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. 2024 Mar 14;29(6):1297. doi: 10.3390/molecules29061297

The Indiscriminate Chemical Makeup of Secondary Metabolites Derived from Endophytes Harvested from Aloe barbadensis Miller in South Africa’s Limpopo Region

Mpho Mamphoka Nchabeleng 1, Thierry Youmbi Fonkui 1, Green Ezekiel 1,*
Editor: Josphat Matasyoh1
PMCID: PMC10974520  PMID: 38542933

Abstract

The efficacy of 23 bacterial isolates obtained from surface-sterilized stems and leaves of three medicinal plants (Aloe barbadensis Miller, Artemisia afra, and Moringa oleifera) was investigated in an endeavour to prevent the growth of Mycobacterium bovis using the cross-streak method. Endophytes were isolated by incubating sterile plant materials on nutrient agar at 30 °C for 5 days. Two isolates showing activity were subsequently utilized to produce the extracts. Whole-genome sequencing (WGC) was used to identify the isolates. Secondary metabolites produced after 7 days of growth in nutrient broth were harvested through extraction with ethyl acetate. The extracts were chemically profiled using gas chromatography–high resolution time-of-flight mass spectrometry (GC–HRTOF-MS). NCBI BLAST search results revealed that the isolated endophytes belonged to the Pseudomonas and Enterobacter genera, based on WGC. Two endophytes, Aloe I4 and Aloe I3–I5 from Aloe barbadensis, exhibited potency based on the cross-streak method. The metabolite profiling of the selected endophytes identified 34 metabolites from Aloe I4, including ergotamine, octadecane, L-proline and 143 other metabolites including quinoline and valeramide, which inhibit microbial quorum sensing. These findings suggest that bacterial endophytes from medicinal plants, particularly Aloe barbadensis, hold promise as sources of antimycobacterial agents for human health applications.

Keywords: endophytes, secondary metabolites, Mycobacterium bovis, tuberculosis, medicinal plants

1. Introduction

The healthcare sector has become greatly concerned with the global growing resistance to antimicrobial and chemotherapeutic medications in recent years. There is a pressing need to explore and create novel anticancer and antimicrobial agents [1]. As a result, there has been a renewed passion for investigating microorganisms as a potential repository of new discoveries. This is based on the remarkable success of bacterial compounds in the production of effective antimicrobial agents, anticancer drugs, and agricultural pesticides [2]. As an alternative to antibiotics, bacterial bioactive chemicals may play a role in microbe-to-microbe and microbe-to-host reactions, according to a growing body of research [3].

The use of medicinal plants to address human health problems dates back many years, and the benefits of medicinal plants in the health care system are undeniable. Plants are important and reliable reservoirs of bioactive compounds, as evidenced by their prolonged and continuous use in addressing the pressing issues in human health [4]. Aloe barbadensis Miller, commonly known as aloe vera, Artemisia afra, known as wormwood, and Moringa oleifera, generally named drumstick tree, are three important medicinal plants used in South Africa to address various clinical conditions. Found in tropical and temperate regions, aloe vera is traditionally used for the treatment and prevention of various ailments such as sunburns, wounds, skin disorders, diabetes, ulcers, and other conditions [5]. Recent pharmacological data indicated that Aloe vera displayed anticancer action, digestive protective activity, and antimicrobial properties [6]. Artemisia afra, on the other hand, is commonly used as an infusion for the treatment of malaria and ailments such as colds, fever, influenza, sore throats, pneumonia, and many other ailments [7,8]. The antibacterial activity of wormwood against Mycobacterium tuberculosis has been documented [9]. Moringa Oleifera is another important medicinal plant harbouring bioactive substances in its seeds, leaves, flowers, and pods that is used to alleviate symptoms such as joint pains, malnutrition, and headaches, with many applications in the food sector [10]. Moringa is considered an effective agent against hypocholesterolaemia and hypolipidemia and has also shown antimicrobial properties against various human pathogens including E. coli and S. aureus [11]. Medicinal plants, like any other plants, are generally susceptible to abiotic and biotic stress conditions. To manage and control these attacks, plants often draw benefit from their symbiotic relationship with microorganisms found in their tissues [12]. Endophytes (bacteria and fungi) are suitable suppliers of bioactive chemicals that do not only benefit these plants but have also found use in disease control.

Bacterial endophytes reside within plants’ internal tissues without any notable signs of negative effects on the host. Endophytes have characteristics which are beneficial to their host plants, such as promoting development, maintaining protection against insects and pests, exhibiting antimicrobial properties against plant pathogens, and aiding in regulating stress. They facilitate global nutrient cycling and the differentiation between host health and disease states [13]. They also serve as valuable sources of biotechnologically significant molecules, with numerous biocompatible and therapeutic substances. These organisms function as reservoirs for distinct bioactive metabolites, including alkaloids, phenolic acids, steroids, and tannins, with antibacterial, insecticidal, and anticancer properties, [14] to name a few.

Secondary metabolites play a crucial role in mediating microbial interactions in natural environments, exhibiting a wide range of activities which can span from competitive to cooperative dynamics. Instances of such interactions encompass the synthesis of bioactive secondary metabolites. It is worth noting that these metabolites exhibit variable degrees of specificity, wherein certain metabolites exhibit a broad-spectrum effect by targeting a large range of molecular targets, while others exhibit a narrow-spectrum effect by targeting a more limited scope of molecular targets [4]. The exploration of endophytic secondary metabolites with bioactive properties has gained increasing attention, owing to their various bioactive capabilities and wide range of structural categories. Currently, only a few plants have been studied to explore their endophytic biodiversity and ability to produce bioactive secondary metabolites. In effect, every medicinal plant species on the planet is a habitat for multiple varieties of endophytic bacteria. Endophytes can undertake species-specific interactions and co-evolve with their plant hosts.

The best-known medicinal usage of Aloe barbadensis Miller, a succulent plant which thrives in dry and subtropical climates, is in Ayurvedic, homoeopathic and allopathic medicine. Throughout history, diverse nations have used aloe vera extensively for various purposes. These include using aloe vera to decrease perspiration, administering the plant orally to manage diabetes, and employing the plant to alleviate a variety of gastrointestinal disorders. It has been utilized for managing burn injuries, minor lacerations, genital herpes, and eczema. The leaves of this medicinal plant are abundant in vitamins, minerals, natural sugars, enzymes, amino acids, and various bioactive molecules. These molecules possess soothing, aperient, anti-inflammatory, antioxidant, antimicrobial, antihelmenthic, antifungal, aphrodisiac, antiseptic and cosmetic properties. Given its healing and rejuvenating properties, cosmetic companies use this plant widely. This study aimed to screen, identify, and characterize the secondary metabolites associated with two bacterial endophytes originating from native medicinal plants in Limpopo, South Africa.

2. Results

In this study, twenty-three (23) bacterial endophytes were isolated from the stems and leaves of the abovementioned medicinal plants. Seven (7) bacterial endophytes were isolated from Moringa oleifera, nine (9) from Artemisia afra and seven (7) from Aloe vera. The isolates were then screened for antimicrobial properties, and the chemical profiles of biologically active isolates were studied and are presented below.

2.1. Antimicrobial Susceptibility of Mycobacterium bovis

The cross-streak method used to measure antimicrobial activity revealed that M. bovis was sensitive to secondary metabolites produced by some bacterial isolates. Of the 23 endophytic bacteria tested, 21 showed no activity against the test organism M. bovis, while two isolates, Aloe I4 and Aloe I3–I5, showed clear areas of inhibition against M. bovis (Figure 1).

Figure 1.

Figure 1

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Bar graph showing the antimicrobial properties of the isolated bacteria endophytes from medicinal plants against Mycobacterium bovis. Twenty-one isolates showed no inhibition and two isolates showed inhibition, with isolate Aloe I3/5 exhibiting total inhibition. (0): no inhibition, (10): inhibition observed, (100): complete inhibition or no growth of M. bovis at all.

An inhibitory zone of 5 mm was recorded with isolate Aloe I4, and no growth of M. bovis was observed in the Petri dish treated with Aloe I3–I5 (Figure 2). The complete absence of growth or total inhibition noted here could be attributed to the chemical profile of the isolate.

Figure 2.

Figure 2

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Photograph of the cross-streak method showing the antimicrobial activity of isolate Aloe I4 (left) with a 5 mm area of inhibition (A: Aloe I4, B: M. bovis; C: Zone of inhibition) and Aloe I3/5 (right) showing no growth of M. bovis after 5 days of incubation (A: Aloe I3–I5; B: M. bovis).

Our findings corroborate similar observations in the literature [14]. Previous studies have proven that endophytes produce active compounds which exhibit antibacterial, antifungal, antiviral, antisuppressant, and antioxidant properties [15]. According to Lertcanawanichakul and Sawangnop [16], the cross-streak method is preferred over the agar well diffusion method, but it can have drawbacks as the margins of the inhibition zones are usually indistinct, making it difficult to obtain quantitative data. This was experienced in this study; however, with the clear zones/areas observed on the plates containing Aloe I4 and Aloe I3–I5, compared to the other 21 isolates (Figure 2), it could be deduced that they have antimicrobial activity against Mycobacterium bovis. Other methods, such as the gel well diffusion method, could be employed to further characterize the isolates’ bioactivity against test species. Endophytes and medicinal plants produce vital secondary metabolites which could be used for human benefit.

2.2. Isolation and Characterization of Endophytes

Surface sterilization aids in the removal of potential pollutants (such as soil) and the promotion of internal microorganismal growth [17]. As no bacterial colonies were seen on any of the control plates, the surface sterilization of all plant parts was deemed successful. Various colonies were observed on the plates, with pigmentation varying between white, cream white, yellow, pinkish, and pale yellow. The endophytic isolates varied in texture between viscid, pasty, moist, mucoid, and dry. In this study, twenty-three (23) bacterial endophytes were isolated from the stems and leaves of the abovementioned medicinal plants. Seven (7) bacterial endophytes were isolated from Moringa oleifera, nine (9) from Artemisia afra, and seven (7) from Aloe vera. Our findings corroborate those of Strobel [18], who reported the successful isolation of endophytes from plant parts such as roots, leaves, and stems of Rhyncholacis penicillata. Microscopy analysis revealed thirteen (13) Gram-negative rods and ten (10) Gram-positive rods and cocci isolates. Host plant species, maturity, territorial and ecological distribution, season of sample collection, exterior sterilization method, and growth conditions are all variables which can impact the diversity and dispersion of bacterial endophytes in plants [19]. Several factors influence the distribution and variety of bacterial endophytes in plants, including host plant species, age, plant tissue type, geographical and habitat distribution, sampling season, surface sterilization method, growth media, and culture conditions [19]. The morphological characteristics mentioned here are insufficient for successfully identifying and characterizing bacterial endophytes or species, so molecular techniques such as whole-genome sequencing (WGS) were used to fully identify the species in terms of phylogeny, morphology, virulence, and other important factors [20].

2.3. Molecular and Phylogenetic Identification of Aloe I4 and Aloe I3–I5

Whole-genome sequence (WGS) analysis has become an important tool for determining bacterial relationships and is commonly utilized for the identification of microorganisms. It is used frequently to determine phylogenetic relationships between organisms [21]. De novo genome assemblies were used in this project for the molecular and phylogenetic identification of the isolates. According to the NCBI BLAST results for the Aloe I4 and Aloe I3–I5 gene sequences, the isolates belong to two different bacterial genera: Pseudomonas and Enterobacter. The WGS results of the isolates (Table 1 and Table 2) were submitted to the NCBI database for genome sequences. Enterobacter sp. I4, the whole genome shotgun project, was deposited on GenBank (https://DDBJ/ENA/GenBank (accessed on 27 March 2022)) and assigned a Bio-sample number SAMN26660210 and Bio-project number PRJNA816151 under the accession JALBUO000000000. Pseudomonas sp. I3–I5, the whole genome shotgun project, was deposited on GenBank (https://DDBJ/ENA/GenBank (accessed on 27 March 2022)) and assigned a Bio-sample number SAMN26660159 and Bio-project number PRJNA816147 under the accession CP093940.

Table 1.

De novo genome assembly summary metrics for Aloe I4. Primary contigs represent pseudo haplotype assemblies, while haplotigs represent fully phased and assembled regions of the genome.

Contig Type Polished Contigs Maximum Contig Length Mean Contig Length Median Contig Length N50 Contig Length Sum of Contig Lengths e-Size (Sum of Squares Number of Circular
Contigs
Primary
contigs		 3 2,598,250 1,572,706 1,434,460 2,598,250 4,718,120 1,966,539 0
Haplotigs 1 10.181 10.181 10.181 10.181 10.181 10.181 N/A
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Table 2.

De novo genome assembly summary metrics for Aloe I3–I5. Primary contigs represent pseudo haplotype assemblies, while haplotigs represent fully phased and assembled regions of the genome.

Contig Type Polished Contigs Maximum Contig Length Mean Contig Length Median Contig Length N50 Contig Length Sum of Contig Lengths e-Size (Sum of Squares Number of Circular Contigs
Primary
contigs		 1 4,840,834 4,840,834 4,840,834 4,840,834 4,840,834 4,840,834 1
Haplotigs 3 17.724 15.141 16.977 16.977 45.425 15.792 N/A
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According to Menpara and Sumitra [22], dominating endophytes, particularly in medicinal plants, belong to genera such as Enterobacter, Acinetobacter, Staphylococcus, and Pseudomonas. The two bacterial endophytes identified in this project belonged to the Enterobacter and Pseudomonas genera. The BLAST results showed that the bacterial endophyte Aloe I4 has 100% similarity to several Enterobacter species, but the top hit was Enterobacter cloacae ATCC 13047. Other species included Enterobacter mori LMG 25706, Enterobacter bugandensis EB-247, and Enterobacter dykesii EIT. It also showed 92% similarity to Enterobacter hormaechei subsp. And Enterobacter Xiangfangensis LMG 27195. Two outgroup species that are closely related to Enterobacter were also revealed, namely Klebsiella quasipneumoniae 01A030 and Kluyvera cryocrescens NBRC 102467 (Figure 3). Enterobacter cloacae (E. cloacae) can be found in numerous places, such as sewage, soil, and food [23]. E. cloacae, E. aerogenes and E. sakazakii are species that are commonly present in clinical materials. Enterobacter colonies are either non-pigmented or yellow pigmented [24]. In this project, the colonies of Aloe I4 appeared yellow pigmented. Due to the 100% phylogenetic similarity of Aloe I4 and Enterobacter cloacae ATCC 13047, it was assigned the strain name Enterobacter cloacae I4.

Figure 3.

Figure 3

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Enterobacter cloacae I4 phylogenetic tree based on de novo genome assembly analysis. The numbers beneath the branches represent bootstrap support levels based on 100 replications.

Pseudomonas species are frequently found in plants and have been isolated from a variety of plant parts and tissues [25]. Isolate Aloe I3–I5 showed 99% similarity to Pseudomonas fulva DSM 17717 and Pseudomonas parafulva DSM 17004. It also showed 85% similarity to Pseudomonas sichuanensis WCHPS060039, 70% similarity to Pseudomonas reidholzensis CCOS 865, and 63% similarity to Pseudomonas putida NBRC 14164 (Figure 4). Because Aloe I3–I5 showed a very close relation to Pseudomonas fulva DSM 17717, it was assigned a strain name of Pseudomonas fulva I3–I5.

Figure 4.

Figure 4

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Pseudomonas fulva I3–I5 phylogenetic tree based on de novo genome assembly analysis. The numbers beneath the branches represent bootstrap support levels based on 100 replications.

2.4. Chemical Profile of Aloe vera I3–I5 and Aloe vera I4

Plants and endophytes produce bioactive chemical substances which could have industrial applications, including pharmaceuticals, cosmetics, agriculture, and food and beverages [26]. In this project, GCMS was used to identify secondary metabolites produced by endophytic isolates with antimicrobial activity against Mycobacterium bovis. Both isolates, namely, Pseudomonas fulva I3–I5 and Enterobacter cloacae I4, produced a variety of active compounds, with functional groups ranging across alkanes, esters, alcohols, ketones, and other organic compounds.

Esters include benzoic acid, n-hexadecanoic acid, propanoic acid, benzeneacetic acid, benzene-propanoic acid, 1.2-Benzenedicarboxylic acid, 4-Hydroxybenzoic acid, and L-proline. Alkanes include tridecane, hexadecane, octadecane, eicosane, and pentadecane. Alcohols include tryptophol, methylalcohol, 2.4-Ditert-butylphenol, 1-octen-4-ol, and phenylethyl alcohol. Ketones include ergotamine, 1-methyl-2-pyrollidinone, and 2-pentanone. Other organic compounds include indole, acetaldehyde, pyridine, 1-hexadecene, bisacrylamide, 2.6-octadiene, bis (2-ethylhexyl phthalate, benzohydrazide, and crotetamide) (Table 3 and Table 4). Endophytes produce a diverse spectrum of metabolites with a variety of bioactivities. These chemical metabolites are beneficial in drug development due to their bioactive effects such as antibacterial, antifungal, immunosuppressive, anticancer, and antioxidant properties, as previously mentioned. It is due to these important functions that endophytes are gaining popularity in terms of research, as they can be employed to treat various diseases which are currently difficult to medicate, owing to the ubiquitous global drug resistance. Examples of active compounds isolated from both Pseudomonas fulva I3–I5 and Enterobacter cloacae I4 ethyl acetate extracts with pharmaceutical or medicinal applications include hexadecane, pyrollo, ergotamine, L-proline, octacosane, phenylethyl alcohol, 1-Methyl-2-Pyrrolidinone, and benzenepropanoic acid.

Table 3.

Unique metabolites with varying abundances from Pseudomonas fulva I3–I5.

R.T. (s) Area% Observed Ion m/z Compound Name Formula Area Similarity Peak S/N
Alcohols
1 738.27 0.035 206.166 2,4-Di-tert-butylphenol C14H22O 117,980 888 650
2 371.83 0.96 122.07 Phenylethyl Alcohol C8H10O 3,250,678 940 3197
3 763.75 0.14 125.06 3-Mercapto-3-methylbutanol C5H12OS 479,614 785 813
4 957.02 0.07 161.08 Tryptophol C10H11NO 224,164 752 328
Alkanes and Alkenes
5 270.83 0.14 127.65 Tridecane, 4-methyl- C14H30 454,419.5 905.5 488.0
6 789.12 1.54 196.04 Hexadecane C16H34 5,258,816 940 3164
7 818.01 0.08 1671.612 2-Dodecanone C12H24O 287,327 843 510
8 832.25 0.52 161.41 Pentadecane C15H32 1,758,233 940 828
9 385.94 0.18 157.31 Tridecane C13H28 603,935 922 821
10 1011.24 0.05 189.44 Octadecane, 4-methyl- C19H40 176,886 927 174
11 1085.77 0.0475 130.6396 1-Iodo-2-methylundecane C12H25I 156,952 934 136.5
12 1111.10 1.06 263.30 Eicosane C20H42 3,600,474 965 1270
13 1328.84 0.14 196.89 Octacosane C28H58 480,809 954 243
14 1225.79 0.62 239.27 Tetracosane C24H50 2,113,643 958 801
15 1098.44 0.16 216.14 5-Eicosene, (E)- C20H40 531,752 842.50 353.50
16 1107.145 0.13 182.14295 3-Eicosene, (E)- C20H40 432,411 954.5 232.5
17 743.364 0.197 206.1307 7-Hexadecene, (Z)- C16H32 666,803 944 439
Esters
18 421.96 0.03 150.07 Benzeneacetic acid, methyl ester C9H10O2 97,981 908 204
19 1061.66 0.0275 186.561 1,2-Benzenedicarboxylic acid, butyl 2-ethylhexyl ester C20H30O4 87,747.5 22,613.625 480
20 1067.12 0.03 135.07 Formic acid, 2-phenylethyl ester C9H10O2 99,162 891 210
21 1080.37 0.02 292.20 Benzenepropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, methyl ester C18H28O3 81,726 715 164
22 1061.69 0.03 187.06 Dibutyl phthalate C16H22O4 98,061 916 275
23 1059.04 0.30 219.63 Ethyl 2-cyano-3-(4-methacryloyloxyphenyl)acrylate C16H15NO4 1,011,529 866 1293
Ketones
24 916.74 0.04 169.09 3-Methyl-1,4-diazabicyclo[4.3.0]nonan-2,5-dione, N-acetyl- C10H14N2O3 143,741 758 493
25 951.08 0.04 154.07 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- C7H10N2O2 145,827 848 459
26 1045.79 0.38 183.43 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- C11H18N2O2 1,302,535 845 995
27 374.04 0.15 145.78 4-Piperidinone, 2,2,6,6-tetramethyl- C9H17NO 514,662 758 1406
Other organic compounds
28 167.99 8.01 56.54 Acetic acid, hydroxy- C2H4O3 27,263,571 869 1676
29 273.27 0.01 121.09 Pyridine, 2,4,6-trimethyl- C8H11N 6216 808 122
30 611.31 0.01 131.07 Indole, 3-methyl- C9H9N 37,823 881 200
31 782.83 0.04 183.10 Dodecanoic acid C12H24O2 111,574 868 186
32 1087.75 0.04 228.20 n-Hexadecanoic acid C16H32O2 124,987 852 159
33 1215.02 0.10 211.11 l-Leucyl-d-leucine C12H24N2O3 312,139 717 322
34 1320.19 0.10 244.62 Ergotaman-3′,6′,18-trione, 9,10-dihydro-12′-hydroxy-2′-methyl-5′-(phenylmethyl)-, (5′a,10a)- C33H37N5O5 351,941 852 643
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Table 4.

Unique metabolites with varying abundances from Enterobacter cloacae I4.

No R.T. (s) Area% Observed Ion m/z Name Formula Area Similarity Peak S/N
Alcohols
1 966.5 0.11 146.8 (3S,6S)-3-Butyl-6-methylpiperazine-2,5-dione C9H16N2O2 913,414 723 559
2 697.2 0.03 155.1 1-Dodecanol C12H26O 285,215 919 140
3 623.9 0.03 155.1 1-Undecanol C11H24O 287,760 871 138
4 951.2 0.07 211.2 2,4-Di-tert-butylphenol C14H22O 536,471 802 366
5 763.4 0.09 127.6 1-Octen-4-ol C8H16O 270,020 790 401
6 520.0 0.05 128.0 2-Ethyl-1-hexanol C8H18O 143,985 839 192
7 786.7 0.03 138.2 2-Undecen-4-ol C11H22O 111,899 795 403
8 763.5 0.07 121.1 3-Mercapto-3-methylbutanol C5H12OS 286,892 789 623
9 974.8 0.03 126 4-Mercaptophenol C6H6OS 236,549 758 244
10 512.7 0.01 137.7 Ethanol, 1-methoxy-, benzoate C10H12O3 126,066 778 138
11 606.2 0.79 166.1 Ethanol, 2-(2-butoxyethoxy)- C8H18O3 6,801,240 886 17,274
12 646.6 0.26 141.4 Ethanol, 2-(2-butoxyethoxy)-, acetate C10H20O4 2,226,963 918 1609
13 281.0 0.10 117.2 Ethanol, 2-(2-ethoxyethoxy)- C6H14O3 650,282 833 365
14 942.1 0.04 133.6 Ethanol, 2-[2-(ethenyloxy)ethoxy]- C6H12O3 306,041 787 227
15 164.1 0.31 48.0 Ethanol, 2,2-dichloro- C2H4Cl2O 967,422 947 142
16 269.7 8,42 32.0 Methyl Alcohol CH4O 16,979,127 926 689
17 1161.2 0.04 139.8 n-Tetracosanol-1 C24H50O 166,939 936 108
18 1393.3 0.04 368.3 Phenol, 2,2′-methylenebis[6-(1,1-dimethylethyl)-4-ethyl- C25H36O2 323,970 889 1769
19 738.2 0.02 206.2 Phenol, 2,5-bis(1,1-dimethylethyl)- C14H22O 34,536 890 161
20 1246.6 0.17 228.1 Phenol′, 4,4′-(1-methylethylidene)bis- C15H16O2 1,403,098 726 378
21 371.7 0.08 122.1 Phenylethyl Alcohol C8H10O 455,009 899 653
Alkanes and Alkenes
22 1258.1 0.01 259.1 1,3-Dioxolane-2-heptanenitrile, a-methyl-d-oxo-2-phenyl- C17H21NO3 65,288 747 150
23 988.2 0.48 124.4 1,5-Heptadiene, 3,4-dimethyl- C9H16 4,164,767 869 1555
24 1206.7 0.04 144.6 1,3-Cyclopentadiene, 5-(trans-2-ethyl-3-methylcyclopropylidene)- C11H14 332,887 723 520
25 877.1 0.02 169.2 1-Docosene C22H44 186,631 874 195
26 296.7 0.05 100.6 2-Decene, 5-methyl-, (Z)- C11H22 55,377 848 128
27 825.5 0.03 172.6 3-Eicosene, (E)- C20H40 51,075 759 166
28 708.5 0.06 182.1 3-Tetradecene, (Z)- C14H28 178,811 932 242
29 964.4 0.12 158.1 4-Heptafluorobutyryloxyhexadecane C20H33F7O2 346,559 941 255
30 673.6 0.07 143.3 5-Esene, (E)- C20H40 269,736 806 316
31 812.1 0.18 150.4 7-Hexadecene, (Z)- C16H32 487,592 940 378
32 974.1 0.09 175.2 9-Eicosene, (E)- C20H40 766,434 909 245
33 1057.7 1.34 237.9 Eicosane C20H42 2,954,435 959 1373
34 726.1 0.04 123.4 Heptadecane, 2,6,10,14-tetramethyl- C21H44 141,931 900 122
35 956.2 0.05 126.8 Heptadecane, 2-methyl- C18H38 161,042 929 160
36 1625.0 0.08 211.4 Heptaethylene glycol C14H30O8 209,475 930 166
37 1452.7 0.08 527.5 Heptasiloxane, hexadecamethyl- C16H48O6Si7 690,174 758 417
38 794.7 0.84 171.9 Hexadecane C16H34 1,956,218 943 1681
39 1486.3 0.07 159.4 Hexaethylene glycol C12H26O7 196,923 898 173
40 1348.6 0.21 182.9 Octacosane C28H58 509,942 953 264
41 931.3 0.06 156.5 Octadecane, 4-methyl- C19H40 135,195 898 164
42 1237.8 0.76 247.1 Tetracosane C24H50 2,390,307 955 855
43 548.8 0.28 153.0 Tridecane C13H28 712,424 918 798
44 868.7 0.04 127.1 Tridecane, 4-methyl- C14H30 116,341 907 130
Esters
45 1005.1 0.06 143.4 1,2-Benzenedicarboxylic acid, butyl 2-ethylhexyl ester C20H30O4 523,317 727 365
46 1092.4 0.03 150.0 1,2-Benzenedicarboxylic acid, dipropyl ester C14H18O4 97,603 886 539
47 1546.4 0.97 294.5 1,2-Benzenedicarboxylic acid, decyl octyl ester C26H42O4 715,862 893 152
48 1056.9 0.01 183.4 1H-Indole-3-ethanol, acetate (ester) C12H13NO2 88,047 829 250
49 1377.6 0.01 195.0 2-Fluoro-3-trifluoromethylbenzoic acid, 3-methylbutyl-2 ester C13H14F4O2 92,029 781 513
50 904.9 0.03 149.5 2-Propenoic acid, 2-methyl-, oxiranylmethyl ester C7H10O3 107,925 792 425
51 668.6 0.04 126.8 2-Propenoic acid, tridecyl ester C10H18N2O2 115,873 798 381
52 1308.8 0.01 273.5 2,2-diphenylpropionic acid, 2,2,2-trifluoroethyl ester C17H15F3O2 101,730 743 173
53 266.8 0.18 86. Acetic acid ethenyl ester C4H6O2 437,551 975 312
54 754.1 0.01 194.1 Benzoic acid, 4-ethoxy-, ethyl ester C11H14O3 82,696 868 365
55 1563.6 0.04 180.0 Carbonic acid, nonyl vinyl ester C12H22O3 339,969 876 107
56 765.8 0.14 202.2 Cyclopropanecarboxylic acid, 2-ethylhexyl ester C12H22O2 1,233,173 811 1009
57 1132.1 0.05 154.1 Di(1-methylcyclobutyl) ether C10H18O 370,096 814 213
58 819.8 0.01 177.7 Diethyl Phthalate C12H14O4 112,334 938 539
59 1404.6 0.06 204.6 Diisooctyl phthalate C24H38O4 67,422 860 340
60 1088.3 1.33 215.4 DL-Alanine, N-methyl-N-(byt-3-yn-1-yloxycarbonyl)-, tetradecyl ester C23H41NO4 11,535,230 890 817
61 892.0 0.08 186.5 Hexanedioic acid, bis(2-methylpropyl) ester C14H26O4 649,583 900 3117
62 1398.2 0.39 282.6 l-Norvaline, n-propargyloxycarbonyl-, nonyl ester C18H31NO4 1,742,412 909 232
63 1039.5 0.64 192.1 L-Proline, N-valeryl-, decyl ester C20H37NO3 5,550,110 788 850
64 1568.0 0.09 237.7 Oxalic acid, allyl decyl ester C15H26O4 809,220 907 186
65 1356.1 0.53 363.7 Phosphoric acid, isodecyl diphenyl ester C22H31O4P 4,518,474 881 5661
66 1367.5 0.03 184.1 Phosphoric acid, tris(2-ethylhexyl) ester C24H51O4P 211,680 804 951
67 1559.3 0.35 293.5 Phthalic acid, 4-chloro-2-methylphenyl tetradecyl ester C29H39ClO4 381,354 863 111
68 1544.1 0.86 294.2 Phthalic acid, 7-methyloct-3-yn-5-yl undecyl ester C28H42O4 771,078 861 152
69 1472.5 0.66 321.1 Phthalic acid, 8-chlorooctyl decyl ester C26H41ClO4 1,124,586 901 2078
70 841.5 0.07 192.1 Propanoic acid, 2-methyl-, 2-phenylethyl ester C12H16O2 564,174 734 532
71 0.00 0.001 515.4 Propanoic acid, 3,3′-thiobis-, didodecyl ester C30H58O4S 456,850 748 487
72 1064.7 0.01 227.2 Tridecanoic acid, methyl ester C14H28O2 61,779 814 144
73 1065.2 0.01 206.6 Undecanoic acid, methyl ester C12H24O2 98,910 813 135
Ketones
74 1166.8 0.04 225.1 1,3-Propanedione, 2-bromo-1,3-diphenyl- C15H11BrO2 320,260 858 575
75 1043.6 0.01 140.1 1-(2-Thienyl)-1-propanone C7H8OS 133,221 731 172
76 921.0 0.16 205.1 2,2-Dimethyl-N-phenethylpropionamide C13H19NO 1,219,924.7 792.4 786.5
77 1233.1 0.0 204.1 2,5-Piperazinedione, 3-(phenylmethyl)- C11H12N2O2 146,740 839 110
78 472.4 0.05 134.0 2-Coumaranone C8H6O2 154,354 877 274
79 743.4 0.06 155.5 2-Dodecanone C12H24O 232,592 831 256
80 432.2 0.05 104.0 2-Pentanone, 4-hydroxy-4-methyl- C6H12O2 123,821 804 337
81 381.7 0.03 143.1 4-Butoxy-2-butanone C8H16O2 76,347 752 153
82 373.5 0.10 145.8 4-Piperidinone, 2,2,6,6-tetramethyl- C9H17NO 419,489 775 613
83 1018.1 0.31 154.1 5-Pyrrolidino-2-pyrrolidone C8H14N2O 1,355,861 709 477
84 1501.5 0.03 116.9 Acetone, 1-[4-(dimethylaminoethoxy)phenyl]- C13H19NO2 52,377 925 258
85 218.6 0.02 94.0 Dimethyl sulfone C2H6O2S 63,528 889 472
86 188.0 0.02 78.0 Dimethyl Sulfoxide C2H6OS 54,269 781 147
87 910.8 0.07 185.1 Cyclopenta[c]quinolin-4-one, 1,2,3,5-tetrahydro- C12H11NO 110,533 807 574
88 1283.8 0.01 260.1 Cyclopentanone, 2,5-bis(phenylmethylene)- C19H16O 33,095 808 182
89 1076.1 0.11 218.0 Diphenyl sulfone C12H10O2S 285,983 818 1552
90 956.1 0.05 154.1 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro- C7H10N2O2 135,989 880 114
91 1048.4 2.08 203.6 Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)- C11H18N2O2 5,818,832 819 2202
Other organic compounds
92 857.6 0.01 161.1 3-Benzyl-5-chloro-1,2,3-triazole 1-oxide C9H8ClN3O 63,591 802 121
93 1112.1 0.02 241.1 Metolachlor C15H22ClNO2 199,899 775 1110
94 793.3 0.05 184.1 3-(2-Phenylethyl)pyridazine C12H12N2 38,560 716 211
95 1121.6 0.10 475.0 3-Isopropoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris(trimethylsiloxy)tetrasiloxane C18H52O7Si7 835,937 718 824
96 917.8 0.09 168.7 3-Methyl-1,4-diazabicyclo[4.3.0]nonan-2,5-dione, N-acetyl- C10H14N2O3 208,480 790 309
97 224.7 0.37 190.5 4-(2-Acetoxyphenyl)-1-ethyl-3-methyl-5-(4-nitrophenyl)pyrazole C20H19N3O4 3,198,062 999 332
98 1105.6 0.03 182.1 9H-Pyrido[3,4-b]indole, 1-methyl- C12H10N2 60,640 878 306
99 1051.0 0.05 235.2 Acetamide, 2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl)- C14H20ClNO2 455,685 760 507
100 177.0 8.87 66.5 Acetic acid, hydroxy- C2H4O3 39,385,471 788 2051
101 267.8 0.11 109.5 Acetic anhydride C4H6O3 322,969 995 273
102 1215.3 0.08 222.1 Benzene, (1,2-dicyclopropyl-2-phenylethyl)- C20H22 696,698 732 371
103 865.4 0.01 132.1 Benzene, (1-azido-1-methylethyl)- C9H11N3 83,422 761 165
104 621.3 0.01 135.1 Benzeneacetamide C8H9NO 100,313 846 217
105 519.7 0.04 146.1 Benzeneethanamine, N-(1-methylethylidene)- C11H15N 125,968 879 135
106 651.1 0.09 165.5 Benzeneethanamine, N-(3-methylbutylidene)- C13H19N 254,728 847 133
107 652.6 0.04 138.1 Benzeneethanol, 4-hydroxy- C8H10O2 309,979 876 356
108 962.9 0.04 212.1 Benzyl Benzoate C14H12O2 361,122 873 444
109 603.3 0.06 134.1 Bicyclo[3.1.0]hex-2-ene, 4-methylene-1-(1-methylethyl)- C10H14 111,702 756 259
110 1309.0 0.02 273.6 Bifenthrin C23H22ClF3O2 135,178 725 284
111 1406.2 0.76 390.6 Bis(2-ethylhexyl) phthalate C24H38O4 6,611,090 900 5447
112 1416.0 0.03 315.1 Bumetrizole C17H18ClN3O 302,918 943 1675
113 518.3 0.05 156.1 Butanamide, N-hexyl- C10H21NO 465,622 724 421
114 181.5 2.30 97.0 Butanoic acid, 3-methyl- C5H10O2 1,955,281 856 632
115 743.7 0.09 220.2 Butylated Hydroxytoluene C15H24O 802,322 931 3077
116 964.6 0.10 137.4 Cyclo-(glycyl-l-leucyl) C8H14N2O2 198,566 760 171
117 1536.4 0.04 86.1 Cyclobutane, methoxy- C5H10O 164,364 891 431
118 877.9 0.09 437.0 Cyclooctasiloxane, hexadecamethyl- C16H48O8Si8 791,787 719 1152
119 1361.3 0.17 287.5 Di-n-octyl phenyl phosphate C22H39O4P 1,439,556 733 5036
120 961.9 0.27 168.8 dl-Alanyl-l-leucine C9H18N2O3 2,322,399 853 247
121 1000.9 0.02 201.7 Dodecanoic acid, 2-methyl- C13H26O2 210,456 857 317
122 860.5 0.03 172.6 Dodecanoic acid C12H24O2 113,150 832 158
123 898.2 0.04 253.3 Dodecyl acrylate C15H28O2 347,139 939 273
124 1330.3 2.56 291.3 Ergotaman-3′,6′,18-trione, 9,10-dihydro-12′-hydroxy-2′-methyl-5′-(phenylmethyl)-, (5′α,10a)- C33H37N5O5 7,364,698 870 2348
125 340.0 0.04 103.6 Glycyl-dl-norvaline C7H14N2O3 362,257 773 431
126 1665.0 0.02 432.1 Hexasiloxane, tetradecamethyl- C14H42O5Si6 170,683 750 131
127 1648.3 0.03 342.3 Indeno[1,2-b]pyridine, 7-methyl-5-(2,2,6,6-tetramethylpiperid-4-ylimino)- C22H27N3 270,671 866 1478
128 522.0 0.01 117.1 Indole C8H7N 63,439 854 329
129 484.7 0.25 146.1 Isobutyramide, N-(3-methylbutyl)- C9H19NO 2,176,992 765 1702
130 708.8 0.00 146.1 c, 1,2,3,4-tetrahydro-1,8-dimethyl- C12H16 14,327 729 124
131 1148.5 0.05 227.5 n-Hexadecanoic acid C16H32O2 258,286 825 177
132 358.7 0.03 106.1 Nonanal C9H18O 110,278 815 162
133 854.5 0.02 167.1 Octanamide, N,N-dimethyl- C10H21NO 162,285 796 836
134 1352.4 0.06 252.0 Octicizer C20H27O4P 46,610 810 122
135 1332.4 0.37 247.6 Oxamide, N-(3-m′thoxypropyl)-N′-cycloheptylidenamino- C13H23N3O3 3,237,962 953 828
136 163.1 1.67 32.0 Oxygen O2 5,306,645 957 488
137 339.0 0.05 103.6 Pentanoic acid C5H10O2 424,336 741 250
138 832.1 0.05 191.1 Phenylacetamide, N-isobutyl- C12H17NO 387,577 819 640
139 922.9 0.10 209.1 Phenylacetamide, N-pentyl- C13H19NO 788,730 836 282
140 263.5 0.09 182.3 Phosphonic acid, (p-hydroxyphenyl)- C6H7O4P 817,511 814 3495
141 1347.1 0.03 260.1 terphenyl, 4,4″-diamine C18H16N2 77,775 817 414
142 793.4 0.03 184.1 Pyridine, 3-(4-tolylamino)- C12H12N2 101,812 729 552
143 1144.4 0.02 184.8 Quinoline, 2-(2-methylpropyl)- C13H15N 180,409 750 238
144 890.7 0.02 191.5 ß-Phenylethyl butyrate C12H16O2 225,091 759 162
145 1344.8 0.12 326.1 Triphenyl phosphate C18H15O4P 1,059,663 917 648
146 938.5 0.06 1878 Undecanoic acid C11H22O2 351,308 852 235
147 537.7 0.34 16.8 Valeramide, N-hexyl- C11H23NO 2,316,095 730 3538
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Hexadecane was one of the active compounds produced by both isolates and has been proven to have antifungal, antibacterial, and antioxidant properties [27]. Symptoms of human tuberculosis caused by M. bovis infection include fever, headaches, heavy sweat, coughing, abdominal pain, weight loss, and diarrhoea [28]. According to [29], ergotamine has vital properties such as antibacterial and antifungal activity. It is also employed in pharmaceuticals for the treatment of fever, headaches, and migraine. Fever is one of the symptoms of TB, and this active compound in isolation could be employed and incorporated in the treatment of TB. Pyrollo was isolated from ethyl acetate extracts of Pseudomonas fulva I3–I5 and Enterobacter cloacae I4, which is used in anti-inflammatory drugs, antibiotics and antitumour agents [30]. Methyl alcohol was extracted from Enterobacter cloacae I4 and is known to possess antimicrobial properties. It is commonly employed in pharmaceuticals to produce cholesterol, as well as antibiotics such as streptomycin and vitamins and hormones.

Another active compound extracted from Pseudomonas fulva I3–I5 and Enterobacter cloacae I4 with bioactive properties was octacosane, which is used in the synthesis of proteins [31]. L-proline was identified in both isolates; it is known to have antibacterial and antifungal properties and is used in pharmaceutical preparations such as injections [32]. Most of the active compounds extracted in this study, such as bifenthrin, bumetrizole, nonanal, metolachlor, and quinolone, have pharmaceutical applications but could also have other biotechnological significances, such as uses in cosmetics, pesticides, and detergents, as well as in food and beverages. This study proves the significance of employing medicinal plants and endophytes in drug discovery and development, to eradicate or decrease the rising microbial resistance taking place globally. Endophyte isolation and the investigation of their active compounds is gaining traction worldwide due to their vital health properties [33].

3. Materials and Methods

Three medicinal plants, namely, Aloe barbadensis Miller (Aloe vera), Artemisia afra (Wormwood), and Moringa oleifera (Drumstick tree) were harvested from Apel village in Ga-Sekhukhune (Limpopo province, South Africa, 24°24′0″ S, 29°44′0″ E). The plants were collected and placed in sterile polyethylene bags and transported to the University of Johannesburg laboratory at 4 °C.

3.1. Antimicrobial Susceptibility of Mycobacterium bovis against Bacterial Endophytes Isolated from Medicinal Plants

3.1.1. Culturing of Mycobacterium bovis

Mycobacterium bovis (Kwikstik, Microbiologics, 01203P) was cultured in middlebrook 7H9 broth with oleic acid albumin dextrose catalase (OADC) supplement at a temperature of 37 °C for five days, while shaking at 150 rpm.

3.1.2. Susceptibility Test of Mycobacterium bovis against Endophytic Bacteria

The cross-streak technique was used to assess the antimycobacterial potency of the 23 isolates against Mycobacterium bovis following the method of Okudo and Wallis [34]. In brief, Muller–Hinton agar (MHA) (Sigma-Aldrich, Johannesburg, South Africa, BCBZ7677) plates were prepared following manufacturer guidelines, and 20 mL of the medium was allowed to solidify in Petri dishes. A line was drawn in the centre to divide the plates into twos at the back of each plate for the inoculation of the 23 isolated endophytes. Perpendicularly to this line, two other lines were drawn for the inoculation of M. bovis. Using an inoculation loop, a loop full of each of the 23 bacterial isolates were streaked throughout the central line on the Petri dishes containing the solidifies Muller–Hinton agar and incubated at 37 °C for 3 days for maximum growth. Then, afterwards, the plates were cross streaked with a loop full of test organism (M. bovis) at a 90° angle (on the two lines drawn prior) to the bacterial isolates and further incubated for 5 days at 37 °C to allow for antimicrobial activity.

Endophytic bacteria were isolated from plant components (fresh, healthy, and disease-free stems and leaves) immediately after therapeutic plants were collected. First, all medicinal plant parts (stems and leaves) were washed under running tap water to eliminate contaminants such as soil and dust debris. Plant components were sliced into small segments and treated with 5% Tween 20 for 5 min while vigorously shaking them, then rinsed with distilled water (dH2O) several times [25]. The next step was disinfecting the plant components with 70% ethanol for 1 min, after which components were cleansed five times with dH2O to remove any ethanol residue. They were then treated with 1% sodium hypochlorite and rinsed five times with dH2O, and the final wash was plated on nutrient agar (NA) as a control. The plant components’ outer surfaces were removed, and they were ground in phosphate-buffered saline (PBS). Before being spread out on nutrient agar plates, all macerated components were serially diluted to a concentration of 10−3 [25]. The plates were incubated at 30 °C for 5 days with the controls, with bacterial growth measured daily. After the incubation period, various colonies were selected and sub-cultured on nutrient agar to yield pure isolates. Pure bacterial isolates were kept at −80 °C with 50 percent glycerol on a 1 mL glycerol–1 mL broth medium ratio overnight [25].

The whole-genome sequencing (WGS) was conducted using PacBio technology by Inqaba Biotec in Pretoria, South Africa. The newly generated genome sequences were compared to the most closely related bacterial species using the Basic Local Alignment Search Tool (BLAST) on the NCBI platform (https://blast.ncbi.nlm.nih.gov, accessed on 30 March 2022).

3.1.3. Processing of Samples for Metabolite Profiling

Following the modified approach proposed by Xu et al. [35] and Daji et al. [36], centrifuge tubes were employed to combine 10 mL of 100% methanol with 1 g of the extracts. Using Scientech 704 from Labotech in Johannesburg, South Africa, samples were vortexed before an ultrasonic-aided extraction was carried out for one hour at 4 °C. Subsequently, samples underwent centrifugation at 3500× g revolutions per minute (rpm) at a temperature of 4 °C for 5 min using an Eppendorf 5702R centrifuge (Merck, Johannesburg, South Africa). The liquid portion was filtered using filter sheets with a pore size of 0.45 micrometres and transferred into dark amber vials. The extraction process was performed in triplicate for each sample.

3.1.4. Analysis Using GC-HRTOF-MS

To achieve a reproducible result, the mass optimization of the instrument (Leco Pegasus GC–HRTOF-MS, St. Joseph, MI, USA) was performed and passed the preceding analysis. The extracts were introduced into the GC–HRTOF-MS system (Gerstel GmbH & Co. KG, Mülheim (Ruhr), Germany). The system was fitted with a column, with dimensions of 30 m in length, 0.25 millimetres in internal diameter, and a film thickness of 0.25 micrometres. A sample of 1 microliter was introduced into the gas chromatography–mass spectrometry (GC–MS) system at a flow rate of 1 millilitre per minute, with helium serving as the carrier gas. The input temperature was set at 250 °C, while the transfer line temperature was set at 225 °C. The initial setting of the oven was set at 70 °C for a duration of 30 s. Subsequently, the temperature was decreased at a rate of 10 °C per minute until it reached 10 °C. It was then increased to 150 °C and maintained for a period of 2 min. Following this, the temperature was once again decreased at a rate of 10 °C per minute until it reached 10 °C. Finally, the temperature was raised to 330 °C for a duration of 180 s. The experimental settings suggested for the analysis of MS data included acquiring 13 spectra per second, a mass-to-charge ratio range of 30–1000 m/z, electron impact at an energy of 70 eV, and maintaining the ion source temperature at 250 °C and the extraction frequency at 1.25 Hz. Three sets of triplicate samples were subjected to three injections each, resulting in a total of nine injections per sample. The data were analysed with ChromaTOF® program. All uses of active compounds and applications were gathered from NCBI PubChem database, which may be found at [37].

4. Conclusions

The findings of this study indicate that Aloe vera, a therapeutic plant, harbours a wide variety of bacteria such as Enterobacter cloacae I4 and Pseudomonas fulva I3–I5, which possess antimicrobial properties and significant biotechnological value. This study also found that endophytes create bioactive compounds which may be useful in sectors such as cosmetics, food, and beverages.

Acknowledgments

The authors express their gratitude to the National Research Foundation (NRF) South Africa for providing the funds used to successfully carry out this work.

Author Contributions

Conceptualization, methodology, resources, and validation, G.E.; formal analysis, investigation, M.M.N.; writing—original draft preparation, T.Y.F.; writing—review and editing, G.E. and T.Y.F.; visualization, supervision, project administration, and funding acquisition G.E. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the University Research Committee of the University of Johannesburg, Project No. 075432.

Footnotes

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