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This study is aimed to assess the antibacterial and antiinflammatory effects of mangosteen (Garcinia mangostana L.) leaves through extracts. Phytochemical tests, disc diffusion assay, and bovine serum albumin denaturation assay are performed to identify and measure biological compounds, the antibacterial, and antiinfammatory effects, respectively. The extract of mangosteen leaves with the hypothesized antibacterial and antiinflammatory effects will be a potential source for treating various infectious and chronic diseases.
Cantho University Research Proposal B1904xxx ASSESSMENT OF ANTIBACTERIAL AND ANTI-INFLAMMATORY EFFECTS OF EXTRACTS OF MANGOSTEEN LEAVES Abstract This study is aimed to assess the antibacterial and anti-inflammatory effects of mangosteen (Garcinia mangostana L.) leaves through extracts Phytochemical tests, disc diffusion assay, and bovine serum albumin denaturation assay are performed to identify and measure biological compounds, the antibacterial, and anti-infammatory effects, respectively The extract of mangosteen leaves with the hypothesized antibacterial and anti-inflammatory effects will be a potential source for treating various infectious and chronic diseases Keywords: Mangosteen, antibacterial effect, anti-inflammatory effect Literature Review Mangosteen (Garcinia mangostana L.) which is part of the Guttiferae family, known as the purple mangosteen or mangosteen, (Stone, 2016) Mangosteen is a tropical fruit typically grown in tropical South East Asian countries such as Malaysia, Indonesia, and Thailand (Aizat et al., 2019) Mangosteen is round with thick skin (or also called pericarp) and ripens seasonally, from green to yellow to pink spotted and finally full purple colored fruit (Abdul-Rahman et al., 2017; Parijadi et al., 2018) The edible portion of the fruit resides within the pericarp, comprising of three to more than eight septa, white in color and having sweet-sour taste It is called ‘‘The Queen of Fruits” being among the best tasting tropical fruits (Stone, 2016) Mangosteen consists of chemical compounds, such as xanthones, phenol, flavonoids, saponins, tannins, anthocyanins, terpenes, vitamins B1, B2, and other bioactive substances supporting its medicinal properties (Aljunaid et al., 2020) In traditional medicine, Mangosteen has been used to treat skin infections and diarrhea Recent scientific studies suggest that mangosteen possesses strong antioxidant, anti-cancer, anti-inflammatory, anti-allergic, antimicrobial, and anti-malarial properties Mangosteen is a fruit as a daily meal of human We can buy these fruits in anywhere and anytime at markets Beside consuming fruits as eating fleshing fruits, some products of fruits such as juices and beverages made Nowadays, many studies indicate that mangosteen fruits have many health benefits for human health Mangosteen is a functional plant because most of the plants can be used as medicine (Suhartati et al., 2019) The parts of a mangosteen plant that have been widely used are flesh, pericarp and stems, while mangosteen leaves have not been widely used as ingredients of traditional medicine (Suhartati et al., 2019) The previous studies have been performed on extracts of mangosteen pericarp (Do et al., 2020; Oh et al., 2020) In fact, mangosteen leaves have not been specifically studied for their antibacterial and anti-inflammatory effects Thus, the study "Assessment of antibacterial and anti-inflammatory effects of extracts of Mangosteen leaves." will carried out The topic would aid in many further study that concern about ingredients of traditional medicine from popular plants Materials and Methods Sample preparation Mangosteen leaves are collected, washed, and dried at room temperature (25 oC) followed by crushing into a fine powder The powdered plant material (about 50 grams) is extracted by using Soxhlet extraction technique The crushed leaves is extracted with 350mL of 95% ethanol The excess ethanol is recovered and the crude extract is filtered with ether solution, which are then preserved at 4oC Phytochemical tests According to Rathod et al (2022), phytochemical tests of crude extract are conducted to identify the presence of phytochemicals by following the standard qualitative assays such as Biuret test, Molisch test, Salkowski test, Murexide test, and others - Test for proteins Biuret test is utilized The extract is added to 1mL of 40% sodium hydroxide and drops of 1% copper (II) sulfate solution A violet color will indicate the presence of proteins - Test for carbohydrates Molisch test is utilized The extract is added to drops of the Molisch reagent (a solution of α-Naphthol in 95% ethanol) The solution is then added to a few drops of concentrated sulfuric acid along the sides of the test tube The formation of a purple or reddish-violet color at the interface of two layers will indicate the presence of carbohydrates - Test for phenolic compound A few drops of 5% ferric chloride solution are added to 1mL of the extract solution A dark green or bluish-black color will indicate the presence of phenols - Test for flanovoids A few drops of dilute sodium hydroxide solution are added to 1mL of the extract An intense yellow color is appeared in the test tube The solution is then added to a few drops of dilute hydrochloric acid solution The resulting solution will be colorless because of the presence of flavonoids - Test for terpenoids A few drops of acetic anhydride are added to 1mL of the extract, and drops of concentrated sulfuric acid are then added The presence of a reddish-brown color will indicate the presence of terpenoids - Test for steroids Salkowski test is utilized The extract is dissolved in an equal volume of chloroform, and a few drops of concentrated sulfuric acid are then added along the sides of the test tube A bluish-red to cherry red color will observe in the upper layer, and the lower layer will show with green fluorescence, indicating the presence of steroids - Test for tannins The extract is added to distilled water A few drops of ferric chloride solution are then added A dark-blue or greenish-black color will indicate the presence of tannins - Test for caffeine Murexide test is utilized A few drops of the extract and an equal volume of nitric acid solution are added in a small porcelain disc The sample is then evaporated until it is dry After that, a few drops of ammonium hydroxide are added A purple color will indicate the presence of caffeine Antimicrobial activity measurement Disc diffusion assay is one method for quantifying the ability of antibiotics to inhibit bacterial growth (Hemeg et al., 2020; Amin et al., 2021; Nayak et al., 2021) The 6-mm-diameter discs of Whatman No filter paper are autoclaved for about 15 minutes at 121°C After that, the discs are dried in a drying oven Besides, the 24 hours grown bacterial cultures are evenly spread on Luria-Bertani agar plates The different extracts of mangosteen leaves are dissolved in the dimethyl sulfoxide solution The dissolved extract is then loaded on the 6-mm-diameter sterilized discs kept over agar surface Plates are incubated at 37°C for 24 hours The zones of inhibition are calculated in millimeters to determine the antibacterial activity Ampicillin is used as the positive control Assessment of anti-inflammatory activity Bovine serum albumin or BSA denaturation inhibition assay is an common assay to detect the preliminary anti-inflammatory effect of plant extracts (Deepa et al., 2015; Amin et al., 2021; Nayak et al., 2021) In this assay, the extracts at different concentrations are added to about 2mL of 1% BSA solution The pH is adjusted to 6.5 using 1N HCl solution The solution is incubated at 37°C for 20 minutes and then heated to about 60°C for 10 minutes and allowed to cool After cooling, the absorbance of the control and the test samples is measured by using a spectrophotometer at 660nm The experiment is conducted in triplicate, and the percent inhibition of BSA denaturation is calculated as follows: The inhibition of BSA denaturation (%) = Where: Ac − As x 100 Ac Ac is the absorbance of the control sample As is the absorbance of the test sample Data Analysis The results of this study will be showed by using One-way analysis of variance (ANOVA) and Tukey's test for Post-hoc analysis Charts and data will be processed by using Microsoft Excel software Anticipated Results In the preceded studies, the mangosteen pericarp has been shown to be plenty of bioactive compounds called xanthone derivatives that have memory-enhancing effects and are potential therapeutic value in Alzheimer’s disease, Parkinson’s disease, and depression (Do et al., 2020; Oh et al., 2020) These lead to an expectation that mangosteen leaves will have similar properties Besides, the hypotheses of the antibacterial and anti-inflammatory effects of mangosteen leaves will be potential to treat infectious diseases Furthermore, these will become an opportunity to substitute for fresh herb sources or the extracted herb products in market References Abdul-Rahman, A., Suleman, N I., Zakaria, W A., Goh, H H., Noor, N M., & Aizat, W M (2017) RNA extractions of mangosteen (Garcinia mangostana L.) pericarps for sequencing Sains Malaysiana, 46(8), 1231-1240 Aizat, W M., Jamil, I N., Ahmad-Hashim, F H., & Noor, N M (2019) Recent updates on metabolite composition and medicinal benefits of mangosteen plant PeerJ, 7, e6324 Aljunaid, M., Hariyani, N., Roestamadji, R I., Ridwan, R D., Kusumaningsih, T., & Qaid, H R (2020) Recent Updates of the Oral Benefits of Mangosteen Plant Extracts Journal of International Dental and Medical Research, 13(2), 752-757 Amin, M Z., Afrin, M., Meghla, N S., Nur, M A., Rahman, M M., & Uddin, M J (2021) Assessment of antibacterial, anti-inflammatory, and cytotoxic effects of different extracts of Gynura procumbens leaf Current Therapeutic Research, 95, 100636 Deepa, M., Darsan, M B., & Ramalingam, C (2015) In Vitro Evaluation of the antioxidant, anti-inflammatory and antiproliferative activities of the leaf extracts of Excoecaria agallocha L International Journal of Pharmaceutical Sciences, 7(11), 346-52 Do, H T T., & Cho, J (2020) Mangosteen pericarp and its bioactive xanthones: Potential therapeutic value in Alzheimer’s disease, Parkinson’s disease, and depression with pharmacokinetic and safety profiles International journal of molecular sciences, 21(17), 6211 Hemeg, H A., Moussa, I M., Ibrahim, S., Dawoud, T M., Alhaji, J H., Mubarak, A S., & Marouf, S A (2020) Antimicrobial effect of different herbal plant extracts against different microbial population Saudi Journal of Biological Sciences, 27(12), 3221-3227 Nayak, S S., Mirgane, N A., Pathade, K B., Shivankar, V S., & Wadhawa, G C (2021) Phytochemical analysis, antioxidant and anti-inflammatory activity of leaves and bark of Ceropegia rollae Hemadri Plant Science Today, 8(3), 425-428 Oh, Y., Do, H T T., Kim, S., Kim, Y M., Chin, Y W., & Cho, J (2020) Memoryenhancing effects of mangosteen pericarp water extract through antioxidative neuroprotection and anti-apoptotic action Antioxidants, 10(1), 34 Parijadi, A A., Putri, S P., Ridwani, S., Dwivany, F M., & Fukusaki, E (2018) Metabolic profiling of Garcinia mangostana (mangosteen) based on ripening stages Journal of bioscience and bioengineering, 125(2), 238-244 Rathod, M R., Rajappa, S K., Minagalavar, R L., Praveen, B M., Bharath, D K., & Kittur, A A (2022) Investigation of African mangosteen leaves extract as an environment-friendly inhibitor for low carbon steel in 0.5 M H2SO4 Inorganic Chemistry Communications, 109488 Stone, D (2016) Meet the mangosteen National Geographic Suhartati, R., Apriyani, F., Virgianti, D P., & Fathurohman, M (2019, July) Antimicrobial activity test of Mangosteen leaves ethanol extract (Garcinia mangostana Linn) against Pseudomonas aeruginosa bacteria In Journal of Physics: Conference Series (Vol 1179, No 1, p 012167) IOP Publishing