Abstract: Background and Objective: The microbial infection is a foremost cause of mortality worldwide owing to the emergence of multidrug-resistant pathogens. Moringa concanensis is an imperative medicinal plant that was extensively utilized to treat the numerous ailments in India. The current research investigation was intended to screen the phytochemical profile and examine the antimicrobial and cytotoxic potentials of M. concanensis flowers. Materials and Methods: The pharmacognostic and phytochemical screenings were done by adopting the standard procedures. The antimicrobial efficiency of M. concanensis flower extracts was inspected through the disk diffusion technique. The Minimum Bactericidal and Fungicidal Concentration (MBC and MFC) was investigated through the tube dilution technique. The MTT cytotoxic assay was executed to inspect the cytostatic activity of chloroform extract of M. concanensis flower. Results: The results of phytochemical screening and fluorescence assay were proved the presence of numerous phytoconstituents in the flowers of M. concanensis. The outcomes of antimicrobial studies displayed that the M. concanensis flowers have potent antimicrobial action against the clinical pathogens. The chloroform extract of the M. concanensis flower possessed a noticeable cytotoxic potential to the human lung cancer (HepG2) cell line. Conclusion: The findings of this investigation is the evidence that the flowers of M. concanensis have potent antimicrobial benefits and cytostatic effects against HepG2 cells.
INTRODUCTION
The global impact of infectious ailments is spoiling the life quality of individuals and becoming a life intimidating problem. Presently, the healing of infectious ailments has become the biggest problem owing to the raising of multidrug-resistant pathogens. There are plentiful causes behind the emerging of antibiotic-resistant pathogens, but the frequent one is the improper and limitless utilization of antibiotics1. The excessive utilization of numerous antibiotics has triggered the rapid emergence of multi-drug resistant pathogens. The morbidity as well as mortality, hospitalization durations, healthcare complications and economical burdens were raised in recent decades that owing to the multi-drug resistant pathogens. The microbial infections were regarded as a major public health problem worldwide in this century2. In case of fungal infection, numerous kinds of anti fungal drugs were available and on the other hand, the raising of multi-drug resistance is hugely hindering the treatment to the infected patients. Among these resistant fungal species the Candida and Aspergillus species displayed an augmented resistance towards the many kinds of drugs. As a result of multi-drug resistant especially the Candida species is a prime challenge to the clinical success rate of infected patients3.
The microbial infectious ailments are unevenly affecting the individuals worldwide, particularly in developing and under-developing countries. For this magnificent prevalence of microbial infections is not only due to the microbes, but also owing to the lack of hygiene among peoples, poor health infrastructure, counterfeit drugs, vaccine errors and health awareness deficits. The preceding research findings were highlighted that numerous bacterial strains were displayed a marked resistance towards the different antibiotics4. For this reason, the urgent need to explore the herbal plants to identify the novel, inexpensive, potential compounds as an alternative approach to the antibiotics utilization4. Since the pre-historic periods the natural products were extensively utilized to heal the countless ailments particularly infectious ailments. The herbal based natural products especially the medicinal plant extracts are conventional reserves for the discovery of novel therapeutic agents to heal the ailments5,6. Cancer was holding the top place in causing frequent deaths worldwide, at present, the cancer was treated via many routes for instance, surgical elimination of tumors, chemotherapy, radiotherapy and medications but those are often failed to augment the well beings of sarcoma victims in many cases ends with death7. Even though the liver sarcoma was well treated via those approaches like surgical exclusion, radiation and medications but the success rate was shockingly low and failed to regain the well beings of victims in that way, less than 15% liver cancer victims were benefited surviving up to 5 years or more8.
It was reported that almost 20 kinds of human illnesses can be treated via utilizing the M. concanensis plant. Preceding report highlighted that the M. concanensis was extensively consumed to heal the skin tumor, tiredness, to reduce blood pressure, aphrodisiac, jaundice, eye-care, diabetes and bloat9. The preceding research findings have revealed that the plant M. concanensis possessed greater biological benefits like anti-inflammatory potential, antianalgesic, antipyretic, antiepileptic, antioxidant activity and sunscreen10-12. Conversely there are few explorations were done on the flowers of M. concanensis to claim their antimicrobial, cytotoxic and free radical scavenging potentials. Consequently, in this research exploration, it was intended to examine the M. concanensis flowers on cytotoxic activity and antimicrobial potential against the clinical pathogens.
MATERIALS AND METHODS
Study area: The study was carried out at the Department of Biotechnology and Department of Microbiology, Bharathidasan University Constituent College from September-December, 2019.
Chemicals: The Dulbecco’s Modified Eagles Medium (DMEM) medium, Fetal Bovine Serum (FBS), Penicillin/Streptomycin, Mueller Hinton Agar (MHA) medium, Sabouraud Dextrose Agar (SDA) medium, Ciprofloxacin and Fluconazole were acquired in Sigma-Chemical Co., St. Louis, USA. The other entire chemicals and reagents that were utilized in this investigation are of diagnostic grade acquired from Hi-Media Lab, Virginia, USA.
Collection and authentication of plant material: The healthy and matured flowers of M. concanensis were gathered from the Esanai village, Perambalur, Tamilnadu, India (Latitude 11.2982°N, Longitude 78.8298°E) during September, 2019. The collected plant specimen was scientifically identified and authenticated by Dr. S. John Britto, Director, The Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph’s College (Autonomous), Tiruchirappalli-620002, Tamilnadu, India. The voucher specimen number is SK001.
Preparation of M. concanensis flower powder: The collected healthy flowers of M. concanensis were dehydrated in a shady cabin at room temperature. Then the dehydrated flowers were uniformly grounded using a mechanical grinder to yield fine powder. Finally, the powdered flowers were utilized to extract preparation.
Preparation of M. concanensis flower extracts: The 15 g of powdered flowers were soaked separately in 100 mL chloroform, diethyl ether and petroleum ether, maintained 2 days at 37°C with occasional stirring. After that, the suspension was sifted with the aid of clean muslin cloth subsequently Whatman No. 1 sift paper. The resulted filtrate was then processed on a rotary evaporator fitted with the vacuum pump. Finally, the extract was stored at 4°C until further utilization.
Fluorescent analysis: A small quantity of dehydrated powder and extract of flowers of M. concanensis were located on a slide separately and then 1-2 drops of freshly prepared reagents were mixed with gentle tilting for 1-2 min. Then the slides were examined in day (visible) light and placed it to the viewing chamber of UV transilluminator and examined in both short UV (254 nm) and long UV (365 nm) radiations. The observed colors via mixing the powder and extract with diverse reagents in several radiations were noted and tabulated13.
Qualitative phytochemical screening: The each extracts were inspected through diverse qualitative phytochemical investigations to establish the phytochemical profile of the M. concanensis flowers. All three extracts were inspected to evidence the occurrence of various phytoconstituents via standard procedure14,15.
Cell culture: The human liver cancer (HepG2) cells were bought in American Type Culture Collection (ATCC), USA. The cells were then cultured in DMEM with 10% FBS and 1% Penicillin-Streptomycin mixture and maintained at 37°C in a moistened atmosphere containing 5% CO2 and 95% air incubation.
MTT cytotoxic assay of M. concanensis flowers: The cytotoxicity action of chloroform extract of M. concanensis flowers were examined via the MTT cytotoxic test16. The HepG2 cell lines were loaded in the 96-well plates in a population of 1×104 cells per well and kept incubation for 24 h at 37°C. To elucidate the cytotoxic potential of chloroform extract of M. concanensis flowers it was dissolved in DMSO and supplemented in different dosages (10-100 μg mL–1) to the HepG2 cells and then kept at incubation for 24 h at 37°C. After 24 h of incubation, the MTT solution (100 μL from 5 mg mL–1) was loaded into every well and then plates were again kept for incubation for 4 h. Then the medium was eliminated from the wells and 100 μL of the serum free medium was replenished into the wells and the developed formazan crystals were liquefied by the addition of dimethyl sulfoxide. Finally, the absorbance value was determined via multi well microplate reader at 570 nm.
Morphological analysis of chloroform extract of M. concanensis flowers treated HepG2 cells: To examine the chloroform extract of M. concanensis flowers treated HepG2 cells, the HepG2 cells were loaded on the 96-well plate at 1×105 cell population per well. Then the HepG2 cells were supplemented with 25-100 μg mL–1 of chloroform extract of M. concanensis flowers and incubated for 24 h. Later after incubation, HepG2 cells were inspected beneath the optical microscope to identify the extract induced morphological alterations17.
Collection and maintenance of bacterial and fungal strains: The bacterial strains i.e., Escherichia coli, Bacillus subtilis, Vibrio cholerae, Staphylococcus aureus and Psuedomonas aeruginosa and fungal strains i.e., Aspergillus flavus, Candida albicans, Trichophyton simii, Trichophyton mentagrophytes and Trichophyton rubrum were acquired from the ATCC, USA. The bacterial and fungal strains were maintained individually in MHA medium at 38°C for bacteria and SDA medium at 28°C for fungi in an incubator and utilized for further investigations.
Examination of antimicrobial potential of M. concanensis flowers via disc diffusion technique: The antimicrobial potential of M. concanensis flowers were investigated through the disk diffusion technique18. Briefly, 100 μL of 24 h old culture of every bacterial strain were loaded to the MHA medium fungal strains were SDA medium and spread over the surface of plates. Then the sterile antibiotic discs in 6 mm diameter were infused with each extracts of M. concanensis flowers then the discs were located on a test plates at equidistance. The plates were then kept undisturbed for 30 min to diffuse the extract on the medium and then incubated at 37°C for 24 h to examine the antimicrobial potential of M. concanensis flowers. Ciprofloxacin antibiotic discs (15 μL/disc) were utilized as a reference control for bacteria and Fluconazole discs (15 μL/disc) were utilized for fungi. Later than the incubation the zone of inhibition (in mm) around the disc were examined and regarded as an antimicrobial efficacy of extracts against the test pathogens.
Determination of Minimum Bactericidal and Fungicidal Concentrations (MBC and MFC) of M. concanensis flowers: The Minimum Inhibitory Concentration (MIC) is the least dose needed to hinder the microbial growth subsequently 24 h incubation. The MIC examination through the tube dilution technique was extensively employed to inspect the antimicrobial potential of test sample19. The growth sensitivity of bacterial and fungal pathogens to the M. concanensis flower extracts was examined via the tube dilution technique to calculate the MBC and MFC. Briefly, strains were loaded to the tube consisting of 1mL of respective broth at the density of 5×105 CFU mL–1 of each test pathogens. Then the tube was supplemented with the various doses (100-1000 μg mL–1) of M. concanensis flower extracts. The tubes were further diluted with the addition of MH broth for bacteria and SD broth for fungi. Finally, the assay tubes consisting of diluted samples were then sustained overnight at their respective temperatures for bacteria and fungi with constant shaking. The growth of the strains was inspected via the turbidity formation. The clear tubes excluding turbidity denote the absence of bacterial and fungal growth. The extra care was taken to make sure the sterility and this investigation was repeated for thrice to establish the precise test values20,21. Later than the completion of the investigation, the utilized microbial strains, growth medium and every plastic ware were sterilized and discarded as per the rules suggested by the Institutional Biosafety Committee (IBC).
Statistical examination: Statistical investigations were carried out with the help of the SPSS statistical tool (version 16). Data were examined through ANOVA subsequently DMRT study to distinguish the variations among the test groups. Data were displayed as Mean±SD of triplicate measurement. Data were regarded as significant if ‘p’ values were less than 0.05.
RESULTS
Characteristics of M. concanensis flower powder: The dehydrated and powdered flowers were brownish and have a characteristic flavor in nature. The flower powder has a defined and solid aroma. The greasy spots were noted while the powder was mechanically pressed along with the filter papers. It eventually denotes the occurrence of fatty acids. The stable froth also monitored during the powder was mixed with water and shaken well. It denotes the occurrence of saponins in the flower.
Fluorescence analysis of Moringa concanensis flower extracts and powder: The outcomes of fluorescence analysis of M. concanensis flower extracts as well as powder, evidently proved that this plant has the immense phytoconstituents. The results displayed the different kinds of fluorescent colors like brown, greenish-brown, violet, green, purple, orange and black when exposed to the sun (day) light and UV light (Table 1). The appearance of numerous kinds of color fluorescence was proving the occurrence of an array of phytoconstituents in the M. concanensis flowers (Table 2).
Qualitative phytochemical analysis of M. concanensis flower: As depicted in Table 3, the outcome of the qualitative phytochemical examination of M. concanensis flower has displayed the occurrence of numerous kinds of phytochemicals. The flowers displayed the occurrence of flavonoids, alkaloids, glycerides, volatile oils, steroids, terpenoids, glycosides, reducing sugars and amino acids. While comparing to other solvents, the chloroform extract of M. concanensis flower displayed the presence of a maximum number of phytochemicals (Table 3).
| Table 1: | Fluorescence analysis of M. concanensis flower extracts |
| Table 2: | Results of fluorescence analysis of Moringa concanensis flower powder |
| Table 3: | Phytochemical screening of M. concanensis flower extracts |
| +: Present in small concentration, ++: Present in moderately high concentration, +++: Present in very high concentration, -: Absent | |
Consequently, the chloroform extract was selected for the further free radical scavenging and cytotoxic investigations.
Antibacterial activity of M. concanensis flower extracts: The outcome of the antibacterial activity of M. concanensis flower extracts displayed the appreciable growth inhibition against the test pathogens (Table 4). The chloroform and petroleum ether extracts were displayed the maximum growth-inhibitory efficacy against the E. coli (18±0.6 mm). The B. subtilis exhibited a 15±0.3 mm inhibition zone in the chloroform extract. Otherwise, the P. aeruginosa displayed resistance to all the extract of M. concanensis flower.
Minimum Bactericidal Concentration (MBC) of the M. concanensis flower extracts: The growth inhibition effectiveness of chloroform, diethyl ether and petroleum ether extracts of M. concanensis flowers were inspected against the five human pathogenic bacterial strains. The strong antibacterial action of M. concanensis flowers was noted against the V. cholerae and E. coli in chloroform extract. The minimum inhibitory concentration was noted against the V. cholerae in chloroform extract, where the 190 μg mL–1 of chloroform extract was appreciably inhibited the growth of V. cholerae, subsequently, 220 μg mL–1 of diethyl ether extract was notably suppressed the B. subtilis growth. The MBC values of chloroform, diethyl ether and petroleum ether extracts were depicted in Table 5.
Antifungal activity of M. concanensis flower extracts: As mentioned in Table 6, the M. concanensis flowers were displayed the appreciable antifungal potential against the tested pathogens. All three extracts were revealed the marked in vitro antifungal effects against the tested human pathogenic fungal strains. Among these strains, the T. simi and T. mentagrophytes (21±0.6 and 21±0.3 mm, respectively) were displayed the maximum sensitivity against the diethyl ether extract. The A. flavus exhibited 19±0.4 and 19±0.5 mm inhibition zones in the chloroform and diethyl ether extracts, respectively (Table 6). Whereas, the T. simii was found to be highly resistant to the petroleum ether extract. On the whole, all the extracts are markedly inhibited the growth of tested pathogenic fungal strains.
Minimum Fungicidal Concentration (MFC) of M. concanensis flower extracts: The outcomes of this investigation proved that the chloroform, diethyl ether and petroleum ether extracts of M. concanensis flower exhibited a remarkable antifungal potential against the tested pathogenic fungal strains.
| Table 4: | Antibacterial activity of crude extracts of Moringa concanensis flower |
Values are depicted as Mean±SD for the triplicate measurements, statistical significance was inspected via one-way ANOVA, subsequently, the DMRT test, where *p<0.05, NZ: No zone formation | |
| Table 5: | MBC values (μg mL–1) of M. concanensis flower extracts |
Values are depicted as Mean±SD for the triplicate measurements, statistical significance was inspected via one-way ANOVA, subsequently, the DMRT test, where *p<0.05 | |
| Table 6: | Antifungal activity of M. concanensis flower extracts |
Values are depicted as Mean±SD for the triplicate measurements, statistical significance was inspected via one-way ANOVA, subsequently, the DMRT test, where *p<0.05, NZ: No zone formation | |
| Table 7: | MFC values (μg mL–1) of M. concanensis flower extracts |
Values are depicted as Mean±SD for the triplicate measurements, statistical significance was inspected via one-way ANOVA subsequently, the DMRT test, where *p<0.05 | |
The strong antifungal outcome was noted against the T. mentagrophytes subsequently C. albicans in chloroform extract, where the 100 μg mL–1 of chloroform extract was noticeably inhibited the growth of T. mentagrophytes, subsequently, 110 μg mL–1 of chloroform extract was notably diminished the growth of C. albicans. The MFC values of chloroform, diethyl ether and petroleum ether extracts were displayed in Table 7.
Effect of M. concanensis flower extract against HepG2 cell line: The result of the MTT cytotoxicity test proved that the chloroform extract of M. concanesis flowers displayed an appreciable cytotoxic potential against the human liver cancer (HepG2) cells. The treatment with the M. concanesis flower extract has drastically diminished the viability of human liver cancer (HepG2) cell lines in a dose-dependent mode. Consequently, it was proved that the chloroform extract of M. concanesis flowers were markedly diminished the viability of HepG2 cells.
| Fig. 1: | Effect of chloroform extract of M. concanensis flower on HepG2 cell viability |
Figure 1 displaying the effect of chloroform extract of M. concanensis flowers HepG2 cell viability. Human liver cancer (HepG2) cells were cultured in DMEM with 10% fetal bovine serum. The cells were then treated with 10-100 μg mL–1 of chloroform extract of M. concanensis flowers. The control and treated cells were subjected to MTT assay and the results were statistically investigated.
Effect of M. concanensis flower extract against the HepG2 cell morphology: The chloroform extract of M. concanesis flowers was subjected to inspect the synergistic effect on the morphology of HepG2 cells. The morphological investigation of M. concanesis flower extract-supplemented HepG2 cells were displayed the drastic alterations on the morphology as well as the total amount of adherent cells.
| Fig. 2(a-e): | Effect of chloroform extract of M. concanensis flower on HepG2 cells (a) Control, (b) 25, (c) 50, (d) 75, (e) 100 μg mL–1 |
There are vast differences were exhibited on the morphology of extract treated HepG2 cells than the untreated control cells. The extract treatment (25-100 μg mL–1) displayed the tremendous morphological alterations that include unequal shape, rounding, cell shrinkages and detachments among cells on the HepG2 cells (Fig. 2a-e).
DISCUSSION
The findings of this investigation was proved that the flowers of M. concanensis was appreciably inhibited the growth of pathogenic microorganisms as well as, it was displayed the potential cytotoxicity against the human liver cancer (HepG2) cells. In this decade, the emergence of multidrug-resistant microbes owing to the serious health consequences among populations worldwide, because they acquired the resistance because of recurrent and mishandling utilization of antibiotics. Consequently, the alternative approaches were needed urgently to counteract these issues with natural sourced novel agents22. The antimicrobial potential of M. concanensis flower extracts was inspected through the disc diffusion technique, MBC and MFC against the human-specific clinical pathogens. The outcomes of qualitative phytochemical screening revealed that the occurrence of flavonoids, alkaloids, volatile oils, cardiac glycosides, carbohydrates, amino acids and steroid triterpenes as the probable active constituents occurs in the M. concanensis flower extracts. The occurrence of these secondary metabolites in plants is accountable to generate the biological effects in humans as well as animals and it is accountable for their utilization as herbals23. These bioactive constituents are also accountable to protect the plants against the infection via microorganisms24,25.
The fluorescence is a phenomenon that is exhibited via reacting with different kinds of phytochemical constituents that occur in plant samples. A few phyto-compounds have displayed the fluorescence in the apparent daylight26. This investigation can assist the researchers to recognize and confirm the phytochemical status in the test plant material. These data may deed as a reference for accurate detection of exact plant specimen along with their phytochemical profile27. The M. concanensis flower extracts have possessed the noticeable antimicrobial and cytotoxic potential. The scientific investigation of plant specimens that have been utilized for conventional remedial purposes is essential to identify the potent antimicrobial agents. The in vitro antimicrobial examination is a prime phase to the inspection of novel antimicrobial agents28. The global impact of infectious ailments is regarded with the treatment failures linked with multidrug-resistant pathogens and now it has become as a global concern to public health29,30. In this current exploration, the antimicrobial potential of flower extracts of M. concanensis was inspected against the different human pathogenic bacterial strains like B. subtilis, S. aureus, E. coli, V. cholerae, P. aeruginosa and the fungal strains were A. flavus, C. albicans, T. simii, T. mentagrophytes and T. rubrum. Among these bacterial species, E. coli has noted their more sensitivity in chloroform and petroleum ether extracts (18±0.6 mm). Whereas, P. aeruginosa found to be highly resistant in all these extracts. The fungal species T. simii and T. mentagrophytes were found to be highly sensitive (21±0.6 and 21±0.3 mm, respectively) in diethyl ether extract whereas, the T. simii was found to be highly resistant in petroleum ether extract.
The MBC of the M. concanensis flower extracts was noted in chloroform and petroleum ether extract against V. cholerea, the 190 μg mL–1 of chloroform extract inhibited the visible growth, subsequently the E. coli, the 220 μg mL–1 of chloroform extract of M. concanensis flower completely inhibited the visible growth of E. coli bacteria. The 220 μg mL–1 of diethyl ether extract inhibited the visible growth of B. subtilis (Table 5). This outcome was proved that the chloroform extract of M. concanensis flower displayed the appreciable antibacterial effectiveness than the other extracts. The P. aeruginosa exhibited a maximum resistance to all the tested extracts. The potent antifungal effect was noted against the T. mentagrophytes subsequently C. albicans in chloroform extract, where the 100 μg mL–1 of chloroform extract was appreciably inhibited the growth of T. mentagrophytes, subsequently, 110 μg mL–1 of chloroform extract was notably diminished the growth of C. albicans. The MFC values of chloroform, diethyl ether and petroleum ether extracts were displayed in Table 7.
The cancer is a foremost ailment accountable for numerous death incidences worldwide31. The many cancer incidences were often diagnosed at the very late stage though the many cancer incidences were no longer treatable for remedial methods. Besides, the effectiveness of existing healing approaches for sarcoma was accountable for additional damages and declining the vast excitable pathways of drug metabolism and resistance to drugs linked to transporting proteins of malignant cells32. The cancer cell death has happened in numerous ways whenever the cells get exposed to cytotoxic agents, like plant extracts and drugs. Normally, two kinds of the cell took place frequently that is apoptosis and necrosis. The apoptosis was undergone due to the many molecular and cellular mechanisms for example; contraction of cells, condensation of cells, fragmentation of DNA, blebbing of membranes as well as generation of apoptotic bodies33-36. In this current investigation, it was proved that the treatment of chloroform extract of M. concanensis flowers was noticeably suppressed the growth of the human liver cancer (HepG2) cells via altering the morphology of cells like cell shrinkages.
The results of the MTT cytotoxicity examination confirmed that the chloroform extract of M. concanesis flowers displayed an appreciable cytotoxic potential against the human liver cancer (HepG2) cell line. The treatment with the M. concanesis flower extracts has drastically diminished the viability of human liver cancer (HepG2) cell lines in a dose-dependent mode. The chloroform extract of M. concanesis flowers were also subjected to investigate morphology altering potential against HepG2 cells. The morphological investigation of M. concanesis flower extract-treated HepG2 cells was displayed the noticeable alterations in the morphology as well as the total amount of cells. The extract treatment exhibited incredible morphological alterations like unequal shape, rounding, cell shrinkages and detachments of HepG2 cells (Fig. 2a-e). From the findings, it was proved that the M. concanesis flowers were revealed the incredible antimicrobial potential against the human-specific pathogenic bacterial and fungal strains and displayed the appreciable cytotoxicity against the human liver cancer (HepG2) cell lines.
CONCLUSION
The novel findings of this research investigation were evidence that the M. concanesis flowers were found to highly effective against the human-specific bacterial and fungal pathogenic strains. The findings were also proved that the M. concanesis flower extract treatment was diminished the cell viability of human liver cancer (HepG2) cell lines via stimulating the morphological alterations like rounding, cell shrinkages and detachments. Accordingly, it was concluded that the M. concanesis flower can be a promising source to identify the enhanced antimicrobial as well as cancer cytostatic agents. However, further research in the future was needed to elucidate the precise therapeutic mechanism of M. concanensis flower against the clinical pathogens.
SIGNIFICANCE STATEMENT
This study was evidenced that the flowers of Moringa concanensis (Nimmo) has the immense phytochemicals and possess the potential antimicrobial activity against the infections causing pathogenic microbes. The findings of this study may disclose the pharmacological benefits of this plant and also help to the researchers to develop a novel antimicrobial compounds.