Introduction  

Sidaguri plant (Sida spp) is a plant in the Malvaceae family that is often used as traditional medicine against various diseases in various countries [1]. This plant can grow in tropical and subtropical areas consisting of about 200 species spread throughout the world [2]. Several species of Sida, such as Sida acuta, Sida cordifolia, Sida rhombifolia, Sida spinosa, and Sida veronicaefolia are widely used as traditional medicine in India (including Ayurvedic and Siddha), America, Africa, China, and Indonesia [3, 4]. In Indonesia, especially on the island of Sumatra, several species of the genus Sida have been found, such as Sida acuta, Sida rhombifolia, Sida subcordata, Sida scabrida, Sida cordifolia, and Sida retusa. Sida retusa is one of the Sida species that grow quite a lot in West Sumatra. Traditionally, this herb is often used as a remedy for rheumatism, neurological disorders (epilepsy), diuretics and fever, asthma [5, 6], cough, flatulence, colic, burning sensation, hemorrhoids, and fatigue [7], Tonic, aphrodisiac, diuretic and inflammation [8]. The chemical content of the Sida retusa plant includes ecdysteroids, alkaloids, steroids, phenolics, and flavonoids [9, 10].

Several studies have been conducted on this species of Sida retusa, among others Sida rhombifolia ssp. retusa seed extract inhibits DEN-induced murine hepatic preneoplasia and carbon tetrachloride hepatotoxicity was investigated in rats [11, 12]. S. rhombifolia subsp. retusa exhibits strong cytotoxic, antibacterial, antitubercular, and antimycotic activities [13, 14]. Aqueous extract of Sida rhombifolia ssp. retusa leaves have hypoglycemic and hypolipidemic effects in diabetic-induced animals [15]. Research on this species of Sida retusa has not been done much, so we are interested in conducting metabolite profiling, testing the total phenol and total flavonoid contents, and testing the antioxidant activity of this plant.

Materials and Methods

Sample and plant identification

Sida retusa L was collected on Perumdos Kampus Universitas Andalas Kel. Limau Manih, Kec. Pauh Padang, West Sumatra, Indonesia (Figure 1). Identification of Sida retusa L plants at the Andalas University Herbarium (ANDA) Department of Biology, Faculty of Mathematics and Natural Sciences, Andalas University Padang, West Sumatera, Indonesia, with collection number ANDA 00038434.

Chemicals, reagents, and materials 

All the chemicals and reagents used in this study were analytical grade and were purchased from Merck and Sigma-Aldrich. Spectrophotometry microplate reader (BioRad Xmark®), UV-Vis Spectrophotometer (Shimadzu PHarmspec 1700®), LC-MS instrument consisting of LC system (Waters Acquity UPLC I-Class), LC column (ACQUITY UPLC®) BEH C8 1.7 m 2.1 x 100 mm, Mass spectrometer (XEVO G2-XS QTof).

Sample preparation

The leaves of Sida retusa L West Sumatra are cleaned and air-dried for 2 weeks. Then it is sorted again and then mashed utilizing a grinder.

Extraction

Extraction was carried out using the maceration method according to the Indonesian Herbal Pharmacopoeia. A total of 50 grams of finely powdered leaves of Sida retusa L were macerated with 50 ml of 70% methanol. Soak for the first 6 hours, stirring occasionally, then let stand for 18 hours. Separate the macerate by filtering then the pulp is macerated again with 25 ml of 70% methanol. The obtained maserate was combined and then concentrated with a rotary evaporator until a thick extract was formed [16, 17].

Determination of total phenolic level

Determination of total phenol content by colorimetric method referring to the procedure of Subramanya et al. (2015) with some modifications with gallic acid (GAE) as standard. A 50 μL sample (1,000 μg/mL) was pipetted into a 96-well plate. Then 50 μL of aquabidest was added, Folin-Ciocalteu reagent 7.5% 50 μL and 1% NaOH 50 μL, then incubated for 60 minutes. The absorbance was measured using a microplate reader at 730 nm. The test was repeated 3 times. Gallic acid was used in the calibration curve to determine the total phenol content [18, 19]. The total phenol content in the extract was expressed as milligram gallic acid equivalent/gram extract (mgEAG/g) [20].

Determination of total flavonoid level

Determination of total flavonoid content by colorimetric method referring to the procedure of Sato et al. (2020) with some modifications with quercetin (QE) as standard. A 50 μL sample (1,000 μg/mL) was pipetted into a 96-well plate. Then AlCl₃ 10% (w/v) 30 μL, NaNO3 5% (w/v) 30 μL, aquabidest 50 μL, then incubated for 5 minutes. Then add 40 μL of aquabidest again, and incubate again for 30 minutes. The absorbance was measured using a microplate reader at 430 nm. The test was repeated 3 times. The total flavonoid content in the extract was expressed as the milligram equivalent of quercetin extract/ gram extract (mgQE/g) [21, 22].

Antioxidant activity test

Antioxidant testing using the DPPH Free Radical Attenuation method [23]. One hundred microliters of extract (range concentration 62.5 – 500 μg/mL) was put in a 96-well plate, and then 100 μL of DPPH solution in methanol was added. As a standard, gallic acid was used with the same treatment as the sample [24]. This mixture was stored at room temperature in a dark place for 30 minutes. The absorbance was measured at 517 nm. Each extract test was carried out 3 times. The IC₅₀ value is calculated using a linear regression equation [25-27].

Results and Discussion

Plant inventory of sida retusa L

The main characteristics and geographic origin of this Sidaguri plant (Sida retusa L) are shown in Table 1. Next for identification, plant samples were prepared under dry herbarium conditions. The determination was made at the Andalas University Herbarium, Padang. The morphology of Sida retusa L West Sumatra verified at Andalas University, Padang Herbarium is shown in Table 2.

Identify the chemical content profile

To identify the chemical content profile of Sida retusa L leaf extract, metabolite profiling was carried out using the LC-MS/MS method with specifications LC System (ACQUITY UPLC®H-Class System (waters, USA); LC Column (ACQUITY UPLC® HSS C18 (1.8 m 2.1). x100 mm) (waters, USA) and Mass Spectrometer Xevo G2-S QTof (waters, USA) at the Bogor Police Forensic Laboratory. Based on the data from the interpretation of compound content analysis using UPLC-QToF-MS, it can be seen that 5 main compounds from Sida retusa L leaf extract as shown in Table 3.

From the LC-MS/MS data, the interpretation of the five main chemical constituents of Sida retusa L, namely epoxypheophorbide A (24.63 %), scortechinone X (12.07 %), stigmastane-3β,5α,6β-triol (8. 25% ), shidasterone B (8.11 %) and linolenic acid (6.99 %) (Figure 2). From the five main chemical constituents of this species, there are several compounds that have been isolated from previous studies. epoxypheophorbide A is a class of phaeophytins compounds isolated from Sida rhombifolia species [28]. Shidasterone B is a class of ecdysteroid compounds, which have been isolated from species Sida cordifolia and Sida rhombifolia. Meanwhile, linolenic acid is a class of aliphatic compounds that have been isolated from species of Sida acuta and Sida rhombifolia [3, 29].