Martynia annua safety and efficacy: heavy metal profile, in silico and in vitro approaches on antibacterial and antidiabetic activities

Abstract Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of methanol extract of Martynia annua seed revealed the presence of haploperozide and austricine. For safety, heavy metals content investigation of plant powder using the Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) technique showed that the toxic metals (Pb: 2.07 mg/kg; Cd: 0.07 mg/kg; and As: 0.18 mg/kg) concentrations were found to be below the permissible limit. The extract demonstrated significant antibacterial activity against E. coli (MIC value 125 g/mL). Furthermore, it was effective in inhibiting both α-glucosidase and α-amylase enzymes with a high percentage and IC50 values were 42.28 ± 0.39 µg/mL and 34.11 ± 0.31 µg/mL, respectively. These findings were supported by a molecular docking study, some of the phytochemicals showed higher docking score values than references. However, Martynia annua seeds are safe to consume because they contain low levels of toxic heavy metals and possess antibacterial and anti-diabetic properties. Graphical Abstract


Introduction
A large number of diverse phytochemicals can be synthesised by medicinal plants because of the harsh environmental circumstances that they face, including regular variations in water, pH of the soil, UV irradiation, salt, tide cycles, high temperature, and humidity. Numerous variables could influence soil metal bioavailability, including pH, metal concentrations, soil oxidation-reduction potential, and other chemical and physical features. Medical plants can become contaminated during transport or storage (Yan et al. 2020). A large number of reports from around the world show that herbal plants can be hazardous and have severe consequences, despite the popular notion that they are inherently innocuous (Merlin et al. 2019). Since ancient times, Mexico's inhabitants have employed Martynia annua L. (Martyniaceae) as a medicinal herb for several therapeutic uses (Alrabie et al. 2019). Dr. John Martyn, a botany professor at Cambridge University, gave the name Martynia, after which the family is called, in the eighteenth century (Kumar et al. 2012). As 'Bichchhu' in Hindi, it is applied to the camel neck TB glands and used to treat epilepsy. Leaf juice is used as a gargle to heal sore throats, fruit is used to treat inflammation, and leaf paste is effective when applied to dangerous bug stings and wounds on domestic animals (Malik and Katare 2020). Tannins, glycosides, carbohydrates, flavonoids, phenols, and anthocyanins were found in Martynia annua extracts after the phytochemical screening. Cyanidin-3-galactosides, p-hydroxybenzoic acid, linoleic acid, pelargonidin 3-5-diglucoside, gentisic acid, apigenin, apigenin-7-o-glucuronide, and arachidic acid were all found in GC-MS analysis of aqueous and ethanol extracts of the plant (Gupta and Deogade 2018). The seeds revealed the presence of fatty acids such as palmitic acid, malvalic acid, stearic acid, oleic acid, linoleic acid, and arachidic acid (Bhardwaj and Dubey 2016). The effectiveness of Martynia annua is mostly attributed to phytochemicals such as flavonoids, alkaloids, essential oils, and other secondary metabolites. An excessively high concentration of heavy metals could pose an even greater risk to human health than its pharmaceutical effects. As a result, heavy metal concentrations should be monitored to maintain the plant's safety and efficiency. Heavy metals and LC-MS analysis of Martynia annua seed phytochemicals appear to be the focus of less coordinated research efforts, according to the literature review. For Martynia annua seeds using hyphenated instruments, there are no published studies from India. Therefore, the goal of this work was to use the LC-MS technique to screen for phytochemicals in the methanol extract of its seeds and test their antibacterial and antidiabetic activity in vitro. It is also necessary to examine the heavy metal content and identify whether or not Martynia annua seeds contain any harmful levels of heavy metals.

Preliminary phytochemical screening
The qualitative phytochemical analysis of methanol extract exhibited the presence of alkaloids, flavonoids, glycosides, phenolic compounds & tannins, terpenoids, saponins, fat & oils as shown in Table S2.

Inductively coupled plasma-mass spectrometry (ICP-MS) analysis
WHO established permissible limits for toxic metals (Hg, As, Pb, and Cd) in medicinal plants. Table S3 summarises the findings of the Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) analysis of Martynia annua seeds. This plant accumulated Cr (2.66 mg/kg), Mn (18.37 mg/kg), Fe (594.44 mg/kg), and Ni (5.82 mg/kg) over allowable levels Cr (0.2 mg/kg), Mn (2 mg/kg), Fe (20 mg/kg), Ni (1.63 mg/kg), and Cu (40 mg/kg) proposed by Food and Agriculture Organization/World Health Organization (FAO/ WHO) for edible plants. Cu permissible limits in medicinal plants have been set at 20 and 150 mg/kg in China and Singapore, respectively (Pejin et al. 2012;Nkuba and Mohammed 2017). Concentrations of Co, Zn, and Cu in this plant were found to be (0.38 mg/kg), (24.30 mg/kg), and (13.11 mg/kg), respectively, which are tolerable levels for edible plants, as determined by FAO/WHO (3.5 mg/kg) and (27.4 mg/kg), respectively. Pb, As, and Cd do not serve any purpose in the human body and are highly poisonous heavy metals (Balali-Mood et al. 2021). Several nations have established national permitted limits for the Cd, As and Pb concentrations in raw herbal material. It has been said that the amounts of Cd, As, and Pb permissible in herbal material in India are 0.3 mg/kg, 3 mg/kg, and 10 mg/kg, respectively (AYUSH. 2011). The levels of Cd, As, and Pb in this investigation were determined to be within the AYUSH permitted limits of 0.07 mg/kg, 0.18 mg/kg, and 2.07 mg/kg, respectively. Our study was successful in proving that the Martynia annua is safe to use. A study by (Selvaraju et al. 2011) reported a significantly high concentration of iron in three parts of Tribulus terrestris gathered from various places in Tamilnadu (Kulhari et al. 2013) found that the levels of Cr in several medicinal plants from Haryana and Rajasthan states in India ranged from 1.87 mg/kg to 4.93 mg/kg. Nickel (Ni) was found to range from 3.72 to 8.48 mg/kg in Centella asiatica and Bacopa monneri taken from two separate Indian regions, according to (Kalpana et al. 2018). One study (Diaconu et al. 2012) looked at the heavy metal content of ten medicinal plants taken from Romanian pollution-free zones and found that Mn concentrations ranged from 15.34 mg/kg to 158 mg/kg.

Liquid chromatography-mass spectrometry (LC-MS) analysis
The result of LC-MS analysis provided more than 19 peaks, 10 peaks in positive mode and negative mode ionisation, respectively, as shown in Figure S1, but only five and six compounds were tentatively identified in the positive mode, and negative mode, respectively, and Figure S2 showed Extracted ion chromatograms (EIC) & mass chromatograms (m/z) of selected phytochemicals. Table S4 showed identified compounds.

Biological activity
2.4.1. Antibacterial activity E. coli was shown to be the most susceptible, while other bacterial strains proved to be more resistant. The extract with a MIC of 125 g/mL strongly suppressed E. coli as compared to the standard antibiotic ampicillin (MIC ¼ 100 g/mL). The extract has a MIC of 200 mg/mL against A. aeroginosa, whereas ampicillin was shown to be ineffective against this pathogen. Figure S3 and Table S5 depict the findings relevant to the plant extract's antibacterial activity. The extract has modest antibacterial activity against S. aureus and S. pyogenes.

Antidiabetic activity
According to the findings, a-glucosidase inhibition of plant extract ranged from 36.24% to 59.13%. The maximal inhibition of a-glucosidase (59.13%) by plant extract was at 50 g/mL, which was greater than the maximum inhibition of metformin (53.70%) at 10 g/mL and IC 50 of the extract was 42.28 ± 039 mg/mL. Plant extract salivary a-amylase inhibition ranged from 7.9 to 73.3%. Maximum salivary a-amylase inhibition (73.3%) was seen at a concentration of 50 g/mL, which was greater than the maximum inhibition of metformin (58.4%) at 10 g/mL and the IC 50 of the extract was 34.11 ± 0.31 mg/mL (Table S6). The results showed that the methanolic extract had a high a-amylase inhibitory effect as well as a-glucosidase enzyme activity Figure S4. (Saiyad and Gohil 2013) concluded that the methanolic extract of the Martynia annua flower showed good antidiabetic activity in STZ and STZ-NIC induced diabetic rats. Elbashir et al. (2018) studied in vitro antidiabetic activity of aqueous and ethanolic extracts of Martynia annua mature fruit using a-glucosidase assay, the result showed that the IC 50 of ethanol extract was found to be 78.78 ± 3.19 mg/mL whereas IC 50 for the standard (Acrobose) was 240 ± 0.03. Martynia annua seeds have never been studied for their antidiabetic properties, and this is the first time that has been done.
The antidiabetic activity shown in this study might be due to the presence of phytochemicals such as flavonoids, terpenoids, and tannins in the methanolic extract.

Molecular docking study
Some phytochemicals obtained from LC-MS analysis of this study, including ampicillin and metformin (reference standards), were docked to the active sites of 3FV5 and 3TOP to determine the binding mode and binding score of the active sites of these two enzymes. Compound molecular docking data can be found in Table S7. The compounds with the best stability and inhibitory properties have lower binding energy and more hydrogen bonds. Figure S5 displayed the binding interaction of Isorhamntin-3,7-di-o-glucoside at the active site of 3TOP. Isorhamntin-3,7-di-O-glucoside (PF) demonstrated the best docking affinity (-8.07 kcal/mol) with protein 3FV5. A Pi-H bond formed with amino acid residue PRO75 as well as three hydrogen bonds with amino acids ASP69, ARG132, and ARG72 as shown in Figure S6. In addition, it showed the best docking affinity (-8.58 kcal/mol) with protein 3TOP. Two H-Pi bonds formed with amino acid residue PHE1560 and TRP1355 as well as three hydrogen bonds with amino acids ASP1157, ASP1279, and ASP1426 as shown in Figure S7.
This study's findings confirmed that Martynia annua seed has antibacterial activity and antidiabetic activity (Efficacy), and the bioactive compounds identified by LC-MS support the traditional medical uses of the plant. Additionally, the ICP-MS analysis revealed that Martynia annua is free of toxic heavy metals and is safe to use (Safety).

Experimental
Detailed information on this section is provided in the Supplementary Material.

Conclusion
This is the first study to investigate the efficacy and safety of Martynia annua seeds bought from a traditional healer in the Aurangabad district. haploperozide and austricine compounds were identified in this plant's methanolic extract for the first time, and further research is needed to isolate them. The plant extract demonstrated significant inhibition of a-amylase and a-glucosidase enzymes and was efficient against E. coli. The ICP-MS analysis revealed that Martynia annua is free of hazardous heavy metals and is safe to use but nutrient metals levels such as Cr, Mn, Fe, and Ni had levels above the permissible limit for edible plants.