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. sample injected was 0.1 L and temperature of the injector was set at 250°C. The bioactive compounds No.of colonies×dilution factor (10n ) CFU/mL = were identified by comparing their mass spectra 0.1mL(volume plated) with the available data in the library of the National Institute Standard and Technology database. Effect of the Extract on the Membrane Integrity The cellular membrane integrity was evaluated by RESULTS AND DISCUSSION measuring the release of the cell constituents into the Antimycobacterial Activity cell suspension at 280 nm. A procedure previously used [22] by Kim et al.[20] was followed with little modification. Following the study by Mariita et al. which considered The suspension of M. smegmatis with a concentration ≤37.5 mg/mL MIC value as positive for plant extracts, of approximately 1.0 × 105 CFU/mL was treated with the present study considered 25.00 mg/mL as a cutoff the MIC, 2X MIC, and 3X MIC of the hexane extract value to evaluate the antimycobacterial activity. of N. ampullaria. A bacterial suspension free of The antimycobacterial activity of N. ampullaria extract sample and bacterial suspension with RIF were extracts showed the obtained MIC and MBC values included as a standard and positive control in the assay, [Table 1]. The result demonstrated that hexane extract of N. ampullaria had the lowest MIC (0.39 mg/mL) respectively. The flasks were then incubated for 8 h followed by ethyl acetate extract of N. ampullaria at 37°C under the shaking condition at 150 rpm. The (1.5 mg/mL) while methanol and water extracts absorbance of cell supernatant was determined using were found to exhibit the same MIC (3.13 mg/mL). a spectrophotometer (Spectra Max 250, Molecular Likewise, the MBC result showed that hexane extract Devices, USA) at 280 nm after the cell suspensions had the lowest MBC (1.56 mg/mL) value followed by were centrifuged (6000 rpm, 10 min). The result was ethyl acetate (3.13 mg/mL), methanol (6.25 mg/mL), expressed as the relative ratio of OD of treated cells 280 and water (12.50 mg/mL) extract. The hexane extract to the untreated cells. of N. ampullaria was seen as the most active extract Effect of the Extract on Morphology of in antimycobacterial activity determination using Mycobacterial Cell Tubular Exchanger Manufacturers Association. It could be as a result of low polar compounds present in To investigate the effect of the extract on the the hexane extract that could possibly cross the lipid morphology of mycobacterial cell, field emission barrier of the mycobacterial cell wall.[23] No report was scanning electron microscopy (FESEM) of the found in literature regarding the antimycobacterial extract-treated and untreated M. smegmatis was screening of N. ampullaria apart from ethnomedicinal carried out. A method previously used by Piroeva reports. Thus, this study would be the first to report [21] et al. was followed with few modifications. the antimycobacterial activity of this plant. Briefly, the inoculum was prepared and treated as described above. The treated M. smegmatis cells Effect of the Hexane Extract of N. ampullaria on were harvested (8, 24, 48, and 72 h after exposure) by Cellular Growth of M. smegmatis centrifugation for 5 min (1 mL) at 600 rpm. After that, Based on the antimycobacterial results, hexane the supernatant was thrown away, and this procedure extract of N. ampullaria was selected for time-kill was repeated twice. The collected pellets were placed assay to investigate its effect on cellular growth of onto sterilized coverslips coated with 0.8% agar and M. smegmatis cells. Unlike the MIC/MBC assay, time- left to stand at room temperature for 30 min. This was kill assay allows the determination of the rapidity of later dehydrated in an oven for 12 h at the temperature bactericidal activity of the selected extract.[24] It was of 37°C. After that, attached bacterial cells were shown that there was progressive increase in cell count dehydrated further in ethanol (10, 25, 50, 75, 96, and from 0 to 72 h for an untreated control [Figure 1]. An absolute 99.99%) for 30 min in each concentration. The hydrated samples were dried for 1 h at 37°C in Table 1: Antimycobacterial activity of Nepenthes an oven. The prepared samples were coated with gold ampullaria extracts and observed using JEOL JSM-7600F FESEM, Japan. Sample MIC (mg/mL) MBC (mg/mL) Gas Chromatography–mass Spectrometry (GC– Water 3.13 12.50 MS) Methanol 3.13 6.25 Ethyl acetate 1.56 3.13 The hexane extract was analyzed using GC-MS-2010 Hexane 0.39 1.56 Plus – Shimadzu to identify the active components Rifampicin (µg/mL) 3.13 6.25 present in the extract. The column (0.25 µm thickness, MIC: Minimum inhibitory concentration, MBC: Minimum bactericidal 30.0 m length, 0.25 mm ID) temperature was set at concentration

Drug Invention Today | Vol 11 • Special Issue 2 • 2019 211 Shuaibu Babaji Sanusi, et al. immediate decline in the cell count after 8 h of exposure et al.[28] which reported that the methanol extract of to the hexane extract of N. ampullaria and RIF at 1X Allium ascalonicum L. displayed bactericidal effect MIC; however, regrowth occurred after 24 h. This is again mycobacteria species within the same contact in accordance with the previous study by Olajuyigbe time with positive control (RIF). It is suggested that and Afolayan[25] which evaluated the in vitro time-kill the crude extracts contain phytoconstituents that might assay of the crude ethanol extract of Erythrina caffra be developed to elicit better antimycobacterial activity against some bacteria in diarrhea. This regrowth may that will be compared favorably with RIF (positive be as a result of the degradation of the plant extracts control) which killed the cells within the same exposure which allow the cell culture to be able to proliferate time although at lower concentration. again.[26] At 2X MIC, hexane extract of N. ampullaria showed bactericidal activity 48 h after exposure, Effects of Hexane Extract of N. ampullaria on while RIF exhibited bactericidal activity after 72 h. Membrane Integrity of M. smegmatis This showed that hexane extract of N. ampullaria at The cell membrane integrity was investigated by 2X MIC displayed bactericidal effect faster than the measuring the cellular constituents (protein materials positive control though at a lower concentration. It was such as amino acids) in the supernatant of M. smegmatis reported by Khara et al.[27] that the synthetic cationic released as a result of leakage of the cell membrane. α-peptide displayed bactericidal effect stronger than Figure 2 shows the ratios of intracellular constituents

RIF, the first-line anti-TB drug. At X3 MIC, both of absorbing at 280 nm (OD280) released by M. smegmatis them demonstrated bactericidal activities just 8 h cells treated for 8 h with 1X MIC, 2X MIC, and 3X after treated. This is in line with a study by Igbokwe MIC of hexane extract of N. ampullaria and RIF as a

10 8 Control 6 4 HNA MIC x1 2

CFU/mL) 0 HNA MIC x2 0 8 16 24 32 40 48 56 64 72 Cell viability (log HNA MIC x3 a Time (hrs)

10 8 Control 6 4 RIF MIC x1 2 CFU/mL) 0 RIF MIC x2 cell viability (log cell viability (log 0 8 16 24 32 40 48 56 64 72 RIF MIC x3 Time (hrs) b Figure 1: Time-kill curves of the extracts (a) hexane extract of Nepenthes Ampullaria, (b) rifampicin (positive control) at X1, X2, and X3 minimum inhibitory concentration against Mycobacterium smegmatis

120

100

80

60 RIF Relative ratio 40 HNA

20

0 MIC X1 MIC X2 MIC X3

Sample treatment

Figure 2: The effect of hexane extract of Nepenthes ampullaria on release of cell constituents absorbing at 280 nm from Mycobacterium smegmatis

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Table 2: Phytocompounds identified in the hexane extract of Nepenthes ampullaria by gas chromatography–mass spectrometry analysis ID RT (min) Name of identified phytocompounds Peak area (%) S/I 3 50.460 Oleyl alcohol, heptafluorobutyrate 0.36 98 4 53.260 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (Phytol) 0.56 92 6 55.335 Chloroacetic acid, pentadecyl ester 2.07 99 7 55.545 Methyl 9-cis,11-trans-octadecadienoate 0.49 89 8 55.735 Z,E-2-Methyl-3,13-octadecadien-1-ol 0.44 93 10 57.464 Octadeca-(3Z,13Z)-dien-1-yl acetate 6.63 99 11 58.916 9-Cycloheptadecen-1-ol 0.32 97 12 60.868 Eicosen-1-ol, cis-9- 2.62 98 13 61.614 Heneicosyl trifluoroacetate 0.44 96 16 65.572 Behenic alcohol 0.23 95 17 65.833 2-Propen-1-one, 1-(2,6-dihydroxy-4-methoxyphenyl)-3-phenyl-, (E)- 1.24 97 21 68.434 Phthalic acid, di(2,4,4trimethylpentyl) ester 1.06 98 32 82.878 Alpha-tocopherol 4.16 98 33 83.398 Succinic acid, di(dodec-9-yn-1-yl) ester 0.22 87 41 90.316 25-Nor-9,19cyclolanostan-24-one, 3-acetoxy-24phenyl- 0.85 95 RT: Retention time, SI: Similarity index

Effects of Hexane Extract of N. ampullaria on Cellular Morphology of M. smegmatis Electron microscopy has been used effectively in examining the morphological changes during the growth of microorganisms. One of the potentials of this instrument which remains largely unexplored is in the a b study of the morphological consequences of exposure of bacteria to antimicrobial agents. For any molecule to effectively inhibit the growth of bacteria, it must act on a critical structure necessary for its growth and survival. In mycobacteria, such crucial target is the cell wall. If an extract can disrupt the cell wall structure, it may possible be a potent drug candidate.[31] In this study, the antimycobacterial effect of the hexane extract of c d N. ampullaria on the morphology of M. smegmatis cells Figure 3: Field emission scanning electron microscopy demonstrated that the untreated cells were rod shaped, image of Mycobacterium smegmatis cells treated with slender with intact and smooth surface [Figure 3a]. The hexane extract of Nepenthes ampullaria (a: 0 h; b: 24 h; cells treated with extract underwent some morphological c: 48 h; d: 72 h). changes. Figure 3b shows the treated cell after exposure for 24 h. As shown in Figure 3b, M. smegmatis treated positive control relative to those released by untreated with the extract for 24 h elongated into filamentous form M. smegmatis. It was observed that the cellular and the cell surface appeared as irregularly wrinkled and constituents released when treated with hexane extract coarse. Dziadek et al.[32] reported that some elongated, of N. ampullaria at 1X MIC, 2X MIC, and 3X MIC filamentous cells that contained bulges were produced increased by 6.6, 7.3, and 5.3 times, respectively, when M. smegmatis subjected to a condition that compared to positive control [Figure 2]. The cell wall accelerates the production of FtsZ, which leads to eventual of mycobacteria plays a significant role in the regulation lyse of the cells. The filamentous cell was considered to be overstressed and dying population.[33] Based on of cellular growth and it is critical virulence factor. Figure 3c, M. smegmatis treated with the extract for 48 h Furthermore, it serves as static barriers against many anti- showed that the cells elongated into filamentous form and TB drugs and biological stresses.[29] A slight changes or the cells appeared to be bulged at the end with the surface breakage in the structural integrity of the cell membranes of the cells appeared irregularly wrinkled and coarse. can detrimentally compromise the cell metabolism and More drastic morphological changes were observed [30] eventually lead to cell death. The analysis of the after exposure to extract for 72 h [Figure 3d]. The cells cellular constituent released in the supernatant as a appeared shrunken, ruptured, amorphous debris, and result of the leakage of the membrane of M. smegmatis ghost cell. The cell surface appeared irregularly wrinkled provides additional insights into the mechanism of and coarse. The treated cells in this study were wrinkled, antimycobacterial action targeting cell wall. cracked, and ruptured, this is in line with the previous

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