Curcubinoyl-conjugated flavonoids from methyl jasmonate-treated 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. Curcubinoyl moiety is structurally similar with methyl jasmonate, which was used as an elicitor. Therefore, our present study suggested that elicitor might increase the content of active constituents and also contribute to the production of new metabolites by participation as precursors in biosynthesis. Taken together, plant cell cultures are useful not only for efficient production of active compounds but also development of new secondary metabolites. NMR were on a Avance and 800 MHz spectrometers using CD 3 OD as Silica gel Sephadex and Diaion HP-20P (Mitsubishi Kasei, Japan) for column chromatography. TLC was performed on silica gel 60 F 254 (0.2 mm, Merck) or silica gel 60 RP-18 F 254S (0.2 mm, Merck), and spots were detected by a 10% vanillin-H 2 SO 4 in EtOH spray reagent. MPLC was performed on a Biotage Isolera Prime chromatography system and a Lichroprep RP-18 column (40-63 μm). Semi-prep HPLC was performed using a Waters system (three 515 pumps and a 996 photodiode array detector) with a Phenomenex Gemini-NX column (USA).


Introduction
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][5][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 4 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.

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 . In addition, a carbonyl signal was observed at δC 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.  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 C33H40O13, 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 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, 9 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. 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.

Conclusion
Conclusively, in vitro tissue culture techniques of plant cells have been utilized not only for stable production with improved biomass and bioactive metabolites accumulation but also for the discovery of new secondary metabolites. In addition, elicitor controls the biosynthesis of metabolite and can contribute to development of new metabolite by participating in biosynthetic pathway as a substrate.

Materials and methods
General experimental procedure IR spectra were obtained using JASCO FTIR 4100 spectrometer in CH3OH 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

Plant Material
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 The root cultures were  Table 1.