Triterpenes isolated from Cleome amblyocarpa: evaluation of antioxidant potential

Abstract

Investigation of the chemical constituents of Cleome amblyocarpa growing wild in Jordan resulted in the isolation and structural elucidation of two new dammarane triterpenes along with 9 other known compounds. The new compounds were identified as 17-hydroxyambylone (1) and 15β-acetoxy-17α-hydroxycabralealactone (2). The known compounds included calycopterine (3), 17α-hydroxycabraleahydroxylactone (4), cleocarpanol (5), ambylone (6), β-sitosterol (7), β-sitosteryl glucoside (8), isorhamnetin-3-O-rhamnoside (9), kaempferol-3,7-O-dirhamnose (10) and 3′-O-methylquercetin-3,7-di-O-rhamnopyranoside (11). Structural elucidation of all isolated compounds was based on thorough investigation of their spectroscopic data including NMR (1D and 2D), HRESIMS, IR, and UV-Vis spectroscopy. LC-MS/MS analysis versus a selected set of authentic samples led to the detection of 21 constituents, including 9 of the isolated compounds. Antioxidant activities of isolated pure compounds were assessed individually and compared to ascorbic acid and α-tocopherol using DPPH^˙ and ABTS^˙+ assay methods. Compounds 9–11 showed strong antioxidant activities whereas compounds 4–6 exhibited low activity. New compounds 1 and 2 demonstrated moderate DPPH^˙ radical scavenging power (IC₅₀ = 14.70 ± 2.52 and 77.90 ± 3.77 μg mL⁻¹, respectively) and a moderate ABTS^˙+ scavenging effect (IC₅₀ = 22.6 ± 0.04 and 78.30 ± 1.13 μg mL⁻¹).

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1. Introduction

The Cleomaceae family, recently separated by Thomson and Cooke from the Capparidaceae family, comprises nine genera.¹ Among them, Cleome is the largest, with more than 200 species of medicinal and ecological importance. In Jordan, three species grow wild including Cleome amblyocarpa Barratte & Murb., Cleome arabica L. and Cleome droserifolia (Forssk.) Del.¹

Cleome species are used globally in folk medicine for the treatment of many ailments like abdominal pain, rheumatism, scabies and diabetes. They are also reported to exert sudorific, irritant and sedative effects,^2,3 and several species are well recognized for their anti-inflammatory, antiseptic, antimicrobial, carminative, anthelmintic, analgesic, antioxidant, and cytotoxicity activities.^4–6 Phytochemical investigation of the selected Cleome species revealed the presence of diverse secondary metabolites, including terpenoids, phenols, alkaloids, flavonoids, anthraquinones, and glucosinolates.^7–13

Cleome amblyocarpa Barratte & Murb. is a glandular-pubescent annual herb that grows up to 80 cm in height. It has a hairy coat that traps a layer of dust and sand grains on its erect multi-branched stem. The leaflets are elliptical, with three-foliolate lower leaves and simple upper leaves. The flowers have a light yellowish color with red-brownish tips. The fruits are flat and pendulous at maturity, with wholly tomentose seeds.¹ This species grows wild in North and Northeastern tropical Africa as well as in West and Southwest Asia. It thrives in deserts and dry shrublands and blooms once or twice a year.¹⁴

In traditional herbal medicine, the leaves of C. amblyocarpa are used for the treatment of gastric and rheumatic diseases.¹⁵ In Tunisia, the plant is prescribed for the treatment of headaches, nausea, vomiting, and stomach pain,¹⁶ while in Egypt, its aerial parts are valued for their antibacterial and anti-inflammatory effects.¹⁷ Previous phytochemical studies on C. amblyocarpa have reported the presence of flavonoids, dammarane-type triterpenes, cembrane derivatives, stigma-4-en-3-one, lupeol, and taraxasterol.^9,18,19

Following our thorough investigations of medicinal plants in Jordan and Mediterranean neighboring countries,^6,20–22 herein we report the isolation and characterization of two new dammarane-type triterpenes along with other nine known compounds from the aerial parts of C. amblyocarpa (Fig. 1). Moreover, LC-MS/MS analysis of the plant extracts versus selected authentic samples resulted in the detection of 21 compounds, including ten of the isolated compounds (1–7, 9–11). The crude extracts (the butanol (CB) and the aqueous methanol (CM)) and the pure isolated compounds (1–6, 9–11) were screened for their in vitro antioxidant activity using DPPH^˙ and ABTS^˙+ scavenging assay methods.

Fig. 1 Structures of compounds isolated from C. amblyocarpa growing wild in Jordan.

2. Result and discussion

The whole air-dried and defatted plant material was subjected to extraction with ethanol. After complete evaporation of the extracting solvent, the obtained dried residue was fractionated in solvents of different polarities according to the procedure described in the literature to obtain the aqueous methanol (CM) and the butanol (CB) fractions.^20,21

2.1. The isolated compounds

Chromatographic separation of CM and BM fractions resulted in the isolation and structural elucidation of two new dammarane triterpenes along with nine other known compounds. The two new compounds were identified as 17-hydroxyambylone (1) and 15β-acetoxy-17α-hydroxycabralealactone (2) (Fig. 1). The other compounds were calycopterine (3),^23,24 17α-hydroxycabraleahydroxylactone (4),^25,26 cleocarpanol (5),^18,25,27 ambylone (6),^19,28 β-sitosterol (7),²⁹ β-sitosteryl glucoside (8),³⁰ isorhamnetin-3-O-rhamnoside (9),³⁰ kaempferol-3,7-O-dirhamnose (10)³⁰ and 3,7-dirhamnose-isorhamnetin (11).³⁰ Structural elucidation of all isolated compounds was achieved based on extensive investigation of their IR, HRESIMS, UV-Vis, 1D and 2D NMR (including COSY, HMQC, and HMBC) spectral data.

Compound 1 was isolated as a white amorphous solid. Inspection of the IR spectrum of compound 1 indicated the presence of hydroxyl (3398 cm⁻¹), lactone (1753 cm⁻¹) and ether (1254 cm⁻¹) groups (Fig. S1). The molecular ion peak observed at m/z 445.2927 in the HREIMS of compound 1 allowed the prediction of the molecular formula C₂₇H₄₂O₅ ([M–H]⁻, calcd for [C₂₇H₄₁O₅]^–: 445.2954). Initial analysis of the ¹H, ¹³C, DEPT, HMQC, COSY and HMBC spectra of compound 1 revealed signals for 27 nonequivalent carbons including 5 methyls, 10 methylenes, one oxygenated methylene (δ_C-19 68.0), 3 methines, 8 quaternary carbons three of which are oxygenated (δ_C-3 98.2, δ_C-17 83.4 and δ_C-20 93.0), in addition to one carbonyl carbon of a lactone moiety (δ_C=O 176.6) (Fig. S3). The ¹H-NMR and HMQC spectra displayed signals for 5 methyl groups resonating at δ_H 0.88 (Me-30), 0.98 (Me-18), 1.03 (Me-29), 1.13 (Me-28) and 1.43 (Me-21); double doublet signals for the oxygenated C-19 (δ_C 68.0) methylene protons at δ_H 4.25 (1H, dd, J = 8.8, 2.7 Hz, H-19a) and 3.75 (1H, dd, J = 8.8, 1.6 Hz, H-19b) in addition to overlapping peaks in the range δ_H 1.42–2.22 assigned to the aliphatic protons of compound 1 (Fig. S2).

The combination of all these spectral data suggested that compound 1 is a damarane triterpene that is very similar to the structure of the damarane triterpenoid ambylone (compound 6), differing only in the detection of an extra hydroxyl group in compound 1.^19,28 The location of this hydroxyl group was suggested to C-17 of the skeleton based on the disappearance of the methine signal δ_H-17 1.24 observed in compound 6. This assignment was further supported by the down field shift observed for C-17 in compound 1 (δ_C 83.4) when compared to C-17 in compound 6 (δ_C 49.1). Careful inspection of the different 2D NMR spectra including COSY, HMQC and HMBC (Fig. 2 and S4–S6) allowed the correct assignment of all oxygenated carbons in compound 1 (δ_C-3 98.2, δ_C-17 83.4 and δ_C-20 93.0). The HMBC spectrum of compound 1 revealed long range correlations between H-19 protons (δ_H 3.75 & 4.25) and each of C-10 (δ_C 32.6), C-5 (δ_C 49.5) and C-1 (δ_C 35.5), thus confirming oxygenation of C-19 and the substitution pattern of ring A. The location of the extra hydroxyl group at C-17 was evidenced from the long-range correlations observed between H-21 protons (δ_H 1.43) and each of C-20 (δ_C 93.0) and C-17 (δ_C 83.4) in addition to the correlation between H-16 (δ_H 1.52) and C-17. These correlations allowed identification of compound 1 as 17-hydroxyambylone.

Fig. 2 HMBC and ¹H–¹H COSY correlation of compounds 1 and 2.

Compound 2 was obtained from subFr. CMIII-3-sub-3 as pure white amorphous solid. The molecular formula C₂₉H₄₄O₆ of 2 was characterized based on its HRESIMS, which revealed a molecular ion peak at m/z 487.3042 [M–H]⁻ (calcd for [C₂₉H₄₃O₆]^–: 487.3060). The IR absorption bands at 3494, 1767, 1740, 1648 and 1295 cm⁻¹ indicated the presence of hydroxyl, lactone, ketone, ester, and ether groups, respectively (Fig. S7).

The 1D spectra (Fig. S8–S11) and 2D (Fig. 2 and S12–S14) spectra (CDCl₃) of compound 2 closely resembled those of the known dammarane triterpene 17α-hydroxycabralealactone (Takhi et al., 2011), except for the presence of an extra acetyl group. The ¹H-NMR spectrum (Fig. 2 and S8) revealed 6 methyl singlets assigned to Me-21 (δ_H 1.47), Me-28 (δ_H 1.17), Me-19 (δ_H 1.09), Me-29 (δ_H 1.04), Me-18 (δ_H 1.01) and Me-30 (0.97). An additional methyl singlet signal resonating at 2.14 was attributed to the CH₃CO protons. The overlapping peaks detected upfield the spectrum in the range δ_H 1.29–2.69 were typical of the different aliphatic protons of the suggested skeleton.⁴ However, the proton spectrum revealed a multiplet signal integrating for one proton and resonating at δ_H 3.81 (1H, m) that was assigned to H-15, where the acetyl group location was suggested.

The spectral data observed in the ¹³C-NMR and DEPT (CDCl₃) experiments of 2 (Fig. S9–S11) were in total agreement with the suggested 17α-hydroxycabralealactone skeleton, with the extra acetyl groupe positioned at C-15. These spectra revealed signals for 29 carbon including 7 Me's, 9 CH₂'s, 3 CH's, one CH–O moiety (δ_C 68.3, assigned for C-15), 3 quaternary carbons in addition to other 3(C=O) groups comprising one ketone (δ_C 218.2), one lactone (δ_C 178.4) and one acetyl (δ_C 176.7) carbons. However, the location of the acetyl group at C-15 was confirmed by detailed analysis of the 2D-NMR spectra (Fig. 2 and S12–S14). The strong ¹H–¹H correlation observed in the COSY spectrum between H-15 (δ_H 3.81) and H-16 (δ_H 2.34) in addition to the long-range correlations observed in the HMBC spectrum between H-15 (δ_H 3.81) and each of C-16 (δ_C 32.5), C-14 (δ_C 49.9) and C-1′ (δ_C 176.7) confirmed the attachment of the acetyl group to C-15. Other important correlations observed between H-16 (δ_H 2.34) and C-17 (δ_C 83.9), H-21 (δ_H 1.47) with each of C-20 (δ_C 92.9) and C-17 (δ_C 83.9) in addition to the correlation observed between H-2′ (δ_H 2.14) and C-1′ (δ_C 176.7) helped in confirming the oxygenation pattern of rings D and E of the proposed structure. Based on these data, compound 2 was identified as 15β-acetoxy-17α-hydroxareabralealactone. Table 1 lists the ¹H and ¹³C-NMR (CDCl₃) chemical shifts for compounds 1, 2 and 6 (known compound ambylone) while Fig. 2 shows the important COSY and HMBC correlations in both new compounds 1 and 2.

Table 1 ¹H NMR (400 MHz) and ¹³C (100 MHz) spectroscopic data (CDCl₃, δ in ppm, J in Hz) for compounds 1, 2 and the known compound 6 (ambylone)

#	1:  17-hydroxyambylone		2: 15β-acetoxy-17α-hydroxareabralealactone		6: ambylone
	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC
1	1.25 (1H, m)	35.5	1.18 (1H, m)	39.9	1.19 (1H, m)	33.3
	1.51 (1H, m)		1.51 (1H, m)		1.50 (1H, m)
2	1.48 (1H, m)	29.5	1.40 (1H, m)	34.0	1.47 (1H, m)	29.2
	2.67 (1H, m)		2.33 (1H, m)		2.68 (1H, m)
3	—	98.2	—	218.2	—	98.0
4	—	40.5	—	47.4	—	40.5
5	1.90 (1H, m)	49.5	1.95 (1H, m)	47.4	1.85 (1H, m)	49.7
6	1.37 (1H, m)	19.8	1.36 (1H, m)	19.6	1.36 (1H, m)	19.7
	2.46 (1H, m)		2.05 (1H, m)		2.56 (1H, m)
7	1.15 (1H, m)	29.7	1.15 (1H, m)	33.9	1.16 (1H, m)	29.5
	1.29 (1H, m)		1.33 (1H, m)		1.24 (1H, m)
8	—	39.8	—	40.8	—	39.2
9	1.51 (1H, m)	46.2	1.42 (1H, m)	50.0	1.48 (1H, m)	45.1
10	—	32.6	—	36.8	—	35.6
11	1.21 (1H, m)	22.6	1.21 (1H, m)	21.9	1.21 (1H, m)	22.6
	1.95 (1H, m)		1.60 (1H, m)		1.60 (1H, m)
12	1.50 (1H, m)	29.3	1.50 (1H, m)	22.8	1.50 (1H, m)	27.0
	1.95 (1H, m)		1.92 (1H, m)		1.92 (1H, m)
13	1.58 (1H, m)	45.1	1.55 (1H, m)	46.1	1.55 (1H, m)	43.3
14	—	49.9	—	49.9	—	50.0
15	1.90 (1H, m)	32.5	3.81 (1H, m)	68.3	1.95 (1H, m)	31.2
	1.14 (1H, m)				1.18 (1H, m)
16	1.54 (1H, m)	23.0	1.78 (1H, m)	32.5	1.78 (1H, m)	25.3
	1.80 (1H, m)		1.97 (1H, m)		1.97 (1H, m)
17	—	83.4	—	83.9	1.24 (1H, m)	49.1
18	0.98 (3H, s)	15.3	1.01 (3H, s)	16.1	0.91 (3H, s)	15.2
19	4.25 (1Ha, dd, 8.8, 2.7); 3.75 (1Hb, dd, 8.8, 1.6)	68.0	1.09 (3H, s)	15.4	4.27 (1Ha, dd, 8.8, 2.7); 3.76 (1Hb, dd, 8.8, 1.6)	68.0
20	—	93.0		92.9		90.0
21	1.43 (3H, s)	22.9	1.47 (3H, s)	22.9	1.38 (3H, s)	22.5
22	1.48 (1H, m)	35.5	1.48 (1H, m)	29.2	1.48 (1H, m)	35.6
	2.53 (1H, m)		2.00 (1H, m)		2.57 (1H, m)
23	2.56 (1H, m)	26.8	2.53 (1H, m)	29.6	2.52 (1H, m)	25.4
	2.65 (1H, m)		2.62 (1H, m)		2.63(1H, m)
24	—	176.7	—	178.4	—	176.4
25	—	—	—	—	—	—
26	—	—	—	—	—	—
27	—	—	—	—	—	—
28	1.13 (3H, s)	29.1	1.17 (3H, s)	26.7	1.05 (3H, s)	26.8
29	1.03 (3H, s)	18.5	1.04 (3H, s)	21.0	1.00 (3H, s)	18.4
30	0.88 (3H, s)	16.5	0.97 (3H, s)	16.9	0.88 (3H, s)	16.9
1′	—	—	—	176.7	—	—
2′	—	—	2.14 (3H, s)	23.4	—	—

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The ECD spectra of compounds 1 and 2 were recorded with a Jasco J-720 spectropolarimeter in chloroform (Fig. S15). The ECD values for 1 and 2 showed positive (300 nm) cotton effects.

Further qualitative analysis of the CB and CM fractions on LC-MS/MS revealed the presence of 21 compounds including 10 of the isolated compounds. Identification of these constituents was based on comparing their mass spectra with our built-in library that includes 59 authentic secondary metabolites isolated and identified in our laboratories. Table 2 lists the compounds identified in both fractions.

Table 2 The LC- MS/MS analysis data of detected metabolites from the CB and CM extracts of C. amblyocarpa from Jordan^a

No.	RT [min]	m/z meas^*	Mwt calcd	Name	Class^#	Molecular formula^a	CB^*	CM^*
a All are reported as [M–H]^−; *CB: butanol fraction, CM: aqueous methanol fraction, ^#Class of secondary metabolite detected: PA: phenolic acid; Fl: flavonoid; DT: dammarane triterpene, St: sterol.
1	2.25	153.0228	154.0301	2,5-Dihydroxybenzoic acid	PA	C7H6O4	+	—
2	2.63	137.0247	138.0317	4-Hydroxybenzoic acid	PA	C7H6O3	+	+
3	4.94	609.1436	610.1509	Luteolin-7,3′-di-O-glucoside	Fl	C27H30O16	+	—
4	5.28	609.1462	610.1535	3-Glu-7-RhaQuercetin	Fl	C27H30O16	+	—
5	5.55	593.1487	594.1585	3-Rha-7-RhaQuercetin	Fl	C27H30O15	+	—
6	5.78	563.1416	564.1488	Apiin	Fl	C26H28O14	+	—
7	5.81	463.0884	464.0957	Hyperoside	Fl	C21H20O12	+	+
8	5.90	413.1452	414.1525	β-Sitosterol (7)	St	C29H50O	+	+
9	6.14	593.1513	594.1585	3-O-Neohesperidoside kaempferol	Fl	C27H30O15	+	+
10	6.33	577.154	578.1612	Kaempferol-3,7-O-dirhamnose (10)	Fl	C27H30O14	+	+
11	6.51	607.1644	608.1731	3′-O-methylquercetin-3,7-di-O-rhamnopyranoside (11)	Fl	C28H32O15	+	+
12	7.53	285.0401	286.0474	3,6,3′,4′-Tetrahydroxyflavone	Fl	C15H10O6	+	—
13	7.74	461.1064	462.1137	Isorhamnetin-3-O-rhamnoside (9)	Fl	C22H22O11	+	+
14	9.97	299.0539	300.0612	Hispidulin	Fl	C16H12O6	+	+
15	10.80	299.0539	300.0612	Ladanetin	Fl	C16H12O6	+	+
16	14.02	373.0903	374.0976	Calycopterine (3)	DT	C19H18O8	+	+
17	16.02	445.2897	446.2969	17-Hydroxyambylone (1)	DT	C27H42O5	—	+
18	18.43	473.2840	474.2914	Cleocarpanol (5)	DT	C30H50O4	—	+
19	20.26	487.2993	488.3066	15β-acetoxy-17α-hydroxycabralealactone (2)	DT	C29H44O6	—	+
20	22.01	429.2944	430.3017	Ambylone (6)	DT	C27H42O4	—	+
21	23.28	431.3171	432.3173	17α-hydroxycabraleahydroxylactone (4)	DT	C27H44O4	—	+

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2.2. Determination of total phenol (TPC) and total flavonoid (TFC) contents

Both, the (CM) and (CB) fractions were assayed for their TPC and TFC according to the procedure listed in the literature.²² The TPC was determined based on the regression equation Y = 7.9501x + 0.0389, R² = 0.9993, established with gallic acid, where Y is the absorbance at 765 nm and x refers to the TPC of the tested fraction. Similarly, the TFC was determined using AlCl₃ method described in the literature,^6,20 and quantitative estimation was based on the linear regression equation Y = 0.6416x + 0.0007, R² = 0.999, established with quercetin, where Y is the absorbance at 510 nm and x is the TFC in the tested fraction. As revealed in the results shown in Table 3, CB fraction is richer in both phenolic compounds (103.53 ± 0.50 GAE) and flavonoids (448.82 ± 6.30 QE) when compared to the CM fraction. These findings supported the antioxidant activity results, in which CB fraction showed higher DPPH^˙ and ABTS^˙+ scavenging potentials when compared to the CM fraction.

Table 3 Results for TPC (mg g⁻¹ GAE) and TFC (mg g⁻¹ QE) of the CB and CM fractions. Data are expressed as the mean ± SD of three independent experiments

Fraction/compound	TPC	TFC
CB	103.53 ± 0.50	448.82 ± 6.30
CM	64.00 ± 1.47	120.53 ± 0.00

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2.3 Antioxidant activity

The two crude fractions (CB and CM) and their isolated pure compounds (1–6, 9–11) were assayed for their antioxidant activities, using the DPPH^˙ and ABTS^˙+ methods, results are shown in Table 4 (Fig. S59 and S60). Compounds 9, 10, and 11 exhibited strong antioxidant activities, with compound 11 being significantly more potent than compounds 9 and 10. This enhanced activity may be attributed to the greater number of hydroxyl groups in compound 11 compared to the other two compounds (9 and 10). The new compounds 1 and 2 showed moderate DPPH^˙ radical scavenging power (IC₅₀: 14.70 ± 2.52 and 77.90 ± 3.77 μg mL⁻¹, respectively), with their activities being markedly lower than those observed for the positive controls; ascorbic acid and α-tocopherol (IC₅₀: 1.58 ± 0.035 and 1.79 ± 0.01 μg mL⁻¹, respectively). Compounds 1 and 2 also exhibited moderate ABTS^˙+ scavenging effect (IC₅₀: 22.6 ± 0.04 and 78.30 ± 1.13 μg mL⁻¹, respectively), which was also lower than those observed for the tested reference compounds, ascorbic acid and α-tocopherol (IC₅₀: 1.78 ± 0.06 and 2.33 ± 0.01 μg mL⁻¹, respectively).

Table 4 Results for the DPPH^˙ and ABTS^˙+ antioxidant IC₅₀ (μg mL⁻¹) values for extracts CB, CM, pure isolated constituents (1–6, 9–11) and reference compounds. Data are expressed as the mean ± SD of three independent experiments

Fraction/Compound	IC50 (μg mL^−1)
	DPPH^˙	ABTS^˙+
CB	16.00 ± 0.50	7.50 ± 2.50
CM	18.00 ± 0.05	11.00 ± 0.50
1	14.70 ± 2.52	22.6 ± 0.04
2	77.90 ± 3.77	78.30 ± 1.13
3	7.10 ± 0.37	7.09 ± 0.70
4	168.00 ± 1.97	n.d
5	271.00 ± 6.14	193.00 ± 1.80
6	n.d	n.d
9	10.50 ± 1.24	7.97 ± 0.58
10	8.32 ± 0.85	2.38 ± 0.11
11	2.55 ± 0.07	1.25 ± 0.04
Ascorbic acid	1.58 ± 0.035	1.78 ± 0.06
α-tocopherol	1.79 ± 0.01	2.33 ± 0.01

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3. Conclusion

Phytochemical investigation of the butanol (CB) and aqueous methanol (CM) fractions obtained from the crude extract of C. amblyocarpa from Jordan resulted in the isolation and identification of two new dammarane triterpenes, namely 17-hydroxyambylone (1) and 15-acetoxy-17-hydroxycabralealactone (2) in addition to other 9 known compounds. LC-MS/MS analysis of the crude extracts versus a selected set of authentic samples led to the detection of 22 constituents. The crude fractions, new compounds 1 and 2, and previously untested known compounds 3–6, 9–11 were all assayed for their DPPH^˙ and ABTS^˙+ scavenging power. Among the tested compounds, isorhamnetin-3-O-rhamnoside (9), kaempferol-3,7-O-dirhamnose (10) and 3′-O-methylquercetin-3,7-di-O-rhamnopyranoside (11) exhibited exceptionally strong antioxidant potentials in both assay methods that were close to the activity observed for the reference controls. 3′-O-methylquercetin-3,7-di-O-rhamnopyranoside (11) was significantly more potent due to the substitution pattern of this flavonol glycoside. Moreover, the new compounds, 1 and 2, exhibited moderate antioxidant potentials in both assay methods, with compound 1 being more active than compound 2. This could mainly be attributed to the presence of the free 3-OH and 17-OH hydroxyl groups, as these groups, although aliphatic, provide accessible hydrogen donor sites. For compound 2, the replacement of the 3-OH group with a carbonyl group in addition to the acetylation at C-15, together eliminate the potential hydrogen donation capacity, increase steric hindrance and hydrophobicity. These structural features reduce radical accessibility, therefore explaining the lower DPPH˙ and ABTS˙⁺ radical scavenging power of compound 2 compared to 1.

Despite that the current results revealed moderate activity of the new compounds, further pharmacological screening of the plant material and the pure constituents may help in revealing their undiscovered therapeutic potentials.

4. Experimental

4.1. General

¹H-NMR spectra were recorded on a Bruker Avance III 400 MHz spectrometer with TMS as an internal standard. ¹³C-NMR spectra were recorded at 100 MHz. High resolution mass spectra (HRESIMS) were acquired by electrospray ionization in the positive mode technique using a Bruker APEX-4 Mass spectrometer. TLC was performed on silica gel 60 GF254 pre-coated glass plates (0.25 or 0.50 mm in thickness, Macherey-Nagel). Compounds were visualized under UV light or spraying with sulfuric acid-anisaldehyde spraying reagent followed by heating. Circular dichroism (CD) spectra were recorded with a Jasco J-720 spectropolarimeter (0.1 nm resolution, 10 accumulations, 1 cm cell) in chloroform. Blank measurement of chloroform provided no ICD effects in the region examined.

LC-MS/MS was conducted on a Bruker Daltonik Impact II ESI-Q-TOF System equipped with a Bruker Daltonik Elute UPLC system (Bremen, Germany), both in the positive [M + H]⁺ and negative [M–H]⁻ electrospray ionization modes. Apollo II ion Funnel electrospray source was used to power this instrument. The capillary capacity was 2500 V, the nebulizer gas pressure was 2.0 bar, the dry gas (nitrogen) flow rate was 8 L min⁻¹, and the dry temperature was 200 °C. The mass resolution was 50 000 FSR (Full Sensitivity Resolution), while the TOF repetition rate was up to 20 kHz. The mass accuracy was <1 ppm.

4.2. Plant material

C. amblyocarpa Barratte & Murb. was collected from Wadi Rum region during the full flowering stage (April-2020). The taxonomic identity of the plant was certified by Prof. Dr Jamil N. Lahham (Department of Biology, Faculty of Science, Yarmouk University, Irbid, Jordan). A reference specimen (CA/C/2020) was stored in Prof. Mahmoud A. Al-Qudah Laboratory, Yarmouk University, Irbid, Jordan.

4.3. Extraction and fractionation

Air dried and powdered plant material (10 kg) was subjected to extraction with petroleum ether to remove fatty acids and waxes (20 L, 10 days, RT). Then, secondary metabolites were extracted from the defatted dried plant reside by maceration in ethanol at room temperature (5 times, 7 days each). Then, the combined and dried ethanolic extract (approximately 500 g) was partitioned as described in the literature^6,20–22 to afford the aqueous methanol (CM; 129.1 g) and butanol (CB; 115.96 g) fractions.

4.4. Determination of TPC, TFC and antioxidant activity

The TPC and TFC of CB and CM fractions were evaluated according to the Folin–Ciocalteu technique and aluminum chloride assay method, respectively, according to the procedure described in the literature.²² Data are expressed as the mean ± SD of three independent experiments.

The antioxidant activity of both fractions and isolated compounds (1–6, 9–11) was assessed using the DPPH^˙ and ABTS^˙+ assay methods, as outlined in the literature.^6,22 Data are expressed as the mean ± SD of three independent experiments.

4.5. Isolation and purification of secondary metabolites

Fraction CM (129.1 g) was absorbed on silica gel (150 g, 230–400 mesh) and then subjected to column chromatography in a column filled with the same adsorbent (600 g, 230–400 mesh) and eluted with a gradient combination of n-hexane/EtOAc of increasing polarity. A total of 211 eluent fractions (500 mL each) were collected and then consolidated into five main groups (CMI – CMV) based on their TLC behavior. These groups were then subjected to a combination of CC, TLC or crystallization to separate their constituents. Fr. CMI (2.5 g) was subjected to CC on a column packed with silica gel (50 g, 230–400 mesh) and eluted with a gradient of n-hexane/EtOAc affording four subfractions (SubFr. CMI-1 to SubFr. CMI-4). SubFr. CMI-2 (500 mg) was purified by crystallization in MeOH to yield 3 (70 mg). Subjecting Fr. CMII (10.80 g) to CC (250 g silica gel, 230–400 mesh) eluted with a gradient mixture of n-hexane/EtOAc afforded five subfractions (SubFr. CMII-1 to SubFr. CMII-5). SubFr. CMII-3 (610 mg) was purified by crystallization in MeOH to yield 4 (55 mg). SubFr. CMII-4 (700 mg) was purified on CC (packed with 5 g silica gel, 230–400 mesh) eluted with n-hexane/EtOAc to yield 5 (122.0 mg). Fraction CMIII (41.7 g) was also subjected to CC (200 g silica gel, 70–230 mesh) eluted with n-hexane/EtOAc mixture of increasing polarity (5% stepwise) to produce six subfractions (SubFr. CMIII-1 to SubFr. CMIII-6). SubFr. CMIII-3 (6.5 g) was chromatographed on a silica gel column (150 g, 230–400 mesh) eluted with n-hexane/CH₂Cl₂ gradient mixture and four subfractions were obtained (SubFr. CMIII-3-sub-1 to SubFr. CMIII-3-sub-4). Consequently, subFr. CMIII-3-sub-1 (1.5 g) was purified on CC (75 g silica gel, 230–400 mesh, eluent: gradient of Bz/EtOAc mixture) to obtain pure 6 (25 mg). SubFr. CMIII-3-sub-2 (1.0 g) was purified by CC (50 g silica gel, 230–400 mesh eluent: gradient of Bz/EtOAc mixture) to obtain 1 (20 mg). Similarly, 2 (40 mg) purification was achieved upon subjecting subFr. CMIII-3-sub-3 (1.75 g) to CC (75 g silica gel, 230–400 mesh) using Bz/EtOAc as an eluent. Compound 7 (1.40 g) was recovered from Fr. CMIV (8.0 g) upon crystallization in MeOH. Fraction CMV (34 g) was subjected to CC (250 g silica gel, 70–230 mesh, eluent system: n-hexane/EtOAc) to generate seven subfractions (subFr. CMV-1 to subFr. CMV-7). SubFr. CMV-4 (2.0 g) was separated on CC (150 g silica gel, 230–400 mesh) eluted with n-hexane/EtOAc mixture of increasing polarity, to yield 8 (711 mg).

The butanol fraction was treated in a similar way. Briefly, CB (115.96 g) fraction was absorbed on silica gel (150 g, 70–230 mesh) and then chromatographed on a column packed with the same adsorbent (550 g) and eluted with a gradient mixture of CHCl₃/MeOH of increasing polarity. The elution process afforded 100 fractions (500 mL each) that were consolidated into three major groups according to their TLC behavior (CBI − CBIII). Each group was then subjected to a combination of CC, TLC and recrystallization to separate its pure constituents. Fr. CBI (30.5 g) was separated on a silica gel column (250 g, 70–230 mesh) eluted with a gradient mixture of CHCl₃/MeOH afforded 4 subfractions (subFr. CBI-1 to subFr. CBI-4). Further chromatographic separation of subFr. CBI-2 (6.5 g) on CC (150 g silica gel, 230–400 mesh, eluent system: gradient of Bz/EtOAc) yielded other four subfractions (subFr. CBI-sub-2-1 to subFr. CBI-2-sub-4). Purification of subFr. CBI-2-sub-2 (1.5 g) on CC (75 g silica gel, 230–400 mesh; eluent: EtOAc/MeOH of increasing polarity) afforded 9 (25 mg). SubFr. CBI-2-sub-3 (0.5 g) was purified on a sephadex LH-20 column to obtain 10 (50 mg). Fr. CBIII (3.0 g) was separated on a silica gel column (170 g, 70–230 mesh; eluent system: isocratic elution with EtOAc) affording 11 (28.5 mg).

4.5.1. 17α-hydroxyambylone (1). Compound 1 was obtained from SubFr. CMIII-3-sub-2 as pure white amorphous solid. IR (KBr) ν (cm⁻¹): 3398, 2966, 1753, 1254. R_f = 0.48 (5% MeOH/CHCl₃) and 0.77 (60% EtOAc/Bz). ¹H-NMR and ¹³C-NMR (CDCl₃) δ ppm, see Table 1. HRESIMS m/z 445.2927 [M–H]⁻ (calcd for [C₂₇H₄₁O₅]⁻: 445.2954).

4.5.2. 15β-acetoxy-17α-hydroxycabralealactone (2). Compound 2 was obtained from subFr. CMIII-3-sub-3 as pure white amorphous solid. IR (KBr) ν (cm⁻¹): 3494, 2947, 1767, 1740, 1648, 1455, 1295, R_F = 0.56 (5%MeOH/CF) and 0.45 (30%EtOAc/Bz). ¹H-NMR and ¹³C-NMR (CDCl₃) δ ppm, see Table 1. HRESIMS m/z 487.3042 [M–H]⁻ (calcd for [C₂₉H₄₃O₆]⁻: 487.3060).

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

Data will be made available on request.

Supplementary information is available. See DOI: https://doi.org/10.1039/d5ra04113a.

Acknowledgements

This work was supported and funded by the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University (IMSIU) (grant number IMSIU-DDRSP2501).

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