A-Type Proanthocyanidins from the Stems of Ephedra sinica (Ephedraceae) and Their Antimicrobial Activities

Phytochemical investigation of the n-BuOH-soluble fraction of the EtOH extract of the herbaceous stems of Ephedra sinica, which is known as Ephedrae Herba in Traditional Chinese Medicine, led to the isolation and identification of 12 A-type proanthocyanidins, containing five dimers, two trimers and five tetramers [i.e., (+)-epigallocatechin-(2α→O→7,4α→8)-(-)-catechin, named ephedrannin D1, a dimer; epigallocatechin-(2α→O→7,4α→8)-epigallocatechin-(4α→8)-catechin (ephedrannin Tr1), a trimer; and epigallocatechin-(2α→O→7,4α→8)-epigallocatechin-(4α→8)-epigallocatechin-(2α→O→7,4α→8)-gallocatechin, named ephedrannin Te1, a tetramer). Tetramers composed of gallocatechin are reported for the first time in Ephedraceae. Catechin, epicatechin, gallocatechin, epigallocatechin and four known dimers were also isolated. The structures were elucidated by extensive spectroscopic analysis. The absolute configurations of the 4α linkages, which were confirmed by NOESY and CD experiments, are the outstanding characteristic of most of these isolated A-type proanthocyanidins. The antimicrobial activities of these compounds were tested by measuring the minimum inhibitory concentrations (MIC) against bacteria (both Gram positive and Gram negative) and fungi, and were found to be in the range of 0.00515–1.38 mM. Compounds 6, 8, 10 and 11 exhibited moderate antimicrobial activities against Canidia albicans.


Introduction
Ephedra sinica Stapf. (Ephedraceae) known as Ephedrae Herba ("Mahuang" in Chinese), has been used as an important medicinal herb in Traditional Chinese Medicine for thousands of years, and it is famous for containing six alkaloids of the ephedrine series [(-)-ephedrine, (+)-pseudoephedrine, (-)-N-methylephedrine, (+)-N-methylpseudoephedrine, (-)-norephedrine, (+)-norpseudoephedrine] [1]. According to the Chinese Pharmacopeia [2] and Japanese Pharmacopeia [3], "Ephedra Herb" is derived from the dried herbaceous stems of Ephedra sinica Stapf., E. intermedia Schrenk et C. A. Mey. or E. equisetina Bge., and is used for the treatment of asthma and cough, and as a diaphoretic. For years, ephedrine alkaloids were considered to be the main pharmacoactive constituents and few non-alkaloid-constituents was reported.
Nowadays, there has been considerable research on the bioactivities of proanthocyanidins, including antibacterial, antiviral, anticarcinogenic, anti-inflammatory, antiallergic, and vasodilatory effects [4][5][6][7], and primarily their antioxidant activity. Tannins, mainly proanthocyanidins, were proved by colorimetric reactions to occur in large amounts in the stems of many species of Ephedra (e.g., Eurasian Ephedra: E. intermedia, E. przewalskii, E. alata, E. distachya and E. fragilis; North American species of Ephedra: E. californica, E. fasciculata, E. nevadensis, E. torreyana, E. trifurca and E. viridis) [8]. The Eurasian Ephedra species contain ephedrine alkaloids, but the North American species of Ephedra, known as "Mormon tea" is believed to not contain significant amounts of ephedrine alkaloids [8]. The phytochemical basis behind the purported stimulant and therapeutic nature (such uses include cough medicines, an antipyretic, an antisyphilitic, a stimulant for poor circulation, and an antihistamine [9]) of "Mormon tea" produced from North American Ephedra is thus likely a result of their proanthocyanidin content [8], and hence proanthocyanidins may also play an important role in Asian species of Ephedra. For example, the stem of E. distachya, contains condensed tannins (including proanthocyanidins) that decrease the effects of uremic toxicity after kidney failure in rats [10]. Ephedranin A and B, both belonging to the A-type proanthocyanidins and considered to possess anti-inflammatory [11] and cytotoxic effects [12], were isolated from the root of E. sinica (called Ephedrae Radix and used as an antiperspirant in Traditional Chinese Medicine). Therefore it is obvious that proanthocyanidins may also play an important role in the pharmacological actions of Ephedrae Herba. However, proanthocyanidins of the stem of Ephedrae Herb and their bioactivities remain unknown.
In this study, four monomers, nine dimers, two trimers and five tetramers of A-type proanthocyanidins were isolated from the n-BuOH-solutable fraction of the EtOH extract of the herbaceous stems of E. sinica, among which the structures of 12 unknown compounds were determined by extensive spectroscopic techniques.

Chemistry
From the n-BuOH-solutable fraction of the EtOH extract of the herbaceous stems of E. sinica, 12 A-type proanthocyanidins 1-12 which are new compounds and include five dimers, two trimers and five tetramers, were isolated and identified together with eight known compounds 13-20. Tetramers composed of gallocatechin are reported for the first time in Ephedraceae.
Compounds 17-20 were identified as catechin (17), epicatechin (18), gallocatechin (19), and epigallocatechin (20) by comparing their NMR spectroscopic data with authentic samples and literature data. The 1 H-and 13 C-NMR chemical shifts of the compounds 1-15 are summarized in Tables 1-3, and their structures are depicted in Figure 1.        Compound 1, an amorphous white powder, on TLC examination showed a typical reddish coloration characteristic of phenols with anisaldehyde-sulphuric acid reagent. The molecular formula was determined to be C 30 H 24 O 13 by HR-ESI-TOF-MS, indicating 1 to be a dimeric proanthocyanidin. Its UV spectrum (HPLC-DAD) presented a band with maximum at 278 nm. All of the above data suggested that it belonged to the group of catechins/proanthocyanidins. The 1 H-NMR spectrum showed an AX system for δ H 4.10 (1H, d, J = 3.6 Hz, H-3) and 4.28 (1H, d, J = 3.6 Hz, H-4) in ring C, and the 13 C-NMR spectrum showed a characteristic signal for a C-2 ketal carbon δ C 100.6, suggested 1 to be an A-type of proanthocyanidin. In the aromatic area of the 1 H-NMR spectrum, three singlets resonating at δ H 6.80 (d, J = 2.0 Hz), 6.76 (d, J = 8.2 Hz) and 6.70 (dd, J = 2.0, 8.2 Hz), were assigned to the ABX system of a catechin moiety. The presence of a singlet at δ H 6.76 integrating for two protons indicated the presence of a gallocatechin group. Further 2D NMR experiments (HSQC, HMBC, and NOESY) enabled the complete identification of the structure. The HMBC spectrum showed cross-peaks between the protons H-2′, 6′ (ring B) of the gallocatechin group and an oxygenated carbon at C-2 (δ C 100.6), and between the H-4 (C ring) and C-2 (δ C 100.6), which confirmed the presence of an epigallocatechin as the upper part (unit I) of compound 1. Therefore, catechin was the terminal part (unit II) of the proanthocyanidin A-type skeleton. The 4→8 interflavanoid bond was confirmed by the key correlation between H-4 (ring C) and C-9 (ring D), H-2 (ring F) and C-9 (ring D). The NOESY experiment showed interactions between H-6 (ring D) and the aromatic protons H-2′, 6′ of ring B, and most importantly the cross-peak between H-3 (ring C) and H-6 (ring D). The latter one is considered to be of diagnostic importance, as it proves further the trans-stereochemistry of the 3,4-bond. The α-orientations at C-4 of the interflavan linkages were deduced from the diagnostic negative Cotton effect observed in the 220-240 nm region of the CD spectrum following the chiroptical rule which permits unambiguous assignment of absolute configuration at these chiral centers [16]. As the absolute configuration at position C-3 was characterized as 3S (β-hydroxyl group), based on the NMR spectroscopic data, the absolute configurations at positions 2, 3, 4 should be 2R, 3S, 4S, respectively. There is no distinguishing difference between compounds 15 and 1 in CD spectra (220-240 nm), but they differ in NMR data, especially in the unit II (Tables 1 and 3), which we can deduce that they are conformers with (+)-catechin or (-)-catechin as their terminal parts. As compound 15 was reported to have (+)-catechin as its terminal part, compound 1 was identified as (+)-epigallocatechin-(2α→O→7,4α→8)-(-)-catechin, a dimer, and named ephedrannin D 1 .
Compound 13, an amorphous white powder, showed a reddish coloration with anisaldehydesulphuric acid reagent on TLC examination. The negative HR-ESI-TOF-MS of 13 showed a [M−H] − peak at m/z 591.1136, which corresponded to a molecular formula of C 30 H 24 O 13 . The 1 H-NMR and 13 C-NMR spectra were similar to those of 1, thus we concluded they were structural isomers. Further 2D NMR experiments (HSQC, HMBC, and NOESY) confirmed that 1 and 13 share the same relative configuration. The strong positive Cotton effect at 238 nm is consistent with the β-orientation of the C-4-flavan-3-ol groups [16], and the weak negative Cotton effect at 271 nm followed by a diagnostic positive effect at 287 nm was thought belonging to the 2α-phenyl (C ring)-2α-phenyl (F ring) structure [17]. On the basis of the relative configuration determination via NMR, the absolute configurations at positions 2, 3, 4 are designated as 2S, 3R, 4R, and compound 13 was identified as (-)-epigallocatechin-(2β→O→7,4β→8)-(-)-catechin, and named ephedrannin D 2 .
Compound 6 was obtained as an amorphous white powder and showed a reddish coloration with anisaldehyde-sulphuric acid reagent on TLC examination. Its molecular formula was determined to be C 45 H 36 O 20 by a HR-ESI-TOF-MS experiment, which suggested 6 to be a trimeric proanthocyanidin. The 1 H-NMR spectrum of 6 revealed signals for three 3′,4′,5′-trisubstituted flavan-3-ol moieties, i.e., three singlets at δ H 6.51, 6.55, 6.76 each integrating for two protons indicated the presence of three gallocatechin groups and two singlets (δ H 5.91 and 6.13) in the aromatic region. Two meta-coupled protons [H-6 (δ H 5.87) and H-8 (δ H 6.00) (J = 2. The HMBC spectrum showed cross-peaks between the protons H-2′, 6′ (ring B) of the gallocatechin group and the oxygenated carbon at δ C 100.6 (C-2), and between H-4 of the C ring and δ C 100.6 (C-2), which confirmed the presence of an epigallocatechin as the upper part (unit I) of compound 6. From the 1 H-NMR data, a gallocatechin group was deduced as the terminal part (unit III) from the presence of two H-4 protons (ring I). Thus, another epigallocatechin was assigned to be the middle unit (unit II) of compound 6. The 4→8 interflavanoid bond was confirmed by the key HMBC correlations between H-4 (ring C) and C-9 (ring D), H-2 (ring F) and C-9 (ring D), H-4 (ring F) and C-9 (ring G), H-2 (ring I) and C-9 (ring G). The NOESY experiment also showed interactions between H-6 (ring D) and H-2′, 6′ (ring B), H-6 (ring G) and H-2′, 6′ (ring E). The CD spectra obtained for compound 6 was characterized by a weak Cotton effect at 275 nm and a strong positive Cotton effect at 238 nm. These bands are ascribed to the 1 L b , 1 L a electronic transitions of the aromatic moieties in the flavan-3-ol rings. The Cotton effect at 238 nm is consistent with the β-orientation of the C-4 flavan-3-ol groups. On the basis of the relative configuration determination via NMR, together with correlation of the Cotton effect previously reported [16], compound 6 was established as epigallocatechin-(2β→O→7,4β→8)-epigallocatechin-(4β→8)-gallocatechin, a trimer, named ephedrannin Tr 1 .
The HR-ESI-TOF-MS data of compound 12 showed the [M−H] − ion at m/z 1197.2148, indicating a tetrameric structure with C 60 H 46 O 27 as its molecular formula. The 1 H-NMR spectrum of 12 revealed signals for three 3′,4′,5′-trisubstituted flavan-3-ol moieties and one 3′,4′-disubstituted flavan-3-ol moiety, i.e., singlets at δ H 6.65, 6.76, and 6.76 each integrating for two protons indicated the presence of three gallocatechin groups, one ABX system with singlets at δ H 6.84, 6.95 and 7.13 indicating the presence of a catechin/epicatechin unit, one AX system for two meta-coupled protons [H-6 (δ H 5.91) and H-8 (δ H 6.06) (J = 2.4 Hz)], and three singlets (δ H 5.76, 5.97 and 6.12) in the aromatic region, and two AX system for [H-3 (δ H 4.19) and H-4 (δ H 4.48) (J = 3.6 Hz) (ring F)] and [H-3 (δ H 4.19) and Literature research showed that only 13 trimers [18,19] and one tetramer [20] with A-type linkages composed of gallocatechin were reported. We reported two trimers and five tetramers of this kind. Furthermore, tetramers composed of gallocatechin are report for the first time in Ephedraceae. A-type proanthocyanidins with 4α linkages, the main type found in E. sinica, are less common in Nature than 4β ones, but in our work, 12 A-type proanthocyanidins with 4α linkages were isolated and identified.
All the tested compounds showed antibacterial and antifungal activities in different levels, which may, to some extent, correspond to the antimicrobial action [22] of Mahuang. Furthermore, compound 15, previous isolated from Quercus ilex L. was reported to have antimicrobial activity (MIC = 0.17 mM) against Pseudomonas aeruginosa [14]. In our results, compound 1, a conformer of compound 15, possessed similar activity (MIC = 0.169 mM) against Pseudomonas aeruginosa.

General
Optical rotations were recorded on a JASCO DIP-140 digital polarimeter (Tokyo, Japan). IR spectra were measured on a Nicolet Nexus 470 infrared spectrometer (Madison, WI, USA). CD spectra were measured on a Jasco-810 CD spectrometer. NMR spectra were taken on Bruker AVANCE DRX 400 spectrometer (Fällanden, Switzerland), with tetramethylsilane (TMS) as an internal standard, and chemical shifts were indicated in δ values (ppm). HR-ESI-TOF-MS measurements were performed on a Waters Xevo G2 Q-TOF mass analyser (Milford, MA, USA). Column chromatography was performed with Amberlite XAD-2 gel (Sigma, Philadelphia, PA, USA) and Toyopearl HW-40C (TOSOH Corp., Tokyo, Japan). TLC was performed on silica gel GF 254 Qingdao,China). Preparative HPLC was conducted on an Inertsil C18 column (20 mm i.d. × 250 mm, 5 μm) on a system equipped with a Shimadzu LC-20AP HPLC pump and a Shimadzu SPD-20A UV/VIS detector (Kyoto, Japan). All other chemical solvents used for isolation were of analytical grade (Beijing Beihua Fine Chemicals, Beijing, China and Wako Pure Chemical Industries, Osaka, Japan).

Plant Material
Dried herbaceous stems of Ephedra Sinica Stapf. were collected from Hangjin banner, Inner Mongolia, China, in May 2010, and the plant material was identified by one of the authors, Prof. Shao-Qing Cai. Its voucher specimen (No.6527) was deposited in the Herbarium of Pharmacognosy, School of Pharmaceutical Sciences, Peking University Health Science Centre (Beijing, China).

Extraction and Isolation
The dried and powdered herbaceous stems of E. sinica (35 kg) were sequentially extracted for 2 h each time under controlled reflux with EtOH-H 2 O (95:5, V/V, 3 × 280 L) and EtOH-H 2 O (1:1, V/V, 3 × 280 L). The combined extract solution was concentrated under reduced pressure to obtain a crude extract (5,880 g), and then the crude extract was suspended in H 2 O and successively partitioned with petroleum ether (60-90 °C), EtOAc, and n-BuOH.
For MIC determination, overnight culture of the bacteria strains were diluted with fresh Mueller-Hinton Broth (Beijing AoBoXing Universeen Bio-Tech Co. Ltd.), and standardized to 2 × 10 4 CFU/mL as bacteria suspension. Two μL of compounds solutions were added to row A of columns 2 to 11 on each 96-well plate containing 40 μL Mueller-Hinton Broth in each well, followed by a 2-fold serial dilution of each compound from row B to row H. Positive and negative controls were set up as described in the primary screening assay. Plates were incubated at 37 °C for 16 h and checked for bacteria growth. MIC here is defined as the lowest concentration of compound that results in inhibition of visible bacterial growth (no turbidity) compared with the positive control antibiotics.