Fistuloates A–C: New antioxidative aromatic compounds isolated from Cassia fistula

Abstract

Three new aromatic compounds named fistuloates: A (3-butyl-6-ethylnonyl-3-(1-phenylethyl) benzoate), B (3-(6-tert-butyl-1-ethoxy-5-ethyl-4-methylnonyl)-5-ethylbenzoic acid isobutyl ester) and C (4,5-diethoxy-2-propylbenzoic acid 7-(6-pentyltetrahydropyran-2-yl)-heptyl ester) were isolated from the ethyl acetate soluble fraction of Cassia fistula Linn. The structural formulae of the isolated compounds A–C were established through interpretation of their spectral data. Compounds A–C exhibited significant antioxidant properties in 2,2-diphenyl-1-picrylhydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and superoxide anion radical scavenging assays that signifies that C. fistula carries potential anti-oxidant constituents and may assist to cure diseases associated with oxidative stress and others.

Introduction

Cassia is a genus of 600 species of flowering plants (angiosperm) in the family Fabaceae, which is also recognized as Leguminosae. It exists in South American countries, Mexico, South Asia, Sri Lanka and China. ¹ Cassia fistula is 10 to 20 m in height and alternates leaves with five to eight pairs of leaflets, 6–15 cm in length, while the lower surface is waxy white. Flowers have hanging axillary racemes which are 80 cm long. The flowers have five yellow-coloured petals. The fruits are narrow, cylindrical pods 30–60 cm in length. The pods are sticky and pulpy, and the pulp is dark brown in colour, sweet in taste and filled with seeds.

The plant extracts of different organs of C. fistula are important sources of secondary metabolites, mainly phenolic compounds. ² Alkaloids and triterpenes are reported from its flowers, ³ and anthraquinone and dihydroxyanthraquinone are reported from its bark. ⁴ Aromatic compounds, rhein, steroids and wax have also been isolated from this plant in addition to anthraquinone-bearing skeletons, isovanillic acid (an aromatic acid), stigmasterols, flavon-3-ol derivatives, reducing sugars, saponin and 2,4-dihydroxybenzaldehyde. ⁵ From its seeds, octacosan-5,8-diol, (+)-catechin, (–)-epicatechin, rhein, glycerides, palmitic acids and stearic acid have been isolated.

Medicinally, different extracts of Cassia species display antimicrobial properties,^6,7 for example, its betulinic acid constituent has strong antimicrobial potential and anti-HIV infection properties. It also possesses antipyretic, anti-inflammatory, antitumor, analgesic and antioxidant potential.^8–10 Phytotherapeutic studies of the genus Cassia indicated that it has been largely used in folk medicine as an antiseptic, a purgative laxative, a diuretic, and to treat leprosy and different types of ulcers and jaundice. ² This plant also serves in the treatment of diseases like liver disorders, tuberculosis, skin infections and diabetes.^9–11 C. fistula has promising medicinal uses in China for the treatment of fungal diseases, ringworm, diarrhoea and skin infections.^12,13 Similarly, plant parts are effective and are used against hypercholesterolaemia and nasal infections. ¹⁴ The leaf extract of C. fistula is also reported to possess anti-tussive and wound healing properties.^15,16 It has been demonstrated that the C. fistula parts exhibited therapeutic properties against the treatment of hypercholesterolaemia partially due to their fibre and mucilage content. ¹⁷ C. fistula is one of the most commonly used plants in Unani and Ayurvedic medicines, and is suggested to be useful in the treatment of haematemesis, pruritus and leucoderm. ¹⁸

All these facts motivated us to further explore the hidden chemical constituents of the C. fistula plant. Here, we describe the isolation and characterization of three new compounds from C. fistula and the evaluation of their antioxidant potential.

Results and discussion

Extraction and structural characterization

The crude methanol extract was concentrated and the residue extracted using different solvents with increasing polarity: n-hexane, CH₂Cl₂, EtOAc (ethyl acetate) and methanol. The results of repeated column chromatography on EtOAc soluble fractions, and experiments performed for isolation showed the presence of three compounds as colourless solids. Their structural formulae were confirmed by different spectroscopic techniques as, infrared (IR), ¹H and ¹³C NMR (nuclear magnetic resonance) spectroscopy, and mass spectrometry. The structures of the compounds are illustrated in Figure 1.

Figure 1.

Structural formulae of compounds A–C isolated from C. fistula.

Fistuloate A was obtained as a colourless solid (m.p. 153–154ºC) having the optical rotation value of [α]²⁵_D  = +32 (c = 0.12, MeOH). The IR spectrum displayed bands at 1460–1500 cm⁻¹ corresponding to the presence of aromatic unsaturation, and at 1715 to 1730 cm⁻¹ due to an ester functionality. In addition, sp₂ C–H stretching occurred at 3005 cm⁻¹. The high-resolution mass spectrometry (HRMS) (electron ionization (EI)) mass spectrum indicated the respective M⁺ signal at m/z (mass-to-charge ratio) = 436.33, suggesting its molecular formula as C₃₀H₄₄O₂, while its calculated mass was 436.67; this represents 9 degrees of overall unsaturation, among which 8 were assigned to 2 benzene rings and 1 for carbonyl unsaturation. Linked scan measurement of the M⁺ ion of fistuloate A revealed that ions at m/z 359, 331, 227, 255, 183 and 85 were generated directly from it.

The ¹H NMR spectrum recorded for fistuloate A indicated a pair of irregular signals at shift ranges from 7.24–7.74 ppm, suggesting the existence of benzene rings. A triplet for two protons at 4.25 ppm (J = 6.5) was assigned to the oxymethylene protons. The signals observed at 1.28–1.42 ppm corresponded to the methylene protons in long chain and branches of compound A, while the protons of the terminal methyl groups showed peaks at shifts ranges from 0.91–0.93 ppm. Only the protons of the methyl at position 14 generated a doublet peak at 1.70 ppm (J = 6.1). The ¹³C NMR spectroscopic values for compound A suggested the existence of 30 carbon atoms through appearance of 28 peaks and the DEPT NMR determined their multiplicities that corresponded to four CH₃ carbons, four 4° carbon centres, 10 methylenic carbons and 12 ‒CH– carbon centres. Highly deshielded signals that appeared at 167.8, 128.8, 130.9 and 132.5 ppm were correlated with the carbonyl and benzene ring carbons, respectively. The peak observed at 68.2 ppm was attributed to the oxymethylene carbon atom. The branching terminal methyl carbons furnished peaks at 10.9 and 14.1 ppm. Based on the evidence obtained, the structure for fistuloate A was suggested as 3-butyl-6-ethylnonyl 3-(1-phenylethyl) benzoate (Figure 1).

Compound B was accessed as a colourless solid (m.p. 159–160ºC) with the optical rotation of [α] ^{25 _D}  = -44 (c = 0.12, MeOH). The IR spectrum revealed absorptions at 1610 and 1730 cm⁻¹ that providing evidence of the presence of an aromatic ring and an ester functionality, while a strong and intense signal appeared at 1196 cm⁻¹ suggesting an ethoxy functionality in compound B. In addition, C–H presence was confirmed through observation of a peak 2830 cm⁻¹. Moreover, a weak absorption due to the aromatic sp₂ C–Hs was observed at 3011 cm⁻¹. Substituents on the benzene ring were confirmed from the presence of bending vibrations ranging from 860 to 750 cm⁻¹. HRMS (EI) indicated the molecular formula of fistuloate B as, C₃₁H₅₄O₃ (m/z = 474.41) (calculated mass was 474.46). This suggests a total of 5 degrees of unsaturation in compound B, where 4 were attributed to a substituted benzene and 1 for the double bond of the carbonyl moiety. Linked scan measurements for the generated M⁺ ion showed that the ions at m/z 417, 401, 269, 211 and 57 arose directly from it.

The ¹H NMR spectrum obtained for compound B exhibited all relevant signals. The signals in the aromatic region (7.55–7.71 ppm) were assigned to the protons of benzene, while a multiplet at 2.89 ppm corresponded to the methine proton at position 10. A doublet at 4.89 ppm (J = 5.9) was assigned to the methylene protons (C-9) attached to oxygen. The other two oxymethylene protons of position 10’ showed a quartet at 3.91 ppm (J = 5.4). A multiplet range in between 0.89 and 1.28 ppm corresponded to –CH₃ protons. Multiplets appearing at 1.39–1.96 ppm corresponded to the protons of methines in the side chain, while the methine at position 1’ showed a triplet at 4.25 ppm (J = 5.7). The ¹³C NMR spectrum for compound B indicated a total of 28 peaks, and their multiplicities were examined through DEPT NMR, where we observed 5 quaternary carbon atoms, 8 methines, 8 methylenes and 10 CH₃ centres. The peaks appeared at 158.9, 125.9, 126.0 and 132.3 ppm indicated the ester functionality and aromatic carbon atoms, respectively. A peak due to the oxymethylene carbons appeared at 69.8 ppm (C-9) and 63.3 ppm (C-10′). The signal exhibited at 33.8 ppm was attributed to the t-butyl quaternary carbon. Based on these evidence, the structural formula of compound B was suggested shown in Figure 1 and named as 3-(6-tert-Butyl-1-ethoxy-5-ethyl-4-methylnonyl)-5-ethylbenzoic acid isobutyl ester.

Fistuloate C was isolated as a colourless solid (m.p. 167–168ºC). The optical rotation value as determined was [α]²⁵_D = +25 (c = 0.12, MeOH). The HRMS (EI) results revealed the peak at m/z = 504.38 (calculated m/z = 504.74), that suggested the molecular formula for the fistuloate C as C₃₁H₅₂O₅. It suggests 6 degrees of unsaturation, where 4 were assigned to the benzene ring, 1 to the ester carbonylic functionality and another indicated an additional ring in the molecule. Linked scan measurements of the obtained M⁺ ion suggested that the observed fragment ions at m/z 461, 269, 253, 141 and 71 were directly generated from it. IR spectral results for the compound C furnished peaks at 1615 and 1716 cm⁻¹ corresponding to the benzene ring and the ester functionality, while an intense band observed at 1103 cm⁻¹ in the fingerprint region corresponded to the bending vibration of the ether C–O. In addition, the aromatic sp₂ C–H were evident from the presence of an absorption at 3010 cm⁻¹. The presence of methyl and methylene sp₃ C–Hs were apparent at 2896 cm⁻¹.

Considering the ¹H NMR spectrum data for compound C, the signal observed at 7.56 ppm corresponded to aromatic protons, while the triplet signal at 4.25 (J = 6.8) and overlapping quartet at 4.32 ppm (J = 6.7) appeared, due to the presence of oxymethylene protons. In addition, the multiplet at 3.52 ppm evidenced the existence of oxymethine protons. A triplet appeared at 2.13 ppm (J = 6.3) and was attributed to the methylene protons attached at C-7. The protons of the terminal methyls were observed at shift range from 0.91 to 0.97 ppm, while the methylene protons appeared at shifts of 1.13–2.13 ppm. The ¹³C NMR spectrum indicated 31 carbons, and their positions were further confirmed through DEPT NMR that corresponded to 5 quaternary centres, 4 methine carbons, 18 methylene carbons and 4 methyl carbons in compound C. The observed signals at 165.47 ppm corresponded to the carbonyl carbon of ester group, while signals ranging from 111.0 to 149.27 ppm corresponded to those of the benzene ring. The signal at 63.9 ppm was due to oxomethylene carbons. In addition, the terminal CH₃ carbons occurred at 13.0, 14.0 and 14.2 ppm. Based on this evidence, structure of fistuloate C was elucidated as given in Figure 1 and assigned the name 7-(6-pentyltetrahydro-2H-pyran-2-yl)heptyl 4,5-diethoxy-2-propylbenzoate. The NMR spectral data for the compounds A and B are given in Table 1, while Table 2 shows the data for compound C.

Table 1.

¹H and ¹³C NMR data of compounds A and B (CDCl₃, δ in ppm). ^a

A			B
H/C	δ (H) (J in Hz)	δ (C)	H/C	δ (H) (J in Hz)	δ (C)
1	Q	130.9	1	Q	137.8
2 CH	4.26 (q, J = 6.1)	38.7	2 CH	7.71 (s)	126.0
3 CH	Q	135.9	3	Q	129.0
4 CH	7.73 (dd, J = 7.6, 2.7)	132.5	4 (CH)	7.75 (m)	125.9
5 CH	7.31 (dd, J = 7.6, 7.3)	130.7	5	Q	143.0
6 CH	7.74 (d, J = 7.3)	126.9	6 (CH)	7.50 (s)	132.3
7	Q	137.8	7 (CH2)	2.81 (q, J = 6.2)	27.6
8 (CH)	7.56 (s)	128.8	8	Q	158.9
9, 13 (CH)	7.24 (m)	126.0	9 (CH2)	4.89 (d, J = 5.9)	69.8
10, 12 (CH)	7.54 (m)	128.7	10 (CH)	2.89 (m)	26.8
11 (CH)	7.57 (m)	130.8	11, 12 (CH3)	0.89 (d, J = 5.8)	14.3
14 (CH3)	1.70 (d, J = 6.1)	22.7	13 (CH3)	1.28 (t, J = 6.2)	14.2
1′	Q	167.7	1′ (CH)	4.25 (t, J = 5.7)	86.9
2′ (CH2)	4.25 (t, J = 6.5)	68.2	2′ (CH2)	1.46 (m)	34.9
3′ (CH2)	1.42 (m)	30.4	3′ (CH2)	1.31 (m)	29.7
4′ (CH)	1.39 (m)	29.7	4′ (CH)	1.96 (m)	34.0
5′ (CH2)	1.28 (m)	28.9	5′ (CH)	1.42 (m)	45.0
6′ (CH2)	1.28 (m)	28.9	6′ (CH)	1.39 (m)	50.1
7′ (CH)	1.39 (m)	38.7	7′ (CH2)	1.31 (m)	27.8
			8′ (CH2)	1.27 (m)	26.0
			9′ (CH2)	1.45 (m)	22.2
8′ (CH2)	1.28 (m)	38.0	10′ (CH2)	3.91 (q, J = 5.4)	63.3
9′ (CH2)	1.37 (m)	22.7	11′	Q	33.8
10′ (CH2)	1.31 (m)	23.8	12′ (CH3)	0.89 (t, J = 5.7)	14.2
11′ (CH2)	1.28 (m)	29.7	13′ (CH3)	0.97 (t, J = 5.6)	14.6
12′ (CH2)	1.28 (m)	23.8	14′ (CH3)	0.87 (d, J = 5.8)	13.0
13′ (CH2)	1.33 (m)	23.0	15′ (CH3)	0.95 (t, J = 5.4)	19.0
14′ (CH3)	0.91 (t, J = 6.9)	14.1	16′, 16′, 16′ (CH3)	0.91 (s)	27.1
15′ (CH3)	0.92 (t, J = 6.7)	14.1
16′ (CH3)	0.93 (t, J = 7.0)	10.9

a

Q represents quaternary carbon.

Table 2.

¹H and ¹³C NMR data of compound C (CDCl₃, δ in ppm). ^a

H/C	δ (H) (J in Hz)	Δ (C)	H/C	δ (H) (J in Hz)	δ (C)
1	Q	121.9	3′ (CH2)	1.70 (m)	31.0
2	Q	131.2	4′ (CH2)	1.40 (m)	25.1
3 CH	7.56 (s)	111.0	5′ (CH2)	1.13 (m)	28.6
4	Q	149.2	6′ (CH2)	1.00 (m)	28.8
5	Q	142.5	7′ (CH2)	1.00 (m)	28.7
6 CH	7.74 (s)	112.8	8′, 14′ (CH2)	1.42 (m)	34.7
7 (CH2)	2.13 (t, J = 6.3)	37.1	9′, 13′ (CH)	3.52 (m)	67.6
8 (CH2)	1.59 (m)	23.0	10′, 12′ (CH2)	1.35 (dd)	29.7
9, 10 (CH2)	4.32 (q, J = 6.7)	63.9	11′ (CH2)	1.59 (dd)	19.3
11, 12 (CH3)	0.97 (t, J = 6.5)	14.2	15′ (CH2)	1.12 (m)	24.9
13 (CH3)	0.95 (t, J = 6.9)	13.0	16′ (CH2)	1.12 (m)	32.2
1′	Q	165.5	17′ (CH2)	1.28 (m)	21.9
2′ (CH2)	4.25 (t, J = 6.8)	63.9	18′ (CH3)	0.91 (t, J = 6.4)	14.0

a

Q represents quaternary carbon.

The mass fragmentation patterns for compounds A–C, which has been discussed above, also support the suggested structures (Figure 2).

Figure 2.

Mass spectral fragmentation patterns of compounds A–C.

In vitro antioxidant properties of compounds A–C

The results of the evaluation of the DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and superoxide anion radicals scavenging properties of compounds A–C are shown in Table 3. As can be seen, each showed outstanding antioxidant properties, with compound A generally showing the highest activities.

Table 3.

Antioxidant activity of compounds A–C.

Compound	IC50 (μM) ± SEM a
	DPPH scavenging	ABTS scavenging	Superoxide anion scavenging
A	14.1 ± 0.10	5.41 ± 0.07	17.2 ± 1.09
B	10.3 ± 0.17	4.0 ± 0.01	9.64 ± 0.37
C	6.31 ± 0.82	2.2 ± 0.43	10.08 ± 0.02
Ascorbic acid b	10 ± 0.11	−	12 ± 0.31
Trolox b	−	2.4 ± 0.17	−

SEM: standard error of mean; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid).

a

Number of experiments conducted = 3.

b

Used as positive control.

It is a fact that oxygen is essential in aerobic life, but an excess of reactive oxygen species is responsible for infections such as inflammation, immune suppression, diabetes, atherosclerosis, neurodegenerative diseases, cancer and others. ¹⁹ To overcome such issues, antioxidants are promising agents, functioning through different mechanisms, that is, chelating transition metals, decomposition of peroxides, donation of hydrogen atoms to the free radicals and radical scavenging. The three assays mentioned are used to estimate the antioxidant agents and disclose their possible pathway of biochemical action. ²⁰

Experiment

Plant material and chemicals

The plant material was obtained from Gomal University, Pakistan. Prof. Jamil Khan, Faculty of Agriculture, Gomal University, identified the plant and the voucher was submitted to herbarium of Gomal University. Aluminium thin-layer chromatography (TLC) plates (0.5 mm thick) pre-coated with silica gel 60 F254 (0.2 mm layer thickness; E. Merck, Darmstadt, Germany) were used for TLC to check the purity of the compounds. Column chromatography (CC) was carried out using silica gel of 70–230 mesh (E. Merck). Optical rotation was determined with Ogawa Seiki (OSK) polarimeter (ATAGO NO. 15533 Tokyo, Japan). The IR spectra (ν max, cm⁻¹) were obtained through Jasco-320-A spectrophotometer in CHCl₃. HRMS (EI) spectra were recorded using an LTQ-Orbitrap LC-MS spectrometer (Thermo Corporation, USA). NMR spectra were recorded with a Bruker Avance (400 MHz for ¹H and 100 MHz for ¹³C spectrometer; Bruker Corporation, Switzerland). A kit for the antioxidant assay bearing catalogue number CS0790 (Sigma-Aldrich, USA), and the solvents and reagents including dimethyl sulfoxide, nitro blue tetrazolium and DPPH used in this research were of analytical grade (E. Merck).

Extraction from C. fistula and isolation of its constituents

The shade-dried (about 2 months) material of C. fistula was transformed into powdered material of about 3.1 kg, extracted with MeOH (3 × 3.5 L), and then, the solvent was removed through evaporation to afford a greenish gummy crude material of about 98.4 g. This methanolic extract was mixed with MeOH/H₂O (dist.) and then distributed into 16.4 g of n-hexane (fraction F1), 18.2 g of CHCl₃ (fraction F2), 22.2 g of EtOAc (fraction F3) and 16.6 g of n-butanol (fraction F4). Column, over silica gel, of the soluble fraction of ethyl acetate (22.2 g) and elution with n-hexane/EtOAc, EtOAc/MeOH and MeOH (following increasing polarity order) gave 10 fractions (F_a–F_j). The soluble fraction (F_f) obtained from mixtures of n-hexane/EtOAc at a polarity difference of 4:6 showed a prominent TLC spot in addition to some impurities. Further purification of this fraction through preparative TLC (silica gel) and a mobile phase system composed of n-hexane and EtOAc in a ratio of 2:8, which afforded 23 mg of fistuloate A as a colourless solid. Fistuloate B was obtained in an amount of 21 mg from the same column using n-hexane/EtOAc, having a polarity ratio of 3:7. It was further purified by preparative thin layer chromatographic card using a 5:5 ratio of n-hexane to EtOAc, which afforded the pure compound as a colourless solid. Similarly, the soluble fractions obtained from mixtures of hexane/AcOEt (2:8) displayed a prominent TLC spot, which was further purified by preparative TLC (silica gel) using a solvent system (1.5:8.5) to yield 18 mg of fistuloate C as a colourless solid.

Anti-oxidant assay (in vitro)

DPPH scavenging properties

This test was conducted by following the protocol described in the literature.^21,22 Thus, to a 900-µL solution of DPPH containing the reaction mixture was added further to 100 µL of the samples where concentration of the test samples were 0.5–20 μM. Thorough mixing well of the reaction mixture and keeping under darkness for about 1 h at a temperature of 37 °C, was followed by measurement of the spectrophotometric absorbance at around 517 nm. As a reference/blank, MeOH (3%) was used. Ascorbic acid (Aa) was applied as a positive control. In the experiment, a 900-µL DPPH was employed as a negative control.

ABTS antioxidant activity

This antioxidant test was carried out using an antioxidant Assay Kit (bearing catalogue number #CS0790) in duplicate while using ultrapure H₂O. A standard inhibitor such as Trolox, and an ABTS substrate working sample solution was made by addition of 3% of H₂O₂ (25 µL) solution to 10 mL of ABTS to achieve the reference curve. 10 µL of the standard mentioned previously was added to the wells, followed by addition of 20 µL of myoglobin solution in order to achieve a standard curve that was followed by adding 10 µL of the test sample in an amount of 0.5–20 μM and working solution (20 µL) to the wells of samples under examination. In the second step, to each well 150 µL of the substrate was added, then incubated at around 25 °C for 5 min, followed by adding a stop solution in amount of 100 µL (bearing catalogue S3446) into each well. The endpoint determination was carried out at 405 nm of absorbance using plate reader method. ²³

Superoxide anion scavenging activity

The assay was conducted according to the method reported in Sabu and Ramadasan. ²⁴ The reaction test mixture was composed of 0.5 mL of the solution of nitro blue tetrazolium (NBT) (1 M NBT in 100 mM buffer of phosphate, pH 7.4). Also, 0.3 mL of NADH (1 M NADH in phosphate buffer (100 mM), pH = 7.4) and 0.1 mL (0.5–20 μM) of test compounds and ascorbic acid (50 mM phosphate buffer pH 7.4) were mixed. The reaction was started by adding 100 µL of PMS (phosphomethazonesulfate) solution (60 µM PMS in 100 mM of phosphate buffer, pH = 7.4 to the mixture). The tubes were uniformly illuminated with an incandescent visible light for 15 min, and the optical density was measured at 530 nm before and after the illumination. The values (in percentage inhibition) obtained on account of superoxide generated in the control and test samples were measured.

Conclusion

Phytochemical studies of C. fistula were performed through chromatographic and spectral techniques. As a result, three new aromatic compounds were isolated from the ethyl acetate soluble fraction. The three new aromatic compounds, fistolates A–C, showed strong antioxidant activity with IC₅₀: 14.1 ± 0.10, 5.41 ± 0.07 and 17.2 ± 1.09 μM; 10.3 ± 0.17, 4.0 ± 0.01 and 9.64 ± 0.37 μM; and 6.31 ± 0.82, 2.2 ± 0.43 and 10.08 ± 0.02 μM; respectively. In most parts of the world, C. fistula is a very common and essential ingredient of traditional medicines. Therefore, further investigations are recommended for the exploration of its hidden medicinal values.