Research Article |
Corresponding author: Weny J. A. Musa ( wenymusa977@gmail.com ) Academic editor: Rumiana Simeonova
© 2023 Weny J. A. Musa, Nurhayati Bialangi, Ahmad Kadir Kilo, Boima Situmeang, Ninik Triayu Susparini, Ilham Dwi Rusydi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Musa WJA, Bialangi N, Kilo AK, Situmeang B, Susparini NT, Rusydi ID (2023) Antioxidant, cholesterol lowering activity, and analysis of Caesalpinia bonducella seeds extract. Pharmacia 70(1): 97-103. https://doi.org/10.3897/pharmacia.70.e96817
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Tombili plant (Caesalpinia bonducella) belongs to the family of Fabaceae. The seed extract of tombili has been empirically used as a traditional medicine. The purpose of this research was to fractionate tombili seed extract and test their antioxidant and cholesterol lowering activities. Extraction was made into fractions using n-hexane, ethyl acetate, and methanol as a solvent. The chemical compound of the ethyl acetate fraction was analyzed using liquid chromatography-mass spectrometry (LC-MS/MS). Antioxidant activity was tested using the DPPH method. The highest antioxidant activity was shown in ethyl acetate fraction with an IC50 value of 86.153 μg/mL. The second was the methanol fraction with an IC50 value of 94.053 μg/mL, and the third was the n-hexane fraction with an IC50 value of 100.933 μg/mL. The cholesterol lowering activity analysis showed that all fractions could inhibit cholesterol. The highest anti-cholesterol activity shown in ethyl acetate fraction with the concentration of 600 μg/mL can inhibit 81.5% of the cholesterol activity. The LC-MS/MS analysis showed that the ethyl acetate fraction contained glucoside, homoplantaginin, and vernolic acid compounds.
antioxidant, cholesterol lowering activity, Caesalpinia bonducella, LC-MS/MS analysis
Cardiovascular diseases (CVDs) are the diseases of heart and blood vessels which considered one of the leading causes of death worldwide. Hypercholesterolemia is a key risk factor for cardiovascular disease and monitors the primary mortality of developing heart disease. The release of toxic free radicals by endothelial cells and vascular smooth muscle cells is the primary pathogenic factor for CVDs (Taleb et al. 2018). The cholesterol present in these matrices also can oxidize in favorable conditions such as the presence of water, temperature, pH, and the form of substrate (
One common plant that has been used in traditional medicine is Caesalpinia bonducella, belongs to the family of Fabaceae (
In an earlier study, the phytochemical screening of Tombili seed showed that tombili seed contains saponins, phytosterols, flavonoids, and phenolics group compounds (Singh and Raghav 2012). This plant has proven pharmacological activity including the reduction of glucose levels (
The tombili seeds collected from Bobohu villages, Gorontalo province, Indonesia. Tombili was identified at the Laboratory of Biology Department (Plant Taxonomy), Faculty of Mathematics and Natural Science, Gorontalo State University, Indonesia with the specimen being identified by Weny JA Musa and verified by the relevant department under specimen number of 021/UN47.B4./LL/2019 as Caesalpinia Bonducella (L.) Fleming. The chemicals used were methanol, ethyl acetate, and n-hexane solvent (Pro Analysis Grade) used for extraction and fractionation. 2,2- diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The cholesterol solution Sigma-Aldrich (St. Louis, MO, USA), sulfate acid (H2SO4), and acetic anhydride were purchased from Merck (Darmstadt, Germany).
The extract was made with 1.2 kg of Tombili seed powder and macerated for three days. To obtain n-hexane and ethyl acetate fractions, 200 of the extract was partitioned by liquid-liquid extraction with n- n-hexane and ethyl acetate at a 1:1 ratio. The rotary evaporator was used to concentrate all fractions. The yield of each solvent was then determined. The fractions were created by combining n-hexane (29.5 g) and ethyl acetate (63.4 g) Antioxidant and cholesterol-lowering activity was assessed in all fractions. The fraction with the highest antioxidant activity and the lowest cholesterol activity was injected into an LC-MS/MS for chemical compound analysis. The external appearance and necessary procedure for the extraction are shown on Fig.
The antioxidant activity of each fraction was tested using the DPPH technique. According to
%inhibition = [(absorbance control – absorbance sample) / absorbance control] × 100%
The fractions’ reduced cholesterol levels were determined by comparing them to the blank absorbance (cholesterol solution). The cholesterol solution stock was created by combining 100 mg of cholesterol in 100 mL of ethanol (1000 ppm). H2SO4 (0.1 mL) and acetic anhydride (2 mL) were added to 0.025, 0.05, 0.1, 0.2, 0.3, and 0.4 mL of cholesterol solution in a cuvette (BRAN UV cuvette) and diluted with ethanol solvent until 5 mL, respectively (blank solutions). To 0.5 mL of cholesterol solutions, 0.025, 0.05, 0.1, 0.2, 0.3, and 0.4 mL of solution (1000 ppm) to concentrations of 100, 200, 400, and 600 ppm sample, H2SO4 (0.1 mL), acetic anhydride (2 mL), and ethanol until 5 mL were added, respectively. After being incubated for 30 minutes at room temperature, the absorbance of the solutions was measured at λ 423 nm (
The extraction efficiency from plant materials is influenced by several factors, including the chemical screening of phytochemicals, the partition method used, the size of sample particles, and the presence of interfering substances (Stalikas 2007). The extractions yield depends on the temperature, extraction time, solvent polarity, and sample content. In this work, the extracts of tombili seed were partitioned by using various range polarity of solvent from nonpolar (n- hexane) to polar (methanol). The final methanol fraction obtained 107.1 g (55.35%), n-hexane (14.7%) while ethyl acetate (31.7%). It can also be seen that the fraction yield of methanol is higher than ethyl acetate. These results show that the extraction yield.
DPPH was used to determine the antioxidant activity of tombili seed extracts. With the highest absorption band at 517 nm, DPPH was used as a stable organic free radical reagent. The absorption of this radical species was lost when the polarity of the solvent used in the extraction process of tombili seed was increased. Primary metabolites (proteins and carbohydrates) have been extracted in water and ethanol, resulting in a higher yield than ethyl acetate and n-hexane.
Accepted, resulting in a purple-to-yellow visual discoloration This radical reacts quickly with a wide range of samples and is sensitive enough to distinguish active ingredients at low concentrations (
Fractions | Concentration (ppm) | % inhibition1 | % inhibition2 | % inhibition3 | IC50 μg/mL | Average (μg/mL) |
---|---|---|---|---|---|---|
n-Hexane | 20 | 6.308 | 5.900 | 6.088 | 100.798 99.505 102.495 | 100,933±1.4995 |
25 | 9.227 | 10.041 | 9.803 | |||
30 | 12.409 | 12.008 | 11.764 | |||
35 | 14.994 | 15.010 | 14.860 | |||
40 | 16.856 | 17.080 | 16.718 | |||
Ethyl acetate | 20 | 4.251 | 5.678 | 6.832 | 88.499 81.286 88.675 | 86.153±4.2161 |
25 | 8.299 | 10.972 | 9.420 | |||
30 | 12.348 | 13.474 | 14.078 | |||
35 | 14.372 | 1 6.554 | 17.184 | |||
40 | 17.712 | 20.692 | 18.840 | |||
Methanol | 20 | 11.334 | 6.308 | 6.617 | 93.551 95.890 92.718 | 94.053±1.6445 |
25 | 12.938 | 9.227 | 10.084 | |||
30 | 16.248 | 12.409 | 11.974 | |||
35 | 18.115 | 14.994 | 15.336 | |||
40 | 22.066 | 17.786 | 18.907 |
Table
Cholesterol lowering activity test of fractions Caesalpinia bonducella seed.
Sample | Concentration (ppm) | % exhibited1 | % exihibited2 | % exihibited3 | Average (%) |
---|---|---|---|---|---|
n-hexane | 100 | 7.9 | 6.1 | 7 | 7 |
200 | 2 | 6.1 | 4.05 | 4.05 | |
400 | 22.8 | 21.2 | 22 | 22 | |
600 | 25.7 | 31.3 | 28.5 | 28.5 | |
Positive Control | 100 | 57.4 | 49.5 | 53.5 | 53.5 |
Ethyl acetate | 100 | 15.9 | 13.8 | 14.9 | 14.9 |
200 | 43 | 40.6 | 41.8 | 41.8 | |
400 | 67.9 | 67.5 | 67.7 | 67.7 | |
600 | 80.9 | 82 | 81.5 | 81.5 | |
Positive Control | 100 | 90.9 | 91.6 | 91.4 | 91.3 |
Methanol | 100 | 15.5 | 7.7 | 11.6 | 11.6 |
200 | 47.1 | 39.5 | 43.3 | 43.3 | |
400 | 68.2 | 65.5 | 66.9 | 66.9 | |
600 | 76.2 | 77.3 | 76.8 | 76.8 | |
Positive Control | 100 | 83.7 | 83.4 | 83.6 | 83.6 |
The amount of cholesterol-lowering salt was reduced dose-dependently. The cholesterol level in the untreated fractions, also known as the negative control, was calculated to be 100%. All experiments were repeated three times (n = 3) and compared to negative and positive controls. Table
It was first reported that tombili fractions have cholesterol-lowering properties. All fractions were carried out at concentrations of 100, 200, 400, and 600 ppm, respectively. The colorimetric experiment revealed an anti-cholesterol effect for n-hexane, ethyl acetate, and methanol fractions. The ethyl acetate component has the highest percentage. More than 50% of cholesterol can be detected in a 600ppm ethyl acetate fraction. Ethyl acetate has a greater effect than n-hexane and methanol.
Caused by the concentration of the chemical compound in fraction with the hydroxyl group that interacted with another functional group through hydrogen bonding. Unlike ethyl acetate, the intramolecular reaction of n-hexane and methanol fractions does not occur.
The ethyl acetate fraction TEA3 was analyzed for its chemical composition using LC-MS/MS (Fig.
Role in its higher activity as an anticancer therapy. These outcomes Liquid chromatography-mass spectrophotometry (LC/MS) analysis of ethyl acetate fraction showed five obvious peaks at retention times of 7.34, 8.63, 9.31, 9.38, and 9.89 min as shown on Fig.
Peak | tR (min) | Formula | Observed actual mass | Neutral mass (Da) | Adduct | Mass error (mDa) | Identification |
---|---|---|---|---|---|---|---|
1 | 8.63 | C23H26O10 | 485.1405 | 462.15260 | +Na | -1.4 | 9,10-dimethoxy-pterocarpan-3β-glucoside |
2 | 7.34 | C22H22O11 | 463.1223 | 462.11621 | +H | -1.2 | Homoplantaginin |
3 | 9.31 | C18H32O3 | 319.2252 | 296.23514 | +Na | 0.8 | Vernolic acid |
4 | 9.89 | C17H16N2O2 | 281.127 | 280.12118 | +H | -0.9 | – |
5 | 9.38 | C26H38O7 | 463.2671 | 462.26175 | +H | -2.0 | – |
The tested cholesterol lowering property of n-hexane, ethyl acetate, and methanol fraction from tombili seed was first reported. The highest antioxidant activity was shown in the ethyl acetate fraction. All fractions showed cholesterol lowering activity. Most importantly, the raised concentration of fractions exhibited a dose-dependent manner. The analysis of chemical content showed that the ethyl acetate fraction contains 9,10-Dimethoxy-pterocarpan-3β-glucoside, homoplantaginin, and vernolic acid compounds as lowering cholesterol agent.
The authors would like to thank the Ministry of Research, Technology, and Higher Education of the Republic of Indonesia, which has provided research funding through PTUPT schemes in 2021. Thank you, Mrs. Sofa Fajriah, in the Indonesian Institute of Sciences for LC/MS measurements.