Study of Metabolites of Cucumis melo subsp. agrestis var conomonby HR-LCMS Q-TOF/MS

Introduction

The Cucumis genus includes ornamentals and several regularly grown crops, making it one of the most important genera of flowering plants economically. Cucumis species are distinguished by their bushy, ascending, trailing growth habits^1.It is widespread in South America and other tropical regions of Asia, yet it is endemic to arid regions of India².Cucumis melo cultivars belong to the Cucurbitaceae family have  bitter taste and  maximum length of 10cm before reaching maturity, Cucumis melo subsp. agrestisis conversely, specific cultivar locally called Mangalore southekaayi is sweet in taste and grows to a maximum size of 20cm to 25cm³. There are several variations in the pulp texture. In Coastal Karnataka, this fruit is known as mage-kaayi or moge-kaayi, a typical vegetable grown on a backyard plot ⁴. It is eaten as wholesome food. It is a great source of preventive and healing bioactive components. Since ancient times, C. melo has been consumed as a traditional meal and used to soothe headaches for its calming properties, constipation, and skin swelling⁵. Additionally, it has revitalizing properties. C melo subsp. agrestis var conomon, also acknowledged as Dosakaya Kani Vellari, is a golden cucumber cultivar that is used to make sambar and pachadi. It has broad green patches that turn dark brown when they mature and white flesh⁶. Local names for this cultivar include golden yellow and Malabar cucumber. This useful climber seed is widely cultivated and is being researched for its antibacterial and antioxidant properties. It is ingested as a powerful nutrient to prevent infections and strengthen immune systems ⁷.

The objective of this analysis was to detect the organic bioactive substances existing in the frothy, soapy substances of Cucumis melo subsp. agrestis var conomon fruit that is sold in the Karnataka market. The comprehensive analytical method for studying metabolite profile in natural product research is mass spectroscopy. The High-Resolution Liquid Chromatography Quadrupole Time of Flight Mass Spectrometry or HR LCMS QTOF-MS was applied to examine acetone extract of the frothy white substance.

Materials and Methods

Compoundsprerequisite for study was procured from Himedia. The HR-LCMS of the sample was done in SAIF, IIT Bombay. Pawai.  Mumbai.

Plant Collection

The C. melo subsp. agrestis var conomon fruit cultivar was gathered from a farm in the village of Kattebelaguli, which is close to Holenarasipura taluk in the Hassan district of Karnataka, India. The white, frothy compounds were extracted using fresh fruits. Cutting the tip into thin piece and rubbing it back against the cucumber in a circular motion the flat surface of the flower and stem ends produced the white frothy substance. Fresh foamy material weighing 100g was gathered, dried, and dissolved in 10ml of acetone. Following onSoxhlet extraction, the residue was dissolved in 2ml of acetone, sealed, and used for analysis. Figure-1

Figure 1: Cucumis melo subsp. agrestis var conomoncultivar and collection foamy extract. Click here to View Figure

Extraction and Separation

Soxhlet extraction of foamy extract was done. Distillation was performed to isolate the solvent; the concentrate was evaporated to desiccation and the remainder was dissolved in 5ml of acetone Preliminary phytochemicals study was done according to Deepika et al⁸.

HRLCMS Analysis

The acetone-prepared extract that was subsequently analysed by HR-LCMS. HR-LCMS analysis was carried out at the IIT Bombay, Pawai, Mumbai, Sophisticated Analytical Instrument Facility (SAIF). Agilent Technologies, USA was able to obtain the metabolite fingerprint of the foamy substance that is C melo subsp. agrestis var conomon. ZORBAX Eclipse Plus-C18, 5micron (Agilent) Mode, 150×2.2 mm. Model G6550A, a high-resolution liquid chromatography and mass spectrometry system with a mass resolution of 0.010%, was employed. The MS acquisition method, with a scanning rate of one spectrum per second, was configured with a minimum range of 60 (m/z) and a maximum range of 1000 Dalton (m/z). By comparing the retention time (RT) and mass of the compounds, Agilent Mass Hunter Qualitative Analysis B.06 was able to identify the bioactive chemicals. With a gas flow rate of 13psi/minute, gas chromatography has been sustained at 250⁰C. Using a hip sampler model G4226A, the auxiliary speed and ejection speed were both set at 100 µl/minute. The injection volume utilized for HR-LCMS was 8 µl, and the flush out factor was 5 µl. In the first minute of the 30-minute acquisition period, the solvent composition flow A: Milli-Q water containing 0.1% formic acid B: Acetonitrile and TOF/6500 series Table-1 and Table-2. By comparing retention times and mass spectra with the respective reference standards, metabolites were found. A composite pattern of major and minor peaks were seen in the chromatogram that was produced. The analysis was completed at the advanced analytical instrument facility located in Mumbai, India, at the Indian Institute of Technology.

Table 1: Solvent Arrangement of HRLCMS

	Channel	Ch 1 Solv	Name 1	Ch2 Sol	Name 2	Selected	Used	Percent
1	A	100.0%Water V.02	0.1%FA in water	100.0% V.02	0.1%FA in Water	Ch 2	yes	95.00%
2	B	100.0% Methanol V.03		100,0% Acetonitrile V. 02		Ch 2	yes	5.00%

Table 2: Time Table.

	Time	A	B	Flow	Pressure
1	1.00min	95.00%	5.00%	0.300mL/min	1200.00bar
2	25.00min	0.00%	100.00%	0.300mL/min	1200.00bar
3	30.00min	0.00%	100.0%	0.300mL/min	1200.00bar
4	31.00min	95.00%	5.00%	0.300mL/min	1200.00bar
5	35.00min	95.00%	5.00%	0.300mL/min	1200.00bar

 

Results

Preliminary Phytochemical Analysis

The examination of phytochemicals Salkowski, Molisch, and Libermann-Buchard test findings were positive, indicating the presence of sugars and steroids in the Cucumis melo subsp. agrestis var conomon cultivar. It was determined that steroids were present after a specific confirmatory test for lactone rings and deoxysugars was carried out. Previous studies have identified the presence of tannins, polysaccharides, terpenoids, saponins, resins, phytosterols, and cardiac glycosides in the phytochemical analysis of C. melo leaves extract in methanol and acetone⁹. The presence of glycosides, tannins, alkaloids, flavonoids, phlobatanin, and anthraquinone saponin were observed in the epicarp, mesocarp, and seeds of mature fruits from Cucumis melo (L.), Lagenaria reviflora (Benth), and Citrullus lanatus (Thunb). The stems of Cucumis melo included cucurbitane-type triterpenoids, and spectroscopic investigation revealed the presence of cucurbitacin B and cucurbitacin. Gallic acid content was demonstrated by chloroform extract from the dried fruit Cucumis melo L., sometimes known as honeydew melon¹⁰.

HRLCMS Study

To analyse the bioactive compounds found in the frothy, soapy substances HRLCMS tied with a Quadrupole ion trap Mass Spectrometer was done. It is the quick investigative technique captivating sensitivity and resolution into consideration the qualitative and quantitative phytochemical composition of acetone extract of the foamy substance of C melo subsp. agrestis var conomon was done. It exhibited 10 peaks,it is composed of manifold classes of metabolites. The first emerged compound wasTyrosyl-glutamate (14.59ppm) of mass 310.112.The second emerged compound was Tyrosyl-glutamate (14.96ppm) of mass 310.1119, fourth was Phaeophorbide (2.5ppm) of mass 606.2493, sixth compound was Hydrocortisone cypionate, (13.9ppm) of mass 486.2914., eighth compound emerged was Hydrocortisone cypionate(14.62ppm) of mass 485.2839, tenth was UDP-N-acetyl-2-amino-2-deoxy-D-glucuronate(2.62ppm) of 666. 0614.The third, fifth, seventh and ninth emerged peaks were not so conspicuous, chromatogram of foamy extract made up of 9 major and 8 minor peaks Figure 2 andFigure 3 totally 10 prominent bioactive compounds were identified along with MS spectrum peak, compound structure, ratio m/z, retaining, time, ppm, molecular formula and organic formula of altogether projecting compounds were identified showed in Table-3. This is the first report on analysis of foamy extract.

Figure 2: High Resolution Liquid Chromatogram study of Foamy extracts of Cucumis melo subsp. agrestis var. conomon.( By SAIF Bombay) Click here to View Figure

Figure 3: Prominent Bioactive Compounds identified in Foamy Extract of Cucumis melo subsp. agrestis var. conomon Click here to View Figure

Table 3: Details of Prominent Phytochemicals of foamy Substance with Mass, MS spectrum peak, M/Z, PPM and compound structure of Cucumis melo subsp. agrestis var. conomon Click here to View Table

A validated RP-HPLC method was used to study and quantify cucurbitacin E in five distinct kinds of melon fruit. A 70:30 (v/v) ratio of acetonitrile to water (1% glacial acetic acid) was used. The results showed that the cucurbitacin E concentration varied amongst five different melon fruit cultivars by 0.0129%w/w to 0.231%w/w The fruit extract with the highest concentration was discovered to be C melo var. flexuosus (L.) Naudin fruit ¹¹. We discovered in our investigation that the fruit froth extract had no cucurbitacins for C. melo subsp. agrestis var. conomon. Using flow-injection analysis and detection based on atmospheric pressure chemical ionization-Fourier transform mass spectrometry, the first proof of the presence of Cucurbitacins D, B, R, and C and their isomeric forms were found in a bitter-tasting fruit of the “Scopatizzo” melon, a landrace of C. melo typical of the Apulia region in Southern Italy ^12. .The fruit extract from C. melo was shown to have a high level of Superoxide Dismutase Activity, which was thought to be caused by the fruit’s cucurbitacin, phenolic compounds, vitamins, minerals, fatty acids, and essential oils. C. melo was widely recognized for its anti-inflammatory properties. The pharmacological characteristics of plants of the Cucumis genus include anti-inflammatory, anticancer, antidiabetic¹³, antiwrinkle, antibacterial, antioxidant, and analgesic effects¹⁴.

Our study showed that foamy extract is rich source of both Tyrosyl Glutamate,a dipeptide of mass 310.112 (14.59ppm), Tyrosyl Glutamate mass 310.1119(14.96ppm), Hydrocortisone cypionate a glucocorticoid of mass 486.2914(13.9ppm) and Hydrocortisone cypionate of mass 485.2839(14.62ppm) Figure-3.

The mechanism of action of hydrocortisone cypionate for the treatment of numerous illnesses was investigated in this context due to its importance in pharmacology. Hydrocortisone is one of the most important glucocorticoids in humans. The homeostatic mechanisms of the body must be regulated. Hydrocortisone has anti-inflammatory properties and can be used to treat inflammation caused by corticosteroid-responsive dermatoses¹⁵.It is distinguished by its ability to attach to the cortisol receptor and start a variety of important effects on the immune system, metabolism, cardiovascular system, and homeostasis. They differ from sex steroids and mineralocorticoids in their receptors, target cells, and modes of action. It suppresses cell-mediated immunity. They function by inhibiting the genes that generate TNF-alpha(Tumor Necrosis Factor), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-8, with IL-2(IL-Interleukin) being the most important. Reduced cytokine synthesis limits T cell proliferation ¹⁶.It affects humoral immunity, which results in decreased IL-2 and IL-2 receptor expression in B cells. This reduces the production of antibodies as well as the growth of B cell clones. Reduced levels of IL-2 also result in a decrease in the activation of T lymphocyte cells^{17, 21}.

Discussion

Information on the action mechanism of hydro cortisone is available. The cytosolic glucocorticoid receptor is the site of hydrocortisone binding. Following its attachment to the receptor, the newly created receptor-ligand complex moved into the nucleus of the cell, where it bound to many glucocorticoid response elements (GRE) in the target genes’ promoter regions^{18, 19.}Following its interaction with fundamental transcription factors, the DNA-bound receptor causes the expression of particular target genes to rise. The anti-inflammatory properties of corticosteroids from lipocortins, phospholipase A2 inhibitory proteins that regulate the formation of prostaglandins and leukotrienes by inhibiting arachidonic acid¹⁰.In particular, lipocortin-1 (annexin-1) is synthesised in response to glucocorticoids, and it attaches to cell membranes to block phospholipase A2 from getting into contact with its substrate, arachidonic acid¹⁶.

The two main products in inflammation Prostaglandins and Leukotrienes are inhibited by the action of Glucocorticoids²⁰.Our study revealed that foamy extract of C, melo subsp. agrestis var. conomoncould be the source of inexpensive bioactive compounds for the treatment of inflammation. Apart from prominent compounds other 50 compounds are also listed in Supplementary Data.

Conclusions

The presence of large number of phytoconstituents have been revealed C. melo var. agrestis commonly known sambar southe it has been used for its several properties  in conventional usage . However, not much work is being performed in foamy extract which has been carried out to show its valuable effects. Hence, this study will provide base for further research in this field. The soapy foamy extract of might be the resource of inexpensive bioactive compounds for the treatment of inflammation in medical field.

Acknowledgement

The authors were thankful for the Sophisticated Analytical Instrument Facility (SAIF), by IIT Bombay, Pawai, Mumbai to execute the HR-LCMS of sample. 

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

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