Curcubinoyl flavonoids from wild ginseng adventitious root cultures

Wild ginseng (Panax ginseng) adventitious root cultures were prepared by elicitation using methyl jasmonate and investigated further to find new secondary metabolites. Chromatographic fractionation of wild ginseng adventitious root cultures led to the isolation of eleven compounds. The chemical structures of isolated compounds were identified as four known flavanone derivatives (1–4), one new curcubinoyl derivative, jasmogin A (5) and six new curcubinoyl-flavanone conjugates, jasmoflagins A-F (6–11) by extensive spectroscopic analysis. Newly isolated curcubinoyl derivatives showed inhibitory activity against lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. Therefore, our present study suggested that elicitor stimulated plant cell cultures might contribute to the production of new metabolites.

Natural products contain a variety of ingredients and have long been used to prevent and treat diseases. However, securing natural products is essential in order to develop these natural products, which is sometimes not easy due to various constraints. Plant tissue culture technology is suggested as a powerful tool for obtaining natural substances [1][2][3] . This is widely used for the production of plant materials because it is less affected by weather and other external conditions than plant cultivation and relatively for a short period of time.
For maximum productivity, culture conditions such as culture medium and the incubation conditions, etc., are optimized when growing plant tissues [4][5][6] . In particular, the use of elicitors is widely used for increased productivity and useful substances. As elicitors, salicylic acid and methyl jasmonate (MJ), which control the immune of plants, are most widely used [7][8] . These elicitors greatly increase the content of biomass and useful metabolites 4,9 . Moreover, new ingredients have been reported in elicitor-stimulated plant cell culture [10][11] . Therefore, plant tissue culture has become an important tool not only for securing plant materials but also finding new metabolites.
Panax ginseng C.A. Meyer (Araliaceae) is commonly known as Korean ginseng. It is one of the most widely used tonic to enhance immune response and consequent health and longevity for over 2000 years in Oriental countries 12 . Various efficacy of P. ginseng, including anti-cancer, anti-inflammatory, anti-diabetic, anti-fatigue and neuroprotective activities have been also reported from a lot of research [13][14][15][16] .
Ginseng grows in wild environment or is cultivated on farm. Cultivated ginseng is systematically grown on farm under the control of growth condition and harvested after 4-6 year cultivation. The wild ginseng, also called mountain ginseng in Korea, grows without human touch in deep areas with low sunlight and temperature changes. This difference in the cultivation environment and genotypes leads to differences in the composition and efficacy of the two specimens. Wild ginseng has been reported to have enhanced host defense components and biological activities. The concentration of ginsenosides and amino acids in wild ginseng were much higher than those of cultivated ginseng 17,18 . However, due to the short supply and consequent high price of wild ginseng has limited its usage despite of beneficial biological activities. Therefore, sufficient production is required for the development as products. As a preparation of wild ginseng, tissue culture system is considered as a valuable tool to achieve rapid and stable production of excellent individual. We previously established efficient adventitious root cultures of wild ginseng with fast growth and stable production 19,20 . In addition, we also demonstrated the increased yield and antioxidant activity of MJ-elicitated wild ginseng adventitious root cultures compared to MJ-untreated samples 21 . In the present study, MJ-treated wild ginseng adventitious root cultures were investigated further to find new secondary metabolites.

Results and discussion
Isolation of compounds from MJ-treated wild ginseng adventitious root cultures. Plant cell cultures were used not only for the stable production but also useful to find new secondary metabolites for better pharmacological activity [9][10][11] . Investigation on the constituents of the adventitious root cultures of P. ginseng yielded eleven compounds including seven new compounds (Fig. 1). Known compounds were identified as naringenin (1), naringenin-4′-O-β-glucoside (2), naringenin-7-O-β-glucoside (3) and hesperetin 7-O-β-glucoside (4) by the analysis of their spectroscopic data and comparison with literature values 22-24 . Structural determination of the new compounds. Compound 25.5 (C-8)]. In addition, a carbonyl signal was observed at δ C 175.9 (C-1) in the 13 C NMR spectrum. In the HMBC spectrum, correlations from H-4 to C-6, 7 and from H-5 to C-3 suggested the presence of cyclopentyl moiety in 5. These NMR spectroscopic data of 5 were quite similar to those of curcurbic acid, a hydroxylated jasmonate derivative 25 , except for the additional hydroxyl group. The position of an additional hydroxyl group was determined to be C-11, which was confirmed by the HMBC correlations from H-11 to C-8, 12 (Fig. 2). The stereochemistry was determined by the NOESY correlations between H-6, H-7 and H-2 and between H-8 and H-11 (Fig. 2). Taken together, compound 5 was defined as shown and named jasmogin A.
Compound 6 was purified as a white amorphous powder and assigned the molecular formula as C 33 H 40 O 13 by its HRESI-MS (m/z 667.2354 [M + Na] + , calcd. C 33 H 40 NaO 13 667.2331). The NMR spectroscopic clearly showed that 6 has hydroxylated curcurbic acid moiety of 5 as a partial structure. Additionally, compound 6 was supposed to be a glycoside from the anomeric signals at   (1) 23 . Taken together, 6 was suggested as a flavanone glycoside consisting of naringenin, glucose and curcurbic acid moieties. The linkage of each unit was determined by cross peaks between H-1″ of glucose and C-7 of naringenin, and between H-6″ of glucose and C-1‴ of curcurbic acid moiety in the HMBC spectrum (Fig. 3). Collectively, compound 6 was defined as shown and named jasmoflagin A. Compound 7 was purified as a white amorphous powder and showed an HRESI-MS ion at m/z 667.2354 ([M + Na] + , calcd 667.2361) for C 33 H 40 NaO 13 . The spectroscopic data of 7 were quite similar to those of 6, which suggested that 7 is also a curcurbinoyl derivative of naringenin glycoside. Careful comparison of 1 H and 13 C NMR data of 7 with those of 6 showed the differences in the chemical shifts of H-11 and H-12. The hydroxymethine   were quite similar to those of 6, except for the disappearance of hydroxymethine proton at δ H 4.05 (H-6‴) of 6. Additional carbonyl signal at δ C 220.1 in the 13 C NMR spectrum suggested that 8 is a jasmonate derivative of naringenin glycoside. Further HMBC correlation from H-4‴, H-5‴, H-7‴ to C-6‴ confirmed the presence of carbonyl moiety at C-6‴ (Fig. 3). Taken together, compound 8 was determined as shown and named jasmoflagin C.
Compound 9 was purified as a white amorphous powder and showed an HRESI-MS ion at m/z 651.2404 ([M + Na] + , calcd 651.2412) for C 33 H 40 NaO 12 . The spectroscopic data of 9 suggested that 9 is also a curcurbinoyl derivative of naringenin glycoside. However, on the contrary to 6 and 7, hydroxymethine signals in curcurbic acid were not observed in the 1 H and 13 C NMR data of 9. Further analysis demonstrated that the hydroxylmethine signals at [δ H 4.62 (1H, m, H-11); δ C 62.5] in 6 were replaced by methylene signals at [δ H 2.06 (2H, m, H-11); δ C 20.1] in 9. In addition, HMBC correlation between H-10‴ and C-11‴ and between H-11‴ and C-12‴ confirmed the detachment of hydroxyl group at C-11 in 6 (Fig. 3). Taken together, compound 9 was determined as shown and named jasmoflagin D.
Compound 10 was purified as white amorphous powder and assigned the molecular formula as C 33 H 40 O 13 , which is same as 6. The spectroscopic data of 10 were quite similar to those of 6 and suggested 10 is comprised of naringenin glucoside and curcurbic acid with a hydroxyl group. Differences in the 1 H and 13 C NMR data of 10 from those of 6 were observed as downfield shift of CH 3 -12‴ from δ H 1.20 to δ H 1.68 and upfield shift of H-8‴ from δ H 2.04, 2.31 to δ H 1.60, 1.70, suggesting the change in the positions of hydroxyl group and double bond in curcurbic acid moiety. The 1 H-1 H COSY correlations of H-8‴/H-9‴ and H-11‴/H-12‴ together with HMBC correlations from H-12‴ to C-11‴ determined the position of hydroxyl group at C-9‴ (Fig. 3). Taken together, compound 10 was determined as shown and named jasmoflagin E.
Compound 11 was obtained as white amorphous powder. The molecular formula of 12 was determined as C 33 H 43 NO 13 by its HRESI-MS (m/z 668.2415 [M + Li] + , calcd. C 33 H 43 NLiO 13 668.2889). The 1 H and 13 C NMR spectrum also proposed 11 as a curcubinoyl derivative of naringenin glucoside. The 1 H and 13 C NMR spectrum of 11 were comparable to those of 10, except for the disappearance of signals for double bond of curcurbic acid in 10. Additional methine signal at [δ H 3.24 (1H, m, H-10‴); δ C 74.5] and methylene signals at [δ H 1.40 (2H, m, H-11‴); δ C 26.2] were observed in 11. These data suggested the presence of amine group to curcubinoyl moiety of 10, which is also confirmed by the presence of nitrogen in 11 from MS analysis. The positions of hydroxyl and amine groups were determined to be C-9‴ and C-10‴, respectively, by the HMBC correlations from H-7‴/8‴ to C-9‴ and from H-8‴/12‴ to C-10‴ (Fig. 3). Taken together, compound 11 was defined as shown and named jasmoflagin F. NO inhibitory activity of isolated compounds. Next, we investigated the anti-inflammatory effects of newly isolated compounds by measuring the production of NO in LPS-stimulated RAW 264.7 macrophages. As shown in Fig. 4, compounds 5, 7 and 10 dose-dependently reduced NO production stimulated by LPS without any significant cytotoxic effects at the concentration ranging from 5 to 50 μM. Compound 5, which has only curcubinoyl moiety, inhibited NO production. However, addition of flavanone moiety to compound 5 reduced the inhibitory activity, as observed in compound 6. Interestingly, among the curcubinoyl flavanone derivatives, compounds 7 and 10 showed stronger inhibitory activity compared to others, which suggested the importance of the position of hydroxyl group in curcurbinoyl moiety.

Conclusion
Fractionation of using various chromatographic techniques yielded eleven compounds from the MJ-treated adventitious root cultures of wild ginseng. The chemical structures of isolated compounds were identified by spectroscopic analysis and further identified seven new compounds. The newly reported compounds are curcubinoyl derivative, named jasmogin A (5) and curcubinoyl-conjugated flavanone derivatives, named jasmoflagins A-F (6)(7)(8)(9)(10)(11). Considering the structural similarity between methyl jasmonate and curcubinoyl moiety, addition of elicitor can affect not only the increase of biosynthesis of active metabolite, but MJ itself also participate in biosynthetic pathway as a substrate, which needs to be clarified by further study.

Materials and methods
General experimental procedure. IR spectra were obtained using JASCO FTIR 4100 spectrometer in CH 3 OH solvent. Optical rotations were measured on a JASCO DIP-1000 polarimeter (Tokyo, Japan). HRESIMS data were measured on maXis 4G (Bruker) and LCQ Fleet (Thermoscientific), respectively. NMR spectra were recorded on a Bruker Avance 400, 500 and 800 MHz spectrometers using CD 3  Adventitious root cultures of wild ginseng (P. ginseng) were produced from a 100-year-old wild ginseng through callus culture as we described previously 20 Table 3. 13 C NMR spectroscopic data for compounds 6-11 (CD 3 OD).