Chinese Journal of

Natural Chinese Journal of Natural 2018, 16(11): 08110828 Medicines

doi: 10.3724/SP.J.1009.2018.00811

Phytochemistry and of genus ZHANG Ben-Mei1∆, WANG Zhi-Bin1∆, XIN Ping1, WANG Qiu-Hong2, BU He1, KUANG Hai-Xue1*

1 Key Laboratory of Chinese Materia Medica (Ministry of Education), University of Chinese , Harbin 150040, ; 2 Department of Natural Medicinal Chemistry, College of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510000, China

Available online 20 Nov., 2018

[ABSTRACT] The genus Ephedra of the Ephedraceae family contains more than 60 species of nonflowering seed distributed throughout Asia, America, Europe, and North Africa. These Ephedra species have medicinal, ecological, and economic value. This review aims to summarize the chemical constituents and pharmacological activities of the Ephedra species to unveil opportunities for future research. Comprehensive information on the Ephedra species was collected by electronic search (e.g., GoogleScholar, Pubmed, SciFinder, and Web of Science) and phytochemical books. The chemical compounds isolated from the Ephedra species include alka- loids, flavonoids, tannins, polysaccharides, and others. The in vitro and in vivo pharmacological studies on the crude extracts, fractions and few isolated compounds of Ephedra species showed anti-inflammatory, anticancer, antibacterial, antioxidant, hepatoprotective, anti-obesity, antiviral, and diuretic activities. After chemical and pharmacological profiling, current research is focused on the antibac- terial and antifungal effects of the phenolic acid compounds, the immunosuppressive activity of the polysaccharides, and the antitumor activity of flavonoids.

[KEY WORDS] Ephedra; Phytochemistry; Pharmacology [CLC Number] R284.1, R965 [Document code] A [Article ID] 2095-6975(2018)11-0811-18

Introduction used to treat cold, bronchial asthma, cough, fever, flu, head- ache, edema and allergies. E. sinica has become a natural Ephedra is a genus of non-flowering seed plants belong- product source of ephedrine, , and other com- [1] ing to the Ephedraceae family , which includes approxi- pounds [3]. In addition, in some western countries, Ephedra mately 67 species, mainly in the desert areas of Asia, America, sinica has been used as a dietary supplement [4]. It can also be Europe and North Africa. Among these species, 15 ones and used to lose weight by increasing sweating and basal metabo- four varieties can be found in China. Ephedra sinica is a lism and by stimulating the central nervous system [5]. More- member of genus Ephedra and is the primary species that has over, it has also been combined with cardiovascular drugs to been used in China for more than 5000 years [2]. treat cardiovascular diseases [6]. Many reports about the side Ephedrae Herba is defined as the stem of Ephedra sinica effects of E. sinica have emerged, especially in western coun- Stapf, Ephedra intermedia Schrenket C. A. Meyer, or Ephedra tries. A number of side effects of over-the-counter drugs con- equisetina Bunge (Ephedraceae) in the Chinese Pharma- taining ephedrine have been reported, such as myocardial copoeia 15th edition. Dried E. sinica stems are traditionally infarction, arrhythmia, epilepsy, loss of consciousness, and death [7]. In 2004, the FDA announced the prohibition of die- [Received on] 28-Dec.-2017 tary supplements containing E. sinica [8]. With the restriction [Research funding] The study was supported by the Major State on the use of E. sinica and its related preparations in the US, Basic Research Development Program (973 Program) of China ( No. other countries have also joined the ban, which has led to 2013CB531800). difficulties in the use and development of ephedra drugs. To [*Corresponding author] Tel: 86-451-87267188, Fax: 86-451-8726- 7188, E-mail: hxkuang@ yahoo.com resolve this problem, novel pharmacological effects of △These authors contributed equally to this work. ephedra should be investigated. In addition to ephedrine al- These authors have no conflict of interest to declare. kaloids, there are other substances in ephedra, such as poly-

– 811 – ZHANG Ben-Mei, et al. / Chin J Nat Med, 2018, 16(11): 811828 saccharides, flavonoids (30–71), tannins (72–118), and mis- more than 145 compounds have been isolated and identified cellaneous compounds (119–145). These substances have from the genus Ephedra, including , flavonoids, anti-inflammatory, anticancer, antibacterial, antioxidant, and tannins, and polysaccharides. The compounds isolated from hepatoprotective activities. We conducted a systematic review each species are documented in Tables 1–6. Their chemical of the literature to summarize the currently reported chemical structures are shown in Figs. 1–5. constituents and pharmacological activities of Ephedra. Alkaloids Alkaloids are the main components of this genus. Historical and Botanical Characterization of Ephedra Twenty-nine alkaloids, 1–29, were isolated from this genus. E. sinica is one of the original species of the Ephedraceae Structural characteristics of these compounds are listed as family that was recorded in the Chinese Pharmacopoeia as follows: (1) the amphetamine-type alkaloids (6–18) are the “Ma Huang”. As a and an anti-asthmatic, E. sinica main active ingredients of E. sinica, including amphetamine has a long medicinal history for the treatment of cold, fever, alkaloids of three stereoisomers (6–11). In addition, com- headache, rhinobyon and other symptoms [9]. In the ancient pound 12, structurally similar to ephedrine, is an anti-in- Han Dynasty, E. sinica was recorded as a stimulant and anti- flammatory compound isolated from Ephedra sinica and be- tussive agent. During the time of the Roman Empire, E. sinica longs to the oxazolone derivatives. Compounds 13 and 14 was well known until it was eventually dropped from medie- may also belong to the oxazolidine of the structure. val European literature [10]. However, in the beginning of the Compounds 15 and 16, structurally similar to pseudoephed- 20th century, Ephedra sinica emerged as a weight loss and rine and ephedrine, are trace alkaloids in E. sinica. Compound performance enhancement drug and gradually attracted inter- 18 is a new amphetamine-type alkaloid, which is isolated est. However, products containing Ephedra were banned in from the ethanol extract from the E. sinica stem. (2) Ephedri- the US in 2004 after several years of high-profile reports of nes isolated from the E. sinica root are mainly macrocyclic adverse events [11]. spermine alkaloids (1–4) and an imidazole alkaloid (5). (3) In E. sinica plants are perennial herbs standing, more than recent years, five new quinoline alkaloids have been isolated one meter tall, with thin stems, no leaves, and a strong smell from other species of Ephedra (19–23). These quinoline al- of pine and astringency [12]. Furthermore, E. sinica is a kaloids have a carboxyl group on the C-2 position and have drought-tolerant shrub distributed in both temperate and sub- oxygen-containing substitutions on the benzene ring (20, tropical arid environments in the northern hemisphere and 22–23). Generally, these substitutions are methoxyl and hy- South America [13]. droxyl groups. (4) A pyrrolidine alkaloid (24) is isolated from Progress in Phytochemical Studies on the Genus the seeds of E. foeminea and E. foliata. (5) Other alkaloids (25–29) have been isolated from other species of Ephedra. Ephedra The sources, names and structures of these alkaloid Until now, phytochemical studies have revealed that compounds are shown in Table 1 and Fig. 1.

Table 1 Alkaloids different species of the genus Ephedra (the structures of main compounds are illustrated in Fig. 1) Classification Chemical name Plant source Ref. Macrocyclic spermine alkaloids Ephedradine A (1) E. sinica (root) [26] Ephedradine B (2) E. sinica (root) [27] Ephedradine C (3) E. sinica (root) [27] Ephedradine D (4) E. sinica (root) [28] Imidazole alkaloid Feruloylhistamine (5) E. sinica (root) [29] Amphetamine-type alkaloids D(–)-Ephedrine (6) E. Sinica, E. nebrodensis, E. major [30-32] L(+)-Pseudoephedrine (7) E. sinica, E. nebrodensis [31, 33] D(–)Norephedrine (8) E. sinica, E. nebrodensis [31, 33] L(+)-Noreseudoephedrine (9) E. sinica [31, 33] D(–)Methylephedrine (10) E. sinica, E. nebrodensis [31, 33] L(+)-Methylpseudoephedrine (11) E. sinica, E. nebrodensis [31, 33] Ephedroxane (12) E. sinica [34-35] 3, 4-Dimethyl-5-pheyloxazolidine (13) E. sinica [17, 35] 2, 3, 4-Trimethyl-5-phenyloxazolidine (14) E. sinica [17, 35] O-benzoyl-L(+)-pseudoephedrine (15) E. sinica [35-36] O-benzoyl-D(–)-ephedrine (16) E. sinica [35-36] Hordenine (17) E. aphylla [35, 37] (S)-N-((1R, 2S)-1-hydroxy-1-phenylpropan-2-yl)-5- E. sinica [35, 38] oxopyrrolidine-2-carboxamide (18)

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Continued Classification Chemicla name Plant source Ref. Quinoline alkaloids Transtorine (19) E. transitoria [39] 6-Methoxykynurenic acid (20) E. pachyclada ssp. sinaica [40] Kynurenic acid (21) E. pachyclada ssp. sinaica [40] 6-Hydroxykynurenic acids (22) E. foeminea ssp. foliata [40] Ephedralone (23) E. Alata, E. aphylla [41-42] Pyrrolidine alkaloid cis-3, 4-Methanoproline (24) E. foeminea ssp. foliate (seed) [43] Other alkaloids Maokonine (25) E. sinica (root) [44] (±)-1-Phenyl-2-imido-1-propanol (26) E. sinica [45] Tetramethylpyrazine (27) E. sinica [46] N-methybenzlamine (28) E. sinica [47] (2S, 3S, 4S)-2-(carboxycyclopropyl)glycine (29) E. altissima [43]

Fig. 1 Alkaloids isolated from the genus Ephedra

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Flavonoids flavonoid glycosides are commonly made up of glucose and Flavonoids are the most common class of secondary me- rhamnose. Formerly, it was believed that most flavonoids in tabolites within this genus. More than forty flavonoids (30–71) the genus were derivatives of the flavonol aglycones kaemp- have been identified from the genus Ephedra, which are clas- ferol, herbacetin or quercetin. Recently, some other aglycones sified as flavonols (30–46), dihydroflavonols (47–48), fla- such as apigenin, catechin, luteolin, and hesperetin have been vonones (49–51), flavanols (52–57), flavones (58–69), and characterized. Most flavonoid glycosides are mono-gly- anthocyans (70–71). Flavones and their glycosides, as well as cosides or di-glycosides. In addition, three glycosides (35) flavonols and their 3-O-glycosides constituents, are the most and four glycosides (69) are also present. Information about common flavonoids in Ephedra. The sugar moieties of these flavonoids is shown in Table 2 and Fig. 2.

Table 2 Flavonoids of different species of the genus Ephedra (the structures of main compounds are illustrated in Fig. 2) Classification Chemical name Plant source Ref. Flavonols Herbacetin (30) E. sinica [48] Kaempferol (31) E. sinica (root) [49] Quercetin (32) E. sinica (root) [49] 33.Herbacetin 7-methyl ether (33) E. sinica [48] 34.Rutin (34) E. campylopoda [17] Herbacetin 8-methyl ether3-O-glucoside-7-O-rutinoside (35) E. alata [50] Herbacetin 7-O-(6″-quinylglucoside) (36) E. alata [50] Herbacetin 3-O-rhamnoside 8-O-glucoside (37) E. aphylla [42] Pollenitin B (38) E. sinica [51] Herbacetin-8-methyl ether 3-O-glucoside (39) E. sinica [48] Herbacetin 7-O-glucoside (40) E. sinica [51] Kaempferol 3-O-rhamnoside 7-O-glucoside (41) E. sinica [51] Herbacetin 7-O-neohesperidoside (42) E. sinica [51] Kaempferol-3-O-glucoside-7-O-rhamnoside (43) E. sinica [51] Kaempferol 3-O-rhamnoside (44) E. alata [50] Quercetin 3-O-rhamnoside (45) E. alata [50] Quercetin-3-O-glucoside (46) E. campylopoda [17] Dihydroflavonol Dihydroquercetin (47) E. sinica (root) [49] 3-Hydroxynaringenin (48) E. sinica (root) [52] Flavonone 3′, 4′, 5, 7-Tetrahydroxy flavanone (49) E. sinica (root) [49] Naringenin (50) E. sinica (root) [52] Hesperidin (51) E. sinica [53] Flavanols (–)-epicatechin (52) E. sinica , E. nebrodensis [51, 54] (–)-epiafzelechin (53) E. sinica (root) [49] Catechin (54) E. sinica [51] Afzelechin (55) E. sinica (root) [49] Leucocyanidin (56) E. sinica [55] Symplocoside (57) E. sinica [51] Flavones Tricin (58) E. sinica [48] Luteolin (59) E. sinica [56] Apigeni (60) E. sinica (root) [49] 3-Methoxyherbacetin (61) E. sinica [48] Apigenin-5-rhamnoside (62) E. sinica [48] 6-C-glycosyl-chrysoeriol (63) E. sinica [57] Swertisin (64) E. sinica [58] Isovitexin-2″-O-rhamnoside (65) E. sinica [51] Apigenin-7-O-glucoside (66) E. campylopoda [17] Vitexin (67) E. sinica [51] Lucenin III (68) E. alata [50] 2″, 2′″-Di-O-β-glucopyranosyl-vicenin II (69) E. aphylla [42, 59] Anthocyan Leucodelphinidin (70) E. sinica [55] Leucopelargonin (71) E. sinica [55]

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Fig. 2 Flavonoids isolated from the genus Ephedra

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Tannins Continued Tannins are also important compounds in Ephedra, which Classification Chemical name Plant source Ref. mainly exist in the form of condensation. Tannins, mainly proan- Trimer proan- Ephedrannin Tr1 (99) E. sinica [65] thocyanidins, have been proven to be present in many Ephedra thocyanidins Ephedrannin Tr2 (100) E. sinica [65] plants (e.g., Eurasian Ephedra: E. przewalskii, E. alata, E. distachya, E. fragilis and E. intermedia; North American Ephedrannin Tr3 (101) E. sinica [65] Ephedra: E. californica, E. nevadensis, E. fasciculata, E. Ephedrannin Tr4 (102) E. sinica [65] trifurca, E. torreyana, and E. viridis) [14]. At present, the con- Ephedrannin Tr5 (103) E. sinica [65] densed tannins of procyanidin A-type (Fig. 4) are the most Ephedrannin Tr6 (104) E. sinica [65] [15] common in Ephedra , containing dimers (72–98), trimers Ephedrannin Tr7 (105) E. sinica [65] (99–110, 112, 113), and tetramers (114–118). These com- Ephedrannin Tr8 (106) E. sinica [65] pounds are made of flavan-3-ol monomer units, which are Ephedrannin Tr9 (107) E. sinica [65] linked together through C4-C6 (107, 108), C4-C8, or C2-O-C7 bonds. Compound 111 belongs to the B-type proanthocyanid- Ephedrannin Tr10 (108) E. sinica [65] ins, which do not contain C-O-C bonds. In addition, all the Ephedrannin Tr11 (109) E. sinica [65] flavan-3-ol monomer units are afzelechin/epiafzelechin, cate- Ephedrannin Tr12 (110) E. sinica [65] chin/epicatechin, and gallocatechin/ epigallocatechin. The Ephedrannin Tr13 (111) E. sinica [65] plant species, chemical names, and structures of tannins are Ephedrannin Tr14 (112) E. sinica [51] shown in Table 3 and Fig. 3. Ephedrannin Tr15 (113) E. sinica [51] Table 3 Tannins compounds of different species of the genus Tetramer Ephedrannin Te1 (114) E. sinica [51] Ephedra proanthocya- nidins Ephedrannin Te2 (115) E. sinica [51] Classification Chemical name Plant source Ref. Ephedrannin Te3 (116) E. sinica [51] Dimer proan- Ephedrannin A (72) E. sinica (root) [60-61] Ephedrannin Te4 (117) E. sinica [51] thocyanidins Ephedrannin B (73) E. sinica (root) [60-61] Ephedrannin Te5 (118) E. sinica [51] Muhuannin A (74) E. sinica (root) [62] Muhuannin D (75) E. sinica (root) [60, 63] Polysaccharides Muhuannin B (76) E. sinica (root) [60] Large-molecule components in Ephedra species have Muhuannin E (77) E. sinica (root) [60] also been studied. Ephedra sinica contains a high amount of polysaccharides, which range from 3% to 5% of the total dry Muhuannin C (78) E. sinica (root) [17] weight [16]. Additionally, it has been reported that the con- Muhuannin F (79) E. sinica (root) [64] centration of uronic acid measured by the phenol sulfuric acid Muhuannin G (80) E. sinica (root) [64] [17] method is 45.9% . A water-soluble arabinan (Mw 6.15 kDa) Muhuannin H (81) E. sinica (root) [64] has been isolated from the E. sinica stems and investigation Muhuannin I (82) E. sinica (root) [64] of its structure has indicated that the polysaccharide consists Muhuannin J (83) E. sinica (root) [64] of (1→5)-Araf, (1→3, 5)-Araf, T-Araf, (1→3)-Araf, and (1→ Muhuannin K (84) E. sinica (root) [64] 2, 5)-Araf residues at proportions of 10 : 2 : 3 : 2 : 1. The Ephedrannin D1 (85) E. sinica [65] structure includes a branched (1→5)-α-Araf backbone where Ephedrannin D2 (86) E. sinica [65] branching exists at the O-2 and O-3 positions of the residues with 7.7% and 15.4% of the 1, 5-linked α-Araf substituted at Ephedrannin D3 (87) E. sinica [51] the O-2 and O-3 positions. The presence of a branched Ephedrannin D4 (88) E. sinica [65] structure contains a much longer linear (1→5)-α-Araf back- Ephedrannin D5 (89) E. sinica [65] bone as a repeating unit. Generally, the tentative structure of Ephedrannin D6 (90) E. sinica [65] the arabinan (ESP-B1H) is shown in Fig. 4 [18]. Another acidic Ephedrannin D7 (91) E. sinica [65] hetero-polysaccharide (ESP-B4) has been isolated from E. Ephedrannin D8 (92) E. sinica [65] sinica and shows an immunosuppressive effect [19]. It contains Ephedrannin D9 (93) E. sinica [65] a repeating unit of [→4]-GalpA-(1→2)-Rhap-(1→) as a hairy Ephedrannin D10 (94) E. sinica [65] region, substituted by arabinose, xylose, galactose, glucose, [20] Ephedrannin D11 (95) E. sinica [65] mannose, and glucuronic acid . Three acidic hetero- poly- saccharides (ESP-A3, ESP-A4, and ESP-B4) have also been Ephedrannin D12 (96) E. sinica [65] isolated and purified from the stem of E. sinica. Their mono- Ephedrannin D13 (97) E. sinica [65] saccharide compositions and corresponding molar ratios are Ephedrannin D14 (98) E. herb [65] shown in Table 4 [21]. Furthermore, a pure polysaccharide

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Fig. 3 Tannins isolated from the genus Ephedra

(ESP-B1) was isolated from E. sinica. It consisted of mannose, (ESP-A1 and ESP-A2) have been isolated from the cold water glucose, galactose, and arabinose. The ratios of the substances extract of E. sinica [23]. Their monosaccharide compositions and are shown in Table 4 [22]. Additionally, two polysaccharides ratios are also shown in Table 4. Moreover, monosaccharide

– 819 – ZHANG Ben-Mei, et al. / Chin J Nat Med, 2018, 16(11): 811828 composition and proportion of polysaccharides from Ephedra and E) are isolated from [25]. Their mono- root have been analyzed. The results are shown in Table 4 [24]. saccharide compositions and proportions have been determined. In addition, five hypoglycemic glycans (ephedrans A, B, C, D, The results are shown in Table 5.

Fig. 4 The tentative structures of the arabinan (ESP-B1H)

Table 4 Monosaccharide composition and proportion of polysaccharides from Ephedra sinica polysaccharides arabinose rhamnose galacturonic acid mannose glucose galactose xylose glucuronc acid Ephedra root polysaccharide 7.59 5.93 9.94 ESP-B1 93.3 3.5 2.2 1.0 ESP-A3 7.5 14 3.8 13.7 1.0 22.3 6.8 10.2 ESP-A4 4.1 5.1 2.2 1.6 1.0 17.3 1.2 3.1 ESP-B4 4.5 2.0 50.0 1.0 1.0 5.5 1.0 1.5 ESP-A1 61.1 12.9 11.1 11.6 3.2 ESP-A2 67.7 5.0 6.7 20.6

Table 5 Monosaccharide composition and proportion of polysaccharides from Ephdra distachya polysaccharides molecular mass rhamnose trehalose arabinose xylose mannose galactose glucose A 1.2 × 106 2.4 1.0 0.8 0.7 0.2 1.0 0.2 B 1.5 × 106 1.0 0.3 0.7 0.9 0.9 1.0 0.1 C 1.9 × 104 0.2 0.4 0.3 1.0 0.2 D 6.6 × 103 0.5 1.0 0.1 1.0 0.2 E 3.4 × 104 0.4 0.4 0.3 1.0 0.7

Miscellaneous compounds (137), caffeic acid (141), chlorogenic acid (142), and a new Three phenolic glycoside compounds (129–131) are isolated glycoside of phenylpropionic acid (143), while 2-hydroxyl- from E. nebrodensis and four phenolic acids (134–136, 140) 5-methoxybenzoic acid (138) and iso-ferulic acid (139) are also are isolated from E. equisetina. Some other phenolic com- isolated from the E. sinica roots. Additionally, six terpenoid pounds which have been isolated from the stems of E. sinica compounds (123–128), a lignin compound (120), and an ester are trans-cinnamic acid (132), syringing (133), quinaldic acid compound (122) are isolated from the E. sinica roots. Further-

– 820 – ZHANG Ben-Mei, et al. / Chin J Nat Med, 2018, 16(11): 811828 more, a new naphthalene derivative, 1-methyl-2, 3-methyle- In addition, a lignin compound (119) is isolated from E. alata. nedioxy-6-naphthalenecarboxylic acid methyl ester (121), and These compounds are shown in Table 6, and their structures two quinones (144–145) are isolated from the E. sinica stems. are presented in Fig. 5.

Table 6 Miscellaneous compounds of different species of the genus Ephedra Classification Chemical name Plant source Ref. Lignans (±)-Syringaresinol (119) E. alata [41] Sesquipinsapol B (120) E. sinica (root) [64] Naphthalenes Methyl-2,3-methylenedioxy-6-naphthalenecarboxylic acid methyl ester (121) E. sinica [45] Esters Ethyl caprylate (122) E. sinica (root) [66] Terpenoid (–)-α-Terpineol-8-O-β-D-glucopyranoside (123) E. sinica (root) [64] (+)-α-Terpineol-8-O-β-D-glucopyranoside (124) E. sinica (root) [64] Geranyl-β-D-glucopyranoside (125) E. sinica (root) [64] Daucosterol (126) E. Sinica (root) [64] Sitosterol (127) E. sinica (root), E. campylopoda [64, 67] Stigmasterol-3-O-β-D-glucopyranoside (128) E. sinica (root) [66] Phenolic acids Nebrodenside A (129) E. nebrodensis [31, 54] Nebrodenside B (130) E. nebrodensis [31, 54] O-coumaric acid glucoside (131) E. nebrodensis [31, 54] Trans-cinnamic acid (132) E. sinica [51] Syringin (133) E. sinica [51] ρ-Coumaric acid (134) E. alata, E. aphylla, E. equisetina [41-42, 68] ρ-Hydroxybenzoic acid (135) E. aphylla, E. equisetina [42, 68] Protocatechuic acid (136) E. aphylla, E. equisetina [42, 68] Quinaldic acid (137) E. pachyclada [69] 2-Hydroxyl-5-methoxybenzoic acid (138) E. sinica (root) [64] Iso-ferulic acid (139) E. sinica (root) [64] Vanillic acid (140) E. equisetina [68] Caffeic acid (141) E. sinica [45] Chlorogenic acid (142) E. sinica [45] (3R)-3-O-β-D-glucopyranosyl-3-phenylpropanoic acid (143) E. sinica [38] Quinones Physcion (144) E. sinica [45] Rhein (145) E. sinica [45]

problem, the pharmacological effects of other components of Pharmacological Properties of Ephedra E. sinica need to be further studied, such as flavonoids, poly- Ephedra sinicahas been used for thousands of years in saccharides, tannins, organic acids and others. The following the treatment of allergies, bronchial asthma, cold, fever, etc. sections mainly describe the pharmacological effects of crude Since Professor Nagayoshi Nagai reported ephedrine analogs extracts and isolated compounds from the genus Ephedra. as the active constituents in E. sinica, most of the pharmacol- Anti-inflammatory activity ogical effects of E. sinica have been attributed to ephedrine As early as 1985, it was reported that ephedrine analogs, analogs, although E. sinica also contains other chemical com- mainly including ephedrine (6), pseudoephedrine (7), and ephe- ponents, such as flavonoids, tannins, and other compounds. droxane (12), had potent anti-inflammatory activity in vivo. However, these ephedrine analogs have some side effects, This anti-inflammatory effect was likely due to the inhibition including insomnia, palpitations, arrhythmia, and hypertension, of prostaglandin E2 biosynthesis [70]. because these compounds can stimulate the sympathetic and Another study on the possible effects of ephedra- monk- parasympathetic nerves. Additionally, the synthetic form of shood-asarum-soup (“Mahuang-Fuzi-Xixin-Tang” in Chinese) ephedrine, amphetamine, is more addictive and toxic. The US on the IgE-mediated anaphylaxic response was measured in Food and Drug Administration banned the use of dietary sup- 2004. Interestingly, only E. sinica and ephedra-monkshood- plements containing ephedrine analogs in 2004. To solve this asarum-soup inhibit IgE-mediated histamine release and

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Fig. 5 Structures of miscellaneous compounds from different species of the genus Ephedra

– 822 – ZHANG Ben-Mei, et al. / Chin J Nat Med, 2018, 16(11): 811828 increase cAMP levels from rat basophilic leukemia (RBL-2H3) Antibacterial and antifungal activities cells. However, the components responsible for the inhibitory The phenolic compounds isolated from the Ephedra effect of E. sinica are not ephedrines [71]. It may be another herbs exhibit substantial antimicrobial activity against Gram- component distinct from the ephedrine derivatives, which positive bacteria and Gram-negative bacteria [76]. Among E. should be studied further. Another study showed that E. sinica strobilacea, E. pachyclada and E. Procera, the E. strobilacea could suppress the expression of cyclooxygenase (COX-2) in showed the highest antimicrobial activity against three mi- C6 rat glioma cells [72]. Prostaglandins (PGs) are major me- croorganisms, including Pseudomonas aeruginosa, a Gram diators in the regulation of inflammation and immune func- negative bacterium, Staphylococcus aureus, a Gram positive tion. COX is the rate-limiting enzyme in PG production. Ac- bacterium, and Aspergillus nigra a fungus. The plant metha- tivation of nuclear factor-κB (NF-κB) participates in the tran- nolic extracts of E. pachyclada show significant antimicrobial scriptional activation of the COX-2 gene. The E. sinica plays activity against Klebsiella pnemoniae, a Gram negative bacte- an anti-inflammatory role by inhibiting the expression of rium, Bacillus subtilis, a Gram positive bacterium and highest COX-2 protein and NF-κB-dependent transcription. antifungal activity against the Candida albicans microorgan- In 2005, the water distillates of E. sinica were injected ism. There is a strong relationship among total phenol content into the ST36 acupoint on each knee joint in adjuvant-induced and antioxidant, antimicrobial activity, as phenols are very arthritic rats. The E. sinica herb-acupuncture showed an anti- important plant constituents because their hydroxyl groups arthritic effect [73]. The experiment was performed using an in can scavenge free radicals [77]. vitro phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide From the n-BuOH-soluble fraction of the EtOH extracts (LPS)-induced human macrophage model and an in vivo ad- from E. sinica, 12 A-type proanthocyanidins are isolated and juvant-induced arthritic (AIA) rat model. After treatment with identified. These compounds are tested by measuring the E. sinica, the mRNA expression of tumor necrosis factor-α minimum inhibitory concentrations (MIC) against bacteria (TNF-α) and interleukin (IL)-6 genes were down- regulated. (both Gram-positive and Gram-negative) and fungi, with a Interestingly, Ephedra did not work in the non-acupoint. It concentration range of 0.00 515–1.38 mmol·L–1 [65]. exerted a medicinal effect when injected only into the appro- Additionally, other secondary metabolites from E. tran- priate acu-point. The E. sinica herb-acupuncture therapy could sitoria have been assessed for their antibacterial activity. One reduce adverse effects associated with the disease. It could be of the isolated quinolone alkaloid (4-quinolone-2-carboxylic an alternative to traditional therapy. acid) (19) exhibits significant inhibitory activity against common Wang et al. found that the acidic polysaccharides from E. bacteria, including Enterobacter cloacae, Escherichia coli, sinica had immunosuppressive activity and confirmed that the Staphylococcus aureus, and Pseudomonas aeruginosa [39]. main acidic polysaccharide ESP-B4 had potential therapeutic Another study evaluated the effects of E. major on fungal effects on rheumatoid arthritis. ESP-B4 reduces the release of growth and aflatoxin (AF) production by Aspergillus para- inflammatory factors and cytokines by inhibiting the TLR4 siticus NRRL 2999. It was found that the methanolic extracts signaling pathway [19]. of plant aerial parts and roots inhibited fungal growth and

Iksoo et al found that in the Ephedra root extracts, ephe- aflatoxin B1 (AFB1) production dose-dependently with IC50 drannin A (72) and ephedrannin B (73) had anti-inflammatory values of 559.74 and 3.98 μg·mL–1 for AFB1, respectively. effects. They could suppress the transcription of TNF-α and Meanwhile, essential oil extracts of plant aerial parts signifi- IL-1β and inhibit LPS-induced inflammation. They suppressed cantly inhibited fungal growth at the highest concentration of the translocation of NF-κB and the phosphorylation of p38 1000 μg·mL–1 without any obvious effect on AFB1 produc- mitogen-activated protein (MAP) kinase [61]. tion at all concentrations used (0–1000 μg·mL–1) [78]. The complement-inhibiting components of E. sinica could Anti-cancer activity treat the second injury after spinal cord injury (SCI). E. sinica An investigation has shown that the water fraction (EW), reduced inflammation and improved motor function after SCI the remaining water phase from the methanol extract of E. by inhibiting the activity of complement. These results showed sinica after fractionation with ethyl acetate and butanol, pos- that E. sinica could inhibit the activity of complement hemo- sesses significant antitumor activity in BDF1 mice containing lysis and myeloperoxidase, decrease the complement deposi- B16-F10 melanoma cells. Administration at 30 mg·kg–1·d–1 tion and improve motor function [74]. Furthermore, Ling et al. showed comparable inhibitory activity on the growth of the extracted and purified the complement-inhibiting component tumor in vivo. The in vitro antitumor activity is due to its anti- of E. sinica. C3 complement is harmful to the neurological angiogenic and anti-invasion effects. At doses up to 30 μg·mL–1, function of patients with subarachnoid hemorrhage (SAH), a non-cytotoxic concentration, EW inhibits tube formation which can cause brain damage, including brain cell death, induced by human umbilical vein endothelial cells and the blood-brain barrier damage, and brain edema. The extracts of invasion of B16F10 melanoma cells through a matrix mem- E. sinica could not only improve the neurological function of brane by more than 90% [79]. rats after brain injury, but could also improve the early A study has reported that herbacetin (30), the aglycon of blood-brain barrier and brain edema after SAH [75]. herbacetin 7-O-neohesperidoside (42) isolated from Ephedra

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[80] plants, has antiproliferative and analgesic effects . Another (CCL4)-induced chronic and acute liver failure. These results study has shown that it could also suppress the HGF-induced have demonstrated that the pathological indexes of hepatic motility of human breast cancer MDAMB-231 cells [81]. The injury, including aspartae aminotransferase (AST) and alanine mechanism of this inhibitory action is through inhibiting ty- aminotransferase (ALT), are significantly reduced in CCL4+ rosine phosphorylation of c-Met and the PI3K/Akt pathway. low/high E. pachyclada groups. In addition, the pathological Additionally, the herbacetin (30) can directly inhibit the tyro- examination showed that hepatic injuries, including inflam- sine kinase activity of c-Met [51, 82]. To further investigate the mation, necrosis and hepatitis, are partially healed with the mechanism of ephedra inhibiting the phosphorylation of Met, treatment of E. pachyclada extracts [88]. Therefore, the hepa- another study has found that Ephedra could promote HGF- toprotective mechanism of E. pachyclada is to inhibit oxida- stimulated MET and p-MET endocytosis followed by its tive stress and inflammation. downregulation in an NSCLC cell line. The degradation of In another D-galactosamine and lipopolysaccharide (LPS)- MET likely occurrs in the late endosome pathway [83]. induced hepatitis model, the hepatoprotective effects of The preparation method of ephedrine alkaloids-free Ephedra Ephedra extracts have been evaluated [89-90]. The Ephedra Herb (EFE) extracts has been established. The EFE extracts extracts markedly alleviates hepatocyte apoptosis and in- have anti-proliferative effects against the H1975 non-small flammatory factor infiltration by reducing of serum alanine cell lung cancer (NSCLC) cell line [84]. Moreover, the anti- aminotransferase (ALT) and total bilirubin (T. Bil) activity, proliferative effects arecomparable to that of the E. herb ex- levels of TNF-α, and the activities of caspase- 8, 9, and 3. tract. Therefore, the EFE, a new herbal medicine with reduced A recent study has found that ephedra non-alkaloids have side effects and retained anticancer effects, may have great therapeutic potential for the treatment of hyperlipidemia in potential for development. diet-induced mice [91-92]. These non-alkaloids can improve Additionally, a study has reported that the methanol ex- lipid metabolism. They may prevent free radical generation tracts from E. herb has inhibitory effects on the X-linked in- and support recuperation of liver function. What is more, they hibitor of apoptosis protein (XIAP). An in vitro fluorescence are relatively safe for use under the maximum tolerated dose. polarization assay and a protein fragment complementation Antiviral activity analysis have confirmed that the extracts of Ephedra contain E. herb extracts can induce the replication of latent hu- an inhibitory component for XIAP. Moreover, silica gel col- man immunodeficiency virus type 1 (HIV-1) in latently in- umn chromatography and precipitation from methanol and fected U1 cells in vitro, through NF-κB activation. The ulti- chloroform mixtures have confirmed that the active ingredi- mate goal is to eliminate the persistent viral reservoirs in in- ents are oligomeric proanthocyanidins. The inhibitory activity dividuals infected with HIV-1. NF-κB is an inducible cellular of oligomeric proanthocyanidins has been evaluated, with an transcription factor that regulates a wide variety of cellular –1 IC50 value of 27.3 μg·mL through in vitro FP assay and pro- and viral gene expression. The E. herb extracts can efficiently tein fragment complementation analysis [85]. induce NF-κB nuclear translocation and activate the NF-κB Antioxidant and hepatoprotective activity promoter [93]. As early as 1985, feruloylhistamine (5), an imidazole al- A study has confirmed that ephedrine alkaloids-free kaloid, was isolated from underground part of Ephedra plants Ephedra Herb (EFE) extracts have anti-influenza virus activ- (Ephedraceae). A series of feruloylhistamine analogs were ity by showing inhibition of MDCK cell infected with influ- [81] also prepared. The study found that the feruloylhistamine enza virus A/WSN/33 (H1N1) . The IC50 values of Ephedra analogs exhibited antihepatotoxic actions [29]. Herb extract and EFE are 8.6 and 8.3 μg·mL–1, respectively. The phenolic and flavonoid constituents from the E. alata The safety assessment of EFE extracts suggests that it is safer plant show an antioxidant effect [86]. As a natural source of potent than Ephedra Herb extract itself. EFE could be a safer alter- antioxidants, the constituents could prevent many diseases native to Ephedra Herb, but further study is necessary. when used in food, cosmetics, and pharmaceutical products [87]. Miscellaneous biological activities An in vitro and in vivo study has revealed that the E. Zhai et al have examined the effects of E. sinica on the pachyclada extracts possess significant protective effects on treatment of bleomycin-induced rat idiopathic pulmonary primary hepatocytes against carbon tetrachloride damage. fibrosis. Pulmonary fibrosis is characterized by early lung Inflammation and oxidative stress are the major causes of injury followed by fibrosis. E. sinica can significantly reduce liver disease. E. pachyclada has anti-inflammatory and anti- alveolitis and pulmonary fibrosis grades [94-95]. E. sinica has oxidant activities. The antioxidant activity of the E. pachy- been used to treat pulmonary fibrosis in the clinic, suggesting clada extracts was measured in vitro by 2, 2'- di- that traditional Chinese medicine had a major impact on idio- phenyl-1-picrylhydrazyl (DPPH) and β-carotene bleaching pathic pulmonary fibrosis. assays. The DPPH scavenging activity of E. pachyclada ex- The hydro-alcoholic extract from the stems of E. pachy- –1 tracts showed an IC50 value of 55.53 ± 0.5 μg·mL . The clada has been shown to be effective in experimentally healing hepatoprotective effects of E. pachyclada extracts were also rat ulcers [96]. E. pachyclada, is frequently cited by Kerman examined in vivo on mouse models of carbon tetrachloride people as an active agent against gastrointestinal disorders.

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As activators of transient receptor potential vanilloid 1 Formerly letters FT-ICR-MS analysis of lignin (TRPV1), a pain receptor, the E. Herb extract has analgesic FT-ICR-MS analysis of lignin [J]. Nat Prod Res, 2012, 26(15): effects [97-98]. However, ephedrine does not show such effects. 1368-1374. 2, 3, 5, 6-Tetramethylpyrazine (TMP) (27) isolated from [2] Xie M, Yang Y, Wang B, et al. Interdisciplinary investigation E. sinica has been evaluated for its depigmenting effect in a on ancient Ephedra twigs from Gumugou Cemetery (3800 B.P.) UVA-induced melanoma/keratinocytes co-culture system. in Xinjiang region, northwest China [J]. Micros Res Techniq, TMP at a 100 μmol·L–1 concentration significantly decreased 2013, 76(7): 663-672. the expression of tyrosinase-related protein 1 (TRP1) and [3] Kilpatrick K, Pajak A, Hagel JM, et al. Characterization of aromatic aminotransferases from Ephedra sinica Stapf [J]. microphthalmia-associated transcription factor (MITF) levels. Amino Acids, 2016, 48(5): 1209-1220. Cytokines, such as TNF-α, IL-1β, IL-8 and granulocyte-ma- [4] Pawar RS, Grundel E. Overview of regulation of dietary sup- crophage colony-stimulating factor (GM-CSF), were signifi- plements in the USA and issues of adulteration with phene- [99] cantly decreased with TMP treatment . TMP could be used as thylamines (PEAs) [J]. Drug Test Anal, 2016, 9(3): 500-517. a possible drug candidate to improve pigmentation of the skin. [5] Yuliana ND, Jahangir M, Korthout H, et al. Comprehensive Additionally, the ephedrannins A (72) and B (73) isolated review on herbal medicine for energy intake suppression [J]. from E. sinica roots exhibit significant inhibitory activity on Obes Rev, 2011, 12(7): 499-514. melanogenesis in B16F10 melanoma cells. These compounds [6] Soni MG, Carabin IG, Griffiths JC, et al. Safety of ephedra: inhibit the expression of the tyrosinase gene in a dose-de- lessons learned [J]. Toxicol Let, 2004, 150(1): 97-110. pendent manner. 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Cite this article as: ZHANG Ben-Mei, WANG Zhi-Bin, XIN Ping, WANG Qiu-Hong, BU He, KUANG Hai-Xue. Phytochem- istry and pharmacology of genus Ephedra [J]. Chin J Nat Med, 2018, 16(11): 811-828.

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