Secondary Metabolites with Anti-complementary Activity from the Stem Barks of Juglans mandshurica Maxim

1. INTRODUCTION

The human complement system plays an important role in the host defense system against foreign invasive organisms such as viruses, bacteria, and fungi, as well as an external wound. Its effects are normally beneficial to the host, but it can also cause adverse effects depending on the site, extent, and duration of complement activation (Cimanga et al., 1995). Activation of the system may lead to pathologic reactions in a variety of inflammatory and degenerative diseases such as multiple sclerosis, systemic lupus erythematosus, sjogren syndrome, dermatological disease, rheumatoid arthritis, and gout. Therefore, the modulation of complement activity is important and there is a need to develop anti-complementary agents from various sources such as plants (Park et al., 1999; Min et al., 2003).

Juglans mandshurica Maxim (Juglandaceae), a fast growing deciduous tree, is widely distributed in China, Siberia and Korean peninsula. The tree has been used as a folk medicinal plant for the treatment of esophageal, gastric, cardiac and lung cancer. It was reported that volatile constituents from the species inhibited the growth of the neighboring plants and can be developed for chemurgy (Min et al., 2003; Kim, 1994). Some of the valuable constituents such as α-tertalonyl glucopyranosides, naphthoquinones, naphthalenyl glycosides, flavonoids, galloyl glucopyranosides and diarylheptanoyl glucopyranosides have already been isolated from this species (Park et al., 2017; Wang et al., 2017; Min et al., 2003; Li et al., 2003). In addition, several studies have reported the anti-complement activity, inhibition of human immunodeficiency virus type 1 reverse transcriptase and ribonuclease H activities of J. mandshurica extract (Bi et al., 2016; Min et al., 2003; Min et al., 2000).

Thus, and as a part of a project aimed at discovering bioactive and structurally novel compounds from plant sources, the chemical constituents of J. mandshurica bark and their anti-complement activity were investigated.

2. MATERIALS and METHODS

2.4. Extraction and Fractionation

The bark of J. mandshurica was air-dried and ground with a Wiley mill. A precisely weighted amount (3.2 kg) was extracted in 70 % acetone aqueous solution (each 20 L for 72 h×3 times) at room temperature. The extracting solutions were filtered, combined, and concentrated with an evaporator under reduced pressure to afford the crude extract, which was suspended in water and then successively submitted to liquid–liquid fractions with in separation funnels using n-hexane, methylene chloride (CH₂Cl₂), ethylacetate (EtOAc) and n-buthanol (n-BuOH) (Si et al., 2017). For the detailed extraction and fractionation procedures see Fig. 1. The soluble fractions in n-hexane (15.6 g, yield 0.5%), CH₂Cl₂ (20.6 g, yield 0.6%), EtOAc (130 g, yield 4.1%) and H₂O (337.6 g, yield 10.6%) were obtained after freezing-drying as powders.

Fig. 1. Extraction, fractionation and isolation procedures of tannins from J. mandshurica stem bark.

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3. RESULTS and DISCUSSION

Compound I, which has never been reported from J. mandshurica previously, was isolated as a colorless amorphous powder with melting point of 112–114 °C and optical rotation of αD20 + 18.5° (c 0.1 in MeOH). On paper TLC, its R_f (Retention Factor) values are 0.68 (solvent A) and 0.77 (solvent B). Its molecular weight 482 and molecular formula C₂₀H₁₈O₁₄ was determined based on the quasi-molecular ion peaks m/z [M+Na]⁺ at 505, [M+K]⁺ at 521, [2M+Na]⁺ at 987 and [2M+K]⁺ at 1003 in the MALDI-TOF-MS spectrum. The presence of phenolic hydroxyl group in the molecule was recognized from grey-green color with ethanolic FeCl₃ solution on TLC(Imakura et al, 1985). In its IR spectrum, absorption bands at 3380, 1738, 1615, 1511, 1435, 1230, 1185, 875, 830, 735 (cm^-1 in KBr) were observed. Its UV spectrum showed maxima at 262 nm (in MeOH). In ¹H- and ¹³C-NMR spectra, A characteristic anomeric mixture of α- and β-D-glucose and connecting 4,4′,5,5′,6,6′,-hexahydroxydiphenoyl (HHDP) residues were observed. However, compound I, a pair of epimers, could not be completely separated by column chromatography though it appeared as only one spot on two-dimensional paper TLC. Its spectroscopic data were identical with those reported by Seikel et al, (1970), thus compound I was identified as 2,3-O-4, 4′,5,5′,6,6′-HHDP-(α/β)-D-glucose.

Compound II was obtained as a pale brown amorphous powder for the first time from this species. The melting point was 135–137°C. We found its optical rotation αD20 of +106° (c 0.1 in MeOH). Its IR and UV absorption bands were similar with those of compound I described above. R_f of this compound are 0.29 (solvent A) and 0.55 (solvent B). The MALDITOF- MS spectrum of compound II gave peaks of m/z [M+Na]⁺ at 807, [2M+K]⁺ at 823, [2M+Na]⁺ at 1591, [2M+K]⁺ at 1607, respectively, corresponding to the molecular formula C₂₀H₁₈O₁₄. Similar to that of compound I, compound II was also an epimeric compound, which was existing in equilibrium as anomeic mixture of α- and β-D-glucoses as described in previously published literatures (Tsujita et al., 2017; Seikel et al., 1970). In the ¹H-NMR spectrum of compound II, two sets of partially overlapped α- and β-D-glucose were confirmed. In addition, four sets of 4,4′,5,5′,6,6′,-hexahydroxydiphenoyl (HHDP) moieties were observed. The ¹³C-NMR of compound II also presented duplicated signals due to the presence of a pair of epimers. These ¹H- and ¹³C-NMR data were coincided with those reported in literature (Seikel et al., 1970) and compound II was elucidated as pedunculagin consequently.

4. CONCLUSION

By successive liquid-liquid fractionation and repeated TLC-monitored purification of 70% aqueous acetone extraction of the J. mandshurica barks, two ellagitannins were isolated and their chemical structures (Fig. 2) were elucidated as 2,3-O-4,4′,5,5′,6,6′-HHDP-(α/β)-D-glucose (I) and pedunculagin (II) based on their chemical and spectroscopic evidences, and a careful comparison with previously published data. Compounds I and II were epimeric ellagitannins and they were isolated as anomeric mixtures of α- and β-D-glucoses in the current work as described in literatures. It is noteworthy that this is the first report of compounds I and II from J. mandshurica.

Fig. 2. Structures of isolated epimeric ellagitannins from J. mandshurica stem bark.

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The activity of the two epimeric ellagitannins (I and II) were evaluated by a classical pathway of complementary system assay, and our investigation results indicated that both the two compounds inhibited excellent effects and could be used as potential anti-complementary agents.