INTRODUCTION:

Inflammation is the basis pathogenesis of infection and non-infection diseases. Inflammation is the bodys physiological response to defend itself against physical trauma caused by chemicals and microbes¹. In the reaction, there is released some pro-inflammatory mediator such as prostaglandin, histamine and leukotriens causing the main cardinal sign of inflammation such as pain, swelling, redness and increase heat^1,2. However, chronic and uncontrolled inflammation have negative effects causing autoimmune hypersensitivity and degenerative disease¹.

Steroidal and non-steroidal anti-inflammatory drugs are frequently used to relieve inflammation in the health services center. Instead they have many side effects such as gastrointestinal disorder and blood clotting³.

In addition herb can be used as an alternative source instead. Several herbs have been investigated for their anti-inflammatory potential but they were few have been reported⁴. Therefore plant exploration is necessary to provide scientific data.

Pulutan (Urena robata) is a plant with medicinal properties. The ethanol extract of Urena lobata (U. lobashi) has a pronounced anti-inflammatory effect⁵. Pre-clinical study showed U. lobata acts antioxidant and antimicrobial related to the active substances such as quercetin and mangiferin⁶. Previous study about U. lobata leaf extracts as anti-inflammation have been reported by in vivo study^7,8. Flavonoid and terpenoid are the main active compound in the herbs predicted having anti-inflammatory activity based on in silico study⁹. Crude extract contains many phytochemical agent therefore it’s difficult to determine anti-inflammatory compound and their mechanism action. Fractionation is one of the method to separate active compound group based on the polarity using some organic solvent. We use non-polar to polar solvent in fractionation and evaluate the pharmacology effect of them.

Protein denaturation inhibition test is one of method used to screen anti-inflammatory activity of herbs. Denaturation of protein indicated tissue damage and they induce inflammation process by releasing cytokine pro-inflammatory¹⁰. They generate inflammation symptoms as mentioned above. The aim of study was to determine the potency of U. lobata leaf extract and its fractions in reducing inflammation through protein denaturation inhibition tests.

MATERIAL AND METHODS:

Sample Preparation:

U. lobata leaf powder were purchased from Laboratory UPT Materia Medika Batu, Malang, Indonesia with certificate number 074/027/101.8/2015. The simplicia powder (50g) was extracted in 250mL of methanol for 6 hours using shaker and repeated three times using fresh solvent. After evaporating the solvent, a part of concentrated extract (15g) was performed fractionation by partition using n-hexane solvent (fraction A), ethyl acetate (fraction B), n-butanol (fraction C) and water (fraction D) respectively. Each of fraction was evaporated to produce a paste form.

Identification of Flavonoid:

Extract or fraction were solved in ethanol therefore added hot methanol 50%, Magnesium (Mg) powder and hydro chloric acid concentrate. The change of colour to yellow, orange and red indicated the presence of flavonoid¹².

Determination of Total Flavonoid (DTF):

Total flavonoid compound was determined by alumunium chloride colorimetric analysis¹¹. An aliquot (1 mL) of crude extracts or fraction or standard solutions of quercetin (20, 40, 60, 80 and 100μg/mL) was added to 10 mL volumetric flask containing 4 mL of distilled water. To the flask was added 0.30mL NaNO₂ 5%, then added 0.3mL of AlCl₃ 10%. After 5minutes, 2mL NaOH 1 M was added and the volume was brought to 10 mL with distilled water. The solution was mixed and absorbance was measured at 510 nm wavelength against the blank. The total flavonoid content was determined from the calibration curve (Figure 1) and stated as mg quercetin equivalents (QE).

Anti-inflammatory bioassay:

The inhibition of protein denaturation test is used to determine the anti-inflammatory properties of medicinal plants and the method refers to William et.al 2008 with minor modifications¹⁰. Bovine Serum Albumin (BSA) 0,2% was solved in Tris Buffer Saline (pH=6,4). Both of sample and control each 100μL were added up to 1000 μL. They were incubated at room temperature 22°C± 2°C about 15 until 20 minutes and then heated to 70°C where the reaction mixture was kept for 5min. Furthermore, it was kept at room temperature for 15 minutes. The asorbance before and after denaturation was measured for each concentration (10μg/mL, 20 μg/mL, 30μg/mL, 40μg/mL and 50μg/mL) using a spectrophotometer at 660nm. Negative control used water, meanwhile ibuprofen was used as reference drugs.

(Abs of control-Abs of test)

% Inhibition = --------------------------------------- x 100 %

Abs of control

Statistic Analysis:

For protein denaturation inhibition, the inhibition percentage are shown as the mean ± SD and the IC₅₀ value was determined using a linear regression curve by SPSS version 16.

RESULTS:

Methanolic extract of U.lobata and fraction B indicated a high total flavonoid content compared to other fractions (Table 1). On the other hand, fraction A did not contain flavonoid compound based on phytochemical screening.

Figure 1: The calibration curve of Quercetine

Table. 1 Total Flavonoid content of U.lobata leaf extract and its fractions

Sample	Screening flavonoid	n	Total Flavonoid (mg QE/g)
Methanolic extract	+	3	90,28 ± 2,16
Fraction A	-	-	-
Fraction B	+	3	73,39 ± 2,43
Fraction C	+	3	36,73 ± 1,49
Fraction D	+	3	16,77 ± 1,39

Note (+): identified (-): not identified

Protein denaturation inhibition of U.lobata leaf extract and its fraction on were evaluated based on IC₅₀ values. The results described anti-inflammatory activity and were showed at table 2.

Table 2: Inhibitory activity on protein denaturation of U. lobata leaf extract and its fractions

Sample	n	IC₅₀ (μg/mL)	Category
Methanolic extract	3	338,80 ±9,8	Weak
Fraction A	3	430,20 ±10,4	Weak
Fraction B	3	491,69 ±11,2	Weak
Fraction C	3	208,75 ± 7,8	Moderate
Fraction D	3	NA	Inactive
Ibuprofen*	3	79,08 ± 4,1	Strong

Note NA: Not applicable *: References drug

Fraction C showed inhibition on protein denaturation stronger than fraction A, B and D (not applicable), the activity was classified into moderate category. Mean while, methanolic extract of U.lobata more weak than fractions C to suppress protein denaturation. Inhibitory potency of U.lobata leaf extract and its fractions are lower than Ibuprofen as reference drug on protein denaturation.

DISCUSSION:

Fraction C has anti-inflammatory activity strongest and was included into moderate category. They used n-butanol as a semi polar solvent and known by amphipathic fraction. Some active compounds soluble in this solvent such as glycosides, saponins, tannins and alkaloids¹³. Glycosides is a pro drugs metabolized by microflora, absorbed in the intestine and then transferred to the liver¹³. Fraction A and B are hydrophobic fraction, therefore any constituents such as oils, terpenoid, flavonoid, lignans, steroid and coumarin soluble in this fraction. Some of them like aglycones have high pharmacology activity^5,13. However, hydrophobic fraction (fraction A and B) in this study indicated a low activity to suppress protein denaturation. The possibility active compound in the hydrophobic fraction cannot soluble in the media, therefore they were not able to protect protein denaturation. Some of phytosterol such as β-sitosterol and stigmasterol were found in U.lobata leaf extract^14,15, they showed anti-inflammatory activity based on in silico study⁹.

Fraction C of U.lobata more potent to suppress protein denaturation than ethanolic extract. This possibility it caused by interaction of active compound which have antagonistic effect^3,16. Beside it, the component of crude extract more complex than fraction, moreover the possibility of interaction between active compound is greater^3,13. The composition of crude extract more complex than reference drug (ibuprofen) as single compound. Therefore, the possibility of interaction between active compound in the extract is greater and affect the activity^3,16, which may show an increase or decrease of the activity of substances.

Total flavonoid content of ethanolic extract and fraction B are higher than fraction C. However, their activity to supress protein denaturation or anti inflammation are lower than fraction C. It indicates the activity are controlled by others compound besides flavonoid. Other substances that is predicted as anti-inflammatory in this herb such as steroid and phenolic. U.lobata contains steroid compounds such as stigmasterols, β-sitosterol and phenolic such as mangiferin, gossypetin⁸. Mangiferin, stigmasterol, β-Sitosterol also indicated anti-inflammatory activity by in silico, in vitro and in vivo test^9,17,18,19. Based on previous research, U.lobata showed anti-diabetic potency by inhibition of dipeptidyl peptidase-4 (DPP-4) activity due to stigmasterol, β-Sitosterol and mangiferin^15,20,21. Other study indicated U.lobata has a low to moderate level of toxicity on zebrafish therefore it is safe to use for therapy²².

CONCLUSION:

Anti-inflammatory activity of n-butanol fraction (fraction C) of U.lobata stronger than its extract and other fraction, however methanolic extract has higher content of total flavonoid .

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

ACKNOWLEDGEMENT:

This work was funded by Research Grant of Ministry Education and Culture Indonesia.

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Received on 21.10.2021 Modified on 23.01.2023

Research J. Pharm. and Tech 2023; 16(11):5406-5409.

DOI: 10.52711/0974-360X.2023.00875