Effect of Nepenthes Ampullaria Jack Extract on the Cell Growth, Cell
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Research Article Effect of Nepenthes ampullaria Jack extract on the cell growth, cell membrane integrity, and morphology of mycobacterial cells Shuaibu Babaji Sanusi1,2, Mohd Fadzelly Abu Bakar1*, Maryati Mohamed1, Siti Fatimah Sabran1, Azizul Isha3 ABSTRACT Objective: The study is aimed to evaluate the antimycobacterial properties of Nepenthes ampullaria extracts and to investigate its effect on cellular growth, cell wall integrity, and morphology of mycobacterial cell. Materials and Methods: N. ampullaria root was obtained from Johor National Park, Endau Rompin, and was extracted with methanol, water, ethyl acetate and n-hexane. The antimycobacterial activity of the extracts was tested using microdilution assay. The time-kill assay, leakage of compound absorbing at 280 nm, and field emission scanning electron microscopy were carried out to investigate the effect of the extract on cellular growth, membrane integrity, and morphology. The bioactive compounds present in the crude extract were investigated using gas chromatography–mass spectrometry (GC–MS). Results: The hexane extract of N. ampullaria demonstrated the best antimycobacterial activity against Mycobacterium smegmatis (minimum inhibitory concentration [MIC] and minimum bactericidal concentration values of 0.39 and 1.56 mg/mL, respectively). At 3-fold of MIC, hexane extract of N. ampullaria killed the entire bacterial cell within 8 h of exposure by causing the cell lysis. The GC–MS analysis revealed the presence of phytoconstituents that might contribute to the antimycobacterial effect. Conclusion: The study demonstrated the antimycobacterial properties of N. ampullaria and further studies could lead to the development of new antituberculosis agents. KEY WORDS: Antimycobacterial activity, Membrane integrity, Mycobacterium smegmatis, Nepenthes ampullaria INTRODUCTION the novel compound from natural sources with little side effects, cost effective, and non-toxic with a novel Tuberculosis (TB) is still seen as one of the major mechanism of action is urgently required to combat this health issues, especially in the low-income countries.[1] menace. Natural occurring pure compounds, as well as Approximately 2 billion persons are said to be infected extracts from plants that have inhibitory activity against with TB, but only about 10% which is around 9 million mycobacterial cells, are widely found in nature.[4] individuals becomes ill with active disease in their Nepenthes species from family Nepenthaceae which is lifetime, and almost 2 million die from it every year.[2] widely known as pitcher plant is a carnivorous plant. It Even though the disease can be treated and cured with produces unique pitcher for catching and digestion of chemotherapy, the treatment course is too lengthy pray (insect) to obtain nutrients at the habitats deprived taking 6–9 months leading to poor compliance of of nitrogen. Nepenthes ampullaria is different from patients. Consequently, result for the selection of drug other pitcher plants as its evolved a detritivore habit resistance including deadly multidrug-resistant TB and for the acquirement of nutrients from leaf litter in extensively drug-resistant TB bacteria.[3] The need for place of insects.[5] Brunei Darussalam, Kalimantan (Indonesia), and Malaysia (Sabah and Sarawak) Access this article online are considered to be the world diversity center of Nepenthes.[6] The root of Nepenthes is traditionally jprsolutions.info 0975-7619 Website: ISSN: used to relieve gastrointestinal discomfort, including 1Department of Technology and Natural Resources, Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia, Pagoh Educational Hub, 84600 Pagoh, Johor, Malaysia, 2Department of Microbiology, Faculty of Science, Kaduna State University, Tafawa Balewa Way, PMB 2339, Kaduna, Nigeria, 3Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia *Corresponding author: Mohd Fadzelly Abu Bakar, Department of Technology and Natural Resources, Faculty of Applied Sciences and Technology, Universiti Tun Hussein Onn Malaysia, Pagoh Educational Hub, 84600 Pagoh, Johor, Malaysia. E-mail: [email protected] Received on: 15-07-2019; Revised on: 19-08-2019; Accepted on: 08-11-2019 Drug Invention Today | Vol 11 • Special Issue 2 • 2019 209 Shuaibu Babaji Sanusi, et al. dysentery, stomachache, and bedwetting,[7] while Preparation of the Inoculum the pitcher has reportedly been used for diabetes The Mycobacterium smegmatis MC2 155 (ATCC [8] treatment. N. ampullaria and Nepenthes gracilis are 700084) used in this study was obtained from [9] the two most common species used in folk medicine. Microbiology Laboratory, FAST, UTHM. The Root decoction of N. ampullaria was understood to pure isolate was prepared and then maintained on be employed in asthma treatment by Orang Asli Middlebrook 7H10 Agar at 37°C. The inoculum from Jakun community in Endau Rompin, Johor, density was adjusted to 0.5 McFarland standards [10] Malaysia. (approximately 1.5 × 108 CFU/mL) which was further diluted to 1100 ratio (approximately 1 × 106 CFU/mL). Several previous studies have reported the biological [11] activity of other Nepenthes species such as antioxidant, Determination of Antimycobacterial Activity antidiabetic,[12] antimalarial,[13] antibacterial,[14] The antimycobacterial activity was evaluated using antifungal,[15] and anti-inflammatory.[16] However, no attention was given phytopharmacological study of microdilution assay. Briefly, 50 µl of broth medium Nepenthes ampullaria, and to the best of our knowledge, was placed to all the 96 wells. Then, equal amount no report has been published on the antimycobacterial of obtained working samples was added and 2-fold activity this species not alone the effects of the extract dilution series were done across the column of the at the cellular level. Thus, this study is aimed to evaluate plate giving the final testing concentrations of 0.098– the antimycobacterial activity of N. ampullaria crude 25 mg/mL. The same procedure was done for RIF extracts and to investigate its effect on cellular growth, to obtain 0.098–50 µg/mL concentration. The broth cell wall integrity, and morphology of mycobacterial cell. medium mixed with extract/RIF and broth medium only were used as sterility controls. Broth medium MATERIALS AND METHODS with inoculum is used as growth control. About 50 µl of diluted bacterial inoculum was added to the entire Plant Materials wells except for sterility test. The prepared 96-well The roots of N. ampullaria Jack were collected from plates were sealed and incubated overnight at 37°C. Johor National Park, Endau Rompin, Mersing, Johor, Afterward, 30 µl of tetrazolium-Tween 80 (MTT) was Malaysia. The sample was authenticated by Dr. Alona added onto the entire wells and reincubated at 37°C Cuevas Linatoc (botanist) in biodiversity laboratory, overnight. The minimum inhibitory concentration Faculty of Applied Sciences and Technology (FAST), (MIC) was interpreted as the lowest extract Universiti Tun Hussein Onn Malaysia (UTHM). After concentration at which no color change of the MTT [18] collection, the sample was dried in a hot oven at 40°C was observed (from yellow to purple). To determine 72 h, ground into powder, and kept in the sealed. minimum bactericidal concentration (MBC), all the wells that showed growth inhibition were plated Preparation of the Extracts on the Middlebrook MH10 Agar and incubated for The powder (100 g) material was macerated sequentially 72 h at 37°C. The MBC was defined as the lowest with 500 mL hexane, ethyl acetate, and methanol room concentration of sample that shows no visible cell temperature for 24 h, filtered using a No. 1 Whatman colony on the plate. filter paper, and then evaporated in a rotary evaporator Effect of the Extract on Cellular Growth of set at 40°C water bath. The water extract, on the other hand, was prepared by soaking the plant material in M. smegmatis distilled water and the mixture was gently heated to the The effect of the extract on cellular growth temperature of 60°C in a water bath until the volume of of M. smegmatis was determine by time-kill the water was brought down to one-fourth of its original assay adopted from by Silva et al.[19] with little volume.[17] Then, the mixture was cooled and strained modifications. About 10 mL of Middlebrook 7H9 (filtered) through the Whatman no. 1 filter paper and broth medium mixed with the hexane extract of the filtrate was frozen at −80°C in a freezer and then N. ampullaria/RIF at MIC, 2X MIC, and 3X MIC freeze-dried at −44°C using a freeze dryer. The stock concentrations were inoculated with a suspension of solutions were prepared by dissolving solvent extracts M. smegmatis (approximately 1.0 × 105 CFU/mL). in dimethyl sulfoxide and water extract in sterile The culture without extract or RIF was used as a distilled water at a concentration of 200 mg/mL which standard. The inoculated flasks were incubated at was stored in −20°C. Working solution was prepared 37°C in shaking incubator (150 rpm). The cells were by diluting the stock solution in sterile distilled harvested at time 0, 8, 24, 48, and 72 h after treatment water to obtained 50 mg/mL concentration. For stock and the serial dilutions were made to determine the solution of rifampicin (RIF), concentration of 1 mg/mL viable cell counts. Ten microliters from the diluted in absolute methanol was prepared and stored in −20°C samples were plated on the Middlebrook 7H10 Agar as well, the stock solution was diluted to prepare the and allowed to dry. The dried inoculated plates working solution with the concentration of 100 µg/mL. were incubated at 37°C for 72 h. Thereafter, the 210 Drug Invention Today | Vol 11 • Special Issue 2 • 2019 Shuaibu Babaji Sanusi, et al. total colony counts were determined. The tests were 50°C for 4 min, which was subsequently increased done in triplicate and the mean log (CFU/mL) was to 300°C at the rate of 3°C/min. The volume of the calculated using the below formula.