Production and Structural Diversification of Withanolides by Aeroponic Cultivation of Plants of Solanaceae: Cytotoxic and Other Withanolides from Aeroponically Grown Physalis coztomatl

Withanolides constitute one of the most interesting classes of natural products due to their diversity of structures and biological activities. Our recent studies on withanolides obtained from plants of Solanaceae including Withania somnifera and a number of Physalis species grown under environmentally controlled aeroponic conditions suggested that this technique is a convenient, reproducible, and superior method for their production and structural diversification. Investigation of aeroponically grown Physalis coztomatl afforded 29 withanolides compared to a total of 13 obtained previously from the wild-crafted plant and included 12 new withanolides, physacoztolides I−M (9–13), 15α-acetoxy-28-hydroxyphysachenolide C (14), 28-oxophysachenolide C (15), and 28-hydroxyphysachenolide C (16), 5α-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (17), 15α-acetoxy-5α-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (18), 28-hydroxy-5α-chloro-6β-hydroxy-5,6-dihydrophysachenolide D (19), physachenolide A-5-methyl ether (20), and 17 known withanolides 3–5, 8, and 21–33. The structures of 9–20 were elucidated by the analysis of their spectroscopic data and the known withanolides 3–5, 8, and 21–33 were identified by comparison of their spectroscopic data with those reported. Evaluation against a panel of prostate cancer (LNCaP, VCaP, DU-145, and PC-3) and renal carcinoma (ACHN) cell lines, and normal human foreskin fibroblast (WI-38) cells revealed that 8, 13, 15, and 17–19 had potent and selective activity for prostate cancer cell lines. Facile conversion of the 5,6-chlorohydrin 17 to its 5,6-epoxide 8 in cell culture medium used for the bioassay suggested that the cytotoxic activities observed for 17–19 may be due to in situ formation of their corresponding 5β,6β-epoxides, 8, 27, and 28.


Introduction
Withanolides, a class of polyoxygenated steroidal lactones frequently encountered in plants of the family Solanaceae [1], are known to exhibit a variety of biological activities including cytotoxic, anti-feedant, insecticidal, trypanocidal, leishmanicidal, antimicrobial, anti-inflammatory, phytotoxic, cholinesterase inhibitory and immune-regulatory activities, and the effects on neurite outgrowth and synaptic reconstruction [2,3]. Despite these interesting and diverse biological activities, studies on withanolides have not proceeded beyond preliminary evaluation in cellular and biochemical assays, arguably due to their supply issues as is the case with many biologically active natural products (NPs), including   Studies with W. somnifera and another Solanaceae species, Physalis crassifolia, also suggested that the plant growth rate, yields of biomass and major withanolides, and the ability to produce structurally-diversified withanolides were improved when cultivated using the aeroponic technique compared to soil cultivation under identical controlledenvironmental conditions. Thus, aeroponic cultivation of W. somnifera resulted in the production of two unusual withanolides, 3α-(uracil-1-yl)-2,3-dihydrowithaferin A and 3β-(adenin-9-yl)-2,3-dihydrowithaferin, in addition to withaferin A (1), 2,3-dihydrowithaferin A-3β-O-sulfate (2), and ten other known withanolides [17] (see Supplementary Data, Figure S2). Significantly, the aeroponic cultivation of P. crassifolia produced eleven new 17β-hydroxywithanolides   [18] together with 15α-acetoxyphysachenolide D, 15αacetoxy-28-hydroxyphysachenolide D, 18-acetoxy-17-epi-withanolide K, and physachenolide D encountered in the wild-crafted/soil-grown plant [19] (see Supplementary Data, Figure S3). We have also had notable success with the aeroponic technique in cultivating other plants of the Solanaceae, such as P. peruviana [20], P. philadelphica [21], P. acutifolia [22], and P. coztomatl (this study) and isolating and characterizing over 33 new withanolides, some with promising activities related to their potential use as anticancer agents. Depicted in Table 1 are some Solanaceae plant species grown using the aeroponic technique and comparison of the number of withanolides produced and the % yields of major withanolides (1-8, Figure 1) obtained from the biomass of the aeroponically grown plants with the wild-harvested and/or soil-cultivated plants. The number of withanolides for wild-crafted/soil-grown plant refers to the referenced study reporting highest yield(s) of the major withanolide(s). b Since 2 is a prodrug of 1 [15], total % yield of withaferin A (1) in aeroponicallygrown plant is 0.93. c Yield not reported. d Not encountered in wild-crafted plants [27,28].
We have previously demonstrated that unlike the most extensively studied cytotoxic withanolides including withaferin A (1) with a β-oriented side chain, 17β-hydroxywithanolides (17-BHWs) such as physachenolide C (8), with an α-oriented side chain, selectively inhibited prostate cancer (PC) cell lines at nanoMolar concentrations without affecting many other cancer cell lines and normal human fibroblast cells [18][19][20][21]. Our recent studies suggested that the 17-BHW, physachenolide C (8), was also capable of potentiating immunotherapy of renal carcinoma and melanoma, when used in combination with the immune adjuvants, tumor necrosis factor-α related apoptosis-inducing ligand (TRAIL) and the ds-RNA mimetic, poly I:C [29][30][31][32][33], respectively. Physachenolide C (7) was also shown to induce complete regression of established murine melanoma tumors via apoptosis and cell cycle arrest [34]. Thus, it was of interest to investigate withanolides belonging to different structural types for their potential anticancer activity. Herein we report the isolation and identification of 12 new (9-20) and 17 known (3-5, 8, and 21-33) withanolides from aeroponically grown Physalis coztomatl Moc. and Sessé ex Dunal (Solanaceae) and in vitro evaluation of withanolides (3-5, and 8-33) against a panel of prostate cancer and renal carcinoma cell lines, and normal human fibroblast cells. Previous studies on P. coztomatl, a plant native to South America, has resulted in the isolation of 13 withanolides in two independent studies [27,28], including six 17-BHWs (3, 4, 25, 26, 30, and 32), all of which were also encountered in the biomass obtained from aeroponic cultivation of this plant.

Isolation and Structure Elucidation
A MeOH extract of the aerial parts of aeroponically grown P. coztomatl on fractionation by solvent-solvent partitioning, and column chromatograpy (CC) employing HP-20SS, C 18 RP, and silica gel followed by purification using prep TLC and HPLC afforded withanolides 3-5, 8 (Figure 1), and 9-33 ( Figure 2). withanolides from aeroponically grown Physalis coztomatl Moc. and Sessé ex Dunal (Solanaceae) and in vitro evaluation of withanolides (3-5, and 8-33) against a panel of prostate cancer and renal carcinoma cell lines, and normal human fibroblast cells. Previous studies on P. coztomatl, a plant native to South America, has resulted in the isolation of 13 withanolides in two independent studies [27,28], including six 17-BHWs (3, 4, 25, 26, 30, and 32), all of which were also encountered in the biomass obtained from aeroponic cultivation of this plant.

Isolation and Structure Elucidation
A MeOH extract of the aerial parts of aeroponically grown P. coztomatl on fractionation by solvent-solvent partitioning, and column chromatograpy (CC) employing HP-20SS, C18 RP, and silica gel followed by purification using prep TLC and HPLC afforded withanolides 3-5, 8 (Figure 1), and 9-33 ( Figure 2). Compounds 9 and 10 were identified as withanolide glycosides from their characteristic NMR data and were named as physacostolides I and J, respectively. The molecular formula of 9 was determined to be C36H48O13 by a combination of its HRESIMS and NMR data, suggesting thirteen degrees of unsaturation. The 1 H NMR spectrum of 9 (Table 2)   Compounds 9 and 10 were identified as withanolide glycosides from their characteristic NMR data and were named as physacostolides I and J, respectively. The molecular formula of 9 was determined to be C 36 H 48 O 13 by a combination of its HRESIMS and NMR data, suggesting thirteen degrees of unsaturation. The 1 H NMR spectrum of 9 (Table 2) showed three singlet methyl signals typical of withanolides  Figure S60) precluded oxygenation of C-19 and C-27 methyl groups. The identity of the sugar moiety was confirmed to be a D-glucose by the acid hydrolysis of 9 to afford a sugar with positive specific optical rotation. The ECD spectrum of 9 showed positive cotton effect at 256 nm (see Supplementary Data, Figure S59), suggesting the R configuration of C-22 [35,36]. Based on the foregoing data, the structure of physacoztolide I was determined as (20S,22R)-18-acetoxy-28β-D-O-glucopyranosyl-14α,20β-dihydroxy-1oxo-witha-2,5,16,24-tetraenolide (9).
The molecular formula of physacostolide J (10) was determined to be C 36 H 48 O 12 from its HRESIMS and NMR data. The 1 H NMR data of 10 ( Table 1) were similar to those of 9, and the difference in molecular formulae between 10 (C 36 H 48 O 12 ) and 9 (C 36 H 48 O 13 ) indicated that 10 may be a deoxygenated analogue of 9. The assignment of the 13 C NMR spectrum ( Table 2) by HSQC and HMBC data (see Supplementary Data, Figures S11 and S60) also revealed the similarities between 9 and 10. The major difference in the NMR data was found to be the absence of oxymethine group at δ C 83.4, which was assigned to C-14 in 9. Instead, 10 showed the presence of a methine group (δ C 57.6). This was confirmed by the up-field chemical shifts (∆δ C : −3.2 ppm for C-8, −5.7 ppm for C-13, and −9.3 ppm for C-15) of carbons located β to C-14 in 10 when compared with those of 9 (Table 2). Acid hydrolysis of 10 gave D-glucose. The ECD spectrum of 10 showed a positive cotton effect at 257 nm (see Supplementary Data, Figure S59), suggesting the R configuration of C-22 [35,36]. Thus, the structure of physacoztolide J was determined as (20S,22R)-18acetoxy-28β-D-O-glucopyranosyl-20β-hydroxy-1-oxo-witha-2,5,16,24-tetraenolide (10).
The HRESIMS, 1 H and 13 C NMR data of physacoztolide K (11) were consistent with the molecular formula, C 30 H 40 O 9 . The 1 H NMR data of 11 (Table 2)  was consistent with 23β-hydroxyphysacoztolide E-type sub-structure [18], suggesting the orientation of OH-23 as β. The positive Cotton Effect at 256 nm in its ECD spectrum (see Supplementary Data, Figure S59) established the 22R configuration [35,36]. On the basis of the foregoing evidence, the structure of physacoztolide K was elucidated as (17R,20S,22R)-18-acetoxy-14α,20β,23β,27-tetrahydroxy-1-oxo-witha-2,5,24-trienolide (11). The molecular formula of physacoztolide L (12) was determined to be C 30 H 42 O 9 from its HRESIMS and NMR data. The 1 H and 13 C NMR data (Table 2) suggested that the ring E of 12 is saturated unlike the other withanolides found to co-occur in this plant which contained an unsaturated E-ring. The 1 H NMR spectrum of 12 (Table 2)  ]. The coupling between the two oxygenated methines suggested possible hydroxylation at C-23 [18]. The 13 C NMR spectrum of 12 (Table 2) displayed thirty carbon signals including an acetyl group (δ C 169.7 and 21.2). The assignment of 13 C NMR spectrum with the help of HSQC and HMBC data suggested that C-17 (δ C 49.9) is not oxygenated like in 11, and ring E is saturated as indicated by the up-field shift of the carbonyl signal (δ C 177.6) compared withanolides bearing an unsaturated E-ring δ-lactone [18]. The remaining 13 [20], which established the trans configuration of H-23 and H-24. The irradiation of H 3 -27 showed an NOE with H-24, suggesting the trans configuration of CH 3 -27 and CH 2 OH-28. These data indicated that the gross structure of ring E of 12 is the same as that of 24,25-dihydro-23β,28dihydroxywithanolide G, which was further supported by their almost identical 13 C NMR chemical shifts for the carbons of the ring E moiety [20]. The absolute configuration of C-22 was determined as R by the positive Cotton effect at 256 nm in its ECD spectrum [28] (see Supplementary Data, Figure S59). The appearance of H-17 as a triplet [δ H 2.73 (t, J = 9.7 Hz)] in its 1 H NMR spectrum established the configuration of the side chain at C-17 as β [37]. Thus, the structure of physacoztolide L was identified as (17S,20R,22R,24S,25R)-18-acetoxy-14α,20β,23β,28-tetrahydroxy-1-oxo-witha-2,5-dienolide (12).
The molecular formula of 14 was established as C 32 H 42 O 12 by its HRESIMS and NMR data. Careful analysis of 1 H NMR and 13 C NMR spectra of 14 (Table 3) suggested that it could be an acetoxy analogue of 28-hydroxyphysachenolide C (13) or an oxygenated analogue of 15α-acetoxyphysachenolide C (27) [29]. Comparison of the NMR data of 14 with those of 13 and 27 confirmed that the signals due to the rings A-D of 14 were identical with those of 27 [18], and the signals of the side chain including ring E of 14 were same as those of 13 suggesting that it could be 15α-acetoxy analogue of 28-hydroxyphysachenolide C.  Figure S59) in its ECD spectrum established the R configuration for C-22 of 14 [35,36]. Thus, the structure of this withanolide was elucidated as 15α-acetoxy-28-hydroxyphysachenolide C [(20S,22R)-15α,18-diacetoxy-5β,6β-epoxy-14α,17β,20β,28-tetrahydroxy -1-oxo-witha-2,24dienolide] (14).
The molecular formula of withanolide 15 was determined to be C 30 H 38 O 10 based on its HRESIMS and NMR data, suggesting twelve degrees of unsaturation. The analysis of the 1 H NMR and 13 C NMR spectra (  Figure S59) in its ECD spectrum established the R configuration of C-22 [35,36]. The structure of 15 was thus established as 28- Based on its HRESIMS and NMR data, withanolide 16 was determined to have the molecular formula C 30 H 40 O 10 . It was suspected to be a glucoside from its molecular formula, C 34 H 46 O 11 , and the presence of a signal due to an anomeric proton at δ H 4.23 (d, J = 8.0 Hz) and the typical 13 C NMR signals (δ C 102.5, 73.3, 75.9, 69.8, 76.4, and 61.5) of the glucose moiety and was named physacoztolide M. The 1 H NMR spectrum of 16 (Table 3)  . The olefinic region of the 1 H NMR spectrum of 16 was found to similar to that of physacoztolide I (9) (see above). The absence of a singlet methyl signal around 2.0 ppm in 16 suggested that it lacked the acetyl group present in 9. These data suggested 16 has a similar skeleton as that of 9 and contained three double bonds at 2(3), 5(6), and 16 (17) positions, and the AcOCH 2 at C-13 in 9 was replaced by a CH 3 group in 16. The assignment of the 13 C NMR spectrum of 16 (Table 3) with the help of the HSQC data (see Supplementary Data, Figure S38) and HMBC data (see Supplementary Data, Figures S39 and S60) and comparison of the 13 C NMR data with those of 9 further confirmed that the AcO group at C-18 of 9 is replaced with a proton in 16. The presence of the double bonds at 2(3) and 5(6), and 16 (17) Figure S60). The long-range HMBC correlation between the anomeric proton of the glucose moiety and C-28 (δ C 67.7) located the O-glycosyl moiety at C-28. The presence of the D-glucose moiety in 16 was further confirmed by the acid hydrolysis and the positive [α] D obtained for the resulting sugar. The ECD spectrum of 16 showed a positive Cotton effect at 256 nm (see Supplementary Data, Figure S59) establishing the R configuration of C-22 [35,36]. Therefore, the structure of physacoztolide M was determined as (20S,22R)-28β-D-O-glucopyranosy-14α,20β-dihydroxy-1-oxo-witha-2,5,16,24-tetraenolide (16).  3 OD was used as the solvent. b CDCl 3 was used as the solvent. c CDCl 3 /CD 3 OD (100:1) was used as the solvent.
A small number of chlorinated withanolides have previously been encountered in plants of Solanaceae as minor metabolites and many of these occur as 5,6-chlorohydrins containing 5α-chloro-6β-hydroxy substituents [25,[44][45][46][47][48]. It has been suggested that the chlorine atom present in these 5,6-chlorohydrins may originate from NaCl present in the plant [2]. However, the occurrence of corresponding 5β,6β-epoxides as major matabolites in their source plants (as in P. costomatl) suggests that 5,6-chlorohydrins of withanolides may be possible artifacts formed from their corresponding 5β,6β-epoxides during the extraction of these plants and/or during the isolation of withanolides. The possibility of formation of withanolide chlorohydrins during the isolation process has previously been suggested [49] for which a probable mechanism involving acid catalyzed opening of the 5β,6β-epoxy moiety to generate 5,6-chlorohydrins has been proposed [25]. To test this, we exposed the major withanolide of P. coztomatl, physachenolide C (8), to 0.5% methanolic HCl at 25 • C for 30 min (TLC control). The investigation of the crude product mixture by HPLC suggested that under these mildly acidic conditions, the 5β,6β-epoxide ring of physachenolide C (8) underwent an acid-catalyzed ring opening to afford the corresponding 5,6-chlorohydrin [5αchloro-6β-hydroxyphysachenolide C (17)], 5α-methoxy-6β-hydroxy analogue [physachenolide A-5-methyl ether (19)] and 5α,6β-dihydroxy analogue [physachenolide A (21)] (see Supplementary Data, Figure S62), all of which were encountered in P. coztomatl. Additional experiments to investigate whether these withanolides are genuine plant metabolites or artifacts are currently in progress.

Biological Activities of Withanolides from P. coztomatl
We have previously discovered that some 17β-hydroxywithanolides, including physachenolide C (8), were capable of selectively inhibiting the proliferation of prostate cancer cells at nanoMolar concentrations without affecting many other cancer cells and normal human fibroblast cells [19]. In this study, withanolides 3-5 and 8-33 obtained from aeroponically grown P. crassifolia were evaluated for their cytotoxic activity against a panel of four human prostate cancer (PC) cell lines, LNCaP and VCaP (androgen-sensitive PC), DU-145 and PC-3 (androgen-independent PC), human renal adenocarcinoma (ACHN) cell line, and normal human fibroblast cells, WI-38. Of those tested, withanolides 8, 10, 13, 15, 17, and 18 showed >50% inhibition against at least one of the cancer cell lines at 5.0 µM concentration. Significantly, all those showing promising activity were 18-acetoxy-17β-hydroxywithanolides and these were then evaluated for their IC 50 s (concentrations required to inhibit 50% of the cells). The IC 50 data obtained are depicted in Table 5.
It is noteworthy that 5α-chloro-6β-hydroxy-5,6-dihdrophysachenolide D (17) containing a trans-fused A/B-ring system exhibited cytotoxic activities very close to those of physachenolide C (8) bearing a cis-fused A/B-ring system, against all the cell lines tested (Table 5). This is somewhat surprising as it contradicts our previous finding that the cis-fused A/B-ring conformation (as in 8) is important for the cytotoxic activity of 17βhydroxywithanolides [33]. This unexpected potent activity of 5α-chloro-6β-hydroxy-5,6dihydrophysachenolide D (17) and other withanolide 5,6-chlorohydrins may be attributed to the possible conversion of these to their corresponding 5β,6β-epoxides in the cell culture medium. To test this, 17 was incubated with the cell culture medium (DMEM) used for the cytotoxicity assays with LNCaP and ACHN cell lines and under the conditions used for the assay (37 • C in a 5% CO 2 incubator). The analysis of the incubation mixture by HPLC at intervals of 0 min, 5 min, 2 h, 8 h, and 24 h, suggested that its conversion to physachenolide C (8) is facile and almost complete in 24 h (Figure 3). Since the cytotoxicity assay involves incubation of the test compound for 72 h in the cell culture medium, it is very likely that the unexpected activity observed for 5α-chloro-6β-hydroxywithanolides is due to the conversion of these into their corresponding 5β,6β-epoxywithanolides.  for the assay (37 °C in a 5% CO2 incubator). The analysis of the incubation mixture by HPLC at intervals of 0 min, 5 min, 2 h, 8 h, and 24 h, suggested that its conversion to physachenolide C (8) is facile and almost complete in 24 h (Figure 3). Since the cytotoxicity assay involves incubation of the test compound for 72 h in the cell culture medium, it is very likely that the unexpected activity observed for 5α-chloro-6β-hydroxywithanolides is due to the conversion of these into their corresponding 5β,6β-epoxywithanolides.

General Methods and Materials
Optical rotations were measured at 25 °C with a JASCO Dip-370 digital polarimeter using MeOH as solvent. UV spectra were recorded in MeOH using a Shimadzu UV-1601 UV-Vis spectrometer. ECD spectra were measured with JASCO J-810 circular dichroism spectropolarimeter. 1D and 2D NMR spectra were recorded on a Bruker Avance III 400 NMR instrument at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR. Chemical shift values (δ) are given in parts per million (ppm), and the coupling constants are in Hz. Highresolution MS were recorded on an Agilent G6224A TOF mass spectrometer. Normal phase column chromatography was performed using Baker silica gel 40 μm flash chromatography packing (J. T. Baker) and reversed-phase chromatography was carried out using BAKERBOND C18 40 μm preparative LC packing (J. T. Baker). Analytical and preparative thin-layer chromatography (TLC) were performed on pre-coated 0.20 mm thickness plates of silica gel 60 F254 (Merck) and RP-18 F254 (Merck). HPLC purifications were carried out using 10 mm × 250 mm Phenomenex Luna 5 μm C-18 column (3 mL/min flow rate) with a Waters Delta Prep system consisting of a PDA 996 detector. MM2 energy minimizations

General Methods and Materials
Optical rotations were measured at 25 • C with a JASCO Dip-370 digital polarimeter using MeOH as solvent. UV spectra were recorded in MeOH using a Shimadzu UV-1601 UV-Vis spectrometer. ECD spectra were measured with JASCO J-810 circular dichroism spectropolarimeter. 1D and 2D NMR spectra were recorded on a Bruker Avance III 400 NMR instrument at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR. Chemical shift values (δ) are given in parts per million (ppm), and the coupling constants are in Hz. High-resolution MS were recorded on an Agilent G6224A TOF mass spectrometer. Normal phase column chromatography was performed using Baker silica gel 40 µm flash chromatography packing (J. T. Baker) and reversed-phase chromatography was carried out using BAKERBOND C 18  The cell culture media used for the bioassays are: RPMI medium with 10% FBS, 1% glutamax, and 100 U/mL penicillin, and 100 µg/mL streptomycin for PC-3 cells; EMEM medium with 10% FBS, 1% glutamax, and 100 U/mL penicillin, and 100 µg/mL streptomycin for DU-145 and WI-38 cells; DMEM medium with 10% FBS and 100 U/mL penicillin, and 100 µg/mL streptomycin for VCaP cells; RPMI medium with 5% FCS, 2 mM L-glutamine, 1× nonessential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, 10mM HEPES, and 5 × 10 −5 M 2-mercaptoethanol for LNCaP and ACHN cells.

Aeroponic Cultivation and Harvesting of P. coztomatl
The seeds of P. coztomatl obtained from Trade Wind Fruit (P.O. Box 1102, Windsor, CA 95492, USA) were germinated in 1.0 inch Grodan rock-wool cubes in a Barnstead Lab-Line growth chamber kept at 28 • C under 16 h of fluorescent lighting and maintaining 25-50% humidity. After ca. 4 weeks in the growth chamber, seedlings with an aerial length of ca. 5.0 cm were transplanted to aeroponic culture boxes for further growth, as described previously for Withania somnifera and Physalis crassifolia [17,18]. Aerial parts of aeroponically grown plants were harvested when fruits were almost mature (ca. 3 months under aeroponic growth conditions). Harvested plant materials were dried in the shade, powdered, and stored at 5 • C prior to extraction.

Extraction, Isolation and Identification of Withanolides
Dried and powdered aerial parts of P. coztomatl (200.0 g) were extracted with MeOH (3.0 L) in an ultrasonic bath at 25 • C for 2 h, and then allowed to stand for overnight. After filtration, the resulting filtrate was concentrated under reduced pressure at 40 • C to afford the crude extract (45.0 g). The crude extract (45.0 g) was subjected to solvent-solvent partitioning between hexanes and 80% aqueous MeOH, and the resulting 80% aqueous MeOH layer was diluted with H 2 O to give 50% aqueous MeOH solution, which was further extracted with CHCl 3 to afford the CHCl 3 extract. These were concentrated to afford hexanes (3.2 g) and CHCl 3 extracts (3.5 g). The 50% aq. MeOH layer obtained above was passed through a column of HP-20SS (Supelco, 200.0 g), washed with MeOH and concentrated yielding the 50% aq. MeOH fraction (0.28 g), which showed a TLC profile similar to the CHCl 3 fraction. Thus, the combined CHCl 3 and 50% aq. MeOH fractions (3.78 g) was subjected to column chromatography (CC) on RP C 18

Acid Hydrolysis of Glycosides 9, 10, and 16
To a solution of each glycoside (9, 10 or 16, 0.5 mg) in MeOH (0.5 mL) was added 2N HCl solution (0.5 mL). The mixture was heated at 100 • C. After 1 h (TLC control), the reaction mixtures were concentrated and the residues thus obtained were chromatographed over a column of silica gel (0.5 g) using CHCl 3 /MeOH (8:2) as the eluent. Fractions containing the sugar were collected based on their TLC profiles, concentrated, dissolved in water for qualitative measurement of [α] D .

Cytotoxicity Assay
A tetrazolium dye-based colorometric (MTT) assay was used for evaluating cytotoxicity of the compounds against cancer cell lines, LNCaP (androgen-sensitive prostate adenocarcinoma), PC-3 (androgen-insensitive prostate adenocarcinoma), DU-145 (androgeninsensitive prostate adenocarcinoma), VCaP (androgen-sensitive metastatic prostate cancer), and ACHN (renal carcinoma), and normal human lung fibroblast cells, WI-38. The cells were plated at 1000-4000 cells/well (depending on the cell growth rate) in 96-well flatbottomed microplates. After incubation at 37 • C for 24 h in an atmosphere of 5% CO 2 , serial dilutions of compounds in DMSO were added to triplicate wells so that the final DMSO concentration in each well is <0.2%. Doxorubicin and DMSO were used as positive and negative controls, respectively. After incubation for 72 h at 37 • C in an atmosphere of 5% CO 2 , MTT solution (2 mg/mL, 25.0 µL) was added to each well, and continued to incubate for 3-4 h at 37 • C. The media were removed and 100 µL/well of DMSO was added before data acquisition using a microplate reader at 570 nm.

Conclusions
Withanolides constitute one of the most interesting classes of natural products due to their diversity of structures and biological activities. The work reported here further supports our previous findings that the application of the aeroponic technique for cultivation of plants of Solanaceae is a convenient, reproducible, and superior method for production and structural diversification of withanolides. Investigation of aeroponically grown Physalis coztomatl afforded 29 withanolides including 12 new withanolides (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and 17 known withanolides (3- 5, 8, and 21-33). Evaluation of these withanolides against a panel of prostate cancer (LNCaP, VCaP, DU-145, and PC-3) and renal carcinoma (ACHN) cell lines, and normal human foreskin fibroblast (WI-38) cells suggested that 8, 13, 15, and 17-19 had potent and selective activity for prostate cancer cell lines. This work also resulted in the discovery that the potent cytotoxic activity of withanolide 5,6-chlorohydrins may be due to their facile conversion into the corresponding 5β,6β-epoxides in the cell culture medium used for the bioassay.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.