TLC-contact bioautography and disc diffusion method for investigation of the antibacterial activity of Melastoma malabathricum L. leaves
Dian Mayasari, Yosi B. Murti, Sylvia U. T. Pratiwi, Sudarsono Sudarsono*
Department of Pharmaceutical Biology, Faculty of Pharmacy,
Universitas Gadjah Mada, Yogyakarta 55281, Indonesia.
*Corresponding Author E-mail: sudarsono@ugm.ac.id
ABSTRACT:
The emergence of multi-resistant strains of bacteria reinforces the need to discover new antibacterial agents that are able to combat resistant microorganisms. Medicinal plants are a valuable natural source of bioactive substances against various infectious diseases. Melastoma malabathricum L. is an important herb that is traditionally used to treat several ailments associated with microbial infection diseases such as wounds, diarrhea, dysentery, and toothache. This study investigated extracts of M. malabathricum L. for antibacterial properties against Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA). Disc diffusion and TLC-contact bioautography techniques were employed to examine antibacterial properties of n-hexane, ethyl acetate, and ethanol extracts with observations of diameter inhibition zones and Rf values. Investigation of active compounds in TLC-bioautography used several reagents including citroboric, cerium (IV) sulfate and 2,2-diphenyl-1-picrylhydrazyl (DPPH), continued by identification of chromatogram profiles through densitometry analysis. The three extracts showed good inhibition against bacterial strains with diameter inhibition zones in the range of 8.0 – 14.0 mm with a number of active spots on TLC-contact bioautography for each extract. This plant may serve as useful source of antibacterial agents for resistant microorganisms and further investigation is needed of its bioactive pure compounds as well as their particular therapeutic potentials and applications.
KEYWORDS: Densitometry, disc diffusion, Melastoma malabathricum L., Methicillin-resistant Staphylococcus aureus, TLC-contact bioautography.
INTRODUCTION:
Infectious diseases are a major global health concern that have high morbidity and mortality caused by pathogenic bacteria1. The discovery of antimicrobial agents was one of the major inventions of the twentieth century. However, over years of applications, the antibiotics have led to the development of antibiotic resistance of several bacterial pathogens. Antibiotic resistance is emerging alarmingly to harmfully high levels in all parts of the world. A growing number of infections such as pneumonia, tuberculosis, and salmonellosis are becoming difficult to treat by the antibiotics typically used to treat them.
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the resistant bacteria that have increased dramatically during the recent decades in both hospital and community settings2. The increased bacterial resistance to different classes of antibacterial agents is a serious and dangerous problem that threatens human health3. The emergence of drug resistance has made management of infectious diseases precarious and unpredictable, and there is an urgent need for new bioactive antimicrobial compounds4. In the efforts to discover new lead compounds for antibacterial activities, numerous studies have screened plant extracts to identify secondary metabolites with relevant biological activities5–8.
Plant-derived substances have gained attention due to their significant biological and pharmacological activities. Since ancient times, people have utilized plants for preventing pathogenic diseases and as food preservatives9. It has long been established that medicinal plants have naturally occurring substances with versatile applications. These plants have advantages in drug discovery due to their significant antimicrobial activities and less toxicity10. Many plants have been utilized because they are the richest bio-source of drugs for traditional medicine, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs11,12. It has been estimated that about 28% of higher plant species are used medicinally and that 74% of pharmacologically active plant derived substances were discovered after following up on ethnomedicinal use of the plants. A number of interesting outcomes have been found with the use of mixtures of natural products for healing ailments, including most notably the synergistic effects and non-toxic pharmacological applications of plant extracts13.
Melastoma malabathricum L. belonging to the family Melastomataceae is a shrub plant used as an alternative medicine due to its numerous therapeutic properties which include antibacterial, antioxidant, antidiabetic, anticytotoxic, anti-inflammatory, antiulcer and immunomodulatory properties14–17. The plant is known to be grow widely and abundantly throughout the tropics including Ocean Island, South and South East Asia, China, Taiwan, Australia, and South Pacific Ocean18. As a traditional medicinal plant, the leaves of M. malabathricum L. are chewed up and pounded to form a poultice applied in topical therapy to stop bleeding and accelerate the dryness of the wounds 18,19. Young leaves are also useful for healing of ulcers, gastric ulcers, scar, and black spots on the skin 20. Combinations of the leaves and roots are applied to wounds and pox scars, while combinations of the leaves and flowers are used in the treatment of cholera, prolonged fever, dysentery and leucorrhea18,21,22.
The discovery of bioactive compounds for antimicrobial agents takes considerable effort to isolate and purify them from plant extracts23. To overcome this problem, the approach called bioactivity-guided fractionation was developed24. Thin layer chromatography (TLC) is a powerful technique for separating the mixture of samples. The TLC combined with the biological detection method is known as TLC bioautography, and is an economical alternative with high efficiency and strong specificity in the selection of a chromatographic process and biological detection system25. This method can be used to reduce the time and labor involved in the process to identify biologically active components of plant extracts. Contact-TLC bioautography connects the steps of separation on the adsorbent layer with biological assay performed directly on it24.
Although there are several studies focusing on the biological activity of M. malabathricum, there are varying outcomes concerning the constituents or bioactive compounds of the plants implying where they grow (habitat) plays a significant role in the phytochemistry of medicinal plants. Therefore, we aimed to assess the antimicrobial activity of three extracts of M. malabathricum L. leaves through TLC-contact bioautography and disc diffusion method.
MATERIALS AND METHODS:
Chemical and reagents:
All solvents: n-hexane, ethanol, ethyl acetate, formic acid, methanol, chloroform were of analytical grade supplied from Merck (Merck, Darmstadt, Germany). Cerium (IV) sulfate and citroboric acid reagent. Extracts were monitored by TLC which was done on pre-coated silica gel 60 F254 plates (Merck). All chemical solvents used were of analytical grades. In addition, Mueller Hinton Agar, Nutrient Agar, and Nutrient Broth were purchased from Oxoid, UK.
Plant material:
The leaves of M. malabathricum were collected in June 2018 from the Kuantan Singingi region of Riau province, Sumatera Island, Indonesia with coordinates: 0º33’2”S and 101º32’11”E. Melastoma malabathricum L. that was used in this study was authenticated by a botanist (Dr. Djoko Santosa) and a voucher specimen was deposited at the Department of Pharmaceutical Biology, Universitas Gadjah Mada, Yogyakarta, Indonesia. Once dried at room temperature (after a week), the leaves were ground and store in cool dry conditions before use.
Preparation of extracts:
The dried powdered leaf of M. malabathricum L. was extracted by the Soxhlet Apparatus by n-hexane for two weeks. After separation of the n-hexane extracts, the residue was macerated by ethyl acetate and finally was macerated by ethanol for three days as well as regularly stirred. The extracts were filtered and concentrated under vacuum in a rotary evaporator. All these extracts and fractions were stored at 4°C in the refrigerator for further use.
Bacterial strains:
The antibacterial activities of the M. malabathricum L. extract were tested with bacterial samples from American Type Culture Collection (ATCC) including Staphylococcus aureus (ATCC 25923) and Methicillin-Resistant Staphylococcus aureus (MRSA) (ATCC 33591). These bacterial samples were supplied from the Microbiology Laboratory, Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada.
Thin layer chromatography (TLC):
A 10 µL or 20 µL aliquots of the n-hexane, ethyl acetate, ethanol extract were applied onto TLC silica gel plate 60- F254. The plates were developed in the mobile phase containing: chloroform 100% for n-hexane extract, chloroform: methanol: ethyl acetate: formic acid (50: 15: 30:5 v/v/v/v) for ethyl acetate extract and n-hexane: ethyl acetate (8: 2 v/v) for ethanol extract in chamber over a distance 6 cm. After development, the plates were dried in a stream of warm air. The plates were scanned subsequently at two scanning wavelengths, 254 nm and 366 nm by the TLC Visualizer (CAMAG). Profile chromatogram was performed via Rf values separated into a wide range of Rf values from 0.00 to 1.00. All data obtained were processed with the software winCATS version 11.4.7.2018 (CAMAG). The plates were used for bioautography assay and other sets for derivatization with several sprayers.
TLC-contact bioautography technique:
Contact or contact bioautography was done according to the protocols explained by26 with slight modifications. The inocula of representative bacterial strains with 108CFU/mL concentration namely MRSA and S. aureus were swabbed onto the Mueller-Hinton agar plates for use in TLC contact bioautography technique. The dried developed TLC plate was then place aseptically onto the seeded Mueller-Hinton agar medium for ± 1 hour to allow diffusion of bioactive compounds. After that, the TLC plate was removed and the inoculated agar plate was further incubated at 37°C for 24 hours in aerobic condition. The spots that exhibited antibacterial activity were located by comparing to the TLC plate previously removed. The bioautographic assay allows the detection of active components in a crude plant extract on the TLC plate by recording of Rf values.
Post chromatographic derivatization:
After development, the plates were dried in room temperature for 30 min. The plates were derivatized with sprayer for preliminary identification the class of compounds. The plates were sprayed citroboric and cerium (IV) sulfate reagent. On the basis of Rf values, the substances of the extract samples could be determined by sprayed analysis. The determination of free radical scavenging activity of separation spots on TLC plates was performed by qualitative DPPH test by preparing 0.2% freshly methanolic solution of DPPH. After spraying the DPPH solution on TLC plates, these were stored in a dark room for 30 min. The yellow spots on purple background revealed the zones where substances with the highest free radical scavenging activity were present.
Agar diffusion method:
The antibacterial activity assay was examined using the disc diffusion method. Petri dishes (diameter size 9 cm) contained 10 mL of Mueller Hinton Agar medium (Oxoid, UK) seeded with 100 µL of the culture suspension of the microorganisms in Broth Heart Infusion (BHI, Oxfoid) using the spread plate technique. The inoculum size was adjusted to bacterial cells 106 colony forming units (CFU/mL) estimated by equivalent of 0.5 McFarland standard or can be accurately measured using a spectrophotometer with a 1-cm light path at 600 nm which corresponding to an absorbance reading of 0.127. Following this step, a sterile paper disk (Whatmann No. 1; 6 mm in diameter) was impregnated with test materials (30 µL to give the final concentration 150 µg/mL) and the disc was placed on the agar medium. The plates were left to dry and after that, the petri dishes were incubated at 37°C for 24 h under aerobic condition. Chloramphenicol (30 µg/mL, Oxoid) and solvent (n-hexane, ethyl acetate, ethanol) were used as positive and negative control, respectively. All disc diffusion tests were performed in triplicate and antimicrobial activity was expressed as the mean diameter of clear zone of growth inhibition (diameter expressed in millimeters) around the disc.
RESULTS AND DISCUSSION:
TLC-contact bioautography:
To perform a rapid screening study of potential antibacterial activity of n-hexane, ethyl acetate and ethanol extracts of M. malabathricum L. leaves against S. aureus and MRSA, TLC-bioautography following a disc diffusion method was applied as the combination method to identify bioactive compounds. The successful TLC bioautography method depends on the mobile phase used to separate a composition of matrix samples in TLC plates of plant extracts. Several mobile phases were examined in optimization of the separation of extracts in TLC plates with the following mixtures of solvents: chloroform: methanol: ethyl acetate: formic acid (50: 10: 35: 5 v/v/v/v); n-hexane: acetone (8: 2 v/v); ethyl acetate: methanol: water: formic acid (100: 13: 10: 2 v/v/v/v); dichloromethane: ethyl acetate: formic acid (50: 45: 5 v/v/v); chloroform (100%); n-hexane: ethanol (8:2 v/v). Starting with TLC, several spots with broad range of polarities were obtained. Therefore, the use of the proper solvent system enables the separation of M. malabathricum L. extract into spots with a wider range of polarities.
TLC-bioautography assay allows target-directed isolation of bioactive components for further examination, hence preventing isolation of inactive compounds28. The results clearly demonstrated that the plant extracts exhibited significant antimicrobial activity with the chromatogram profiles of TLC (Figure 1) and the active spots on TLC plate of three extracts showed in their Rf values (Table 1).
Figure 1. TLC chromatogram profiles of n-hexane (A), ethyl acetate (B) and ethanol (C) extract. TLC-contact bioautography of n-hexane (D), ethyl acetate (E) and ethanol (F) extract against S. aureus while G, H and I showed the activity of n-hexane, ethyl acetate, ethanol extract against MRSA with loading mass of samples: 100µg (1) and 50µg (2). Mobile phase used on the separation TLC: chloroform 100% for n-hexane extract, ethyl acetate extract: chloroform: methanol: ethyl acetate: formic acid (50: 15: 30: 5 v/v/v/v) for ethyl acetate extract and n:-hexane: ethyl acetate (8:2 v/v) for ethanol extract.
Table 1. Rf value of bioactive compounds of n-hexane, ethyl acetate and ethanol extract against MRSA and S. aureus
|
Extract |
Concentration |
Methicillin-resistant Staphylococcus aureus |
Staphylococcus aureus |
Active spot |
|
n-hexane |
100µg |
0.00 – 0.16 |
0.00 – 0.25 |
1 |
|
|
50µg |
0.00 – 0.15 |
0.00 – 0.25 |
|
|
ethyl acetate |
100µg |
0.00 – 0.24 0.72 – 1.00 |
0.00 – 0.25 0.75 – 1.00 |
2 |
|
|
50µg |
0.00 – 0.17 0.75 – 1.00 |
0.00 – 0.22 0.80 – 1.00 |
|
|
ethanol |
100µg |
0.00 – 0.28 0.29 – 0.50 |
0.00 – 0.29 0.30 – 0.51 |
2 |
|
|
50µg |
0.00 – 0.27 0.28 – 0.48 |
0.00 – 0.27 0.28 – 0.50 |
|
This study employed the bioautographic method to examine bacterial growth inhibition by visually observing a clear zone and Rf values. The samples were showing the zone of inhibition confirming that they possess good inhibitory activity against test organisms29. Ethyl acetate and ethanol showed excellent antimicrobial activity with two active spots in Rf values of 0.00 to 0.24 and 0.72 to 1.00 for ethyl acetate and 0.00 to 0.28 and 0.29 to 0.50 for ethanol extracts in concentration 100µg against MRSA. However, n-hexane showed moderate antimicrobial activity with one active spot in Rf value of 0 to 0.16 and 0.00 to 0.25 against MRSA and S. aureus, respectively. There is different significance of antibacterial properties of the three extracts against MRSA and S. aureus. Inhibition of MRSA growth had a broad range of Rf values compared to the S. aureus particularly in n-hexane extract.
The most significant advantage of the TLC bioautography technique is the antibacterial active spots can be located and separated by planar chromatography for investigation of the antimicrobial effects in complex samples. The plates are placed on the surface of nutrient agar plates inoculated with microorganism so antimicrobial compounds are transferred from the TLC plate to an inoculated agar plate by direct contact. It is based on the diffusion of compounds, already separated by TLC, from adsorbent or paper to the agar medium30. This is in contrast with work using agar dishes, which does not distinguish between active and inactive components found together in zones of inhibitions. In this case, only the bioactive sum of a sample is indicated, not the activities of single compounds. There are some examples of the application of this methodology for screening antibacterial, antifungal, xanthine oxidase, or free radical scavenging activities31.
TLC is generally employed as a fast, efficient and inexpensive tool for a screening method in different stages of monitoring processes including synthesis, isolation, and other biological studies32. Several studies have documented the application of TLC for the phytochemical profiling, fingerprint analyses of plant extracts33. TLC spots can be analyzed both qualitatively and quantitatively by densitometry in ultraviolet (UV) absorbance as shown in Figure 2. For non-UV absorbing or non-fluorescent compounds, densitometry can be applied after derivatization using chromogenic agents34. The results of densitometry analysis are computerized by chromatogram profiles and area under peak35 following detection under UV lamp of 254 nm and 366 nm.
Figure 2. TLC 3D-profile densitograms of n-hexane, ethyl acetate, and ethanol extract under UV 254 nm light (A, C, E) and n-hexane, ethyl acetate, and ethanol extract under UV 366 nm light (B, D, F), respectively.
Figure 3. TLC plates of n-hexane (1), ethyl acetate (2), and ethanol (3) extracts after spraying with 2,2-diphenylpicryl hydrazyl (DPPH) (A), cerium sulfate (CeSO4) (B), and citroboric acid reagent (C).
Important steps of thin layer chromatography analysis include the detection of investigated substances of the plant extracts. Besides providing information about separated compounds by retardation factors, TLC can also identify the class of compounds by traditional visualization. Figure 3 summarizes the derivatization of TLC plates represented by color descriptions of the separated spots on the TLC visualized under visible light after sprayed with DPPH, citroboric, and cerium (IV) sulfate. The separated components by TLC can be detected with individual colors of substances or fluorescence of substances in UV light and color reaction of separated compounds of TLC with visualizing reactions. These extracts showed activity in the range of UV light and can be directly detected and determined on the chromatographic plate, and hence by densitometric analysis. The methods were found to be equivalent on the basis of repeatability when the results from densitometric measurements were compared with those obtained. The TLC separation was followed by derivatization of DPPH in methanol (0.02% w/v). The compounds possessing radical scavenging activity were detected as bright yellow bands against a purple backgrounds.
Color reaction on the reagent spray cerium (IV) sulfate mechanism showed the cerium (IV) sulfate consisting of concentrated sulfuric acid and acetic anhydride, where sulfuric acid has a destructive and oxidative effect. Cerium (IV) sulfate reagent is used for detecting components of organic compounds36. Citroboric acid is one of the specific sprayers for identification of the presence of flavonoid compounds. The samples that contained flavonoid group exhibit yellow spots on TLC plate37. Based on chromatogram in Figure 3, TLC profiles showed some spots that have Rf values of 0.55 in ethyl acetate extract with brownish yellow in visible light after sprayed by citroboric reagent. For DPPH analysis, the active compound on TLC plates exhibits yellow bands against a purple background. It indicated the presence of phenolic compounds as well as flavonoid compounds. In the present study, ethyl acetate extract showed high content of phenolic compounds. Flavonoids is one of the phenolic compound class that have antioxidant and antibacterial activity. The antimicrobial activity of the M. malabathricum L. is suggested to be due to the presence of phenolic, flavonoid and other semipolar compounds of the mixture samples extracts.
Disc diffusion method:
The results of the disc diffusion method of M. malabathricum L. extracts are in line with the data obtained from TLC-bioautography. The anti-MRSA activity of chloramphenicol was more significant than the ethanolic extract. Ethanol extract showed the greater antimicrobial activity than n-hexane and ethyl acetate extracts. The antimicrobial activities for the three extracts using the disc diffusion method are shown in Table 2.
Table 2. Disc diffusion method of antibacterial activity of extracts against MRSA and S. aureus
|
Extract |
Diameter of inhibition zone (mm) |
|
|
Methicillin-Resistant Staphylococcus aureus |
Staphylococcus aureus |
|
|
n-Hexane |
11.0 ± 0.2 |
8.0 ± 0.5 |
|
Ethyl acetate |
10.0 ± 0.5 |
12.2 ± 0.4 |
|
Ethanol |
12.5 ± 0.4 |
14.0 ± 0.8 |
|
Chloramphenicol |
16.2 ± 0.1 |
25.0 ± 1.5 |
The disc diffusion assay showed that ethanol extract had strong growth inhibition activity against both S. aureus and MRSA with diameter 14 ± 0.8 and 12.5 ± 0.4, respectively. Ethanol extract showed the highest antibacterial activity against S. aureus and MRSA with the big clear zone. Previous study revealed that ethanol extract was the most effective solvent for extracting a broad spectrum of antimicrobial38,39 and polymicrobial biofilm40 substances derived from plants. However methanol extract exhibited moderate antimicrobial activity against six out of seven bacterial microorgabisms41. Ethyl acetate extract exhibited the moderate activity against S. aureus and MRSA with the diameters of the inhibition zone of 12.2 ± 0.4 and 10.0 ± 0.5, respectively. Extract of n-hexane also had moderate inhibition activity against S. aureus and MRSA with diameters 8.00 ± 0.5 and 11.0 ± 0.2, respectively. The clear zone of positive control chloramphenicol was 25.0 ± 1.5 and 12.2 ± 0.4 against S. aureus and MRSA, respectively.
The presence of the clear zone indicates the inhibitory activity of the examined compounds against the bacterial growth. According to previous report, extracts with inhibition zone diameter over >11 mm are assumed to have strong antimicrobial activity, 6-11 mm categorized as moderate activity with < 6 mm as weak activity. The antibiotic chloramphenicol was used as the reference drug (positive control) in this study. The antibacterial potential of the extracts can be assumed as good and shows they have potential activity as antibacterial agents although the zone diameters are much lower than that of antibiotics42. In the disc diffusion technique, the disc was used as reservoirs containing the solutions of the examined samples and solutions with a low activity needed a large concentration or volume43.
Antibacterial properties M. malabathricum L. extract might be due to the presence of bioactive compounds that have been reported in the plants. The bioactive constituents included the phenolic compounds such as tannins: malabathrin A, pedunculagin, strictinin, nobotanin, flavonoid including quercetin, kaempferol, quercitrin, rutin, kaempferol glycosides, phenolic acids such as p-hydroxyl benzoate, gallic acid, and few terpenoids and alkaloids14,15,18,44. In fact, the presence of phenolic compounds has generally contributed to the antimicrobial activity. Polyphenols are well documented to have antimicrobial activity against a huge number of pathogenic bacteria. In addition, oxidized phenols also have an inhibitory effect against bacterial growth45,46. The hydroxylation process increased with the increasing number of hydroxyl group, which in turn increased the antimicrobial activity. The mechanisms involved in polyphenols as antimicrobial agents include envelope transport proteins, inhibition of hydrolytic enzymes and non-specific interactions with the carbohydrates. In addition, flavonoids and tannins could be able to bind or form precipitates with various proteins47.
CONCLUSIONS:
The results of this study confirmed the presence of the various bioactive compounds in the M. malabathricum L. leaves responsible for their therapeutic activities. We have identified several active spots on TLC plate in TLC-contact bioautography technique against S. aureus and MRSA. The results of disc diffusion and TLC- contact bioautography showed excellent activity of the three M. malabathricum L. extracts and supports the potential development of these novel therapeutic antimicrobial agents from M. malabathricum L. attributed to its traditionally use as anti-infection treatment of diseases. Future studies are directed towards the development of purified bioactive substances to improve existing drugs or to create new agents of antibacterial activities. Additionally, considering all of the inhibitory effects of the plant extracts, it may have potential for further development as a natural agent in prevention of infectious diseases. Therefore, it could serve as potential sources of industrial drugs useful in some therapy against bacterial infections.
ACKOWLEDGEMENTS:
We are thankful to the Deputy of Research Reinforcement and Innovation, The Ministry of Research and Technology/National Agency for Research and Innovation of Indonesia by funded this project through PMDSU (Master’s Education towards Doctorate) scholarship program (Grant No. 2944/UNI.DITLIT/DIT-LIT/LT/2019).
CONFLICT OF INTEREST:
The authors declare that there is no conflict of interest in this article.
REFERENCES:
1. Chen L, Cheng X, Shi W, Lu Q, Go VL, Harber D, Ma L. Inhibition of growth of Streptococcus mutans, Methicillin-Resistant Staphylococcus aureus, and Vancomycin-Resistant Enterococci by kurarinone, a bioactive flavonoid isolated from Sophora flavescens. Journal of Clinical Microbiology. 2005; 43(7): 3574-3575.
2. Ibrahim A, Aqel AA, Aljamal A. Effect of the methanol extracts of Salvia libanotica, Rosmarinus officinalis, Capparis spinosa and Achillea fragrantissima against two strains of Staphylococcus aureus. African Journal of Microbiology Research. 2013; 7(29): 3750-3753.
3. Fisher JF, Meroueh SO, Mobashery S. Bacterial resistance to β-lactam antibiotics: compelling opportunism, compelling opportunity. Chemical Reviews. 2005; 105(2): 395-424.
4. Demain AL, Sanchez S. Microbial drug discovery: 80 years of progress. The Journal of Antibiotics. 2009; 62(1):5-16.
5. Subramani R, Narayanasamy M, Feussner K-D. Plant-derived antimicrobials to fight against multi-drug-resistant human pathogens. 3 Biotech. 2017; 7(3):172.
6. Agarwal P, Agarwal N, Gupta R, Gupta M, Sharma B. Antibacterial activity of plants extracts against Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus faecalis. Journal of Microbial and Biochemical Technology. 2016; 8(5). 404-407.
7. Sundar S, Padmalatha K, Helasri G, Vasanthi M, Narmada BS, Lekhya B, Jyothi RN, Sravani T. Anti-microbial activity of aqueous extract of natural preservatives: cumin, cinnamon, coriander and mint. Research Journal of Pharmacy and Technology. 2016; 9(7): 843-847.
8. Das SK, Dhake AS, Nayak A, Das NB, Pandeya SN. Antibacterial and antifungal activity of aerial part of plant Ammannia baccifera Linn. Research Journal of Pharmacy and Technology. 2011; 4(3): 430-432.
9. Durbar US, Geetha RV. Antibacterial effects of South Indian spices on oral microbes. Research Journal of Pharmacy and Technology. 2015; 8(8): 1135-1136.
10. Singh DD. Efficacy of Antimicrobial properties of Acalypha indica against clinical isolates of human Pathogen. Research Journal of Pharmacy and Technology. 2019; 12(9): 4231-4234.
11. Hammer KA, Carson CF, Riley TV. Antimicrobial activity of essential oils and other plant extracts. Journal of Applied Microbiology. 1999; 86(6): 985-990.
12. Ncube NS, Afolayan AJ, Okoh AI. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. African Journal of Biotechnology. 2008; 7(12): 1797-1806.
13. Gibbons S. An overview of plant extracts as potential therapeutics. Expert Opinion on Therapeutic Patents. 2003; 13(4): 489-497.
14. Susanti D, Sirat HM, Ahmad F, Ali RM. Bioactive constituents from the leaves of Melastoma malabathricum L. Jurnal Farmasi Ilmiah. 2008; 5(1): 1-6.
15. Wong K-C, Hag Ali DM, Boey P-L. Chemical constituents and antibacterial activity of Melastoma malabathricum L. Natural Product Research. 2012; 26(7): 609-618.
16. Alnajar ZAA, Abdulla MA, Ali HM, Alshawsh MA, Hadi AHA. Acute toxicity evaluation, antibacterial, antioxidant and immunomodulatory effects of Melastoma malabathricum. Molecules. 2012; 17(3): 3547-3559.
17. Balamurugan K, Nishanthini A, Mohan VR. Antiulcer activity of Melastoma malabathricum L. leaf extracts (Melastomataceae). International Journal of Advanced Research. 2013; 1(5): 49-52.
18. Joffry SMohd, Yob NJ, Rofiee MS, Affandi MMRM, Suhaili Z, Othman F, Akim AM, Desa MNM, Zakaria ZA. Melastoma malabathricum (L.) Smith ethnomedicinal uses, chemical constituents, and pharmacological properties: a review. Evidence-Based Complementary and Alternative Medicine. 2012; 2012: 1-48.
19. Zakaria ZA, Raden Mohd. Nor RNS, Hanan Kumar G, Ghani ZDFA, Sulaiman MR, Devi GR, Jais AMM, Fatimah CA. Antinociceptive, anti-inflammatory and antipyretic properties of Melastoma malabathricum leaves aqueous extract in experimental animals. Canadian Journal of Physiology and Pharmacology. 2006; 84(12): 1291-1299.
20. Lohézic-Le Dévéhat F, Bakhtiar A, Bézivin C, Amoros M, Boustie J. Antiviral and cytotoxic activities of some Indonesian plants. Fitoterapia. 2002; 73(5): 400-405.
21. Perry LM, Metzger J. Medicinal Plants of East and Southeast Asia: Attributed Properties and Uses. MIT Press, Cambridge. 1980.
22. Sharma HK, Chhangte L, Dolui AK. Traditional medicinal plants in Mizoram, India. Fitoterapia. 2001; 72(2): 146-161.
23. Sun Z-L, Liu T, Wang S-Y, Ji X-Y, Mu Q. TLC-bioautography directed isolation of antibacterial compounds from active fractionation of Ferula ferulioides. Natural Product Research. 2019; 33(12): 1761-1764.
24. Choma I, Jesionek W. TLC-direct bioautography as a high throughput method for detection of antimicrobials in plants. Chromatography. 2015; 2(2): 225-238.
25. Dewanjee S, Gangopadhyay M, Bhattacharya N, Khanra R, Dua TK. Bioautography and its scope in the field of natural product chemistry. Journal of Pharmaceutical Analysis. 2015; 5(2): 75-84.
26. Choma IM, Grzelak EM. Bioautography detection in thin-layer chromatography. Journal of Chromatography A. 2011; 1218(19): 2684-2691.
27. Bandet T, Whitehead S, Blondel-Hill E, Wagner K, Cheeptham N. Susceptibility of clinical Moraxella catarrhalis isolates in British Columbia to six empirically prescribed antibiotic agents. Canadian Journal of Infectious Diseases and Medical Microbiology. 2014; 25(3): 155-158.
28. Suleimana M, McGaw L, Naidoo V, Eloff J. Detection of antimicrobial compounds by bioautography of different extracts of leaves of selected South African tree species. African Journal of Traditional Complementary and Alternative Medicine. 2009; 7(1): 64-78.
29. Patel SR, Suthar AP, Shah AM, Hirpara HV, Joshi VD, Katheria MV. Antimicrobial Activity of Methanolic Extracts of Mucuna pruriens, Semecarpus anacardium, Anethum graveolens by Agar Disc Diffusion Method. Research Journal of Pharmacy and Technology. 2009; 3(1): 165-167.
30. Botz L, Nagy S, Kocsis B. Detection of microbiologically active compounds. Springer Scientific, Hungary. 2001.
31. Marston A. Thin-layer chromatography with biological detection in phytochemistry. Journal of Chromatography A. 2011; 1218(19): 2676-2683.
32. Šegan S, Opsenica D, Milojković-Opsenica D. Thin-layer chromatography in medicinal chemistry. Journal of Liquid Chromatography & Related Technologies. 2019; 42(9-10): 238-248.
33. Tambunan AP, Bahtiar A, Tjandrawinata RR. Influence of extraction parameters on the yield, phytochemical, TLC-densitometric quantification of quercetin, and LC-MS profile, and how to standardize different batches for long term from Ageratum conyoides L. leaves. Pharmacognosy Journal. 2017; 9(6): 767-774.
34. Mroczek T. Qualitative and quantitative two-dimensional thin-layer chromatography/high performance liquid chromatography/diode-array/electrospray-ionization-time-of-flight mass spectrometry of cholinesterase inhibitors. Journal of Pharmaceutical and Biomedical Analysis. 2016; 129: 155-162.
35. El-Aleem AEBA, Khalile SM, Badawy AM, El-Naggar OK. HPLC and TLC-densitometric methods for the determination of some antimigraine drugs in bulk powder and in pharmaceutical preparations. Research Journal of Pharmaceutical Dosage Forms and Technology. 2013; 5(5): 288-294.
36. Susilo S, Suciati. R. Studies of morphological and secondary metabolites variety of mosses (bryophyta) in Cibodas, West Java. Indonesian Journal of Advanced Research. 2016; 4(12): 1397-1402.
37. Jork W, Funk W, and Wimmer H. Thin-Layer Chromatography: Reagents and Detection Methods. Vol. 1 a: Physical and Chemical Detection Methods Fundamentals, Reagents I. VCH, Weinheim. 1990.
38. Thota P, Bhogavalli PK, Chenreddy SK. Phytochemical Investigation and Antibacterial Screening of Leaf Extracts of Sapindus saponaria Vahl. Research Journal of Pharmacy and Technology. 2012; 5(2): 273-276.
39. Jayashree V, Anju KC, Ragavendran MP, Ravichandiran V. In vitro antimicrobial activity using ethanolic extract of flower and stem extract of Cassia auriculata linn. Research Journal of Pharmacy and Technology. 2015; 8(7): 901-905.
40. Pratiwi SUT, Hamzah H. Inhibition and degradation activity of (Sapindus rarak seeds) ethanol extract against polymicrobial biofilm. Research Journal of Pharmacy and Technology. 2020; 13(11): 5425-5430.
41. Kumar AS, Mazumder A, Vanitha J, et al. Antibacterial activity of methanolic extract of Sesbania Grandiflora (Fabaceae). Research Journal of Pharmacy and Technology. 2008; 1(1): 59-60.
42. Mohapatra DP, Thakur V, Brar SK. Antibacterial efficacy of raw and processed honey. Biotechnology Research International. 2010; 2011: 1-6.
43. Venkatachalam T, Kumar VK, Kumar PS, Kalaiselvi P, Chitra M, Kumar NS. In-vitro antioxidant and antimicrobial activities of ethyl acetate extract of Evodia lunu-Ankenda (Gaertn) Merr. Bark. Research Journal of Pharmacy and Technology. 2009; 1(3): 201-203.
44. Yoshida T, Nakata F, Hosotani K, Nitta A, Okudat T. Dimeric hydrolysable tannins from Melastoma malabathricum. Phytochemistry. 1992; 31(8): 2829-2833.
45. Karou D, Dicko MH, Simpore J, Traore AS. Antioxidant and antibacterial activities of polyphenols from ethnomedicinal plants of Burkina Faso. African Journal of Biotechnology. 2005; 4(8): 823-828.
46. Abas FZ, Zakaria ZA, Ani FN. Antimicrobial properties of optimized microwave-assisted pyroligneous acid from oil palm fiber. Journal of Applied Pharmaceutical Sciences. 2018; 8(7): 65-71.
47. Cowan MM. Plant products as antimicrobial agents. Clinical Microbiology Reviews. 1999; 12(4): 564-582.
Received on 31.12.2020 Modified on 28.04.2021
Accepted on 06.07.2021 © RJPT All Right Reserved
Research J. Pharm. and Tech 2021; 14(12):6463-6470.
DOI: 10.52711/0974-360X.2021.01117