Total Phenolic and Flavonoid Content, Antioxidant, and Toxicity Test with BSLT of Meistera chinensis Fruit Fraction from Southeast Sulawesi

Meistera chinensis is one of the new generations of the Zingiberaceae family. Zingiberaceae have a large number of species and still need research to reveal their chemical content. Meistera chinensis, a local plant that is widespread in Konawe Regency, Southeast Sulawesi. Empirically, M. chinensis is used as a flavor enhancer in food, aches, and increases body immunity. There are no reports of chemical content or its biological activity. In this study, the fruit was tested for phytochemicals, antioxidants using the ABTS test, total phenolic, total flavonoid content, and toxicity test with BSLT. Meistera chinensis was extracted with ethanol and fractionated by ethyl acetate solvent use vacuum liquid chromatography. Phytochemical screening was carried out qualitatively by using the calorimetric method. The fraction toxicity was monitored by a lethal test for brine shrimp (BSLT). These fractions for the ABTS method obtained fractions 1-8 (F1-F8) and ascorbic acid were used as controls. The results showed radical scavenging activities fraction of M. chinensis fruit was a very strong activity with IC50 of 42.7±3.53 mg/L (F8). The total phenolic and flavonoid contents were 30.72±1.07 mgGAE/g and 8.02±0.48 mgQE/g, respectively. The phytochemical evaluation contains terpenoids, saponins, phenolics, steroids, alkaloids, and flavonoids. The BSLT toxicity test was found to be very toxic with IC50 of 5.2±0.72 mg/L. These findings indicate that the fruit of M. chinensis acts as an antioxidant and toxicity agent.


INTRODUCTION
The evidence from the last several decades strongly Previous research Zingiberaceae species such as Etlingera elatior (ginger torch) function as antioxidants 8 , anticancer 9 , antibacterial, antifungal, cytotoxic, tyrosinase inhibitory activity 10 , and immunomodulatory activity 11 . Literature studies suggest that E. elatior has pharmacological activities such as anti-tumor, anti-hyperglycemic, antiinflammatory, and anti-hyperuricemic 12 .
Meistera chinensis is one of the species that belong to the Zingiberaceae family. Meistera chinensis, which is the local plant of Southeast Sulawesi and found in the Konawe Regency. Empirically, it is used as a flavor enhancer in food, aches, and increases body immunity.
The safety of potential toxic effects in drug research is vital to ensure their use. The low toxicity and clinical effectiveness of natural compounds are one of the researchers' aims to obtain plant biological activity 13 .
Toxicity testing is the first step in drug safety parameters before it becomes a drug product used in humans. One of the toxicity testing methods is Brine Shrimp Lethality Test (BSLT). This method can identify the toxicity of natural ingredients 14 .
The evidence base of researchers has found that the antioxidants in various plants can be used to treat disease and maintain human health 15

Extraction
About 3,000 g of M. chinensis fruit powder dissolved with 95% ethanol in a closed glass container for 3 x 24 hours.
The maceration process was carried out for three days and filtered and replaced the solvent. The filtrate was collected and concentrated by using a rotary vacuum evaporator at 50°C to obtain a viscous extract of 150 g.

Fractionation by vacuum liquid chromatography
Separation of chemical compounds was carried out by thin-layer chromatography (TLC) to find suitable eluents as references in vacuum liquid chromatography.
Columns made using 60 GF254 silica gel with a ratio of 1:2.
Filter paper as a barrier and silica in the column, then  The LC50 score is defined as the concentration of a compound causing 50% mortality of shrimp larvae. Data were analyzed by probit in linear regression y = a + bx.
The level of toxicity of a compound is classified according to Meyer et al. 25 . Very toxic when the LC50 of ≤30 µg/mL, moderate toxic with LC50 of >30-1000 µg/mL, and low toxic with LC50 of >1000 µg/mL.

Statistical analysis
Total flavonoids and phenolic content were performed by UV-Vis spectrophotometry. Each sample analyzed was replicated three times and were presented as mean (± SD) at least three independent experiments using SPSS version 20.0. Statistical analysis was with ANOVA with statistical significance set at p <0.05.

Fractionation by vacuum liquid chromatography
The initial fraction with TLC aims to find a solvent system to separate the compound, so its pattern can be seen. Each The chromatogram shows differences in compounds from each fraction as indicated by the difference in the Rf value on the appearance of the spots formed (Figure 3).

Visualization of detection of separation results is essential
in TLC analysis. The UV detection involves using a UV active compound (indicator) put into a stationary phase.
Shortwave UV light (254 nm) with an indicator (manganese-activated zinc silicate) will produce a light green color, while the compounds will form dark spots.
Longwave UV light (366 nm) with an indicator will produce a purple color. Compounds that absorb 366 nm of UV light will appear as different spots, depending on the compound type. Compounds that do not absorb UV light at 254 nm or 366 nm are spray detected using the oxidizing reagent CeSO4 in H2SO4 to form black spots 28 .
The Rf value of the spot appearance of each fraction is presented in the Table I.  Table II shows the presence of a phytochemical.  Based on Table III Table IV. The total flavonoid content expressed as mg equivalent per g of dry sample was 8.02±0.48 mgQE/g (Table IV).
This results in sync with the total phenolic content

Toxicological Evaluation Brine Shrimp Lethality Assay
The toxicity of the M. chinensis fruit extract was measured using the BSLT method. The mortality percentage and LC50 of ethyl acetate fraction of M. chinensis are shown in Table V. The LC50 is a value that indicates the concentration of a toxic compound that causes the death of organisms up to 50%. The toxicity test for larvae mortality (Table V) showed an LC50 value of about 5.20±0.72 mg/L and was included in the category highly toxic (≤30 ppm). The LC50 of ≤30 mg/L is defined as highly toxic; LC50 of ≤1,000 mg/L as toxic; and LC50 of >1,000 mg/L as non-toxic.
Toxicity testing is a common method that can be used to discover new types of drugs 37 .
The BSLT method is based on an extract's toxicity test, which is simple, fast, inexpensive, and accurate 38 . The   (Table   II), such as flavonoids, phenolics, and terpenoids.
Zingiberaceae is a widespread plant group in Indonesia