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Extraction of phenolic components from an Aloe vera (Aloe barbadensis Miller) crop and their potential as antimicrobials and textile dyes
Sustainable Chemistry and Pharmacy 14 (2019) 100168
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Sustainable Chemistry and Pharmacy journal homepage: http://www.elsevier.com/locate/scp
Extraction of phenolic components from an Aloe vera (Aloe barbadensis Miller) crop and their potential as antimicrobials and textile dyes Gonzalo Canche-Escamilla a, Pamela Colli-Acevedo a, Rocio Borges-Argaez a, *, �ceres-Farfan a, Patricia Quintana-Owen b, J. Fernando May-Crespo c, Mirbella Ca Jesus Alejandro Yam Puc a, Pablo Sansores-Peraza a, Blanca Marina Vera-Ku a a
Centro de Investigacion Cientifica de Yucatan, Calle 43 No. 130 � 32 y 34, Col. Chuburna de Hidalgo, C.P. 97205, Merida, Yucatan, Mexico Department of Applied Physics, Cinvestav-Unidad M�erida, Carretera Antigua a Progreso, Km. 6, CP. 97310, M�erida, Yucatan, Mexico National Council of Science and Technology (CONACYT-El Colegio de Michoacan), Cerro de Nahuatzen 85, Fraccionamiento Jardines Del Cerro Grande, CP. 59370, La Piedad, Michoacan, Mexico b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Aloe vera Dyes Anthraquinones Antimicrobial
The present work focuses on the use of solid and agricultural residues from Aloe vera crops, as a source of antimicrobial agents and textile dyes. The roots from an A. vera plantation post-harvest were extracted with ethyl acetate, purified and phytochemically characterized to obtain five metabolites: aloesaponarin-I (1), deoxyery throlaccin (2), lacaic acid D methyl ester (3), aloesaponarin-II (4), and aloesaponol-I (5). Acid hydrolysis of the solid industrial residue gave aloe-emodin (6) as the main product with a good yield. All of the components were tested for the first time against phytopathogenic bacteria strains, and deoxyerythrolaccin and lacaic acid D methyl ester were active against Xanthomonas campestris with MIC values of 46.86 and 93.75 μg/mL, respec tively. Aloesaponarin-I and aloe-emodin, the main products, were tested as dyes for polyester fabrics using different mordants and pH bath conditions. The colour of each material was investigated in terms of the CIELAB L*, a* and b* values, and the colour fastness to light and washing was investigated according to the Mexican standard methods (NMX-A-074-INNTEX-2005; NMX-A-105-B02-INNTEX-2010). Aloesaponarin-I dyed polyester bright yellow but the final colour was very sensitive to the pH of the dye bath. Aloe-emodin dyed polyester deep yellow, and the fabrics showed good colour fastness to light and to domestic laundering. This study provides evidence that the phenolic components obtained from agricultural residues of the aloe industry can be useful organic alternatives as antimicrobial agents and textile dyes.
1. Introduction The agricultural industry is a plentiful source of lignocellulosic ma terials (roots, stems and leaves), which are not generally used and are a source of contamination in fields or in disposal areas (Singh and Bajaj, 2017; Panesar et al., 2015). In addition to structural components such as lignin, cellulose and hemicellulose, lignocellulosic materials include a large number of low molecular weight compounds generally referred to as secondary metabolites, which can be used for various applications (Soto et al., 2019; Sharma et al., 2019). Aloe vera (Aloe barbadensis Miller, Liliaceae family) is a plant with a large worldwide market that generates a high amount of agricultural and industrial residues. This shrub is mainly distributed in tropical
regions of Africa, Madagascar, and southern Arabia and was introduced to America by the Spanish conquerors. It grows well in semi-arid zones in temperatures from 21 � C to 27 � C with an annual rainfall of 590 to 4030 mm, but it is very resistant to drought conditions and high tem peratures (Silva et al., 2010). The leaves are the main commercial product obtained from the plant. The leaves contain gel, which is of high commercial value, since it is used in the manufacture of cosmetics, pharmaceuticals and beverages (Dalia et al., 2017; Maan et al., 2018). Some pharmaceutical industries use aloin, obtained from the yellow exudate of the leaves, for the preparation of diacerein, a drug used for treating osteoarthritis (Bartels et al., 2010). More than 200 compounds obtained from A. vera have been reported with a variety of biological activities, including for gastrointestinal conditions, burns, skin
* Corresponding author. Centro de Investigaci� on Científica de Yucatan, Calle 43 No. 130 � 32 y 34, Col. Chuburna de Hidalgo, C.P. 97205, Merida, Yucatan, Mexico. E-mail address: [email protected] (R. Borges-Argaez). https://doi.org/10.1016/j.scp.2019.100168 Received 17 February 2019; Received in revised form 10 August 2019; Accepted 18 August 2019 Available online 5 September 2019 2352-5541/© 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Aloe vera crop without an irrigation system and remaining roots after the leaf exudate collection (Maxcanu, Yucatan, Mexico).
regeneration, inflammation, kidney infections, bladder conditions, flu, asthma, bronchitis and arthritis (Drudi et al., 2018; Salah et al., 2017; Baruah et al., 2016; Radha and Laxmipriya, 2015; Liu et al., 2019). Worldwide, thousand tons of aloe leaves are produced and A. vera base products generate 110 billion dollars in revenue (Ahlawat and Khatkar, 2011). Among the North and South American countries that export A. vera, including Brazil, Argentina, Costa Rica and Venezuela, Mexico occupies an important position. In southeast Mexico, the commercialization of aloe products for the cosmetics and food industries is based on the concentrated gel from the leaves. Aloin is also collected from the leave exudate for the pharmaceutical industry, generating approximately 400 kg of solid waste per month in local agroindustry in �n, Mexico. Acid hydrolysis of the complex solid residue gives Yucata aloe-emodin as the main product in good yield (88%). On the other hand, the root of this plant is generally discarded during plantation maintenance and could provide another source of phenolic components. It has been previously reported that the anthraquinones aloesaponarin-I and aloesaponarin-II, together with the tetrahydroanthracenes aloesaponol-I, aloesaponol-II, aloecrisone and aloebarbendol, can be isolated from the roots of this species (Abdissa et al., 2014). Addition ally, the dimers aspodelin and dibenzopyran have been isolated from these roots (Van Wyk et al., 1995). One of the main quinones present in the roots is aloesaponarin-I, which can be isolated as orange crystals (5% w/w) after a simple fractionation of the ethyl acetate or etanolic root extracts. Some aloe metabolites have been tested against several mi croorganisms such as bacteria and fungi (Nejatzadeh-Barandozi, 2013; Laib et al., 2019), but they have never been tested on phytopathogenic bacteria such as Xanthomonas campestris pv. carotae, Pectobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. pisi. While the dyeing properties of quinones are well-known (Siva et al., 2012; Yusuf et al., 2017; Nagia and El-Mohamedy, 2007), the dyeing proper ties of aloesaponarin-I and aloe-emodin have never been reported. To find new potential uses for and encourage the comprehensive utilization of this crop, the objective of the present study was to test the antimi crobial properties of the root and solid waste metabolites, and the dyeing properties of the two main components, aloesaponarin-I and aloe-emodin, by direct and post-mordant methods.
from Fluka and Sigma-Aldrich. The synthetic fibres were all commer cially obtained. 2.2. Plant material The roots of A. vera were collected twice, in August 2012 and in August 2015, from a plantation located in Maxcanu, Yucatan, Mexico that did not have an irrigation system (Fig. 1). The roots were washed with tap water and then, air-dried. When the material was crispy, it was milled, weighed and stored under refrigeration until its extraction. 2.3. Phytochemical purification of A. vera root extract The dried roots (2.70 kg) were cut, ground, and extracted three times with cold methanol (MeOH) for 72 h. The extracts were combined, and the solvent was removed under reduced pressure to produce 300 g of crude methanolic extract. The MeOH extract (200 g) was then sus pended in a 1:1 mixture of water/MeOH (ca. 150 mL of solution per 20 g of crude methanolic extract) and the resulting suspension was extracted with ethyl acetate (EtOAc) to yield a medium polarity fraction (35.00 g). The EtOAc extract (2.52 g) was subjected to flash column chromatog raphy (CC) eluting with n-hexane/EtOAc containing increasing amount of EtOAc to afford 70 fractions. The fraction eluted with 3% EtOAc in nhexane afforded aloesaponarin I (1, 111.50 mg). The fraction eluted with 50% EtOAc in n-hexane was further purified by Sephadex LH-20 using CH2Cl2/MeOH 1:1 to afford deoxyerythrolaccin (2, 21 mg) and lacaic acid D methyl ester (3, 19.70 mg). Subsequent flash column chromatography (CC) of additional amounts of EtOAc extract (9.90 g) using a gradient elution with mixtures of n-hexane/EtOAc/MeOH and n-hexane/acetone resulted in the isola tion of aloesaponarin II (4, 18.20 mg) in pure form. Additionally, a combination of CC and Sephadex LH-20 with CH2Cl2/MeOH 1:1 and 100% MeOH, respectively, resulted in the isolation of aloesaponol-I (5, 16.30 mg). The purity of the isolates were determined by GC-MS analysis. 2.4. Identification of the isolated metabolites
2. Material and methods
1
H NMR (400 MHz) and 13C NMR (100 MHz) spectra were obtained either on CDCl3 or [CD3]2CO and [CD3]2SO on a Bruker Avance 400 spectrometer using the residual solvent signal as a reference. IR were recorded in KBr disc on an FT-IR Nicolet Proteg�e. Melting points were determined on a Thermocouple apparatus. UV spectra were recorded on an UV–Vis spectrophotometer Thermo Scientific Genesys 10S. EI-mass spectra were recorded on an Agilent Technologies 6890 N gas chro matograph interfaced to an Agilent Technologies 5975B inert mass se lective detector (MDS). The analysis was performed on an Ultra 1 capillary column (25 m � 0.321 mm i. d.; 0.52 μm; 100% dimethylpo lysiloxane). Helium was used as the carrier gas at a constant pressure (1.5 mL/min). The initial oven temperature was kept at 180 � C for 5 min � and then ramped up to 300 � C at 10 C/min. HRMS were recorded on an Agilent 1190 liquid chromatograph equipped with an API1400 (triple quadrupole) mass spectrometer (AB SCIENX).
2.1. General experimental procedures Analytical TLC was carried out on precoated aluminium plates (E.M. Merck, 60F254, 0.2 mm thickness). Detection of the components was performed using a UV cabinet (λ 254 and 366 nm) and by using a so lution of phosphomolybdic acid (20 g) and ceric sulfate (2.5 g) in 500 mL of sulfuric acid (5%), followed by heating. A specific reagent for quinone €rntrager reagent) by spraying an aqueous solu detection was used (Bo tion of 5% KOH on TLC analytical plates. The chromatographic sepa ration was performed using silica gel 60 (0.040–0.063 mm; Merck) and gel permeation column chromatography was carried out using Sephadex LH-20 from Pharmacia. The chemical solvents, reagents, and mordants such as [KAl(SO4)2�12H2O] and K2Cr2O7 were commercially obtained 2
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2.5. Solid industrial residue treatment
the fabrics were washed with tap water and dried at room temperature. In the post-mordanting method, the fabrics were kept dyeing with the sample for 50 min at 98 � C, and then the mordant was added to the bath and incubated for 10 min. Aluminium sulfate hydrate [KAl (SO4)3⋅12H2O] and potassium dichromate (K2Cr2O7) were used as mordants.
The dry solid industrial residue (2 kg) was supplied by Tecnologías Innovadoras Industry from Yucatan, Mexico. This solid was obtained by spray drying of the mother liquor generated in obtaining aloin by recrystallization of the yellow exudate from the leaves. The material (named ‘A29’) was kept under dry conditions in a black plastic bag in order to avoid chemical deterioration of its components before the analysis. A solution of the solid waste (4 g) and ferric chloride (10 g) in water (200 mL) was heated to refluxing at 100 � C for 21 h, until a darkbrown solid formed. This solid was separated from the hot solution and washed twice with distilled water (200 mL). The solid obtained after the ferric chloride oxidation was purified by re-crystallization in toluene (Vittori and Collins, 1997). A red-orange solid was obtained and dried under vacuum. A sample of this solid was identified by its 1H NMR spectra and by comparison of those reported on literature.
2.8. Colour strength and depth measurements The colour strengths of the stained fabric samples with aloesaponarin-I and aloe-emodin, with and without mordant, were measured by applying the light reflectance technique. A light source D65 with a deuterium-halogen lamp (AvaLight DH-S-BAL) and an optical fibre was used. The reflected light was collected using an integrating sphere (Labsphere USRS-99-010) and sent to the spectrometer with an optical fibre (AvaSpec-2048). The reflectance spectra were obtained from 200 nm to 800 nm. The reference was an Ocean Optics WS-1-SL. The colour strength (K/S) value was evaluated following the approach of Bhuyan and Saikia (2005), using the Kubelka-Munk equation:
2.6. Antimicrobial activity test The antimicrobial activity was tested for all the isolated compounds against the phytopathogenic bacteria Xanthomonas campestris pv. carotae (ATCC 10547), Pectobacterium carotovorum subsp. carotovorum (ATCC 138) and Pseudomonas syringae pv. pisi (ATCC 11043) using a disc diffusion test for preliminary antimicrobial detection (Hood et al., 2003). The tested compounds were dissolved in either acetone or chlo roform, according to their solubility, to a concentration of 1.5% w/v. An aliquot of 10 μL was taken and placed on a filter paper disc (7 mm diameter) such that each paper disc contained 150 μg of pure compound. The solvent was used as negative control and a neomycin solution (0.002 μg/disc) was used as a positive control. Trypticase Soy Agar (TSA) petri dishes were inoculated with 150 � 106 cfu/mL, according to a 0.5 McFarland Nephelometer. The impregnated paper discs were placed on the plate and incubated at 36 � C for 24 h. After incubation, the inhibition zones were measured in mm. All the tests were performed in triplicate. The compounds that showed positive results were then tested to deter mine their Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) using 96 well plates (Moulari et al., 2006). The active compounds selected were dissolved to a concentration of 6% w/v in DMSO. The test dilutions for each tested compound were 1500, 750, 375, 187.5, 93.75, 46.87, 23.43 and 11.72 μg/mL after inoculation with 150 � 106 cfu/mL. All the tests dilutions were per formed in triplicate. A control with Muller Hinton (MH) media, a posi tive control with neomycin (4 μg/ml) and a 10% v/v DMSO vehicle control were included. The plates were incubated at 36 � C for 18 h. After incubation, 50 μL of a 1% w/v solution of 2,3,5-triphenyltetrazolium chloride (TTC) was added to the plates and incubated for 1 h. The absence of colour was interpreted as growth inhibition. The MIC value is the first concentration at which there is no colour. To obtain the MBC, a loop from each well on the plate was inoculated on a MH agar plate. The MBC is the concentration at which there is no bacterial growth. If the MIC shows bacterial growth, a bacteriostatic effect is established. If there is no growth, a bactericidal effect is demonstrated for that compound.
K/S ¼ (1-R)2/2R Where R is the reflectance of the dye sample, K is the absorption coef ficient and S is the scattering coefficient. The colour measurements were described in terms of the L*a*b* colour space, defined by CIE-1976, where the parameter L* is the ach romatic lightness and there are two chromatic components a* (green-red axis) and b* (blue-yellow axis). From the obtained data for the two chromatic components, the hue (H) and chroma or colour saturation (C) values were calculated. Hue is determined by the dominant wavelength and identifies a colour as found in its pure state in the light spectrum. It is represented by the hue-angle in the a* - b* plane, and increases by counter clockwise rotation around the a*, b* axis such that 0� is red, 90� is yellow, 180� is green, and 270� is blue. Chroma denotes the saturation of a colour and is a measure of how much grey and white light is mixed with the pure focal colour. Chroma ranges from neutral to brilliant and is represented as the length from the origin of the axis on the a* - b* plane. Thus, hue and chroma were calculated using the following formulae: H ¼ arctan (b*/a*) C ¼ (a*2 þ b*2)1/2 Finally, to obtain the depth of the colour, the total colour difference ΔE was calculated using the natural fabric (polyester) as a reference and was defined as: ΔE ¼ [(ΔL*)2 þ (Δ a*)2 þ (Δb*)]1/2 The K/S values and colour coordinates were obtained from three replicate measurements. 2.9. Fastness testing The colour fastness to light was determined for the polyester fibres according to the Mexican standard NMX-A-105-B02-INNTEX-2010 in a Fadeometer Ci3000 þ using a xenon arc lamp. The exposure time for the sample was 20 h. Additionally, the colour fastness to domestic laun dering was determined according to the Mexican standard method NMXA-074-INNTEX-2005 which is consistent with the ISO-105-C06 stan dard. The colour fastness test was performed at 70� C for 30 min using 50 steel balls. A multi-fibre adjacent fabric containing wool, cellulose ac etate, polyamide, cotton, acrylic and polyester was also used to test the staining of the polyester. The colour change in the fabric was given by grey scale numbers from one to five, with five as the best value.
2.7. Dyeing processes €isa €nen et al., 2001), 1 mg of the an Following Raisanen et al. (Ra thraquinones aloesaponarin I and aloe emodin were (separately) used for each 100 mg of textile. The samples were first dissolved in a solution of sodium phosphate 0.1 M and 10% of Glauber salt. The liquor to fabric ratio was 20:1 and the dyeing was performed using a Thermo Park heater equipped with a digital thermometer. The fabrics were dyed using baths with different pH values (4, 7, 10) adjusted with buffer so lutions. Initially, the dyeing bath temperature was raised from 50 � C to 98 � C. The fabrics were then added to the bath and kept there for 50 min. Subsequently, the dye bath was allowed to cool for 20 min. Afterwards, 3
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Fig. 2. Chemical structures of the components obtained from the A. vera roots (1–5) and solid waste (6).
3. Results and discussion
acetone with a TLC Rf value of 0.23 using n-hexane:acetone 8:2 as the €rntrager eluent. This compound showed an orange colour with the Bo reagent. The 1H NMR spectrum of aloesaponarin-II was similar to aloesaponarin-I except for the lack of a methoxycarbonyl group at the C2 position. Instead of this signal, a doublet at δ 6.99 with meta coupling (J ¼ 2.0 Hz) corresponding to the H-2 proton was observed. The 1H NMR spectrum of compound 4 was similar to the data reported by Cui et al. (2006) for aloesaponarin-II (4). Aloesaponol-I (5) was recovered as pale yellow needles soluble in DMSO. This compound had an Rf value of 0.19 with the mobile phase nhexane: EtOAc 1:1, and a light brown coloration when developed with €rntrager reagent. In its 1H NMR spectrum, a singlet was observed the Bo at δ 2.70, in addition to a singlet at δ 3.85 corresponding to a methyl group (CH3-11) and a methoxycarbonyl group (OCH3-13), respectively. In the aromatic region, two singlets were observed at δ 6.93 and 6.95, together with four doublets of doublets at δ 2.69 (J ¼ 16.7, 7.1 Hz), 2.90 (J ¼ 15.8, 7.1 Hz), 2.95 (J ¼ 17.2, 3.6 Hz) and 3.12 (J ¼ 15.8, 3.4 Hz). These coupling constants are consistent with geminal and vicinal coupling, indicating the presence of four methylene protons separated by an intermediate methine proton. The presence of a multiplet at δ 4.25 confirms the signal of the methine proton. Finally, two large singlets with interchangeable protons, belonging to hydroxyl groups, were observed at δ 5.19 and 10.76 in addition to a signal at δ 15.27 from a hydroxyl group in the β-position to a carbonyl. Since this compound showed different signals than any compound that had been previously isolated, we performed 13C NMR and 2D NMR experiments to confirm its structure. In the 13C NMR spectrum of Aloesaponol-I (5), seventeen carbons were detected. Two signals between δ 205–168 corresponded to carbonyl groups and 10 signals between 167–108 corresponded to ar omatic carbons. In addition, two signals between δ 50–70 typical of oxygenated carbons, were detected, together with three signals below δ 50, identified as saturated carbons. The number of signals detected in the 1H NMR and 13C NMR spectra, the type of couplings observed, and the chemical shifts obtained were similar to methyl 3,6,9-trihydroxy-imethyl-8-oxo-5,6,7,8-tetrahydro-2-anthracenecarboxylate, known as aloesaponol-I (5) (Dagne et al., 1992). Finally, aloe-emodin (6), the main product of the oxidative hydro lysis of the A29 solid residue, was obtained as a reddish orange powder with a TLC Rf value of 0.66 using EtOAc:CHCl3 1:1 as the eluent. In the 1 H NMR spectrum, the following signals were observed: a singlet at δ 4.55, corresponding to the methylene protons of a primary benzyl alcohol (CH2-11), and, in the aromatic region, singlets at δ 7.20 (H-2) and δ 7.59 (H-4) together with three characteristic signals of the aro matic protons in an ABC spin system at δ 7.62 (dd, J ¼ 0.90, 7.40 Hz, H5), δ 7.72 (t, J ¼ 7.90 Hz, H-6), δ 7.30 (J ¼ 0.90, 8.30 Hz, H-7). Finally, two singlets corresponding to the phenolic protons in positions 1 and 8
3.1. Compounds identification All the compounds obtained from the root (1–5) extract and the solid industrial residue (6), were characterized using their 1H NMR spectra, IR, EI-MS, UV data and compared to those in the literature (Fig. 2). Aloesaponarin I (1) consisted of orange needles that were soluble in €rntrager chloroform, which showed a red-orange coloration with the Bo reagent and a TLC retention factor (Rf) of 0.6 with an n-hexane:EtOAc 1:1 mobile phase. This compound was the main compound isolated with a 5% yield from the EtOAc root extract. In the 1H NMR spectrum of aloesaponarin I (1), two singlets were detected, one at δ 2.96 indicating the presence of a methyl group (CH3-11), and another at δ 4.06 corre sponding to a methoxycarbonyl group (OCH3-13). In the aromatic re gion two protons with ortho and meta couplings were present. Each signal appears as a double of doublets at δ 7.30 (J ¼ 8.3, 1.0 Hz) and δ 7.76 (J ¼ 7.4, 1.1 Hz), respectively. In the same region, a triplet at δ 7.62 with ortho coupling to the vicinal protons (J ¼ 7.9 Hz) and a singlet at δ 7.78 were also observed. Finally, in the low field region, two signals were observed, a broad singlet characteristic of a hydroxyl group at δ 10.42 and a singlet at δ 12.92, typical of a hydrogen-bond to the carbonyl group of an anthraquinone ring. The 1H NMR data of aloesa ponarin I (1) were similar with those reported by Yagi et al. (1977). Deoxyerythrolaccin (2) was isolated as an orange amorphous powder soluble in acetone, which showed an orange coloration when developed €rntrager reagent (TLC Rf of 0.55 using n-hexane:EtOAc [1:1] with the Bo as the eluent). In the 1H NMR spectrum of deoxyerythrolaccin, a singlet was observed at δ 2.75, characteristic of a methyl group (CH3-11) and four signals appeared as doublets at δ 6.62 (J ¼ 2.5 Hz), δ 7.05 (J ¼ 2.5 Hz), δ 7.16 (J ¼ 2.3 Hz) and δ 7.55 (J ¼ 2.8 Hz), typical of aro matic protons with meta couplings. A singlet corresponding to a hy droxyl group, analogous to that observed in aloesaponarin-I, was detected (δ 13.30 ppm). Comparing the spectroscopic data to the liter ature indicated that compound 2 was 1,3,6-trihydroxy-8-methyl-9, 10anthraquinone, also known as deoxyerythrolaccin (2) (Yagi et al., 1977). Lacacic acid D methyl ester (3) consisted of an orange amorphous powder soluble in acetone, which was visualized on the TLC as a violet €rntrager reagent (Rf of 0.33 in spot when it was developed with the Bo the solvent system n-hexane:EtOAc 1:1). The 1H NMR spectrum showed signals similar to those observed for aloesaponarin-I, except for the presence of two doublet signals located in the aromatic region at δ 6.64 and δ 7.17 (J ¼ 2.5 Hz) from two protons with meta coupling (H-5 and H7), suggesting the presence of an intermediate hydroxyl group at posi tion 6. The signals observed were similar to those reported for the lacacic acid D methyl ester (3) (Yagi et al., 1977). Aloesaponarin-II (4) was isolated as orange needles soluble in 4
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OH), 11.83 (1H, s, 8-OH), 7.72 (1H, dd, J ¼ 8.1, 7.7 Hz, H-6), 7.62 (1H, dd, J ¼ 7.4, 0.9 Hz, H-5), 7.59 (1H, s, H-4), 7.30 (1H, dd, J ¼ 8.3, 0.9 Hz, H-7), 7.20 (1H, s, H-2), 4.55 (2H, s, CH2-11). 1 H NMR spectral data are available online as Supplementary data.
Table 1 Inhibition zone diameter, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of metabolites 2 and 3 against X. campestris. Compound
Inhibition diameter (mm) at 10 μg/mL
MIC value (μg/ ml)
MBC value (μg/ ml)
2 3
14.50 � 0.99 10.00 � 0.57
46.87 93.75
373 1500
3.2. Antimicrobial activity results In this work, the antimicrobial activity of compounds 1–6 were evaluated via disc diffusion test and broth microdilution assays against the phytopathogenic bacteria Xanthomonas campestris pv. carotae, Pec tobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. pisi. The disc diffusion test was first used to identify the active compounds, and subsequently the plate microdilution test was used to establish the effective dose of active compounds. In the disc diffusion test, only deoxyerythrolaccin (2) and lacaic acid D methyl ester (3) showed activity against X. campestris while being inactive against P. carotovorum and P. syringe. In the plate microdilution bioassay, deoxyerythrolaccin (2) showed an MIC value of 46.86 μg/mL whereas lacaic acid D methyl ester (3) had an MIC value of 93.75 μg/mL against X. campestris (Table 1). To distinguish if the antibacterial activity is bactericidal or bacteriostatic, an aliquot of each compound concentra tion was inoculated into solid medium MH after a prior 18 h of incu bation with X. campestris. Bacterial growth indicates a bacteriostatic effect, while the absence of growth is classified as a bactericidal effect. As seen in Table 1, deoxyerythrolaccin (2) showed bactericidal activity at 375 μg/mL, whereas lacaic acid D methyl ester (3) had a bactericidal effect at 1500 μg/mL. Related to this, it has been reported that anthra quinones can inhibit the activity of the enzymes nicotinamide adenine dinucleotide and mitochondrial succinate oxidase as well as the elec tronic transfer from the bacterial respiratory chain, and the process of dehydrogenation in the bacterium, which can damage the integrity of the membrane, causing loss of the cytoplasm (Cowan, 1999; Balachan dran et al., 2016). This inhibition could be related to the observed ac tivity of deoxyerythrolaccin (2) and lacaic acid D methyl ester (3) in the antimicrobial test. These results suggest some preliminary structure-activity relationships for the anthraquinone structure. The presence of an OH group at C-6 position, as in deoxyerythrolaccin (2) and lacacic acid D methyl ester (3), could influence the antimicrobial activity of both compounds, since this activity was not found on the other compounds, which lack of this group. In contrast, the presence of a methoxycarbonyl in position 2 had a negative effect on the antimicrobial activity, since lacaic acid D methyl ester (3) was less active than deox yerythrolaccin (2). These results arle consistent with previous reports of the activities of rhubarb anthraquinones against Aeromonas hydrophila, where it was observed that the presence of polar groups at the C-3 and C-6 positions in rhein and emodin make these anthraquinones more active than those with less polar groups such as physion and crisophanol (Lu et al., 2011). To the best of our knowledge, there are no previous reports about the antibacterial activity of these compounds against X. campestris with the exception of aloesaponarin-II, which has been reported to be active against X. oryzae pv. oryzae, pathogenic bacteria that affect rice crops, with an MIC value of 38.40 μg/mL (Donghua et al., 2013). However, as
*Neomycin (0.002 μg/mL) was used as positive control.
of the aromatic rings were observed at δ 11.89 and δ 11.83. Based on these results, compound 6 was identified as aloe-emodin as previously reported by Cui et al. (2008). The chemical data of the isolated com pounds are listed below: Aloesaponarin- I (1): Orange neddles; m. p: 199–203 � C; UV λMeOH m� ax : 270, 415, 435 nm. IR (KBr): 3380, 1740, 1700, 1675, 1630 cm 1.1H NMR (CDCl3, 400 MHz) δ 12.92 (1H, s, 8-OH), 10.42 (1H, s, 3-OH), 7.78 (1H, s, H-4), 7.76 (1H, dd, J ¼ 7.4, 1.1 Hz, H-5), 7.62 (1H, t, J ¼ 7.9 Hz, H-6), 7.30 (1H, dd, J ¼ 8.3, 1.0 Hz, H-7), 4.06 (3H, s, H-13), 2.96 (3H, s, H-11). EI-MS m/z 312.06 [Mþ]. Deoxyerythrolaccin (2): Orange amorphous powder; m. p: above 300 � C; UV λMeOH m� ax : 284, 432 nm. IR (KBr): 3400, 1670, 1630, 1600 cm 1.1H NMR ([CD3]2CO, 400 MHz) δ 13.30 (1H, s, 8-OH), 7.55 (1H, d, J ¼ 2.8 Hz, H-5), 7.16 (1H, d, J ¼ 2.3 Hz, H-4), 7.05 (1H, d, J ¼ 2.5 Hz, H-7), 6.62 (1H, d, J ¼ 2.5 Hz, H-2), 2.75 (3H, s, H-11); HRMS m/z 270.053 [Mþ] (calculated for C17H16O6, 270.2369). Lacacic acid D methyl ester (3): Orange amorphous powder; m. p: 270–275 � C; UV λMeOH m� ax : 220, 270, 285, 420, 435 nm. IR (KBr): 3300, 1740, 1715, 1670, 1630 cm 1.1H NMR ([CD3]2CO, 400 MHz) δ 13.29 (1H, s, 8-OH), 7.68 (1H, s, H-4), 7.17 (1H, d, J ¼ 2.5 Hz, H-5), 6.64 (1H, d, J ¼ 2.5 Hz, H-7), 3.93 (3H, s, H-13), 2.69 (3H, s, H-11). EI-MS m/z 328.05 [Mþ]. Aloesaponarin-II (4): Orange neddles; m. p: 250–254 � C; UV λMeOH m� ax : 280, 410 nm. IR (KBr): 3400, 1670, 1630 cm 1.1H NMR ([CD3]2CO, 400 MHz) δ 12.92 (1H, s, 8-OH), 7.59 (2H, m, H-6 and H-7), 7.47 (1H, d, J ¼ 2.3 Hz, H-4), 7.17 (1H, dd, J ¼ 7.6, 2.1 Hz, H-5), 6.99 (1H, d, J ¼ 2.3 Hz, H-2), 2.66 (1H, s, CH3); HRMS m/z 254.056 [Mþ] (calculated for C15H10O4, 254.2375). Aloesaponol-I (5): Yellow neddles; m. p: 248–250 � C; UV λMeOH m� ax : 228, 242, 270, 278, 308, 320, 380 nm. IR (KBr): 3350, 3150, 1710, 1625, 1610 cm 1.1H NMR ([CD3]2SO, 400 MHz) δ 15.27 (1H, s, 6-OH), 10.76 (1H, s, 6-OH), 6.95 (1H, s, H-10), 6.93 (1H, s, H-5), 5.19 (1H, s, 3-OH), 4.25 (1H, m, H-3), 3.85 (3H, s, H-13); 3.12 (1H, dd, J ¼ 15.8, 3.4 Hz, H4b), 2.95 (1H, dd, J ¼ 17.2, 3.6 Hz, H-2b), 2.90 (1H, dd, J ¼ 15.8, 7.1 Hz, H-4a), 2.70 (3H, s, H-11), 2.69 (1H, dd, J ¼ 16.7, 7.1 Hz, H-2a). 13C NMR ([CD3]2SO, 100 MHz) δ 204.18 (C-1), 168.72 (C-12), 166.45 (C-9), 155.59 (s, C-6), 141.32 (s, C-4b), 137.74 (s, C-4a), 137.16 (s, C-8), 125.95 (s, C-7), 117.08 (d, C-10), 115.88 (s, C-8a), 110.76 (s, C-8b), 108.07 (d, C-5), 64.98 (d, C-3), 64.98 (d, C-3), 52.61 (q, C-13); 46.96 (t, C-2), 38.06 (t, C-4), 21.06 (q, C-11). HRMS m/z 316.0941 [Mþ] (calculated for C17H16O6, 316.3124). Aloe-emodin (6): Reddish orange powder; m. p: 223–224 � C; UV MeOH λm�ax : 210, 220, 280 nm. 1H NMR (CDCl3, 400 MHz) δ 11.89 (1H, s, 1-
Table 2 Colour parameters for the fabrics dyed with aloesaponarin-I and aloe-emodin with and without mordants. Compound
Without mordents (direct)
With mordents (post-mordanting method) KAl (SO4)2⋅12H20
1 6
K2Cr2O7
pH
L*
a*
b*
C
H
L*
a*
b*
C
H
L*
a*
b*
C
H
4 7 10 4 7 10
94.74 95.90 99.47 89.81 89.68 88.52
5.03 0.26 10.88 2.10 0.57 1.08
12.52 7.57 38.71 32.70 26.15 19.09
13.50 7.58 40.23 32.15 26.16 19.13
111.91 268.12 285.69 86.27 91.24 86.76
95.73 95.90 98.59 87.45 89.29 83.05
5.28 1.97 5.45 3.05 2.41 6.81
9.83 2.15 27.44 29.44 30.82 25.63
11.17 2.94 27.98 29.60 30.92 26.52
118.26 225.63 281.24 84.08 85.52 75.10
94.26 94.78 98.01 92.46 88.61 88.88
2.29 0.37 5.41 0.47 3.60 2.49
9.64 5.76 25.61 10.65 14.48 14.79
9.92 5.77 26.18 10.66 14.97 15.00
103.62 266.25 281.92 92.98 75.73 80.35
5
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Fig. 3. K/S values of aloesaponarin-I and aloe-emodin.
has been mentioned, aloesaponarin-II did not show antibacterial activity against any of the strains evaluated. The antibacterial activities observed for deoxyerythrolaccin (2) and lacacic acid D methyl ester (3) show that these compounds have potential as natural controls of X. campestris strains.
molecule in solution and also on the affinity of the dye towards the fibre [Drivas et al., 2011]. The colorimetric data, colour strength and colour obtained at different bath pH conditions are shown in Table 2. The (þ) colour values for a* indicate a reddish colour, while negative a* values indicate a greenish colour. Conversely, positive b* values indicate a yellow colour, while negative *b values indicate a blue colour. As can be seen from Table 2, when aloesaponarin-I was applied, negative a* values were observed at pH 4 and 7, indicating that the colour obtained was more greenish compared to the colour obtained at pH 10. Additionally, high luminosity L* values within a small range (from 94 up to 99) were obtained for the three applied pH bath conditions, which slightly increased at higher pH. The b* values went from a yellowish colour for the acidic condition of pH 4 (12.52) to almost colourless with negative values ( 38.73) at basic pH. The effect on the depth of shade data
3.3. Dye properties of aloesaponarin I and aloe-emodin on polyester fabric 3.3.1. Effect of dye bath pH Different bath pH conditions were used for dyeing with aloesaponarin-I and aloe-emodin, in order to compare the shades ob tained and the best conditions for uptake of the material. It is known that the dye bath pH has a strong relationship on the stability of the dye
Fig. 4. Colour shades from aloesaponarin-I and aloe-emodin under different pH bath conditions, without mordants (WM) and by post-mordanting methods using KAl (SO4)2⋅12H2O (M1) and K2Cr2O7 (M2) as mordants. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 6
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Table 3 Fastness properties of the polyester fabrics dyed with aloesaponarin-I and aloe-emodin. Compound
pH
Washing fastness (NMX-A-074-INNTEX-2005) Without mordents
1 6
4 7 4 7 10
4–5 4–5 4 4–5 3–4
Light fastness (NMX-A-105-B02-INNTEX-2010)
With mordents KAl (SO4)2⋅12H20
K2Cr2O7
4–5 4–5 4 4–5 4–5
4–5 4–5 3–4 4 3–4
(expressed as K/S) of aloesaponarin I is shown in Fig. 3, where it is evident that the values decrease as the pH increases. This behaviour suggests that protonation of the functional groups of both the dye and the fibre controlled the dye uptake. Conversely, aloe-emodin yielded yellow shades independent of the bath solution pH (positive values of a* and b*), suggesting a better interaction between the OH groups and the aromatic rings of the dye with the polyester material. Polyester is made of a polymer formed from terephthalic acid and ethylene glycol, which has periodic phenylene groups along the polymer chain. These groups can interact with the hydroxyl groups present in aloesaponarin I and aloe-emodin, leading to hydrogen bonds, and it is even possible that there are π-π interactions between the two materials (fibre-dye). It is clear that an increase in the pH, which prevents the formation of hydrogen bonds, causes a decrease in the colour intensity using aloesaponarin-I. The better adsorption properties of aloe-emodin may be related to the structural size of the molecule, its additional OH group, as well as a better interaction between the terephthalic acid unit of the fibre and the π-electron system of the anthraquinone ring (R€ ais€ anen et al., 2001).
Without mordents
With mordents KAl (SO4)2⋅12H20
K2Cr2O7
4–5 4–5 4 4–5 4–5
4–5 4–5 4 4–5 4–5
4–5 4–5 4–5 4–5 4
polyester fabrics dyed with aloe-emodin (at all the pH values tested) and aloesaponarin-I (at pH 4 and 7). The values showed that no differences in the washing and light fastness of the un-mordanted and mordanted polyester fabrics were seen with aloesaponarin-I, and the dyeing ob tained has excellent colour values (4–5 units on the grey scale where 5 is the highest rating). In the case of aloe-emodin mordanted with KAl (SO4)3⋅12H2O, a noticeable improvement in light fastness and fastness to domestic and industrial washing was observed at pH 7 and 10. In contrast, under the same pH conditions K2Cr2O7 showed a medium value of 3–4 in washing fastness. This may be due to complexation be tween the mordant and the quinone dye instead of the dye attaching to the polyester surface. These results were compared with the values for emodin, an anthraquinone similar to aloe-emodin, but differing due to €is€ the presence of a methyl group at the 3 position (Ra anen et al., 2001). Accordingly, the colour fastness to light and to washing gave similar results for the un-mordanted polyester dyed fabric, and showed uniform colouring with good yields, similar to aloe-emodin. In contrast, it was not possible to perform the colour fastness test on aloesaponarin-I at pH 10 because of its poor fixation properties (Fig. 4). From these results, we conclude that dyeing with aloesaponarin-I with or without mordants at pH 4 gives excellent light and washing fastness on polyester fabrics, whereas for aloe-emodin, aluminium sulfate hydrate is the best mordant for improving its fastness properties.
3.3.2. Effect of the mordants used Aloesaponarin-I and aloe-emodin are phenolic compounds that dissociate to form phenoxide monovalent and/or divalent ions under different pH bath conditions. Improved solubility was observed for both compounds as the pH increased. According to Drivas et al. (2011), who worked with the anthraquinones purpurin and alizarin, this change in colour and increase in solubility is due to the ionization of the hydroxyl groups, forming salts with better solubility in water. However, the ionization of the functional groups may be favourable or unfavourable depending on their interaction with the structure of the fibre. This interaction can be improved by the presence of metallic mordants, giving better colour fixation with the fibre, deeper shades and better colour fastness properties. In this study, a post-mordanting method was used and KAl(SO4)3⋅12H2O and K2Cr2O7 were used as mordants. From the colorimetric data for the aloe-emodin staining, deeper yellow shades were obtained using KAl(SO4)3⋅12H2O compared with less intense yel low shades on polyester obtained using K2Cr2O7 as the mordant (Fig. 4). The brightness of the shades may be due to better absorption of the dye and easy formation of metal complexes between the mordant and the fabric. However, the fabrics dyed with aloe-emodin showed deeper yellow shades with positive b* values (from 10.65 up to 32.07) for all the methods studied (Fig. 4). The strongest colour was found with KAl (SO4)3⋅12H2O as the mordant. In contrast, when using aloesaponarin-I greenish yellow shades were obtained using K2Cr2O7, suggesting a bet ter interaction between this mordant and the anthraquinone structure. This interaction becomes weaker as the solubility of aloesaponarin-I increased (negative b* values), becoming almost colourless. Thus, by using a post-mordanting method, better colour fixation can be achieved in polyester fabric dyeing with aloe emodin using KAl(SO4)3⋅12H2O as the mordant and aloesaponarin-I and K2Cr2O7 as the dyes.
4. Conclusions The root of Aloe vera and the industrial residue produced during the aloin extraction are rich in phenolic components, and five metabolites were extracted of the root and one of the industrial waste. Desoxyery throlacin and lacaic acid D methyl ester, which are present in its roots showed antimicrobial properties against the phytopathogenic bacteria X. campestris. Additionally, the main products aloesaponarin-I and aloeemodin have excellent dyeing and colour fastness properties for poly ester fabric. Aloesaponarin-I dyes polyester bright yellow and aloeemodin produces a deeper yellow colour. The uptake of aloesaponarin-I was found to be more sensitive to pH than the uptake of aloe-emodin. Declaration of interest None. Author contributions RBA and GCE obtained the financial support, designed all the ex periments and wrote the manuscript. PCA performed the antimicrobial tests and dyeing experiments. MCF and BMVK gave technical support to PCA for the antimicrobial assays. PQO and FMC analysed the colori metric data and CIELAB parameters. JAYP and PSP supervised the isolation and identification of the metabolites. All the authors have read and approved the final manuscript.
3.3.3. Fastness properties of the dyed fabrics Table 3 shows the fastness rating with respect to light and to do mestic and industrial washing of the un-mordanted and mordanted 7
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Acknowledgements
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Extraction of phenolic components from an Aloe vera (Aloe barbadensis Miller) crop and their potential as antimicrobials and textile dyes Gonzalo Canche-Escamilla a, Pamela Colli-Acevedo a, Rocio Borges-Argaez a, *, �ceres-Farfan a, Patricia Quintana-Owen b, J. Fernando May-Crespo c, Mirbella Ca Jesus Alejandro Yam Puc a, Pablo Sansores-Peraza a, Blanca Marina Vera-Ku a a
Centro de Investigacion Cientifica de Yucatan, Calle 43 No. 130 � 32 y 34, Col. Chuburna de Hidalgo, C.P. 97205, Merida, Yucatan, Mexico Department of Applied Physics, Cinvestav-Unidad M�erida, Carretera Antigua a Progreso, Km. 6, CP. 97310, M�erida, Yucatan, Mexico National Council of Science and Technology (CONACYT-El Colegio de Michoacan), Cerro de Nahuatzen 85, Fraccionamiento Jardines Del Cerro Grande, CP. 59370, La Piedad, Michoacan, Mexico b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Aloe vera Dyes Anthraquinones Antimicrobial
The present work focuses on the use of solid and agricultural residues from Aloe vera crops, as a source of antimicrobial agents and textile dyes. The roots from an A. vera plantation post-harvest were extracted with ethyl acetate, purified and phytochemically characterized to obtain five metabolites: aloesaponarin-I (1), deoxyery throlaccin (2), lacaic acid D methyl ester (3), aloesaponarin-II (4), and aloesaponol-I (5). Acid hydrolysis of the solid industrial residue gave aloe-emodin (6) as the main product with a good yield. All of the components were tested for the first time against phytopathogenic bacteria strains, and deoxyerythrolaccin and lacaic acid D methyl ester were active against Xanthomonas campestris with MIC values of 46.86 and 93.75 μg/mL, respec tively. Aloesaponarin-I and aloe-emodin, the main products, were tested as dyes for polyester fabrics using different mordants and pH bath conditions. The colour of each material was investigated in terms of the CIELAB L*, a* and b* values, and the colour fastness to light and washing was investigated according to the Mexican standard methods (NMX-A-074-INNTEX-2005; NMX-A-105-B02-INNTEX-2010). Aloesaponarin-I dyed polyester bright yellow but the final colour was very sensitive to the pH of the dye bath. Aloe-emodin dyed polyester deep yellow, and the fabrics showed good colour fastness to light and to domestic laundering. This study provides evidence that the phenolic components obtained from agricultural residues of the aloe industry can be useful organic alternatives as antimicrobial agents and textile dyes.
1. Introduction The agricultural industry is a plentiful source of lignocellulosic ma terials (roots, stems and leaves), which are not generally used and are a source of contamination in fields or in disposal areas (Singh and Bajaj, 2017; Panesar et al., 2015). In addition to structural components such as lignin, cellulose and hemicellulose, lignocellulosic materials include a large number of low molecular weight compounds generally referred to as secondary metabolites, which can be used for various applications (Soto et al., 2019; Sharma et al., 2019). Aloe vera (Aloe barbadensis Miller, Liliaceae family) is a plant with a large worldwide market that generates a high amount of agricultural and industrial residues. This shrub is mainly distributed in tropical
regions of Africa, Madagascar, and southern Arabia and was introduced to America by the Spanish conquerors. It grows well in semi-arid zones in temperatures from 21 � C to 27 � C with an annual rainfall of 590 to 4030 mm, but it is very resistant to drought conditions and high tem peratures (Silva et al., 2010). The leaves are the main commercial product obtained from the plant. The leaves contain gel, which is of high commercial value, since it is used in the manufacture of cosmetics, pharmaceuticals and beverages (Dalia et al., 2017; Maan et al., 2018). Some pharmaceutical industries use aloin, obtained from the yellow exudate of the leaves, for the preparation of diacerein, a drug used for treating osteoarthritis (Bartels et al., 2010). More than 200 compounds obtained from A. vera have been reported with a variety of biological activities, including for gastrointestinal conditions, burns, skin
* Corresponding author. Centro de Investigaci� on Científica de Yucatan, Calle 43 No. 130 � 32 y 34, Col. Chuburna de Hidalgo, C.P. 97205, Merida, Yucatan, Mexico. E-mail address: [email protected] (R. Borges-Argaez). https://doi.org/10.1016/j.scp.2019.100168 Received 17 February 2019; Received in revised form 10 August 2019; Accepted 18 August 2019 Available online 5 September 2019 2352-5541/© 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Aloe vera crop without an irrigation system and remaining roots after the leaf exudate collection (Maxcanu, Yucatan, Mexico).
regeneration, inflammation, kidney infections, bladder conditions, flu, asthma, bronchitis and arthritis (Drudi et al., 2018; Salah et al., 2017; Baruah et al., 2016; Radha and Laxmipriya, 2015; Liu et al., 2019). Worldwide, thousand tons of aloe leaves are produced and A. vera base products generate 110 billion dollars in revenue (Ahlawat and Khatkar, 2011). Among the North and South American countries that export A. vera, including Brazil, Argentina, Costa Rica and Venezuela, Mexico occupies an important position. In southeast Mexico, the commercialization of aloe products for the cosmetics and food industries is based on the concentrated gel from the leaves. Aloin is also collected from the leave exudate for the pharmaceutical industry, generating approximately 400 kg of solid waste per month in local agroindustry in �n, Mexico. Acid hydrolysis of the complex solid residue gives Yucata aloe-emodin as the main product in good yield (88%). On the other hand, the root of this plant is generally discarded during plantation maintenance and could provide another source of phenolic components. It has been previously reported that the anthraquinones aloesaponarin-I and aloesaponarin-II, together with the tetrahydroanthracenes aloesaponol-I, aloesaponol-II, aloecrisone and aloebarbendol, can be isolated from the roots of this species (Abdissa et al., 2014). Addition ally, the dimers aspodelin and dibenzopyran have been isolated from these roots (Van Wyk et al., 1995). One of the main quinones present in the roots is aloesaponarin-I, which can be isolated as orange crystals (5% w/w) after a simple fractionation of the ethyl acetate or etanolic root extracts. Some aloe metabolites have been tested against several mi croorganisms such as bacteria and fungi (Nejatzadeh-Barandozi, 2013; Laib et al., 2019), but they have never been tested on phytopathogenic bacteria such as Xanthomonas campestris pv. carotae, Pectobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. pisi. While the dyeing properties of quinones are well-known (Siva et al., 2012; Yusuf et al., 2017; Nagia and El-Mohamedy, 2007), the dyeing proper ties of aloesaponarin-I and aloe-emodin have never been reported. To find new potential uses for and encourage the comprehensive utilization of this crop, the objective of the present study was to test the antimi crobial properties of the root and solid waste metabolites, and the dyeing properties of the two main components, aloesaponarin-I and aloe-emodin, by direct and post-mordant methods.
from Fluka and Sigma-Aldrich. The synthetic fibres were all commer cially obtained. 2.2. Plant material The roots of A. vera were collected twice, in August 2012 and in August 2015, from a plantation located in Maxcanu, Yucatan, Mexico that did not have an irrigation system (Fig. 1). The roots were washed with tap water and then, air-dried. When the material was crispy, it was milled, weighed and stored under refrigeration until its extraction. 2.3. Phytochemical purification of A. vera root extract The dried roots (2.70 kg) were cut, ground, and extracted three times with cold methanol (MeOH) for 72 h. The extracts were combined, and the solvent was removed under reduced pressure to produce 300 g of crude methanolic extract. The MeOH extract (200 g) was then sus pended in a 1:1 mixture of water/MeOH (ca. 150 mL of solution per 20 g of crude methanolic extract) and the resulting suspension was extracted with ethyl acetate (EtOAc) to yield a medium polarity fraction (35.00 g). The EtOAc extract (2.52 g) was subjected to flash column chromatog raphy (CC) eluting with n-hexane/EtOAc containing increasing amount of EtOAc to afford 70 fractions. The fraction eluted with 3% EtOAc in nhexane afforded aloesaponarin I (1, 111.50 mg). The fraction eluted with 50% EtOAc in n-hexane was further purified by Sephadex LH-20 using CH2Cl2/MeOH 1:1 to afford deoxyerythrolaccin (2, 21 mg) and lacaic acid D methyl ester (3, 19.70 mg). Subsequent flash column chromatography (CC) of additional amounts of EtOAc extract (9.90 g) using a gradient elution with mixtures of n-hexane/EtOAc/MeOH and n-hexane/acetone resulted in the isola tion of aloesaponarin II (4, 18.20 mg) in pure form. Additionally, a combination of CC and Sephadex LH-20 with CH2Cl2/MeOH 1:1 and 100% MeOH, respectively, resulted in the isolation of aloesaponol-I (5, 16.30 mg). The purity of the isolates were determined by GC-MS analysis. 2.4. Identification of the isolated metabolites
2. Material and methods
1
H NMR (400 MHz) and 13C NMR (100 MHz) spectra were obtained either on CDCl3 or [CD3]2CO and [CD3]2SO on a Bruker Avance 400 spectrometer using the residual solvent signal as a reference. IR were recorded in KBr disc on an FT-IR Nicolet Proteg�e. Melting points were determined on a Thermocouple apparatus. UV spectra were recorded on an UV–Vis spectrophotometer Thermo Scientific Genesys 10S. EI-mass spectra were recorded on an Agilent Technologies 6890 N gas chro matograph interfaced to an Agilent Technologies 5975B inert mass se lective detector (MDS). The analysis was performed on an Ultra 1 capillary column (25 m � 0.321 mm i. d.; 0.52 μm; 100% dimethylpo lysiloxane). Helium was used as the carrier gas at a constant pressure (1.5 mL/min). The initial oven temperature was kept at 180 � C for 5 min � and then ramped up to 300 � C at 10 C/min. HRMS were recorded on an Agilent 1190 liquid chromatograph equipped with an API1400 (triple quadrupole) mass spectrometer (AB SCIENX).
2.1. General experimental procedures Analytical TLC was carried out on precoated aluminium plates (E.M. Merck, 60F254, 0.2 mm thickness). Detection of the components was performed using a UV cabinet (λ 254 and 366 nm) and by using a so lution of phosphomolybdic acid (20 g) and ceric sulfate (2.5 g) in 500 mL of sulfuric acid (5%), followed by heating. A specific reagent for quinone €rntrager reagent) by spraying an aqueous solu detection was used (Bo tion of 5% KOH on TLC analytical plates. The chromatographic sepa ration was performed using silica gel 60 (0.040–0.063 mm; Merck) and gel permeation column chromatography was carried out using Sephadex LH-20 from Pharmacia. The chemical solvents, reagents, and mordants such as [KAl(SO4)2�12H2O] and K2Cr2O7 were commercially obtained 2
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2.5. Solid industrial residue treatment
the fabrics were washed with tap water and dried at room temperature. In the post-mordanting method, the fabrics were kept dyeing with the sample for 50 min at 98 � C, and then the mordant was added to the bath and incubated for 10 min. Aluminium sulfate hydrate [KAl (SO4)3⋅12H2O] and potassium dichromate (K2Cr2O7) were used as mordants.
The dry solid industrial residue (2 kg) was supplied by Tecnologías Innovadoras Industry from Yucatan, Mexico. This solid was obtained by spray drying of the mother liquor generated in obtaining aloin by recrystallization of the yellow exudate from the leaves. The material (named ‘A29’) was kept under dry conditions in a black plastic bag in order to avoid chemical deterioration of its components before the analysis. A solution of the solid waste (4 g) and ferric chloride (10 g) in water (200 mL) was heated to refluxing at 100 � C for 21 h, until a darkbrown solid formed. This solid was separated from the hot solution and washed twice with distilled water (200 mL). The solid obtained after the ferric chloride oxidation was purified by re-crystallization in toluene (Vittori and Collins, 1997). A red-orange solid was obtained and dried under vacuum. A sample of this solid was identified by its 1H NMR spectra and by comparison of those reported on literature.
2.8. Colour strength and depth measurements The colour strengths of the stained fabric samples with aloesaponarin-I and aloe-emodin, with and without mordant, were measured by applying the light reflectance technique. A light source D65 with a deuterium-halogen lamp (AvaLight DH-S-BAL) and an optical fibre was used. The reflected light was collected using an integrating sphere (Labsphere USRS-99-010) and sent to the spectrometer with an optical fibre (AvaSpec-2048). The reflectance spectra were obtained from 200 nm to 800 nm. The reference was an Ocean Optics WS-1-SL. The colour strength (K/S) value was evaluated following the approach of Bhuyan and Saikia (2005), using the Kubelka-Munk equation:
2.6. Antimicrobial activity test The antimicrobial activity was tested for all the isolated compounds against the phytopathogenic bacteria Xanthomonas campestris pv. carotae (ATCC 10547), Pectobacterium carotovorum subsp. carotovorum (ATCC 138) and Pseudomonas syringae pv. pisi (ATCC 11043) using a disc diffusion test for preliminary antimicrobial detection (Hood et al., 2003). The tested compounds were dissolved in either acetone or chlo roform, according to their solubility, to a concentration of 1.5% w/v. An aliquot of 10 μL was taken and placed on a filter paper disc (7 mm diameter) such that each paper disc contained 150 μg of pure compound. The solvent was used as negative control and a neomycin solution (0.002 μg/disc) was used as a positive control. Trypticase Soy Agar (TSA) petri dishes were inoculated with 150 � 106 cfu/mL, according to a 0.5 McFarland Nephelometer. The impregnated paper discs were placed on the plate and incubated at 36 � C for 24 h. After incubation, the inhibition zones were measured in mm. All the tests were performed in triplicate. The compounds that showed positive results were then tested to deter mine their Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) using 96 well plates (Moulari et al., 2006). The active compounds selected were dissolved to a concentration of 6% w/v in DMSO. The test dilutions for each tested compound were 1500, 750, 375, 187.5, 93.75, 46.87, 23.43 and 11.72 μg/mL after inoculation with 150 � 106 cfu/mL. All the tests dilutions were per formed in triplicate. A control with Muller Hinton (MH) media, a posi tive control with neomycin (4 μg/ml) and a 10% v/v DMSO vehicle control were included. The plates were incubated at 36 � C for 18 h. After incubation, 50 μL of a 1% w/v solution of 2,3,5-triphenyltetrazolium chloride (TTC) was added to the plates and incubated for 1 h. The absence of colour was interpreted as growth inhibition. The MIC value is the first concentration at which there is no colour. To obtain the MBC, a loop from each well on the plate was inoculated on a MH agar plate. The MBC is the concentration at which there is no bacterial growth. If the MIC shows bacterial growth, a bacteriostatic effect is established. If there is no growth, a bactericidal effect is demonstrated for that compound.
K/S ¼ (1-R)2/2R Where R is the reflectance of the dye sample, K is the absorption coef ficient and S is the scattering coefficient. The colour measurements were described in terms of the L*a*b* colour space, defined by CIE-1976, where the parameter L* is the ach romatic lightness and there are two chromatic components a* (green-red axis) and b* (blue-yellow axis). From the obtained data for the two chromatic components, the hue (H) and chroma or colour saturation (C) values were calculated. Hue is determined by the dominant wavelength and identifies a colour as found in its pure state in the light spectrum. It is represented by the hue-angle in the a* - b* plane, and increases by counter clockwise rotation around the a*, b* axis such that 0� is red, 90� is yellow, 180� is green, and 270� is blue. Chroma denotes the saturation of a colour and is a measure of how much grey and white light is mixed with the pure focal colour. Chroma ranges from neutral to brilliant and is represented as the length from the origin of the axis on the a* - b* plane. Thus, hue and chroma were calculated using the following formulae: H ¼ arctan (b*/a*) C ¼ (a*2 þ b*2)1/2 Finally, to obtain the depth of the colour, the total colour difference ΔE was calculated using the natural fabric (polyester) as a reference and was defined as: ΔE ¼ [(ΔL*)2 þ (Δ a*)2 þ (Δb*)]1/2 The K/S values and colour coordinates were obtained from three replicate measurements. 2.9. Fastness testing The colour fastness to light was determined for the polyester fibres according to the Mexican standard NMX-A-105-B02-INNTEX-2010 in a Fadeometer Ci3000 þ using a xenon arc lamp. The exposure time for the sample was 20 h. Additionally, the colour fastness to domestic laun dering was determined according to the Mexican standard method NMXA-074-INNTEX-2005 which is consistent with the ISO-105-C06 stan dard. The colour fastness test was performed at 70� C for 30 min using 50 steel balls. A multi-fibre adjacent fabric containing wool, cellulose ac etate, polyamide, cotton, acrylic and polyester was also used to test the staining of the polyester. The colour change in the fabric was given by grey scale numbers from one to five, with five as the best value.
2.7. Dyeing processes €isa €nen et al., 2001), 1 mg of the an Following Raisanen et al. (Ra thraquinones aloesaponarin I and aloe emodin were (separately) used for each 100 mg of textile. The samples were first dissolved in a solution of sodium phosphate 0.1 M and 10% of Glauber salt. The liquor to fabric ratio was 20:1 and the dyeing was performed using a Thermo Park heater equipped with a digital thermometer. The fabrics were dyed using baths with different pH values (4, 7, 10) adjusted with buffer so lutions. Initially, the dyeing bath temperature was raised from 50 � C to 98 � C. The fabrics were then added to the bath and kept there for 50 min. Subsequently, the dye bath was allowed to cool for 20 min. Afterwards, 3
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Fig. 2. Chemical structures of the components obtained from the A. vera roots (1–5) and solid waste (6).
3. Results and discussion
acetone with a TLC Rf value of 0.23 using n-hexane:acetone 8:2 as the €rntrager eluent. This compound showed an orange colour with the Bo reagent. The 1H NMR spectrum of aloesaponarin-II was similar to aloesaponarin-I except for the lack of a methoxycarbonyl group at the C2 position. Instead of this signal, a doublet at δ 6.99 with meta coupling (J ¼ 2.0 Hz) corresponding to the H-2 proton was observed. The 1H NMR spectrum of compound 4 was similar to the data reported by Cui et al. (2006) for aloesaponarin-II (4). Aloesaponol-I (5) was recovered as pale yellow needles soluble in DMSO. This compound had an Rf value of 0.19 with the mobile phase nhexane: EtOAc 1:1, and a light brown coloration when developed with €rntrager reagent. In its 1H NMR spectrum, a singlet was observed the Bo at δ 2.70, in addition to a singlet at δ 3.85 corresponding to a methyl group (CH3-11) and a methoxycarbonyl group (OCH3-13), respectively. In the aromatic region, two singlets were observed at δ 6.93 and 6.95, together with four doublets of doublets at δ 2.69 (J ¼ 16.7, 7.1 Hz), 2.90 (J ¼ 15.8, 7.1 Hz), 2.95 (J ¼ 17.2, 3.6 Hz) and 3.12 (J ¼ 15.8, 3.4 Hz). These coupling constants are consistent with geminal and vicinal coupling, indicating the presence of four methylene protons separated by an intermediate methine proton. The presence of a multiplet at δ 4.25 confirms the signal of the methine proton. Finally, two large singlets with interchangeable protons, belonging to hydroxyl groups, were observed at δ 5.19 and 10.76 in addition to a signal at δ 15.27 from a hydroxyl group in the β-position to a carbonyl. Since this compound showed different signals than any compound that had been previously isolated, we performed 13C NMR and 2D NMR experiments to confirm its structure. In the 13C NMR spectrum of Aloesaponol-I (5), seventeen carbons were detected. Two signals between δ 205–168 corresponded to carbonyl groups and 10 signals between 167–108 corresponded to ar omatic carbons. In addition, two signals between δ 50–70 typical of oxygenated carbons, were detected, together with three signals below δ 50, identified as saturated carbons. The number of signals detected in the 1H NMR and 13C NMR spectra, the type of couplings observed, and the chemical shifts obtained were similar to methyl 3,6,9-trihydroxy-imethyl-8-oxo-5,6,7,8-tetrahydro-2-anthracenecarboxylate, known as aloesaponol-I (5) (Dagne et al., 1992). Finally, aloe-emodin (6), the main product of the oxidative hydro lysis of the A29 solid residue, was obtained as a reddish orange powder with a TLC Rf value of 0.66 using EtOAc:CHCl3 1:1 as the eluent. In the 1 H NMR spectrum, the following signals were observed: a singlet at δ 4.55, corresponding to the methylene protons of a primary benzyl alcohol (CH2-11), and, in the aromatic region, singlets at δ 7.20 (H-2) and δ 7.59 (H-4) together with three characteristic signals of the aro matic protons in an ABC spin system at δ 7.62 (dd, J ¼ 0.90, 7.40 Hz, H5), δ 7.72 (t, J ¼ 7.90 Hz, H-6), δ 7.30 (J ¼ 0.90, 8.30 Hz, H-7). Finally, two singlets corresponding to the phenolic protons in positions 1 and 8
3.1. Compounds identification All the compounds obtained from the root (1–5) extract and the solid industrial residue (6), were characterized using their 1H NMR spectra, IR, EI-MS, UV data and compared to those in the literature (Fig. 2). Aloesaponarin I (1) consisted of orange needles that were soluble in €rntrager chloroform, which showed a red-orange coloration with the Bo reagent and a TLC retention factor (Rf) of 0.6 with an n-hexane:EtOAc 1:1 mobile phase. This compound was the main compound isolated with a 5% yield from the EtOAc root extract. In the 1H NMR spectrum of aloesaponarin I (1), two singlets were detected, one at δ 2.96 indicating the presence of a methyl group (CH3-11), and another at δ 4.06 corre sponding to a methoxycarbonyl group (OCH3-13). In the aromatic re gion two protons with ortho and meta couplings were present. Each signal appears as a double of doublets at δ 7.30 (J ¼ 8.3, 1.0 Hz) and δ 7.76 (J ¼ 7.4, 1.1 Hz), respectively. In the same region, a triplet at δ 7.62 with ortho coupling to the vicinal protons (J ¼ 7.9 Hz) and a singlet at δ 7.78 were also observed. Finally, in the low field region, two signals were observed, a broad singlet characteristic of a hydroxyl group at δ 10.42 and a singlet at δ 12.92, typical of a hydrogen-bond to the carbonyl group of an anthraquinone ring. The 1H NMR data of aloesa ponarin I (1) were similar with those reported by Yagi et al. (1977). Deoxyerythrolaccin (2) was isolated as an orange amorphous powder soluble in acetone, which showed an orange coloration when developed €rntrager reagent (TLC Rf of 0.55 using n-hexane:EtOAc [1:1] with the Bo as the eluent). In the 1H NMR spectrum of deoxyerythrolaccin, a singlet was observed at δ 2.75, characteristic of a methyl group (CH3-11) and four signals appeared as doublets at δ 6.62 (J ¼ 2.5 Hz), δ 7.05 (J ¼ 2.5 Hz), δ 7.16 (J ¼ 2.3 Hz) and δ 7.55 (J ¼ 2.8 Hz), typical of aro matic protons with meta couplings. A singlet corresponding to a hy droxyl group, analogous to that observed in aloesaponarin-I, was detected (δ 13.30 ppm). Comparing the spectroscopic data to the liter ature indicated that compound 2 was 1,3,6-trihydroxy-8-methyl-9, 10anthraquinone, also known as deoxyerythrolaccin (2) (Yagi et al., 1977). Lacacic acid D methyl ester (3) consisted of an orange amorphous powder soluble in acetone, which was visualized on the TLC as a violet €rntrager reagent (Rf of 0.33 in spot when it was developed with the Bo the solvent system n-hexane:EtOAc 1:1). The 1H NMR spectrum showed signals similar to those observed for aloesaponarin-I, except for the presence of two doublet signals located in the aromatic region at δ 6.64 and δ 7.17 (J ¼ 2.5 Hz) from two protons with meta coupling (H-5 and H7), suggesting the presence of an intermediate hydroxyl group at posi tion 6. The signals observed were similar to those reported for the lacacic acid D methyl ester (3) (Yagi et al., 1977). Aloesaponarin-II (4) was isolated as orange needles soluble in 4
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OH), 11.83 (1H, s, 8-OH), 7.72 (1H, dd, J ¼ 8.1, 7.7 Hz, H-6), 7.62 (1H, dd, J ¼ 7.4, 0.9 Hz, H-5), 7.59 (1H, s, H-4), 7.30 (1H, dd, J ¼ 8.3, 0.9 Hz, H-7), 7.20 (1H, s, H-2), 4.55 (2H, s, CH2-11). 1 H NMR spectral data are available online as Supplementary data.
Table 1 Inhibition zone diameter, minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of metabolites 2 and 3 against X. campestris. Compound
Inhibition diameter (mm) at 10 μg/mL
MIC value (μg/ ml)
MBC value (μg/ ml)
2 3
14.50 � 0.99 10.00 � 0.57
46.87 93.75
373 1500
3.2. Antimicrobial activity results In this work, the antimicrobial activity of compounds 1–6 were evaluated via disc diffusion test and broth microdilution assays against the phytopathogenic bacteria Xanthomonas campestris pv. carotae, Pec tobacterium carotovorum subsp. carotovorum and Pseudomonas syringae pv. pisi. The disc diffusion test was first used to identify the active compounds, and subsequently the plate microdilution test was used to establish the effective dose of active compounds. In the disc diffusion test, only deoxyerythrolaccin (2) and lacaic acid D methyl ester (3) showed activity against X. campestris while being inactive against P. carotovorum and P. syringe. In the plate microdilution bioassay, deoxyerythrolaccin (2) showed an MIC value of 46.86 μg/mL whereas lacaic acid D methyl ester (3) had an MIC value of 93.75 μg/mL against X. campestris (Table 1). To distinguish if the antibacterial activity is bactericidal or bacteriostatic, an aliquot of each compound concentra tion was inoculated into solid medium MH after a prior 18 h of incu bation with X. campestris. Bacterial growth indicates a bacteriostatic effect, while the absence of growth is classified as a bactericidal effect. As seen in Table 1, deoxyerythrolaccin (2) showed bactericidal activity at 375 μg/mL, whereas lacaic acid D methyl ester (3) had a bactericidal effect at 1500 μg/mL. Related to this, it has been reported that anthra quinones can inhibit the activity of the enzymes nicotinamide adenine dinucleotide and mitochondrial succinate oxidase as well as the elec tronic transfer from the bacterial respiratory chain, and the process of dehydrogenation in the bacterium, which can damage the integrity of the membrane, causing loss of the cytoplasm (Cowan, 1999; Balachan dran et al., 2016). This inhibition could be related to the observed ac tivity of deoxyerythrolaccin (2) and lacaic acid D methyl ester (3) in the antimicrobial test. These results suggest some preliminary structure-activity relationships for the anthraquinone structure. The presence of an OH group at C-6 position, as in deoxyerythrolaccin (2) and lacacic acid D methyl ester (3), could influence the antimicrobial activity of both compounds, since this activity was not found on the other compounds, which lack of this group. In contrast, the presence of a methoxycarbonyl in position 2 had a negative effect on the antimicrobial activity, since lacaic acid D methyl ester (3) was less active than deox yerythrolaccin (2). These results arle consistent with previous reports of the activities of rhubarb anthraquinones against Aeromonas hydrophila, where it was observed that the presence of polar groups at the C-3 and C-6 positions in rhein and emodin make these anthraquinones more active than those with less polar groups such as physion and crisophanol (Lu et al., 2011). To the best of our knowledge, there are no previous reports about the antibacterial activity of these compounds against X. campestris with the exception of aloesaponarin-II, which has been reported to be active against X. oryzae pv. oryzae, pathogenic bacteria that affect rice crops, with an MIC value of 38.40 μg/mL (Donghua et al., 2013). However, as
*Neomycin (0.002 μg/mL) was used as positive control.
of the aromatic rings were observed at δ 11.89 and δ 11.83. Based on these results, compound 6 was identified as aloe-emodin as previously reported by Cui et al. (2008). The chemical data of the isolated com pounds are listed below: Aloesaponarin- I (1): Orange neddles; m. p: 199–203 � C; UV λMeOH m� ax : 270, 415, 435 nm. IR (KBr): 3380, 1740, 1700, 1675, 1630 cm 1.1H NMR (CDCl3, 400 MHz) δ 12.92 (1H, s, 8-OH), 10.42 (1H, s, 3-OH), 7.78 (1H, s, H-4), 7.76 (1H, dd, J ¼ 7.4, 1.1 Hz, H-5), 7.62 (1H, t, J ¼ 7.9 Hz, H-6), 7.30 (1H, dd, J ¼ 8.3, 1.0 Hz, H-7), 4.06 (3H, s, H-13), 2.96 (3H, s, H-11). EI-MS m/z 312.06 [Mþ]. Deoxyerythrolaccin (2): Orange amorphous powder; m. p: above 300 � C; UV λMeOH m� ax : 284, 432 nm. IR (KBr): 3400, 1670, 1630, 1600 cm 1.1H NMR ([CD3]2CO, 400 MHz) δ 13.30 (1H, s, 8-OH), 7.55 (1H, d, J ¼ 2.8 Hz, H-5), 7.16 (1H, d, J ¼ 2.3 Hz, H-4), 7.05 (1H, d, J ¼ 2.5 Hz, H-7), 6.62 (1H, d, J ¼ 2.5 Hz, H-2), 2.75 (3H, s, H-11); HRMS m/z 270.053 [Mþ] (calculated for C17H16O6, 270.2369). Lacacic acid D methyl ester (3): Orange amorphous powder; m. p: 270–275 � C; UV λMeOH m� ax : 220, 270, 285, 420, 435 nm. IR (KBr): 3300, 1740, 1715, 1670, 1630 cm 1.1H NMR ([CD3]2CO, 400 MHz) δ 13.29 (1H, s, 8-OH), 7.68 (1H, s, H-4), 7.17 (1H, d, J ¼ 2.5 Hz, H-5), 6.64 (1H, d, J ¼ 2.5 Hz, H-7), 3.93 (3H, s, H-13), 2.69 (3H, s, H-11). EI-MS m/z 328.05 [Mþ]. Aloesaponarin-II (4): Orange neddles; m. p: 250–254 � C; UV λMeOH m� ax : 280, 410 nm. IR (KBr): 3400, 1670, 1630 cm 1.1H NMR ([CD3]2CO, 400 MHz) δ 12.92 (1H, s, 8-OH), 7.59 (2H, m, H-6 and H-7), 7.47 (1H, d, J ¼ 2.3 Hz, H-4), 7.17 (1H, dd, J ¼ 7.6, 2.1 Hz, H-5), 6.99 (1H, d, J ¼ 2.3 Hz, H-2), 2.66 (1H, s, CH3); HRMS m/z 254.056 [Mþ] (calculated for C15H10O4, 254.2375). Aloesaponol-I (5): Yellow neddles; m. p: 248–250 � C; UV λMeOH m� ax : 228, 242, 270, 278, 308, 320, 380 nm. IR (KBr): 3350, 3150, 1710, 1625, 1610 cm 1.1H NMR ([CD3]2SO, 400 MHz) δ 15.27 (1H, s, 6-OH), 10.76 (1H, s, 6-OH), 6.95 (1H, s, H-10), 6.93 (1H, s, H-5), 5.19 (1H, s, 3-OH), 4.25 (1H, m, H-3), 3.85 (3H, s, H-13); 3.12 (1H, dd, J ¼ 15.8, 3.4 Hz, H4b), 2.95 (1H, dd, J ¼ 17.2, 3.6 Hz, H-2b), 2.90 (1H, dd, J ¼ 15.8, 7.1 Hz, H-4a), 2.70 (3H, s, H-11), 2.69 (1H, dd, J ¼ 16.7, 7.1 Hz, H-2a). 13C NMR ([CD3]2SO, 100 MHz) δ 204.18 (C-1), 168.72 (C-12), 166.45 (C-9), 155.59 (s, C-6), 141.32 (s, C-4b), 137.74 (s, C-4a), 137.16 (s, C-8), 125.95 (s, C-7), 117.08 (d, C-10), 115.88 (s, C-8a), 110.76 (s, C-8b), 108.07 (d, C-5), 64.98 (d, C-3), 64.98 (d, C-3), 52.61 (q, C-13); 46.96 (t, C-2), 38.06 (t, C-4), 21.06 (q, C-11). HRMS m/z 316.0941 [Mþ] (calculated for C17H16O6, 316.3124). Aloe-emodin (6): Reddish orange powder; m. p: 223–224 � C; UV MeOH λm�ax : 210, 220, 280 nm. 1H NMR (CDCl3, 400 MHz) δ 11.89 (1H, s, 1-
Table 2 Colour parameters for the fabrics dyed with aloesaponarin-I and aloe-emodin with and without mordants. Compound
Without mordents (direct)
With mordents (post-mordanting method) KAl (SO4)2⋅12H20
1 6
K2Cr2O7
pH
L*
a*
b*
C
H
L*
a*
b*
C
H
L*
a*
b*
C
H
4 7 10 4 7 10
94.74 95.90 99.47 89.81 89.68 88.52
5.03 0.26 10.88 2.10 0.57 1.08
12.52 7.57 38.71 32.70 26.15 19.09
13.50 7.58 40.23 32.15 26.16 19.13
111.91 268.12 285.69 86.27 91.24 86.76
95.73 95.90 98.59 87.45 89.29 83.05
5.28 1.97 5.45 3.05 2.41 6.81
9.83 2.15 27.44 29.44 30.82 25.63
11.17 2.94 27.98 29.60 30.92 26.52
118.26 225.63 281.24 84.08 85.52 75.10
94.26 94.78 98.01 92.46 88.61 88.88
2.29 0.37 5.41 0.47 3.60 2.49
9.64 5.76 25.61 10.65 14.48 14.79
9.92 5.77 26.18 10.66 14.97 15.00
103.62 266.25 281.92 92.98 75.73 80.35
5
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Fig. 3. K/S values of aloesaponarin-I and aloe-emodin.
has been mentioned, aloesaponarin-II did not show antibacterial activity against any of the strains evaluated. The antibacterial activities observed for deoxyerythrolaccin (2) and lacacic acid D methyl ester (3) show that these compounds have potential as natural controls of X. campestris strains.
molecule in solution and also on the affinity of the dye towards the fibre [Drivas et al., 2011]. The colorimetric data, colour strength and colour obtained at different bath pH conditions are shown in Table 2. The (þ) colour values for a* indicate a reddish colour, while negative a* values indicate a greenish colour. Conversely, positive b* values indicate a yellow colour, while negative *b values indicate a blue colour. As can be seen from Table 2, when aloesaponarin-I was applied, negative a* values were observed at pH 4 and 7, indicating that the colour obtained was more greenish compared to the colour obtained at pH 10. Additionally, high luminosity L* values within a small range (from 94 up to 99) were obtained for the three applied pH bath conditions, which slightly increased at higher pH. The b* values went from a yellowish colour for the acidic condition of pH 4 (12.52) to almost colourless with negative values ( 38.73) at basic pH. The effect on the depth of shade data
3.3. Dye properties of aloesaponarin I and aloe-emodin on polyester fabric 3.3.1. Effect of dye bath pH Different bath pH conditions were used for dyeing with aloesaponarin-I and aloe-emodin, in order to compare the shades ob tained and the best conditions for uptake of the material. It is known that the dye bath pH has a strong relationship on the stability of the dye
Fig. 4. Colour shades from aloesaponarin-I and aloe-emodin under different pH bath conditions, without mordants (WM) and by post-mordanting methods using KAl (SO4)2⋅12H2O (M1) and K2Cr2O7 (M2) as mordants. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 6
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Table 3 Fastness properties of the polyester fabrics dyed with aloesaponarin-I and aloe-emodin. Compound
pH
Washing fastness (NMX-A-074-INNTEX-2005) Without mordents
1 6
4 7 4 7 10
4–5 4–5 4 4–5 3–4
Light fastness (NMX-A-105-B02-INNTEX-2010)
With mordents KAl (SO4)2⋅12H20
K2Cr2O7
4–5 4–5 4 4–5 4–5
4–5 4–5 3–4 4 3–4
(expressed as K/S) of aloesaponarin I is shown in Fig. 3, where it is evident that the values decrease as the pH increases. This behaviour suggests that protonation of the functional groups of both the dye and the fibre controlled the dye uptake. Conversely, aloe-emodin yielded yellow shades independent of the bath solution pH (positive values of a* and b*), suggesting a better interaction between the OH groups and the aromatic rings of the dye with the polyester material. Polyester is made of a polymer formed from terephthalic acid and ethylene glycol, which has periodic phenylene groups along the polymer chain. These groups can interact with the hydroxyl groups present in aloesaponarin I and aloe-emodin, leading to hydrogen bonds, and it is even possible that there are π-π interactions between the two materials (fibre-dye). It is clear that an increase in the pH, which prevents the formation of hydrogen bonds, causes a decrease in the colour intensity using aloesaponarin-I. The better adsorption properties of aloe-emodin may be related to the structural size of the molecule, its additional OH group, as well as a better interaction between the terephthalic acid unit of the fibre and the π-electron system of the anthraquinone ring (R€ ais€ anen et al., 2001).
Without mordents
With mordents KAl (SO4)2⋅12H20
K2Cr2O7
4–5 4–5 4 4–5 4–5
4–5 4–5 4 4–5 4–5
4–5 4–5 4–5 4–5 4
polyester fabrics dyed with aloe-emodin (at all the pH values tested) and aloesaponarin-I (at pH 4 and 7). The values showed that no differences in the washing and light fastness of the un-mordanted and mordanted polyester fabrics were seen with aloesaponarin-I, and the dyeing ob tained has excellent colour values (4–5 units on the grey scale where 5 is the highest rating). In the case of aloe-emodin mordanted with KAl (SO4)3⋅12H2O, a noticeable improvement in light fastness and fastness to domestic and industrial washing was observed at pH 7 and 10. In contrast, under the same pH conditions K2Cr2O7 showed a medium value of 3–4 in washing fastness. This may be due to complexation be tween the mordant and the quinone dye instead of the dye attaching to the polyester surface. These results were compared with the values for emodin, an anthraquinone similar to aloe-emodin, but differing due to €is€ the presence of a methyl group at the 3 position (Ra anen et al., 2001). Accordingly, the colour fastness to light and to washing gave similar results for the un-mordanted polyester dyed fabric, and showed uniform colouring with good yields, similar to aloe-emodin. In contrast, it was not possible to perform the colour fastness test on aloesaponarin-I at pH 10 because of its poor fixation properties (Fig. 4). From these results, we conclude that dyeing with aloesaponarin-I with or without mordants at pH 4 gives excellent light and washing fastness on polyester fabrics, whereas for aloe-emodin, aluminium sulfate hydrate is the best mordant for improving its fastness properties.
3.3.2. Effect of the mordants used Aloesaponarin-I and aloe-emodin are phenolic compounds that dissociate to form phenoxide monovalent and/or divalent ions under different pH bath conditions. Improved solubility was observed for both compounds as the pH increased. According to Drivas et al. (2011), who worked with the anthraquinones purpurin and alizarin, this change in colour and increase in solubility is due to the ionization of the hydroxyl groups, forming salts with better solubility in water. However, the ionization of the functional groups may be favourable or unfavourable depending on their interaction with the structure of the fibre. This interaction can be improved by the presence of metallic mordants, giving better colour fixation with the fibre, deeper shades and better colour fastness properties. In this study, a post-mordanting method was used and KAl(SO4)3⋅12H2O and K2Cr2O7 were used as mordants. From the colorimetric data for the aloe-emodin staining, deeper yellow shades were obtained using KAl(SO4)3⋅12H2O compared with less intense yel low shades on polyester obtained using K2Cr2O7 as the mordant (Fig. 4). The brightness of the shades may be due to better absorption of the dye and easy formation of metal complexes between the mordant and the fabric. However, the fabrics dyed with aloe-emodin showed deeper yellow shades with positive b* values (from 10.65 up to 32.07) for all the methods studied (Fig. 4). The strongest colour was found with KAl (SO4)3⋅12H2O as the mordant. In contrast, when using aloesaponarin-I greenish yellow shades were obtained using K2Cr2O7, suggesting a bet ter interaction between this mordant and the anthraquinone structure. This interaction becomes weaker as the solubility of aloesaponarin-I increased (negative b* values), becoming almost colourless. Thus, by using a post-mordanting method, better colour fixation can be achieved in polyester fabric dyeing with aloe emodin using KAl(SO4)3⋅12H2O as the mordant and aloesaponarin-I and K2Cr2O7 as the dyes.
4. Conclusions The root of Aloe vera and the industrial residue produced during the aloin extraction are rich in phenolic components, and five metabolites were extracted of the root and one of the industrial waste. Desoxyery throlacin and lacaic acid D methyl ester, which are present in its roots showed antimicrobial properties against the phytopathogenic bacteria X. campestris. Additionally, the main products aloesaponarin-I and aloeemodin have excellent dyeing and colour fastness properties for poly ester fabric. Aloesaponarin-I dyes polyester bright yellow and aloeemodin produces a deeper yellow colour. The uptake of aloesaponarin-I was found to be more sensitive to pH than the uptake of aloe-emodin. Declaration of interest None. Author contributions RBA and GCE obtained the financial support, designed all the ex periments and wrote the manuscript. PCA performed the antimicrobial tests and dyeing experiments. MCF and BMVK gave technical support to PCA for the antimicrobial assays. PQO and FMC analysed the colori metric data and CIELAB parameters. JAYP and PSP supervised the isolation and identification of the metabolites. All the authors have read and approved the final manuscript.
3.3.3. Fastness properties of the dyed fabrics Table 3 shows the fastness rating with respect to light and to do mestic and industrial washing of the un-mordanted and mordanted 7
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Acknowledgements
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