10 5 M. At the same time, verbascoside (109), 2-(3,4-dihydroxyphenethy)-O-α-arabinopyranosyl-(1-2)-α-L-rhamnopyranosyl-(1-3)-6-O-β-D-glucopyranoside (110), cistanoside E (111) and lavandulifolioside (112) were also isolated and identified. 3.8. Nonapeptides In 1997, cycloleonuripeptides A, B, C, D (114–117), four new proline-rich cyclic decapeptides, were isolated from Leonurus heterophyllus (Morita et al., 1997). Then, in 2006, cycloleonuripeptides E (118) and F (119), as cyclic nonapeptides, were isolated and identified from Leonurus heterophyllus(Morita et al., 2006). 3.9. Essential oils Xiong (2013a) analyzed and studied the chemical composition of the essential oils from the herb (Yimucao) and the fruit (Chongweizi) of Leonurus japonicus with GC–MS and its antibacterial activity with micro-dilution assays. The results of GC–MS demonstrated that “Yimucao oil” was mainly composed of diterpenes (32.77%) and sesquiterpenes (45.37%), with phytone (19.02%), phytol (13.75%), caryophyllene oxide (11.49%) and β-caryophyllene (9.89%) being the most significant constituents. Other sesquiterpenes present in appreciable amounts were spathulenol (5.31%), α-caryophyllene (3.38%) and isocaryophyllene (3.00%). The primary structures belonged to the caryophyllane, aromadendrane and cadinane classes. Additionally, monoterpene compounds were present. “Chongweizi oil” was characterized by large amounts of aliphatic compounds (43.04%), including 12 aliphatic hydrocarbons, two aldehydes and five esters. Among them, aliphatic hydrocarbons constituted 32.7%, followed by nhexadecane (9.65%), n-tridecane (7.72%) and n-pentadecane (4.36%), most of which were not detected in “Yimucao oil.” In the aliphatic compounds, the monoterpene fraction was noteworthy (15.11%), with bornyl acetate (7.33%) and bornyl acrylate (1.81%) as the main constituents, which were also absent in “Yimucao oil.” With regard to the sesquiterpenes and diterpenes in “Chongweizi oil,” the fractions were relatively small when compared to “Yimucao oil,” accounting for only 10.45% and 8.45%, respectively. The results of a microdilution assay indicated that “Yimucao oil” had antibacterial activity against various Gram-positive bacteria. In particular, β-caryophyllene had wide-spectrum activity against Gram-positive bacteria with minimum inhibitory concentrations (MICs) from 0.032 to 0.256 mg/ ml and minimum bactericidal concentrations (MBCs) from 0.064 to 0.256 mg/ml. By contrast, the oil of “Chongweizi” was inactive in the antibacterial assay. 3.10. Others In 2005 and 2009, benzoic acid (120), salicylic acid (121), syringic acid (122), adenosine (123) and stigmasterol (124) were isolated from Leonurus heterophyllus and Leonurus japonicus (Cai, 2005; Zhang et al., 2009). Also identified from Leonurus heterophyllus were 2, 6-dimethyl-2E, 7-octadiene-1, 6-diol (125), β-sitosterol (126) and daucosterol (127) (Cong, 2004). Xiong et al. (2013b) isolated five compounds from Leonurus japonicus: (2S,5S)2-hydroxy-2,6,10,10-tetramethyl-1-oxaspiro[4,5]dec-6-en-8-one (128), 3-oxo-α-ionone (129), (þ)-dehydrovomifoliol (130), (þ)-3hydroxy-β-ionone (131) and chamigrenal (132). At the same time, Kuchta et al. (2012a) determined the phenolic compounds of

22


Leonurus japonicus using HPLC and HPTLC methods: chlorogenic acid (137), caffeic acid (138), ferulic acid (139), cichoric acid (140), hyperoside (81), rutoside (63) and apigenin-7-O-D-glucoside (66).

4. Pharmacology 4.1. Effects of chemical compounds Pharmacological studies have demonstrated that many of the chemical compounds in Leonurus japonicus show good bioactivities in vivo or in vitro. The alkaloids of this plant especially show marked cardioprotective effects as well as effects on the uterus. The different chemical components of Leonurus japonicus will provide a good foundation for further development. In Table 3, we listed the active compounds of Leonurus japonicus and their bioactivities, except for leonurine and stachydrine, which are discussed in detail below. 4.1.1. Leonurine The bioactivities of leonurine, such as the effect on the uterus and the cardioprotective effects, continue to be discovered and investigated. First, Yeung et al. (1997) isolated leonurine from Leonurus artemisia and suggested that it had uterotonic properties on the isolated rat uterus. Then, Li (2009) studied the effect and mechanism of action for leonurine on incomplete abortions induced by mifepristone (8.3 mg/kg) and misoprostol (100 mg/kg) in the early stages of pregnancy in rats. Their results showed that compared with the model group leonurine at three concentrations (0.5, 1.5 and 4.5 mg/kg) decreased the weight (compare 0.89 g for the model group with 0.56, 0.39 and 0.41 g, respectively, for the three concentrations of leonurine) and the index related to the weight of uterus (model, 0.0032; leonurine, 0.0019, 0.0014 and 0.0015), reduced the bleeding volume (model, 0.29 ml; leonurine, 0.28, 0.26 and 0.20 ml) and bleed time (model, 100.5 h; leonurine,

56, 53 and 51 h), and increased the uterine contraction rate (model, 6.62/10 min; leonurine, 9.12, 9.38 and 9.62/10 min), uterine tension (model, 4.50 g; leonurine, 5.32, 6.15 and 6.28 g), uterine contractility (model, 30.10 /g; leonurine, 49.1, 57.62 and 60.42 /g) and the level of estradiol (model, 27.85 pg/ml; leonurine, 61.23, 63.99 and 71.83 pg/ml) in the sera of rats. The results from the histopathological examination suggested that the uterine tissue from the leonurine group was improved (P o0.01). Further study showed that leonurine increased uterine contractions and up-regulated GnRH protein in the hypothalamus and α ER and β ER mRNA in uterine tissue. Leonurine also decreased LN protein, elevated serum E2 and ET/NO in uterine tissue (Po0.01), and improved the hypothalamus-pituitary-ovarian axis function. Liu (2009) conducted a comprehensive in vivo and in vitro study examining the cardioprotective effects and mechanisms of action of leonurine isolated from Leonurus japonicus using oxidatively stressed H9c2 cells as well as rat models of hypoxic neonatal cardiomyocytes, acute myocardial infract and heart failure. Their results showed that leonurine had direct cardioprotective effects mediated via antioxidants. In addition, leonurine (10 6 mol/l) lowered intracellular calcium overload, up-regulated Bcl2 gene expression and inhibited mitochondrial apoptosis by blocking Bax protein translocation from the cytoplasm to mitochondria (P o0.05). Leonurine also protected against myocyte injury caused by hypoxia and ischemia induced by ROS (Po 0.05). Liu (2011) reported that leonurine had neuroprotective effects in an experimental ischemic stroke induced in male adult rats by a permanent middle cerebral artery occlusion (MCAO). In addition, compared to the sham group, leonurine (7.5 and 15 mg/kg) protected the ischemic cortex through increasing SOD, CAT, UCP4 and Bcl2 activities and decreasing Bax and MDA expression (Po0.05). Cheng et al. (2010) studied the effects of a combination of leonurine and stachydrine against acute myocardial ischemia in mice. After injecting isoproterenol to induce a mouse model of acute myocardial ischemia, leonurine and stachydrine were

Table 3 The activities of some compounds from Leonurus japonicus. Compounds

Synonym

Effects

In vitro

Reference

Arteannuin B

Leonurus japonicus Leonurus japonicus

Antibacterial activity Antibacterial activity

Xiong et al. (2013a)

15, 16-Epoxy-3αhydroxylabda-8, 13(16), 14-trien-7-one

Leonurus japonicus

Anti-inflammatory activity

Leonoside E Leonoside F Verbascoside Cistanoside E Leojaponin

Leonurus japonicus

Hepatoprotective activity

It showed antibacterial activity against Escherichia coli and Enterobacter aerogenes with the MIC values of 25 μg/ml and 50 μg/ml It showed antibacterial activity against three Gram-positive strains, including Macrococcus caseolyticus, Staphylococcus auricularis and Staphylococcus aureus (MIC 25, 50, and 200 μg/ml, respectively). It showed marked anti-inflammatory activity, and it could suppress LPSinduced on the inflammatory mediators, i.e., iNOS, COX-2, and TNF-α secretion, via the inactivation of the NF-κB, MAPK, and Akt signaling pathways It exhibited hepatoprotective activity against D-galactosamine-induced toxicity in HL-7702 cells at concentration of 1 10 5 M. And the inhibitions were 30%, 30.9%, 24.4% and 31.7% compare to Bicyclol 25.3%

Leonurus japonicus

Cytoprotective activity

(2S,5S)-2-Hydroxy-2,6,10,10tetramethyl-1-oxaspiro[4.5] dec-6-en-8-one

Leonurus japonicus

Against platelet aggregation

Prehispanolone

Leonurus Platelet activating heterophyllus factor receptor antagonist

Leoheteronin A Leopersin G

Leonurus Cholinesterase heterophyllus inhibitory activity

Chamigrenal

15,16-Epoxy-8,17-dinor-9-oxo- Leonurus sibiricus 7,9-seco-13(16),14labdadien-7-oic acid

Estrogen sulfotransferase inhibitory activity

It exhibited significant cytoprotective activities against glutamate-induced toxicity, exhibiting cell viability of about 50%, at concentrations ranging from 0.1 μm to 10 μm It inhibited abnormal increase of platelet aggregation induced by ADP at a concentration of 10 μm, with the maximum aggregation ratio of 42.0 7 15.63% (Po 0.01). While the ratio of the blank control was 61.4 7 9.44% It inhibited the binding of [3H]-platelet activating factor (PAF) to rabbit platelets with potencies closely resembling that of inhibition of PAFinduced aggregation, and the integrity of the tetrahydrofuran ring of prehispanolone is critical for its interaction with the PAF receptor Leoheteronin A and leopersin G showed cholinesterase inhibitory activity in a dose dependent manner with IC50 values of 11.6 and 12.9 mm, respectively. Tacrine was used as a positive control with an IC50 value of 170.2 nm It showed the strongest activity with an IC50 value of 7.9 mm, which is comparable to the activity of the positive control, meclofenamic acid (IC50 5.4 mm)


Khan et al. (2012)

Li et al. (2012)

Moon (2010)


Lee et al. (1991)

Hung et al. (2011)

Narukawa et al. (2014)


administered orally for 7 days. Compared to the model group, the combination groups (leonurine 2.5 mg/kg þstachydrine 5 mg/kg and leonurine 5 mg/kg þstachydrine 10 mg/kg) markedly inhibited the change in the T wave and reduced the content of MDA and the activity of LDH. This combination therapy also ameliorated the pathological damage associated with the myocardial ischemia induced by isoproterenol. These results suggested that the combination of leonurine and stachydrine has a marked protective effect against acute myocardial ischemia in mice. Chao et al. (2006) suggested that leonurine and stachydrine could be used as a moderate diuretic, retaining potassium. After orally administering 2.5 mg/100 g to rats, stachydrine increased the excretion of urine significantly more than leonurine did, with the effect of both alkaloids peaking in 2 h (3.565 and 2.825 ml, respectively). The analysis of ions in the urine indicated that both stachydrine and leonurine increased the secretion of Na þ (3097 and 3120 ppm, respectively) and Cl (69.99 and 69.87 mg/ml), whereas the levels of K þ were decreased (1452 and 1310 ppm, respectively).

4.1.2. Stachydrine Because stachydrine is used to monitor the quality of Leonurus japonicus, its activities have been widely studied. The effect of stachydrine intervention on the hypertrophy of myocardial cells in neonate rats induced by norepinephrine was investigated in 2010 (Zhao et al., 2010). The results demonstrated that along with the extended treatment time with stachydrine (10 4, 10 5 and 10 6 M), the surface area, protein/DNA ratio and ANP of myocardial cells were markedly increased (P o0.01). By contrast, the content of BNP in the supernatant from cell cultures was decreased (Po 0.05). These results suggested that stachydrine can inhibit myocardial cell hypertrophy induced by norepinephrine in a time-dependent manner. To study the mechanism for this action, the anti-hypertrophy activity was examined by evaluating the levels of ROS and NF-κB in myocardial cell hypertrophy induced by angiotensin II in rats. After treating with stachydrine, the number of ROS-positive myocardial cells was decreased and the level of p-IκB (ser 32) protein in the cytosol and NF-κB (p65) protein in the nucleus were suppressed. Stachydrine showed marked antihypertrophic effects by inhibiting the NF-κB signaling pathway (P o0.05) (Guo et al., 2012). Shan et al. (2013) studied the inhibitory effects of stachydrine from Leonurus heterophyllus on the expression of a fetal gene in the cardiac myocyte hypertrophy induced by norepinephrine. In this test, neonatal ventricular cardiomyocytes were isolated from rats (1–2 days old) using collagenase type II enzyme. The cardiomyocytes were treated with norepinephrine (1 μmol/l) for 72 h in the presence or absence of carvedilol (positive control, 1 μmol/l), or stachydrine (10 μmol/l). The results showed that stachydrine attenuated cardiomyocyte hypertrophy by abrogating the changes induced by norepinephrine, and significantly decreased the cell area, protein content, protein/DNA ratio, β-MHC expression and β/α-MHC ratio in cultured cardiomyocytes (P o0.05). Qin et al. (2013) studied the effects of leonurine and stachydrine on rat uterine contractions in vitro. After treatment with leonurine (0.03, 0.06 and 0.09 mg/ml), the extent of the uterine smooth muscle contraction induced by oxytocin was significantly reduced compared to that before the treatment (Po0.01), and the frequency of contractions was reduced. By contrast, after treatment with stachydrine (0.18 mg/ml), the extent of the uterine smooth muscle contraction induced by oxytocin was significantly increased compared to that before treatment (Po 0.01), and the frequency of contractions was increased. Therefore, the authors suggested that leonurine can inhibit the contraction of rat uterine smooth muscle in a contraction model induced by oxytocin in vitro, but stachydrine can enhance the contraction. The authors

23

also demonstrated that stachydrine promoted the protein expression of IL-12 and IL-6 as well as the mRNA expression of T-bet and RORγt, while inhibiting the mRNA expression of GATA-3 and Foxp3. Therefore, the Th1/Th2/Th17/Treg paradigm in mice subjected to an RU486-induced abortion shifted to Th1 and Th17 after stachydrine administration. In addition, administering stachydrine significantly reduced the volume of uterine bleeding during the RU486-induced abortion (Po 0.01) (Li et al., 2013). 4.2. Effects of crude extract The effects of the crude extract of Leonurus japonicus are shown in Table 4. 4.2.1. Effects on the uterus As a famous folk medicine in China, Leonurus japonicus was historically used primarily to treat some diseases in women, such as menoxenia, dysmenorrhea, amenorrhea, lochia, edema of the body, etc. These therapies all indicated that this medicine might directly target the uterus or that its activity may be associated with the uterus. Therefore, researchers focused their investigations of the effect of Leonurus japonicus on the uterus and on the mechanisms of this action. 4.2.1.1. In vivo test. By investigating the degree and frequency of uterine contractions induced by oxytocin in guinea pigs, the dysmenorrheal effect of Leonurus artemisia was studied. After administering an aqueous extract of the aerial part of Leonurus artemisia (10 g/kg), the frequency as well as the degree (from 1.75 g to 2.84 g; P o0.05) of uterine contractions in normal guinea pigs increased; however, the oxytocin- or F2α-induced writhing reaction in mice, auricular edema in mice and inflammation of the uterus in rats were reduced or alleviated (P o0.05). In addition, the contractions induced by PGF2α (1.592 ng/10 g compared to the control group 2.134 ng/10 g) and the absorbance of PGE2 (21.0 compared to the control group 60.1) in uterine smooth muscle and the increased serum progesterone level in rats were reduced by Leonurus artemisia (P o0.05). Thus, these authors suggested that Leonurus artemisia may play an important role in treating dysmenorrhea by relaxing uterine spasms, decreasing inflammation, reducing PGF2α and PGE2 concentrations in uterine smooth muscle and increasing the serum progesterone level (Jin et al., 2004). Xu et al. (2007) reported that an injection of Leonurus heterophyllus (20 mg/ml, total alkaloids from the aerial part, Chengdu First Pharmaceutical Company, China) had a marked effect on uterine contraction and hemostasis during cesarean section and after surgery. In 2009, the effect of Leonurus heterophyllus and the mechanism mediating its reduction in the bleeding of the tunica intima in endometritis of postpartum rats were investigated (Ye, 2009). After treating with the aqueous extract of the aerial part of Leonurus heterophyllus (1.8 g/kg), compared to the model group (55.84 pg/ml and 2854.33 mg/l), the levels of tumor necrosis factor-α (TNF-α; 45.66 pg/ml) and transforming growth factor-β1 (TGF-β1; 1313.13 mg/l) in the postpartum inflamed uterus and the expression of TIMP-1, all of which function to stop bleeding and start healing, were reduced (Po 0.05). In addition, the degradation of the extracellular matrix (ECM) and postpartum uterine involution were accelerated. 4.2.1.2. In vitro test. After culturing human myometrial smooth muscle cells (MSMCs) in vitro, the effect of the aqueous extract of the aerial part of Leonurus heterophyllus on the proliferation, apoptosis and expression of calponin in MSMCs induced by LPS was studied. The results showed that the cytoactivity after treating

24

Table 4 The activities of crude extract from Leonurus japonicus. Synonym Effect on uterus Leonurus artemisia

Parts/extracts

Doses

Aqueous extract of the aerial part

10 g/kg

1 ml (20 mg/ml) 1.8 g/kg

Leonurus heterophyllus Aqueous extract of the aerial part

0.2, 2, 20, 200 mg/ml

Leonurus heterophyllus Alkaloids of the aerial part Cardioprotective activity Leonurus heterophyllus Flavones and alkaloids extracts of the aerial part

100 μl (1 ml/0.02 g)

Leonurus heterophyllus Alkaloids extracts of the aerial part

60, 30 and 6 g/kg

Leonurus heterophyllus Total alkaloids of the aerial part

1.8 mg/100 g b.w. (20 mg/ml)

2 ml/100 g and 0.5 ml/100 g (1 ml equal to 2 g raw material)

After treating with the medicine, the cytoactive was significantly lower than LPS group (Po 0.05), but the apoptosis was higher. Meanwhile, the expression of Calponin was significantly lower than the normal group and LPS group (Po 0.01) It has the marked effects on uterine muscle with enhancement rates of intensity and motoricity 63% and 109%

In vivo

References

Jin et al. (2004) After administrating the extract, the degree and frequency of uterine constriction of normal guinea pigs were increased from 1.75 g to 2.84 g (Po 0.05), and the twisting reaction induced by oxytocin or 15 M-PGF2α and the auricular edema in mice and inflammation of uterus in rats were reduced and alleviated (Po 0.05). Meanwhile, PGF2α (1.595 ng/10 g compared to control group 2.134 ng/10 g) and PGE2 (21.0 compared to control group 60.1) in uterine smooth muscle and increased serum progesterone level in rats were reduced (P o0.05) It has the marked effect on uterine contraction and hemostasis Xu et al. (2007) during cesarean section and after operation Ye (2009) After treating with the medicine, compared to the model group (55.84 pg/ml and 2854.33 mg/l), the levels of TNF-α (45.66 pg/ml), TGF-β1 (1313.13 mg/l) on postpartum inflammatory uterine, and the expression of TIMP-1 to stop bleeding and start the repair were reduced (P o0.05). Meanwhile, the degradation of ECM and speed up post-partum uterine involution were accelerated Zeng (2007)

Cong (2004)

After administering orally the extracts for 7 days, compared the Li et al. (2006) model group, J point have not been marked moved in the assay of II lead electrocardiogram, and the left ventricular systolic pressure (LVSP) and the left ventricular pressure changing rate of maximum ( 7 dp/dtmax) have been recovered compared to normal group (Po 0.05). Meanwhile, the activities of CPK and lactic LDH in blood serum of rats were inhibited (Po 0.05). And the level of MDA were reduced and the activity of SOD was increased in the cardiac muscle tissue (P o 0.05) After administrating for three weeks, compared with the normal Jiang et al. (2006) group and sham operation group, the acute myocardial infarction model group was increased in left ventricular end diastolic pressure (Po 0.05), but the maximal rate of rise and the left ventricular pressure were decreased and fall. And middle dose (30 g/kg) of the alkaloid is better than high (60 g/kg) and low doses (6 g/kg). That is because that the high dose of alkaloid might be induced the toxicity of body at equal pace to treat the diseases After injecting, it could improve renal function in renal ischemia Zheng (2004) reperfusion injury and diminished the renal tubule damage (Po 0.05). And this activity may be related to scavenge oxygen free radicals, reduce lipid peroxidation, increase SOD and ATPase activity, and attenuate Ca2 þ overload and NO content (Po 0.05). The further study showed that the levels of CK and MDA in the plasma of Leonurus heteropyllus and verapamin (positive drug) group were lower than the model group (Po 0.05)


Leonurus heterophyllus Total alkaloids of the aerial part Leonurus heterophyllus Aqueous extract of the aerial part

In vitro

Anti-oxidative activity Leonurus heterophyllus Aqueous extract of the aerial part

Leonurus heterophyllus Flavones extract of the aerial part Anti-cancer activity Leonurus japonicus Ethanol extract of aerial part

400 mg/kg/day

0.5%, 1%,1.5%, 2% and 2.5%

It displayed the stronger scavenging effect on ONOO and inhibitory effect on lipid peroxidation than ascorbic acid and Trolox (P o 0.01). Meanwhile, it has the marked capacity to scavenge ABTS þ radicals, the average TEAC values were 562% and 588% when ascorbic acid and Trolox were taken as standards, respectively

Sun et al. (2005a) After administering orally the extract from 1 week before and continuing until 3 weeks after myocardial infarction, the drug group has a higher survival rate (55.4%) than rats in the model group (40.5%). However, the anti-oxidant effects were exerted only under the condition of oxidative stress by selectively preserving the activities of superoxide dismutase and glutathione peroxidase, as well as depressing the formation of MDA (Po 0.05), especially in the acute phase of acute myocardial infarction (Po 0.01) It the marked activity on scavenging of hydroxyl radicals with the Luo and Huang dose-dependent manner (5–25% inhibition) (2006)

Neuroprotective activity Leonurus heterophyllus Alkaloid extract of aerial 3.6, 7.2, part 14.4 mg/kg

Analgesic and anti-inflammatory activity Leonurus sibiricus Methanol extract of the aerial part

Antibacterial activity Leonurus sibiricus

Liang et al. (2011) Compared with model group, the extracts (7.2 and 14.4 mg/kg) could significantly decrease neurological deficit scores and reduce the infarct volume on rats with focal cerebral ischemic injury and the MPO content in ischemic brain (Po 0.05). Meanwhile, it could significantly decrease the NO level compared with the model group (P o0.05) at the concentration of 14.4 mg/kg. In addition, the extract significantly decreased the apoptosis ratio of nerve fiber compared with the model group (Po 0.05) Islam et al. (2005) After injecting intraperotoneally the extract, it showed a significant analgesic effect in acetic acid-induced writhing in mice with the inhibition 69.68% and 44.15%, respectively. And the positive drugs the diclofenac sodium displayed the inhibition 74.67%. Meanwhile, after giving orally to rats the extract (400 and 200 mg/kg), it showed a significant anti-inflammatory activity against carrageenin induced rat paw edema in rats (Po 0.05)

500, 250 mg/kg, and 400, 200 mg/kg

Anthelminthic activity 100 mg/kg Leonurus heterophyllus Methanol extract, petroleum extract, dichloromethane extract and propanone extract Leonurus artemisia Aqueous extract of the aerial part

500 mg/disc Carbon tetrachloride, chloroform, acetone and methanol extracts of the aerial part

Tao et al. (2009)

The methanol extract (100%) has the best anthelminthic activity than petroleum extract (60%), dichloromethane extract (55.7%) and propanone extract (0%) by inhibiting the activity of Car E and Ach E. And it could be used as nerve toxicant due to this activity After treating to Oncomelania hupensis for 1–2 days, the activity of esterase isozyme were higher than the compared group, and reached the highest level at 3–4 days. Whereas dealt for 5 days, the activity of esterase remarkably reduced, and some new enzyme belt appeared and some original enzyme belt vanished Carbon tetrachloride and chloroform extracts had a broad spectrum of antibacterial activity. And at 500 mg/disc, the zone of inhibitions of the CCl4 and chloroform extracts against Staphylococcus aureus, S. epidermis, Streptococcus pyogenes, Escherichia coli, Vibrio cholera, Shigella dysenteriae and Shigella boydii were 20, 20, 30, 25, 13, 16, 17 and 19, 14, 28, 20, 15, 17 and 19 mm, respectively. The positive drugs kanamycin (30 mg/disc) was 30, 32, 40, 39, 30, 34 and 38 mm

Xu et al. (1994)


It caused cell death in a dose-dependent and time-dependent fashion in both positive and negative breast cancer cells. IC50 for suppression of cell proliferation at 24 h and 48 h were 96.2, 89.1, and 67.7, 53.4 mg/ml for MCF-7 and MDA-MB231 cells, respectively. And low concentrations of ethanol extract caused cell cycle arrest at G2/M phase. Meanwhile, morphology, hoechst 33342 staining and flowcytometry evidence all indicated the cell death is not in an apoptotic nature

Zhang et al. (2007)

Ahmed et al. (2006)

25

Moon (2010) It inhibit glutamate-induced toxicity in primary cultured rat cortical cells with cell viability 18.4%, 29.4% and 52.4% at the concentration of 0.1, 1 and 20 mm Glutamate-induced toxicity Leonurus japonicus Methanol extract of aerial part

The protective effect of benign prostatic hyperplasia Leonurus japonicus Total alkaloids of the 19, 37.5 and aerial part from 75 mg/kg

Synonym

Table 4 (continued )

Parts/extracts

Doses

In vitro

In vivo

Gao (2010) After administrating the extract, the prostate exponent and the content of T, DHT, and the expression of bFGF, EGF, IGF-1 were reduced (Po 0.05), but the expression of TGF-β1 and TGF-β1/bFGF which could restrain BPH were increased in model mice or rats. Meanwhile, the prostate ultrastructure and relieve the histological anomaly of the model animals0 prostate constitution were improved by the total alkaloids (Po 0.05)


References

26

with Leonurus heterophyllus þLPS group was significantly lower than that in the LPS-treated group (Po 0.05), but apoptosis was increased. In addition, the expression of calponin in the Leonurus heterophyllus- and stachydrine-treated groups was significantly lower than that in the normal group and the LPS-treated group (P o0.01). These results indicated that the promotion of uterine involution by Leonurus heterophyllus may be mediated by reducing abnormal proliferation and down-regulating the expression of calponin in MSMCs and that stachydrine may be the active compound (Zeng, 2007). The effect of six different extracts (70% ethanol extract, acetic ether extract, 60% ethanol extract dissolved in water, 60% ethanol extract in ethanol, 30% ethanol extract and alkaloids) of Leonurus heterophyllus on uterine muscle contraction in rats was also studied in vivo. The results showed that the alkaloid extract had the strongest effects on the uterine muscle, with enhancements in the intensity and rate of contractility by 63% and 109%, respectively (Cong, 2004).

4.2.2. Cardioprotective activity Except for the uterus, the efficacy of Leonurus japonicus in the treatment of menoxenia, dysmenorrhea, amenorrhea and other diseases was thought to be mediated primarily through activating blood circulation to dissipate blood stasis. This indicated that Leonurus japonicus might have a protective effect on the cardiovascular system. Ischemic heart diseases, especially acute myocardial infarction (AMI), are important cardiovascular system diseases that have remained the leading cause of death in both developed and developing countries during the past quarter century. Li et al. (2006) studied the effects of treating myocardial ischemia in rats with the flavone and alkaloid extracts of the aerial part of Leonurus heterophyllus. After orally administering the flavone and alkaloid extracts for 7 days, compared with the model group, the J point was not markedly changed in the II-lead electrocardiogram, and the left ventricular systolic pressure (LVSP) as well as the maximum rate of change in the left ventricular pressure ( 7dp/dtmax) were recovered compared with the normal group (P o0.05). The activity of creatine phosphate kinase (CPK) and lactic dehydrogenase (LDH) in the blood serum of these rats was inhibited (P o0.05). In addition, the level of MDA was reduced and the activity of SOD was increased in the cardiac muscle tissue compared with that in the model group (P o0.05). Together, these results indicate that the Leonurus japonicus extract could significantly protect the cardiac muscle tissue and reduce the ischemic injury induced by isoprenaline. Jiang et al. (2006) reported that the alkaloid extract of the aerial part of Leonurus heterophyllus could improve heart function in rats after an acute myocardial infarction. After three weeks of administration, the hemodynamic parameters and histological changes in the myocardium were measured by inserting catheters. Compared with the normal group and sham-operated group, the acute myocardial infarction model group showed an increase in the left ventricular end diastolic pressure (P o0.05); however, the maximal rate of rise decreased and the left ventricular pressure fell. The middle dose (30 g/kg) of the alkaloid extract of Leonurus heterophyllus was better than either the high (60 g/kg) or low dose (6 g/kg), perhaps because the high dose might additionally induce toxicity. In 2004, the protective effects and mechanism of action of an Leonurus heterophyllus injection (20 mg/ml, total alkaloids from the aerial part, Chengdu First Pharmaceutical Company, China) in renal and myocardial ischemia-reperfusion injury were studied. The results showed that this injection (1.8 mg/100 g body weight) could improve renal function in renal ischemia reperfusion injury and diminish the renal tubule damage (Po0.05). These actions may be related to scavenging of oxygen free radicals, reducing lipid peroxidation,


increasing SOD and ATPase activity, and attenuating the Ca2 þ overload and NO content (Po0.05). Further study showed that the levels of CK and MDA in the plasma of groups treated with Leonurus heterophyllus or verapamin (a positive control drug) were lower than those in the model group (Po0.05). Thus, Zheng (2004) suggested that Leonurus heterophyllus exerted a significant protective effect on the myocardial ischemia reperfusion injury and could restore the myocardial constriction during reperfusion. 4.2.3. Anti-oxidative activity In ischemic heart diseases, reactive oxygen species (ROS) have played an important role. ROS may possess highly reactive and toxic properties and generate enzymes to exacerbate the degree of myocardial damage sustained by the ischemic myocardium. In addition, because the production of free radicals override the scavenging effects of antioxidants leading to oxidative stress, other diseases such as diabetes and stroke may develop (Ferrari et al., 1998; Wattanapitayakul and Bauer, 2001; Zhu et al., 2004). In 2005, the anti-oxidative stress effects of the aqueous extract of the aerial part of Leonurus heterophyllus on ischemic rat hearts were studied in vitro and in vivo (Sun et al., 2005a). For the in vivo experiment, after orally administering the extract (400 mg/kg/day) starting 1 week prior and continuing until 3 weeks after myocardial infarction, the rats in the treated group had a higher survival rate (55.4%) than those in the model group (40.5%). However, the antioxidant effects of Leonurus heterophyllus were exerted only under the condition of oxidative stress by selectively preserving the activities of superoxide dismutase and glutathione peroxidase as well as by depressing the formation of malondialdehyde (Po0.05), especially in the acute phase of acute myocardial infarction (Po0.01). The effects of scavenging free radicals and inhibiting the formation of reactive oxygen species may play a key role in protecting the endogenous antioxidant system from oxidative stress in vivo. For in vitro experiment, the extract displayed a stronger effect than ascorbic acid and Trolox on scavenging ONOO and inhibiting lipid peroxidation (Po0.01). In addition, the extract had the capacity to markedly scavenge ABTS þ radicals; the average TEAC values were 562% and 588% when ascorbic acid and Trolox were used as standards, respectively. Finally, Luo and Huang (2006) reported that the flavones extract of the aerial part of Leonurus heterophyllus markedly increased the activity of scavenging of hydroxyl radicals in a dose-dependent manner. 4.2.4. Anti-cancer activity To confirm the anti-cancer action of motherwort, Nagasawa et al. (1990) studied the effect of Leonurus sibiricus on preneoplastic and neoplastic mammary gland growth in multiparous GR/A mice. Chronic ingestion of the methanol extract of the aerial part in drinking water at a concentration of 0.5% enhanced the development of both pregnancy-dependent mammary tumors (PDMT) and mammary cancers originating from PDMT. By contrast, the treatment markedly suppressed the development of the mammary cancers that originated from the hyperplastic alveolar nodules (HAN) associated with the decreased formation of HAN (P o0.05). The incidence of uterine adenomyosis was also inhibited in mice given the extract. The urinary excretions of allantoin, creatine and creatinine and glucose tolerance were stimulated, which may at least partly contribute to the inhibition of mammary cancers originating from the HAN. The study further evaluated the chemopreventive role in lesions of the mammary gland and uterus of GR/A mice and the effects on these lesions of the adsorbed (MW1) and unabsorbed (MW2) fractions separated by ion-exchange resins. The incidence of palpable mammary tumors was suppressed and their growth was retarded by both MW1 and MW2 (P o0.05). Together, these findings indicate the importance of the synergistic action of several components, specified and

27

unspecified, for the full manifestation of the effects of Chinese medicine (Nagasawa et al., 1992). Tao et al. (2009) studied the anti-cancer action of the ethanol extract of the aerial part of Leonurus japonicus at the cellular level. Their results demonstrated that the cytotoxicity of the extract in human breast cancer cells was non-apoptotic and estrogen receptor independent. The ethanol extract caused cell death in a dose-dependent and time-dependent fashion in both positive and negative breast cancer cells. The IC50 for suppression of cell proliferation at 24 h and 48 h was 96.2 and 89.1 mg/ml for MCF-7, and 67.7 and 53.4 mg/ml for MDA-MB231 cells, respectively. Low concentrations of the ethanol extract caused cell cycle arrest at the G2/M phase. The evidence from morphology, Hoechst 33342 staining and flow cytometry all indicated that the cell death was not apoptotic. The anti-cancer activity of the aqueous extract of the aerial part of Leonurus heterophyllus on MCF-7 and MDA-MB 453 human breast cancer cells was demonstrated in 2003 and 2008 (Chinwala et al., 2003; Mazzio and Soliman, 2009). 4.2.5. Neuroprotective activity Given that neuronal damage following cerebral ischemia is a serious risk to stroke patients, Liang et al. (2011) studied the neuroprotective effects of an alkaloid extract of the aerial parts from Leonurus heterophyllus on cerebral ischemic injury. After 24 h of reperfusion following ischemia for 2 h induced by middle cerebral artery occlusion, rats were intraperitoneally administered increasing doses of this alkaloid extract (3.6, 7.2, 14.4 mg/kg, respectively). Their results indicated that compared with model group, the extracts (7.2 and 14.4 mg/kg) could significantly decrease neurological deficit scores and reduce the infarct volume in rats with focal cerebral ischemic injury and the MPO content in the ischemic brain (Po0.05). The extract also significantly decreased the NO level compared with that in the model group (Po0.05) at a concentration of 14.4 mg/kg. In addition, the extract significantly decreased the apoptosis ratio in nerve fibers compared with that in the model group (Po0.05). Therefore, these authors suggested that the alkaloid extract from Leonurus heterophyllus may be used for the treatment of ischemic stroke as a neuroprotective agent. 4.2.6. Analgesic and anti-inflammatory activity In 2005, the analgesic and anti-inflammatory activities of Leonurus sibiricus in vivo was studied. Injecting the methanol extract of the aerial parts (500 and 250 mg/kg, intraperitoneally) caused a significant analgesic effect in acetic acid-induced writhing in mice with an inhibition of 69.68% and 44.15%, respectively; the positive control drug diclofenac sodium displayed an inhibition of 74.67%. In addition, the oral administration of the extract (400 and 200 mg/kg) in rats showed a significant antiinflammatory activity against carrageenan-induced paw edema (P o0.05) (Islam et al., 2005). Shin et al. (2009) studied the anti-inflammatory activity of Leonurus sibiricus on the secretion of inflammatory cytokines in a human mast cell line (HMC-1) after treating with phorbol 12-myristate 13-acetate (PMA) plus calcium ionophore A23187 prior to the activation of the HMC-1 cells. The aqueous extract of the aerial part of Leonurus sibiricus (1 mg/ml) inhibited PMA plus A23187-stimulated gene expression and production of TNF-alpha, IL-6, and IL-8. The PMA plus A23187-induced NF-kappa B activation in HMC-1 cells was also inhibited by the extract. These results indicate that this extract may be helpful in regulating inflammatory diseases. 4.2.7. Anthelminthic activity Xu et al. (1994) studied the anthelminthic activity and the mechanism for this action of the methanol extract of the aerial

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part from Leonurus heterophyllus against Panonychus citri McGregor and Myzus persicae. The results demonstrated that at a dose of 100 mg/kg, the methanol extract (100%) demonstrated stronger anthelminthic activity mediated by the inhibition of the activity of Car E and Ach E than the petroleum extract (60%), dichloromethane extract (55.7%) or propanone extract (0%). This activity suggests that the methanol extract could also be used as a nerve toxicant. Zhang et al. (2007) studied the anthelminthic activity of Leonurus artemisia against Oncomelania hupensis. Their result showed that after administering the aqueous extract of the aerial part of Leonurus artemisia (0.2, 0.4, 0.6, 0.8 and 1.0 g/l) against to Oncomelania hupensis for 1–2 days, the activity of the esterase isozyme was higher than that of the compared group and reached the highest activity at 3–4 days. By contrast, application of the aqueous extract for 5 days remarkably reduced the activity of the esterase, and new isozymes appeared, while some of the original isozymes vanished. When treating with stachydrine (0.60 and 0.8 g/l), the level of glycogen increased from 9.15% to 58.72% and from 11.68% to 76.16%, respectively. These results indicated that stachydrine may disrupt Oncomelania hupensisps by affecting energy metabolism. 4.2.8. Antibacterial activity Ahmed et al. (2006) investigated the antibacterial activity of the carbon tetrachloride, chloroform, acetone and methanol extracts of the aerial part of Leonurus sibiricus in vitro. Their results demonstrated that the carbon tetrachloride and chloroform extracts have a broad spectrum of antibacterial activity. At a concentration of 500 mg/disc, the zone of inhibition for the CCl4 and chloroform extracts against Staphylococcus aureus, Staphylococcus epidermis, Streptococcus pyogenes, Escherichia coli, Vibrio cholera, Shigella dysenteriae and Shigella boydii were 20, 20, 30, 25, 13, 16, 17 and 19 mm for the CCl4 extract and 14, 28, 20, 15, 17 and 19 mm for the chloroform extract, respectively. The positive control drug kanamycin (30 mg/disc) demonstrated a zone of inhibition for these same species of 30, 32, 40, 39, 30, 34 and 38 mm, respectively. 4.2.9. Others Gao (2010) studied the effect of the total alkaloids (19, 37.5 and 75 mg/kg) from the aerial part of Leonurus japonicus in mouse and rat models of benign prostatic hyperplasia (BPH). Their results showed that the prostate exponent, the contents of T and DHT, and the expression of bFGF, EGF, IGF-1 were reduced (P o0.05), but the expression of TGF-β1 and TGF-β1/bFGF that could restrain BPH was increased in these models. The ultrastructure and the histological anomalies observed in the prostate in the animal models were improved by the total alkaloids (P o0.05). This research suggested that total alkaloids from Leonurus japonicus are effective in animal models of BPH and that the mechanisms from the total alkaloid actions may be associated with growth factors or with the regulation of the proportions of androgens and estrogens. Hung et al. (2011) reported that the 70% ethanol extract of the aerial part of Leonurus heterophyllus significantly inhibited cholinesterase activity (P o0.05). The results from another study demonstrated that the methanol extract of the aerial part of Leonurus japonicus might inhibit glutamate-induced toxicity in primary cultured rat cortical cells, with cell viabilities of 18.4%, 29.4% and 52.4% at concentrations of 0.1, 1 and 20 mM, respectively (Moon, 2010). Schmidt et al. (2013) studied the effect of treating the typical symptoms of type 2 diabetes mellitus with extracts from the aerial part of Leonurus sibiricus. The results demonstrated that the insulin released from INS-1E cells into the culture medium over 24 h was significantly increased in the presence of 500 mg/l of either an aqueous or methanolic extract (P o0.05). Acute

application of the aqueous extract resulted in depolarization of the cell membrane potential paralleled by an initial increase and subsequent decline and silencing of the action potential frequency, inhibition of the KATP channel, persisting depolarization and an increase in the concentration of intracellular calcium. The electrophysiological effects were comparable to those of 100 μM tolbutamide. Furthermore, all extracts of Leonurus sibiricus stimulated INS-1E cell proliferation. Thus, these authors suggested that the extract of Leonurus sibiricus can enhance insulin secretion and/or foster cell proliferation and may provide possible explanations for the underlying therapeutic principles in the empirical use of Leonurus sibiricus-containing compounds for diabetes mellitus and related disorders.

5. Analysis of active constituents and quality control As a famous traditional Chinese medicine, Leonurus japonicus was historically applied widely in the clinic to treat diseases in women such as those associated with reproduction and pregnancy known as mothers0 diseases. By 2010, more than 15 preparations with Leonurus japonicus as the primary and active ingredient were listed in the Chinese Pharmacopoeia and approved by the government. Medicines such as “Ba Zhen Yi Mu Wan,” “Yi Mu Cao Ke Li,” “Kun Ning Ke Li” and “Kang Gong Yan Pian” (Table 1) were used to promote blood circulation to restore menstrual flow and treat menoxenia induced by blood stasis and reduced menstruation as well as other women0 s diseases. To control the quality of the raw materials and the preparations of Leonurus japonicus, stachydrine has been used as the active standard compound with TLC and HPLC methods by the Chinese Pharmacopoeia since 1990. To examine the content of stachydrine in different parts of Leonurus japonicus, Tang et al. (2008) used HPLC to determine the content of stachydrine in flowers, stems and leaves. Their results showed that the content of stachydrine was highest in leaves (0.83%), followed by flowers (0.33%) and then stems (0.25%) using a Spherisorb SCX column, 20 mmol/l sodium dihydrogen phosphate (containing 0.04% triethylamine and 0.15% phosphoric acid, mobile phase) and a detection wavelength of 192 nm. These results were confirmed by Lin (2011) who also demonstrated that the content of stachydrine in Leonurus japonicus differed in plants obtained from different regions of China. The content of stachydrine in the leaves, flowers and stems was highest in plants from Beijing city (3.15%, 1.82% and 0.98%, respectively), followed by Liaoning province (2.06%, 1.54% and 0.87%), Shandong province (1.72%, 1.25% and 0.68%) and Henan province (1.22%, 0.86% and 0.43%). Zhang et al. (2007) compared the content of stachydrine in the leaves and stems of Leonurus japonicus from 12 different regions in China using TLC scanning. They found that Qingdao city of Shandong province had plants with the highest stachydrine content in the leaves and stems (0.92% and 0.56%, respectively), followed by Cangshan county (0.91% and 0.59%), Mengyin county of Shandong province (0.90% and 0.61%), Linbao city of Henan province (0.88% and 0.79%), Chaoyang district of Beijing city (0.83% and 0.11%), Ningan county of Heilongjiang province (0.73% and 0.22%) and Tianshui city of Gansu province (0.64% and 0.03%). To precisely determine the content of stachydrine in Leonurus japonicus, Kuchta et al. (2013) developed a highly reproducible instrumental HPTLC method that used postchromatographic derivatization and changed the absorbance into a detectable derivative at 517 nm with an automatic HPTLC system using scanner and analysis software. This method was shown to be precise with respect to concentration and yielded highly reproducible data over numerous inter-day repetitions. Not only did the independent evaluation of the scanned HPTLC sheets for stachydrine peak area and height result in almost identical values for all samples but also


the results of a parallel-developed direct quantitative 1H-NMR procedure using the N-CH3 singlet δ 3.03 ppm in comparison with the singlet of the two vinylic protons of the internal standard maleic acid at δ 6.18 ppm were always within the standard deviation of the HPTLC data. These measurements of the sample revealed that the content of stachydrine (w/w) was from 0.2% to 1.0% for the aerial parts of Leonurus japonicus. Thus, these authors suggested that instrumental HPTLC may be a powerful tool for quality assurance of stachydrine-containing plants and herbal drugs, especially for industrial routine protocols. Recently, because of additionally detected pharmacologic actions of leonurine, this compound was included with stachydrine for use in monitoring the quality of Leonurus japonicus. Chen et al. (2010b) developed a UPLC-DAD-MS method to determine the content of leonurine in Chinese motherwort (Leonurus japonicus). Their results indicated that the extracted amount of leonurine was 0.15 mg/g when UPLC-DAD quantification was performed on a reversed-phase Acquity UPLC BEH C18 column (100 mm 2.1 mm) with 1.7 μm spherical porous particles. A linear gradient elution of A (ammonium formate buffer, pH 4.0) and B (methanol) was performed as follows: 0 min, 2% B; 15 min, 65% B; 17 min 95% B; 20 min, 2% B; v/v. The flow rate was 0.2 ml/min. The column temperature was 30 1C, and injected volume was 2 μl. DAD was used for the detection, and the wavelength for quantification was set at 277 nm. The UPLC-ESI-MS analysis was operated in positive ESI mode. The electrospray needle voltage was 4 kV, and the capillary temperature was 250 1C. Typical background source pressure was 1.2 10 5 Torr as read by an ion gauge. The drying gas was nitrogen. Following ion trapping, ions were mass analyzed and detected in the electron multiplier at 880 V. The LCQ mass analyzer was scanned to 2000 m/z in positive mode to confirm peak identity by observation of the corresponding ionized molecule ([Mþ H] þ ). The collision gas for the MS-MS experiment was He. Normalized collision energy was 32.5%. Then, Kuchta et al. (2012b) developed a highly reproducible RP-HPLC method using a special octadecyl-bonded stationary phase and an acetonitrile/water gradient (adjusted to pH 2.5 by phosphoric acid) as the mobile phase (DAD/277 nm). That found that the reversed-phase packing with the hydrophilic endcapping clearly contributed to an improved peak shape for leonurine, and clearly enhanced the selectivity of separation compared to classical RP-phases. This method was shown to be precise with respect to concentration, exhibiting a linear response in the range of 2.5–12.5 μg/ml leonurine, a detection limit well below 0.5 μg/ml, and correlation coefficients constantly higher than 0.99 (5 levels, n¼3) over numerous inter-day repetitions, and demonstrating the robustness of the newly developed HPLC protocol. Their results showed that the content of leonurine in the samples of the aerial part of Leonurus japonicus obtained from China and Japan was from 0.001% to 0.049%, while that of Leonurus japonicus from domestic cultivation in Japan displayed significantly higher amounts of at least 0.1%. Thus, the authors thought that the HPLC method described above could be used for the quality control of leonurine contained in TCM and in pharmacopeial analytics for the differentiation of Leonurus japonicus samples.

6. Toxicity According to the experiences gathered using folk applications and to the records contained in the classical books on traditional Chinese medicine, Leonurus japonicus appeared innocuous. However, modern toxicological studies have indicated that it has some toxic and side effects. Sun et al. (2005b) studied renal toxicity in rats administered Leonurus japonicus. After the oral administration of drugs containing Leonurus japonicus (90 and 30 g/kg) to rats for

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90 days, the urine levels of routine factors and microalbuminuria were significantly changed; the levels of blood ALT, AST, Cr and BUN were increased by the high concentration (Po 0.05). In addition, the histopathological analysis showed that 50% of the rats demonstrated fibroplasia in the interstitial tissue of the renal medulla, and 40% of the rats had a significant cloudy swelling in the epithelium of the renal tubule. These results indicated that Leonurus japonicus could cause a variable degree of renal damage in rats. Then, using long-term administration of the alcohol extract of Leonurus japonicus in rats, Huang and Sun (2010) studied the mechanism of the nephrotoxicity. In their study, after orally administering the extract (120, 60 and 30 g/kg) to rats for 45 days, the histopathology of nephridial tissue and the content of MDA, total SH, GSH and the activity of SOD and GSH-Px in serum were investigated. In the histopathological examination, the alcohol extract induced a variable degree of renal tubular injury in a dose-dependent manner. Compared with the control group, the contents of total-SH (controls, 3.39 μmol/l; treated, 5.83, 4.93 and 4.25 μmol/l) and MDA (controls, 0.39 nmol/ml; treated, 0.53, 0.49 and 0.46 nmol/ml) in serum were increased, and the activities of GSH (controls, 417.14 mg/l; treated, 247.08, 313.19 and 352.71 mg/l), SOD (controls, 526.00 U/ml; treated, 376.30, 417.20 and 436.70 U/ml) and GSH-Px (controls, 57.40 U/ml; treated, 40.67, 47.09 and 50.05 U/ml) were decreased in serum (P o0.05). The authors thought that some of the pathological changes observed following the administration of Leonurus japonicus may be caused by oxidative damage. To investigate the toxic components in Leonurus japonicus, Luo et al. (2008, 2010) studied the toxic effects of total alkaloids and the petroleum ether extract. After administering the alkaloid extract intragastrically to mice at doses of 0.615 or 1.23 g/kg/day for 15 days, the subacute toxic effects on the liver and kidney were studied. Their results showed that at the high dose, the alkaloid extract significantly altered the activity of AST (P o0.05) but with no significant differences in other indexes of liver and renal function at either dose. Thus, the oral administration of the alkaloid extract produced no significant hepatotoxicity or nephrotoxicity in mice, whereas a high dose was associated with a slight toxic effect on the function of the liver. After administering the petroleum ether extract intragastrically to rats at a dose of 60 g/kg/ day for 15 days, the subacute toxic effects on the liver and kidney were studied. The results demonstrated that compared to control group, the extract significantly increased the serum ALT (controls, 7.78 U/l; treated, 31.13 U/l), AST (controls, 9.37 U/l; 85.48 U/l), BUN (controls, 4.49 mmol/l; treated, 9.29 mmol/l) and Cr (controls, 91.67 μmol/l; treated, 200.63 μmol/l) as well as the urinary protein (controls, 50.53 mg/l; treated, 66.49 mg/l) and urinary NAG (controls, 16.83 U/l; treated, 32.56 U/l). In addition, pathological injury to the liver and kidney, such as cellular swelling and inflammatory infiltration, was observed. After recovery, the level of urinary protein remained increased, but the level of the other liver and renal functional parameters decreased (Po0.05). Moreover, the pathological injury was ameliorated. Thus, the toxicity of the petroleum ether extract was higher than that of the aqueous extract, and the toxicity of the petroleum ether extract on urinary protein was irreversible during the short time tested. However, the other toxic effects were reversible. Thus, the authors suggested that the toxic component(s) were mainly in the petroleum ether extract. By comparing the acute toxicity of 95% alcohol extracts from fresh, dry and stir-frying with wine Leonurus japonicus, Huang et al. (2010) investigated the effects of the processing method and optimized the processed products from Leonurus japonicus. The acute toxicity of the various processed products from the highest to the lowest is fresh, dry and stir-frying with wine, and the LD50 of the fresh and dry extracts are 83.089 and 102.93 g/kg, respectively. These results indicated that the toxicity of Leonurus japonicus could be

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decreased after processing, and the amount of the attenuation depended upon the processing method.

7. Conclusion Because of its marked effects in treating various women0 s diseases, Leonurus japonicus was widely used as an herbal remedy called Leonuri for thousands of years in the clinics of some countries of East Asia, especially in China, Korea and Japan. In the Chinese Pharmacopoeia (2010 edition), alkaloids were used to control the quality of Leonurus japonicus and should be at a minimum of 0.04% stachydrine and 0.004% leonurine (Committee for the Pharmacopoeia of P.R. China, 2010). However, in Europe, because Leonurus cardiaca was more widely distributed and more popular than Leonurus japonicus, the aerial part of this plant was approved for wide use in the clinic by the government as the herbal remedy Leonuri or motherwort. According to the European Pharmacopoeia (7th edition), flavonoids were used to monitor the quality, and Leonurus cardiaca should contain a minimum of 0.2% hyperoside (Wojtyniak et al., 2013). Thus, the differences in chemical composition and pharmacology between Leonurus japonicus and Leonurus cardiaca should be investigated further, especially the differences in active compounds or active compositions and the monitoring of quality for the herbal remedies. Different from other species of TCM, species of Leonurus such as Leonurus cardiaca in Europe, Leonurus japonicus in Asia and Leonurus leonurus in South Africa were historically used as a folk medicine to treat some women0 s diseases. At that time, due to the lag in academic communication and other historic issues, people in the various regions used different Latin derivations of Leonurus japonicus, and the nomenclature for the species became confused. According to the website www.theplantlist.org, there are 12 synonyms, with 6 synonyms having three-star confidence levels. However, in China, only four synonyms, Leonurus artemisia (Lour.) S.Y. Hu, Leonurus heterophyllus Sweet and Leonurus sibiricus auct. pl. and Stachys artemisia Lour., were thought to be synonyms of this species, whereas the others may be varieties of species or subspecies (http://frps.eflora.cn). Therefore, we suggest that a single name should be determined for this plant. In the current review, we used the four synonyms listed above that had been accepted and approved in China to describe this plant. To date, more than 130 chemical components have been isolated and identified from Leonurus japonicus. Pharmacological studies demonstrated that many of these components have good bioactivities in vivo or in vitro. Leonurine and stachydrine especially have been widely investigated and demonstrate marked pharmacological effects. The alkaloid components are thought to be the active ingredients of this plant. However, the activity of the primary components of the plant, the labdane diterpenoids and flavones, have not been investigated extensively. Thus, we suggest a stronger focus on these compounds to discover and develop different and marked bioactivities. In China, because of its marked effect on treating women0 s diseases, Leonurus japonicus was called a superior medicine for thousands of years. It was also thought for a long time to be innocuous. However, the results of modern toxicological studies have demonstrated that it has renal toxicity and is slightly hepatotoxic following chronic administration. In the past, it was forbidden to treat diseases of pregnant women in the clinic due to the potential of stimulating the uterus. In addition, women may respond differently during the various phases of their menstrual cycles, becoming affected easily by environmental factors or drugs. Therefore, comprehensive and systemic toxicological research should be conducted and further developed. In summary, the phytochemical and pharmacological studies of Leonurus japonicus have received much interest. New extracts have

been developed, and active compounds have been isolated and proven to affect the uterus as well as to exert cardioprotective and anti-oxidative actions. Similar actions were empirically identified during the traditional use of Leonurus japonicus for treating various women0 s diseases. However, the issues with toxicity and the confusion in nomenclature have slowed the development of Leonurus japonicus as a modern therapeutic agent. Further research, especially on its toxicity, effect on the uterus and cardioprotective actions, should have priority.

Acknowledgments This work was financed by The Special Fund of the Chinese Central Government for Basic Scientific Research Operations in Commonweal Research Institutes (No. 1610322014011) and the Special Fund for Agro-scientific Research in the Public Interest (201303040-14). The authors would also like to express their gratitude to Dr. Allan Grey at Lanzhou University for correcting the English in this paper.

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