Anti-leishmanial physalins—Phytochemical investigation, in vitro evaluation against clinical and MIL-resistant L. tropica strains and in silico studies

Abstract

Cutaneous leishmaniasis (CL) is a major health problem in over 98 countries of the world, including Pakistan. The current treatments are associated with a number of adverse effects and availability problem of drugs. Therefore, there is an urgent need of easily available and cost effective treatments of CL- in Pakistan. The bioassay-guided fractionation and purification of crude extract of Physalis minima has led to the isolation of a new aminophysalin B (1), and eight known physalins, physalin B (2), 5ß,6ß-epoxyphysalin B (3), 5α-ethoxy-6ß-hydroxy-5,6-dihydrophysalin B (4), physalin H (5), 5ß,6ß-epoxyphysalin C (6), and physalin G (7), K (8), and D (9). It is worth noting that compound 1 is the second member of aminophysalin series, whereas compound 6 was fully characterized for the first time. The structures of compounds 1–9 were elucidated by spectroscopic techniques Whereas, the structural assignments of compounds 1 and 8 were also supported by single-crystal X-ray diffraction studies. The anti-leishmanial activity of isolated physlains 1–9 was evaluated against Leishmania major and Leishmania tropica promastigotes. Compounds 2, 3, and 5–7 (IC₅₀ = 9.59 ± 0.27–23.76 ± 1.10 μM) showed several-fold more potent activity against L. tropca than tested drug miltefosine (IC₅₀ = 42.75 ± 1.03 μm) and pentamidine (IC₅₀ = 27.20 ± 0.01 μM). Whereas compounds 2, 3 and 5 (IC₅₀ = 3.04 ± 1.12–3.76 ± 0.85 μM) were found to be potent anti-leishmanial agents against L. major, several fold more active than tested standard miltefosine (IC₅₀ = 25.55 ± 1.03 μM) and pentamidine (IC₅₀ = 27.20 ± 0.015 μM). Compounds 4 (IC₅₀ = 74.65 ± 0.81 μM) and 7 (IC₅₀ = 39.44 ± 0.65 μM) also showed potent anti-leishmanial ativity against the miltefosine-unresponsive L. tropica strain (MIL resistant) (miltefosine IC₅₀ = 169.55 ± 0.78 μM). Molecular docking and predictive binding studies indicated that these inhibitors may act via targeting important enzymes of various metabolic pathways of the parasites.

Introduction

Cutaneous leishmaniasis, a neglected tropical disease (NTD), infects 700,000 to 1.2 million poorest people in over 98 countries of Africa, Asia, the Middle East and America annually. Poverty, migration, poor hygiene, and malnutrition are the major risk factors associated with endemicity of various forms of leishmaniasis in the developing world [1–3]. About 70 types of sand flies are known to be responsible to transmit the Leishmania parasite geographically distributed Old (Asia, Africa, and the Middle East) and New world i.e. western hemisphere countries. Broadly they belong to the phlebotomine and Lutzomyia genera. The diagnosis of leishmaniasis, both parasitic (direct) and immunological (indirect), is often challenging in low-resource countries [1]. Pakistan is among the endemic countries with CL cases in almost every part of the country. In 2020, as per WHO Global Leishmaniasis Surveillance Report, Afghanistan, Algeria, Brazil, Colombia, Iraq, Pakistan and Syria, are the seven countries, each reported >6,000 CL cases, representing >80% of cases globally [4]. CL is not a life-threatening infection but cause extreme psychological trauma, and stigma as infected people pushed to be excluded from public life.

The available therapeutic options are now losing effectiveness due to the emergence of resistance [5, 6]. Furthermore, these available drugs are expensive, and some have toxic effects on liver and kidneys [7]. Medicinal plants have immense potential to yield compounds that are capable of treating many ailments. In recent days, pharmaceutical industries have reformed their attention towards plant-based medicine and formulation because of their least toxicity and more efficacy [8–10]. Physalis minima Linn is a member of the Solanaceae family, commonly known as native gooseberry, and reported for anti-cancer, anti-diabetic, anti-oxidant, anti-ulcer, analgesic, antipyretic, inhibition of amylase, lipase and alpha-glucosidase enzymes, as well as anti-gonorrhoeal, and anti-inflammatory properties [11–15]. Previous research on P. minima led to the isolation of bioactive withanolides and physalins, which showed potent anti-inflammatory and cytotoxic activities [16–19]. We have reported the in vitro anti-leishmanial activity of the Physalis minima plant extracts and its pure constituents physalin B (2), 5ß,6ß-epoxyphysalin B (3), and physalin H (5) against promastigotes of L. major for the first time [20–22]. The presented study deals with the isolation, structure determination, and anti-leishmanial effect of a new aminophysalin B (1) along with eight known physalins, physalin B (2), 5ß,6ß-epoxyphysalin B (3), 5α-ethoxy-6ß-hydroxy-5,6-dihydrophysalin B (4), physalin H (5), 5ß,6ß-epoxyphysalin C (6), and physalin G (7), K (8), and D (9) in vitro against promastegotes of L. tropica. Compounds 1, 4, 6, 7, 8 and 9 were also evaluated against promastegotes of L. major but no significant activity was observed. Moreover, the effect of compounds 2–9 were also evaluated for their potential against the MIL- resistant L. tropica. These steroidal lactones 1–9 were also subjected to in-silico studies to predict their mechanisms of action.

Materials and methods

General

Precoated TLC plates (silica gel, 20×20, 0.25 mm thick PF₂₅₄, Merck, Germany) were used for thin layer chromatography, and stained by spraying with Dragendroff’s and vanillin reagents. Column chromatography was carried out on silica gel (70–230 mesh, Merck, Germany) [23]. Recycling preparative separation was performed on a HPLC JAI LC-908W (Japan) instrument, equipped with GAIGEL-SIL, D-60-10, CHCl₃-MeOH as the mobile phase, with UV detection at 254 nm. JMS-600H mass spectrometer (JEOL, Japan) was used to record the mass spectra (EI- and HREI-MS) in m/z (relative abundance %). Electrospray ionization mass spectra (ESI-MS) were recorded on Amazon speed ion trap low-resolution mass spectrometer (Bruker Daltonics, Germany) and high resolution electrospray ionization mass spectra (HRESI-MS) were measured on MaXis II ESI–QTOF Ultra High-Resolution Mass Spectrometer (Bruker Daltonics, Germany).Ultraviolet (UV) experiments were performed in methanol on Hitachi U-3200 spectrophotometer (Hitachi, Japan). Infrared (IR) analysis was performed in KBr on FT-IR-8900 (Shimadzu, Japan), and Bruker VECTOR 22 spectrophotometers (Bruker, France). UV Spectra (nm) were measured in methanol and chloroform on Evolution 300 UV visible spectrophotometer. FT-IR-8900 spectrophotometer was used to record IR spectra (cm^-1) (JASCO, Japan). Optical rotations were measured in methanol with a digital polarimeter JASCO P-2000 (JASCO, Japan) by using 10 cm cell tube. ¹H- and ¹³C-NMR spectra were recorded on Avance NMR spectrometer at 500 and 75–150 MHz in CDCl₃ or CD₃OD, respectively (Bruker, Zurich, Switzerland). Standard pulse sequences were used for DEPT, and 2D-NMR experiments. The ¹H-NMR, ¹³C-NMR, COSY, HMBC, HMQC, and NOESY spectra were recorded in deuterated chloroform, and methanol on Bruker Avance spectrometers (Bruker, Zurich, Switzerland). X-Ray crystallography data were collected on Bruker D8 venture diffractometer. Cu Kα radiation (1.54178 Å) was used for X-ray diffraction data collection. SAINT program was employed for data integration and reduction; Direct method was utilized for structure solution with full-matrix least square method with aid of SHELXTL (SHELX2016/6).

Plant material

Fresh plants (50 kg.) of Physalin minima (L. Var. indica), were collected from Karachi, Pakistan, and identified by a plant taxonomists, Department of Botany, University of Karachi. A voucher specimen (G. H. 68261) was deposited at the herbarium.

Extraction and isolation

The air-dried plants (11 kg) were extracted with EtOH (100 L) for 10 days. After evaporation of the solvent, a brownish residue (1.7 kg) was obtained that was allowed to be suspended in distilled H₂O (5 L), and defatted with petroleum ether (10 L) [24]. The defatted aqueous extract was further extracted with CH₂Cl₂ (35 L). The CH₂Cl₂ extract was concentrated under reduced pressure to obtain a greenish dried powder (85 g) that was subjected to column chromatography and eluted with Pet ether: CH₂Cl₂, CH₂Cl_2, CH₂Cl_2: MeOH (10:90…90:10, 100, 99:1–97:3) to afford four main fractions A (2.14 g), B (1.54 g), C (1.14 g), and D (24 g), respectively. The repeated column chromatography of fraction A (2.14 g) afforded one major fraction a (300 mg, petroleum: acetone 80: 20) which was further purified on recycling reverse phase HPLC to obtain purified compound 2 (10 mg, CHCl₃: MeOH-99:1, Flow rate: 4 mL/min, R_T = 16 min) and 3 (60 mg, CHCl₃: MeOH-99:1, Flow rate: 4 mL /min, R_{T =} 18min). Fraction B (1.54 g) yielded sub fraction b (500 mg, petroleum: acetone 70: 30) which was further subjected to recycling reverse phase HPLC to obtain compound 5 (10 mg, CHCl₃: MeOH-99:1, Flow rate: 4 mL/min, R_T = 16 min). Fraction C (1.14 g) afforded one major sub fraction c (petroleum: acetone 80: 20) which yielded and purified compound 6 (5 mg, CHCl₃: MeOH-99:1, Flow rate: 4 mL/min, R_T = 16 min) via recycling reverse phase HPLC. Fraction D (24 g) was subjected to column chromatography and three major sub fractions were obtained. Subtraction d (1.41) (petroleum ether: acetone-55:45), e (1.0) (Petroleum ether: acetone-45:55) and f (20 g) (petroleum: acetone-20:80) which were purified by using recycling reverse phase HPLC to obtain compounds 1 (3 mg, CHCl₃: MeOH-97:3, Flow rate: 3 mL/min, R_T = 24 min) and 4 (4 mg, CHCl₃: MeOH-96:4, Flow rate: 4 mL/min, R_T = 24 min), 7 (10mg, CHCl₃: MeOH-97:3, Flow rate: 3 mL/min, R_T = 30 min), 8 (6 mg, CHCl₃: MeOH-97:3, Flow rate: 3 mL/min, R_T = 24 min), and 9 (6 mg, CHCl₃: MeOH-97:3, Flow rate: 3 mL/min, R_T = 32 min.

Aminophysalin B (1)

Colorless solid (3 mg): [α] _D = -65 (c = 0 .01, MeOH), UV (MeOH) λ_max nm (log∈): 312 (3.2). IR (KBr) ν_max cm^-1: 3463 (OH), 1732 (C = O), 1656 (C = C-C = O), ¹H-NMR (CDCl₃, 500 MHz): Table 1. ¹³C-NMR (CDCl₃, 175 MHz): Table 1. HRESI-MS m/z: 526.2059 [M+H]⁺(C₂₄H₃₁NO₉+H requires 526.2072).

Table 1. ¹H-NMR and ¹³C-NMR chemical shift data of compounds 1 and 6 (δ in ppm, J in Hz).

C. No.	1a	1b	6a	6b
1	-	208.8	-	204.4
2	6.00 (d, J 2,3 = 10.0)	119.8	5.99 (dd, J2,3 = 10.0, J2,4 = 2.5)	145.5
3	6.98 (dd, J 3,4 = 9.5, J 3,4 = 6.0)	140.9	6.83 (ddd, J3,2 = 10.0, J3,4β = 2.5, J3,4α = 6.0)	128.6
4	6.12 (d, J 3,4 = 5.5)	125.5	2.93, m; 2.04, m	32.9
5	-	155.6	-	61.8
6	4.49, m	70.4	3.24 (d, J3,4 = 2.5)	64.6
7	2.10, m; 2.53, m	29.4	2.35, m; 1.71, m	29.8
8	3.13, m	39.4	2.22, m	39.1
9	2.57, m	34.3	2.51, m	35.5
10	-	52.0	-	49.8
11	2.35, m; 1.65, m	28.2	2.26, m; 1.14, m	22.4
12	2.54, m; 2.04, m	29.5	2.64, m; 1.76, m	26.4
13	-	79.4	-	79.7
14	-	210.7	-	102.1
15	-	171.5	-	210.8
16	2.52, m	56.5	2.50, m	54.6
17	-	83.3	-	81.8
18	-	173.0	-	171.8
19	1.54, s	23.0	1.21, s	14.3
20	-	80.0	-	82.7
21	1.85, s	21.7	1.92, s	20.9
22	4.50, m	77.3	4.58, (dd, J22,23 = 4.5, J22,23 = 2)	76.8
23	2.05, m; 1.97, m	31.6	1.99, m; 2.11, m	31.7
24	-	29.1	-	36.6
25	2.36, m	42.2	-	139.3
26	-	164.9	-	163.7
27	4.17 (dd, J27,27 = 10.0, J27,25 = 20.5)	47.7	4.20 (6.64, s; 5.72, s)	133.3
	4.46 (J27,27 = 21)
28	1.04, s	28.2	1.58, s	27.3

^a 500 MHz, CDCl₃

Single-crystal X-ray diffraction data. Empirical formula = C₂₈ H₃₁ NO_9. H₂O, Mr = 543.55, Crystal system: orthorhombic, space group: P2₁2₁2₁, Unit cell dimensions: a = 9.7870(4) Å, b = 9.7870(4) Å, c = 16.3581(7) Å, Volume: 2412.66(18) ų, Z = 4, ρ_calc = 1.496 Mg/m3, F(000) 1152, Crystal size: 0.900 x 0.060 x 0.030 mm, θ range for data collection: 5.266 to 68.135 deg. Total 15,452 reflections were collected, out of which 4,364 were found unique (R_int = 0.0945). Final R indices were R₁ = 0.0551for [I>2σ (I)], wR₂ = 0.1446, R indices were R₁ = 0.0699, wR₂ = 0.1547 for all data, largest diff. peak and hole: 0.434 and -0.369 e. Å^-3, CCDC No. 2177422.

Physalin B (2)

Colorless solid (10 mg): [α] _D = -257 (c = 0 .05, MeOH), UV (MeOH) λ_max nm (log∈): 230 (3.6). IR (KBr) ν_max cm^-1: 3410 (OH), 1748 (lactone), 1653 (C = C-C = O), ¹H-NMR (CDCl₃, 500 MHz): 1.19 (s, H₃-28), 1.25 (s, H₃-19), 1.95 (s, H₃-21), 4.35 (t, overlapped J_22,23 = 2.5 Hz, H-22), 3.75 (d, J_27,27 = 12.5 Hz, H₂-27), 4.50 (dd overlapped, J_27,27 = 13.5 Hz, J_27,25 = 4.5, H₂-27), 5.91 (dd, J_2,3 = 10.0 Hz, J_{2, 4ß} = 2 Hz, H-2), 6.77 (ddd, J_3,2 = 10.0 Hz, J_3,4a = 5.0 Hz, J_3,4ß = 2.5, H-3), 5.56 (d, J_6,7 = 6.0 Hz, H-6), EIMS m/z (rel.int. %): 510 (M⁺), 492 (100.0), 477 (21.7), 464 (48.0), 173 (46.2), 159 (70.9), 145 (26.1), 91 (26.6), 43 (18.1).

5β, 6β-epoxyphysalin B (3)

White solid (60 mg), [α] _D = -108 (c = 0 .05, MeOH), UV (MeOH) λ_max nm (log∈): 230 (3.5). IR (KBr) ν_max cm^-1: 3422 (OH), 1778 (lactone), 1656 (C = C-C = O), ¹H-NMR (CDCl₃, 500 MHz): 1.24 (s, H₃-28), 1.29 (s, H₃-19), 1.93 (s, H₃-21), 3.25 (d, J_{6α, 7β} = 3.0 Hz, H-6), 4.52 (t, overlapped J_22,23 = 1.5 Hz, H-22), 3.74 (d, J_27,27 = 13.5 Hz, H-27), 4.50 (dd overlapped, J_27,27 = 14.0 Hz, J_27,25 = 4.5 Hz, H-27), 5.99 (dd, J_2,3 = 10.5 Hz, J_{2, 4ß} = 2.0 Hz, H-2), 6.84 (ddd, J_3,2 = 10.0 Hz, J_3,4α = 5.0 Hz, J_3,4ß = 2.5 Hz, H-3), EI-MS m/z (rel. int. %): 526 (6.7, M⁺), 498 (100.0), 480 (15.7), 360 (16.6), 159 (17. 0), 133 (20.6), 91 (14.6), 55 (15.0).

5α-ethoxy-6ß-hydroxy-5,6-dihydrophysalin B (4)

Colorless solid (4 mg): [α] _D = -58 (c = 0 .05, MeOH), UV (MeOH) λ_max nm (log∈): 230 (3.2). IR (KBr) ν_max cm^-1: 3405 (OH), 1787 (lactone), 1667 (C = C-C = O), ¹H-NMR (CDCl₃, 500 MHz): 0.98 (t, J = 7 Hz, CH₃CH₂O), 1.24 (s, H₃-28), 1.50 (s, H₃-19), 1.98 (s, H₃-21), 4.00 (m, H-6), 4.54 (t, overlapped J_22,23 = 1.5, H-22), 3.76 (d, J_27,27 = 13.5 Hz, H-27),4.49 (dd, J_27,25 = 4.5 Hz H-27), 5.85 (dd, J_2,3 = 10.0 Hz, J_{2, 4ß} = 2.5 Hz, H-2), 6.57 (ddd, J_3,2 = 10.0 Hz, J_3,4α = 5.0 Hz, J_3,4ß = 2.0 Hz, H-3), EI-MS m/z (rel. int. %): 572 (7.4, M⁺), 544 (10.8), 526 (24.8), 508 (60.1), 454 (30.3), 171 (79.3), 147 (77.1), 133 (100.0), 109 (89.9), 91 (64.3), 55 (75.6).

Physalin H (5)

Colorless solid (10 mg): [α] _D = -260 (c = 0 .01, MeOH), UV (MeOH) λ_max nm (log∈): 230 (3.8). IR (KBr) ν_max cm^-1: 3426 (OH), 1778 (lactone), 1673 (C = C-C = O), ¹H-NMR (CDCl₃, 500 MHz):1.35 (s, H₃-19), 1.25 (s, H₃-28), 1.99 (s, H₃-21), 4.10 (d, J_{6α, 7β} = 3.0 Hz, H-6), 4.54 (t, overlapped J_22,23 = 2.5 Hz, H-22), 3.75 (d, J_27,27 = 12.5 Hz, H₂-27), 4.50 (dd, overlapped, J_27,27 = 13.5 Hz, J_27,25 = 4.5 Hz, H₂-27), 5.94 (dd, J_2,3 = 10.0 Hz, J_{2, 4ß} = 2.0 Hz, H-2), 6.71 (ddd, J_3,2 = 10.0 Hz, J_3,4α = 5.0 Hz, J_3,4ß = 2.5 Hz, H-4) HRESI-MS m/z: 563.1674 [M+H]⁺ (C₂₈H₃₁ClO₁₀+H requires 563.1679).

5β, 6β-epoxyphysalin C (6)

White solid (5 mg): [α] _D = -168 (c = 0 .01, MeOH), UV (MeOH) λ_max nm (log∈): 230 (3.1). IR (KBr) ν_max cm^-1: 3427 (OH), 1782 (lactone), 1656 (C = C-C = O), ¹H-NMR (CH₃OD, 500 MHz): Table 1. ¹³C-NMR (CH₃OD, 175 MHz): Table 1. HRESI-MS m/z: 527.1907 [C₂₉H₃₀O₁₀+H; 527.1912].

Physalin G (7)

White solid (10 mg): [α] _D = -92 (c = 0 .01, MeOH), UV (MeOH) λ_max nm (log∈): 312 (5.8).IR (KBr) ν_max cm^-1: 3418 (OH), 1776 (lactone), 1622 (C = C-C = O), ¹H-NMR (CDCl₃, 500 MHz):1.25 (s, H₃-28), 1.93 (s, H₃-19), 1.51 (s, H₃-21), 4.49 (m, H-6), 4.52 (m, H-22), 3.74 (J_27,27 = 13.0 Hz, H₂-27), 4.50 (m, H₂-27), 5.93 (d, J_2,3 = 5.5 Hz, H-2), 6.82 (dd, J_3,2 = 9.5 Hz, J_3,4 = 5.5 Hz, H-4), 6.03 (d, J_4,3 = 6.0 Hz), EIMS m/z (rel. int. %): 526 (2.8, M⁺), 508 (55.1), 482 (79.8), 454 (40.1), 185 (38.9), 159 (27.6), 159 (98.6), 134 (67.1) 109 (43.5), 91 (27.0), 55 (32.4).

Physalin K (8)

Colorless solid (6 mg): [α] _D = -266 (c = 0 .01, MeOH), UV (MeOH) λ_max nm (log∈): 229 (3.0). IR(KBr) ν _max cm^-1: 3407 (OH), 1775 (lactone), 1630 (C = C-C = O), ¹H-NMR (CDCl₃, 500 MHz):1.12 (s, H₃-28), 1.20 (s, H₃-19), 1.90 (s, H₃-21), 3.90 (d, J_{6α, 7β} = 3.0, H-6), 4.55 (m, H-22), 3.76 (d, J_27,27 = 13.5 Hz, H₂-27), 4.49 (dd, J_22,23α = 13.5 Hz, J_22,23β = 5.0 Hz, H-22), 6.65 (dd, J_3,2 = 6.5 Hz, J_3,4 = 8.0 Hz, H-3), 7.00 (dd, J_4,2 = 1.0 Hz, J_4,3 = 8.5 Hz, H-4), HRESI-MS m/z: 559.1800 [C₂₈H₃₀O₁₂+H; 559.1810].

Single-crystal X-ray diffraction data. Empirical formula = C₂₈ H₃₀ O_12. H₂O. CH₃OH, Mr = 358.46, Crystal system: Monoclinic, space group: P2₁, Unit cell dimensions: a = 7.4821(5)Å, b = 11.7099(7)Å, c = 15.2299(11)Å, Volume: 1327.52(15)ų, Z = 2, ρ_calc = 1.522 Mg/m3, F(000) 776, Crystal size: 0.270 x 0.130 x 0.010 mm, θ range for data collection: 4.773 to 68.237 deg. A total of 17,151 reflections collected, out of which 4,824 reflections were judged observed (Rint = 0.1337), Final R indices were, R₁ = 0.0521for [I>2σ (I)], wR₂ = 0.1156, R indices were R₁ = 0.0816, wR₂ = 0.1300 for all data, largest diff. peak and hole: 0.349and—0.269e. Å^-3, CCDC No. 2177423.

Physalin D (9)

Colorless solid (6 mg): [α] _D = +78.4 (c = 0 .01, MeOH), UV (MeOH) λ_max nm (log∈): 230 (3.2).IR (KBr) ν_max cm^-1: 3377 (OH), 1788 (lactone), 1653(C = C-C = O),¹H-NMR (CDCl₃, 500 MHz):1.25 (s, H₃-28),1.29 (s, H₃-28), 1.98 (s, H₃-28), 3.76 (m, H-6), 4.53 (m, H-22), 3.74 (m, H₂-27), 4.50 (d, J_27,25 = 5.0 Hz, H₂-27), 6.65 (ddd, J_3,2 = 10.5, Hz, J_3,4a = 4.5 Hz, J_3,4ß = 2.5, H-3), 5.90 (dd, J_2,3 = 10.5 Hz, J_{2, 4ß} = 2.5 Hz, H-2), HRESI-MS [M+H]⁺ m/z: 545.2013 [C₂₈H₃₂O₁₁+H; 545.2004].

Promastigote growth and inhibition assay

L. major and L. tropica (Clinical isolates) promastigotes were grown at 22 ± 25°C in RPMI-1640 (Sigma) medium containing 10% of heat-inactivated (56°C for 30 min) fetal bovine serum. Cell viability was initially evaluated by wet mount method by observing the live cells under compound microscope. Parasite growth was assessed by counting live / motile promastigotes in a Neubauer chamber. Viable cell count was used for the further experimentations (treatments) [25].

For the estimation of particular concertation at which tested compound caused 50% inhibition (IC₅₀) of resistant cell proliferation concerning untreated controls, the MTT assay was employed [26–28]. The promastigotes in their log phase were used in 96-well plates. 1 × 10⁶ wild-type and resistant promastigotes were dispensed in 96 96-well plates, and incubated with tested compounds at a range concentration of 200–10 μM at 27°C for 72 h. After incubation of 72 h, MTT dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) was added and further incubate it for 3–4 h. Amphotericin B, pantamidine and miltefosine were used as the positive control, untreated promastigotes were used as negative control moreover DMSO control was also added in the study as a stock solution of the compounds that were prepared in DMSO. All the experiments were performed in triplicates. After completion of the experiment the absorbance by Multiskan ascent plate reader at 452 nm and percent inhibition was calculated by the following formula.

Cytotoxic concentration (%) = [100- (Absorbance of test)/ (Absorbance of solvent control) ×100] %.

MIL-resistant line

Promastigote cultures tL. tropica (Clinical isolates) were maintained at 26°C in RPMI-1640 medium containing 15% FBS and 1% penicillin–streptomycin mixture.

Generation of miltefosine-unresponsive strain was carried under high MIL pressure by in vitro passage with a stepwise increase in the MIL concentration. At each step, parasites were cultured and passaged every 3–4 days at an initial concentration of 5x10⁵ promastigotes/mL in order to achieve -stable growth as compared to the wildtype isolate. Growth rates were measured for resistant populations and compared with the WT strain. Parasites were counted at an initial concentration of 5x10⁵ parasites/mL and growth was measured daily using a Neubauer chamber until the population reached the stationary phase. Furthermore, the fluorescence microscopic investigations via DAPI stain were also carried out to supplement the study [29, 30].

Cytotoxicity against BJ and 3T3 cell lines

The cytotoxicity of the test compounds against normal cell lines was evaluated through MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide] colorimetric assay in 96-well flat-bottomed microplates [31]. The 3T3 (mouse fibroblast) and BJ (human fibroblast) were cultured Dulbecco’s Modified Eagle Medium, supplemented with 5% of fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 μg/mL of streptomycin in 75 cm2 flasks, and then incubated in 5% CO₂ incubator at 37°C. The growing cells were counted by using a haemocytometer, and diluted with the particular medium. The 5x10⁴ cells/mL of the cell culture was prepared, and a sample (100 mL) was introduced into each well. The medium was removed after overnight incubation, and 200 μL of fresh medium was added with different concentrations of compounds (1–30 μM). After 48 hours, 200 μL MTT (0.5 mg/mL) was added, and the incubation was further continued for 4 hrs. After that time, 100 μL of DMSO was added to each well. The absorbance was measured using a micro plate reader (Spectra Max Plus, Molecular Devices, CA, USA) at 570 nm for the extent of MTT reduction to formazan within the cells. The cytotoxicity was recorded as CC50 against cells by using Soft- Max Pro software. Doxorubicin and cycloheximide were used as positive control. The experiments were run in triplicate.

Docking studies

In order to predict the tentative binding affinities and the possible mode of inhibition via these compounds, molecular docking studies against various leishmanial metabolic enzymes that have role in the growth and survival of the parasite were conducted.

Protein preparation

For docking studies, the selected protein targets i. e., pteridine reductase (PDB:1E92), pyruvate kinase (PDB:3PP7), glyceraldehyde-3-phosphate dehydrogenase (PDB:1I32), phosphoglucose isomerase (PDB:1T10), dihydroortate dehydrogenase (PDB:4EF8), fructose-1,6-bisphosphatase (PDB:5OFU), SAM-dependent methyltransferase (PDB:1XTP), mitochondrial fumarate hydratase (PDB: 6MSO), N-myristoyltransferase (PDB: 6QDB), and MAP kinase (PDB: 3UIB), were prepared by Protein Preparation Wizard in Maestro Schrödinger 12 [32, 33]. During preparation, the missing hydrogens were added, and partial charges were assigned using OPLS-3e force field. Hydrogens and heavy atoms were optimized by restrained minimization.

Ligand preparation and database generation

The 2D structures of compounds were converted to 3D structures, via the Ligprep module in Maestro Schrödinger 12 [34]. Ligprep was used to correct the protonation, and ionization states of the compounds, and assigned proper bond orders. Afterwards, the tautomeric and ionization states were created for each ligand.

Receptor grid generation and molecular docking

The grid box was defined by selecting the co-crystallized molecules in the binding site of abovementioned selected protein targets to keep the center of each docked ligand with the same dimensions of binding box. Rigid receptor docking protocol was run in standard precision (SP) mode of Glide based on OPLS-3e force field [35–37]. During the process of docking, the protein was fixed, while ligands were flexible.

The molecular mechanics-generalized Born surface area (MM-GBSA) method in Prime was used for rescoring the docked pose of ligand in the binding site of the selected target protein [38]. These poses were taken as inputs for the energy minimization of the protein-ligand complexes (E_complex), free protein (E_protein), and free ligands (E_ligand). The binding free energy ΔG_bind was determined according to the following equation:

$$\Delta Gbind=Ecomplex\left(minimized\right)-Eligand\left(minimized\right)-Ereceptor\left(minimized\right)$$

Statistical analysis

Three replicates were used in each experiment, unless otherwise stated. All results were presented as means standard deviations. A one-way ANOVA was used to analyze statistically differences at a P-value 0.05 (95% confidence interval) in conjugation with the Dunnett test using Graph pad prism version 9.4.1 (California. San Diego).

Results

Isolation and structural characterization

The whole plant of P. minima Linn. yielded nine compounds 1–9, including a new aminophysalin (1). These compounds were characterized by spectroscopic techniques (Fig 1).

Fig 1. The structures of compounds 1–9 isolated from Physalis minima.

Compound 1 was obtained as a colorless crystalline solid. The ESI-MS of 1 exhibited the [M+H]⁺ at m/z: 526.2059 [C₂₈H₃₂NO₉+H; 526.3072]. The IR spectrum showed a strong absorption at 3463 cm^-1, indicating the presence of OH groups. The absorption band at 1732 cm^-1 is a characteristic absorption of physalin, which appeared due to a five-membered lactone ring. Furthermore, the IR absorption at 1656 cm^-1 was assigned to an α,β-unsaturated ketone [39–41]. The¹H-NMR spectrum (Table 1) of compound 1 showed three characteristic methyl singlets, resonated at δ 1.54, 1.85, and 1.04 and assigned to H-19, H-21, and H-28 methyl respectively. The appearance of three mutually coupled olefin carbon signals appeared at δ 6.00 (d, J_2,3 = 10.0 Hz), 6.98 (dd, J_3,4 = 9.5 Hz, J_3,4 = 6.0 Hz) and 6.12 (dd, J_4,3 = 5.5 Hz) indicated a 2-ene-1-one system in ring A. The presence of nitrogen-containing bridge between C-15, and C-27 (i.e entirely different from other physalin) was inferred from the appearance of a pair of characteristic signals δ 4.17 (dd, J_27,25 = 20.5 Hz, J_27,27 = 10.0 Hz) and δ 4.46 (J_27,27 = 21) for H₂-27 [42]. An overlapped signal also appeared at δ 4.49 indicating the hydroxylation at C-6 carbon [43]. ¹³C-NMR spectra (Table 1) showed 28 carbon signals, assigned to eleven quaternary, nine methine, five methylene, and three methyl carbons. There were four carbon signals with chemical shift above δ 170, and assigned to two ketone carbonyls [δ_c 210.7 (C-1), δ_c 208.8 (C-14)] and two ester carbonyls [δ_c 171.3 (C-18), δ_c 173.0 (C-26)]. The presence of an additional signal at δ 164.9 (C-15), and downfield shift of C-27 at δ 47.7 suggesting the nitrogen-containing bridge between C-15 and C-27. The presence of carbonyl carbon at C-14 was supported by key HMBC correlations of δ 3.13 (H-8) and 2.52 (H-16) with δ_c 208.8 (C-14) and 164.9 (C-15), respectively (Fig 2). The HMBC correlation of H₂-27 (δ 4.46, 4.17) with 164.9 (C-15) supported the nitrogen-containing bridge. COSY correlation between H-25 and H-27 was also supporting the proposed structure. The OH at C-6 was deduced based on HMBC correlations of H-4 (δ 6.12) with C-6 (δ 70.4) (Fig 2).

Fig 2. Key COSY (——) and HMBC (---->) correlations in compounds 1 and 6.

The structure of compound 1 was unambiguously deduced based on single-crystal X-ray diffraction analysis (Figs 4 and 5). The ORTEP diagram depicted that the molecule consists of seven fused rings, i.e. rings A (C1-C5/C10), B (C5-C10), C(C8/C9/C11-17), D (C13/C17/C18/C20/O9/O10), E (C16/C17/C20/C22-C24), F (C22-C26/O-9), and G (C-15/C-16/C-24/C27-N1). Rings A and B transfused with each other through C5/C10 linkage and exists in half chair and chair conformations, respectively. Ring C (C8/C9/C11-17) appeared in twisted-boat-chair conformation with the cleavage of ether linkage between rings D and E as appeared in other reported physalins. Tetrahydropyridine moiety i.e. ring G (C15/C16/C24/C27N1) is completely different from reported physalin [39, 40]. The result showed that C6-OH and 28-CH₃ were on the same side and assigned to be β-oriented. Whereas C13-OH, C17-OH and C21-CH₃ were deduced to be α-oriented (Fig 3). In the crystal lattice molecules found to be linked to form a three-dimensional network (Fig 4). Previously reported, aminophysalin A [42] was the first physalin having an unusual structural feature in with a nitrogen atom. Based on spectroscopic compound 1 was s found to be the second member of aminophysalin series.

Fig 4. Crystal packing diagram of compound 1.

Fig 5. Key NOESY correlations in compounds 6.

Fig 3. ORTEP view of compound 1 at 50% probability level.

Compounds 2–5 were identified as previously reported physalin B (2) [44, 45], 5β, 6β-epoxyphysalin B (3) [45], 5α-ethoxy-6ß-hydroxy-5,6-dihydrophysalin B (4) [42, 46], and physalin H (5) [47] by comparison of the reported spectroscopic data. These compounds had first reported from P. angulata, P. alkekengi, and P. minima, respectively.

Compound 6 was isolated here first time from Physalis minima. Its structure was previously reported only on a tentative assignment based on the fragmentation pattern studied through UHPLC-QTOF-MS/MS analysis Physalis alkekengi L. var. franchetii (Mast.) [41]. Therefore, this is the first detailed report of the isolation and validated structure characterization of 5ß,6ß-epoxyphysalin C (6). Compound 6 was obtained as a white solid. The ESI-MS [M+H]⁺m/z: 527.1907 [C₂₈H₃₂NO₉+H; 527.1912]. The IR spectrum showed strong absorption at 3427, 1782, and 1656 indicating the presence of OH groups, five-membered lactone ring and α,β-unsaturated ketone, respectively. The ¹H-NMR spectrum (Table 1) showed three characteristic methyl singlets at δ1.21, 1.92, and 1.58, and assigned to H₃-19, H₃-21, and H₃-28 methyl respectively. Two mutually coupled olefin proton signals, appeared at δ 5.99 (dd, J_2,4 = 10.0 Hz, J_2,3 = 2.5 Hz) and δ 6.83 (ddd, J_2,3 = 3.0 Hz, J_3,4 = 6.0 Hz, J_3,4 = 10.0 Hz) indicated a 2-ene-1-one system in ring A. A one-proton downfield broad singlet at δ 3.24 (d, J = 3.0 Hz) was assigned to the C-6 methine proton, geminal to oxy group. The assigned C-6 proton at δ 3.24 was further supported by HMBC correlations with C-8 (δ 39.1) and C-7 (δ 26.4) (Fig 2). HMBC correlation of H-3 (δ 6.83) with C-5 (δ 61.8) supported the presence of an epoxide between C-5 and C-6. The low J value (J = 3.0 Hz) and comparison of the chemical shift with a known physalin (5β,6β-epoxyphysalin B) suggested α-orientation of H-6 [32]. Two characteristic singlets at δ 6.64 (s) and 5.72 (s) indicating the absence of oxygen containing bridge between C-14 and C-27. An olefin at C-27 was inferred by HMBC correlations of H₂-27 (δ 6.64, δ 5.72) with C-26 (δ 163.7) and C-24 (δ 36.6). The ¹³C-NMR data (Table 1) showed 28 carbon signals assigned to twelve quaternary, seven methine, six methylene, and three methyl carbons. The signal at δ 64.4 was assigned to C-5, and indicated the epoxidation at C-6. Two additional quaternary signals at δ 102.1 and 139.3 were assigned to C-14 and C-24, respectively. NOESY correlation between the H-9 (δ 2.51) and H-6 (δ 3.24) indicated a β epoxide (Fig 5). Compound 6 was identified as 5β, 6β-epoxyphysalin C based on spectroscopic analysis.

Compounds 7 and 9 were identified as known physalins G (7) and physalin D (9) through comparison of their reported spectroscopic data [43, 48].

The first report for the isolation of physalin K (8) was from Physalis minima in 1980 [43]. However the structure was extensively revised later based on spectroscopic techniques in 1995 [49]. We have studied the structural parameters of physalin K (8) based on single-crystal X-ray diffraction data and found it in good agreement with the revised structure.

Like other physalin, the molecular skeleton was found to have a rigid framework, consisting of eight condensed rings. Two six-membered rings i.e. rings A (C1/C2/C5/C10/O2/O3) and B (C2-C5/O2/O3) linked together through epi-dioxy linkage at C2 and C5 adopts screw boat conformation. Ring C (C5-C10) is transfused with ring A through C5/C10 bonds, and exists in chair conformation. The eight-membered ring D (O7/C8/C9/C11-C13/C17/C14) and adopts a boat-chair conformation, connected with two spiro-fused rings E (C13/C17/C18/C20/O9/O10) and F (C14-C17/O7) through O7/C13/C14/C17- /O7, while dimethyl substituted six-membered ring G (C-16/C-17/C-22—C-24) is connected with two lactone ring moieties of H (C14-C16/C24-C27/O6) and I (C22-C26/O11/O12) along C16/C23-C25 linkage (Figs 6 and 7).

Fig 6. ORTEP view of compound 8 at 50% probability level.

Fig 7. Crystal packing diagram of compound 8.

Anti-leishmanial activity

In order to evaluate the anti-leishmania activity, Physalis minima extracts (Ethanol/water, Pet ether, Dichloromethane, and Ethyl acetate), active fractions (A-D), and pure physalins 1–9 were incubated in various concentrations with axenic cultures of L. tropica and L. major in comparison to three commercially available drugs, amphotericin B, pentamidine, and miltefosine (Tables 2 and 3). These drugs were used as a positive control to broaden the range due to different mode of actions. The IC₅₀ values (Table 3) indicated that chloro substituted physalin H (5) was the most active member (IC₅₀ = 9.59 ± 0.27 μM) (P-value = 0.0009 for pantamidine while for miltefosine and amphotericin B the P-value was = < 0.0001), followed by physalins B (2, IC₅₀ = 13.33 ± 0.098 μM), physalins G (7, IC₅₀ = 19.49 ± 0.02 μM), and 5ß,6ß-epoxyphysalin C (6, IC₅₀ = 23.76 ± 1.10 μM) (P-value = < 0.0001 for all three tested standards)against L. tropica and showed a more potent activity than the standards pentamidine (IC₅₀ = 27.20 ± 0.01 μM) and miltefosine (IC₅₀ = 42.75 ± 1.03 μM). The anti-leishmanial activity of physalins B (2), 5ß,6ß-epoxyphysalin B (3) and physalin H (5) was found in agreement with previous data reported by us [23] against L. major, in comparison to amphotericin B. In the current study, the comparison of the anti-leishmanial activity of physalins B (2), 5ß,6ß-epoxyphysalin B (3) and physalin H (5) against L. major include comparison with pentamidine and miltefosine. The results indicate that growth inhibition potential of physalins B (2, IC₅₀ = 3.04 ± 1.12 μM) (P-value = 0.9001 for amphotericin B while for miltefosine and pantamidine the P-value was = < 0.0001), 5ß,6ß-epoxyphysalin B (3, IC₅₀ = 3.76 ± 0.85 μM) (P-value = 0.9997 for amphotericin B while for miltefosine and pantamidine the P-values was = < 0.0001) and physalin H (5, IC₅₀ = 3.34 ± 0.64 μM) (P-value for amphotericin B, miltefosine and pantamidine the P-value was = < 0.0001). These were several-fold more potent than the standard drugs pentamidine (IC₅₀ = 27.20 ± 0.015 μM) and miltefosine (IC₅₀ = 25.55 ± 1.03 μM). Compounds 1, 4, and 8 appeared to be inactive against both L. major and L. tropica strains (Table 3). Compounds 3, 5, and 6 showed low selectivity index (SI) values of 0.19, 0.119, and 0.007, respectively against L. major tested on a 3T3 cell line. However, compound 2 showed SI value of 3.28. All these compounds showed <1 SI index against L. tropica cultured on 3T3 cells. Moreover, on the human fibroblast cell line (BJ), compounds 2, 3, 5, and 6 showed ≥1 SI values of 3.5, 7.34, 3.11, and 1.19 against L. major, respectively. While compounds 3, 5, and 6 also showed SI values of 1.48, 1.08, and 4.20 against L. tropica. The SI value higher than 1 indicated that these compounds are theoretically more effective and safe drugs. However, compound 2 demonstrated a SI value of 0.8 on L. tropica. (Tables 4–7).

Table 2. Anti-leishmanial activity of extracts and fractions against L. major and L. tropica.

		L. major				L. tropica
		IC50 (μg/ml ± SD)	p-value Amphotericin	p-value Pentamidine	p-value Miltefosine	IC50 (μg/ml ± SD)	p-value Amphotericin	p-value Pentamidine	p-value Miltefosine
Extracts	Ethanol/water	>100	-			>100	-
	Pet ether	>100	-			>100	-
	Dichloromethane	19.01±0.7	<0.0001	<0.0001	<0.0001	19.53±1.3	<0.0001	<0.0001	0.0021
	Ethyl acetate	>100	-			>100	-
Fractions	A	6.67±0.1	<0.0001	<0.0001	<0.0001	10.16±2.0	<0.0001	0.0568	<0.0001
	B	9.95±1.9	<0.0001	0.8898	0.5084	6.41±0.074	<0.0001	0.0001	<0.0001
	C	6.30±0.018	<0.0001	<0.0001	<0.0001	6.34±0.045	<0.0001	<0.0001	<0.0001
	D	6.37±0.015	<0.0001	<0.0001	<0.0001	6.37±0.015	<0.0001	0.0001	<0.0001
Standard drugs	Amphotericin	3.14 ± 0.04				3.16 ± 0.04
	Pentamidine	9.25 ± 0.005				9.25 ± 0.005
	Miltefosine	10.4 ± 0.42				17.4 ± 0.42

Table 3. Anti-leishmanial activity of compounds 1–9 against L. major and L. tropica.

Compounds	L. major				L. tropica
	IC50 (μM± SD)	p-value Amphotericin	p-value Pentamidine	p-value Miltefosine	IC50 (μM± SD)	p-value Amphotericin	p-value Pentamidine	p-value Miltefosine
1	Inactive	-	-	-	Inactive	-	-	-
2	3.04 ± 1.12	0.9001	<0.0001	<0.0001	13.33 ± 0.098	<0.0001	<0.0001	<0.0001
3	3.76 ± 0.85	0.9997	<0.0001	<0.0001	18.53 ± 0.28	<0.0001	<0.0001	<0.0001
4	Inactive	0.9999	<0.0001	<0.0001	Inactive	<0.0001	<0.0001	<0.0001
5	3.34 ± 0.64	<0.0001	<0.0001	<0.0001	9.59 ± 0.27	<0.0001	0.0009	<0.0001
6	84.79 ± 1.56	<0.0001	<0.0001	<0.0001	23.76 ± 1.10	<0.0001	<0.0001	<0.0001
7	52.28 ± 1.18	0.9001	<0.0001	<0.0001	19.49 ± 0.02	<0.0001	<0.0001	<0.0001
8	inactive	-	-	-	inactive	-	-	-
9	inactive	-	-	-	55.24± 0.01	<0.0001	<0.0001	<0.0001
Amphotericin B	3.39 ± 0.043				3.42 ± 0.04
Pentamidine	27.20 ± 0.015				27.20 ± 0.01
Miltefosine	25.55 ± 1.03				42.75 ± ± 1.03

Table 4. Selectivity index of pure compounds against BJ fibroblast cell line.

	Samples	BJ cell line	L. major	L. tropica	L. major	L. tropica
		CC50 (μM± SD)	IC50 (μM± SD)	IC50 (μM± SD)	SI (CC50/ IC50)	SI (CC50/ IC50)
Extracts	Ethanol/water	>100	>100	>100	-	-
	Pet ether	>100	>100	>100	-	-
	Dichloromethane	26.5±1.1	19.01±0.7	19.53±1.3	1.39	1.35
	Ethyl acetate	>100	>100	>100	-	-
Fractions	Fraction-A	10.4 ± 1.0	6.67±0.1	10.16±2.0	1.49	1.02
	Fraction-B	>100	9.95±1.9	6.41±0.074	-	-
	Fraction-C	>100	6.30±0.018	6.34±0.045	-	-
	Fraction-D	>100	6.37±0.015	6.37±0.015	-	-

Table 7. Selectivity index of pure compounds against 3T3 mouse fibroblast cell line.

	Samples	3T3 cell line	L. major	L. tropica	L. major	L. tropica
		CC50 (μM± SD)	IC50 (μM± SD)	IC50 (μM± SD)	SI (CC50/ IC50)	SI (CC50/ IC50)
Extracts	Ethanol/water	>100	>100	>100	-	-
	Pet ether	>100	>100	>100	-	-
	Dichloromethane	4.5 ± 0.5	19.01±0.7	19.53±1.3	0.23	0.23
	Ethyl acetate	>100	>100	>100	-	-
Fractions	Fraction-A	12.2 ± 1.5	6.67±0.1	10.16±2.0	7.30	1.20
	Fraction-B	4.8 ± 1.6	9.95±1.9	6.41±0.074	0.48	0.74
	Fraction-C	2.7 ± 0.3	6.30±0.018	6.34±0.045	0.42	0.42
	Fraction-D	3.3 ± 0.3	6.37±0.015	6.37±0.015	0.51	0.51

Table 6. Selectivity index of pure compounds against 3T3 mouse fibroblast cell line.

Samples	3T3 Cell line	L. major	L. tropica	L. major	L. tropica
	CC50 (μM± SD)	IC50 (μM± SD)	IC50 (μM± SD)	SI (CC50/ IC50)	SI (CC50/ IC50)
1	-	Inactive	Inactive	-	-
2	10 ± 0.4	3.04 ± 1.12	13.33 ± 0.098	3.28	0.007
3	0.72 ± 0.5	3.76 ± 0.85	18.53 ± 0.28	0.19	0.038
4	19.6± 0.8	Inactive	Inactive	-	-
5	0.4 ± 0.07	3.34 ± 0.64	9.59 ± 0.27	0.119	0.041
6	0.6 ± 0.04	84.79 ± 1.56	23.76 ± 1.10	0.007	0.025
7	Non cytotoxic	52.28 ± 1.18	19.49 ± 0.02	-	-
8	Non cytotoxic	Inactive	Inactive	-	-
9	Non cytotoxic	Inactive	55.24± 0.01	-	-

Anti-leishmanial activity against MIL-resistant L. tropica

The safe, non-toxic and effective drug, miltefosine was selected after evaluating its cytotoxicity on BJ cell line. The selection of miltefosine depends upon its potential for the treatment of Leishmaniasis along with the rapid acquisition of resistance. Several studies have reported miltefosine for the treatment of L. tropia. It also works well during the study presented against locally isolated strains of L. tropica. Furthermore, compounds 2–9 were also evaluated for their potential against the Miltefosine-unresponsive strain (MIL resistant) L. tropica. Compounds 4 (IC₅₀ = 74.65 ± 0.81 μM) and 7 (IC₅₀ = 39.44 ± 0.65 μM) (P-value was = < 0.0001) were found to be more potent anti-leishmanial agents against the MIL resistant promastigotes than the standard Miltefosine (IC₅₀ = 169.55 ± 0.78 μM). It is interesting to be noted that compound 4 found to be inactive against clinical isolates of L. major and L. tropica (Table 8). Any possible alteration in nucleus and cytoplasm caused by the acquisition of MIL resistance was evaluated by DAPI staining. No morphological changes or any other distortion in growth of resistance lines were observed after microscopic observations and supported the propagation and survival of Leishmania parasite in higher concentration of drugs. Collectively, the microscopic observations based on DAPI staining strongly supported that the viability and dynamics of resistant line were similar to that of wild type.

Table 8. Anti-leishmanial activity of compounds 2–9 against the MIL-resistant L. tropica.

		MIL Resistant
		L. tropica
	Compounds	IC50 (μM± SD)
Pure Compounds	2	inactive
	3	inactive
	4	74.65 ± 0.81
	5	inactive
	6	inactive
	7	39.44 ± 0.65
	8	inactive
	9	inactive
Standard drug	Miltefosine	169.55 ± 0.78

Cytotoxicity of physalin against BJ (Human fibroblast) cell lines

Cytotoxicity Physalis minima extracts (Ethanol/water, Pet ether, Dichloromethane, and Ethyl acetate), active fractions (A-D), and pure physalins 2–9 were evaluated against normal fibroblast (3T3) and BJ (human fibroblast) cells lines, and all tested compounds were found to be non-cytotoxic in nature (Tables 4 and 5).

Table 5. Selectivity index of crude fractions against BJ fibroblast cell line.

Samples	BJ cell line	L. major	L. tropica	L. major	L. tropica
	CC50 (μM± SD)	IC50 (μM± SD)	IC50 (μM± SD)	SI (CC50/ IC50)	SI (CC50/ IC50)
1	Non cytotoxic	Inactive	Inactive	-
2	10.8 ± 1.5	3.04 ± 1.12	13.33 ± 0.098	3.5	0.8
3	27.6	3.76 ± 0.85	18.53 ± 0.28	7.34	1.48
4	14.2 ± 0.8	Inactive	Inactive	-	-
5	10.4 ± 1.0	3.34 ± 0.64	9.59 ± 0.27	3.11	1.08
6	>100	84.79 ± 1.56	23.76 ± 1.10	1.19	4.20
7	>100	52.28 ± 1.18	19.49 ± 0.02	-	-
8	>100	Inactive	Inactive	-	-
9	>100	Inactive	55.24± 0.01	-	-
Amphotericin	16.059	3.39 ± 0.043	3.42 ± 0.04	4.37	4.69

Docking studies

Compounds with significant in vitro anti-leishmanial activities proceeded for docking studies against various therapeutically important enzymes involved in different metabolic pathways For this purpose, various enzymes related to major metabolic pathways including glycolysis (pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglucose isomerase), folate pathway (pteridine reductase), gluconeogenesis (fructose-1,6-bisphosphatase cytosolic and mitochondrial fumarate hydratase), pyrimidine biosynthesis (dihydroortate dehydrogenase), lipid metabolism (N-myrositoyltransferase), posttranslational modification (SAM-dependent methyltransferase), and cell signalling (glycogen synthase kinase, MAP kinase) were selected. Among listed targeted enzymes, compounds 2, 3, 5, 6, 7, and 9 showed the best binding interaction with phosphoglucose isomerase and N-myrositoyltransferase. The docking scores were in the range of -4.3 to -5.7 Kcal/mol (Fig 8), while the binding energies were in the range of -11 to -75 Kcal/mol (Fig 9).

Fig 8. Docking scores of selected compounds (2, 3, 5, 6, 7, and 9) against leishmanial phosphoglucose isomerase (carbohydrate metabolism).

Fig 9. Docking scores of selected compounds 2, 3, 5, 6, 7, and 9 against leishmanial N-myristoyltransferase (lipid metabolism).

Discussion

In last few decades, extensive research on medicinal plants has contributed to increased of natural products against health disorders globally. In continuation of our work to explore the biological properties of medicinal plants, our group has previously reported the in vitro anti-leishmanial effect of physalins B (2), 5ß,6ß-epoxyphysalin B (3) and physalin H (5), and other isolated physalins from -Physalis minima against promastigotes of L. major [21, 22], in comparison to amphotericin B. Amphotericin B is the drug of choice for the treatment of various parasitic and fungal infections, including leishmaniasis. However, the clinical use of amphotericin B is limited because of systematic toxicities, and lack of accessibility and affordability [50] The recommended least toxic, non-conventional amphotericin B formulations, such as Fungizone®, AmBisome^®, Abelcet^®, and Amphocil^® are expensive, and, therefore, out of the reach of the poor patients [51, 52]. Unfortunately, the first line pentavalent antimonials are also not available in many leishmanial endemic countries. Therefore, alternatives such as pentamidine, and most recently miltefosine are the recommended US-FDA approved drugs, prescribed by dermatologists for CL. One of the recommended treatment regimens for leishmaniasis includes the combination of amphotericin B and miltefosine. A study conducted in Ethiopia, one of the CL which is caused by L. aethiopica, was found to be more severe and difficult to treat as compared to CL caused which is caused by other species. As far as treatment options are concerned, Miltefosine is the only oral anti-leishmanial drug, with a favourable side-effect profile compared to routinely available sodium stibogluconate (SSG) [53, 54]. Therefore, in the current study of the inhibition abilities of isolated physalins towards promastigotes of L. major and L. tropica were studied in vitro, and compared with amphotericin B, pentamidine, and miltefosine. The physalins B (2), 5ß,6ß-epoxyphysalin B (3), and chloro substituted Physalin H (5) were previously reported by us as -promising candidates with significant abilities to reduce the growth of promastigotes of L. major, in comparison to amphoterin B [21]. The published results were further validated in the present study and we further conclude that in addition to amphotericin B, these tested compounds also have several-fold more inhibition potential against promastigotes of L. major than pentamidine (IC₅₀ = 27.20 ± 0.015 μM), and miltefosine (IC₅₀ = 25.55 ± 1.03 μM). In the current study, the test compound also showed potently anti-leishmanial abilities against L. tropica promatigotes in vitro, and appeared to be several fold more active than tested standards pentamidine (IC₅₀ = 27.20 ± 0.01 μM), and miltefosine (IC₅₀ = 42.75 ± 1.03 μM). The activity of some selected compounds was also evaluated against a lab generated MIL-resistant line. Compound 2 showed SI value of >1 while compounds 3, 5, and 6 showed <1 SI against L. major on 3T3 cell line. Compounds 3, 5, and 6 showed >1 SI values against both L. major and L. tropica cultured on BJ cell line. However, compound 2 demonstrated >1 SI value only against L. tropica.

The alkylphosphocholine drug, miltefosine possesses its potential against several parasitic species and different cancer cells, along with activity against various pathogenic fungi and bacteria [55]. Knowledge of experimental MIL resistance in Leishmania is limited to defects in drug internalization (defective inward translocation of MIL) and increased drug efflux [56]. A major potential drawback in the use of miltefosine for the treatment of leishmaniasis could be the relatively rapid generation of drug resistance in vitro [57]. Miltefosine is considered a breakthrough in the treatment, making it feasible to eliminate a regional disease, unfortunately the acquisition of resistance is of major concern [58]. Mechanisms that are responsible for the resistance acquisition in leishmaina parasite against miltefosine includes reduction in drug uptake, increased efflux and alteration in permeability of the plasma membrane [59–63]. Due to this type of data and research highlights there is a necessity to find out the alternative therapeutic options for leishmaniasis. During the study presented the resistant strain was generated and libraries of compounds were evaluated against the developed line. L. tropica MIL-unresponsive / resistant parasites were generated by using the step-wise selection of the drug-miltefosine up to the concentration of 196 μM. No marked difference in growth pattern were also analyzed between WT and MIL-resistant strain. Compounds 4 and 7 were observed as significantly potent as compared to the miltefosine, revealing the potential of these natural compounds against drug–unresponsive strains. However, these results of the current study demonstrated the promising anti-leishmanial potential of physalins against L. major and L. tropica promastigotes that need to be further validated by in vivo studies as an anti-leishamanial agent for oral use.

Targeting the metabolic and biochemical pathways of Leishmania is one of the most appropriate therapeutic strategy. Following this rationale, compounds 2, 3, 5–7, and 9 with significant in-vitro anti-leishmanial activity were validated through docking studies against various metabolically important enzymes, in order to predict their mechanism of action. Compounds 2, 3, 5–7, and 9 showed better binding affinities against phosphoglucose isomerase and myrositoyltransferase with docking scores in the range of -2 to -7 Kcal, while the binding energies were in the range of -11 to -75 Kcal/mol. The results were then compared with the docking scores and binding energies of anti-leishmanial clinical drugs including amphotericin B, pentamidine, and miltefosin, and found to be comparable with them. The results further indicated that the compounds might affect the glycolytic and lipid metabolic pathways by targeting phospoglucose isomerase and N-myristoyltransferase, respectively. Phosphoglucose isomerase (PGI) is considered a promising target for the development of anti-parasitic drugs, as it acts on two essential metabolic pathways, glycolysis and gluconeogenesis. It is an aldose-ketose isomerase that catalyzes the reversible interconversion of G6P into fructose-6-phosphate (F6P) [64].

Conclusion

Leishmaniasis, a neglected tropical disease is a rapidly growing infection in more than 98 countries of the world. The presented study concluded with the identification of natural steroidal lactones i.e. physalins as potential candidates to be explored further against CL. The complex structures of all natural compounds 1–9 were elucidated via combined use of MS, IR, and NMR spectroscopic techniques. The assigned structures of compounds 1, and 8 were further supported by single-crystal X-ray diffraction studies. Anti-leishmanial activities of isolated physlains were evaluated against L. major and L. tropica . The literature survey revealed that all of the physalins possess—anti-leishmanial activity against L. tropica (clinical isolate of Pakistan), reported for the first time. The promising results of compounds 4 and 7 against the miltefosine-unresponsive L. tropica strain (MIL resistant) concluded the anti-leishmanial potential of tested compounds against resistant strain. The compounds were also able to interact with therapeutically relevant enzymes of leishmania including phosphoglucose isomerase, and N-myrositoyltransferase.

Supporting information

S1 Fig. EI-MS of compound 1.

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S2 Fig. HREI-MS of Compound 1.

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S3 Fig. 1H-NMR spectrum of compound 1.

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S4 Fig. 13C-BB spectrum of compound 1.

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S5 Fig. 13C-DEPT 135 spectrum of compound 1.

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S6 Fig. 13C-DEPT 90 spectrum of compound 1.

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S7 Fig. COSY spectrum of compound 1.

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S8 Fig. NOESY spectrum of compound 1.

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S9 Fig. HMBC spectrum of compound 1.

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S10 Fig. HSQC spectrum of compound 1.

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S11 Fig. EI-MS of compound 6.

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S12 Fig. HREI-MS of compound 6.

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S13 Fig. 1H-NMR spectrum of compound 6.

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S14 Fig. 13C-BB spectrum of compound 6.

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S15 Fig. 13C-DEPT 135 spectrum of compound 6.

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S16 Fig. 13C-DEPT 90 spectrum of compound 6.

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S17 Fig. HMBC spectrum of compound 6.

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S18 Fig. NOESY spectrum of compound 6.

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S19 Fig. HSQC spectrum of compound 6.

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S20 Fig. COSY spectrum of compound 6.

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S21 Fig. EI-MS of compound 8.

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S22 Fig. HREI-MS of compound 8.

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S23 Fig. 1H-NMR spectrum of compound 8.

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S1 Data

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S2 Data

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S2 File

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S3 File

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Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

“Sammer Yousuf and M. Iqbal Choudhary, researcher leading to these results has, in part, received funding from UK Research and Innovation via the Global Challenges Research Fund under grant agreement ‘A Global Network for Neglected Tropical Diseases’, grant number MR/P027989/1.” “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”