INTRODUCTION:

Indonesia is a nation surrounded by tropical rainforests with a diverse range of plants that are utilized in traditional medicine. The richness of Indonesia's vegetation leads researchers to look for natural plant sources that may be utilized to treat a range of illnesses. ^1,2Natural materials have been widely utilised as drugs because they contain biochemical compounds that provide therapeutic effects for the body. 30-40% of medicinal products in circulation today are either directly or indirectly derived from natural materials.

The development of phytochemicals either for complementary or alternative medicine, to prevent or overcome diseases has been widely reported in recent decades. 80% of the world's plant population is believed to have medicinal properties.³ One plant that has been widely recognised by the public is the red dragon fruit (Hylocereus polyrhizus).

The peel of red dragon fruit is a high source of carotenoids, anthocyanins, flavonoids, and phenolic compounds—all of which are plant-based antioxidants. Anthocyanin levels, pectin components, and other substances including galacturonic acid, manossa, galactose, xylose, and ramnose are also present in the peel of red dragon fruit.^4,5 The results of research by Wahdaningsih et al. (2017) showed that the total flavonoid content of methanol extract, ethyl acetate soluble fraction and ethyl acetate insoluble fraction were 0.5139, 46.54 and 11.3811µgQE/g extract, respectively.⁶ Among the eight thousand naturally occurring phenolic chemicals, flavonoids represent the biggest category of plant phenolics.⁷ Flavonoids, a group of polyphenolic compounds found in fruits, vegetables, and grains, have been linked to several health benefits when consumed in meals.^8,9 A plant pigment called quercetin is a strong antioxidant flavonoid, particularly a flavonol. It is a versatile antioxidant with protective properties against tissue damage brought on by a variety of medication toxicities.¹⁰ Furthermore, quercetin has anti-inflammatory, antineoplastic, and immunomodulatory properties.^11,12

One technique for separating substances included in a mixture on a TLC plate is thin layer chromatography (TLC). The variations in a compound's affinity for and interaction with the stationary phase and mobile phase cause separation. two phases: mobile and stationary. The spots that emerge during elution may be qualitatively analyzed to determine the Rf (retardation factor) value. Furthermore, the RSD (relative standard deviation) value, the amount of compounds present on the TLC plate, and the compound recovery can all be quantitatively determined using this approach. (relative standard deviation), as well as recovery that demonstrates precision and accuracy.¹³ In cases where it comes to identifying the active ingredients in herbs, HPTLC is a great alternative to HPLC because of its many advantages, which include ease of use, precision, affordability, and speed of findings.¹⁴ While TLC has been used for flavonoid analyses since the early 1960s, the advancement of equipment that permits coupling of the. Although flavonoid studies have been conducted using TLC since the early 1960s, the advancement of equipment that permits the combination of densitometry and HPTLC separations of flavonoids. Different combinations of HPTLC stationary phases and developing solvents were utilized for the study of various flavonoid classes (flavonoid glycosides, polar flavonoid aglycones, and non-polar flavonoid aglycones), different combinations of HPTLC stationary phases and developing solvents were used.¹⁵ Quercetin analysis has been done in a number of studies, including the following: quantitative analysis of quercetin in Psidium guajava leaves using the HPTLC method and analysis of quercetin in the ethyl acetate fraction of Cosmos caudatus leaves and Centella asiatica plants.^16–18 However, research related to quercetin analysis in red dragon fruit peel extract using the HPTLC method is still rare. Based on the description above, it is necessary to conduct research on the qualitative and quantitative analysis of quercetin using the HPTLC-Densitometry method on red dragon fruit peel extract.

MATERIAL AND METHODS:

Materials:

Hylocereus polyrhizus peel samples were cleaned and then dried at room temperature for several days. After drying, it was blended and sieved with 60 mesh size. The 10kg sample powder was macerated with redistilled technical methanol at room temperature for 3 x 24 hours. The entire filtrate was collected and concentrated with an evaporator, then weighed. The methanol extract was then fractionated with n-hexane, dichloromethane, ethyl acetate and methanol. The fractionation results were then concentrated with an evaporator and weighed the yield.

Qualitative Analysis of Quersetin:

Red dragon fruit peel extract was measured as 500mg and dissolved in 1ml ethanol. Macerate the solution for 24hours then centrifuged to take the supernatant. Spotted the sample as much as 50µL on a silicagel 60 F₂₅₄ plate, include quercetin comparator. The plate was inserted into a saturated chamber with mobile phase n-hexane: ethyl acetate: formic acid (6:4:0.1), then elute until the limit mark and observe under UV light.

Quantitative analysis of quersetin¹⁹:

a) Quersetin standard solution preparation:

Weigh 10mg of standard quercetin, then put it into a 10 mL flask, add ethanol until the limit mark. Standard curve was prepared with concentration series of 5; 10; 25; 50; 75 and 100µg/mL.

b) Sample preparation:

100 mg of sample, were weighed then dissolved it with ethanol. The solution was vortexed and sonicated for 60 minutes. The sample was centrifuged and the supernatant was taken and then duplicated. The supernatant that has been obtained is filtered using a 0.45µm syringe

c) Determination of total quersetin content:

HPTLC was performed on Xbridge C18 5µm, 4.6x250 mm HPTLC plates with phosphoric acid 0,5%: methanol (60:40) as a mobile phase. The sample was injected into the HPLC and included the quercetin comparator. The sample was analysed for 20 minutes at a wavelength of 370 nm. This methods was triplicated.

RESULTS:

Qualitative Analysis Of Quersetin:

A stain parallel to the quercetin standard used was observed by ocular inspection, UV light at 254nm and 365nm, and the TLC test that was performed using the chosen mobile phase. Additionally, a retention value (Rf) of 0.26 was achieved, meeting the 0.2–08 Rf value criteria.²⁰ Qualitative test results can be seen in Figure 1.

Figure 1. Chromatograms obtained from separation of Hylocereus polyrhizus peel methanol extract and standard (Q). Visualization was under UV light of wavelength 254 nm (A), 365 nm (B), and visible light (C)

Quantitative Analysis of Quersetin:

a) Quercetin standard solution preparation:

The relationship between quercetin content and area gave a line equation of y = 38.41x+7.1237 with a correlation coefficient (R²) = 1, where y is the area formed (AUC) and x is the quercetin level (µg/mL). The regression curve of standard quersetin is shown in Figure 2.

Figure 2. Regression curve of standard quercetin

b) Determination of total quercetin content:

The HPTLC report of quercetin from the methanol extract of red dragon fruit peel (retention time 6.243-6.245) at 370nm showed well-resolved peaks when compared with the standard (Figure 3). Total quercetin levels were then quantified using the equation from the regression of the standard curve and obtained levels as shown in Table 1.

HPTLC results of red dragon fruit peel methanol extract at a wavelength of 370nm have shown well-resolved peaks when compared with standard as depicted in Table 1 and Figure 3.

Table 1. HPTLC analysis results of Hylocereus polyrhizus peel methanol extract

Obtained Parameters	Red dragon fruit peel methanol extract			Quercetin standard
Obtained Parameters	1	2	3	Quercetin standard
Retention Factor	36.414	36.938	36.944	36.90
Retention Time	6.243	6.245	6.244	6.252

Figure 3. Chromatogram of Hylocereus polyrhizus peel methanol extract. in the determination of levels, three repetitions were carried out to obtain the average total content. (Q) Standard quercetin

DISCUSSION:

The flavonoid class of polyphenolic chemicals, which includes quercetin, is a natural source of antioxidants. The antioxidant, anti-carcinogenic, anti-inflammatory, and cardioprotective effects of quercetin.^21,22 HPTLC is a helpful technique for evaluating quality when it comes to the identification and measurement of chemical compounds present in different types of green vegetables and plants.²³Based on the analysis that has been done, red dragon fruit peel extract positively contains quersetin compounds with an average total content of 206.76µg/G sample. The outcomes of this method's development can be compared with those of other studies that examined quercetin in a variety of plant samples. In these studies, the development of TLC analysis necessitates taking into account the number of sample spots, the wavelength of analysis, the combination of mobile phases, and the stationary phase that is used.¹⁸

Table 2. Total content of quercetin in Hylocereus polyrhizus peel methanol extract by triple replication.

Sample	Weight (g)	Amount (µg/g)	Mean (µg/g)
*Hylocereus polyrhizus* peel methanol extract	0,1026	226.46	206.76
	0,1022	226.52
	0,1018	167.29

CONCLUSION:

Our work sheds light on the components found in the methanol extracts of the peel of the red dragon fruit (Hylocereus polyrhizus), which were verified by straightforward techniques like TLC and HPTLC, for both inexperienced professionals and the area of pharmacognosy.

ACKNOWLEDGEMENT:

All authors have made substantial contributions to the work reported in the manuscript. Authors would like to thank all the individuals for their expertise and assistance throughout all aspects of our study and for their help in writing the manuscript.

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Received on 25.11.2023 Revised on 15.03.2024

Accepted on 10.06.2024 Published on 20.01.2025

Available online from January 27, 2025

Research J. Pharmacy and Technology. 2025;18(1):63-66.

DOI: 10.52711/0974-360X.2025.00010

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