Antiparasitic and Antimicrobial Indolizidines from the Leaves of Prosopis glandulosa var. glandulosa

Abstract

A new indolizidine alkaloid, named Δ^1,6-juliprosopine (1), together with previously known indolizidine analogs (2–6), was isolated from the leaves of Prosopis glandulosa var. glandulosa, collected from Nevada, USA; while two other known indolizidines juliprosopine (6) and juliprosine (7) were isolated from P. glandulosa leaves collected in Texas, USA. The structures of compound 1 and 7 were determined using a combination of NMR and MS techniques. Compound 7 exhibited potent antiplasmodial activity against Plasmodium falciparum D6 and W2 strains with IC₅₀ values of 170 and 150 ng/mL, respectively, while 1 was found to be less active (IC₅₀ values 560 and 600 ng/mL). Both the compounds were devoid of VERO cells toxicity up to a concentration of 23800 ng/mL. The antileishmanial activity of indolizidines was evaluated against Leishmania donovani promastigotes, axenic amastigotes and amastigotes in THP1 macrophage cultures. When tested against macrophage cultures, the tertiary bases (1, 3, 6) were found to be more potent than quaternary salts (2, 5, 7), displayed IC₅₀ values between 0.8–1.7 μg/mL and 3.1–6.0 μg/mL, respectively. In addition, compound 7 showed potent antifungal activity against Cryptococcus neoformans and antibacterial activity against Mycobacterium intracellulare, while 1 was potent only against C. neoformans and weakly active against other organisms.

Introduction

Prosopis glandulosa Torrey var. glandulosa (Fabaceae), commonly known as Honey Mesquite, is a medium-sized flowering tree native to the southwestern United States and Mexico [1, 2]. The plant is a folk remedy for dyspepsia, eruptions, eyes, hernias, skin ailments, sore throat and umbilical ailments [3, 4]. Earlier investigations on this plant reported various types of biological activities such as antibacterial [5, 6], antifungal [7], anti-infective and antiparasitic activities [8, 9], which are attributed to piperidinyl indolizidine alkaloids. We have previously reported [8] several potent antiparasitic and antimicrobial diastereoisomeric tertiary and quaternary indolizidine alkaloids from P. glandulosa collected in Nevada. In continuation of our previous investigation, we now have isolated a new tertiary indolizidine alkaloid, named Δ^1,6- juliprosopine (1), and large amounts of five known indolizidines (2–6) [8]. In addition, we also have examined a sample of P. glandulosa var. glandulosa, obtained from Texas, which yielded two known indolizidines, namely the tertiary juliprosopine (6) and the quaternary juliprosine (7), and interestingly devoid of compounds 1–5. We herein report the isolation and structure elucidation of 1, as well as the antiplasmodial and antimicrobial activities of 1 and 7, and a detailed antileishmanial activity of isolated compounds (1–3, 5–7) against Leishmania donovani promastigotes, axenic amastigotes and amastigotes in THP1 macrophage cultures.

Materials and Methods

General

Optical rotations were measured in CHCl₃ or MeOH using an AUTOPOL IV instrument at ambient temperature. IR spectra were obtained using a Bruker Tensor 27 instrument. NMR spectra were acquired on a Varian Mercury 400 spectrometer (Varian Inc.; Palo Alto, CA) with standard pulse sequences operating at 400 MHz in ¹H NMR and 100 MHz in ¹³C NMR in CDCl₃ or CD₃OD (Cambridge Isotope Laboratories, Inc.) using the residual solvent as internal standard. TLC was carried out on aluminum oxide IB-F plates (Baker-flex) using CH₂Cl₂-MeOH-NH₃·H₂O (8:2:0.1) as solvent. Centrifugal preparative TLC (CPTLC, Chromatotron^®, Harrison Research Inc., model 8924) was carried out on 1, 2 and 4 mm alumina (Analtech, Inc.) rotors, using CH₂Cl₂-MeOH-NH₃·H₂O as solvent. The compounds were visualized by observing under UV light at 254 or 365 nm, followed by spraying with Dragendorff’s or 1% vanillin-H₂SO₄ spray reagents. All positive controls for biological experiments are commercially available with declared purity from known vendors.

Plant Material

The leaves of P. glandulosa var. glandulosa were collected from Nevada, USA (voucher # NIX 230506/1/A) in May 2006 and Texas (voucher # 3794/11062008/PRGLM) in June 2008, and were identified by Dr. Vaishali C. Joshi, NCNPR, School of Pharmacy, University of Mississippi. Voucher specimens are deposited at the Herbarium of NCNPR, University of Mississippi.

Extraction and isolation

The air dried powdered leaves of P. glandulosa (Nevada, 1.8 kg) were extracted by percolation (95% EtOH, 2 L × 3, 48 h) and resulted EtOH extracts was evaporated to dryness (273 g). The dried extracts (130 g) was dissolved in aqueous 0.1 N HCl and partitioned successively with n-hexane (1L × 3) followed by CH₂Cl₂ (600 mL × 3). The aqueous acidic layer was then basified with 0.1 N NH₄OH to pH 11 (based on pKa 10.2 of tryptamine), followed by partitioning with CH₂Cl₂ (500 mL × 4). The pH of the remaining basic layer was then adjusted to 12 by NH₄OH, followed by partitioning successively with CH₂Cl₂ (500 mL × 3) and EtOAc (300 mL × 3). The CH₂Cl₂ fraction (2.8 g) was subjected to CPTLC, using 4 mm Al₂O₃ rotor with CH₂Cl₂-MeOH-NH₃ ·H₂O as solvent to yield 13 fractions. Further purification by repeated CPTLC afforded compound 1 (37 mg), 2 (60 mg), 3 (53 mg), 4 (35 mg) 5 (94 mg) and 6 (84 mg) (Fig. 4S in Supporting Information). Compounds 2–6 were identified by ¹H and ¹³C NMR spectral data, as well as by direct comparison with authentic samples of prosopilosidine, prosopilosine, isoprosopilosine, isoprosopilosidine and juliprosopine, respectively, previously isolated from the same plant [8].

A sample of P. glandulosa (1 Kg, air dried) leaves collected in Texas was extracted (95% EtOH, 1.5 L × 3, 48 h) and evaporated to dryness (yield 149 g). The EtOH extract was fractionated using acid/base treatment to give alkaloidal enriched fraction (67 g), and then processed according to procedure as described previously [8]. The TLC characteristic of alkaloidal fraction was found to be significantly different to those of the samples from Nevada. CPTLC separation of alkaloidal fraction, using above method, yielded compound 6 (200 mg) and juliprosine (7; 225 mg). The identity of compound 6 was established by ¹H and ¹³ NMR spectral data and direct comparison with an authentic sample of juliprosopine.

Δ^1,6-juliprosopine (1)

Colorless gum; [α]²⁰_D +6.9 (MeOH, c 0.5), R_f 0.63 [Al₂O₃/CH₂Cl₂-MeOH-NH₃·H₂O (96:4:0.1)]; UV (CH₃OH) λ_max (log ε) 287.0 (2.81), 204.5 (3.48); IR (film) ν_max, cm⁻¹ 3270 (OH, NH), 2924, 2852, 1462 (C-H) and 1657 (C=N); ¹H and ¹³C NMR, see table 1; HRESIMS m/z 626.5628 [M + H]⁺ (calcd for C₄₀H₇₁N₃O₂, 626.5624).

Table 1.

¹H and ¹³C NMR Data with HMBC correlation (J values in Hz, in parenthesis) for 1.

Position	δH	δCa	HMBC (H-C)	Position	δH	δCa	HMBC (H-C)
2, 2′	3.53 br s	56.1 d	C-4/4′, C-6/6′, C-7/7′	9‴	1.40-1.2b	27.1 t	C-6‴′, C-10‴
3, 3′	3.83 br s	66.3 d	C-4/4′, C-5/5′, C-7/7′	10″	1.94 t (7)	35.1 t	C-9″, C-5‴′, C-6‴′, C-7‴′
4, 4′	1.82 m, 1.73m	25.6 t	C-3/3′, C-6/6′	10‴	1.4-1.2b	27.9 t	-b
5, 5′	2.27m, 2.31m	29.5 t	C-6/6′, C-3/3′, C-1‴	1‴′	1.4-1.15 b m	33.1 t	C-3‴′, C-8‴′, C-8a‴′
6, 6′	-	171.1 s	-	2‴′	1.83 m; 1.73 m	21.4 t	C-1‴′, C-8a‴′
7, 7′	1.23b	17.5 q	C-2/2′, C-3/3′	3‴′	3.15 m, 2.10 m	54.5 t	C-1‴′, C-5‴′, C-8a‴′
1″, 1‴	2.15 t (7.2)	40.7 t	C-6/6′, C-5/5′	5‴′	3.28 d (15.2) 2.60 d (15.2)	55.2 t	C-3‴′, C-6‴′, C-7‴′, C-8a‴′, C-10″
2″-8″, 2‴-8‴	1.40-1.2 b	29.3, 29.4 (x 2), 29.5 (x 2), 29.6, 30.1 (all triplets)	-b	6‴′	-	136.0 s	-
				7‴′	5.32 s	123.9 d	C-5‴′, C-8‴′, C-8a‴′, C-10″
				8‴′	1.99 m	42.5 d	C-6‴′, C-7‴′, C-8a‴′
9″		27.1 t	C-10″, C-8‴′	8a‴′	1.77 m	65.5 d	-

Spectra recorded in CDCl₃ at 400 MHz (¹H) and 100 MHz (¹³C).

^aMultiplicities were determined by DEPT and HSQC experiments.

^bOverlapped signals.

Juliprosine (7)

Colorless gum; [α]²⁰_D +5 (CHCl₃, c 0.5) [Lit. 21: [α]_D 11, CHCl₃], R_f 0.22 [Al₂O₃/CH₂Cl₂-MeOH-NH₃·H₂O (96:4:0.1)]; UV (CH₃OH) λ_max (log ε) 277 (3.81), 226 (3.79), 222 (3.85); IR (film) ν_max, cm⁻¹ 3291 (OH, NH), 2925, 2853, 1458 (C-H), and 1506 (C-C); The ¹H and ¹³C NMR spectral data were in agreement with those reported previously by Daetwyler et al. [21], HRESIMS: m/z 628.5854 [M]⁺ (calcd. for C₂₀H₂₇O₄, m/z 628.5854).

Biological activity

Antileishmanial assay

The in vitro antileismanial assay was done on a culture of Leishmania donovani promastigotes and axenic amastigotes by Alamar Blue assay [13]. In a 96 well microplate the samples with appropriate dilution were added to the leishmania promastigotes/axenic amastigote culture (2×10⁶ cell/mL). The compounds were tested in duplicates at six concentrations ranging from 40 to 0.0128 μg/mL. The plates were incubated at 26 °C for 72 hours (37 °C for amastigote) and growth of leishmania promastigotes/amastigote was determined. IC₅₀ and IC₉₀ values were computed from the dose response curves. The in vitro amastigote assay in macrophages is described in Fig. 5S, Supporting Information Section. The compounds were also tested against VERO cells and PMA transformed THP1 cells by Neutral Red assay [22].

Antiplasmodial assay

The in vitro antiplasmodial activity, as described previously [8], was measured by a colorimetric assay that determines the parasitic lactate dehydrogenase (pLDH) activity [14]. All experiments were carried out in duplicates.

Antimicrobial assay

All microorganisms were obtained from the ATCC (Manassas, VA) and include the fungi Candida albicans (ATCC 90028), C. krusei (ATCC 6258), C. glabrata (ATCC 90030), Cryptococcus neoformans (ATCC 90113), and Aspergillus fumigatus (ATCC 204305) and the bacteria Staphylococcus aureus (ATCC 29213), methicillin-resistant S aureus (MRSA, ATCC 33591), and Mycobacterium intracellulare (ATCC 23068). For all organisms, excluding M. intracellulare and A. fumigatus, susceptibility testing was performed using a modified version of the CLSI (formerly NCCLS) methods [15, 16] and optical density was used to monitor growth. Media supplemented with 5% Alamar Blue (BioSource International, Camarillo, CA) was utilized for growth detection of M. intracellulare [17] and A. fumigatus [18]. Concentrations that afford 50% inhibition (IC₅₀s) relative to controls were calculated using XLfit 4.2 software (IDBS, Alameda, CA) using fit model 201 based on duplicate readings. Minimum fungicidal or bactericidal concentrations were determined by removing 5 μL from each clear well, transferring to agar, and incubating until growth was seen. Drug controls [Ciprofloxacin (ICN Biomedicals, Ohio, 99.3% purity) for bacteria and Amphotericin B (ICN Biomedicals, Ohio, 94.8% purity) for fungi] were included in each assay.

Results and Discussion

Compound 1 (colorless gum) was analyzed for a molecular ion of m/z 626.5628 [M+H]⁺ (calcd. 626.5624) by HRESIMS for C₄₀H₇₁N₃O₂. The IR (film) spectrum showed the presence of OH/NH group(s) (ν_max 3270 cm⁻¹) and C=N (ν_max 1657 cm⁻¹) absorption bands. The NMR spectra of 1 revealed the presence of a 6,7-dehydroindolizidine nucleus [10, 11, 19, 20] consisting of four triplets, three doublets, and a singlet (Table 1, Fig. 2S in supporting information). In addition, NMR spectra displayed oxymethine, methine adjacent to nitrogen, methylene, and methyl signals at δ_C 66.3, 56.1, 25.6, 29.5, and 17.5, respectively, assigned to two piperidinyl rings, as well as 10 methylenes for two decanyl moieties. Comparison of NMR spectra of 1 with spectra of other dehydroindolizidines such as juliprosopine [8, 12] suggested close similarities with that of juliprosopine (6). However, there is only one notable difference for the piperidinyl rings of 1 (δ_C 56.1, 66.3, 25.6, 29.5, 171.1, and 17.5; C-2/2′-C-7/7′) with that of juliprosopine (δ_C 57.2, 67.8, 26.1, 25.8, 55.8, and 18.4; C-2/2′-C-7/7′) at C-6/6′. The downfield value observed for C-6/6′ (δ_C 171.1, quaternary carbon) when compared with juliprosopine (δ_C 55.8, tertiary carbon) suggested the presence of a double bond between 1 and 6 position (nitrogen and carbon), which allowed us to conclude that the piperidinyl ring was dehydrogenated at this position. The HMBC experiment of 1 (Table 1) showed correlations between H-7‴′ and C-5‴′, C-8‴′, C-8a‴′, and C-10″; H-10″ and C-5‴′, C-6‴′, and C-7‴′; H-8‴ and C-6‴′, C-7‴′, and C-8a‴′ confirming a 6,8-dialkylated 6,7-dehydroindolizidine nucleus of juliprosopine (6). The NOESY experiment showed correlation between H-2/2′ and H-3/3′, suggesting that these protons were cis and on the α-face of the molecule, thereby the C-2/2′ methyl group and C-3/3′ OH group were on the β face (3R) of the molecule, hence two 2β-methyl-3β-hydroxy-6-decanylpiperidine moiety with the same configuration for both ring was confirmed. On the basis of above discussion, the structure of 1 was deduced as shown (Figure 1), and it was named as Δ^1,6-juliprosopine.

Figure 1.

Compounds (1–7) isolated from P. glandulosa.

During the course of isolation of 1, five other indolizidines 2-6 (Fig. 1) were isolated from P. glandulosa from Nevada. Their structures were confirmed by NMR [8] and direct comparison with authentic samples (available in our laboratory) as prosopilosidine (2), prosopilosine (3), isoprosopilosine (4), isoprosopilosidine (5) and juliprosopine (6). Examination of EtOH extracts of P. glandulosa leaves, collected from Texas, showed weak in vitro antiinfective and antiparasitic activities. Its alkaloidal profile, by TLC, was found to be substantially different with those observed for Nevada sample. This prompted us to carry out an acid/base fractionation. The bioactivity of the EtOH extract was significantly increased in the alkaloid enriched CH₂Cl₂ fraction. A CPTLC of the CH₂Cl₂ fraction resulted in the isolation of compounds 6 [8] and 7 [21], in the yields of 0.044% and 0.050%, respectively. The structure of compound 7 was confirmed by comparing its physical and spectral data (¹H and ¹³C NMR and MS) with those reported previously for juliprosine [21].

Δ^1,6-juliprosopine (1) and juliprosine (7) were tested for in vitro antiplasmodial, antileishmanial, antibacterial and antifungal activities. The dihydroindolizinium salt (7) exhibited potent antiplasmodial activity and high SI (Selectivity Index) (Table 2) against P. falciparum (IC₅₀ values of 170 and 150 ng/mL, against chloroquine sensitive (D6) and chloroquine resistant (W2) strains, respectively). The activity of 7 was found to be similar to those of chloroquine (IC₅₀= 150 and 140 ng/mL) against P. falciparum W2 strain. However, the antiplasmodial activity of 7 was found to be less potent compared to its diastereoisomer prosopilosidine (2), previously isolated from same plant [8]. On the other hand, the dehydroindolizidine base Δ^1,6-juliprosopine (1) was less potent (IC₅₀ = 560 and 600 ng/mL against D6 and W2 strains) than 7 and juliprosopine (6) [8], with no toxicity against mammalian VERO cells like 7, and less toxic than 6 (IC₅₀ = >23800 ng/mL for both 1 and 7 vs. 5000 ng/mL for 6). Therefore, 2 and 7 exhibited higher SI’s against P. falciparum D6 and W2 strains (SI >610, >250 and SI >140, >159, respectively) than 1 and 6 (SI >43, 23 and SI 40, 13 respectively), confirming our previous observation that the quaternary alkaloids (i.e., 2, 7) are better candidates than the tertiary bases (i.e., 1, 6) for further antiplasmodial studies.

Table 2.

Antiplasmodial activities of 1 and 7 and two analogues.

Compound	P. falciparum (ng/mL)				VERO (ng/mL)
	D6a		W2b		IC50
	IC50	SIc	IC50	SIc
1	560±5	>43	600±5	>40	>23800
2d	39±2	>610	95±2	>250	>23800
6d	220±25	23	380±50	13	5000
7	170±35	>140	150±60	>159	>23800
Chloroquine	17		140		>23800
Artemisinin	16		17		>23800

^aChloroquine-sensitive clone;

^bChloroquine-resistant clone;

^cSelectivity index= IC₅₀ VERO cells/IC₅₀ P. falciparum;

^dData reported previously [8], and used here for comparison with 1 and 7.

The antileishmanial activities of tertiary (1, 3, 6) and quaternary (2, 5, 7) indolizidines were evaluated against promastigotes and axenic amastigotes [13], and amastigotes in macrophage cultures of L. donovani (Table 3). The promastigotes/amastigotes activity of 3 and 6 were found to be equally potent to standard drug pentamidine (IC₅₀= 0.18/1 and 0.3/2 μg/mL for 3 and 6 vs. 0.8/2.1 μg/mL for pentamidine). However, the quaternary salts (2, 5 and 7) were found to be less potent, but also less toxic towards VERO cells than tertiary bases (3 and 6). Interestingly, the presence of a C-1,2 double bond in both piperidine rings, as observed in 1, retained potent antileishmanial activity (IC₅₀= 0.8 and 1.8 μg/mL) against both L. donovanii promastigotes and axenic amastigotes, respectively, while decreased its VERO toxicity, compared to 3 and 6. When tested against L. donovanii in THP1 macrophage cultures, the tertiary bases (1, 3, 6) were found to be more potent than quaternary salts (2, 5, 7), displayed IC₅₀ values between 0.8-1.7 μg/mL vs. 3.1–6.0 μg/mL. The activities of tertiary bases in THP1 were also superior to those of pentamidine (IC₅₀ = 3.0 μg/mL).

Table 3.

Antileishmanial activities of P. glandulosa extracts and compounds 1–7.

Extract/Compound	Leishmania donovani						Cytotoxicity (Transformed THP1 cells)
	Promastigotes		Axenic Amastigotes		Macrophage-Amastigotes
	IC50	IC90	IC50	IC90	IC50	IC90	IC50	IC90
P. glandulosa (from Nevada, EtOH extract)	44.2 ± 7.3	91.4 ± 5.2	NT	NT	NT	NT	NT	NT
P. glandulosa (from Texas, EtOH extract)	15.4 ± 2.1	31.9 ± 3.5	NT	NT	NT	NT	NT	NT
P. glandulosa (From Nevada, alkaloid fr.)	18.7 ± 1.9	34.7 ± 2.2	NT	NT	NT	NT	NT	NT
1	0.83 ± 0.11	1.67 ± 0.21	1.83 ± 0.24	5.89 ± 0.76	1.67 ± 0.23	2.48 ± 0.62	8.47 ± 1.32	>20
2	0.91 ± 0.17	4.17 ± 0.54	2.08 ± 0.30	25.4 ± 5.3	3.23 ± 0.54	8.74 ± 1.33	9.49 ± 2.25	>20
3	0.17 ± 0.05	6.33 ± 1.07	0.98 ± 0.12	6.49 ± 0.84	1.08 ± 0.21	1.65 ± 0.22	2.33 ± 0.51	3.89 ± 0.51
5	1.22 ± 0.21	4.52 ± 0.74	2.14 ± 0.32	24.8 ± 3.7	5.87 ± 0.76	9.49 ± 1.14	7.92 ± 1.22	19
6	0.29 ± 0.05	1.14 ± 0.21	1.92 ± 0.19	6.25 ± 0.47	0.79 ± 0.11	1.34 ± 0.24	3.43 ± 0.32	7.89 ± 1.2
7	1.38 ± 0.23	5.85 ± 0.72	2.58 ± 0.37	29.8 ± 4.3	3.08 ± 0.43	7.89 ± 1.91	19.5 ± 1.0	>20
Pentamidine	1.06 ± 0.32	3.86 ± 0.41	2.09 ± 0.37	14.7 ± 2.2	2.97 ± 0.44	6.87 ± 2.11	>20	>20
Amphotericin B	0.09 ± 0.01	0.24 ± 0,04	0.32 ± 0.05	0.97 ± 0.14	0.14 ± 0.02	1.74 ± 0.22	3.34 ± 0.41	8.05 ± 1.33

The results (IC₅₀/IC₉₀) are presented as μg/mL. NT= not tested. Values are mean ± S.D. of three observations.

Finally, compound 7 showed potent in vitro antifungal activity against C. neoformans, and antibacterial activity against M. intracellulare with IC₅₀/MIC values of 0.17/0.63, and 0.80/1.25 μg/mL, and 1 against C. neoformans (IC₅₀/MIC = 0.66/1.25 μg/mL, respectively) (Table 4). The minimum fungicidal concentration (MFC) of 7 against C. neoformans was equipotent to amphotericin B. Both the compounds showed weak activities against S. aureus and MRSA, while 1 also displayed weak activity against C. albicans, C. krusei and M. intracellulare. The antifungal activity of a mixture of juliprosine (7) and isojuliprosine were reported previously against various pathogenic fungi [7], however juliprosine had not been reported previously for biological activity as a single pure compound.

Table 4.

Antimicrobial activity of 1 and 7.

Compound	IC50/MIC/MFC or MBC (μg/mL)
	C. albicans	C. glabrata	C. krusei	C. neoformans	A. fumigatus	S. aureus	MRSA	M. intracellulare
1	6.7/20/20	-/-/-	5.4/10/10	0.66/1.25/1.25	-/-/-	5.58/10/20	5.36/10/10	9.72/10/20
7	-/-/-	-/-/-	-/-/-	0.17/0.63/0.63	-/-/-	3.0/5.0/10	3.03/5.0/10	0.80/1.25/5
Amphotericin B	0.13/0.63/0.63	0.14/0.63/0.63	0.54/1.25/1.25	0.32/1.25/1.25	0.72/1.25/2.5	NT	NT	NT
Ciprofloxacin	NT	NT	NT	NT	NT	0.1/0.5/-	0.08/0.25/1	0.25/0.5/-

IC₅₀ is the concentration that affords 50% inhibition of growth; MIC (minimum inhibitory concentration) is the lowest test concentration that allows no detectable growth; MFC/MBC (minimum fungicidal/bactericidal concentration) is the lowest test concentration that kills the organism; NT = Not tested; (-) = Not active at the highest test concentration of 20 μg/mL.

This appears to be the first report of Δ^1,6-juliprosopine (1) from either a natural or synthetic sources. Compound 1 is a new addition to our previously reported indolizidines (2–6) [8] from P. glandulosa, collected in Nevada. During this investigation, we also analyzed P. glandulosa sample from Texas, which afforded only 6 and 7 and devoid of indolizidines 1–5. This work suggests that the qualitative and quantitative nature of the bioactive alkaloidal profile in P. glandulosa varies significantly due to geographical location.