1. INTRODUCTION
Portulaca grandiflora is one of the potential ornamental plants centered on esthetic value and nutritional value. P. grandiflora is a species of the Portulacaceae family that has been shown to have various pharmacological effects [1]. Apart from highlighting the attractiveness of its morphology, P. grandiflora has various bioactive compounds that are helpful in folk medicine and maintain the health of the body’s organs [2]. Ethnobotanical reviews noted that P. grandiflora has edible leaves [3]. Regarding its medicinal use, P. grandiflora functions as anti-inflammatory, antifungal, antiseptic, antidiabetic, and antispasmodic [4]. Furthermore, P. grandiflora is utilized to treat various mild-to-severe ailments [5].
As a medicinal and therapeutic plant, P. grandiflora has numerous phytonutrient compounds. P. grandiflora has a typical class of compounds named Portulacanones. Yan et al. [6] found that Portulacanones compounds have cytotoxic activity against human cancer cells. Besides phenolic compounds, the results of biochemical screening of P. grandiflora performed by Devi et al. [2] showed the presence of other compounds such as glycosides, phytosterols, saponins, and isoprenoids. Like most medicinal plants, P. grandiflora has a significant role in warding off free radicals. The capacity of plants to stave off free radicals is closely related to the presence of phenolic compounds [7].
Optimizing the potential of antioxidant activity in medicinal plants is necessary needed considering the number of emerging diseases nowadays. The antioxidant capacity of plants improves with the increase in the production of polyphenolic compounds [8,9]. Plant biochemical studies have reported considerable benefits of polyphenolic compounds on human health [10-12]. Different studies related to optimizing the plant growth environment contribute to the increase in plant secondary metabolites [13-17]. Moreover, improvement of the nutritional and biochemical properties of plants can be achieved through genetic improvement of plants. Mutation breeding, a combination of plant breeding programs and mutation techniques, is important in improving plant genetic potential [18]. One of the outcomes of the mutation breeding program is a new cultivar with more improved nutrition.
Artificial mutation technique using gamma-ray irradiation, ionizing radiation, is frequently utilized to increase plants’ morphological diversity and phytochemical content. Hong et al. [19] reported an increase in the activity of antioxidant enzymes and the phenolic content of wheat due to short and long exposure to gamma irradiation. El-Beltagi et al. [20] conveyed that the 5 kGy gamma-ray irradiation dose increased the glucose, fructose, flavonoid, and phenolic content of date fruits. Due to gamma-ray irradiation, the proline and polyphenols content improved significantly in Terminalia arjuna [21]. The recurrent irradiation method is one of the four methods of applying artificial mutations that plant mutation breeders prefer to maximize plant genetic diversity [22]. Several studies and reviews have reported increased morphological variations caused by gamma-ray mutagenesis using the recurrent method. Nevertheless, changes in biochemical composition due to recurrent irradiation treatment are still severely limited.
A plethora of comprehensive research on exploring and improving the biochemical compounds of P. grandiflora, particularly the polyphenol content, has been conducted [1-5]. However, to the best of our knowledge, no studies report the alteration of polyphenolic contents and the antioxidant capacity of P. grandiflora obtained from the gamma-ray mutation. This research aims to obtain information on the phenolic and flavonoid contents along with the antioxidant activity of three selected mutants of P. grandiflora in an advanced population (MV8 population), which is obtained through recurrent induced mutation using gamma-ray irradiation.
2. MATERIALS AND METHODS
2.1. Study Region
The process of harvesting three test genotypes of P. grandiflora was carried out in the experimental field of Sabisa Farm, IPB University, Sindang Barang, Bogor, West Java, and the biochemical analysis of P. grandiflora was situated at Tropical Biopharmaca Research Center, IPB University, Bogor, West Java.
2.2. Plant Material Preparation and Extraction Method
In this study, three P. grandiflora genotypes that arose out from the recurrent irradiation method were selected to measure the phenolic, flavonoid, and antioxidant activity. The underlying reason for selecting the three potential genotypes of P. grandiflora was based on the emergence of new morphological characters after eight generations of mutation breeding using the gamma-rays recurrent method [Table 1]. The whole plant part of the P. grandiflora was extracted as the test sample solution. The sample extraction was using the maceration method with slight modifications [23]. Beginning with weighing the fresh weight, then the three selected genotypes of P. grandiflora were dried using an oven until the water content reached less than 10%. Soon after, the dry weight is weighed, and the dry sample is formed into a powder. The powder samples of P. grandiflora were immersed in aqueous ethanol (70% v/v) for 48 h. The ratio between extract and solvent is 1:10. The filtrates were evaporated to obtain the sample in the form of a paste.
Table 1: Phenotypic descriptions of three selected P. grandiflora mutants.
| Genotypes | Sample code | Flower color | Stem color | Leaves color | Plant length (cm) | Flower diameter (cm) |
|---|---|---|---|---|---|---|
| PGA50+35 3.1.23 | PA | Pink-purple | Orange-brown | Green | 29.51–32.15 | 3.0–3.2 |
| PGB50+45 11.1.8 | PB | Pink-purple | Orange-brown | Green | 27.43–31.37 | 2.8–3.2 |
| PGC75+55 8.1.22 | PC | Pink | Orange-brown | Green | 21.13–25.04 | 2.5–3.0 |
2.3. Total Phenolic Content (TPC) Analysis
The phenolic content of P. grandiflora is estimated using the Folin–Ciocalteu method with modifications [24]. Folin–Ciocalteu solution consisting of 160 ml of purified water, 10 ml of Folin–Ciocalteu reagent (10%), and 20 ml of Na2CO3 (10%) was mixed with P. grandiflora extract and incubated for 30 min. The absorbance of the sample was measured at a wavelength of 750 nm using a microplate reader (Epoch BioTek, USA). The TPC was defined as mg gallic acid equivalent per gram of extract (mg GAE g-1).
2.4. Total Flavonoid Content (TFC) Analysis
The flavonoid assay was carried out using the aluminum chloride (AlCl3) solution method with a slight change [25]. The test solution consisted of a mixture of 10 ml of P. grandiflora sample, 60 ml of methanol, 10 ml of AlCl3 (10%), 10 ml of potassium acetate (CH3COOK) with a concentration of 1 molar, and 120 ml of distilled water. The sample mixture was stored in the dark for 30 min. The sample wavelength was measured at 415 nm. Total flavonoids in the sample were expressed in mg of quercetin equivalent per gram of extract (mg QE g-1).
2.5. 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Radical Scavenging Activity Assay
Antioxidant capacity analysis using the stable free radical DPPH referring to Nurcholis et al. [26] with modification. A 200 ml of extract consisting of each of the six concentration levels (0. 31.25, 62.5, 125, 250, 500, 1000, and 2000 ppm) was combined with 2 ml of DPPH (0.1 mM) and 0.8 ml of methanol. The test solution was homogenized for 1 h. Absorbance is measured using a microplate reader (Epoch BioTek, USA) at a wavelength of 517 nm.
2.6. Data Analysis
The fresh weight and dry weight data of three P. grandiflora genotypes are presented in a bar chart processed with Microsoft Excel 2010. The mean biochemical properties of P. grandiflora were tested statistically by analysis of variance (ANOVA) and followed by Tukey’s test at a 5% significance level to express a significant difference. Correlation analysis was performed by R studio software (corrplot package).
3. RESULTS AND DISCUSSION
3.1. Fresh and Dry Weight
Figure 1 provides information on the fresh and dry weights of the three selected genotypes of P. grandiflora. It is readily apparent that PA was recorded with the least fresh and dry weight, while PC showed the highest weight among the three genotypes. After the drying process, the three genotypes of P. grandiflora experienced an extreme reduction in weight. The highest dry weight was recorded in the PC genotype with 84 gr, and the lowest weight was recorded in the PA genotype with 69 g. Engagingly, PC genotype, plants with pink flowers, showed the lowest plant length and flower diameter compared to the other two genotypes (PA and PB) but had the highest weight among the two different genotypes.
 | Figure 1: Comparison of fresh and dry weights among three P. grandiflora genotypes. Different letters display significant differences based on the Tukey post hoc test at the 5% level. [Click here to view] |
Although exogenous factors influence plants’ fresh and dry weight, genetic factors cannot be separated from their contribution to plant mass. Vanani et al. [27] have explored the variety of fresh and dry weight in Lolium perenne genotypes. Sujatha et al. [28] reported their findings that the weight measurement of the genotypes of Gerbera, one of the well-known ornamental plants, proved to be significantly different. Chaimala et al. [29] found the genetic basis of the six test genotypes of Jerusalem artichoke to affect plant growth characteristics such as total biomass and shoot and tuber dry weight. In this study, the genotype diversity in fresh and dry mass was derived from the induced artificial mutation using gamma-ray irradiation, even though external factors also play a role in plant conditions.
3.2. Phenolics, Flavonoids, and Antioxidant Activity
The antioxidant, total phenolic, and total flavonoid activities of selected genotypes of P. grandiflora are summarized in Table 2. In this study, we measured the antioxidant effect of P. grandiflora extract through the reaction capacity with the stable free radical DPPH. Dzoyem et al. [30] explained that the smaller the IC50 value of an extract, the better the antioxidant potential. The IC50 value of the ethanolic extract of three genotypes of P. grandiflora showed that the PB sample had the highest antioxidant activity (270.56 g mL-¹), followed by the PA sample (301.15 g mL-¹), and last, the PC sample (327.59 g mL-¹). Bi et al. [31] classifying IC50 values based on the efficacy of antioxidant activity: Weak (IC50 >500 g mL-1), medium (IC50,100–500 g mL-1), and strong (IC50 >500 g mL-1). Referring to the former category, the antioxidant activity of the three selected genotypes of P. grandiflora was moderate. However, the IC50 of ascorbic acid, one of the positive controls frequently used for in vitro assays, far greater compared to the IC50 value of the three P. grandiflora genotypes (3.77 μmg-¹). The antioxidant capacity of the three potential genotypes of P. grandiflora was higher than Apium graveolens and Sonchus arvensis that had been analyzed by Sukweenadhi et al. [32]; three genotypes of Curcuma aeruginosa reported by Nurcholis et al. [26]; and four extracts of medicinal plants evaluated by Laryea [33]. Nevertheless, the methanol extract of Caesalpinia volkensii, Vernonia lasiopus, and Acacia hockii had more promising antioxidant activity than the three examined extracts of P. grandiflora [34].
Table 2: The mean values of phenolic, flavonoid, and antioxidant capacity of three selected genotypes of P. grandiflora.
| Sample | Genotypes | IC50 (g mL-¹) | TPC (mg EAG 100 g-¹) | TFC (mg EK 100 g-¹) |
|---|---|---|---|---|
| PA | PGA50+35 3.1.23 | 301.15a | 4.72 | 1.11 |
| PB | PGB50+45 11.1.8 | 270.56a | 4.57 | 1.16 |
| PC | PGC75+55 8.1.22 | 327.59a | 3.70 | 1.04 |
| A | Ascorbic acid | 3.77b | - | - |
Different letters display significant differences based on the Tukey post hoc test at the 5% level.
Phenolic content (TPC) in the three selected genotypes of P. grandiflora ranged from 3.70 to 4.72 mg EAG 100 g-¹, PC genotype had the lowest TPC content, while PA genotype was recorded with the highest TPC. Likewise, with regard to TFC, The PC genotype had the lowest flavonoid content (1.04 mg EK 100 g-¹), while in contrast, the PB genotype possessed the highest flavonoid content (1.16 EK 100 g-¹). Interestingly, in terms of morphology, the genotype of P. grandiflora with pink petals tended to have lower polyphenol content and antioxidant properties than the genotype with pink-purple petals. Interestingly, in terms of morphology, the genotype of P. grandiflora with pink petals tended to have lower polyphenol content and antioxidant properties than the genotype with pink-purple petals. Our findings agree with Gonçalves et al. [35] who found that the pink petal orchid had low phenolic content. Nonetheless, in particular cases, the phytochemical content in pink petal plants is greater than in colorless petals. Hallman [36] studied the polyphenol content in pink and white Robinia flowers and found that pink flowers contain more phytochemicals than white flowers. Another study conducted by Yang and Lu [37] revealed that white petal Macadamia flowers contain higher polyphenols and antioxidants than pink petal flowers. These unique phenomena occur based on the availability of different active secondary metabolites in each species. It is necessary to conduct further analysis, metabolite profiling, to compare the widespread secondary metabolites contained between pink petal and pink-purple petal of P. grandiflora.
3.3. Correlation Analysis
Figure 2 illustrates the relationship among the observed biochemical constituents of P. grandiflora. TPC and TFC have a solid positive correlation value of 0.84. This result aligns with the fact that flavonoid is one of the compounds deriving from phenolic compounds. These results correspond to the findings of Calvindi et al. [24] who informed a positive correlation between TPC and TFC in winged wings, but in contrast with Wairata et al. [38] who discovered a negative correlation between TPC and TFC in Garcinia forbesii. Moreover, regarding the antioxidant potential of P. grandiflora, TPC and TFC have strong negative correlation values with IC50 of –0.76 and −0.99, respectively. The negative correlation values, particularly between phenolic, flavonoid, and IC50, imply that the higher the phenolic and flavonoid content of P. grandiflora, the lower the IC50 value. Potent antioxidant expressed with a low IC50 value. Essence, TPC, and TFC are markedly responsible for the strength of the antioxidant properties of P. grandiflora.
 | Figure 2: Correlation matrix on the biochemical properties of P. grandiflora. [Click here to view] |
4. CONCLUSION
The three mutated genotypes of P. grandiflora resulting from artificial irradiation using gamma-rays had antioxidant capacity derived from phenolic and flavonoid compounds. These favorable genotypes have the prospect of being new multifunctional cultivars.
5. ACKNOWLEDGMENTS
The present research is fully supported by PDUPT research program (186/E5/PG.02.00.PT/2022) from the Ministry of Research, Technology and Higher Education of the Republic of Indonesia.
6. AUTHORS’ CONTRIBUTIONS
Waras Nurcholis conducted the research design, conducted research, analyzing the data, wrote manuscript draft, and improved the manuscript. Syarifah I. Aisyah conducted research designs and wrote drafts, Regina A. M. Saraswati conducted the research, analyzing the data, and wrote manuscript draft, and Yoshua S. Yudha wrote draft, improved the manuscript, and analyzing the data.
7. FUNDING
The Ministry of Research, Technology and Higher Education of the Republic of Indonesia through PDUPT research program with number of 186/E5/PG.02.00.PT/2022.
8. CONFLICTS OF INTEREST
State that the authors have no conflicts of interest.
9. ETHICAL APPROVALS
This study does not involve experiments on animals or human subjects.
10. DATA AVAILABILITY
All data generated and analyzed are included within this research article.
11. PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
REFERENCES
1. Ilakkiya V, Dhanalakshmi B, Kaviya V. Effect of physical treatment on the physicochemical, rheological and functional properties of yam meal of the cultivar “Ngumvu“from
2. Devi M, Komal S, Logeshwari B. Preliminary phytochemistry and antidiabetic activity of
3. Ao K. Investigation of antioxidant activity (
4. Ningrum AG, Frety EE, Diah I, Shabran ZH, Setiani RE, Dewi ER. Antioxidant activity of purslane (
5. Chowdhary CV, Meruva A, Naresh K, Elumalai RK. A review on phytochemical and pharmacological profile of
6. Yan J, Sun LR, Zhou ZY, Chen YC, Zhang WM, Dai HF,
7. Jorkesh A, Hamidoghli Y, Olfati J, Samizadeh H, Bakhshi D, Palá-Paúl J. Morphological and biochemical variability of
8. Suleria HA, Barrow CJ, Dunshea FR. Screening and characterization of phenolic compounds and their antioxidant capacity in different fruit peels. Foods 2020;9:1206. [CrossRef]
9. Kumar PS. Phytochemical and pharmacological overview of
10. Nurhasnawati H, Sundu R, Sapri S, Supriningrum R, Kuspradini H, Arung ET. Antioxidant activity, total phenolic and flavonoid content of several indigenous species of ferns in East Kalimantan, Indonesia. Biodivers J Biol Divers 2019;20:576-80. [CrossRef]
11. Chepel V, Lisun V, Skrypnik L. Changes in the content of some groups of phenolic compounds and biological activity of extracts of various parts of heather (
12. Rahman MM, Islam BM, Biswas M, Khurshid Alam AH.
13. Rahmah NN, Rafi M, Syukur M, Nurcholis W. Influence of combined NPK and manure on improving growth, photosynthetic characteristic and yield of
14. Marlin M, Simarmata M, Salamah U, Nurcholis W. Effect of nitrogen and potassium application on growth, total phenolic, flavonoid contents, and antioxidant activity of
15. Moradzadeh S, Siavash SM, Rahimi A, Pourakbar L, El Enshasy HA, Sayyed RZ. Bio-chemical fertilizer improves the oil yield, fatty acid compositions, and macro-nutrient contents in
16. Nurcholis W, Ambarsari L, Purwakusumah ED. Curcumin analysis and cytotoxic activities of some
17. Fitria R, Seno DS, Priosoeryanto BP, Hartanti, Nurcholis W. Volatile compound profiles and cytotoxicity in essential oils from rhizome of
18. Purente N, Chen B, Liu X, Zhou Y, He M. Effect of ethyl methanesulfonate on induced morphological variation in M3 generation of
19. Hong MJ, Kim DY, Jo YD, Choi HI, Ahn JW, Kwon SJ,
20. El-Beltagi HS, Aly AA, El-Desouky W. Effect of gamma irradiation on some biochemical properties, antioxidant and antimicrobial activities of sakouti and bondoky dry dates fruits genotypes. J Radiat Res Appl Sci 2019;12:437-46. [CrossRef]
21. Akshatha, Chandrashekar KR, Somashekarappa HM, Souframanien J. Effect of gamma irradiation on germination, growth, and biochemical parameters of Terminalia arjuna Roxb. Radiat Prot Environ 2013;36:38-44. Availabe from:https://www.researchgate.net/publication/289683867_Effect_of_gamma_irradiation_on_germination_growth_and_biochemical_parameters_of_Terminalia_arjuna_Roxb
22. Syukur M, Sastrosumarjo S. Plant Cytogenetics. 2nd ed. Bogor:IPB Press;2015.
23. Sahid ZD, Syukur M, Maharijaya A, Nurcholis W. Total phenolics, flavonoids, antioxidant activity, and a-glucosidase inhibitory activity of ornamental pepper and several other lines. Ornamental Hortic 2022;28:230-8. [CrossRef]
24. Calvindi J, Syukur M, Nurcholis W. Investigation of biochemical characters and antioxidant properties of different winged bean (
25. Khumaida N, Syukur M, Bintang M, Nurcholis W. Phenolic and flavonoid content in ethanol extract and agro-morphological diversity of
26. Nurcholis W, Khumaida N, Syukur M, Bintang M. Evaluation of free radical scavenging activity in ethanolic extract from promising accessions of
27. Vanani FR, Shabani L, Sabzalian MR, Dehghanian F, Winner L. Comparative physiological and proteomic analysis indicates lower shock response to drought stress conditions in a self-pollinating perennial ryegrass. PLoS One 2020;15:e0234317. [CrossRef]
28. Sujatha S, Aswath C, Shilpashree VM. Assessing resource use in gerbera genotypes for precision nutrient use in protected condition. J Plant Nutr 2020;43:2200-13. [CrossRef]
29. Chaimala A, Jogloy S, Vorasoot N, Toomsan B, Jongrungklang N, Kesmala T,
30. Dzoyem JP, McGaw LJ, Eloff JN.
31. Bi W, He C, Ma Y, Shen J, Zhang LH, Peng Y,
32. Sukweenadhi J, Yunita O, Setiawan F, Kartini K, Siagian MT, Danduru AP,
33. Laryea MK, Borquaye LS. Antimalarial, antioxidant, and toxicological evaluation of extracts of
34. Guchu BM, Machocho AK, Mwihia SK, Ngugi MP.
35. Gonçalves F, Gonçalves JC, Ferrão AC, Correia P, GuinéRP. Evaluation of phenolic compounds and antioxidant activity in some edible flowers. Open Agric 2020;5:857-70. [CrossRef]
36. Hallmann E. Quantitative and qualitative identification of bioactive compounds in edible flowers of black and bristly locust and their antioxidant activity. Biomolecules 2020;10:1603. [CrossRef]
37. Yang WH, Lu CZ. Phenolic composition and antioxidant properties of 2 taxa of macadamia flowers. Nat Prod Commun 2022;17:1-10. [CrossRef]
38. Wairata J, Fadlan A, Purnomo AS, Taher M, Ersam T. Total phenolic and flavonoid contents, antioxidant, antidiabetic and antiplasmodial activities of