Chemical Constituents from the Roots and Rhizomes of Asarum heterotropoides var. mandshuricum and the In Vitro Anti-Inflammatory Activity

Anti-inflammatory compounds were investigated from the ethanol extract of the roots and rhizomes of Asarum heterotropoides var. mandshuricum, a traditional Chinese medicine called Xixin and used for pain and inflammatory. Nine new compounds were isolated, including six new lignans, neoasarinin A–C (1–3), neoasarininoside A and B (4 and 5), and asarinin B (7), and one new monoterpene, asarincin A (8), two new amides, asaramid II and III (10 and 11), and one new natural monoterpene, asaricin B (9), along with 37 known compounds (6, 12–47). Their structures and absolute configurations were elucidated on the basis of spectroscopic methods and chemical analyses. This is the first report of the absolute configuration of asarinin A (6). The 8-O-4′ neolignans (1–5) were reported in the genus Asarum for the first time. The 15 compounds 17, 19, 22–25, 28, 31, 36, 40, 42, 43, 45–47 were isolated from the genus Asarum, and compounds 16, 32, 33, 37 and 39 were isolated from A. heterotropoides var. mandshuricum for the first time. Thirty-seven of the isolates were evaluated for anti-inflammatory activity against the release of β-glucuronidase in polymorphonuclear leukocytes (PMNs) induced by the platelet-activating factor (PAF), and compounds 1, 4, 7, 8, 14, 17–19, 22, 24, 25, 29, 30, 32, 33, 40–43, 45, and 46 showed potent anti-inflammatory activities in vitro, with 27.9%–72.6% inhibitions at 10−5 mol/L. The results of anti-inflammatory assay suggested that lignans obtained from the CHCl3 extract might be the main active components of Xixin.

In this anti-inflammatory activity assay in vitro, we also tested the inhibitory activity of β-glucuronidase release of the different polarity extracts, including Pet. extract, CHCl 3 extract, EtOAc extract, and n-BuOH extract obtained from the ethanol extract of roots and rhizomes of A. heterotropoides var. mandshuricum. The results showed that the CHCl 3 extract displayed strong inhibitory activity with a rate of 76.8% (p < 0.01) at 10 µg/mL, while the other three extracts exhibited no anti-inflammatory activity (Figure 7). Moreover, by detailed analysis of the 21 anti-inflammatory active compounds, it was found that 13 of them were isolated from the CHCl 3 extract, comprising nine lignans (1, 7,  14, 17-19, 22, 24, and 25), two benzene derivatives (41 and 45), one monoterpene (8), and one fatty glyceride (46). According to the results of the anti-inflammatory study, it was inferred that the lowly polar components that existed in the CHCl 3 extract, especially lignans, as well as benzene derivatives, were the main active anti-inflammatory components of this traditional Chinese medicine.
In addition, the anti-inflammatory active compound 14 was found to be the most abundant among all isolates-the amount obtained from 231 g CHCl 3 extract was about 10 g, likely responsible for most of the anti-inflammatory effect of the CHCl 3 extract. Based on these results, it is possible that one of the mechanisms for the anti-inflammatory effect of A. heterotropoides var. mandshuricum is due to the inhibition of the release of β-glucuronidase in PMN cells. However, a number of mediators and mechanisms are also involved in inflammatory reaction, so additional investigations are required to find the anti-inflammatory mechanisms and active components of A. heterotropoides var. mandshuricum.

General
Melting points were obtained on an XT-4A micromelting point apparatus (Shanghai Hui Tong Optical Instrument Co., Ltd., Shanghai, China) without correction. Optical rotations were determined on a Perkin-Elmer 243B digital polarimeter (Boston, MA, USA). UV spectra were carried out on a Cary 300 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). CD spectra were measured on a JASCO J-810 spectropolarimeter (Jasco, Tokoyo, Japan). A Nicolet NEXUS-470 FTIR spectrophotometer (Madison, WI, USA) was used for scanning IR spectroscopy. NMR spectra were recorded on Varian INOVA-500 (Walnut Creek, CA, USA) and JOEL JNM-ECA600 spectrometers (JEOL, Tokyo, Japan). The chemical shifts are expressed as δ (ppm) values using solvent as an internal standard, and coupling constant, J, are in Hz. Mass spectra were detected with Bruker APEX IV FT (Bruker, Billerica, MA, USA) and ABI Q-STAR mass spectrometers (Navarre, FL, USA). GC was carried out on a Shimadzu GC-2010 series system fitted with a FID detector (Shimadzu Corporation, Kyoto, Japan) and performed with a DB-1701 column (30 m × 0.25 mm i.d., 0.25 μm film thickness, Agilent Technologies, Santa Clara, CA, USA). Semi-preparative HPLC was conducted on an Alltima C18 column (10 mm i.d. × 250 mm, 10 μm, Alltech, Nicholasville, KY, USA) equipped with an Alltech 426 HPLC pump and an Alltech UVIS 2000 detector (Alltech, Nicholasville, KY, USA). Column chromatography was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) or Sephadex LH-20 gel (Pharmacia Co., Ltd., Shanghai, China). TLC analysis was performed on silica gel (400 mesh, Qingdao Marine Chemical Co., Ltd., Qingdao, China) and precoated polyamide plates (0.2 mm, Zhejiang Siqing Biochem Co., Ltd., Taizhou, China) plates. D-glucose was obtained from Beijing Chemical Reagent Company. All other chemical solvents used for isolation were of analytical grade (Beijing Beihua Fine Chemicals Co., Ltd., Beijing, China). Fractions were monitored by TLC and spots were detected by UV illumination or visualized by heating silica gel plates sprayed with 10% H2SO4 in 95% EtOH.

General
Melting points were obtained on an XT-4A micromelting point apparatus (Shanghai Hui Tong Optical Instrument Co., Ltd., Shanghai, China) without correction. Optical rotations were determined on a Perkin-Elmer 243B digital polarimeter (Boston, MA, USA). UV spectra were carried out on a Cary 300 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). CD spectra were measured on a JASCO J-810 spectropolarimeter (Jasco, Tokoyo, Japan). A Nicolet NEXUS-470 FTIR spectrophotometer (Madison, WI, USA) was used for scanning IR spectroscopy. NMR spectra were recorded on Varian INOVA-500 (Walnut Creek, CA, USA) and JOEL JNM-ECA600 spectrometers (JEOL, Tokyo, Japan). The chemical shifts are expressed as δ (ppm) values using solvent as an internal standard, and coupling constant, J, are in Hz. Mass spectra were detected with Bruker APEX IV FT (Bruker, Billerica, MA, USA) and ABI Q-STAR mass spectrometers (Navarre, FL, USA). GC was carried out on a Shimadzu GC-2010 series system fitted with a FID detector (Shimadzu Corporation, Kyoto, Japan) and performed with a DB-1701 column (

Acid Hydrolysis of Compounds 4 and 5, and GC Analysis
Compounds 4 (1 mg) and 5 (2 mg) in 2 N HCl (3 mL) were refluxed at 80 • C for 1 h. After cooling, the reaction mixture was neutralized with NaHCO 3 and successively extracted with CHCl 3 (4 × 3 mL). The aqueous layer was then evaporated to dryness and the residue was dissolved in anhydrous pyridine (200 µL), and L-cysteine methyl ester hydrochloride (0.06 mol/L, 200 µL) was added. The mixture was stirred at 60 • C for 1 h. Then 150 µL trimethylsilylation reagent hexamethyldisilazane-trimethyl chlorosilane (HMDS-TMCS, 3:1) was added, and the mixture was stirred at 60 • C for an additional 30 min. After centrifugation, the supernatant was concentrated under a stream of N 2 . The residue was portioned between n-hexane and H 2 O (0.2 mL each), and the n-hexane layer (2 µL) was analyzed by GC for sugar identification (detector, FID; injection temperature, 260 • C; detector temperature, 280 • C; temperature gradient system for the oven, 160 • C for 1 min and then raised to 230 • C at a rate of 5 • C/min; carrier gas, N 2 ; flow rate, 1 mL/min). D-Glucose was identified for compounds 4 and 5 by comparison with retention time of authentic D-glucose (t R = 20.76 min) after treatment in the same manner.

Anti-Inflammatory Activity Assay
Compounds and extracts used to test were dissolved in DMSO at the concentration of 10 −5 mol/L and 10 µg/mL. A suspension of rat polymorphonuclear leukocytes (PMNs) (2.5 × 10 6 cells/mL, 250 µL) was incubated at 37 • C for 15 min with the presence of the test sample (2.5 µL) (n = 3) [71]. Then cytochalasin B (1 mmol/L, 2.5 µL, Sigma-Aldrich, Shanghai, China) was added and incubated for 5 min, followed by activating with platelet-activating factor (PAF) (1 µmol/L, 2.5 µL, Sigma-Aldrich, Shanghai, China). After 10 min, the reaction was terminated in an ice bath. The supernatant was obtained by centrifugation at 4000 rpm for 5 min (4 • C), and then the supernatants (25 µL) and phenolphthalein glucuronic acid (2.5 mmol/L, 25 µL, Sigma-Aldrich, Shanghai, China) were incubated with acetic acid buffer (0.1 mol/L, 100 µL, pH 4.6) at 37 • C, 5% CO 2 , for 18 h. Finally, NaOH (0.2 mol/L, 150 µL) was added to terminate the reaction. The results were quantified by reading the absorbance at 550 nm by BIO-RAD model 450 enzyme-labeling instrument. The inhibitory rate (I.R.) was obtained by the following formula: OD model , OD sample , and OD control refer to the average absorbance of the four wells of PAF, the three wells of the test sample, and the control group, respectively.

Conclusions
The roots and rhizomes of A. heterotropoides var. mandshuricum are used in traditional Chinese medicine for the treatment of pain and inflammatory diseases [1]. Previous research proved that some compositions of the volatile oil, monoterpenes, lignans, and amides isolated from the genus Asarum showed potential anti-inflammatory activity [5,15,16,[24][25][26]. In this paper, 47 compounds, including nine new compounds, one new natural compound, and 37 known compounds, were isolated and identified from the ethanol extract of this plant. Meanwhile, 8-O-4 neolignans were reported in this genus for the first time. In order to evaluate the potential anti-inflammatory effects of non-volatile constituents separated from the roots and rhizomes of A. heterotropoides var. mandshuricum, 37 compounds were tested to assess the inhibitory rate of the release of β-glucuronidase in PMNs cells induced by PAF. The results showed that 10 of the lignans (1, 4, 7, 14, 17-19, 22, 24, and 25), two of the flavonones (29 and 30), three of the monoterpenes (8, 32 and 33), five of the benzene derivatives (40-43 and 45) and one fatty glyceride (46) possessed varying degrees of anti-inflammatory activity. The lignans, especially the tetrahydrofuran ring in their structures (7, 14, 17-19, 22, and 24), displayed significant anti-inflammatory activity. From an integrated analysis of previous research [5,25,26], it could be inferred that lignans are the main non-volatile constituents with anti-inflammatory activity.
Additionally, the flavonones are commonly found in the genus Asarum [9,55,57,72,73], but there is no study reporting whether the flavonones obtained from this genus possess anti-inflammatory activity since this type of compound had been proved to be potentially useful for inhibiting the inflammatory response [74,75]. This study demonstrated that two flavonones (29 and 30) showed anti-inflammatory activity against the release of β-glucuronidase in PMNs cells, which is the first report of the anti-inflammatory effects of flavonones from this genus. Interestingly, (-)-asarinin (14), the effects of which had been reported for LPS-activated NO production in macrophages RAW264.7 cells and PAF-induced β-glucuronidase release in PMN cells in the literature [5] and in this paper, respectively, was the most abundant in this plant (Xixin) among all isolates. This primary finding provides support for further study of this compound for the development of a novel anti-inflammatory agent. The results provide a scientific explanation for the use of this plant as an herbal medicine in the treatment of inflammatory diseases.