GC-MS analysis of phytochemical compounds of Opuntia megarrhiza (Cactaceae), an endangered plant of Mexico

Opuntia megarrhiza is an endemic plant used in Mexican traditional medicine for the treatment of bones fractures in humans and domestic animals. One of the most used technique for the detection and characterization of the structure of phytochemical compounds is the Gas Chromatography Coupled to Mass Spectrometry. The goals of the present study were to identify and characterize the phytochemical compounds present in wild individuals of O. megarrhiza using this analysis. We used chloroform and methanol extracts from cladodes, and they were analyzed by gas chromatography-electron impact-mass spectrometry. We obtained 53 phytochemical compounds, 19 have been previously identified with some biological activity. Most of these compounds are alkanes, alkenes, aromatic hydrocarbons, fatty acids, and ketones. We detected some fragmentation patterns that are described for the first time for this species. The variety of metabolites presents in O. megarrhiza justifies the medicinal use of this plant in traditional medicine and highlight it as a source of phytochemical compounds with potential in medicine and biotechnology. Subjects Organic Chemistry (other), Organic Compounds, Photochemistry


INTRODUCTION
The members of Cactaceae represent a diverse evolutionary lineage endemic to America, over 1,450 species belonging to ca. 127 genera (Barthlott & Hunt, 1993;Hunt, Taylor & Charles, 2006;Hernández-Hernández et al., 2011). They are successful plants adapted to arid and semiarid environments, where the conditions imply a constant stress, so they have developed different phytochemical compounds with an important biological activity such as alkaloids, amino acids, antioxidant phenol components (betalains and flavonoids), carotenoids, coumarins, esters, fibers, phytosterols, tannins, terpenes, The main chemical analysis for the detection and characterization of the structure of phytochemical compounds are the Thin-Layer Chromatography, the UV-Vis spectrophotometry, the Nuclear Magnetic Resonance, the liquid chromatography-mass spectrometry (LC-MS), and the Gas Chromatography Coupled to Mass Spectrometric (GC-MS) (Robertson, 2005;Marquet, 2012). The last one is the most used in metabolomics research since it is a very selective technique for the detection and characterization of metabolites (Fiehn, 2016). The LC-MS is a robust technique for general unknown screening, however its major drawback is the lack of universal reference libraries obtained with different instrument types (Marquet, 2012), as in GC-MS. The GC-MS together with the metabolomic analysis are a key for the profiling of metabolites in plants since they perform the qualitative and quantitative characterization of all the molecules (metabolites) present in their cellules (Harrigan & Goodacre, 2012).
Opuntia megarrhiza Rose is a species endemic to Mexico, locally known as "nopal camote" (Fig. 1). It is restricted to some regions of the Chihuahuan Desert, particularly in the State of San Luis Potosi (Hernández, Gómez-Hinostrosa & Bárcenas, 2001), and it is listed as endangered in the IUCN Red List (Hernández et al., 2013). It grows in different habitats as xerophytic scrubs, oak forest, and other mountain forests (Hernández, Gómez-Hinostrosa & Bárcenas, 2001;Segura-Venegas & Rendón-Aguilar, 2016). This species is characterized by its massive roots, which are succulent, gross, and deeply buried in ground, 30 to 60 cm long and 5 to 10 cm diameter (Bravo-Hollis & Sánchez-Mejorada, 1991;  Hernández & Godínez, 1994). The cladodes are relatively small contrasting with other Opuntia species. The flowers are yellowish-green to pink, 3 to 5.5 cm long and 2.5 to 6 cm diameter at anthesis. Fruits are ovoid, 2.5 cm long, and the seeds are ca. 4 mm diameter (Bravo-Hollis & Scheinvar, 1999;Hernández, Gómez-Hinostrosa & Bárcenas, 2001). Opuntia megarrhiza is used by locals in the treatment of bones fractures, both animals and humans (Hernández, Gómez-Hinostrosa & Bárcenas, 2001). In Cerro El Borrego (Guadalcazar, San Luis Potosi), the root is applied directly in the fractures, but in other localities like Xoconoxtle (Zaragoza, San Luis Potosi) people use cladodes or the whole plant, and sometimes is mixed with parts of Cylindropuntia spp., to make a paste that is applied with bandages in the injury (Segura-Venegas & Rendón-Aguilar, 2016). Given the ethnopharmacological value of O. megarrhiza, previously highlighted by the empirical traditional medicine, the goals of the present study were to identify and characterize the phytochemical compounds present in cladodes from wild individuals of the species, using GC-MS. There are not previous studies about the bioactive phytochemical compounds in this species, so its phytochemical characterization could contribute to increase the knowledge of the species and its potential biotechnological applications, but also improving bio-valorization and environment.

Sampling
All the protocols involving plants were adhered to relevant ethical guidelines and permissions for plant sampling from herbaria JAAA and SLPM (SGPA/DGGFS/712/0501/ 18) were used. Non-lethal samples of cladodes were obtained from ten wild individuals, sampling randomly, of Opuntia megarrhiza located at Cerro San Pedro (San Luis Potosi). We do not collect whole plants and the identification was conducted in field by taxonomists Arturo De Nova and Eleazar Carranza form herbarium SLPM and registered with photographs then confirmed with Research Grade in iNaturalist (46236747, 46978331). The Fig. 1A show a reference herbarium voucher from the studied locality for identification (SLPM 22132).

Extracts
The extractions were conducted at Laboratorio de Biotecnologia de la Facultad de Agronomia y Veterinaria, UASLP and Centro Regional de Biociencias, UASLP. Cladode fragments were cleaned using brushes and distillated water to eliminate possible associated microorganisms. We used 95 g of sample, which was macerated to make a semisolid paste, then either 25 ml of methanol (MeOH) for extraction 1 or 25 ml of chloroform (CHCl 3 ) for extraction 2. These solutions were vortex mixed one hour to prevent conglomeration and sedimentation of small particles. The extracts were filtered three times using Whatman paper, grade 1, 5 and 6 in order, in a vacuum chamber. The volumes were adjusted to 10 mg/ml for all extracts. Subsequently, we made a dilution for each solution using acetone as solvent (1:1): (1) acetone-chloroform ((CH 3 ) 2 CO/CHCl 3 ), and (2) acetone-methanol ((CH 3 ) 2 CO/MeOH). Before depositing in a vial, extracts were filtered through a polytetrafluoroethylene (PTFE) or polyvinylidene (PVDF) membranes (with different hydrophobic adsorption ranges and size exclusion pores), and transferred to a vial for analysis in the GC-MS.

GC-MS analysis scan mode
This process was conducted at Coordinacion para la Innovacion y la Aplicacion de la Ciencia y la Tecnologia, UASLP. We used the Hewlett Packard gas chromatograph HP 6890, coupled to mass spectrometry detector with electronic impact HP 5973 (Agilent Technologies, Palo Alto, CA, USA). The column exerted was an absorbed silica capillary column of 95% methyl-poly-siloxane and 5% phenyl (HP-5MS; length: 60 m, diameter: 0.25 mm and film 0.25 µm). Helium was used as carrier gas, and the flow rate was 1 ml/min. The GC oven temperature gradient was: 60 C (hold for 3 min) initially, then increased 5 C each minute until 300 C, this final temperature was hold 5 minutes. The transfer line temperature was 280 C. The ion source temperature was 230 C and the MS was scanned at 50 to 550 mass range. The essays were processed in the ChemStations software (Houston, TX, USA) to generate the chromatograms for the interpretation of the spectra.

Identification of phytochemical compounds
The identification was performed by comparing the spectrum of unknown compounds with the spectrum of known compounds in the National Institute of Standards and Technology (NIST98) mass spectral library. Compound name, synonyms, molecular weight, and the mass spectrum for each compound were obtained from NIST Standard Reference 69 and PubChem databases to confirm the GC-MS results. Nomenclature for all compound names was standardized with IUPAC rules. Relative quantification of the compounds present in each sample was obtained from the relative area of the peaks in the chromatograms. Biological activity for each identified compound was obtained with an exhaustive search in scientific publications and from the Dr. Duke phytochemical and ethnobotanical databases. The identity of five compounds showing similarity above 90% (as recommended in Mangas-Marín et al., 2018) with phytochemical compounds previously reported with biological activity was verified by using the commercial pure standards: The used standards were 1,3-benzothiazole (Purity: minimum 97.0%), henicosane (Purity: minimum 99.5%), hentriacontane (Purity: minimum 98.0%), methyl hexadecanoate (Purity: minimum 98.5%), triacontane (Purity: minimum 98.0%), were purchased from Sigma (St. Louis, MO, USA). All standards were diluted in acetone as the final solvent at a concentration of 5 ppm and were analyzed in the GC-MS using the same parameters than in the samples.

RESULTS
A total of 53 phytochemical compounds were detected based on the analyses of the obtained chromatograms (Tables 1-4). The (CH 3 ) 2 CO/MeOH extract showed 11 peaks with the PVDF membrane filter and 12 with the PTFE. The (CH 3 ) 2 CO/CHCl 3 extract showed 23 peaks with PVDF and seven with PTFE (Fig. 2).

DISCUSSION
The use of Opuntia megarrhiza in traditional medicine in Mexico has been reported previously (Segura-Venegas & Rendón-Aguilar, 2016), however, this is the first study that demonstrate the presence of phytochemical compounds with biological activities.
Opuntia species are used in the world as local medicinal interventions for chronic diseases and as food sources, mainly because they possess nutritional properties and biological activities that has been recently reviewed (Aruwa, Amoo & Kudanga, 2018). Here we report for the first time, the identification of several phytochemical compounds in O. megarrhiza with biological activities. Our findings highlight the relevance of this species in developing of new drugs, trough future chemical studies, and encourage of planting this species once this one is listed as endangered in the IUCN Red List. Biotechnological methods are reliable and provide continuous sources of raw material and natural products for food, pharmaceutical, and cosmetic industries (Rao & Ravishankar, 2002;Nalawade et al., 2003;Julsing, Quax & Kayser, 2007). Previously, it has been indicated that more than 50,000 plant species are used in phytotherapy and medicine, and around 66% of them are harvested from nature leading to local extinction of many species or degradation of their habitats (Tasheva & Kosturkova, 2012). Alternatives to protect these useful plants, should be directed to both preservation of the plant populations and elevating the level of knowledge for sustainable utilization of these plants in medicine have been previously indicated (WHO, 2010, http://www.who.int/mediacentre/ factsheets/fs134/en/). Biotechnological methods offer possibilities not only for faster cloning and conservation of the genotype of the plants (Verpoorte, Contin & Memelink, 2002;Tripathi & Tripathi, 2003) but for modification of their gene information, regulation, and expression for production of valuable substances in higher amounts or with better properties (Rao & Ravishankar, 2002;Khan et al., 2009).
GC-MS is one of the most precise methods to identify various metabolites present in plant extracts (Fiehn et al., 2000;Roessner et al., 2000;Roessner et al., 2001;Kopka, 2006a;Kopka, 2006b;Fiehn, 2006;Fernie, 2007;Saito & Matsuda, 2010;Tiago et al., 2016;Dinesh-Kumar & Rajakumar, 2018) since some of these chromatographs include preloaded libraries or databases (NIST and WILEY) that allows to know the possible identity of the metabolites by comparing the resulting mass spectra with those found as reference in these libraries (Kim et al., 2019;Wei et al., 2014). Several studies indicate that Opuntia plants contain different phytochemical groups such as phenolic acids, sterols, esters, coumarins, terpenoids, and alkaloids with several health benefits (Piattelli, Minale & Prota, 1965;Stintzing, Schieber & Carle, 2001;Strack, Vogt & Schliemann, 2003;Paiz et al., 2010;Osorio-Esquivel et al., 2011;Aruwa, Amoo & Kudanga, 2018). However, the nature of the compound extracted depends largely on their solubility in the extraction solvent, the degree of polymerization of the phenols, and the interaction of the phenols with other constituents of the plant (Choi et al., 2002;El Cadi et al., 2020). But the use of different membrane filters allows to identified chemical compounds with different hydrophobicity and molecule sizes. Previously, it has been indicated that PTFE has less hydrophobic adsorption but more size exclusion (Xiao et al., 2014).
In addition, identity of five of the compounds found was corroborated using pure commercial standards. The ions obtained from each of the standards corresponded to those found in the extracts according to the NIST base of the equipment. GC-MS has a library of mass spectra, which makes it easy to obtain compounds that have the most similar mass to the library spectrum (Kim et al., 2019). However, the attribution of a GC-MS chromatographic peak should be confirmed whenever possible by comparison with a standard compound analyzed under the same experimental conditions (Sturaro, Parvoli & Doretti, 1994). We identified five compounds in the extracts performed through the use of standards. In this context, the analytical standard is used as a reference in the qualitative, quantitative and identity determinations of a compound, it must also have high purity and stability (Sun et al., 2015).

CONCLUSIONS
The GC-MS analysis of cladode extracts of Opuntia megarrhiza conducted here proves the presence of several phytochemical compounds responsible for biological activities previously reported support the medicinal use of this plant in traditional medicine. In particular, the anti-inflammatory activity in compounds with a high similarity percentage in our results (e.g., hexadecanoic acid, 2,4-ditert-butylphenol, hentriacontane, 1,3-benzothiazole, and methyl hexadecanoate) supports its use for treating bone fractures. Hence, O. megarrhiza represents a source for finding phytochemical compounds with potential use in medicine and biotechnology. Our results represent an advance in the knowledge of an endangered plant, not previously studied, and with ethnical uses, and support future target studies through the identification of compounds with biotechnological potential using certified standard and additional tools.

ADDITIONAL INFORMATION AND DECLARATIONS
Funding Madeleyne Cupido received a grant of CONACYT (Graduate Studies Scholarship 1007054). This research was funded by the international cooperative research of Rural Development Administration (RDA) from Republic of Korea, (Grant PJ012429012016 to Pablo Delgado-Sánchez) and CONACyT (Grant 2014-243454 to JADN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: CONACYT: 1007054. International Cooperative Research of Rural Development Administration (RDA), Republic of Korea: PJ012429012016. CONACyT: 2014-243454.