Peruranolides A–D, four new withanolides with potential antibacterial and cytotoxic activity from Physalis peruviana L.

Background : Many drugs for anti-tumour have been developed, nevertheless, seeking new anticancer drug is the focus of ongoing investigation. Withanolides have been reported to possess potent antiproliferative activity. Literature findings revealed that a diversity of withanolides were obtained from Physalis peruviana , however, the antitumor activity of these bioactive compounds is still unclear. Methods : The EtOAc fraction of P. peruviana were decolorized on Middle Chromatogram Isolated (MCI) Gel column, repeatedly subjected to column chromatography (CC) over sephadex LH-20, preparative High Performance Liquid Chromatography (HPLC) and silica gel to afford compounds. Their chemical structures of the new isolates were elucidated through analyzing spectroscopic and HRESIMS data. All these obtained metabolites were appraised for their potential antiproliferative activity against the human breast cancer cell line MCF-7 by MTT assay, and in vitro antibacterial activity of the isolated compounds ( 1 – 7 ) were evaluated against E. coli , B. cereus and S. aureus. Results : Four new withanolides, including one withaphysalin-type withanolide (peruranolide A, 1 ), two 13,14-seco-withaphysalins (peruranolides B–C, 2 – 3 ), as well as one normal withanolide (peruranolide D, 4 ), were purified and separated from P. peruviana L.. Compound 5 was discovered to exhibit potent cytotoxic effect with an IC 50 value of 3.51 µ M. In vitro antibacterial activities, compounds 1 – 7 had no obvious inhibitory activity against E. coli , but had moderate inhibitory activities against B. cereus and S. aureus . Conclusions : Our findings might offer valuable clues for the utilization of withanolides as lead compounds for antineoplastic or antibacterial drug development.


Introduction
In the past decade, considerable attention has been paid to tumour, which was the primary leading cause of premature death (age between 30 and 69 years) [1].At present, many drugs for anti-tumour have been developed, such as alkylating agents, anti-metabolic drugs and anti-tumor an-tibiotics; nevertheless, seeking new anticancer drug is the focus of ongoing investigation.
The genus Physalis, containing approximately 120 species around the world, distributed mostly in tropical and temperate regions of America.Physalis peruviana L., as a traditional folk medicine, has been extensively used for a variety of therapeutic purposes [10].For example, P. peruviana has been exploited as heat-clearing and detoxifying, antiphlogistic, diuretic, applied in Sore throat, swollen gums, pemphigus, eczema, etc. [11].Literature findings revealed that a diversity of physalins, C28 steroidal lactones as well as withanolides were obtained from P. peruviana [12,13], however, the antitumor activity of these bioactive compounds is still unclear [14,15].This indicated the strong possibility that withanolides obtained from P. peruviana have a tendency to anticancer.
Herein, the detailed isolation and structural characterization of these four novel withanolides along with three known ones from the title plant, as well as their antiproliferative toward the human breast cancer cell line MCF-7 and in vitro antibacterial activity against E. coli, B. cereus and S. aureus were presented.

General experimental procedures
The materials and instruments for the purification and for the spectroscopic measurements of the compounds from the title plant are detailed in the Supporting Information.

Cytotoxicity assay
The human breast cancer cell line (MCF-7) was purchased from the Kunming Institute of Zoology.Cells were supplemented with streptomycin, 10% fetal bovine serum and penicillin (Gibco, USA) in DMEM medium.Cytotoxic assays were proceeded using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method followed by the reported protocol [16].In the experiment, the compounds were prepared into stock solution with dimethyl sulfoxide (DMSO), and then an appropriate amount of secondary mother solution was prepared into 100 µM with culture medium, which was then diluted by doubling.Cells were seeded in 96-well microplates at a density of 1 × 10 4 cells/well and then treated with compounds for 24 h at 3.13, 6.25, 12.5, 25, 50 and 100 µM.The volume of different concentrations of compounds added to the corresponding wells was 100 µL.Doxorubicin was chosen as the positive control.The final concentration of DMSO in the culture medium was <0.05% [17].After the addition of 20 µL of the MTT solution (5 mg/mL) to each well, the plate was incubated for 4 h under the same conditions to stain live cells.The supernatants were removed and the crystals were dissolved in 150 µL of DMSO.The absorption was measured at 490 nm.
The cytotoxic activity of each compound was calculated and expressed as the concentration of compound that achieved 50% inhibition (IC 50 ) of the cells.

Antibacterial assay in vitro
In vitro antibacterial activity of the isolated compounds (1-7) were studied against three bacteria strains using broth microdilution technique.The bacteria tested were purchased from Microbial Culture Preservation Center, Guangdong Institute of Microbiology.These include Escherichia coli (E. coli ATCC8739), Bacillus cereus (B.cereus CMCC63302), Staphylococcus aureus (S. aureus CMCC26003).Minimum inhibitory concentration (MIC) of the compounds were carried out following the procedure described by the reported protocol [18].Briefly, stock solutions were prepared with DMSO at a certain concentration.In the 96-well plate, add 100 µL of the mixture of diluted bacteria solution and citrate indicator to the first and eighth rows of Wells.An appropriate amount of solution sample and MH liquid medium (200 µL in total) were added to the first row of well plates.After evenly mixing the solution, move 100 µL of the solution to the corresponding wells in the second row and dilute successively to the eighth row. 2 DMSO-d6 was used as solvent.
3 CDCl3 was used as solvent.
Each of these solutions was then serially diluted (8 times) in 200 µL of nutrient broth in a 96 well plates to the desired concentrations (100, 50, 25, 12.5, 6.25,3.16,1.56 and 0.78 µg/mL).Finally, the 96-well plates were incubated in a 37 ℃ constant temperature incubator for 18 h, and the color changes of the bacteria liquid were observed.Vancomycin was chosen as the positive control.All equipment and culture media were sterilised before use.
Compound 4 possessed a molecular formula of C 30 H 40 O 9 , was purified as a yellow amorphous powder.The 1 H and 13 C NMR data of 4 were discovered to be very similar to those of physaminimin F [20], except for the resonances arising from C-5 and C-6.The HMBC correlations from H-3 at δ Additionally, on the basis of molecular formula and the degree of unsaturation, an epoxy moiety placed at C-5 and C-6 was proposed.In general, the R-O-6 in withanolide-type compounds adopted β-orientation, and thus the H-6 resonance with a small coupling constant and then showed as a broad singlet.In the case of 4, the H-6 pro- ton appeared as a double doublet with J values of 2.5 and 7.7 Hz, suggesting R-O-6 being α-oriented.This deduction was confirmed by the observed NOESY correlation of H-6/H-8.The correlation from H-6 to H-4 was also observed in the NOESY experiment, indicating the α-orientation HO-4.Similarly, the H-4 signal showed as a doublet with a large J value of 6.4 Hz, implying an axial position of H-3 and therefore HO-3 was β-oriented.Thus, the structure of 4 was established as 15α-acetoxy-5α,6α-epoxy-3β,4α,14αtrihydroxy-1-oxo-witha-16,24-dienolide, named as peruranolide D.
In the previous investigation, the plants from Physalis were verified to be the major sources for the exploitation of new antitumor drugs [28][29][30].All isolated metabolites were therefore appraised for their cytotoxicity against the human breast cancer cell line MCF-7 by MTT method.As shown in Table 4, compound 5 exhibited potent inhibitory activity with an IC 50 value of 3.51 µM, comparable to that of the positive control doxorubicin at 0.90 µM.On the other hand, compounds 6-7 showed moderate with IC 50 values at 36.89 and 48.64 µM, respectively.Additionally, in vitro antibacterial activities of the compounds 1-7 were tested.MIC results of the compounds were shown in the Table 5.As a result, compounds 1-7 had moderate inhibitory activities against B. cereus and S. aureus.The MIC of 1 were 12.5 and 25 µg/mL, and the others were 25 and 50 µg/mL, respectively.However, they had no obvious inhibitory activity against E. coli with MIC of 100 µg/mL, compared with 1.56 µg/mL of vancomycin.

Conclusions
In summary, four novel withanolide-type compounds (1-4), together with three known analogues (5-7), were obtained from P. peruviana L.. Compounds 2-3 possess a 13,14-seco-withaphysalin skeleton, while others were two withaphysalin-type withanolides (1, 7) and three normal withanolides (4)(5)(6).The cytotoxic activities of these isolated compounds were evaluated against MCF-7.Compound 5 exhibited potent activity with an IC 50 value of 3.51 µM and compounds 5-7 showed moderate inhibitory effect.In vitro antibacterial activities, compounds 1-7 had no obvious inhibitory activity against E. coli, but had moderate inhibitory activities against B. cereus and S. aureus.Overall, our findings might offer valuable clues for the utilization of withanolides as lead compounds for antineoplastic or antibacterial drug development.

2
DMSO-d6 was used as solvent.3CDCl3 was used as solvent.by two downfield signals at δ H 4.57 and δ H 4.48.Their positions were established to be at C-6 and C-15, respectively, as inferred by the correlations from δ H 4.57 to C-4 (δ C 118.6), and from δ H 4.48 to C-14 (δ C 61.0) and C-16 (δ C 37.8) in the HMBC spectrum.Interpretation of its 1 H and 13 C NMR data suggested the chemical structure of 1 closely resembles that of physaminimin E[20], differing by the substituent pattern of C-18.Interestingly, it was found that the chemical shifts of C-18 could occur downfield at ≥108 ppm when the hydroxyl group attached at C-18 was etherified[19,20].In our case, a naked hydroxy linked at C-18 was determined by its upfield chemical shifts occurring at 103.1 ppm.The configurations of chiral carbons in 1 were determined to be the same as those in physaminimin E by ROESY experiment and biogenetic considerations.For instance, the observed ROESY correlations from Me-19 to H-8, from H-8 to H-15, from H-15 to H-16β, and from Me-21 to H-18 supported the pro- posed α-orientation of HO-15 and β-orientation of HO-18.

Table 5 . In vitro antibacterial activities of Compounds 1-7 (µg/mL).
Compound 2 was isolated as a yellow amorphous powder and the molecular formula of C 28 H 38 O 8 was assigned by the [M-H] − at m/z 501.2493 (calcd.for 501.2494) in HR-ESI-MS, implying an unsaturation equivalence of ten.Two characteristic signals appeared at δ C 101.0 and δ C 104.9 in the