Comparison of bioactive compounds content in leaf extracts of Passiflora incarnata, P. caerulea and P. alata and in vitro cytotoxic potential on leukemia cell lines

ABSTRACT

Passiflora caerulea L., P. alata Curtis and P. incarnata L. (synonym for P. edulis Sims), are the most popular representatives of the Passiflora genus in South America. In recent years, a growing attention is paid to the biological activity and phytochemical profiles of crude extracts from various species of Passiflora in worldwide. The aim of this study was to evaluate and to compare of anti-leukemic activity of the dry crude extracts from leaves of three Passiflora species from greenhouse of Poland in two human acute lymphoblastic leukemia cell lines: CCRF-CEM and its multidrug resistant variant. Two systems of liquid chromatography in order to assessment of phytochemical composition of extracts were applied. Extracts of P. alata and P. incarnata showed the potent inhibitory activity against human acute lymphoblastic leukemia CCRF-CEM, while P. caerulea not showed activity (or activity was poor). Despite similarities in quality phytochemical profile of extracts from P. caerulea and P. incarnata, differences in quantity of chemical compounds may determine their various pharmacological potency. For the activity of P. alata extract the highest content of terpenoids and a lack of flavones C-glycosides are believed to be crucial. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.

Keywords:
Passion flower; HPLC-ESI-MSn/UPLC-PDA; Flavones C-glycosides; Terpenoids; Anti-leukemic activity

Introduction

Passiflora incarnata L., P. caerulea L. and P. alata Curtis belong to the family Passifloraceae, consisting of eighteen genera and approximately 630 species (Perez et al., 2007). Passiflora plants are used in traditional medicine not only in South America (Dhawan et al., 2004), but also in the Netherlands, Spain, Italy and Poland (Ozarowski and Thiem, 2013). They exhibit various pharmacological activities and possess complex, biologically active compounds (Miroddi et al., 2013). P. incarnata (purple passion flower), originating from North America, is one of the most important medicinal plants, and is a source of valuable herbal substance (Passiflorae herba) described in European Pharmacopoeia and by European Medicines Agency (EMA, 2014). According to the international plant list, P. incarnata is a synonym of P. edulis Sims. P. edulis is scientific name accepted (The Plant List, 2017). This herb has been commonly used in traditional phytotherapy in sleep disorders, nervousness, and anxiety for a long time throughout the world (Miroddi et al., 2013). Passiflora alata (winged-stem passion flower), native to the Amazon, from Peru to eastern Brazil, is officially recognized as a medicinal plant described in the Brazilian Pharmacopoeia (Farmacopeia Brasileira, 2010). Besides of P. alata, P. edulis is also in the Brazilian Pharmacopoeia. The edible fruits of this species are economically important for food industry. Passiflora caerulea (blue passion flower), native to Brazil, is possibly the most known species of the Passifloraceae family, but, to date, there are any data on the complete phytochemical profile of the extract from P. caerulea leaves. The first time the plant material from P. caerulea has been recognized as equivalent to the P. incarnata by Oswiecimska (1956).

Recent study demonstrated that the ethanolic extract of P. caerulea showed anti-inflammatory, anti-diarrhoeal and spasmolytic activities in experimental colitis model (Anzoise et al., 2016). Moreover, it was observed that a flavone chrysin (from P. caerulea) increased libido and sperm count in rats (Dhawan et al., 2002).

In the recent years, a growing attention is paid to the biological activity and phytochemical profiles of extracts from different species of Passiflora genus (Pereira et al., 2004; Ozarowski and Thiem, 2013; Farag et al., 2016; Wosch et al., 2017). Moreover, very interesting is chemical composition of leaf extracts from plants growing in greenhouse controlled conditions in polish terms and the relationship between the presence of active compounds in the extracts of P. incarnata, P. alata and P. caerulea and their anti-leukemic activity.

To the best of our knowledge, the crude extracts from leaves of P. incarnata, P. alata and P. caerulea were not tested on the acute human leukemia cell lines. Acute leukemias are malignant clonal disorders of hematopoietic precursor cells, in which leukemic stem cells acquire mutations that confer self-renewal capabilities, altered hematopoietic differentiation, and increased proliferative capacity (Zhou et al., 2013). Therefore, in vitro screening of plant extracts containing various chemical compounds may essentially contribute to the discovery and development of new, clinically useful drugs of natural origin as an alternative strategy for prevention and management of leukemia.

Materials and methods

Plant material

Leaves of Passiflora incarnata L., P. alata Curtis, P. caerulea L., Passifloraceae, were obtained from plants grown in the greenhouse of Department of Medicinal and Cosmetic Natural Products, University of Medical Sciences, Poznan. Controlled condition of greenhouse as follows: temperature range from 25 ºC to 40 ºC, 60–70% humidity. The plants grew on a substrate composed of peat: gravel (3:1) plus Substral Osmocote^® Universal.

The material was identified at the Department of Medicinal and Cosmetic Natural Products, Faculty of Pharmacy, Poznan University of Medical Sciences. The voucher specimens (no. 15.174, no. 15.175, no. 15.176 respectively) have been deposited in the Herbarium of the Institute of Natural Fibers and Medicinal Plants in Poznan, Poland.

Chemicals and reagents

Adriamycin and MTT solution were obtained from Sigma–Aldrich (St. Louis, MO, USA). Solvents for extraction and LC–MS analyses (methanol, acetonitrile, formic acid and ultrapure water) were obtained from Sigma–Aldrich (Poznan, Poland).

Preparation of the extracts

Dry leaves (5 g) after drying oven with air circulation (25 ºC, 24 h) were extracted with methanol pure PA (1:10, m/V) three times for 1 h by reflux. Next, the extract was concentrated under vacuum to eliminate the methanol content. The yields of the dry extracts were 32.6% for P. caerulea, 27.9% for P. alata, 21.9% for P. incarnata.

Metabolite identification with LC–MS systems

Identification of secondary metabolites presented in the extracts of leaves from Passiflora species were performed using two complementary LC–MS systems. The first system HPLC-DAD-MSⁿ consisted of Agilent 1100 HPLC instrument with a photodiode-array detector PDA (Palo Alto, CA, USA) and Esquire 3000 ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) with the X Bridge C18 column (150 mm × 2.1 mm, 3.5 µm particle size) and the MSn spectra were recorded in the negative and positive ion modes. The injection volume was 10 µl. The elution was conducted with water containing 0.1% formic acid (solvent A) and acetonitrile (solvent B). The gradient elution was started at 8% of B and linearly changed to 10% of B in 10 min, then to 25% of B in 30 min and to 98% of B over 10 min, followed by the return to stationary conditions and was re-equilibrated for 10 min. The most important MS parameters were as follow: the ion source ESI voltage −4 kV or 4 kV; nebulization with nitrogen at a pressure of 30 psi at a gas flow rate 9 l/min. Ion source temperature at 310 ºC, skimmer 1: −10 V. The spectra were scanned in the range of 50–3000 m/z.

Second system consisted of UPLC (the Acquity system, Waters, Milford, USA) hyphenated to Q Exactive hybrid MS/MS quadrupole – Orbitrap mass spectrometer. Injection volume was 10 µl. Chromatographic separation for this system were carried out using a water acidified with 0.1% formic acid (solvent A) and acetonitrile (solvent B) with mobile phase flow of 0.4 ml/min in the following gradient: 0–5 min from 10% to 25% B, 5–13 min to 98% B and maintained this conditions for 14.5 min. Up to 15 min system returned to starting conditions and was re-equilibrated for 3 min. Q-Exactive MS operated upon following settings: the HESI ion source voltage −3 kV or 3 kV. The sheath gas flow 48 l/min, auxiliary gas flow 13 l/min, ion source capillary temperature 250 ºC, auxiliary gas heater temperature 380 ºC. The CID MS/MS experiments were performed using collision energy 15 eV. Calibration of this MS/MS in both ionization modes was carried out using calibration solutions: Pierce LTQ Velos ESI Positive ion Calibration Solution and Pierce LTQ Velos ESI Negative ion Calibration Solution (Thermo Scientific). Data were analyzed with Xcalibur version 3.0.63.

The MSn (up to the MS5) and MS/MS spectra were recorded in negative and positive ion modes using a previously published approach (Piasecka et al., 2015, 2017; Ozarowski et al., 2016). The individual compounds were identified via comparison of the exact molecular masses with mean error less than 5 ppm, mass spectra and retention times to those of standard compounds, online available databases (PubChem, ChEBI, Metlin and KNApSAck) and literature data.

Chromatographic data pre-processing

Chromatographic data were analyzed with Empower 2 Chromatography Data Software (Waters, Milford, USA). Normalization by the mass of sample and integration of peaks by ApexTrack Algorithm was done. Additional manual integration events were added in the points of chromatograms where automatic integration did not separate peaks well. Area of each peak expressed as percent of total area of chromatogram were exported in ASCII format and served for statistical analysis. Bar chart that shows the area percent of identified peaks was performed using function ggplot in R package ggplot2.

Venn diagram was implemented in R script using function venn in package gplots.

Cytotoxicity of the extract against human acute lymphoblastic leukemia cell lines

Cell lines

Two human acute lymphoblastic leukemia cell lines were used as the experimental model: CCRF-CEM (ATCC^®: CCL-119™) and its multidrug resistant (MDR) variant CCRF-ADR5000 overexpressing ABCB1. MDR cells were obtained by selection with the use of adriamycin from their wild-type counterparts described previously (Paszel et al., 2011).

Viability test

Viability of the human acute lymphoblastic leukemia cells treated with the tested extracts was assessed by the MTT assay described by Mossman (1983). The test was performed as described previously (Paszel et al., 2011). Briefly, CCRF-CEM cells were exposed for 24, 48 and 72 h to the studied extracts in a concentration range of 3.125–100 µg/ml. As a reference compounds four flavonoids: vitexin, isovitexin, apigenin and luteolin were used. CCRF-CEM and CCRF-ADR5000 cell lines were treated with these compounds for 24, 48 and 72 h in the concentration range of 3.125–100 µM. The solvent, methanol in a concentration of 0.25%, was also applied as a control and was identified as not cytotoxic in the used concentration. Experiments were performed in duplicates and were repeated three times. Based on the results from MTT test IC₅₀ values were calculated using CalcuSyn software (BioSoft, Cambridge, UK).

Statistical analysis

All values of phytochemical determinations and results of the study were expressed as means ± SEM. Moreover, results obtained from leukemia cell lines were calculated using T-Student test (p < 0.05).

Results and discussion

Phytochemical studies

Two complementary spectrometric systems: high resolution MS and an ion trap MS enabled to identify eighty two secondary metabolites in crude extracts of Passiflora leaves (Fig. 1; Table 1). Previous study showed, that the highest amount of phenolic compounds (expressed as mg of gallic acid equivalent/g of extract) was measured in P. alata (8.21 ± 0.003 mg/g), P. caerulea (6.23 ± 0.000 mg/g) and P. incarnata (4.85 ± 0.003 mg/g) (Hadaś et al., 2017). Phenolic acid derivatives constituted 18%, 11.5% and 5% of the area of identified peaks of P. alata, P. caerulea and P. incarnata, respectively. The majority of the metabolites belonged to flavonoids. In particular, glycosides of flavonols quercetin and myricetin were found in P. cearulea and P. alata whereas glycosides of flavones apigenin, luteolin and chrysin were the most abundant phytochemicals in all studied plants.

Table 1
Secondary metabolites identified in Passiflora incarnata, P. alata, P. caerulea leaves by HPLC-UV-MSⁿ .

No.	rt [min]	Compound name	Fragmentation pathway [m/z]		Chemical formula		Molecular mass of ions [M-H]- or [M-H]+		Δ ppm	λ [nm]	P. caerulea	P. incarnata	P. alata	Id l	CAS	Ref
			ESI (-)	ESI (+)			Calculated	Measured
1	3.2	Phenylalanine		166, 120	C9H12NO2		166.086	166.0863	-1.3614	260	*	*	*	1	63-91-2	Std
2	3.5	Caffeic acid-glucoside	341, 179, 161, 113		C15H18O9			Not detected in HR MS		304sh	*			2		Parveen et al., 2013
3	4.3	Pinoresinol	357, 241, 151,		C20H21O6		357.13474	357.1344	1.059	275			*	2	487-36-5	Ye et al., 2005; Eklund et al., 2008
4	4.4	Dihydroxybenzoyl-hexoside	315, 153	317, 155	C13H15O9		315.0718	315.0722	-1.1026	281	*	*		2		Parveen et al., 2013
5	4.9	Shikimate-hexoside	319, 157		C13H19O9		319.10342	319.1035	-0.111	275, 300		*		3		Parveen et al., 2013
6	5.3	Tryptophan	203, 159, 116	205, 188	C11H13O2N2		205.097	205.0972	-0.9744	285	*	*	*	1	73-22-3	Std
7	7.1	Dihydroxybenzoyl-pentoside	285, 153	287, 155	C12H13O8		285.0616	285.0616	-0.0215	286	*	*	*	3		Parveen et al., 2013
8	7.3	Dihydroxybenzoyl-glucosylpentoside	447, 267, 153		C18H23O13		447.11441	447.1144	-0.0078	284	*		*	3		Parveen et al., 2013
9	8.8	Luteolin 6,8-di-C-glucoside-7-O-glucoside	771, 651, 609, 489, 399, 369		C33H39O21		771.1978	771.1963	-2.0038	269, 335		*		3		Ferreres et al., 2003
10	10.5	Isovitexin 8-C-arabonoside-7-O-glucoside	725, 635, 605, 563, 473, 443, 353	727, 709, 643, 559	C32H37O19		725.1924	725.1907	-2.3184	270, 332	*	*	*	3		Ferreres et al., 2003
11	10.5	Quercetin 3'-O-glucoside-7-O-glucosyldeoxyhexoside	771, 609, 301		C33H39O21		771.1978	771.1978	-0.0252	272, 345	*		*	3		Kachlicki et al., 2008
12	10.6	Isovitexin 2"-O-glucoside-7-O-glucoside (apigenin 6-C-glucoside 2"-O-glucoside-7-O-glucoside)	755, 593, 455, 413, 293	757, 595, 577, 529, 445, 379, 337, 293	C33H39O20		755.2029	755.1998	-4.1733	269, 332		*		3		Simigriotis et al., 2013
13	11.2	Luteolin 6-C-hydroxybenzoylpentoside-di-O-glucoside	861, 699, 647, 327		C39H41O22		861.21027	861.2095	0.899	269, 340			*	3		Ferreres et al., 2003; Ye et al., 2005
14	11.4	Luteolin 6,8-di-C-glucoside	609, 489, 369, 399	611, 593, 473, 408, 353, 299	C27H29O16		609.1456	609.1477	-3.521	270, 344	*	*		2	29428-58-8	Ferreres et al., 2003; Waridel et al., 2001
15	11.9	Apigenin 6,8-di-C-glucoside	593, 473, 383, 353	595, 577, 511, 457	C27H29O16		593.1501	593.1497	-0.7389	269, 333	*	*		2	23666-13-9	Ferreres et al., 2003; Waridel et al., 2001
16	12.3	Apigenin 6, 8-di-C-glucoside-7-O-glucoside	755, 593, 473, 383, 353		C33H39O20		755.2029	755.1998	-4.1733	268, 334	*	*		3		Ferreres et al., 2003
17	13.5	Luteolin 6-C-pentoside-8-C-glucoside	579, 489, 459, 399, 367		C26H27O15		579.135	579.1376	-2.751	270, 241	*			3		Ferreres et al., 2003
18	14.2	Vitexin 6"-O-glucoside (apigenin 8-C-glucoside 6"-O-glucoside)	593, 473, 431, 311	595, 577, 529, 433, 337, 267, 283	C27H29O15		593.1501	593.1477	-4.0371	271, 335	*			3	639850-16-1	Ferreres et al., 2007; Wojakowska et al., 2013
19	14.6	Isoorientin 7-O-glucoside (luteolin 6-C-glucoside 7-O-glucoside)	609, 489, 447, 327		C27H29O16		609.145	609.1423	-4.3948	272, 345	*	*		2	35450-86-3	Ferreres et al., 2007; Simigriotis et al., 2013
20	15.2	Isoorientin (luteolin 6-C-glucoside)	447, 327	449, 431, 383, 353, 299	C21H19O11		447.0922	447.0907	-3.2633	272, 345		*		1	4261-42-1	std
21	15.4	Orientin 7-O-deoxyhexoside (luteolin 8-C-glucoside 7-O-deoxyhexoside)	593, 473, 429, 377, 299	595, 577, 529, 449, 367	C27H29O15		593.1989	593.1479	-3.9288	268, 344	*			3		Ferreres et al., 2007
22	16.1	Apigenin 6,8-di-C-pentoside	533, 473, 443, 413, 383, 353	535, 499, 481, 379, 283	C25H27O13		535.1458	535.1446	2.2345	269, 332	*	*		3		Ferreres et al., 2003
23	16.6	Isoscoparin 2"-O-glucoside (chrysoeriol 6-C-glucoside 2"-O-glucoside	623, 443, 323, 309	625, 463, 445, 391, 313	C28H31O16		623.1612	623.1634	-3.5303	243, 271, 349		*		3	97605-25-9	Ferreres et al., 2007
24	17.4	Vitexin 6"-O-pentoside (apigenin 8-C-glucoside 6"-O-pentoside)	563, 473, 443, 413, 341, 311	565, 499, 427, 313	C26H27O10		563.1395	563.1414	3.246	269, 332	*			3		Ferreres et al., 2007
25	17.6	Orientin 6"-O-glucoside (luteolin 8-C-glucoside 6"-O-glucoside)	609, 489, 429, 357, 327	611, 593, 545, 473, 425, 395, 341, 299	C27H29O16		609.1456	609.1477	-3.521	272, 345		*		3		Piasecka et al., 2015; Ferreres et al., 2007
26	18.1	Isovitexin 7-O-glucoside (apigenin 6-C-glucoside 7-O-glucoside)	593, 431, 311		C27H29O15		593.1501	593.1477	-4.0371	269, 335		*		3	20310-89-8	Ferreres et al., 2007
27	18.2	Quercetin glucosyldeoxyhexoside	609, 301		C27H29O16		609.145	609.1424	-4.2946	273, 350	*			3		Cuyckens et al., 2001
28	18.6	Luteolin 7-O-glucosyldeoxyhexoside	593, 447, 413, 285, 257		C27H29O15		593.1501	593.1477	-4.0317	268, 345		*		3		Cuyckens et al., 2001
29	18.8	Apigenin 6-C-pentoside-8-C-glucoside	563, 473, 443, 353, 383		C26H27O14		563.1395	563.1411	3.779	269, 333	*	*		3		Ferreres et al., 2003
30	19	Isovitexin 6"-O-deoxyhexoside (apigenin 6-C-glucoside 6"-O-deoxyhexoside)	577, 413, 311	579, 433, 415, 367, 337, 283	C27H29O14		577.15625	577.1563	-0.05	270, 335		*		3		Ferreres et al., 2007
31	19.2	Apigenin 6-C-deoxyhexoside-8-C-pentoside	547, 487, 457, 427, 367, 337, 309, 281	549, 531, 483, 465, 375	C26 H27O13		547.1457	547.1449	-1.488	268, 334	*			3		Ferreres et al., 2003
32	19.6	Isoscoparin 2"-O-deoxyhexoside (chrysoeriol 6-C-glucoside 2"-O-deoxyhexoside)	607, 443, 323, 307	609, 463, 397, 313	C28H31O15		607.1663	607.1697	-2.751	246, 272, 349	*		*	3		Ferreres et al., 2007
33	20.8	Apigenin C-glucose C-deoxyhexose	577, 503, 473,443,383, 353	565, 547, 499, 403, 295	C27H29O14		577.1552	577.1533	-3.3312	268, 335	*	*		3		Piasecka et al., 2015; Ferreres et al., 2003
34	21	Isovitexin 2"-O-dideoxyhexoside (apigenin 6-C-glucoside 2"-O-dideoxyhexoside)	561, 413, 251		C27H29O13		561.16187	561.1614	0.902	329, 333			*	3		Xu et al., 2013
35	21.1	Vitexin 2"-O-glucoside (apigenin 8-C-glucoside 2"-O-glucoside)	593, 473, 413, 357, 293	595, 577, 457, 283	C27H29O15		593.1501	593.1477	-4.0371	270, 334	*	*		3	61360-94-9	Ferreres et al., 2007
36	21.8	Luteolin 6-C-[6"-O-deoxyhexoside]- dideoxyhexoside	561, 327		C27H29O13		561.29169	561.2916	0.071	271, 344		*		3		Ferreres et al., 2007; Xu et al., 2013
37	21.8	Orientin 6"-O-deoxyhexose (luteolin 8-C-glucoside 6"-O-deoxyhexose)	593, 473, 327, 357		C27H29O16		593.1501	593.1497	-0.7389	269, 346			*	3		Ferreres et al., 2007
38	22	Chrysin 6-C-glucoside-8-C-pentoside	547, 457, 427, 337, 309	549, 513, 413, 309	C26H27O13		547.276	547.2766	1.115	264, 330	*			3		Ferreres et al., 2003; Zucolotto et al., 2011
39	23.2	Apigenin 6-C-[2"-O-deoxyhexoside] - pentoside	547, 457, 383, 293	549, 265, 221	C26H27O13		547.276	547.2767	1.334	269, 335	*			3		Ferreres et al., 2007
40	23.4	Luteolin 7-O-[6"-acetyl]-deoxyhexoside	473, 327, 285		C23H21O11		473.10907	473.1089	0.286	269, 346			*	3		Simigriotis et al., 2013
41	24.4	Chrysin 6-C-glucoside	415, 295, 267		C21H19O9		415.1035	415.1037	0.5863	271, 346	*			3	28368-57-2	Zucolotto et al., 2011
42	24.5	Isoorientin 2"-O-glucoside (luteolin 6-C-glucoside 2"-O-glucoside)	609, 489, 429, 357, 327, 309	611, 449, 431, 353, 299	C27H29O16		609.145	609.1424	-4.2946	271, 344		*		3	55196-48-0	Piasecka et al., 2015; Waridel et al., 2001
43	24.9	Chrysin 6-C-[2"-O-glucoside]-glucoside		579, 417, 351	C27H29O14		577.1563	577.1564	0.1618	266, 331	*			3		Wojakowska et al., 2013; Zucolotto et al., 2011
44	25.5	Chrysin 6-C-glucoside-7-O-glucosyldeoxyhexoside		725, 561, 417, 351	C33H39O18		723.2142	723.214	-0.2739	272, 329	*			3		Wojakowska et al., 2013; Zucolotto et al., 2011
45	25.7	Blumenol C-glucoside		373, 211, 193	C19 H15O8		371.0772	371.0772	-0.0018	253	*	*		3	135820-80-3	Peipp et al., 1997; Marino et al., 2008
46	25.8	Vitexin (apigenin 8-C-glucoside		433, 367, 337, 309	C21H19O10		431.0978	431.0988	0.001	269, 333		*		1	3681-93-4	std
47	26	Isovitexin (apigenin 6-C-glucoside)	431, 311	433, 415, 367, 283	C21H19O10		431.09833	431.0984	-0.093	269, 334		*		1	38953-85-4	std
48	26.2	Isovitexin 7-O-deoxyhexoside (apigenin 6-C-glucoside 7-O-deoxyhexoside)	577, 413, 311		C27H29O14		577.1563	577.1544	3,2222	269, 335		*		3		Cuyckens et al., 2001
49	26.3	Pinoresinol O-glucosyldeoxyhexoside-O-glucoside	827, 519, 357, 151		C38 H51 O20		827.29913	827.2979	1.466	278		*		3		Eklund et al., 2008
50	26.5	Isovitexin 2"-O-deoxyhexoside (apigenin 6-C-glucoside 2"-O-deoxyhexoside)	577, 413, 293		C27H29O14		577.1563	577.1544	3.2222	269, 335	*	*	*	3		Zucolotto et al., 2011
51	26.5	Chrysin di-O-glucoside		579, 417, 255	C27H29O14		577.15686	577.1563	1.007	265, 330		*		3		Piasecka et al., 2015; Zucolotto et al., 2011
52	26.6	Apigenin 7-O-glucosyldideoxyhexoside	561, 269		C27H29O13		561.16144	561.1614	0.137	269, 335		*		3		Xu et al., 2013; Wojakowska et al., 2013
53	26.8	Apigenin 7-O-glucosyldeoxyhexoside	577, 269		C27H29O14		577.1563	577.1563	0.779	268, 335		*		3		Kachlicki et al., 2008
54	26.8	Apigenin 6-C-[6"-acetyl-2"-O-deoxyhexoside]-glucoside	619, 559, 455, 293		C29H31O15		619.1668	619.1658	-1.7311	275, 339	*			3		Simigriotis et al., 2013
55	26.9	Quercetin 7-O-glucoside		465, 303	C21H21O12		465.10345	465.1038	-0.858	245, 271, 351		*		1	491-50-9	std
56	27	Scoparin 2"-O-glucoside (chrysoeriol 8-C-glucoside 2"-O-glucoside)	623, 503, 443, 323, 298		C28H31O16		623.1612	623.1636	3.85	242, 269, 348		*		3	124902-15-4	Ferreres et al., 2007; Wojakowska et al., 2013
57	27	Apigenin 7-O-glucosylpentoside	563, 269		C26H27O10		563.1395	563.1414	3.246	269, 335		*		3		Cuyckens et al., 2001
58	27.2	Feruloylquinic acid diglucoside	691, 659, 335, 317, 273	693, 675, 643, 504, 359, 337	C29H39O19		691.2091	691.2079	-1.753	325sh	*	*		2		Sakalem et al., 2012
59	27.4	Chrysin 6-C-[6", 2"-di-O-deoxyhexoside]-glucoside	707, 453, 277	709, 563, 417, 321	C33H39O17		707.2193	707.2193	0.039	275, 332	*			3		Ferreres et al., 2007; Zucolotto et al., 2011
60	27.6	Luteolin 7-O-glucoside		449, 285	C21H21O11		449.10797	449.1089	-2.148	269, 345		*		1	5373-11-5	std
61	29.6	Chrysin 6-C-[2"-O-pentoside]-glucoside	547, 277, 175	549, 417, 399, 351, 321, 267	C26H27O13		547.276	547.277	1.8979	265, 331	*			3		Ferreres et al., 2007; Zucolotto et al., 2011
62	30.3	Chrysin 6-C-[2"-O-deoxyhexoside]-glucoside	561, 277, 175	563, 417, 399, 351, 321, 267	C27H29O13		561.1614	561.1613	-0.203	269, 331	*	*		3		Ferreres et al., 2007; Zucolotto et al., 2011
63	32.7	Blumenol C		211, 175, 133, 119	C13H23O2		211.16896	211.1693	-1.404	255		*		2	36151-02-7	Peipp et al., 1997; Marino et al., 2008
64	34.2	Phillygenin O-glucoside-O-pentoside	665, 503, 371		C32H41O15		665.24664	665.25	2.325	274			*	3		Eklund et al., 2008
65	34.7	Chrysin 6-C-glucoside		417, 351	C21H19O9		415.1035	415.1035	0.0716	265, 333	*			3	28368-57-2	Zucolotto et al., 2011
66	35.4	Isoorientin 2"-O-deoxyhexoside (luteolin 6-C-glucoside 2"-O-deoxyhexoside)		595, 449, 413, 329, 299	C27H29O15		593.1501	593.1522	3.58	269, 346		*		3		Piasecka et al., 2015; Zucolotto et al., 2011
67	36.1	Luteolin O-glucoside-O-deoxyhexoside		595, 449, 431, 287	C27H29O16		593.1507	593.1522	3.58	271, 348		*		3		Kachlicki et al., 2008
68	36.5	Blumenol B glucoside	387, 369, 225, 207		C19H31O8		387.20255	387.2024	0.281	256			*	3		Marino et al., 2008
69	37.4	Apigenin-6-C-deoxyhexoside-7-O-glucoside	577, 415, 311, 283		C27H29O14		577.1552	577.1533	-3.3312	269, 335	*			3		Simigriotis et al., 2013
70	37.5	Blumenol C pentoside	341, 209		C18H29O6		341.19672	341.197	-0.709	255			*	3		Peipp et al., 1997; Marino et al., 2008
71	37.5	orientin 2"-O-dideoxyhexoside (luteolin 8-C-glucoside 2"-O-dideoxyhexoside		579, 431, 413, 335, 299	C27H29O14		577.15668	577.1563	0.695	269, 344		*		3		Piasecka et al., 2015; Xu et al., 2013
72	38.6	Chrysin O-deoxyhexoside-O-glucoside		563, 417, 255	C27H29O13		561.1614	561.1617	0.5668	264, 331	*			3		Wojakowska et al., 2013; Zucolotto et al., 2011
73	39.1	Myricetin glucosyldeoxyhexoside	625, 463, 317		C27H45O16		625.2713	625.2718	0.77	271, 355	*			3		Simigriotis et al., 2013
74	40.1	Cyclopassifloic acid glucoside	697, 535, 517		C37H61O12		697.41718	697.4169	0.473	244		*		3		Yoshikawa et al., 2000
75	41.7	Luteolin 6-C-deoxyhexoside 7-O-glucoside	593, 431, 327	595, 449, 431, 413, 329, 299	C27H29O15		593.1989	593.1479	-3.9288	269, 347		*		3		Zucolotto et al., 2011
76	42.7	Apigenin O-deoxyhexoside-O-glucoside	577, 415, 269		C27H29O14		577.15649	577.1563	0.366	269, 333		*		3		Cuyckens et al., 2001
77	43.6	Dicaffeoylquinic acid	515, 353	517, 193	C25 H39 O11		515.25018	515.2498	0.766	315		*		2	89919-62-0	Clifford et al., 2005
78	44.4	Chrysin 6-C-deoxyhexoside-7-O-glucoside	561, 399, 295		C27H29O13		561.16199	561.1614	1.1106	264, 331		*		3		Zucolotto et al., 2011
79	46	Apigenin 6-C-[2"-O-deoxyhexoside]-deoxyhexoside	561, 397, 293		C27H29O13		561.16144	561.1614	0.1317	269, 335		*		3		Xu et al., 2013; Zucolotto et al., 2011
80	46.7	Chrysin 6-C-glucoside-6"-O-deoxyhexoside-	561, 457, 295	563, 401, 365, 311	C27H29O13		561.16199	561.1614	1.115	266, 333		*		3		Zucolotto et al., 2011
81	47.1	Apigenin-8-C-deoxyhexoside-7-O-glucoside	577, 473, 415, 353, 311, 283	579, 417, 399, 355	C27H29O14		577.15729	577.1563	1.752	269, 334		*		3		Xu et al., 2013
82	47.7	Luteolin 8-C-[6"-O-glucoside]-dideoxyhexoside	577,473, 415, 357, 327		C27H29O14		577.15753	577.1563	2.168	269, 346		*		3		Ferreres et al., 2003; Ferreres et al., 2007; Xu et al., 2013

• Legend: ^aMetabolite identification level according to Metabolomics Standards Initiative recommendation (Sumner et al., 2007). The levels include: 1. Identified compounds; 2. Putatively annotated compounds without chemical reference standards, based upon physicochemical properties and spectral similarity with public spectral libraries; 3. Putatively characterized compound classes based upon characteristic physicochemical properties of a chemical class of compounds, or by spectral similarity to known compounds of a chemical class; 4. Unknown compounds - although unidentified or unclassified these metabolites can still be differentiated and quantified based upon spectral data.Std, identification on the basis of standard compound fragmentation; italic, main peak detected in MSn experiment; ref, references.MW, molecular mass.

Fig. 1
UV chromatograms recorded at 280 nm by UPLC-MS/MS of metabolites detected in extract of: A. Passiflora caerulea; B. P. incarnata; C. P. alata.

The diversity of glycosylation site in Passiflora species concerns all identified flavonoids (Fig. 2). O,C-glycosides were in the highest content in all Passiflora species. Interestingly, C-glycosides were observed in high content in P. caerulea (more than 30% of peaks area) and in P. incarnata (more than 20% of peaks area) in set of parameters described in section 2.4. Glucose, deoxyhexose, pentose and dideoxyhexose were found among glycosidic substituents of flavone aglycons. The MS analysis in both negative and positive ionization modes enabled to distinct 6-C- and 8-C-glycosidic bond as well as 2"-O- and 6"-O-glycosides.

Fig. 2
Percentage ratios for groups of identified compounds in the extracts of Passiflora caerulea, P. incarnata, P. alata.

In addition, terpenoidal structures such as blumenols B and C (megastigmanes) and cyclopassifloic acid glucoside were also identified (in the group “others” in Fig. 2) in Passiflora species. The highest content of terpenoids was identified in P. alata for which 38% of the area of identified peaks is not the origin of the phenylpropanoid pathway (Fig. 2).

Detailed MS analysis for all detected chemical compounds is presented in Table 1.

Chromatograms of P. caerulea and P. incarnata showed greater similarities in metabolites content than P. alata (Fig. 3). Earlier study (Farag et al., 2016) using hierarchical cluster analysis of Passiflora species showed that they share comparable metabolite profiles. Moreover P. caerulea appeared to be the closed to P. incarnata to terms of overall chemical composition. Five metabolites were common to all species (precursors of phenylpropanoids and terpenoids phenylalanine and tryptophan, as well as dihydroxybenzoyl-pentoside, apigenin 6-C-glucoside 8-C-arabonoside-7-O-glucoside and isovitexin 2"-O-deoxyhexoside). P. alata and P. incarnata had the lower number of common metabolites. Metabolites of P. alata were only partially identified, however the unknown metabolites should not have an impact on similarieties among Passiflora species. The unknown metabolites belonged tentatively to terpenoids rather than phenylpropanoids due to characteristic of UV maximum of absorption and MS fragmentation.

Fig. 3
Venn diagram: similarities in metabolites content between extracts of Passiflora caerulea, P. incarnata, P. alata.

The MS analysis in both negative and positive ionization modes enabled to distinct 6-C- and 8-C-glycosidic bond as well as 2"-O- and 6"-O-glycosides. Two different C,O-diglycosides of the flavone luteolin (metabolites 19 and 21, Table 1) are substituted in a different manner. The most abundant product ion at m/z = 447 observed during fragmentation of metabolite 19 in the negative ionization corresponded to the [M−H-162]⁻ indicating the presence of an O-glucoside (Figs. A.1A, A.9). An analogous [M−H-146]⁻ ion for 21 revealed the substitution with an O-deoxyhexose (Figs. A.1B, A.9). The [Agly+42-H]⁻ and the [Agly+72-H]⁻ product ions are the same for 19 and 21, however, present in a different ratio. The most abundant [Agly+42-H]⁻ ion of 19 indicated the 6-C-glycoconjugate of luteolin (isoorientin), whereas the most abundant [Agly+72-H]⁻ ion of 21 referred to the 8-C-glycoconjugate of luteolin (orientin) (Ferreres et al., 2003). On the basis of the above information metabolite 19 and 21 were identified as isoorientin 7-O-glucoside and orientin 7-O-deoxyhexoside, respectively.

Apigenin substituted with deoxyhexose and glucose was identified in six isoforms: compounds 30, 33, 50, 53, 69 and 76. Analysis in negative ionization enabled to distinguished compounds 30 and 50 on the basis of their main product ions in MS2 and MS3 according to Piasecka et al. (2015). The first mentioned compound was characterized by the major product [Agly+42-H]⁻ ion typical for C-glycosides of flavones and the deprotonated [M−H-164]⁻ and [M−H-266]⁻ ions indicated on deoxyhexose substituted to glycosidic carbon (Figs. A.2A, A.9). The only one possible place of substitution with such a fragmentation is C-[6"-O-deoxyhexosyl]-glucosides. The former compound has the major product [Agly+(42–18)-H]⁻ ion characteristic for structure of the C-[2"-O-deoxyhexosyl]-glucosides of flavones (Figs. A.2B, A.9). The deprotonated [M−H-164]⁻ ion confirmed that deoxyhexose is substituted to glycosidic moiety. Thus, 30 and 50 were identified as isovitexin 6"-O-deoxyhexoside and isovitexin 2"-O-deoxyhexoside, respectively. Detachment of fragments 74, 104, 90 and 120 amu as well as the major product [Agly+84-H]⁻ ion at m/z = 353 of isomeric 33 indicated on structure of di-C-glycosides of flavone according to Ferreres et al. (2003) (Figs. A.2C, A.9). The intensity of deprotonated ions formed after detachment of glucose and deoxyhexose moiety have similar intensity, thus the distinction of particular substituents between 6-C and 8-C of aglycon moiety was rather problematic. Therefore, 33 was tentatively identified as apigenin C-glucose C-deoxyhexose. Metabolites 53 and 76 have the major deprotonated product ion at m/z = 269 similar to apigenin standard and therefore indicated on O-type glycosylation on flavone skeleton (Figs. A.2D and E, A.9). The differences in fragmentation pattern in MS2 and MS3 between compounds 53 and 76 enabled to distinguish their glycosidic patterns. In 53 the [M−H-308]⁻ ion indicated on O-glucosyldeoxyhexoside substituent according to Kachlicki et al. (2008). Proportionally high intensive [M−H-164]⁻ ion indicated on deoxyhexosyl(1→2)glucosidic bond. In 76 a high intensive [M−H-162]⁻ and [M−H-162-146]⁻ ions reflected successive detachment of glucose and deoxyhexose which suggested substitution of both glycosides on separate hydroxyl groups on aglycon (Cuyckens et al., 2001) (Figs. A.2E, A.9). Thus, 76 was identified as apigenin O-deoxyhexoside-O-glucoside. Compound 69 represented another isomeric structure of apigenin deoxyhexosylglycoside on the basis of the major product [Agly+42-H]⁻ ion. The high intensive [M−H-162]⁻ ion indicated on O-glucose substituted to aromatic ring of aglycon and [M−H-162-104]⁻ ion indicated on C-deoxyhexose (Ferreres et al., 2007) (Figs. A.2F, A.9). Therefore 69 was assumed as apigenin 6-C-deoxyhexoside 7-O-glucoside. Metabolites 36, 52, 71 and 82 were identified as flavone structures with poorly known dideoxyhexosides. Similar structures were previously isolated and identified as 8-C-β-digitopyranoside and 8-C-β-boivinopyranoside of luteolin and apigenin by NMR from P. edulis leaves and stems (Xu et al., 2013) (for P. edulis, P. incarnata is recognized as synonym). The main product ion at m/z = 327 in negative ionization mode of 36 was typical for C-glycosides of luteolin as described Ferreres et al. (2007) (Fig. A.3A). The most abundant [Agly+42-H]⁻ ion indicated on 6-C-glycosides rather than 8-C isomer. The [M−H]⁻ ion of compound 36 showed losses of 234 amu (88 + 146) reflecting the structure of 6-C-dideoxyhexose (1→6)deoxyhexose. Precise defining of certain glycoside residue reacquires further NMR analysis since three isomeric deoxyhexosides: rhamnose, fucose and chinovose have been reported in Passiflora edulis fo. flavicarpa (Li et al., 2011) (Chinovose is isomer of rhamnose and is defines as isorhamnose or 6-deoxy-α-D-glucopyranose). Therefore, compound 36 was identified as luteolin 6-C-[6"-O-deoxyhexoside]-dideoxyhexoside. The fragmentation of deprotonated compound 52 yielded losses of 292 amu (162 + 130) derived from the glucosyl-dideoxyhexoside moiety giving the [Agly-H]⁻ product ion, typical for apigenin. The place of glycosidic substitution was determined as 7-OH at aglycon moiety on the basis of similarities in fragmentation scheme with other flavone derivatives described in literature (Piasecka et al., 2015; Ferreres et al., 2007). Thus, the diglycosides was identified as apigenin 7-O-glucosyldideoxyhexoside. Compounds 66 and 71 was detected only in positive ionization in which protonated [M+H-146]⁺ and [M+H-130]⁺ ions of compounds 66 and 71, respectively followed by fragmentation adequate for C-glucoside allowed to assume that both compounds have structure of 6-C-[2"-O-glycoside]-glucoside of luteolin according to Piasecka et al. (2015). Thus, 66 was identified as isoorientin 2"-O-deoxyhexoside and 71 as isoorientin 2"-O-dideoxyhexoside. Further investigation is necessary to study diversity of O- and C-dideoxyhexosyl substituents in Passiflora species. Dideoxyhexose in structure of 82 were identified as C-linked to aglycon moiety, because glucose fragment 162 amu corresponding to entire glucose moiety and main [Agly+42-H]⁻ product ion was similar to 23 (Fig. A.3B). Glucosides and pentosides of phenolic acids such as dihydroxybenzoic and shikimic acids (2, 5 and 7) were observed in lesser proportion. These compounds have been tentatively identified to be phenolic acids glucosides and not the respective esters on the basis of the MS fragmentation pattern. Losses of 162 amu constitute a typical fragmentation pattern of the glucose moiety of compounds 4, 5, as well as losses of 132 amu adequate for pentose moiety in 7 in the negative ion mode. In addition, glucoside of blumenol C (45), a megastigmane terpenoid widely distributed in plant kingdom (Peipp et al., 1997) was also detected. Their UV maximum absorbance is about 250 nm, thus presence chromatographic peak for the compound in 280 nm indicated on relative high amount of the compound. The fragmentation of 45 in both ionization modes is similar to fragmentation of hydroxyferulic acid glucoside (Piasecka et al., 2015) (Fig. A.4A) which can lead to misidentification of the isobaric structures. Nevertheless, determination of accurate masses of 45 in high resolution mass spectrometer confirmed the structure of blumenol C glucoside (Fig. A.4B) which is identified for the first time in Passiflora species.

Cytotoxicity of the extracts and chemical compounds

Several studies showed that flavonoids can play important beneficial roles in chemoprevention and the usage of plant-derived natural compounds is a promising approach for the therapy of malignant disorders via various mechanism of pharmacological action (Sak, 2014). Today, many flavonoids are known to exert anticancer potential (e.g. antileukemic activity) both in in vitro and in vivo models (Caxito et al., 2015), for example apigenin, luteolin, quercetin, chrysin, myricetin. According to Moghaddam et al. (2012) the hydroxyflavones (luteolin, apigenin) exerted comparable antiproliferative activities against malignant cells. Although it was previously reported that the extract of P. incarnata (synonym of P. edulis), the most important medicinal plant from Passifloraceae, possesses cytotoxic activity against cancer cells (Ehrlich Ascites Carcinoma) (Sujana et al., 2012), however still very little is known about the antitumor/antileukemic potential of extracts from plants belonging to the genus Passiflora. This prompted us to assess the cytotoxic potential of crude extracts of P. incarnata, P. alata and P. caerulea leaves against human leukemic cells. Due to the fact, that the multidrug resistance of neoplastic cells is a phenomenon which is one of the most important causes of chemotherapy failure in malignant diseases (Paszel et al., 2011), we decided to investigate the cytotoxic activity of the tested extract in multidrug resistant ABCB1 expressing – CCRF-ADR5000 leukemia cells.

Results showed that the most potent against human acute lymphoblastic leukemia CCRF-CEM cells was extract from P. alata leaves with the IC₅₀ value of 91.2 µg/ml after 72 h of treatment (Table 2). Calculation of IC₅₀ factor for the shorter exposure times (24 and 48 h) was not possible, due to the low activity of the extract.

Table 2
IC₅₀ values obtained for CCRF-CEM cells treated with crude extracts of Passiflora incarnata and P. alata during 24, 48 and 72 h.

	IC50 values ± SD (µg/ml)
	CCRF-CEM
	24 h	48 h	72 h
Extract of P. incarnata leaves	nd	nd	nd
Extract of P. alata leaves	nd	nd	91.2 ± 1.1

• nd, not detected.

After 72 h of treatment with 100 µg/ml of P. alata leaves extract we observed a 60% growth inhibition of CCRF-CEM cells (p < 0.001) (Fig. 4). These results may correspond to our previous study in which it was shown that P. alata leaves extract contained the highest concentration of phenolic compounds (Hadaś et al., 2017). Moreover, quantitative analysis (HPLC-DAD) showed that this extract contained the highest level of apigenin (9.51 mg/100 g dry weight of extract) in comparison with extracts of P. caerulea and P. incarnata (Hadaś et al., 2017). In addition, the biological activity of P. alata extract can be explained by the highest content of terpenoids. The lower concentrations of the extract were significantly less active. In shorter treatment times we did not detect any significant cell viability reduction. Extract of P. incarnata leaves was less potent against CCRF-CEM cells and the highest concentration (100 µg/ml) induced only a 25% inhibition of cells growth (p < 0.01) (Fig. 5), whereas the extract from leaves of P. caerulea did not show any statistically significant effect (data not shown). Moreover none of the studied extracts were active against multidrug resistant CCRF-ADR5000 cells. Probably the lack of activity in multidrug resistant cells may be the result of the removal of its active components into the extracellular environment by transmembrane protein ABCB1 (data not shown).

Fig. 4
Influence of the extract of Passiflora alata on leukemic cells (CCRF-CEM). Legend: CCRF-CEM was treated with different concentrations of the extract (concentration range of 3.125–100 µg/ml for 72 h. Each point represents the mean ± SD of four independent experiments performed in duplicate (^***p < 0.01).

Fig. 5
Influence of the extract of Passiflora incarnata on leukemic cells (CCRF-CEM). Legend: CCRF-CEM was treated with different concentrations of the extract (concentration range of 3.125–100 µg/ml for 72 h). Each point represents the mean ± SD of four independent experiments performed in duplicate (^**p < 0.01).

Because the most active was P. alata extract containing apigenin and luteolin, in this study tests with using pure compounds were carried out. It was observed that apigenin showed cytotoxic activity in concentration range of 50–100 µM against CCRF-CEM and CCRF-ADR5000 cells (Figs. A.5, A.6) with the IC₅₀ value of 99.6 µM and 68.7 µM after 72 h of treatment, respectively (Table 3). Apigenin was more potent against multidrug resistance CCRF-ADR5000 cells after 48 and 72 h of treatment (Fig. A.6). Compound used in the concentration of 100 µM reduced the population of viable CCRF-ADR5000 cells to 36% after 48 h and to 23% comparing to untreated control cells. Luteolin contained in P. alata extract in concentration of 0.78 mg/100 g dry weight of extract (Hadaś et al., 2017) was the most active chemical compound (Figs. A.7, A.8). It showed a significant cytotoxic activity against CCRF-ADR5000 cells in the whole range of concentrations. Even the lowest concentration of the compound (3.125 µM) caused a significant decrease in CCRF-ADR5000 cells viability. Observed effect was time and dose dependent and after 72 h of treatment with apigenin (100 µM) the population of viable CCRF-ADR5000 cells was reduced to 4%. Values of IC₅₀ factor obtained for multidrug resistant CCRF-ADR5000 cells treated with luteolin were significantly lower comparing to IC₅₀ calculated for wild-type cells (Table 3). This observation indicates the multidrug resistance reduction potential of the compound in the tested leukemic cells. Furthermore, vitexin and isovitexin (contained in passiflora extracts) did not exert any effect in both cell lines (data not shown). Viability of the cells treated with these compounds was not reduced within 72 h of treatment.

Table 3
IC₅₀ values obtained for CCRF-CEM and CCRF-ADR5000 cells treated with apigenin and luteolin during 24, 48 and 72 h.

	IC50 values ± SD (µM)
	CCRF-CEM				CCRF-ADR5000
	24 h	48 h	72 h		24 h	48 h	72 h
Apigenin	nd	101.2 ± 2.1	99.6 ± 0.8		nd	83.4 ± 1.4a	68.7 ± 1.7a
Luteolin	91.2 ± 1.1	78.2 ± 1.4	49.4 ± 0.5		84.2 ± 0.9a	65.1 ± 0.7a	25.5 ± 0.5a

• nd, not detected.
• ^a
  p< 0.001 comparison between both cell lines in the same time point.

Conclusions

Our studies showed that crude extracts from leaves of P. alata showed the most potent and statistically significant viability reduction activity against human acute lymphoblastic leukemia CCRF-CEM. However, the crude extract of P. incarnata (synonym of P. edulis) showed only poor activity and crude extract of P. caerulea leaves did not exert any effect. Our results of phytochemical analysis, similarly as Farag et al. (2016), suggest that P. caerulea may be taken into account as a substitute of the P. incarnata, although our pharmacological studies indicated that activity of P. caerulea extract differs from the extract P. incarnata. Thus, despite similarities in quality phytochemical profile, quantity differences in chemical compounds between two extracts may determine their pharmacological (antileukemic) potency. The highest activity of P. alata extract can be related to the highest content of phenolic compounds, terpenoids, and also apigenin and luteolin. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.

• Appendix A. Supplementary data
  Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjp.2018.01.006.

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Publication Dates

• Publication in this collection
  Mar-Apr 2018

History

• Received
  19 Oct 2017
• Accepted
  23 Jan 2018
• Published
  10 Mar 2018