Chemical profiling and in vitro biological effects of Cardiospermum halicacabum L. (Sapindaceae) aerial parts and seeds for applications in neurodegenerative disorders

Figures & data

Table 1. Extractive yield (%) of C. halicacabum samples.

C. halicacabum	Sample	Plant materials (g)	Total extract (%)	Methanol fraction (%)	n-Hexane fraction (%)
Aerial parts	PN	227.0	10.9	8.7	2.1
	PS	78.0	23.1	20.8	2.4
Seeds	SN	103.0	6.3	3.5	2.8
	SS	50.0	28.6	8.8	19.8

PN: aerial parts – Naviglio extractor; PS: aerial parts – Soxhlet apparatus; SN: seeds – Naviglio extractor; SS: seeds – Soxhlet apparatus.

Table 2. Tentative identification of phenolic and flavonoid compounds in aerial parts and seeds of C. halicacabum by HPLC analysis.

		Flavonoids
C. halicacabum	Phenols*	Total content†	Api‡	Api-7-Glc‡	Lut‡	Rut‡	Flavonoids/Phenols
Aerial parts
PN	154.3	9.7	0.13	0.01	0.08	–	0.06
PS	192.7	23.4	0.94	0.08	0.71	0.10	0.05
Seeds
SN	2.1	0.6	–	–	–	–	0.29
SS	9.7	2.9	–	–	–	–	0.30

PN: aerial parts – Naviglio extractor; PS: aerial parts – Soxhlet apparatus; SN: seeds – Naviglio extractor; SS: seeds – Soxhlet apparatus.

*mg equivalents of chlorogenic acid/g plant materials.

†mg equivalents of quercetin/g plant materials.

‡Quantification by HPLC analysis (mg/g plant materials). Api: apigenin; Api-7-Glc: apigenin-7-O-glucoside; Rut: rutin; Lut: luteolin.

Figure 1. GC-MS chromatogram of the n-hexane fractions of the seeds of C. halicacabum obtained by (a) Naviglio extractor and (b) Soxhlet apparatus.

Table 3. Main non-polar constituents tentatively identified by GC-MS in aerial parts and seeds of C. halicacabum.

		Abundance %
Compound	R^t*	PN	PS	SN	SS	Identification method†
2-Heptenal	8.80		tr	0.3	0.5	1,2
2,4-Heptadienal	9.68	0.4	1.1	0.1	0.1	1,2
1-Octanol	10.02			0.2		1,2
Nonanal	10.71			0.8	0.5	1,2
Caprylic acid methyl ester	10.84			tr		1,2
2-Nonenal	11.71			0.6		1,2
2-Decenal	12.9			1.4	1.1	1,2
Capric acid methyl ester	13.21			0.2		1,2
2,4-Decadienal	13.71			0.6	0.8	1,2
2-Undecenal	13.94			0.4	0.5	1,2
Pentadecane	14.22	0.6	0.5	tr	0.1	1,2,3
Lauric acid	15.66			0.2		1,2
1-Octadecanol	16.10				0.3	1,2
Neophytadiene	17.45	6.4	7.2	tr	tr	1,2
1-Octadecene	17.72	0.8	1.0	1.2	2.7	1,2
Phytol	17.8	5.1	3.8	0.2	0.2	1,2
9,12,15-Octadecatrienal	17.91			0.3	0.5	1,2
6,10,14-Trimethyl-2-pentadecanone	17.96	1.0	1.5			1,2
Methyl palmitate	18.62	tr	1.8	0.3	0.9	1,2,3
Palmitic acid	19.10	tr	3.5	0.6	0.2	1,2,3
1-Nonadecene	19.30			tr	0.2	1,2,3
Phytol acetate	20.01	2.6	2.9	tr	0.1	1,2
Methyl oleate	20.11			2.9	2.7	1,2
Methyl stearate	20.14	0.6	0.6	0.4	0.5	1,2
Methyl linoleate	20.19	0.9	0.7	1.0	0.9	1,2
Methyl linolenate	20.33	0.5	0.9	0.5	0.5	1,2
Oleic acid	20.60			3.1	4.2	1,2
Linoleic acid	20.67	0.9	0.6	1.6	1.7	1,2,3
Methyl 11-eicosenoate	21.53			13.4	14.7	1,2
Methyl 11,13-eicosadienoate	21.60			2.4	1.1	1,2
Seselin	21.71		tr			1,2
8-Methoxypsoralen	21.82	1.5	14.0			1,2
Osthol	22.02	0.6	10.8			1,2
Eicosanoic acid	22.11			39.6	41.7	1,2
Methyl erucate	22.78			2.6	2.0	1,2
Isopimpinellin	23.18		tr			1,2
Heptacosane	23.33	3.5	1.4	1.2	0.4	1,2,3
Erucic acid	23.80			3.5	0.6	1,2
Marmesin	23.85		tr			1,2
Nonacosane	24.94	2.2	0.8	0.6	0.4	1,2,3
Stigmasterol	37.20	5.5	5.4			1,2,3
β-Sitosterol	39.36	7.8	8.3			1,2,3
β-Amyrin	42.79	19.3	14.8			1,2
Hop-22(29)en-3β-ol	45.81	2.3	2.7			1,2

*Retention time (minutes). PN: aerial parts – Naviglio extractor; PS: aerial parts – Soxhlet apparatus; SN: seeds – Naviglio extractor; SS: seeds – Soxhlet apparatus.

†1 – comparison of retention times; 2 – comparison of mass spectra with MS libraries, 3 – comparison with authentic compounds; tr: concentration less than 0.05%.

Table 4. Antioxidant activity of total extracts and fractions of C. halicacabum.

					β-Carotene bleaching test (IC^50 µg/mL)
C. halicacabum	Sample	DPPH test (IC^50 µg/mL)	ABTS test (IC^50 µg/mL)	FRAP test* (µM Fe(II)/g)	30 min	60 min
Aerial parts	PN
	Total	242.2 ± 4.2†	157.8 ± 1.8†	10.1 ± 0.5†	22.7 ± 1.1†	24.1 ± 0.9†
	n-Hexane	65.1 ± 1.1†	662.0 ± 1.9†	6.8 ± 0.1†	23.1 ± 0.4†	33.4 ± 1.1†
	Methanol	34.2 ± 0.5†	90.1 ± 1.3†	12.8 ± 0.7†	8.1 ± 0.3†	8.1 ± 0.9†
	PS
	Total	53.6 ± 1.3†	68.2 ± 0.9†	9.6 ± 0.3†	6.9 ± 0.8†	6.5 ± 0.6†
	n-Hexane	121.2 ± 2.4†	716.5 ± 3.2†	7.0 ± 0.1†	27.2 ± 1.0†	42.2 ± 1.0†
	Methanol	33.1 ± 0.9†	40.2 ± 1.8†	7.7 ± 0.3†	24.7 ± 0.9†	35.5 ± 0.6†
Seeds	SN
	Total	279.1 ± 1.8†	171.3 ± 2.9†	5.8 ± 0.2†	10.8 ± 0.2†	10.3 ± 0.5†
	n-Hexane	118.4 ± 2.5†	692.3 ± 2.6†	2.9 ± 0.5†	36.4%*	25.0%*
	Methanol	40.6 ± 0.7†	128.1 ± 1.8†	4.9 ± 0.2†	7.5 ± 0.8†	8.1 ± 1.1†
	SS
	Total	87.3 ± 3.2†	252.0 ± 2.4†	4.2 ± 0.4†	50.5 ± 1.4†	67.2 ± 1.8†
	n-Hexane	84.0 ± 1.5†	340.2 ± 1.8†	0.4 ± 0.01†	79.8 ± 1.8†	79.8 ± 1.3†
	Methanol	34.1 ± 0.3†	169.1 ± 2.2†	6.2 ± 0.3†	49.7 ± 1.5†	27.7 ± 0.9†
Ascorbic acid		5.0 ± 0.8	1.0 ± 0.03	–
BHT		–	–	63.2 ± 4.5
Propyl gallate					1.0 ± 0.04	1.0 ± 0.03

Data are expressed as media ± SD (n = 3). Positive control: ascorbic acid, BHT, and propyl gallate.

*Samples tested at the concentration of 2.5 mg/mL. PN: aerial parts – Naviglio extractor; PS: aerial parts – Soxhlet apparatus; SN: seeds – Naviglio extractor; SS: seeds – Soxhlet apparatus. DPPH test: One-way ANOVA ***p < 0.0001 (F = 54130, R²= 0.999) Dunnet’s Multiple Comparison Test †p < 0.001. ABTS test: One-way ANOVA ***p < 0.0001 (F = 4949, R²= 1.0) Dunnet’s Multiple Comparison Test †p < 0.001. FRAP test: One-way ANOVA ***p < 0.0001 (F = 1236, R²= 0.998) Dunnet’s Multiple Comparison Test †p < 0.001. β-Carotene bleaching test after 30 min of incubation: One-way ANOVA ***p < 0.0001 (F = 2727, R²= 0.999) Dunnet’s Multiple Comparison Test †p < 0.001. β-Carotene bleaching test after 60 min of incubation: One-way ANOVA ***p < 0.0001 (F = 2020, R²= 0.999) Dunnet’s Multiple Comparison †p < 0.001.

Table 5. Cholinesterases and tyrosinase inhibitory activity of (IC₅₀ µg/mL) of total extracts and fractions of C. halicacabum.

C. halicacabum	AChE	BChE	Tyrosinase
PN
Total	38.7%*	220.4 ± 2.4†	10.8 ± 0.5‡
Methanol	419.5 ± 2.9†	875.8 ± 3.6†	64.1 ± 1.8†
n-Hexane	410.3 ± 3.2†	59.8 ± 1.2†	294.4 ± 3.9†
PS
Total	37.6%*	861.2 ± 2.7†	47.0 ± 1.9†
Methanol	339.2 ± 2.5†	132.9 ± 1.3†	100.0 ± 1.5†
n-Hexane	231.2 ± 1.9†	117.9 ± 1.8†	394.1 ± 4.2†
SN
Total	905.7 ± 3.7†	150.4 ± 1.4†	87.2 ± 1.3†
Methanol	40.5%*	636.3 ± 2.9†	23.9 ± 0.7†
n-Hexane	826.5 ± 4.1†	57.9 ± 1.1†	20.9 ± 0.6†
SS
Total	35.3%*	112.4 ± 1.5†	78.4 ± 2.2†
Methanol	889.8 ± 2.9†	118.0 ± 2.8†	23.5 ± 1.1†
n-Hexane	503.1 ± 2.9^†	73.2 ± 0.9†	21.0 ± 0.4†
Physostigmine	0.2 ± 0.02	2.4 ± 0.04
Kojic acid			10.8 ± 0.9

Data are expressed as media ± SD (n = 3). PN: aerial parts – Naviglio extractor; PS: aerial parts – Soxhlet apparatus; SN: seeds – Naviglio extractor; SS: seeds – Soxhlet apparatus.

*Samples tested at the concentration of 0.9 mg/mL. Physostigmine was used as positive control in AChE and BChE assay while kojic acid was used in Tyrosinase assay. AChE test: One-way ANOVA ***p < 0.0001 (F = 63740, R²= 1.0) Dunnet’s Multiple Comparison Test †p < 0.001. BChE test: One-way ANOVA ***p < 0.0001 (F = 155400, R²= 1.0) Dunnet’s Multiple Comparison Test †p < 0.001. Tyrosinase test: One-way ANOVA ***p < 0.0001 (F = 12580, R²= 0.999) Dunnet’s Multiple Comparison Test †p < 0.001, ‡p > 0.05.