Major chemical constituents from Illicium griffithii Hook. f. & Thoms of North East India and their cytotoxicity and antimicrobial activities

Illicium griffithii Hook. f. & Thoms is an endemic medicinal plant of North East India found in the Eastern Himalayan region of biodiversity mega centre. Herein, chemical investigation of I. griffithii, afforded five compounds and their structures were determined through extensive use of NMR, HRMS, and FT-IR spectroscopy. The complete proton-proton, proton-carbon coupling network of compound 1 was determined using 1H-1H COSY, HSQC and NOESY NMR experiments. All the compounds were evaluated for their cytotoxic activity by MTT assay and antimicrobial activity by Agar well diffusion method. Compound 1 exhibited significant cytotoxicity activity against Lung cancer (A549) and pancreatic cancer (MIAPaCa2) cell lines with IC50 values of 15.01 ± 2.69 μg/mL and 47.77 ± 2.38 μg/mL, respectively. Further, the compound 1 exhibited good antimicrobial activities against Escherichia coli and Candida albicans with MIC 7.50 ± 0.28 μg/mL and 7.50 ± 0.86 μg/mL, respectively. The other isolated compounds along with the extracts of I. griffithii also displayed moderate anticancer and antimicrobial activities against respective strains. To the best of our knowledge, this is the first study of isolation of compounds from bark, wood, and leaf along with cytotoxicity and antimicrobial activities of I. griffithii from the North Eastern region of India and could be a potential herbal medicine in near future.

, we have investigated the leaves, bark, and wood of this plant and the results are presented herein. However, there is no report available till now to identify these bioactive compounds with proper spectroscopic analyses from the parts of I. griffithii.

General
Fractionation and isolation of compounds were done by column chromatography using 60-120 mesh silica gels. TLC experiments were carried out using precoated Silica gel 60 F 254 sheets (Merck, Darmstadt, Germany). 1D and 2D NMR were recorded with a Bruker AVANCE DPX 500 MHz NMR spectrometer, Switzerland with tetramethylsilane (TMS) as the internal reference. Melting points were measured using BUCHI M-560 capillary melting point apparatus. High-Resolution Electro-Spray Ionization Mass spectra (HR-ESI-MS) were recorded using Waters XEVO G2-Xs QT LC-MS system. FT-IR spectra were recorded in Elmer FT-IR 2000 spectrometer on a thin film using chloroform. A549 Lung cancer and MIAPaCa2 pancreatic cancer cell lines were purchased from NCCS, Pune, India. MTT was purchased from Sigma-Aldrich Co (St Louis, MO, USA). The absorbance was measured on an ELISA plate reader (FilterMax F3 Multi-Mode Microplate Readers, Molecular Devices) with a test wavelength of 570 nm and a reference wavelength of 630 nm.

Plant material
The leaves, bark, and wood of I. griffithii were collected by Prof. M. Bordoloi from Arunachal Pradesh, India in July 2016. The plant material was identified by Prof. M. Bordoloi and a voucher specimen (No. NPC/298-300) was deposited at the CSIR-North East Institute of Science and Technology, Jorhat, India.

Measuring cell viability
The cell lines A549 and MIAPaCa2 were cultured in respective complete media DMEM, Ham's F12k and MEM, respectively and supplemented with 10% Foetus Bovine Serum, 1% Gentamycin (antibiotics), 10% Penstrep. However, cells (1 × 10 6 per mL) were seeded in tissue culture grade multi-well plates in complete medium. The plates were incubated under standard conditions in 37 °C humidified atmosphere containing 5% CO 2 for 24 h. After incubation, the whole medium was replaced with FBS free medium and incubated overnight. Afterwards, the cells were treated with the samples of I. griffithii in different concentrations in each well and incubated for 24 h. Well holding medium alone (untreated cells) served as a control.
By using in vitro MTT assay, the cytotoxicity study was evaluated. At first, each well was mixed with 10 mL of MTT (5 mg/mL), and incubated for 4 h. After observation of dark purple crystals of formazan at the bottom of the wells by an inverted microscope, 0.04 N HCl with Isopropanol (100 mL) was mixed to each well and prepared the solution suitable for absorbance measurement. The effect of the samples on the proliferation of cells was expressed as the % cell viability. The half maximal inhibitory concentration (IC 50 ) of the samples was determined from the dose response plotted curves [21]. 2.6. Antimicrobial assay 2.6.1. General The extracts and isolated compounds were tested for antimicrobial activity against microorganisms using the reported method [22]. Four microbes Escherichia coli (ATCC ® 11229 TM ), Staphylococcus aureus (ATCC ® 11632 TM ), Pseudomonas aeruginosa (ATCC ® 27853 TM ), and Candida albicans (ATCC ® 90028 TM ) for antimicrobial activity were purchased from HiMedia. Mueller Hinton Agar (MHA) media was prepared by dissolving 3.8 g of MHA in 100 mL distilled water. After solidification of the media, inoculums of approximately 1 × 10 8 CFU/mL were spread over the plate. Then, 6mm wells were made in the solidified media using a cork borer. Hundred µL of test samples were poured into the required wells and plates were incubated. For bacteria, incubation time is 48 h at 37 °C and for fungus 18 h at 30 °C. The experiment was carried out in triplicates. Antimicrobial activities were evaluated by measuring the zone of inhibition against test microbes and results were presented as mean.

Minimum inhibitory concentration (MIC)
Minimum inhibitory concentration was determined by resazurin reduction assay in 96 well microtitre plates (Nunc™, Thermo Fisher Scientific Inc). At first 100 µL of sterile broth was added to all 96 wells and test samples were serially diluted. Then 20 µL of bacterial and fungal suspension was added to each well to achieve a concentration of 5 × 10 5 CFU/mL. Finally, 20 µL of resazurin indicator was added to each well. Plates were incubated at 30 °C for 18-42 h and colour change was then assessed visually. The plates were observed visually for the colour change of the indicator. The colour change from purple to colourless indicates the growth of microbes. MIC of test samples was determined as the lowest concentration of the compound at which no microbial growth was observed [23].

Results and discussions
In this study, we describe the isolation of five compounds (1-5) from I. griffithii as well as cytotoxicity and antimicrobial activity (Figure 1). Compound 1 was obtained as colourless oil from ethanol extracts of bark. Its molecular formula was determined as C 15 H 18 O 3 by elemental analysis and HR-MS with m/z 247.1451 for pseudo-molecular ion [M+H] + . In FT-IR spectrum, the absorption band observed at 1726.1 cm -1 is due to the presence of a carbonyl group in the structure. In the 1 H NMR spectrum, the two singlets at δ 5.74 and 5.73 ppm each integrated into one proton are assigned to a methylenedioxy group. Further, two singlets that appeared at δ 5.52 and 5.35 ppm are assigned to H-6 and H-3 protons of C 6 ring respectively. Two-three proton singlets at δ 1.50 and 1.55 were assigned to methyl connected to a double bond. The double doublet signals at δ 2.17 with J = 14.0 and 7 Hz and 2.44 ppm with J = 14.0 and 7 Hz each integrating to one proton attributed to a methylene group attached to a prenyl moiety. The presence of an allyl moiety was indicated by two single proton signals at δ 2.13 with J = 13.5 and 7.5 Hz and 2.53 ppm with J = 13.5 and 7 Hz and two olefinic signals at δ 4.87 with J = 1.5 and 10.5 Hz and 4.93 ppm with J = 1.5 and 17 Hz along with a multiplate at 5.49 ppm integrated to one proton. Thus, this proton spectrum is very similar to the compound isolated from I. anisatum as reported in the literature [25]. The structure of compound 1 is further confirmed by its 13 C NMR spectrum. The presence of a carbonyl group was confirmed by 13  carbonyl group was confirmed by 13 C NMR signal at δ C 186.57. Based on this evidence and by comparison with the spectral data of the compound reported from I. tashiroi [26], the structure of the molecule was confirmed as Illicinone G (2  14.03], suggesting that compound 5 is long-chain alkene. The structure was confirmed by comparison with the spectral data of literature determined as 1-Eicosene (3) [27].
Compound 4 was obtained as colourless oil. Its molecular formula, C 16 H 22 O 3 , as determined by elemental analysis and HR-MS with m/z 263.1465 for pseudo-molecular ion [M+H] + . The IR spectra displayed an absorption band attributable to the methoxy group at 2853.8 cm -1 . The NMR spectrum exhibited the presence of two methoxy groups at δ H 3.84 and δ C 56.02. The 1 H NMR data indicated the presence of two aromatic protons H-2 and H-6 at δ H 6.40. An allyl group showed signals including a multiplet at δ H 5.97 integrating to one proton of H-8, a doublet at δ H 5.09 integrating to two protons at H-9, a doublet at δ H 3.34 integrating to two protons at H-7. The presence of a prenyl moiety displayed signals including two singlets at δ H 1.67 and 1.75 for two methyls, a doublet at δ H 4.46 integrating to two protons at H-10 and a triplet of septets at δ H 5.58 integrating to one proton at H-11. The structure was confirmed by comparison with the spectral data of the compound reported from I anisatum [25] as 1-allyl-3,5-dimethoxy-4-(3-methylbut-2-enyloxy)benzene (4). .70], suggesting that compound 5 is long-chain alkene. The structure was confirmed by comparison with the spectral data of literature determined as Tridec-1-ene (5) [28]. In this study, two prenylated C 6 -C 3 (1, 2) and one olefin (3) were isolated from the bark, and one phenolic (4) & one long chain hydrocarbon 5 were isolated from the leaf of I griffithii. Prenylated compound 1 was also isolated from wood of I griffithii. All five compounds were isolated from this Illicium species for the first time.
The cytotoxicity of I. griffithii was established by MTT assay against lung cancer (A549) and pancreatic cancer (MIAPaCa2) cell lines. The control cells showed high proliferation that has been taken as 100%. There is a decrease in the percentage of viable cells with an increase in doses of samples. A treatment with a 100 µg/mL dose of Compound 1 to A549 and MIAPaCa2 cells shows a significant decrease in the viable cells by almost -5.176% and 2.021% respectively at 24 h. Compound 1 induced significant cytotoxicity against A549 and MIAPaCa2 with IC 50 values of 15.01 ± 2.69 µg/mL and 47.77 ± 2.38 µg/ mL, respectively. The similar types of compounds (2, 4) exhibited moderate activity against A549 and MIAPaCa2 with IC 50 values ranging from 67.69-240.42 µg/mL and 74.60-262 µg/mL, respectively. For long-chain hydrocarbons compound 3 & 5 exhibited moderate anticancer activity against A549 and MIAPaCa2 with IC 50 240.42 ± 0.54 µg/mL; 262.00 ± 2.03 µg/ mL and 174.12 ± 1.12; 292.25 ± 0.92 µg/mL, respectively. Earlier, we have found that such types of compounds inhibit A549 lung cancer cells through PI3K pathway inhibition. So, it may be possible that these compounds from this plant showed anticancer activity through AA pathways of PI3 kinase pathway of cancer growth [23]. The results were compared with standard Doxorubicin with IC 50 values of 1.62 ± 1.86 and 0.47 ± 2.63 against A549 and MIAPaCa2 respectively ( Table 1). The cell lines have shown morphological changes in 24 h treatment induced by samples in comparison to control (Figure 2).
The study of antimicrobial activity of the I. griffithii against four microbes E. coli, C. albicans, S. aureus, and P. aeruginosa by Agar well diffusion method ( Table 2). Among these microbes, E. coli and C. Albicans were found to be sensitive for the compounds (1)(2)(3)(4)(5), whereas the extracts were exhibited activity against S. aureus and P. aeruginosa. The compound 1 is appeared to be most active with zone of inhibition (ZOI) 9.16 ± 0.28 mm and 12.50 ± 0.50 mm against E. coli and C. albicans respectively compared with standards Amikacin and Fluconazole ( Table 3). The MIC values of compound 1 are 7.50 ± 0.28 and 7.50 ± 0.86 µg/mL against E. coli and C. albicans whereas MIC values of Amikacin and Fluconazole are 4.37 ± 0.50 and 7.50 ± 0.36 µg/mL, respectively (Figure 3). Similarly, compounds 2 and 4 displayed moderate activity against E. coli with ZOI = 6.23 ± 0.50 mm and 8.36 ± 0.22 mm and against C. albicans with ZOI = 8.50 ± 0.46 mm and 7.45 ± 0.34 mm, respectively. In our earlier study, we have found that long-chain hydrocarbons demonstrated antimicrobial activities [19,23]. Here, two long-chain alkene compounds 3 and 5 showed moderate activity against C. albicans with ZOI = 9.15 ± 0.26 mm and 8.45 ± 0.37 mm, respectively. To the best of our knowledge, this is the first study of isolation of compounds from bark, wood, and leaf of I. griffithii along with cytotoxicity and antimicrobial activities from North East India.