Abstract

   Enhancing the production of economically and medically important plant
   metabolites by genetic and metabolic manipulation is a lucrative
   approach for enhancing crop quality. Nevertheless, the task of
   identifying suitable biosynthetic pathways related to certain
   bioactivities has proven to be challenging due to the intricate
   interconnections of the major metabolic and biochemical processes in
   commercially important plants. The commercial significance of plants
   belonging to the genus Aloe stems from their extensive utilization
   across several industries, such as cosmetics, pharmaceuticals, and
   wellness items, due to their medicinal properties. In the present
   study, we have utilized a reverse association approach to identify
   potential target metabolic pathways for enhancing the production of
   commercially important metabolites of Aloe spp., based on their
   metabolic pathway enrichment profile. The leaves of five highly
   utilized Aloe sp. were subjected to untargeted gas chromatography-mass
   spectrometry analysis followed by testing of free-radical scavenging
   effects against components of the Fenton and Haber-Weiss reaction.
   Through the application of appropriate bioinformatics tools, we
   identified distinct phytochemical classes and determined the enrichment
   of their corresponding biosynthetic pathways, associated the pathways
   with bioactivities, and also identified the inter-relation between the
   commonly enriched pathways. The strong association between metabolic
   pathways and antioxidant potentials suggested the necessity to enhance
   distinct but closely related metabolic pathways in order to enhance the
   quality of Aloe spp. and maximize their antioxidant effects for
   commercial exploitation in cosmetic industries.

   Keywords: Aloe, Metabolite, Photochemical, Antioxidant, Metabolic
   pathway, Metabolomics

Highlights

     * •
       Plants belonging to the genus Aloe are extensively used in the
       cosmetic industry.
     * •
       Identification of the key pathways could guide metabolic
       engineering strategies.
     * •
       Major metabolite classes were identified as relevant to cosmetic
       products.
     * •
       Highly enriched common metabolic and biosynthetic pathways were
       identified.
     * •
       Target pathways pertaining to commercially relevant bioactivities
       were recognized.

1. Introduction

   The genus Aloe comprise of the largest group of pants (over 650 spp.)
   under the family Xanthorrhoeaceae [[25]1]. The Aloe spp. contains over
   75 micronutrient components and over 250 different phytochemicals,
   including flavonoids, saponins, anthraquinones, and lignin, that
   exhibit various bioactivities, including antiviral, antibacterial,
   antifungal, anticancer, anti-inflammatory, moisturizing, anti-aging,
   immunostimulatory, anti-radiation, and wound healing properties
   [[26]2,[27]3]. Currently, these plants are considered to be highly
   significant in terms of their economic value in the field of medicine
   and pharmacology. They are frequently utilized in primary healthcare
   and traditional medicinal practice to effectively cure a wide range of
   disorders by modifying biochemical and molecular pathways
   [[28]4,[29]5]. In recent years, there has been a considerable increase
   in the use of A. vera in the cosmetic industry, in addition to a
   widespread acceptance of Aloe-based products by consumers due to its
   low dermatological sensitivity, and skin smoothening and moisturizing
   properties [[30]2,[31]6]. Especially due to potent antioxidant effects,
   an essential pre-requisite for cosmetic products, Aloe-based
   formulations are often included in commercial products [[32]7,[33]8].
   For instance, various Aloe compositions have been demonstrated to
   possess anti-aging effects [[34]9], UV-protective effects [[35]10], and
   anti-proliferative effects [[36]11] through potent free-radical
   scavenging and antioxidant properties. The amounts of Aloe plant
   components in cosmetic products can vary significantly, ranging from
   less than 1 % to as high as 20 % [[37]12]. Due to the presence of a
   large variety of non-polar constituents, components of Aloe gel, upon
   topical application, are claimed to profoundly enter the deeper layers
   of the skin, facilitating the restoration of lost moisture and
   replenishment of the fatty layer, while neutralizing the free-radicals
   [[38]13].

   The composition of non-polar secondary metabolites, such as essential
   oils, found in medicinal plants are closely linked to their therapeutic
   effectiveness and can be targeted for enhancing crop quality. One can
   gain an understanding of how these substances are regulated
   metabolically by identifying the genes and enzymes involved in their
   biosynthesis pathways. Research on economically significant
   phytometabolites is accelerating as researchers aim to understand the
   pathways responsible for the manufacture of key metabolites and
   manipulate the phytochemical composition to match commercial demands
   [[39]14]. Plant species belonging to the genus Aloe are commonly
   considered safe and serve as valuable resources for traditional
   remedies, pharmaceuticals, nutraceuticals, aromatherapy, preservatives,
   beverages, scents, cosmetics, and botanical pesticides [[40]1].
   Quantity and diversity of the metabolites are influenced by their
   biosynthetic pathways. To improve the production of industrially
   relevant phytochemicals, metabolic and genetic engineering techniques
   have been employed. For instance, by exploring full-scale functional
   genomics, one study generated a whole transcriptome sequence database
   with a focus on the metabolic specificity of A. vera, to identify the
   pathways related to secondary metabolite formation, metabolic
   regulation, and signal transduction, that contributes to the growth and
   physiology of the plant [[41]15]. However, for the practical
   application of these strategies critical for crop improvement, it is
   necessary to first identify the specific biochemical pathways
   associated with the secondary metabolite formation. However, it is
   challenging since many of the plant's biosynthetic pathways are
   interconnected through a shared carbon pool [[42]16,[43]17]. Instead of
   focusing on individual genes responsible for a particular industrially
   significant phytochemical, it is more effective to enhance the complete
   biosynthetic route or multiple co-occurring biosynthetic pathways. This
   approach is more efficient and smarter strategy for developing
   economically valuable phytochemicals [[44]14,[45]18]. To achieve this
   objective, it is necessary to first identify the co-occurring plant
   biosynthetic pathways responsible for producing specific metabolites
   associated with distinct bioactivities.

   Therefore, in the present study, we intended to identify the
   predominant and inter-correlated metabolic and biochemical pathways
   that commonly dictate the formation of the volatile secondary
   metabolites in the Aloe spp. This was based on the hypothesis that
   metabolome-based reverse association strategies for identification of
   the predominant biosynthetic pathways could lead to crop improvement
   through metabolic engineering approaches. For this purpose, we selected
   Aloe vera (L.) Burm.f., AristAloe aristata (Haw.) Boatwr. & J.C.Manning
   (aka, Aloe aristata), Aloe jucunda Reynolds, Aloe aspera Haw., and Aloe
   albiflora Guillaumin, which are among the highly utilized plants in the
   cosmetic industry that belong to the genus Aloe [[46]19]. The volatile
   and non-polar metabolome of these plants were fingerprinted in an
   untargeted manner, followed by the study of the chemical-class
   enrichments and pathways analysis. The bioactivity potential was
   evaluated by testing the free-radical scavenging effects against the
   Fenton and Haber-Wess reaction-associated intracellular free-radical
   formation. Collectively, several clusters of enriched co-occurring
   metabolic pathways were detected, which could be potentially targeted
   using metabolic engineering strategies to enhance the antioxidant
   properties of plants belonging to the genus Aloe.

2. Methodologies

2.1. Plant material collection

   The plant materials were collected from the medicinal garden of North
   Bengal University and processed as described previously [[47]5]. In
   brief, disease-free leaves of A. vera, A. aristata, A. jucunda, A.
   aspera, and A. albiflora were hand-picked. The plant materials were
   identified and authenticated with accession numbers [48]09781,
   [49]09766, [50]09767, [51]09753, and [52]09779. The voucher specimens
   of the leaves were stored in the herbarium of North Bengal University.

2.2. Sample preparation and derivatization

   The collected whole leaves were rinsed with double distilled water
   three times, chopped into 3–5 mm pieces with a sterile scalpel, and
   placed in a dehydrating incubator (37 °C) for 7-d until completely
   dried. Dried leaves (100 mg) were separately mixed with 3 mL n-hexane
   in recti vials and incubated in the dark for 24 h at 25 °C and 120 rpm.
   Next, 40 μL N,O-Bis(trimethylsilyl)trifluoroacetamide with
   trimethylchlorosilane (BSTFA + TMS) was added into the mixture and
   incubated under the same condition for 6 h. The mixture was then passed
   through activated charcoal and Na[2]SO[4] (1:2, w/w) in a mini-column.
   The filtrate was spun at 15,000 rpm for 20 min at 25 °C, filtered
   through a 0.2 μm membrane syringe filter, and the resultant was used
   for GCMS analysis.

2.3. Gas chromatography-mass spectrometry

   The processed and derivatized samples were analyzed in a Trace 1300 Gas
   Chromatography instrument and ISQ QD single quadrupole mass
   spectrophotometer (Thermo-Scientific) as per prior standardized
   protocol [[53]14,[54]20]. The separation column used was TG-5MS with a
   dimension of 30 m × 0.25 mm × 0.25 μm. Samples were injected (1 μL) in
   splitless mode using AI-1310 auto-sampler (Thermo-Scientific). The
   inlet port temperature was set at 250 °C, the initial column
   temperature was 60 °C with a solvent delay of 5 min (4 min hold), and
   the final temperature was 290 °C with 4 min hold at the end. The
   temperature ramp was set at 5 °C/min, achieving a total run time of
   54 min (1 mL/min flow of 99.99 % helium passed through hydrocarbon and
   dehydrating columns). The transfer line for the mass spectrometer was
   set at 290 °C and an ion source temperature was kept at 230 °C
   (electron ionization mode). MS analyzer range was 50–650 amu and the
   samples were analyzed at electron energy 70 eV (vacuum pressure of 2.21
   x 10^−5 torr). MS data for identified peaks was analyzed using AMDIS
   (V2.7) where the major peaks were identified based on the base peak and
   molecular peak patterns of the library references using MS Interpreter