CHEMICAL COMPOSITION OF BIOLOGICAL ACTIVE EXTRACTS OF Tapirira guianensis (ANACARDIACEAE)

Abstract

This work describes the chemical composition of MeOH seeds and flowers extracts of Tapirira guianensis, a known tree that occurs in the Brazilian Atlantic forest. The CH₂Cl₂ soluble fraction of seeds extract was submitted to chromatographic procedures to obtain a mixture of new alkenyl dihydrobenzofuranoids which were identified as 2-[(10’Z)-dodec-10’-enyl]-dihydro-1-benzofuran-5-ol, 2-[(10’Z)-tridec-10’-enyl]-dihydro-1-benzofuran-5-ol e 2-[(10’Z)-pentadec-10’-enyl]-dihydro-1-benzofuran-5-ol (1 - 3) besides β-sitosterol. From the EtOAc soluble fraction of flowers MeOH extract quercetin, quercitrin and gallic acid were obtained by chromatographic techniques. The fatty acid composition of oils from leaves and seeds were also determinate and the leaves’ oil is composed by 63.94% of saturated and 36.04% of unsaturated fatty acids while in the seeds the oil present 42.13% of saturated and 57.87% of unsaturated. All compounds and derivatives were identified by their spectrometric data analysis. The brine shrimp test of the extracts showed the seed CH₂Cl₂ and the EtOAc and BuOH soluble fractions of the flowers were actives and, the alkyl phenols are the responsible for this moderate activity. The antioxidant tests of the extracts indicated EtOAc soluble fraction of MeOH extract of flowers showed better results possibly due the presence of flavonoids and gallic acid.

Keywords:
Tapirira guianensis ; alkenyl dihydrobenzofuranoids; cytotoxic activity; quercetrin; quercetin; fatty acid composition

INTRODUTION

Anacardiaceae is a family comprising 800 species distributed in 81 genera and these species are predominantly found in tropical and subtropical areas.¹ In Brazil, the family Anacardiaceae is represented by 14 genera and 54 species, being 13 of them endemics.¹ Several species of this family show significant economic importance due to their woods for carpentry, fruits, and edible seeds, all of them with expressive commercial value. For example, from Anacardium occidentale L. (“cajú” or cashew) is obtained the worldwide known cashew nut, besides juices, jams and other processed food.²

Besides the economic importance, Anacardiaceae is a family well known due to the existence of some toxic species. Studies indicate that about 25% of the genera of this family have species that are characterized as toxic and they are responsible for severe allergic contact dermatitis.³ It is more likely this toxic effect of some species of Anacardiaceae is due to the presence of secondary metabolites known as non-isoprene phenolic lipids or alkyl/alkenyl phenols, present in the leaf surface or are secreted from the resin of these plants.³

The genus Tapirira consists of 35 known species found from the south of Mexico to all South America.⁴ Brazil contributes with four endemic species of this genus, Tapirira obtusa (Benth.) J.D.Mitch., T. pilosa Sprague, T. retusa Ducke and T. guianensis Aubl..⁵ Between them, T. guianensis is the most well-known native species occurring in all Brazilian biomes and popularly named as “pau-pombo”or”peito-de-pombo”.⁶ Tapirira guianensis is a 8-14 m tree present in humid soils of Brazil, especially in Atlantic forest areas.⁷ The leaves of this species are employed in Brazilian folk medicine in the treatment of leprosy, diarrhea and syphilis.⁸ Previous chemical studies dealing with this species related the presence of cytotoxic alkylphenols from the seeds besides alkyl ester ferulates, triterpenes, steroids, norisoprenoids and flavonoids isolated from the barks and leaves.^8,9

This paper describes the reexamination of organic extracts of seeds and flowers a specimen of T. guianensis as well as, the fatty acid composition, evaluation of cytotoxic and antioxidant activities.

EXPERIMENTAL

General procedures

The solvents (MeOH, EtOAc, CH₂Cl₂, CHCl₃ and hexane) employed in extraction and chromatographic techniques were all PA purchased from Quimex. In the CC were used Sigel 60 (0.063-0.200 and 0.040-0.063 mm) from Merck and Acros and, in TLC 60 F₂₅₄ (Merck) precoated plates. The TLC monitoring were revealed by UV (254 and 365 nm) lamps, iodine fumes and Lieberman-Burchard reagent, prepared by the mixture of 10 mL sulfuric acid, 10 mL acetic anhydride in 50 mL of cold EtOH. The IR spectra were registered in the FT Shimadzu spectrophotometer mod IRAffinity-1 employing dry KBr plates or pellets. The ¹H and ¹³C NMR spectra (mono and bidimensional) were carried out in the Varian^® (GEMINI 2000) and Bruker (DRX 500) spectrometer using traditional pulse sequences for the homonuclear and heteronuclear correlation experiments. Optical rotations were performed using a Perkin-Elmer model 341 Polarimeter employing a microcell. The mass spectra were obtained on a negative ESI LCMS Shimadzu spectrometer mod. 2010A and in the EI (70 eV) HP mass detector mod. 5973. The UV spectra were recorded in a CARY I Varian spectrophotometer. The HPLC/DAD analysis were recorded in a Dionex equipment (mod. UltiMate 3000) with an injection volume of 10 µL. A DIONEX C18 column (120 Å 3.0 x 75 mm) with 5µm particles diameter was used. As eluent system, gradient mixtures MeOH (B) and 0.2% of aqueous acid formic (A): 30-50% of B for 15 min and 50-100% B from 15 to 20 min of running time at 0.5 mL min^-1 flow rate. The detection was registered in 254, 265, 290 and 330 nm UV region.

Plant material

The seeds and flowers of T. guianensis were collected Universidade Federal da Bahia campus (13º00’02.0”S, 38º30’26.1”W), Salvador (BA), Brazil and a plant voucher (# 032912) is deposited in the Herbarium Alexandre Leal Costa of Instituto de Biologia da UFBA. The access to the species was registered in the Sistema Nacional de Gestão do Patrimônio Genético e Conhecimento Tradicional Associado (SisGen) under number A56BAD6.

Extraction and isolation of the constituents of T. guianensis seed extracts

The seeds of T. guianensis were previously dried in a forced circulating oven for 48 h at 40 ºC, and then ground in a blender. The powdered seeds (316.15 g) were submitted to extraction by maceration by 3x employing hexane for 48 h each one. The extract was concentrated in rotaevaporator furnishing 6.62 g of hexane extract and, sequentially it was submitted to liquid-liquid partition between hexane and MeOH:H₂O (95:5). This procedure permitted to obtain the hexane soluble fraction (2.23 g) and the hydroalcoholic one (3.62 g). The hydromethanolic fraction was dissolved in MeOH:H₂O (1:1) and also submitted to partition between CH₂Cl₂, furnishing 3.15 g of the soluble fraction in CH₂Cl₂.

The CH₂Cl₂ soluble fraction was chromatographed over silica gel 60 (0.063-0.200 mm) and eluted with pure CH₂Cl₂ and gradient of mixtures of CH₂Cl₂:MeOH. The CC furnished 19 fractions of 20 mL each and they are monitored and grouped by silica gel TLC (UV lamps and iodine fumes). The first fractions eluted with CH₂Cl₂ (158.35 g) was then submitted to flash CC, also employing silica gel (0.040-0.063 mm). The fractions eluted with mixtures of CH₂Cl₂:MeOH furnished the mixture of the new compound identified as 2-[(10’Z)-dodec-10’-enyl]-dihydro-1-benzofuran-5-ol, 2-[(10’Z)-tridec-10’-enyl]-dihydro-1-benzofuran-5-ol e 2-[(10’Z)-pentadec-10’-enyl]-dihydro-1-benzofuran-5-ol (1 - 3, 20 mg) besides β-sitosterol (4, 25 mg).

Extraction and isolation of the constituents of the extracts of the flowers of T. guianensis

The flowers were previously dried at room temperature, and then ground in a blender and the powered material (344.04 g) was submitted to extraction 3x by MeOH, with about 48 hours for each extraction. The MeOH extract was diluted with H₂O to furnished a MeOH:H₂O (6:4) solution and sequentially it was partitioned with CH₂Cl₂, obtaining 6 g of material and, then, EtOAc furnishing 2.50 g of the soluble fraction of this solvent.

The CH₂Cl₂ soluble fraction was submitted to a silica gel 60 (0,063-0,200 mm) CC and eluted with mixtures of CH₂Cl₂:MeOH, obtaining 20 fractions of 100 mL each. The fraction eluted with 20% of MeOH (479 mg) was submitted to another CC and the fraction eluted with hex:EtOAc (3:2) furnished quercetin (5, 10 mg).

The silica gel 60 CC of the soluble fraction of EtOAc (2.50g) eluted with mixtures of CH₂Cl₂:MeOH, furnished 16 fractions of 100 mL each and the fractions eluted with 30% MeOH (203 mg) was subjected to gel permeation in Sephadex LH 20 eluted with MeOH. This procedure permitted to obtain quercitrin (6, 20 mg) and gallic acid (7, 25 mg).

2-[(10’Z)-Dodec-10’-enyl]-dihydro-1-benzofuran-5-ol, 2-[(10’Z)-tridec-10’-enyl]-dihydro-1-benzofuran-5-ol e 2-[(10’Z)-pentadec-10’-enyl]-dihydro-1-benzofuran-5-ol, (1 - 3). Oil. ESIMS(negative mode) m/z 301, 315 and, 343 ( C₂₀H₃₀O₂, C₂₁H₃₂O₂ e C₂₃H₃₆O₂). [α]²⁵_D +13º (c.1.5, CH₃OH). IR_{νmax film} (cm^-1): 3347; 3005; 2919; 2850; 1492; 1468; 1199; 862; 814. ¹H NMR (400 MHz, CDCl₃, δ_H) and ¹³C NMR (75 MHz, CDCl₃, δ_C): Table 1.

Table 1
¹H (400 MHz) and ¹³C (75 MHz) NMR data and long-range correlations (HMBC) of 1- 3[CDCl₃, J (Hz), δ (ppm)]

position	δC	δH	HMBC
2	83.7	4.74 (m)
3	36.1	3.22 (dd, 8.8, 8.8)
2.83 (dd, 8.0, 8.0)	153.6, 128.2, 83.7, 35.9
3a	128.2	-	-
4	112.4	6.68 (brs)*	153.6, 149.4, 114.1, 36.1
5	149.4	-	-
6	114.1	6.58 (d, 8.4)	153.6, 149.4, 112.4,
7	109.1	6.62 (d, 8.4)	153.6, 149.4, 128.2
7a	153.6	-	-
1’	35.9	1.82 (m)
1.63 (m)	-
2’- 8’	29.8-29.6	1.32 (m)
9’	26.9	2.04 (m)	129.9
10’/11’	129.9	5.36 (m)	27.2
12’	27.2	2.04 (m)	129.9
13’	32.0	1.32 (m)
14’	22.4	1.32 (m)
15’	14.2	0.91 (t, 6.4)	22.4, 32.0

• ^*
  brs broad singlet.

Thioalkylation of 1-3

The derivatives were prepared based on a previously described methodology.⁸ Briefly, the mixture of 1-3 (3.0 mg) was dissolved in dimethyl disulfide (DMDS 0.2 mL), and iodine (1.0 mg) was added to the solution. The resulting mixture was kept at room temperature for 24 h. Subsequently, the reaction was quenched with aqueous Na₂S₂O₃ (5%), and the mixture was extracted with n-hexane; the hexane layer was concentrated to provide a mixture of DMDS derivatives.

Analysis of fatty acids from leaves and seeds of T. guianensis

The hexane soluble fraction of seeds and the hexane extract of leaves (prepared employing 35 g of dried and ground T. guianensis leaves and 100 mL of hexane furnishing 1.19 g of extract) for T. guianensis were submitted to transesterification with a solution sodium methoxide (0.5 mol L^-1) in 2.0 mL of MeOH and 25 mg of the triglycerides/fatty acids mixture and the reaction was stirred during 30 min. In sequence, H₂O was added, and the methyl esters were extracted by partition between hexane.

The methyl ester derivatives were analyzed injecting a solution of the hexane extracts in a GC-MS system (Shimadzu CG-2010/QP-2010 high efficiency, coupled with quadrupole mass detector and a Rtx^®-1 MS (Crossbond^® 100% dimethyl polisiloxane) column of 30 m, Φ 0.25 mm and 0.25 µm of thickness). The speed of carrier gas was set to 40 cm s^-1; the carrier gas flow in the column to 0.8 mL min^-1; the injection mode was split with a rate of 1:30; the temperature of the injector was set to 280 ºC, interface temperature of 260 ºC and the oven temperature gradient set to 80 ºC (at 2.0 min), rising at 1.5 ºC min^-1 then 10 ºC min^-1 to 140 ºC, 3 ºC min^-1 to 200 ºC (10 min) and 5 ºC min^-1 to 250 ºC; with the transfer line set to 250 ºC; the ion source to 250 ºC and an impact energy of 70 eV and voltage detector of 1.3 KV. The methyl ester constituents were identified by molecular ion and analyzing the fragmentation pattern in the mass spectra and comparison with NIST 147 and WYLEY 8 databases.

Brine shrimp test

For an initial screening on the bioactivity, the adapted Brine Shrimp Lethality assay described by Meyer and co-worker¹⁰ was employed to study the general cytotoxicity of the compounds 1-6. Alive brine shrimp (Artemia salina Leach) cysts (500 mg) were transferred to a conical flask containing 3500 mL of artificial seawater. The flasks were aerated with the aid of an air pump and kept at 28 ºC under a bright light and the nauplii hatched after 48 hours approximately. The compounds and extracts were separately dissolved artificial seawater and DMSO to reach the final concentration of 1 mg mL^-1. Solutions of 50, 100, 150, 200, 300 e 400 µg mL^-1 of extracts and for the 1-3 each compound (25, 50, 75, 100, and 150 µg mL^-1) were transferred to vials, which already contained seawater (5 mL) and 10 nauplii. Controls containing DMSO, seawater, and 10 nauplii also performed. The assays were carried out in triplicate. After 24 hours of incubation, the number of live nauplii was counted. The mortality was defined as the absence of controlled forward motion during 30 seconds of observation. The lethal concentration doses for 50% of the brine shrimp (LC₅₀) and the respective 95% confidence intervals were determined by using the GraphPad Prism version 5.0 (GraphPad, La Jolla, CA) software.

DPPH scavenging activities

Radical scavenging activities of plant extracts were determined through spectrophotometry using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging radical assay (MeOH, 40 µg mL^-1) employing 3 mL of DPPH solution and 1 mL of the samples. The radical scavenging ability was calculated by the formula, % I = [(AbsB − AbsA) / AbsB] × 100, and the IC₅₀ was determined from the linear decrease in the inhibition percentage at λ = 517 nm immediately and after 30 min of incubation at room temperature and the solutions protected from light. Gallic acid was employed as positive control and the extracts were evalueted at different concentrations (50, 100, 150, 200 e 250 µg mL^-1).

RESULTS AND DISCUSSION

From the CH₂Cl₂ soluble fraction of the hexane extract of the T. guianensis seeds were isolated by CC an inseparable mixture of three new alkyl dihydrobenzofuranoid (1-3, Figure 1) by usual chromatographic techniques. Besides them, β-sitosterol, quercetin, quercitrin and, gallic acid were also obtained. The TLC analysis of this mixture showed just one spot however the negative ESIMS showed [M-H] molecular ions at m/z 301, 315, and 343. These data combined with the peaks observed in the IR, ¹H, and ¹³C spectra, including hydrogen integration and carbon counts, permitted to assign C₂₀H₃₀O₂, C₂₁H₃₂O₂, and C₂₃H₃₆O₂ as the molecular formula. The IR spectrum of 1-3 showed characteristic stretching bands of OH groups of phenolics (υ 3347 and 1199 cm^-1) besides aromatic and aliphatic C-H (3005, 2919 and 2850 cm^-1), C=C aromatic stretchings between 1492 and 1468 cm^-1 and C-H out-of-plan angular deformation of trisubstituted aromatic ring at 862 and 814 cm^-1. These findings were corroborated with ¹H NMR spectra data, the coupling constants and integration of the peaks at δ 6.68, 6.62, and 6.58 confirmed the substitution pattern of the aromatic ring. The AMX spin coupling system could be confirmed by ¹H-¹H-COSY experiment, besides H-4 is registered as a broad singlet, the COSY spectrum showed a correlation with δ 6.58 (H-6). The multiplet at δ 5.36 (2H) is characteristic of hydrogens of aliphatic double bond carbons, similar to olefinic hydrogens of unsaturated fatty acids with Z configuration. The presence of an alkenyl chain was also confirmed by the hydrogen peaks at δ_H 0.91-2.03 ppm. The ¹³CNMR spectra confirmed all these structural patterns of the compounds. Besides, the peak at δ_C 83.65 (C-5) was considered as oxymethine carbon once the nature of the hydroxyl group present was determined as phenolic. Thus, the carbon bearing the phenolic OH group is a non-hydrogenated one displayed at δ_C153.62 (C-7a). This spectrum also confirmed the Z configuration of the double bond (δ_C 129.89 and 129.90) of the alkenyl chain by the observed deshielded chemical shifts of the allylic carbons at δ_C 27.23 and δ_C 26.95 (C-10’/C-11’).¹¹

Figure 1
Isolated compounds from T. guianensis seeds

The position groups attached in the structure were partially determined by HMBC. The correlations of methylene hydrogens (H-3) at δ_H 2.83 and 3.22 and the C-4 (δ_C 112.40), C-3a (δ_C 128.22), C-7a (δ_C 153.62) and the oxymethine carbon (C-2) are indicative both ring junction (Table 1).

The localization of the OH group at C-5 was just allocated by empirical calculations using the effect of the substituent groups on the aromatic ring described in the literature.¹² The HMBC correlations were useless for this kind of assignment. The obtained theoretical values indicated the position of OH in C-5 were closer than the experimental data.

The location of the double bond in each compound was established through the mass spectral analyses of the dimethyl disulfide (DMDS) derivatives. The EIMS of the DMDS derivatives showed ion peak fragments at m/z 321 (C₁₉H₂₉O₂S) due to the bond cleavage between the carbon bearing the methylthio groups of C-10’ and C-11’. These evidences permitted to identify the mixture 1, 2 and 3 as being 2-[(10’Z)-dodec-10’-enyl]-dihydro-1-benzofuran-5-ol, 2-[(10’Z)-tridec-10’-enyl]-dihydro-1-benzofuran-5-ol and 2-[(10’Z)-pentadec-10’-enyl]-dihydro-1-benzofuran-5-ol, respectively. These compounds present optical activity which indicates they are not artifacts but the absolute configuration of C-2 of these compounds could not be determinate by NMR and remains unknown.

The know isolated compounds sitosterol, quercetin (5), quercitrin or quercetin-3-O-rhaminosyl (6) and gallic acid were identified by NMR, UV spectrometry and comparison with literature data and direct comparison with standard by HPLC (Supplementary Material).¹³

Methanolysis of the mixture of triglycerides from hexane extracts of leaves and seeds yielded a mixture of two fatty acid methyl ester mixtures which were identified by GC-MS and FID GC comparison with a commercial F.A.M.E standard (Table 2). The fatty acid profile of the oil’s seeds indicated it is rich of unsaturated while the leaves’ profile indicates a predominance of saturated fatty acid.

Table 2
Fatty acid composition isolated from T. guianensis leaves and seeds

Fatty acid derivatives	% ± SD (leaves)	% ± SD (seeds)
Myristic (14:0)	9.22 ± 0.63	n.d.
Isopalmitic (i-16:0)	5.48 ± 0.01	n.d.
Palmitic (16:0)	24.71 ± 2.48	17.64 ± 0.75
Linoleic (18:2)	12.26 ± 0.50	29.88 ± 0.39
Linolenic (18:3)	3.53 ± 0.38	n.d.
Oleic (18:1)	20.25 ± 1.25	27.99 ± 1.08
Stearic (18:0)	24.53 ± 1.06	24.49 ± 1.06
Saturated fatty acids	63.94	42.13
Unsaturated fatty acids	36.04	57.87
Total identified	100	100

• SD: standard deviation; n.d. not detected.

The Brine Shrimp Test has been employed as a screening of cytotoxic compounds or extracts. The LD₅₀ of CH₂Cl₂ soluble fraction of seeds (> 1000 µg mL^-1) and flowers (615.46 µg mL^-1) and, EtOAc (166.36 µg mL^-1) and BuOH (249.37 µg/mL) indicated that except the seed CH₂Cl₂ of T. guianensis all of them are moderately active (LD₅₀= 100-1000 µg mL^-1).¹⁴ However, the mixture 1-3 (LD₅₀= 174.06 µg mL^-1) showed considerable activity. These compounds are present in minor concentrations in the seed oils, which justifies the absence of activity in the CH₂Cl₂ soluble fraction of seeds.

The most common and practical methodology to determine antioxidant is quenching of free radical DPPH (2.2-diphenyl-1-picryl-hydrazil).¹⁵ The organic extracts of the flowers of T. guianensis were evaluated and the EtOAc soluble fraction (EC₅₀ = 69.22 µg mL^-1) is more active than the BuOH (EC₅₀ = 116.99 µg mL^-1) comparing with the positive standard (EC₅₀ =26.45 µg mL^-1). This activity probably in accordance with the presence of flavonoids and gallic acid in the EtOAc soluble fraction.

CONCLUSIONS

The chemical study of the organic extracts of seeds of T. guianensis permitted to isolate known compounds and three new alkenyl dihydrobenzofuranoids (1-3) presenting cytotoxic activity in Artemia salina nauppli. Besides the fatty acid profile of hexane extracts of seeds and leaves indicated the oil from leaves is predominantly saturated and the seed oils were composed predominantly by unsaturated fatty acids. The presence of toxic alkylphenols in nonpolar fractions of the seed extracts could not indicate the T. guianensis seed oils for food purposes. However, more investigations must be performed.

• SUPPLEMENTARY INFORMATION
  Some spectra, spectrometric data, chromatograms are available as supplementary material free of charge at http://quimicanova.sbq.org.br

ACKNOWLEDGEMENTS

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) under Grant # 406427/2018-6. The authors also thank CAPES and CNPq for the scholarships.

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Publication Dates

• Publication in this collection
  28 Oct 2020
• Date of issue
  Sept 2020

History

• Received
  25 Mar 2020
• Accepted
  22 June 2020
• Published
  07 Aug 2020