Cytotoxic and other withanolides from aeroponically grown Physalis philadelphica

Abstract

Eleven withanolides including six previously undescribed compounds, 16β-hydroxyixocarpanolide, 24,25-dihydroexodeconolide C, 16,17-dehydro-24-epi-dioscorolide A, 17-epi-philadelphicalactone A, 16-deoxyphiladelphicalactone C, and 4-deoxyixocarpalactone A were isolated from aeroponically grown Physalis philadelphica. Structures of these withanolides were elucidated by the analysis of their spectroscopic (HRMS, 1D and 2D NMR, ECD) data and comparison with published data for related withanolides. Cytotoxic activity of all isolated compounds was evaluated against a panel of five human tumor cell lines (LNCaP, ACHN, UO-31, M14 and SK-MEL-28), and normal (HFF) cells. Of these, 17-epi-philadelphicalactone A, withaphysacarpin, philadelphicalactone C, and ixocarpalactone A exhibited cytotoxicity against ACHN, UO-31, M14 and SK-MEL-28, but showed no toxicity to HFF cells.

1. Introduction

The genus Physalis (Solanaceae) contains over 75 species some of which are used as foods and medicines (Whitson and Manos, 2005; Kindscher et al., 2012). Of these, the most widely distributed and cultivated species is Physalis philadelphica Lam. (Solanaceae) also known as tomatillo (Wang et al., 2012). Tomatillo is one of the basic ingredients of fresh and cooked Mexican and Central American green sauces. In South American traditional medicine, the fruits of P. philadelphica are used to relieve fever, cough, and amygdalitis, the leaves as a remedy for gastrointestinal disorders, and the calices for the treatment of diabetes (Maldonado et al., 2011). Plants of the genus Physalis have also received considerable attention because of their constituent withanolides, a class of polyoxygenated steroids based on C₂₈ ergostane skeleton (Glotter, 1991). Withanolides, which also occur in other genera of Solanaceae including Acnistus, Datura, Dunalis, Jaborosa, and Withania are classified into two main classes, one containing the parent skeleton of withaferin A (group I) and the other with a modified skeleton (group II) (Yang et al., 2016). Many withanolides belonging to group I contain a 2,3-enone moiety in ring A and a δ-lactone moiety in their side chains. Based on their oxygenation patterns and the orientation of the side chain, group I withanolides belong to four basic classes to include withaferin A, withanolide A, withanolide D, withanolide E and their structural analogues. Among these, only those of withanolide E class contain an α oriented side chain and a β-hydroxy group at C-17 and are referred to as 17β-hydroxywithanolides.

Withanolides have been reported 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 effects on neurite outgrowth and synaptic reconstruction (Chen et al., 2011). Among these, the most widely investigated are cytotoxic and other activities related to their potential use as anticancer agents. These studies have suggested that unlike the most extensively investigated withanolide, withaferin A, withanolides belonging to withanolide E (13) class (17β-hydroxywithanolides) were selectively cytotoxic to only certain cancer cell lines and that these two classes of withanolides may have different molecular targets (Xu et al., 2015, 2017; Tewary et al., 2017). Thus, it was of interest to investigate the cytotoxic activity of withanolides belonging to different structural types.

Previous phytochemical investigations of P. philadelphica, also known as P. ixocarpa Brot. (Hudson, 1986), have resulted in the isolation of several withanolides, including physalin B (Subramanian and Sethi, 1973), withaphysacarpin (Subramanian and Sethi, 1973; Kennelly et al., 1997; Su et al., 2002), ixocarpalactone A (Kirson et al., 1979; Su et al., 2002; Gu et al., 2003; Maldonado et al., 2011), ixocarpalactone B (Kirson et al., 1979; Su et al., 2002), ixocarpanolide (Abdullaev et al., 1986; Maldonado et al., 2011), 2,3-dihydro-3-methoxywithaphysacarpin and 24,25-dihydrowithanolide D (Kennelly et al., 1997), philadelphicalactone A (Su et al., 2002; Maldonado et al., 2011), philadelphicalactone B, 18-hydroxywithanolide D and withanone (Su et al., 2002), philadelphicalactone C (Maldonado et al., 2011), philadelphicalactone D (Maldonado et al., 2011), 2,3-dihydro-3β-methoxyisocarpalactone A (Gu et al., 2003), 2,3-dihydro-3β-methoxyisocarpalactone B and 4β,7β,20R-trihydroxy-1-oxowitha-2,5-dien-22,26-olide (Gu et al., 2003). In our continuing interest on new and/or biologically active withanolides (Wijeratne et al., 2014; Xu et al., 2015, 2016, and 2017), we have investigated aeroponically grown P. philadelphica and herein we report the isolation, identification, and cytotoxic activity of six new and five known withanolides, some of which have not been encountered in wild-crafted plants. The new withanolides were identified as 16β-hydroxyixocarpanolide (1), 24,25-dihydroexodeconolide C (2), 16,17-dehydro-24-epi-dioscorolide A (3), 17-epi-philadelphicalactone A (4), 16-deoxyphiladelphicalactone C (5), and 4-deoxyixocarpalactone A (6). Comparison of spectroscopic data with those reported led to the identification of the known withanolides as withaphysacarpin (7) (Kennelly et al., 1997; Su et al., 2002), philadelphicalactone A (8) (Su et al., 2002; Maldonado et al., 2011), philadelphicalactone C (9) (Maldonado et al., 2011), and ixocarpalactones A (10) and B (11) (Kirson et al., 1979; Su et al., 2002).

2. Results and discussion

The aerial parts of aeroponically grown P. philadelphica were extracted with methanol and the resulting extract was fractionated by solvent-solvent partitioning with hexanes and 80% aq. methanol followed by 50% aq. methanol and chloroform to obtain hexanes, 50% aq. methanol and chloroform fractions. Of these only the chloroform fraction was found to contain withanolides. Thus, the chloroform fraction was further fractionated by silica gel and reversed phase column chromatography followed by preparative HPLC to afford withanolides 1–11 (see Fig. 1) as white amorphous powders.

Fig. 1.

Structures of withanolides 1–11 from aeroponically grown P. philadelphica, ixocarpanolide (12), withanolide E (13), and 4β-hydroxywithanolide E (14).

2.1. Structure elucidation of the new withanolides 1–6

The ¹H and ¹³C NMR spectroscopic data of withanolides 1–3 (Tables 1 and 2) suggested that these are structurally related to each other and contain a carbon skeleton bearing 2,3-enone, 5α-hydroxy, 6α,7α-epoxy moieties in rings A/B and δ lactone moiety in the side chain similar to ixocarpanolide (12) (Abdullaev et al., 1986). Withanolide 1 possessed a molecular formula of C₂₈H₄₀O₇ as determined by its HRMS and NMR data and indicated nine degrees of unsaturation. The ¹H NMR spectrum of 1 (Table 1) displayed signals due to three methyl groups attached to non-protonated carbons [δ_H 1.18 (3H, s, H₃-19)], 1.19 (3H, s, H₃-18), and 1.32 (3H, s, H₃-21)], two methyl groups on protonated carbons [δ_H 1.14 (3H, d, J = 6.8 Hz, H₃-28) and 1.23 (3H, d, J = 6.8 Hz, H₃-27)], two vicinal protons of an epoxide moiety [δ_H 3.04 (1H, d, J = 4.0 Hz, H-6β), 3.31 (1H, dd, J = 2.0, 4.0 Hz, H-7β)], two overlapping oxygenated methines [δ_H 4.61 (2H, m, H-16 and H-22)], and two vicinal vinyl protons [δ_H 5.83 (1H, dd, J = 2.4, 10.0 Hz, H-2), 6.58 (1H, ddd, J = 2.0, 5.2, 10.0 Hz, H-3)]. Its ¹³C NMR spectrum (Table 2) exhibited 28 signals corresponding to five methyls (δ_C 14.2, 14.7, 14.8, 21.1, and 22.3), 2,3-enone moiety [δ_C 129.0 (C-2), 139.7 (C-3), 203.1 (C-1)], two oxygenated methines [δ_C 73.4 (C-16) and 81.2 (C-22)], two oxygenated carbons of an epoxide moiety [δ_C 56.3 (C-6) and 57.1 (C-7)], two oxygenated tetrasubstituted carbons [δ_C 73.2 (C-5) and 78.4 (C-20)], and a carbonyl carbon of a δ lactone [δ_C 175.9 (C-26)]. The presence of the 5α-hydroxy-6α,7α-epoxy moiety in rings A and B of 1 was inferred from the characteristic methine resonances at δ_C 56.3 (C-6) and 57.1 (C-7), and that due to oxygenated carbon at δ_C 73.2 (C-5) (Yu et al., 2017). The planar structure of 1 was established by the analysis of its HMBC spectrum which showed key correlations for H₃-19/C-1, H₃-19/C-5, H-6/C-4 (δ_C 36.7), and H-6/C-10 (δ_C 51.0) for the A/B ring system, H₃-18/C-14 (δ_C 49.6), H₃-18/C-17 ((δ_C 58.3), H₃-18/C-12 (δ_C 40.6), H-17/C-16, and H₃-21/C-17 for the C/D ring system, H₃-27/C-26, H₃-28/C-23 (δ_C 31.3), and H₃-21/C-22 for the ring E (Fig. 2). The NOESY correlations (Fig. 3) of H₃-19/H-4β [δ_H 2.68 (brd, J = 18.8 Hz)], H₃-19/H-8β (δ_H 1.84, m), H-4α [δ_H 2.52 (dd, J = 5.2, 18.8 Hz)]/H-6β, H-6β/H-7β, and H-7β/H-8β confirmed the orientation of the 6,7-epoxide group as α. The orientations of H-14, OH-16 and H-17 were confirmed to be α, β, and α respectively, by the NOESY correlations of H-7β/H-15α [δ_H 2.42 (dt, J = 12.4, 7.6 Hz)], H-15α/H-16α, H-16α/H-14α (δ_H 1.23, m), H-16α/H-17α (δ_H 1.28, m), and H₃-21/H-12β (δ_H 2.13, m). The ECD spectrum of 1 exhibited a negative Cotton effect at 335 nm confirming the configuration and the trans-linkage of A and B rings (Begley et al., 1976), and a positive Cotton effect at 242 nm confirming the R configuration of C-22 (Abdullaev et al., 1986). Almost the same ¹³C NMR chemical shifts observed for C-20 and the carbons of D and E rings of 1 and withaphysacarpin (7) (Kennelly et al., 1997), also encountered in this work, suggested that the configuration of C-20 of 1 is the same as that of 7. Thus, the structure of 1 was elucidated as 16β-hydroxyixocarpanolide [(20R,22R,24S,25R)-5α,16β,20-trihydroxy-6α,7α-epoxy-1-oxowitha-2-enolide].

Table 1.

¹H NMR (400 MHz) spectroscopic data for withanolides 1–6 (CDCl₃, δ in ppm, J in Hz).^a

Position	1	2b	3	4	5b	6b
2	5.83 dd (2.4, 10.0)	5.78 dd (2.0, 10.0)	5.83 dd (2.0, 10.0)	6.16 d (10.0)	6.11 d (10.0)	5.97 dd (2.8, 10.0)
3	6.58 ddd (2.0, 5.2, 10.0)	6.54 ddd (2.0, 4.8, 10.0)	6.57 ddd (2.0, 4.8, 10.0)	6.89 dd (2.0, 10.0)	6.89 dd (2.4, 10.0)	6.80 ddd (2.0, 6.0, 10.0)
4 α	2.52 dd (5.2, 18.8)	2.46 dd (4.8, 19.0)	2.51 m	3.70 dd (2.0, 5.6)	3.59 dd (2.4, 5.6)	1.85 dd (6.0, 18.8)
β	2.68 brd (18.8)	2.62 dt (19.0, 2.0)	2.67 brd (18.8)			2.93 brd (18.8)
6	3.04 d (4.0)	2.98 d (3.6)	3.02 d (3.6)	3.18 brs	3.10 brs	3.08 d (2.4)
7	3.31 dd (2.0, 4.0)	3.19 dd (2.0, 3.6)	3.36, dd (2.0, 3.6)	1.25 m	1.18 m	1.18 m
				2.15 ddd (2.4, 3.6, 14.8)	2.06 ddd (2.4, 3.6, 14.8)	1.99 m
8	1.84 m	1.64 m	1.94 m	1.58 m	1.40 m	1.62 m
9	1.54 m	1.52 dt (3.2, 11.8)	1.65 dt (3.2, 10.4)	0.95 m	0.79 m	1.06 m
11	1.37 m	1.18 m	1.40 m	1.45 m	1.37 m	1.48 m
	2.74 ddd (3.6, 7.4, 13.2)	2.67 ddd (3.6, 6.8, 13.2)	2.85 ddd (3.2, 6.8, 13.2)	1.81 m	1.62 m	1.99 m
12	1.28 m	1.63 m	1.49 m	1.65 m	1.07 m	1.15 m
	2.13 m	1.77 m	1.72 m	1.89 m	1.88 m	2.05 m
14	1.23 m	2.13 m	1.73 m	1.52 m	1.45 m	0.72 m
15	1.55 m	1.63 m	2.03 m	1.36 m	1.08 m	1.28 m
	2.42 dt (12.4, 7.6)	1.80 m	2.28 ddd (14.6, 6.4, 3.2)	1.62 m	1.57 m	2.16 m
16	4.61 m	4.24 brd (7.6)	5.51 brs	1.66 m	1.49 m	4.39 m
				2.05 m	1.83 m
17	1.28 m				0.86 m	1.27 m
18	1.19 s	0.79 s	0.85 s	0.87 s	0.73 s	1.09 s
19	1.18 s	1.11 s	1.19 s	1.37 s	1.31 s	1.20 s
20		2.11 m	2.45 dq (7.2, 6.0)
21	1.32 s	0.96 d (7.2)	1.08 d (7.2)	1.25 s	1.13 s	1.28 s
22	4.61 m	4.62 ddd (3.2, 4.0, 10.8)	4.29 ddd (4.1, 6.0, 11.6)	4.64 brd (8.8)	4.02 dd (5.2, 11.2)	3.94 brs
23	1.54 m	1.63 m	1.92 dd (11.6, 14.6)	1.65 m	1.75–1.85 m	4.41 m
	1.98 m	1.85 m	2.05 dd (4.1, 14.6)	2.03 m
24	1.83 m	1.62 m		1.71 m		2.23 m
25	2.22 dq (9.6, 6.8)	2.15 m	2.55 q (7.2)	2.12 m	2.40 q (6.8)	2.62 m
27	1.23 d (6.8)	1.15 d (6.8)	1.24 d (7.2)	1.17 d (6.8)	1.12 d (6.8)	1.11 d (6.8)
28	1.14 d (6.8)	1.07 d (7.2)	1.38 s	1.08 d (6.8)	1.26 s	1.17 d (6.8)

^aAssignments based on ¹H-¹H-COSY, HSQC, HMBC and NOESY data.

^bCDCl₃/CD₃OD (100:1) was used as solvent.

Table 2.

¹³C NMR (100 MHz) spectroscopic data for withanolides 1–6 and 9 (CDCl₃, δ in ppm).^a

Position	1	2b	3	4	5b	6b	9b
1	203.1, C	203.6, C	203.1, C	202.1, C	202.5, C	203.6, C	202.2, C
2	129.0, CH	128.8, CH	129.1, CH	132.4, CH	132.3, CH	129.2, CH	132.4, CH
3	139.7, CH	139.9, CH	139.5, CH	141.9, CH	142.7, CH	144.5, CH	142.5, CH
4	36.7, CH2	36.6, CH2	36.7, CH2	69.8, CH	69.7, CH	32.8, CH2	69.8, CH
5	73.2, C	73.2, C	73.4, C	63.9, C	63.8, C	62.1, C	63.9, C
6	56.3, CH	56.1, CH	56.0, CH	62.1, CH	61.1, CH	63.2, CH	61.3, CH
7	57.1, CH	56.9, CH	57.4, CH	31.2, CH2	30.9, CH2	30.9, CH2	31.0, CH2
8	34.7, CH	35.5, CH	34.5, CH	30.6, CH	29.1, CH	28.9, CH	29.4, CH
9	35.6, CH	34.9, CH	36.1, CH	43.6, CH	43.9, CH	44.6, CH	43.4, CH
10	51.0, C	50.9, C	51.2, C	47.7, C	47.7, C	48.4, C	47.7, C
11	21.6, CH2	21.2, CH2	21.5, CH2	22.2, CH2	21.1, CH2	23.1, CH2	21.0, CH2
12	40.6, CH2	32.9, CH2	34.6, CH2	33.4, CH2	39.3, CH2	40.1, CH2	31.7, CH2
13	44.,1 C	49.3, C	47.7, C	50.0, C	42.4, C	43.1, C	46.9, C
14	49.6, CH	44.0, CH	52.7, CH	51.3, CH	56.3, CH	54.1, CH	50.3, CH
15	36.2, CH2	34.7, CH2	30.6, CH2	25.1, CH2	23.6, CH2	36.9, CH2	22.8, CH2
16	73.4, CH	75.2, CH	123.6, CH	34.8, CH2	21.8, CH2	72.9, CH	31.8, CH2
17	58.3, CH	82.3, C	155.6, C	87.4, C	54.4, CH	57.5, CH	89.9, C
18	14.8, CH3	14.8, CH3	16.5, CH3	16.7, CH3	13.3, CH3	14.6, CH3	15.8, CH3
19	14.7, CH3	14.6, CH3	14.9, CH3	17.2, CH3	16.5, CH3	14.9, CH3	16.5, CH3
20	78.4, C	42.8, CH	36.3, CH	78.9, C	74.9, C	79.1, C	75.7, C
21	22.3, CH3	9.6, CH3	17.2, CH3	19.9, CH3	19.7, CH3	22.2, CH3	22.0, CH3
22	81.2, CH	77.3, CH	77.3, CH	81.0, CH	79.8, CH	72.9, CH	80.8, C
23	31.3, CH2	32.7, CH2	41.6, CH2	31.8, CH2	39.2, CH2	78.4, CH	30.0, CH2
24	31.1, CH	31.2, CH	71.0, C	31.2, CH	70.1, C	41.7, CH	30.7, CH
25	40.7, CH	40.5, CH	44.9, CH	40.4, CH	44.9, CH	40.0, CH	40.2, CH
26	175.9, C	177.2, C	174.1, C	175.1, C	175.3, C	180.9, C	175.1, C
27	14.2, CH3	14.1, CH3	8.8, CH3	14.1, CH3	8.4, CH3	14.0, CH3	13.8, CH3
28	21.1, CH3	20.8, CH3	29.0, CH3	21.0, CH3	28.2, CH3	12.9, CH3	20.8, CH3

^aAssignments based on DEPT, HSQC and HMBC data.

^bCDCl₃/CD₃OD (100:1) was used as solvent.

Fig. 2.

Key HMBC (→) correlations of withanolides 1, 4, and 6 and key ¹H–¹H COSY (−) correlations of 4.

Fig. 3.

Key NOESY correlations of withanolides 1, 4, and 6.

The HRMS and NMR data of withanolide 2 indicated that it has the same molecular formula (C₂₈H₄₀O₇) as that of 1. Comparison of the ¹H and ¹³C NMR spectroscopic data of 1 and 2 suggested that they had closely related structures and contained the 2,3-enone, OH-5α, 6α,7α-epoxide and ring E δ lactone moieties. The ¹H NMR spectrum of 2 (Table 1) analyzed with the help of ¹H–¹H COSY data displayed three CH₃–CH moieties instead of two in 1, suggesting that C-20 in 2 is a methine carbon. Analysis of the HMBC spectrum of 2 (see Figs. S9 and S35, Supplementary data) revealed that both H₃-21 [δ_H 0.96 (3H, d, J = 7.2 Hz)] and H₃-18 [δ_H 0.79 (3H, s)] showed correlations with an oxygenated tetrasubstituted carbon at δ 82.3, suggesting that C-17 is oxygenated. The presence of OH-16 in 2 was deduced from the HMBC correlations of H-16 [δ_H 4.24 (1H, brd, 7.6 Hz)]/C-14 (δ_C 44.0) and H-16/C-20 (δ_C 42.8). The α orientations of OH-16 and OH-17 were determined by the NOESY correlations of H₃-18/H-20 and H₃-18/H-16. The ECD spectrum of 2 exhibited a negative Cotton effect at 336 nm confirming the configuration and the trans-linkage of A and B rings (Begley et al., 1976), and a positive Cotton effect at 242 nm confirming the R configuration of C-22 (Abdullaev et al., 1986). Thus, withanolide 2 was identified as 24,25-dihydroexodeconolide C [(20S,22R,24S,25R)-5α,16α,17α-trihydroxy-6α,7α-epoxy-1-oxowitha-2-enolide].

The molecular formula of withanolide 3 was determined as C₂₈H₃₈O₆ using a combination of HRMS and ¹³C NMR data and indicated ten degrees of unsaturation. Analysis of its 1D and 2D NMR spectroscopic data suggested that it contained 2,3-enone, OH-5α, 6α,7α-epoxide and ring E δ lactone moieties as in 1 and 2. However, proton splitting patterns and carbon chemical shifts of those belonging to rings D and E of 3 were found to be different from those of 1 and 2. A broad singlet proton signal at δ_H 5.51 suggested that 3 contained an additional double bond bearing an olefinic proton. This double bond in 3 was found to be at C-16(17) based on the HMBC correlations of H₃-18 [δ_H 0.85 (3H, s)]/C-17 (δ_C 155.6), H₃-21 [δ_H 1.08 (3H, d, J = 7.2 Hz)]/C-17, H-16/C-13 (δ_C 47.7), and H-16/C-14 (δ_C 52.7) (see Figs. S15 and S35, Supplementary data). Among the five methyl proton signals in the ¹H NMR spectrum of 3, three appeared as singlets (δ_H 1.38, 1.19, and 0.85) and two as doublets [δ_H 1.24 (d, J = 7.2 Hz) and 1.08 (d, J = 7.2 Hz)] suggesting that either C-24 or C-25 should be hydroxylated. The attachment of OH to C-24 was established by the HMBC correlations of H₃-27 [δ_H 1.24 (3H, d, J = 7.2 Hz)]/C-26 (δ_C 174.1), H₃-27/C-24 (δ_C 71.0), and H-22 [δ_H 4.29 (1H, m)]/C-24. The orientation of the methyl groups at C-24 and C-25 were found to be same (α and α, respectively) as those in 1 and 2, by the NOESY correlations of H₃-28/H-22α and H₃-28/H-25 [δ_H 2.55 (1H, m)] (see Figs. S17 and S35, Supplementary data). Similar to 1 and 2, the ECD spectrum of 3 exhibited a negative Cotton effect at 336 nm confirming the configuration and the trans-linkage of A and B rings (Begley et al., 1976), and a positive Cotton effect at 242 nm confirming the R configuration of C-22 (Abdullaev et al., 1986). Accordingly, withanolide 3 was identified as 16,17-dehydro-24-epi-dioscorolide A [(20S,22R,24R,25R)-5α,24-dihydroxy-6α,7α-epoxy-1-oxowitha-2,16-dienolide].

The HRMS and NMR data of withanolide 4 were consistent with the molecular formula C₂₈H₄₀O₇ indicating nine degrees of unsaturation. Its ¹H NMR spectrum (Table 1) exhibited typical resonances of withanolides belonging to the group I type (Yang et al., 2016) comprising of two olefinic protons [δ_H 6.89 (1H, dd, J = 2.0, 10.0 Hz, H-3), 6.16 (1H, d, J = 10.0 Hz, H-2)], two oxygenated methines [δ_H 3.70 (1H, dd, J = 2.0, 5.6 Hz, H-4), 4.64 (1H, brd, J = 8.8 Hz, H-22)], a proton of the 5β,6β-epoxide [δ_H 3.18 (1H, brs, H-6)], and five methyl groups [δ_H 0.87 (3H, s, H₃-18), 1.37 (3H, s, H₃-19), 1.25 (3H, s, H₃-21), 1.17 (3H, d, J = 6.8 Hz, H₃-27), 1.08 d (3H, d, J = 6.8 Hz, H₃-28)]. The ¹³C NMR data (Table 2) indicated the presence of 28 carbons including two carbonyls of 2,3-enone (δ_C 202.1, C-1) and δ-lactone (δ_C 175.1, C-26)] moieties, two oxygenated methines (δ_C 69.8, C-4; 81.0, C-22), two oxygenated tertiary carbons (δ_C 87.4, C-17; 78.9, C-20), oxygenated carbons of the 5β,6β-epoxide moiety (δ_C 63.9, C-5; 62.1, C-6), two quaternary carbons (δ_C 47.7, C-10; 50.0 C, C-13), together with five methylene and five methyl carbons. The HMBC data for 4 (Fig. 2) together with the above ¹H and ¹³C NMR data suggested that its planar structure is the same as that of philadelphicalactone A (9) previously encountered in soil-grown P. philadelphica (Su et al., 2002) and also found to co-occur in this extract (see below). The orientations of OH-4 and 5,6-epoxide in 4 were shown to be β by the NOESY correlations of H₃-19/OH-4 [δ_H 2.99 (1H, brs)] and H-4/H-6. The NOESY correlations of H₃-18 [δ_H 0.87 (3H, s)]/OH-17 [δ_H 3.13 (1H, brs)] and H₃-21 [δ_H 1.25 (3H, s)/H₂-12 [δ_H 1.65 (1H, m) and 1.89 (1H, m)] suggested that OH-17 is β-oriented. The orientations of the methyl groups at C-24 and C-25 of ring E were found to be α and β respectively, similar to those of 1 and 2 and the known withanolides 7, 9, and 12 based on the NOESY correlations of H₃-28 [δ_H 1.08 (d, J = 6.8 Hz)]/H-22α and H-22α/H-25. The ECD spectrum of 4 exhibited positive Cotton effects at 339 and 242 nm, indicating the configuration and the cis linkage of the A and B ring systems (Begley et al., 1976) and the R absolute configuration of C-22 (Abdullaev et al., 1986), respectively. The foregoing data suggested that 4 and 9 were identical to each other except for the orientation of OH-17. Since the structure of 9 has been confirmed by X-ray analysis and the orientation of OH-17 has been shown to be α (Su et al., 2002), OH-17 in 4 should be β oriented. Previous studies have demonstrated applicability of the ¹³C NMR γ-gauche effects on the chemical shifts of C-12, C-18, and C-21 to determine the orientation of OH-17 in a number of withanolides containing epimeric OH-17β and OH-17α groups including perulactone B (17β-OH) and perulactone H (17α-OH) (Fang et al., 2012), withanolide E (17β-OH) and 17-isowithanolide E (17α-OH) (Gottlieb and Kirson, 1981), and 17-epi-withanolide K (17β-OH) (Choudhary et al., 1995) and withanolide K (17α-OH) (Gottlieb and Kirson, 1981). The ¹³C NMR data for these isomeric pairs of 17-hydroxywithanolides have suggested that the change in orientation of OH-17 from β to α caused a decrease (up-field shift) in the chemical shifts of C-12 and C-18, and an increase (down-field shift) in the chemical shift of C-21 due to the γ-gauche effect. The chemical shift differences observed for 4 and 9 (Δ = δ₉−δ₄) were found to be −1.7 ppm for C-12, −0.9 ppm for C-18, and +2.1 ppm for C-21, further confirming the orientation of OH-17 in 4 to be β. Based on the foregoing evidence the structure of withanolide 4, named as 17-epi-philadelphicalactone A, was determined as (20R,22R,24S,25R)-4β,17β,20-trihydroxy-5β,6β-epoxy-1-oxowitha-2-enolide (4). To the best of our knowledge, this constitutes the first report of the occurrence of a 17β-hydroxywithanolide in P. philadelphica.

The molecular formula of withanolide 5 was determined as C₂₈H₄₀O₇ by a combination of HRMS and ¹³C NMR data and indicated nine degrees of unsaturation. The ¹H NMR data of 5 (Table 1) showed characteristic signals for H-2 [δ_H 6.11 (1H, d, J = 10.0 Hz)] and H-3 [δ_H 6.89 (1H, dd, J = 2.4, 10.0 Hz)] of 2,3-enone, H-4 [δ_H 3.59 (1H, dd, J = 2.4, 5.6 Hz)] of 4β-hydroxymethine, H-6β of 5β,6β-epoxide [δ_H 3.10 (1H, brs)], H-22 [δ_H 4.02 (1H, dd, J = 5.2, 11.2 Hz)], H₃-18 [δ_H 0.73 (3H, s)], H₃-19 [δ_H 1.31 (3H, s)], and H₃-21 [δ_H 1.13 (3H, s)], in addition to the two methyl signals at δ_H 1.12 (3H, d, J = 6.8) and 1.26 (3H, s). These data suggested that one of the methyl groups in the δ lactone side chain of 5 is attached to a protonated carbon and the other to an oxygenated carbon. This oxygenated carbon was determined to be C-24 by the HMBC correlations of H₃-27/C-24 (δ_C 70.1), H₃-27/C-26 (δ_C 175.3), H₃-28/C-25 (δ_C 44.9), and H₃-28/C-23 (δ_C 39.2) (see Figs. S27 and S35, Supplementary data). The orientations of OH-4 and 5,6-epoxide moiety in 5 were determined to be β by NOESY correlations of H-6α/H₂-7 [δ_H 1.18 (1H, m), 2.06 (1H, ddd, J = 2.4, 3.6, 14.8 Hz)], and H-6α/H-4α (see Fig. S29, Supplementary data). The orientation of the side chain was deduced to be β by the NOESY correlation of H₃-18/H₃-21. The NOESY correlations observed for H-22α/H₃-28 and H₃-28/H-25 [δ_H 2.40 (1H, q, J = 6.8 Hz)] suggested that CH₃-27 is β oriented whereas CH₃-28 is α oriented as in withanolides 1–4 found to co-occur in this extract. The ECD spectrum of 5 exhibited positive Cotton effects at 339 and 242 nm, confirming the configuration and the cis linkage of A and B rings (Begley et al., 1976) and the R absolute configuration of C-22 (Abdullaev et al., 1986), respectively. Thus, withanolide 5 was identified as 16-deoxyphiladelphicalactone C [(20R,22R,24R,25R)-4β,20,24-trihydroxy-5β,6β-epoxy-1-oxowitha-2-enolide].

The HRMS and NMR data of withanolide 6 were consistent with the molecular formula C₂₈H₄₀O₇ and indicated nine degrees of unsaturation. The ¹H NMR data of 6 (Table 1) exhibited resonances due to two olefinic protons [δ_H 6.80 (1H, ddd, J = 2.0, 6.0,10.0 Hz, H-3), 5.97 (1H, dd, J = 2.8, 10.0 Hz, H-2)], three oxygenated methines [δ_H 4.39 (1H, m, H-16), 4.41 (1H, m, H-23), 3.94 (1H, brs, H-22)], and five methyls [δ_H 1.09 (3H, s, H₃-18), 1.20 (3H, s, H₃-19), 1.28 (3H, s, H₃-21), 1.11 (3H, d, J = 6.8 Hz, H₃-27), 1.17 (3H, d, J = 6.8 Hz, H₃-28)]. The ¹³C NMR spectrum of 6 (Table 2), assigned with the help of HSQC data, supported the presence of 2,3-enone (δ_C 203.6, C-1; 129.2 C-2; 144.5, C-3), 5β,6β-epoxide (δ_C 62.1, C-5; 63.2, C-6), and lactone carbonyl (δ_C 180.9) moieties similar to ixocarpalactone A (10) (Su et al., 2002) with a ring E γ-lactone side chain moiety. Comparison of the NMR data of 6 with those of 10 and the analysis of its HMBC data (Fig. 2) indicated that the CHOH-4 in 10 was replaced by a CH₂-4 in 6. The orientation of the 5,6-epoxide of 6 was confirmed by 1D NOESY correlations of H₃-18/H-4β and H-4α/H-6α (Fig. 3). The orientation of the side chain in 6 was found to be β as in 10 by the NOESY correlations of H-23/H₃-28 and H₃-28/H-25 [δ_H 2.62 (1H, m)]. A positive Cotton effect at 361 nm in the ECD spectrum of 6 confirmed the configuration and cis-linkage of A and B rings (Begley et al., 1976). Based on the foregoing evidence, the structure of withanolide 6 was determined as 4-deoxyixocarpalactone A [(20R,22R,23R,24R,25R)-16β,20,22-trihydroxy-5β,6β-epoxy-1-oxowitha-2-en-23,26-olide].

2.2. Cytotoxic activities of withanolides

Previous studies by us and others have shown interesting cytotoxic activity for a number of withanolides, some exhibiting selectivity for certain cancer cell lines (Chen et al., 2011; Maldonado et al., 2011; Wijeratne et al., 2014; Xu et al., 2015, 2016 and 2017). Most noteworthy is the potency and selectivity shown by some 17β-hydroxywithanolides against prostate cancer cell lines, LNCaP and PC-3 (Xu et al., 2015). Withanolides 1–11 encountered in this study were evaluated for their cytotoxic activity in a panel of five selected tumor cell lines consisting of LNCaP (androgen-sensitive human prostate adenocarcinoma), ACHN (human renal adenocarcinoma), UO-31 (human kidney carcinoma), M14 (human melanoma), SK-MEL-28 (human melanoma), and normal HFF (human foreskin fibroblast) cells. Withanolide E (13) and 4β-hydroxywithanolide E (14), the 17β-hydroxywithanolides which we have previously shown to exhibit selective and potent activity for LNCaP cells (Xu et al., 2017), were included for comparison purposes. The IC₅₀ data for the active withanolides are presented in Table 3. Of those tested, withanolides 4, 7, 9, and 10 showed selective cytotoxicity against human renal and kidney carcinoma, and melanoma cell lines compared to normal HFF cells. It is noteworthy that all active withanolides contain 2,3-enone and 5β,6β-epoxide moieties in rings A and B. However, ixocarpalactone B (11) with these moieties had no cytotoxic activity up to concentration of 10.0 μM suggesting the importance of side chain structure for this activity. The lack of activity of the 17β-hydroxywithanolide, 17-epi-philadelphicalactone A (4), for LNCaP cells up to a concentration of 2.0 μM compared to its analogue 14 (IC₅₀ 0.16 ± 0.40), with 24,25-ene moiety and OH-14α further supports our previous finding that the reduction of the 24,25-double bond of the side chain and/or absence of OH-14α lead to a decrease in cytotoxic activity (Xu et al., 2017).

Table 3.

Cytotoxicity data for withanolides 4, 7, 9 and 10 from aeroponically cultivated P. philadelphica, withanolide E (13) and 4β-hydroxywithanolide E (14) against a panel of selected tumor cell lines and normal cells.^a

Compound	Cell linesb
	LNCaP	ACHN	UO-31	M14	SK-MEL-28	HFF
4	>2.0	6.9 ± 0.0	7.3 ± 0.4	>10.0	>10.0	>10.0
7	>2.0	4.2 ± 0.2	5.3 ± 0.3	3.8 ± 0.2	5.6 ± 0.2	>10.0
9	>2.0	4.4 ± 0.3	3.6 ± 0.1	>10.0	4.6 ± 0.2	>10.0
10	>2.0	4.8 ± 0.1	6.0 ± 0.2	2.9 ± 0.1	8.7 ± 0.4	>10.0
13	0.06 ± 0.01	0.46 ± 0.06	NT	>2.0	>2.0	>10.0
14	0.2 ± 0.0	>2.0	NT	>2.0	>2.0	>10.0
Doxorubicin	0.02 ± 0.01	0.02 ± 0.01	NT	0.09 ± 0.01	0.25 ± 0.01	0.15 ± 0.03

^aResults are expressed as IC₅₀ values in μM. Doxorubicin and DMSO were used as positive and negative controls. Withaferin A (13) and withanolide E (14) were used for comparison purposes. NT = not tested. Withanolides 1–3, 5–6, 8, and 11 had no activity up to 10 μM.

^bKey: ACHN = human renal adenocarcinoma; UO-31 = human kidney carcinoma; M14 = human melanoma; SK-MEL-28 = human melanoma; HFF = human foreskin fibroblast.

3. Conclusions

Chemical investigation of aeroponically grown P. philadelphica led to the isolation of six new and five known withanolides further demonstrating the applicability of this environmentally-controlled cultivation technique for their efficient production. One of the new natural products encountered, 17-epi-philadelphicalactone A (4), belongs to 17β-hydroxywithanolide group of withanolides known to have potent and selective cytotoxicity to certain cancer cell lines and it is significant that this is the first report of the occurrence of this group of withanolides in P. philadelphica. When evaluated for cytotoxic activity, withanolides 4, 7, 9, and 10 exhibited selective cytotoxicity against human renal carcinoma (ACHN), kidney carcinoma (UO-31), and melanoma (M14 and SK-MEL-28) cell lines compared to normal human foreskin fibroblast (HFF) cells. These findings provide additional support that investigation of withanolide producing plants and application of aeroponic technique for their cultivation may lead to the discovery of new withanolide analogues with potent and selective cytotoxicity to some tumor cell lines.

4. Experimental

4.1. General experimental procedures

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 recorded in MeOH on a Jasco 810 spectrometer at 25 °C, using a quartz cell with 1.0 mm optical path length. The 1D and 2D NMR spectra were recorded on a Bruker Avance III 400 NMR instrument at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR. Chemical shift values (δ) are given in parts per million (ppm), and the coupling constants are in Hz. Low-resolution and high-resolution MS were recorded on Shimadzu LCMS-QP8000α and Agilent G6224A TOF mass spectrometers, respectively. 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 plates (0.20 mm thickness) of silica gel 60 F₂₅₄ (Merck) and RP-18 F_254S (Merck). The HPLC purifications were carried out on a Phenomenex Luna 5 μm C₁₈ column (10 × 250 mm) with a Waters Delta Prep system consisting of a PDA 996 detector. The MM2 energy minimizations of possible conformations of compounds were performed using Chem3D 15.0 from PerkinElmer Inc.

4.2. Aeroponic cultivation and harvesting of P. philadelphica

The seeds of Physalis philadelphica Lam. (Solanaceae) obtained from Trade Wind Fruit (P.O. Box 1102, Windsor, CA 95492) were germinated in 1.0 inch Grodan rock-wool cubes in a Barnstead LabLine growth chamber kept at 28 °C with 16 h of fluorescent lighting and 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 cultivation boxes in green houses at the University of Arizona Natural Products Center, 250 E. Valencia Rd., Tucson, AZ 85706 (GPS coordinates: Latitude 32.132620, Longitude −110.965136) for further growth, as described previously for Withania somnifera (Xu et al., 2011) and Physalis crassifolia (Xu et al., 2016). A herbarium sample was deposited at the University of Arizona Natural Products Center (accession number NPCDB-11/6/10). Aerial parts of areoponically grown plants were harvested when fruits were almost mature (ca. 2 months under aeroponic growth conditions). Harvested plant materials were dried in the shade, powdered, and stored at 5 °C prior to extraction.

4.3. Extraction and isolation

The dried and powdered aerial parts of aeroponically grown P. philadelphica (300 g) were extracted with MeOH (2.0 L) in an ultrasonic bath at 25 °C for 2 h, allowed to stand for 8 h and filtered. The resulting filtrate was concentrated to afford the crude MeOH extract (62.8 g). The crude extract was subjected to solvent-solvent partitioning between hexanes and 80% aqueous MeOH. The 80% aqueous MeOH layer was diluted with H₂O to provide 50% aqueous MeOH solution, which was further extracted with CHCl₃. Investigation of the hexanes, CHCl₃ and 50% aqueous MeOH fractions by TLC suggested that only the CHCl₃ fraction contained withanolides. The CHCl₃ fraction (9.8 g) was therefore subjected to column chromatography over RP C₁₈ (80 g), and eluted with 60%, 70%, 80%, 90% aqueous MeOH and MeOH (300 mL each) to give eight fractions (A–H) according to their TLC profiles. The fraction B (116 mg) was subjected to further fractionation by RP C₁₈ HPLC by eluting with 55% aqueous MeOH. Five sub-fractions (B-1–B-5) corresponding to the peaks at retention times (t_Rs) 20.5, 22.7, 32.0, 37.6, and 44.7 min were collected. The sub-fraction B-1 (14.1 mg) was further separated by a silica gel (6 g) column chromatography by eluting with 1:1 (v/v) EtOAc-CHCl₃ to give 9 (9.8 mg). The sub-fraction B-2 (31.9 mg) was further separated by a silica gel (6 g) column chromatography by eluting with 1:1 and 2:1 (v/v) EtOAc-CHCl₃ to give 4 (31.4 mg) and 1 (0.7 mg). The sub-fraction B-3 (4.1 mg) was further purified by a silica gel (6 g) column chromatography eluted with 2:1 (v/v) EtOAc-CHCl₃ to give 3 (1.0 mg). The sub-fraction B-4 (2.6 mg) was purified by a preparative silica gel TLC [95:5 (v/v) CHCl₃-MeOH] to give 6 (1.3 mg, R_f = 0.3), and the B-5 (3.2 mg) was purified by a silica gel (6 g) column chromatography by eluting with 95:5 (v/v) CHCl₃-MeOH to give 2 (2.9 mg). The fraction C (215 mg) was subjected to a RP C₁₈ HPLC byeluting with 52% aqueous MeOH, and 7 (19.8 mg, t_R = 29.0 min) was obtained. A portion of fraction D (100 mg from total of 289 mg) was subjected to a RP C₁₈ HPLC to afford 10 (57.5 mg, t_R = 25.0 min, 55% aqueous MeOH). The fraction E (571 mg) was crystallized in MeOH to afford the colorless crystals of 11 (272 mg) and a mother liquid, which was subjected to a RP C₁₈ HPLC eluted with 50% MeOH to give 5 (36.8 mg, t_R = 27.1 min, 50% aqueous MeOH) and additional amount of 11 (31.6 mg, t_R = 29.5 min, 50% aqueous MeOH). The fraction F (145 mg) was fractionated again on a RP C₁₈ (10 g) column eluted with 50% aqueous MeOH to afford four sub-fractions, F-1–F-4. The sub-fraction F-2 (32.1 mg) was crystallized in MeOH to give 8 (9.1 mg).

4.3.1. 16β-hydroxyixocarpanolide (1)

White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{25}-21$ (c 0.07, MeOH); UV (MeOH) λ_max (log ε) 223 (3.66) nm; ECD (MeOH) [θ] + 1173 (242 nm), −5340 (335 nm); ¹H and ¹³C NMR data, see Tables 1 and 2, respectively; positive HRESIMS m/z 511.2657 [M+Na]⁺ (calcd for C₂₈H₄₀O₇Na, 511.2667).

4.3.2. 24,25-Dihydroexodeconolide C (2)

White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{25}-13$ (c 0.29, MeOH); UV (MeOH) λ_max (log ε) 225 (3.80) nm; ECD (MeOH) [θ] + 694 (242 nm), −5877 (336 nm); ¹H and ¹³C NMR data, see Tables 1 and 2, respectively; positive HRESIMS m/z 511.2687 [M+Na]⁺ (calcd for C₂₈H₄₀O₇Na, 511.2667).

4.3.3. 16,17-Dehydro-24-epi-dioscorolide A (3)

White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{25}-2.0$ (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 227 (3.67) nm; ECD (MeOH) [θ]) 426 (242 nm), −5875 (336 nm); ¹H and ¹³C NMR data, see Tables 1 and 2, respectively; positive HRESIMS m/z 493.2557 [M+Na]⁺ (calcd for C₂₈H₃₈O₆Na, 493.2561).

4.3.4. 17-Epi-philadelphicalactone A (4)

White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{25}+3.0$ (c 2.7, MeOH); UV (MeOH) λ_max (log ε) 217 (3.67) nm; ECD (MeOH) [θ] + 1930 (243 nm), +4458 (339 nm); ¹H and ¹³C NMR data, see Tables 1 and 2, respectively; positive HRESIMS m/z 511.2668 [M+Na]⁺ (calcd for C₂₈H₄₀O₇Na, 511.2667).

4.3.5. 16-Deoxyphiladelphicalactone C (5)

White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{25}+19$ (c 1.1, MeOH); UV (MeOH) λ_max (log ε) 215 (3.85) nm; ECD (MeOH) [θ] + 3157 (232 nm), +3938 (340 nm); ¹H and ¹³C NMR data, see Tables 1 and 2, respectively; positive HRESIMS m/z 511.2676 [M+Na]⁺ (calcd for C₂₈H₄₀O₇Na, 511.2667).

4.3.6. 4-Deoxyixocarpalactone A (6)

White amorphous powder; $[\alpha {]}_{\mathrm{D}}^{25}+42$ (c 0.13, MeOH); UV (MeOH) λ_max (log ε) 222 (3.71) nm; ECD (MeOH) [θ] – 779 (241 nm), +403 (276 nm), +666 (361 nm); ¹H and ¹³C NMR data, see Tables 1 and 2, respectively; positive HRESIMS m/z 511.2681 [M+Na]⁺ (calcd for C₂₈H₄₀O₇Na, 511.2667).

4.4. Cytotoxicity assay

The ACHN (human renal adenocarcinoma), M14 (human melanoma), SK-MEL-28 (human melanoma) and UO-31 (human kidney carcinoma) cells were all obtained from the Developmental Therapeutics Program (DTP) (NCI, Frederick, MD). LNCaP (androgen-sensitive human prostate adenocarcinoma) and HFF (human foreskin fibroblast) cells were purchased from ATCC (Manassas, VA). Cell lines were maintained as recommended by the source institution. Cell numbers were estimated using the MTS dye assay (Promega, Madison, WI) (Brooks et al., 2010). The MTS dye assay was used for evaluating cytotoxicity of withanolides 1–11, 13 and 14 against LNCaP, ACHN, UO-31, M14 and SK-MEL-28 cell lines, and HFF cells. Doxorubicin and DMSO were used as positive and negative controls, respectively. For LNCaP, ACHN, UO-31 and HFF cells, the assay was performed in RPMI (Roswell Park Memorial Institute) medium with 5% FCS, 2.0 mM l-glutamine, 1 × nonessential amino acids, 1.0 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES [4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid] and 5 × 10⁻⁵ M 2-mercaptoethanol. For M14 and SK-MEL-28 cells, DMEM (Dulecco's Modified Eagle) medium replaced RPMI medium. Briefly, 10,0 cells (LNCaP) or 5000 cells (ACHN, UO-31, M14, SK-MEL-28, and HFF) were incubated overnight at 37 °C in 96-well microtiter plates. Serial dilutions of compounds in DMSO or vehicle control (DMSO) were added to triplicate wells and the microtiter plates were incubated for a further 72 h. Viable cell number was determined by the addition of CellTiter 96^® AQueous One Solution Cell Proliferation Assay solution (MTS), plates were incubated for 2 h and then the absorbance at 490 nm was measured. Cytotoxicity was calculated as in the following formula: % Cytotoxicity = [(A_Media-A_Treatment)/A_Media)]*100. The IC₅₀ values and standard deviations (±) were determined using Microsoft Excel software from dose-response curves obtained from at least three independent experiments.