Quantification of cucurbitacin E in different varieties of melon (Cucumis melo L.) fruit through validated RP-HPLC method

Abstract The different varieties of melons (Cucumis melo L.) have been used in various traditional systems of medicine for decades to treat different ailments, including inflammation, cancer, cardiovascular, diabetes, edema, etc. The present study was designed for the quantification of cucurbitacin E in five different varieties of melon fruit through a validated RP-HPLC method. A solvent system is being optimized with a 70:30 (v/v) ratio of acetonitrile: water (1% glacial acetic acid) at a 1 mL/min flow rate and scanning spectrum (λmax) of 230 nm. A calibration curve for standard cucurbitacin E was generated and found to be linear (1–100 µg/mL). The variation of cucurbitacin E content among five different varieties of melon fruits is 0.0129% w/w– 0.231% w/w. This precise and reproducible method may be beneficial in addressing the quality-related aspects of medicinal food plants of Cucurbitaceae and its derived products or formulations. Graphical Abstract


Introduction
Melon (Cucumis melo L.), is a fruit of Cucurbitaceae family that is widely harvested and consumed throughout the world (Girelli et al. 2018).Cucumis melo L. is among the most polymorphic fruit and horticultural varieties of cucurbitaceae food plants, having genetic diversity within this species (Nuñez-Palenius et al. 2008).It is cultivated in temperate climates as well as dry regions all over the globe (Silva et al. 2020).The infraspecific classification of melon has been described into 19 varieties based on the phenotypic properties (Pitrat 2016).The different varieties of melons have been reported in the management of heart disease along with other different therapeutic application including diuretic, stomachic, antioxidative, anti-inflammatory, antiulcer activity, urease enzyme inhibitory potential, and also used as a first-aid therapy for burns and small cuts (Chen et al. 2005;Mallek-Ayadi et al. 2017;Moustafa et al. 2021).The fresh pulp of melons contains water and sugar, approximately 98% of the total weight.Nevertheless, the fruit contains biologically active compounds such as essential micronutrients (minerals and vitamins), non-essential phytoconstituents (phenolic acids), and signature phytocompounds, e.g. 12 types of cucurbitacins (A-T), especially cucurbitacin E as shown in Figure 1 (Amaro et al. 2015;Bajcsik et al. 2017).In a study, seed oils (23.9%) of Cucumis melo L. exhibited potent antioxidant activity due to the presence of 85.2-83.5% total unsaturated fatty acids, with linoleic acid (62.4-72.5%)being the most abundant, followed by oleic acid (10.8-22.7%)and palmitic acid (10.8-22.7%)(Jorge et al. 2015).Cucurbitacin E is the most widely distributed chemical constituent in food plants of the Cucurbitaceae family.The phytoconstituent has been reported to possess anti-inflammatory, anti-angiogenic, immunomodulatory, cytotoxic, cytostatic, and hepatoprotective properties in both in vitro and in vivo models.Despite the potential therapeutic activity, its chronic exposure is undesirable due to its toxicological effects and ascertained to be lethal (Kaushik et al. 2015).To determine the optimal concentration of bio-active constituents present in herbs and herbal products, standardization and chemoprofile are of great importance (Mukherjee et al. 2008;Mukherjee et al., 2015;Rivera-Mondrag on et al. 2019).As it is evident that the content of phytomarkers differs within the species/groups/varieties due to genotypic differences, external growth conditions (climate, temperature, soil, fertilizers, and irrigation), and plant growth conditions (Rehm et al. 1957;Devendra et al. 2012;Maietti et al. 2012;Ibrahim et al. 2019;Zhang et al. 2019) thus it is important to understand the content variation of the phytoconstituents from these fruits for ensuring the quality and safety issues.In addition, the difference in bio-active composition in plants within the species or groups or even varieties may vary in nutritional and pharmacological effects (Chen et al. 2019).In this context, the present study was performed to quantify the cucurbitacin E in five different varieties of melon fruits using a validated RP-HPLC method.The RP-HPLC method has been validated in terms of accuracy, specificity, linearity, and sensitivity according to the guidelines of the International Conference on Harmonization (ICH 1996(ICH , 2005)).

Results and discussion
The cucurbitacin E is a commonly found phytomolecule in medicinal food plants of Cucurbitaceae family (Bajcsik et al. 2017) and exerts significant therapeutic potential, including anticancer, hepatoprotective, immunomodulatory, and anti-inflammatory properties (Gry et al. 2006;Abdelwahab et al. 2011;Attard and Martinoli 2015;Saeed et al. 2019).In this RP-HPLC method, linearity was detected in the range of 1-100 lg/ mL.The correlation coefficient (r) from the calibration curve was found to be >0.99,which suggests the data is closer to the best-fit side.The LOD and LOQ of cucurbitacin E were measured at 2.63 and 7.98 mg/mL respectively.The recovery value of cucurbitacin E was found to be 95.10-97.98%,indicating that the procedure was accurate (Supplementary material Table S3).The intra and inter-day precision of response (AU) and retention time (R t ) was found to be < 2%, which confirms the high repeatability of this method (Supplementary material Table S4).The robustness of this experiment was found to be within the acceptable range, as there was little variance (%SD < 2%).The present study found a variation of cucurbitacin E content (0.0129-0.0231% w/w) in five varieties of melon (Cucumis melo L.) fruit extracts.The highest cucurbitacin E content was found in Cucumis melo var.flexuosus (L.) Naudin (0.0231% w/w), whereas the lowest cucurbitacin E content showed in Cucumis melo var.agrestis Naudin (0.0129% w/w) compared to the other melon varieties in this study.The concentrations of cucurbitacin E in five different varieties of melon have been shown in Supplementary material Table S5.RP-HPLC chromatogram of standard cucurbitacin E and five different melon fruit extracts is represented in Supplementary material Figure S1.The RP-HPLC analysis was also found to be simple, precise, robust, and reproducible for the identification and quantification of cucurbitacin E within a small range.The different factors, including heavy nitrogen, mild or extreme heat, grafting, plant growth regulators, and alterations in the physicochemical characteristics of soil can cause a coordinated activation of several genes and stimulate cucurbitacin synthesis (Kano et al. 2001;Shang et al. 2014).A previous reported study that the application of high nitrogen fertilizer can alter the cucurbitacin synthesis in cucumber (Kano and Goto 2003).A study revealed that the methylovalerate pathway was activated to synthesize cucurbitacins during summer cultivation through oxidation and decomposition of phospholipids in pumpkin rootstocks (Zhang et al. 2019).In the present study, the variation of cucurbitacin E content among five varieties of Cucumis melo fruits may be due to morphological characteristics, environmental conditions (climate, temperature, soil), and cultivation practices.Melon (Cucumis melo L.) plant shows a particular sensitivity towards the salinity, as well as the magnesium content in the soil (Maietti et al. 2012).Several reports also stated that the quantitative variation in metabolite composition in plants is due to different geographic origins and cultivars (D'Urso et al. 2019;Zamljen et al. 2021).The metabolite profile is unique to individuals within a species or a nearby taxonomic group.Still, it can be altered, because the biosynthetic pathways may be influenced by environmental conditions, (Zheng et al. 2016).In this study, five varieties of melon (Cucumis melo L.) fruits were collected from Kolkata, West Bengal, India to determine the quantity of cucurbitacin E. In West Bengal, six different seasonal changes were observed.As per our study, the highest cucurbitacin E content was found in Cucumis melo var.flexuosus (L.) Naudin and the lowest concentration in Cucumis melo var.agrestis Naudin.Cucurbitacin contents may vary in response to drought-related stresses (Mashilo et al. 2018).Different studies showed that the different environmental conditions, including the fluctuating temperature, heat, pH, salt stress, and seed longevity have less or no impact on seed germination of Cucumis melo var.agrestis Naudin, which may reduce the production of cucurbitacin E in the fruits.For Cucumis melo var.flexuosus (L.) Naudin, stringent environmental conditions are required for cultivation, which may increase the content of cucurbitacin E (Maestro and Alvarez 1988;Sohrabi et al. 2016;Kesh and Kaushik 2021).So, the content of cucurbitacin E in the medicinal plants of Cucurbitaceae may vary with the same varieties cultivated in different geographical locations throughout the world (Rehm et al. 1957;Devendra et al. 2012;Maietti et al. 2012;Ibrahim et al. 2019;Zhang et al. 2019).Melon fruit is a commercially important crop in many countries and is also used for food and culinary purposes.The contents of cucurbitacins are negligible in commercial cultivars, whereas in wild cultivars the concentration is higher.Cucurbitacins levels reach their highest concentration in mature fruits (Amaro et al. 2015).But it is also evident that cucurbitacin contents may vary in response to drought-related stresses (Mashilo et al. 2018).This study was designed to understand the content variation of cucurbitacin E in different varieties of melon (Cucumis melo L.) fruits to address the quality-related safety concerns.

Experimental
See Supplementary Material.

Conclusion
The present study explores the variation of cucurbitacin E content in five varieties of melon cultivated in India.This scientifically validated pr ecised chromatographic method may be beneficial for quality evaluation and standardization of Cucumis melo L. fruits and its derived products/formulations in industrial aspect.

Figure 1 .
Figure 1.The molecular structure of cucurbitacin E.