Antibacterial and Antioxidant Compounds from the Root Extract of Aloe debrana

This study was conducted to isolate and identify the chemical compounds from the roots of Aloe debrana (L.) and evaluate their antioxidant and antibacterial activities. From the acetone (99.5%) extract of the roots of this plant, four anthraquinones, such as chrysophanol (1), asphodeline (2), aloesaponarin I (5), and laccaic acid D-methyl ester (6), and a new catechol derivative, 5-allyl-3-methoxybenzene-1,2-diol (3), were isolated and elucidated by different chromatographic and spectroscopic methods together with linoleic acid (4), respectively. Compounds 2, 3, and 4 were reported here for the first time from this plant and compound 3 from the genus Aloe. The compounds were evaluated for their antioxidant activity using H2O2 and DPPH assays and bactericidal activity against S. aureus and E. coli. Compounds 3 and 6 showed highest antioxidant activities with IC50 values of 19.38 ± 0.64 and 32.81 ± 0.78 μg/mL in DPPH, and 28.52 ± 1.08 and 27.31 ± 1.46 μg/mL in H2O2, respectively. The isolated compounds also demonstrated considerable activity towards S. aureus. Among these compounds, compound 3 exhibited the highest activity (91.20 ± 0.12% and 9.14 ± 0.93 mm at 1.0 mg/mL) against this bacterium. The overall results suggest that the isolated compounds may be considered as potential sources of the bioactive agents to be used in the pharmacological, food, and other industries. Moreover, their high sensitivity against S. aureus may also support the use of A. debrana plant in the traditional medicine to treat wounds. Therefore, the isolated compounds are responsible for medicinal properties of this plant.


Introduction
Aloe (Family: Asphodelaceae) has found wide recognition for its medicinal and cosmetic uses [1].Many researchers from diferent countries have shown interest to study on Aloe species because of their bioactive compounds [2], which are responsible for medicinal properties of the plants and manysided activities.Te genus is widespread in the Madagascar, Arabian Peninsula, Jordan, various Indian Ocean islands, and many African countries, and its few species are cultivated in Japan, India, Australia, America, Hawaiian Islands, Caribbean, and Mediterranean regions [3][4][5].Approximately 83 Aloe plants occur in Eastern Africa [6], of which 46 grow naturally in dry and grasslands of Ethiopia with 16 of them being endemic [7].Aloe debrana Christian is a stemless evergreen endemic medicinal Aloe plant of Ethiopia, which commonly grows in the areas of grassland on thin soil overlying basalt, usually on gentle slopes between 2,400 and 2,700 m above sea level in Shewa, Gojam, and Wello regions [4,8].
In Ethiopian traditional herbal medicines, A. debrana is used for the treatment of wounds, eye infammation, malaria, excessive pain, gastrointestinal, and dermatological problems [8].It is useful in water and soil conservation [9], to stop breastfeeding, and it was examined as good thickening agent for printing polyester and cotton with disperse dyes.Te leaf latex of A. debrana is used traditionally as laxative, antidiabetic, and antimalarial agents.It is also used for cleansing the blood, healing of wounds, and cleaning of eyes injured accidently [4].Farmers also use this to cure the wound of the nape of their oxen made during plough [7].
Previously, various types of natural compounds, such as alkaloids, anthraquinones, pre-anthraquinones, naphthoquinones, anthrones, oxanthrones, steroids, chromones, pyrenes, and favonoids, were isolated from Aloe plants [2,5].Moreover, only few compounds were identifed from A. debrana plant.Terefore, the objectives of this work were to isolate compounds from the acetone extract of A. debrana roots and elucidate their structure by using chromatographic and spectroscopic methods, respectively.In addition, the antioxidant and antibacterial potentials of the compounds, which may be useful in foods, pharmaceuticals, and other industries, were also assessed and reported.

General.
Column chromatography (CC): silica gel 200-400 mesh Merck.Sephadex chromatography (SC): LH-20 (200 g).Tin-layer chromatography (TLC): a readymade 0.2-mm-thick layer of silica gel GF 254 (Merck) coated on aluminium plate: detection by UV light at 254 nm, and by using vanillin solution and heating for few minutes or by using iodine vapour.UV-Vis spectra were recorded on Perkin-Elmer Lambda 750 UV/VIS NIR spectrophotometer (200-600 nm).IR spectra were obtained by Perkin-Elmer Spectrum 400 FT-IR/FT-FIR spectrometer.NMR spectra were performed on a Bruker Avance Neo 500 MHz NMR spectrometer in either CDCl 3 or DMSO-d 6 solutions with TMS as internal standard.

Plant Material.
Te fresh roots of A. debrana were collected from Kube Bedesa Koricho, Weliso Woreda, Oromia, Ethiopia, which is 117 km far from south-west of Addis Ababa near Weliso town (located at 8 °32′N 37 °58′E latitude and longitude, respectively) in April 2019.Te plant material was authenticated by Professor Legesse Negash, and a voucher specimen (No. 00A1) was deposited at Ethiopian National Herbarium of the Addis Ababa University.

Antioxidant Activities.
Antioxidant activities of the isolated compounds were evaluated by using DPPH and H 2 O 2 assays at the fnal concentrations within the range of 31.25 to 1000 μg/mL.Ascorbic acid, a well-known antioxidant compound, was used as a positive control in all the assays.DPPH and H 2 O 2 were obtained from School of Pharmacy of the Faculty of Pharmaceutical Sciences, Shoolini University, India.Te assay was also carried out at this school.

DPPH Assay.
Te antioxidant properties of the isolated compounds were determined by DPPH assay [11].Tree millilitres of standard solution of each of the concentrations from 31.25 to 1000 μg/mL was mixed with 1.0 mL of 90 μM DPPH solution in MeOH to make the test solutions.Ascorbic acid was prepared in same way as the test samples.A mixture of 3 mL of MeOH and 1 mL of DPPH solution was used as negative control.Each assay was performed three times, and the prepared samples were incubated in the dark at 37℃ for about 30 min; then, the absorbance for each was determined at a wavelength of 515 nm using a spectrophotometer.Antioxidant activity of all the test samples was expressed as IC 50 (μg/mL).

H 2 O 2 Assay.
Te scavenging activity of the isolated compounds was also investigated three times by H 2 O 2 assay [12].Te concentrations from 31.25 to 1000 μg/mL of each of the test samples and the reference antioxidant compound, and ascorbic acid in deionized water was dissolved in 3.4 mL of 0.10 M phosphate bufer of pH 7.4 and mixed with 0.60 mL of 40 mM H 2 O 2 solution.After few minutes, the absorbance of the mixture was determined at 230 nm using a spectrophotometer.Negative control was prepared by replacing the test samples with distilled water.Antioxidant activity of all test samples was expressed as IC 50 (μg/mL).Tese strains were obtained from KPC Medical College, and the assay was carried out at the Bose Institute, Kolkata, India.

Bacterial Growth Inhibition
Weighed aliquots of each dry sample were dissolved in DMSO to give diferent concentrations (0.25, 0.50, 0.75, and 1.0 mg/mL).From an overnight grown culture in Luria-Bertani (LB) broth media at 37℃, each of 5, 10, 15, and 20 μL of inoculums was separately added to 1 mL fresh culture medium.LB with only samples was considered as blank and LB with only inoculums as controls in the experiments.All the test samples were then incubated for about 48 h at 37℃.Finally, the growth of the bacteria was measured using a UV-Vis spectrophotometer at 600 nm [13].Te sensitivity of the bacterial species to the samples was determined by measuring the percent inhibition of the bacterial growth.Additionally, disc difusion analysis was also performed according to the National Committee for Clinical Laboratory Standards (NCCLS) [14] against the same pathogens, to assess the bactericidal activity of the compounds 3 and 6, which showed good antibacterial activity using the method previously described.For this method, 6-mm-diameter sterilized Whatman No. 1 flter paper discs were saturated with diferent concentrations (0.50 and 1.0 mg/mL) of these samples and placed on nutrient agar (NA) plates.Te plates were pre-inoculated with each of the test strain in suspension (10 7 -10 8 CFU/mL) of bacteria and then incubated for about 24 h at 37 ℃.After incubation, diameters of their inhibition zones (DIZ) in millimetres were measured.Te antibiotic gentamicin was used as a control (positive) against the selected bacterial strains.

Statistical Analysis.
All experimental results were expressed as mean value and standard deviation (x ± SD) of repeated trials (three times for all) and determined using Excel software.Te IC 50 values were also determined using Excel software by plotting inhibition-concentration curves.A comparison of the group means and the diference between the groups (p values <0.05) were verifed by Student's t-test.

Characterization of the Isolated Compounds.
Structure elucidation of the compounds was performed by employing various spectroscopic techniques and by comparing with spectral data reported for the same compounds.Compound 1 was isolated as yellow amorphous solid.By comparing its physical properties, UV (MeOH), IR (KBr, v), and NMR data with the literature values, the compound was identifed as chrysophanol [15].Chrysophanol is a known anthraquinone (phenolic compound) isolated from various organs and species, which shows diverse biological activities that include antimutagenic, anti-infammatory, antiprotozoal, immuno-stimulatory, spasmolytic, antidiabetic, antigenotoxic, and antimicrobial efects [6,10,16,17].It is also active against HIV-1 protease and inhibits the replication of poliovirus, induced necrosis in human liver cancer cells, and well-known potent photosensitizer [10,17].Compound 2 was obtained as orange powder.By comparing its physical properties, UV (MeOH), IR (KBr, v), and NMR data with the literature values, the compound was identifed as asphodeline [18].
Compound 4 was isolated as colourless oil.By comparing the physical properties, IR and NMR data of this compound with the literature values, it was identifed as linoleic acid [21].Linoleic acid is a known useful unsaturated (omega-6) fatty acid that has been reported from various medicinal plants, including Artemisia integrifolia L. [22] and Mesua ferrea L. [23].Compound 5 was obtained as yellow powder.By comparing physical properties, UV (MeOH), IR (KBr, v), and NMR data of this compound with those reported in the literature, it was identifed as aloesaponarin I and it is reported to show moderate antiplasmodial activity [6,24].
Compound 6 appeared as a yellow amorphous solid.Its UV spectrum (MeOH) exhibited absorption maxima at 219, 285, 345, and 433 nm, the typical characteristic of anthraquinones [24].Its IR (KBr, v) spectrum showed the presence of hydroxyl group (3404 cm −1 ), aromatic ring (1568, 1441 cm −1 ), ester carbonyl (1728 cm −1 ), and ketone carbonyl (1639 cm −1 ) functionalities. 1 H-NMR spectrum (500 MHz) of this compound (Table 2) showed one chelated OH group at δ H 13.10, two methyls, and only three aromatic nonequivalent methine protons.Tese methine protons are Evidence-Based Complementary and Alternative Medicine meta-coupled protons at δ H 7.07 (1H, d, J � 2.5 Hz, H-5) and δ H 6.60 (1H, d, J � 2.5 Hz, H-7), consistent with the presence of OH group at C-6, and the third proton at δ H 7.60 (1H, s, H-4) was assigned to H-4 of a 1,2,3-tri-substituted benzene ring.Evidence of a substituent at C-1 was deduced from the presence of a methyl (δ H/C 2.61 (s, 3H)/20.3). 13C-NMR spectrum (Table 2) along with DEPT-135 displayed 17 carbon signals as in compound 5.In the same way as compound 5, the presence of methyl ester at C-2 was confrmed from a methoxy signal at δ H/C 3.87 (s, 3H)/53.0 and δ C 167.7 for ester carbonyl.Te only diference of the two is that, in compound 6, one of the aromatic ring methines of compound 5 was changed to oxygen-bearing aromatic methine carbon (δ C 164.7, C-6).Tis and the connectivity of the protons and carbon resonances of 6 were also supported by a series of the 2D-NMR ( 1 H-1 H COSY, HSQC, and HMBC) spectra.Tere is a correlation only between H-5 (δ 7.07) and H-7 (δ 6.60) in the COSY spectrum, indicating the absence of a proton on C-6 and H-4 is on the tri-substituted anthraquinone benzene ring (Table 2, Figures 1 and 2).Te HMBC spectrum of this compound showed strong correlation between chelated OH group proton at δ 13.10 with the carbons at δ 108.8 (C-7), 165.0 (C-8), and 110.7 (C-12) establishing the site of attachment of the chelated OH to C-8 of benzene ring.Another key strong correlation observed was between aromatic proton signal of tri-substituted anthraquinone benzene ring at δ 7.60 (H-4) with methyl ester substituted aromatic carbon signal at δ C 130.2 (C-2), ketone carbonyl carbon at δ 182.4 (C-10), and other aromatic carbon signal at δ 123.0 (C-13), which established the site of attachment of methyl ester to the aromatic ring (Figure 1).Te NMR spectral data of compound 6 were found in agreement with the NMR spectral data reported in the literature for laccaic acid D-methyl ester [25].Laccaic acid D-methyl ester is an anthraquinone (phenolic compound) previously identifed from A. secundifora roots and reported that it has no cytotoxicity [6].Compounds 2, 3, and 4 were reported here for the frst time from this plant and 3 from the genus Aloe and other plants. .However, compounds 1 and 2 exhibited lowest activities.Te high antioxidant activity of compound 3 may be due to its hydroxyl groups, and that of compounds 5 and 6 may be attributed to their number of hydroxyl and carbonyl groups as clearly discussed in the study published by Ben Ammar et al. [26].

Antioxidant Activities of the
According to the available literature, phenolic compounds or their derivatives were reported to exhibit antioxidant activities [27].Researchers demonstrated that chrysophanol has no activity against DPPH and ABTS + radicals [28].However, other researchers showed that the compound had a scavenging efect on DPPH radical (IC 50 value of 26.56 μg/mL).Tis big diference may be from the errors in the operation and the excessive diferences in experimental conditions [29].When compared to the results obtained in our study, compound 1 and its dimer (2) were less active against the radicals.However, to the best of our knowledge and according to literature survey, there was no previous antioxidant activity report for other compounds.

Bacterial Growth Inhibition of the Isolated Compounds.
In Tables 4 and 5, results of the bactericidal activities (bacterial growth inhibition) of the isolated compounds against the investigated strains of bacteria are shown.Percent inhibition of the bacterial growth demonstrated that all the compounds inhibited the mean growth of a Gram (+) bacterium, S. aureus (13.82 ± 0.27 to 91.20 ± 0.12% inhibition), whereas they showed weak growth inhibition of E. coli (3.06 ± 1.10 to 7.18 ± 1.01% inhibition) evaluated at the fnal concentrations within the range of 0.25 to 1.0 mg/mL.Among the identifed compounds, the highest inhibition was observed for compounds 3 and 5 against the growth of S. aureus in all the tested concentrations.Te diameter of inhibition zones (DIZ) of compounds 3 and 5 was 6.87 ± 0.93 and 9.14 ± 0.93 mm, as well as 6.55 ± 0.87 and 8.21 ± 1.24 mm for S. aureus at 0.5 and 1.0 mg/mL concentrations, respectively.Te results indicate the susceptibility of this bacterium to the compounds.Moreover, compound 3 demonstrated activity (2.91 ± 1.06 mm) towards E. coli at 1.0 mg/mL.However, compound 5 showed no activity towards E. coli at both concentrations.
Phenolic compounds or their derivatives were reported to show antibacterial activities [27].Literature searches on the antibacterial activities of the isolated compounds in the present study indicated that compounds 1 and 5 have been reported to possess antibacterial activities [10,30].Te result for compound 1 is almost comparable (moderately active) with the results reported for the same compound against S. aureus with DIZ of 10 mm at 50 mg/mL [30] and 13 mm at 1.0 mg/mL [10].It was also moderately active
On the other hand, Abdissa et al. [30] reported the greatest antibacterial potential for compound 5 evaluated at 50 mg/ mL concentration.Te compound was highly active towards B. subtilis (DIZ, 27 mm) than the reference antibiotic drug, gentamicin (DIZ, 25 mm).It was also more active against E. coli, P. aeruginosa, and S. aureus with DIZ of 22, 21, and 18 mm, respectively.When compared to the results obtained in our study, this compound was less active (8.21 ± 1.24 mm) against S. aureus and not active towards E. coli at 1.0 mg/mL.Tese variations may be due to the concentrations used for testing the activities.However, to the best of our knowledge, there is no prior report on antibacterial activity of compounds 2 and 3 against any bacterial strains and compound 6 against S. aureus and E. coli.

Conclusion
In this study, six compounds were isolated and elucidated from A. debrana roots.Compounds 2, 3, and 4 were reported here for the frst time from this plant and compound 3 from the genus Aloe and other plants.Te compounds such as 3, 5, and 6 exhibited high antioxidant activities.In addition, the tested compounds demonstrated appreciable growth inhibition of S. aureus.Among them, the highest inhibition observed was for compounds 3 and 5.However, no signifcant activity was reported for any of the isolated compounds against E. coli.Te overall results suggest that the isolated compounds may be useful in foods, pharmaceuticals, and other industries.Moreover, their high sensitivity against S. aureus may also support the use of A. debrana plant in the traditional medicine to treat wounds.Terefore, the isolated compounds are responsible for medicinal properties of this plant.Furthermore, studies on in vivo efcacy tests and toxicity of A. debrana plant would be required to ensure its use for the treatment of wounds and other diferent ailments (supplementary fles) (available here).
Assay.Te bacterial growth inhibition assay of the isolated compounds was performed using cultures of the Gram (+) (Staphylococcus aureus ATCC 2 Evidence-Based Complementary and Alternative Medicine 25923) and the Gram (−) (Escherichia coli ATCC 25922).

Figure 2 :
Figure 2: Structures of compounds isolated from A. debrana roots.

Table 3 :
Antioxidant efect of the compounds isolated from A. debrana roots and standard (DPPH and H 2 O 2 ) assays.

Table 4 :
Antibacterial activity of the compounds isolated from A. debrana roots.

Table 5 :
Diameter of inhibition zones of compounds 3 and 5.Results are presented as mean ± SD (n � 3).DIZ, diameter of inhibition zone; NI, no inhibition.
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