Identification of Secondary Metabolite Compounds in Two Varieties of Young Winged Beans (Psophocarpus tetragonolobus L.) at Two Harvest Ages

The winged bean (Psophocarpus tetragonolobus L.) is a member of the Fabaceae family (beans). Winged bean is developed as a vegetable, and young winged bean pods can be consumed fresh, steamed, fried, or pickled. In Southeast Asia, winged bean pods are generally cooked or consumed as side dishes or salad. Winged bean contains high protein content equivalent to soybean seed. Besides being rich in protein, vitamins, and minerals, the winged bean also has secondary metabolites, including phenolics and flavonoids. This study aimed to identify secondary metabolites in young pods of two varieties, “Fairuz” and “Sandi”, at two different harvest ages of 8 and 10 days after anthesis (DAA) and provide the biological activity on each identified compound. The experiment was conducted at the IPB experimental field at Leuwikopo, Bogor, from September 2020 to February 2021. The identification of secondary metabolites of the young green pods “Fairuz”, and the young purple pods “Sandi”, was performed using the GCMS method at the Regional Health Laboratory (KESDA) DKI Jakarta. The results of the GCMS analysis showed that 1,2-Benzedicarboxylic acid, mono (2-Ethylhexyl) ester was the most abundant compound identified from pods harvested at 8 DAA in both varieties, namely 42.26% in “Fairuz” and 26.66% in “Sandi”. Other compounds, 9,12,15-Octadecatrienoic acid, ethyl ester, (Z, Z, Z) (Linoleic acid ester), were found in “Fairuz”, whereas (9E,12E)-9,12-Octadecadienoic acid (Linoleic acid) was found in “Sandi”; these compounds are hydroxyl group and phenolic glucoside compounds and are found in pods harvested at 10 DAA.


Introduction
Winged bean (Psophocarpus tetragonolobus L.) is a member of the Fabaceae family (beans). Winged beans thrive in hot and humid tropical regions (Mohanty et al., 2013). India, Thailand, Papua New Guinea, and Indonesia (Khan, 1976;Krisnawati, 2016). Winged bean seed contains high protein content equivalent to soybean plants and can be substituted for soybean needs (Amoo et al., 2006;Handayani, 2013;Alalade et al., 2016). Winged bean has much potential as a protein source because, in addition to stems and roots, almost all parts of the plant, including young leaves, flowers, young pods, dried seeds, and tubers, can be consumed (Tanzi et al., 2019;Mohanty et al., 2020). Young winged pods can be consumed fresh, steamed, fried, or pickled, and in Southeast Asia, winged bean pods are generally cooked or consumed as a side dish or salad (Sriwichai et al., 2021). Therefore, winged bean has great potential to be developed as a useful legume crop (Handayani, 2013).
Young winged bean pods contain 3.99% carbohydrates, 0.16% fat, and 93.50% water content (Pangestu, 2020). Young pods contain around 6.1% albumin, globulin, and glutelin, and have a high lysine content, so young pods contain good quality protein compared to other pods (Tadera et al., 1984). Besides being rich in protein, vitamins, and minerals, winged beans also contain secondary metabolites, including phenolics and flavonoids (Singh et al., 2019), and have high tannin content (Mohanty et al., 2013). Immature winged bean pods contain amino acids such as isoleucine, leucine, lysine, methionine, phenylalanine, tyrosine, tryptophan, and valine. The pods also contain 5.0% sucrose, 1.0% raffinose, 2.5% stachyose (NRC, 1981). Traditionally, winged beans can be used to prevent cancer, diabetes, asthma, muscle weakness, overcome migraines, eye diseases, and strengthen the immune system (Singh et al., 2019). The total antioxidant capacity ranges from 1,278 to 1,806 mg AsA/g so winged beans can serve as a good source of antioxidants for humans and animals (Olaiya et al., 2018).
From the many chemical components ingredients in winged bean, studies related to the complete profile of secondary metabolites is not widely known, particularly of the newly developed varieties. Therefore, this study was carried out to investigate the secondary metabolites' profile in young pods of the winged beans. Gas Chromatography-Mass Spectrophotometry (GCMS) was used to analyze the range of secondary metabolites in winged beans. GCMS is one of the best techniques for identifying the constituents of volatiles, long-chain, hydrocarbons, branched-chain, alcohols, acids, and esters (Sermakkani dan Thangapandian, 2012). GCMS has successfully identified the bioactive compounds present in many plant species (Karthikeyan et al., 2019).
There are two new varieties of winged beans, namely "Fairuz" (green pods) and "Sandi" (purple pods). The color difference can indicate the difference in the phytochemical compounds contained in the pods. The phytochemical compounds can also be different from pods harvested at various ages. This study aims to identify phytochemical compounds in young pods of winged bean and compare the results between the two harvest ages 8 and 10 days after anthesis (DAA).

Experimental Site and Materials
The experiment was conducted at the IPB experimental field Leuwikopo, Bogor, West Java, Indonesia, from September 2020 to February 2021. The analysis of secondary metabolites in winged bean pods was conducted at the Regional Health Laboratory (KESDA), DKI Jakarta.
Two winged bean varieties were used to identify the secondary metabolites in young pods, namely "Fairuz" (green pod) and "Sandi" (purple pod). The young pods were harvested 8 or 10 days after anthesis (DAA).

Sample Preparation
Sample preparation was carried out before injecting test substances into each tool. Samples were dried in an oven at 60°C for approximately 3 hours or completely dry.

Sample Extraction
The dried samples were mashed using a blender and then macerated with methanol for 5x 24hours. Each macerated sample was then put into a 10 ml tube using a pipette and dried for 1 hour at 60°C. After drying, it was dissolved with the remaining 200 μl of the macerated extract, followed by chemical analysis using a Gas Chromatography-Mass Spectrometer (GCMS).

Analysis of Secondary Metabolites using GCMS
The analysis procedure conducted by the Regional Health Laboratory followed the Research Institute for Spices and Medicinal Plants method (Utami et al., 2017). The GCMS was used to analyze the content of secondary metabolites in the Agilent 7890 gas chromatography technology with automatic sampling and 5975 mass selective detectors and data systems. A total of 5 μl samples of young winged pods was injected into the GCMS, which has a capillary column size of HP Ultra 2 with a length of 30 m x 0.20 mm x 0.11 m film thickness. The oven temperature was set at an initial temperature of 80°C, then rose at 3°C/ min to 150°C, held for 1 minute, and finally increased 20°C/min to 280°C and held for 26 minutes. The injection temperature was 250°C, the ion source temperature was 230°C, the interface temperature was 280°C, and the quadrupole temperature was 140°C. Helium gas was used as the carrier gas at a constant flow rate of 1.2 mL/min (8:1 split).

Result and Discussion
GCMS had successfully identified the secondary metabolites or phytochemicals of the winged bean pods (Figure 1, 2). Thirteen (13) 13 different compounds were identified in "Fairuz" harvested at 8 and 10 DAA, and 17 and 15 compounds identified in "Sandi" harvested at 8 and 10 DAA, respectively.
Identification of the secondary metabolites was confirmed based on the peak area, retention time (RT), and molecular formula. The retention time depends on the molecular weight of the compounds; our samples had the retention times of 20-30 minutes. In "Fairuz" harvested at 8 DAA, the first compound with a lower retention time was Hexadecanoic Acid, Ethyl Ester (27.831 minutes). In contrast, the last phytochemical compound identified that required the longest retention time was gamma-Sitosterol  minutes), while the phytochemical compound that required the longest retention time was Pyrene, hexadecahydro-(37.174 minutes) ( Table 1).
In "Sandi" harvested at 8 DAA, the secondary metabolite compound identified with the shortest retention time was Hexadecanoic Acid, Ethyl Ester (27.845 minutes). In contrast, the phytochemical compound identified with the most prolonged retention was gamma-sitosterol (37.250 minutes). In "Sandi" pods harvested at 10 DAA, 2,5-Furandione (3.794 minutes) had the lowest retention time, while stigmasterol (35.809 minutes) was the longest retention time ( Table 2).
The phytochemical compounds identified in "Fairuz" and "Sandi" from two harvest dates have biological activity. Therefore, "Fairuz" and "Sandi" are rich sources of secondary metabolites with biological activities (Konovalova et al., 2013).
The abundance distribution of identified secondary metabolites from these two varieties of winged bean at two harvest ages is shown in the heatmap. The heatmap was based on the distribution of phytochemical with an abundance > 1% ( Figure  3). The highest content of secondary metabolite compound from pods harvested at 8 DAA from these two varieties was 1,2-Benzenedicarboxylic acid mono (2-Ethylhexyl) ester. In contrast, at 10 DAA harvest age, the highest abundance compound was 9,12,15-Octadecatrienoic acid, ethyl ester, (Z,Z,Z) in the "Fairuz" and (9E,12E)-9,12-Octadecadienoic acid in the "Sandi".

Discussion
From the results of the GCMS analysis, it has been shown that the identified compounds in the two wingead bean varieties and at harvest ages are very diverse. The compounds with the highest content found in both varieties at each harvest were derived from the linoleic acid compound group of fatty acids. These compounds have biological activity as antiinflammatory, hypocholesterolemic, antihistaminic, antiarthritic, antiacne products, as well as nematicidal and insecticidal properties (Sermakkani dan Thangapandian, 2012).
There are compounds in the "Sandi" harvested at 10 DAA that were not found in those harvested at 8 DAA, or in the "Fairuz" harvested at 8 and 10 DAA. These compounds are from the hydroxyl group and phenolic glucoside, with one of their activities being as antioxidants (Uchegbu et al., 2017;Li et al., 2019). "Sandi" has a purple pod, and the color is darker when harvested at 10 DAA than it is at 8 DAA. The darker color can indicate higher content of metabolite compounds. Previous studies reported that purple, red or blue fruits, and vegetables generally contain anthocyanin pigments (Juansah et al., 2013;Reswari      . The anthocyanin pigments are a group of flavonoids whose activity is an antioxidant (Nomer et al., 2019), which in this study was demonstrated in pods harvested at 10 DAA. Many phytochemical compounds with biological activities make young winged pods have good health benefits, and they can be consumed as functional vegetables.

Conclusion
The secondary metabolite compounds mostly from the linoleic acid group were identified from the young green pods "Fairuz" harvested at 8 and 10 DAA. Similar compounds were found in the purple pods "Sandi". The purple pods harvested at 10 DAA have additional compounds with antioxidant activity other than those from the linoleic acid group. 1,2-Benzenedicarboxylic acid, mono(2-Ethylhexyl) ester was the most abundant compound identified from pods harvested at 8 DAA in both varieties. Therefore, young winged bean pods have benefits when consumed as vegetables.