Coronarin K and L: Two Novel Labdane Diterpenes From Roscoea purpurea: An Ayurvedic Crude Drug

The main objective of cancer treatment with chemotherapy is to kill the cancerous cells without affecting the healthy normal cells. In the present study, bioactivity-guided purification of the n-chloroform soluble fraction from the methanol extract of Roscoea purpurea resulted in the identification of two new labdane diterpenes: coronarin K (1) and coronarin L (2), along with eight known compounds, coronarin A (3), bisdemethoxycurcumin (4), kaempferol 3-O-methyl ether (5), kaempferol (6), fenozan acid (7), 3-(3-methoxy,4-hydroxyphenyl)-2-propenoic acid ferulic acid (8), caffeic acid (9), and gallic acid (10). The structural identification of new compounds (1 and 2) were determined by detailed analysis of 1D (1H and 13C) and 2D NMR (COSY, HSQC, and HMBC) spectroscopic data. The relative configurations of 1 and 2 were determined with the help of NOESY correlations and comparison of optical rotations with known labdane diterpenes, with established stereochemistry, while structure of known compounds was established by direct comparison of their NMR data with those reported in the literature. This is the first report of isolation of this labdane diterpenes and phenolic classes of secondary metabolites in R. purpurea. In the preliminary screening, the methanol extract and its fractions were tested for the cytotoxic activity against a panel of four cancer cell lines (A549, HCT-116, Bxpc-3, and MCF-7); extract and its chloroform fraction were found to be active against the lung cancer cell line, A-549, with IC50 value <25 μg/ml. Owing to the notable cytotoxic activity of the chloroform fraction, the compounds (1–5) were screened for their cytotoxicity against all the cell lines by MTT assay. Coronarin K, 1 showed significant cytotoxic potential against lung cancer cell lines (A-549), with IC50 value of 13.49 μM, while other compounds did not show activity below 22 μM.


INTRODUCTION
In our continuing research program to discover bioactive natural products from natural resources, especially from high-altitude Himalayan endangered medicinal plants, with profound biological activities, our attention was focused on the rhizomes of Roscoea purpurea, commonly known as kakoli, which belongs to the family of Zingiberaceae that is abundantly available in alpine grassland, grassy hillsides, and stony slopes of central to eastern Himalaya from Uttarakhand to North East states, up to an altitude of 3,300 m. It is an essential ingredient of an important Ayurvedic preparation, known as Astavarga, which is a group of eight plants claimed to be useful for healing weakness, body overweight, bone fractures, high fever, and diabetic situations, as well as for healing vata, pitta, and rakta doshas (Dhyani et al., 2010). The groups of Astavarga plants are considered as a very excellent Rasayana, with health-promoting and rejuvenating properties, and are recognized to support our immunity and have the capacity for cell renewal (Balkrishna et al., 2012). Astavarga plants are also known to restore human health and work as strong antioxidants in our body (Mathur, 2003;Pandey, 2005;Sharma and Balkrishna, 2005). Among the eight Astavarga plants, R. purpurea is one of the essential ingredients of several herbal formulations like tonic and Chyawanprash. Traditionally, it is used for the cure of diabetes, diarrhea, hypertension, fever, immunostimulant, inflammation, etc. In Nepal, its tubers are boiled for an edible purpose and are also used in traditional veterinary medicine (Singh and Rawat, 2011). In view of its significance in the Ayurvedic medicinal system, substantial pharmacological and phytochemical works have not been carried out. Previous phytochemical studies on R. purpurea have described the isolation of two principal groups of compounds: steroids and phenolic derivatives (Misra et al., 2015;Srivastava et al., 2015;Rawat et al., 2016). To date, only a few compounds have been identified and quantified through high-performance liquid chromatography (HPLC) analysis from tubers of this plant by Singh and co-workers, and they are presumed to be associated with its potent antioxidant activity (Gopal et al., 2014;Rawat et al., 2014). Alcoholic extracts of the plant have shown in vitro anti-cancer , antioxidant (Rawat et al., 2016), and immunomodulatory activities (Sahu et al., 2010). In our preliminary pharmacological study, cytotoxic activity was found for the methanol extract of R. purpurea against lung cancer cell line at IC 50 25.71 (µg/ml). On bioassay-guided fractionation, activity was localized in a chloroform-soluble fraction. Further work led to the isolation of two new compounds, along with eight known compounds for the first time from this plant species. In the present paper, we have described isolation and structure elucidation of two new compounds from the rhizomes of R. purpurea and the anti-cancer activities of plant extract, fractions, and isolated compounds.

Plant Material
Rhizomes of R. purpurea Sm. (Syn: Roscoea procera Wall.) were collected in September 2013 from Dhanaulti, Mussorie in district Tehri Garhwal of Uttarakhand, India (between 2,500 and 3,200 m asl). The crude herb was authenticated by Dr. Sumeet Gairola, and the voucher specimen (CDR No. 4027) was deposited in the Crude Drug Repository of CSIR-IIIM, Jammu.

Cell Proliferation Assay
Colorimetric MTT assay was used to carry out cell viability studies (Kumar et al., 2013). The plant extract and isolates were screened at different concentrations with a maximum concentration of 100 µM. The cells were rinsed and incubated with 20 µl of MTT dye after treatment at different time intervals. The medium was removed, and DMSO was added to each well after incubation for the solubility of purple formazan crystals. Microplate Reader (BioTek Synergy HT) was used to measure the absorbance at 570 nm. GraphPad Prism Software (La Jolla, CA, United States) was used to determine cell viability percentage and IC 50 values.

Detection of Reactive Oxygen Species (ROS) Accumulation
Lung cancer cell line (A-549) seeded in six-well plates (2 × 10 5 ) was treated with coronarin K 1 (7, 13, 18 µM) and incubated for 48 h. H 2 O 2 was added to a well containing a medium for 2 h before termination. The cells were further incubated with 10 µM DCF for 30 min at 37 • C in the dark. The cells were washed with PBS and analyzed with a fluorescence microscope (Olympus-1X53 magnification). The fluorescent intensity was measured at the emission and excitation wavelength of 480/530 nm (Banskota et al., 2015).

Identification of Compounds
The dried and powdered rhizomes of R. purpurea were extracted with methanol and dried at 40-45 • C. The obtained crude extract was sequentially fractionated with pet ether, CHCl 3, and n-butanol. The chloroform soluble fraction was purified, using chromatographic techniques, which afforded two new compounds (1-2), together with 8 known compounds. The structures of both novel secondary metabolites were established, using detailed NMR spectroscopic studies, whereas the structures of known compounds were identified in comparison with the previously reported literature data. The 1 H and 13 C NMR spectra of compounds 1 and 2 have some common structural features of diterpenes based on the NMR data and gave a positive copper acetate test for diterpenes.
The backbone of the bicyclic ring system was established, using detailed analysis of HMBC correlations from the three methyl signals: δ H 1.02, 1.22, and 1.18, which obtained as a singlet in the 1 H NMR spectrum and showed HMBC correlation with the H 3 -18 and H 3 -19 to C-3, 4, 5; H 3 -20 to C-1, 5, 8, and 10. The 1 H NMR signals of the exocyclic methylene (δ H 5.05, 4.77, δC 110.3) revealed strong HMBC correlations with H 2 -17 to C-7, 8, 9. The methylene H-9 also showed HMBC to C-5, 7, 10, 17, 20. Along with these, a correlation from H-9 to the oxymethine carbon (δ C 65.0) is also revealed by HMBC. The presence of a decalin ring system was established with COSY correlations indicating a coupling from H 2 -6 to H-5 and H-7 and from H 2 -1 to H 2 -2 and H 2 -3.
All above HMBC and COSY correlations established connection of the bicyclic ring with furan ring. The other signal to be assigned was a methylene group CH 2 -11 (δ H 1.84; δ C 32.7). HMBC correlations from the H 2 -11 signal to C-12, 13 and H-12 to C-14 and 16 confirmed the connection of the C-9 to C-10 and C-12 to C-13 furan ring. Thus, the structure of 1 was characterized as Coronarin K, a new labdane diterpene. The relative signs of 1 were established by NOE correlations observed in the NOESY experiment (Figure 3) and observed optical rotation +23.3 • (c.23, CHCl 3 ), which is consistent with published data for a similar skeleton (Itokawa et al., 1988). NOE correlations from H 3 -20 to H-11, and H 3 -18, together with correlations from H 3 -19 to H-5, H-9; H-5 to H-9; H-9 to H-8; H 2 -17 and H-12 to H-16, established the decalin ring system. Thus, the structure of coronarin K (1) is defined as 5S * , 7S * , 9R * , 10S * , 12S. Coronarin L (2) was isolated as a white solid. The molecular formula of Coronarin L (2) was established as C 20 H 28 O 3 , based on the ESIMS and NMR data. The cyclic lactone group in 2 was confirmed in the IR spectrum (1,750 cm −1 ) by the presence of C=O-stretching bands (Itokawa et al., 1988). NMR data comparison (Table 1) revealed that 2 was structurally similar to 1, except for the presence of a diene system [δ H 6.88 (1H, dd, J = 15.8, 9.8 Hz Hz), δ C 135.9 (C-11); δ H 6.08 (1H, d, J = 15.6 Hz),  Frontiers in Chemistry | www.frontiersin.org δ C 121.0 (C-12)] in place of oxymethine at C-12 as it was missing in 13 C spectrum. Furthermore, changes in the proton and carbon chemical shifts of the furan ring suggested oxidation of the C-14-C-15 double bond to a carbonyl group. HMBC correlations from the methine signals at δ H 4.87 (2H, s, H-16) to C-15 (C=O) and C-14 (δ H 7.10, s), together with HMBC correlation with diene C-12, confirmed the presence of a lactone ring at C-12 adjacent to a double bond. A COSY correlation, together with the large 1 H coupling constant (J = 15.8 Hz), between H-11 and H-12, established the E geometry of the C-11-C12 double bond. NOE correlations in 2 (Figure 2) and observed optical rotation +36.9 • (c.20, CHCl 3 ) were identical to that of 1; therefore, the structure of coronarin L (2) was defined as 5S * , 7S * , 9R * , 10S * .

Anti-cancer Activity
Secondary metabolites, which were isolated from the plants of the Zingiberaceae family, have wide use as a new pharmacophore in anti-cancer drug discovery. In our bio-assay-guided isolation of novel anti-cancer agents from R. purpurea, the methanol extract and respective fractions were screened for their anticancer activity, using the MTT assay. The methanol extract and its CHCl 3 fraction showed cytotoxic activity against lung cancer cell line (A549) at IC 50 27.71 and 21.35 (µg/ml), respectively. After purification of chloroform fraction, compounds 1-10 were isolated. Only compounds 1-5 were evaluated for different cancer cell lines. Among the tested compounds, Coronarin K (1) showed prominent activity against lung cancer cell line (A-549) and also showed moderate cytotoxic activity against colon cancer cell line HCT-116 (IC 50 value of 26.03 µM) but had no effect on Bxpc-3 and MCF-7 in acceptable limits. Compounds 2 and 3 were ineffective against all the cell lines except pancreatic cancer cell line (Bxpc-3) at IC 50 value of 26.03 µM. The remaining tested compounds displayed less activity ( Table 2). In a further study, the A-549 cells were treated with compound 1 at 7.0, 13.0, 18.0 µM, and ROS generation was detected in 48 h. Hydrogen peroxide was taken as a positive control. After exposing the cells with compound 1, the fluorescence intensity of DCF was increased in a dose-dependent manner. The results showed that compound 1 exhibited prominent cytotoxicity against human lung cancer cell line (A-549), with IC 50 value of 13.49 µM (Figure 3) and thereby confirmed the ROS generation is crucial to cell death.

CONCLUSION
In the present study, by the cytotoxic activity-guided isolation of R. purpurea extract, we have made an unusual finding of two new labdane diterpenes coronarin K (1) and coronarin L (2), together with the other known compounds 3-10. All the compounds have been isolated physically for the first time from this plant. Furthermore, this is the first report of the natural origin (Fenozan acid 7) of a synthetic di-tert-butyl derivative in plants. In addition to this, we have evaluated the anti-cancer activity of extract, chloroform fraction, and isolated compounds. Among all the tested compounds, only coronarin K (1) showed prominent anti-cancer activity against lung cancer cell line (A-549). Our study also suggest that the antiproliferative effect of the compounds is due to ROS generation and also led to the impaired cell migration. Finally, our study strongly suggests that screening and detailed chemical study of endangered Astavarga plants would be valuable.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
PG conceptualized and wrote the paper. VS and BL performed isolation and characterization of compounds. PK and SG helped in the collection and the identification of the plant material. JS and SS performed the anti-cancer activity. All the authors read and approved the final manuscript.