Impact of Herbal Extracts in Biocontroling of Four Human Pathogenic Bacteria- an in-vitro Study

Harsha Sharma¹, Arun Karnwal²*

¹Bhojia Institute of Life Sciences, Baddi, Himachal Pradesh, India-173405.

²School of Bioengineering and Biosciences, Lovely Professional University, Jalandhar-Delhi G.T. Road, National Highway 1, Phagwara, Punjab, India 144411.

*Corresponding Author E-mail: arunkarnwal@gmail.com

ABSTRACT:

Plants are among the most prominent resources of herbal remedies. The principal advantages of herbal plants in a number of diseases are usually their safeness over antibiotics. The existing research was aimed to examine the antibacterial action associated with crude concentrate of Ocimum sanctum (Tulsi), Syzygium aromaticum (Clove), and Azadirachta indica (Neem) against four human pathogenic bacteria spp. collected from IMTECH. The crude extract of three plants was prepared by pulverizing dried plant material followed by ethanolic extraction in 500 mL of ethyl alcohol. Antimicrobial action of crude extract was analysed by applying agar well diffusion approach against S. aureus MTCC 96, B. subtilis MTCC 441, L. casei MTCC 1423, and E. coli MTCC 1302 In-vitro. MIC and MBC as also determined in present study by applying varios concentration of plant extract against pathogenic bacteria. The extract of Tulsi, Clove, and Neem showed antibacterial activity against the Gram-positive S. aureus MTCC 96, B. subtilis MTCC 441, L. casei MTCC 1423 and Gram-negative E. coli MTCC 1302. Clove extract recorded with the maximum antibacterial activity towards all pathogens during well diffusion (13-17 mm zone of inhibition), minimum inhibitory concentration (7. 5%w/v-15%w/v), and minimum bactericidal concentration (7. 5%w/v-15%w/v) assays. Tulsi and Neem extracts even shown a remarkable area of growth inhibition (10-13mm and 9-11mm, respectively), minimal inhibitory concentration (15%w/v-30%w/v) and minimal bactericidal concentration (7. 5%w/v-20%w/v). Almost all three extracts was furthermore analyzed for their efficiency as antiseptic by means of minimum contact time parameter. It was fond that clove extract required minimum time to exert the antiseptic effect on selected pathogens (1 min at 20%w/v concentration) while other two chosen extracts showed antiseptic effect at 15% and 20%w/v concentration with minimal contact time 1 min. At last, It can be concluded that the ethanol extract of tulsi, clove and neem is effectively potent as natural antiseptics against human pathogens.

KEYWORDS: Antibacterial; Herbs; MIC; MBC; antiseptic; plant extract.

INTRODUCTION:

Antibiotics are the confidant for humans against many human pathogens. However, excessive use of antimicrobial drugs has developed into the serious issue for the appearance and spreading associated with multi-drug tolerant microbial strains of various kinds of micro-organisms¹.

Therefore, because of the facts involving in fast worldwide spread of antibiotic tolerant clinical isolates, the requirement to discover new antimicrobial agents is of critical significance^2,3. Plants are usually abundant in a broad range of secondary metabolites such as tannins, alkaloids and flavonoids, that have been discovered in vitro to own anti-bacterial traits⁴. The application of plants and plant products in medications might be followed dating back with the start of human civilization. The earliest written information concerning the usage of medicinal plants is documented in “Rigveda”: an ancient Indian collection of Vedic Sanskrit hymns, and believed as the most ancient repository of human knowledge^5,6.

In developing countries, various therapeutic herbal plants are utilized for healing diseases as alternate remedies. Several plant extracts as well as essential herbal oils possess biological anti-activity in vitro and in vivo, which often justifies the exploration requirement of plant sourced drugs and aimed for the characterization of antimicrobial action of such plants⁷. A large number of drugs in use is derived from plants^8,9, like morphine from Papaver somniferum, Aswagandha from Withania somnifera, Ephedrine from Ephedra vulgaris, Atropine from Atropa belladonna, Reserpine from Roulphia serpentina etc. As a medicine, each plant part or its extract (i.e. rhizome, outer crust, shoot, leaves and seeds) could be utilized for the treatment of diseases and could be considered as potent and powerful medicines. These plant chemicals could be classified within essential natural oils, alkaloids, acids, steroids, tannins, saponins and so forth¹⁰. As outlined by World Health Organization, herbal plants might be considered as the most effective source intended for obtaining several drugs for the medication of human as well as plant pathogens. Plant derived chemical substances and solutions i. e. neem oil, jojoba oil, cinnamon oil, rosemary essential oil, soybean oil, compost tea, horsetail plant; giant knotweed, have been reported considerably efficient towards various pathogens i.e. P. fluorescens, S. liquefaciens, B. thermosphacta, C. piscicola, L. curvatus, and L. sake^11,12.

The goal of current research was to examine the antibacterial action associated with crude concentrate of Ocimum sanctum (Tulsi), Syzygium aromaticum (Clove), and Azadirachta indica (Neem) against four pathogenic bacterial strains.

MATERIAL AND METHODS:

Plant material:

The three herbal plants, Ocimum sanctum (Tulsi), Syzygium aromaticum (Clove), and Azadirachta indica (Neem), samples were examined against four human pathogen bacteria. The herbal plants tested for present research was procucered from local market of Baddi, Himachal Pradesh, India. Plant products (Ocimum sanctum : leaves; Syzygium aromaticum : flower bud; Azadirachta indica : leaves) were rinsed and cleaned with sterilized distilled water for removal of almost all unwanted contaminants, air-dried and preserved in a aseptic air-tight container until further use.

Collection of test Organism and inoculum preparation

Collection of test Organism and inoculum preparation The bacterial isolates chosen in existing analysis were originally obtained from Institute of Microbial Technology (IMTECH), Chandigarh, India. The Human pathogenic bacteria were used is Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC 441, Lactobacillus casei MTCC 1423, and Escherichia coli MTCC 1302. Bacterial sub-culturing was performed by transfering bacterial culture on Nutrient broth at 37° for 24h of incubation. Nutrient medium used for present study was purchased from Hi Media, India and prepared as mentioned in manufacturer instructions.

Preparation of herbal extract:

The dried portion was pulverized in a mortar and pestle to get fine powder of herbal material. Briefly, for preparation of herbal extract 200g of dry and pulverized plant material was immersed in 500 mL of ethyl alcohol and placed within incubator shaker at 100rpm for overnight. The resulting extracts were centrifuged at 3000rpmfor five min at 4 °C. Immediately after centrifugation, supernatant has been filtered by means of Whatman filter No . 1 and residues were put into a second phase extraction along with 300 mL of ethanol. Following the second phase of extraction, the resulting filtrates were concentrated by using decreased pressure with a rotary evaporator at 50 °C. The particular concenterated extracts were collected and permitted to evaporate over a water bath until finally dehydrated extract along with constant weight was received. The extract was kept in fridge at 4ºC until time of use.

The extract percentage yields (w/w) calculated by using: (extract weight ÷ Initial plant material weight) × 100%

Preparing Bacterial Suspension:

All bacterial strains were subultured on blood agar and incubated at 35°C for 24h. The bacterial suspension was prepared by transferring loopful bacterial culture in to normal saline and retained the density equivalent to the 0.5 McFarland standard turbidity that of 10⁸ cfu/ml.

Antibacterial activity:

The leaf extracts were tested for antibacterial activity using agar diffusion method. The 100 µL of individual bacterial suspension was transferred in sterile petri plates having melted Mueller-Hinton agar (Hi media). Once mixture of bacterial suspension and media became solidify, hole with a diameter of 6 to 8 mm was punched aseptically with a sterile cork borer for testing the plant extract. The extract applied in the experiment was prepared by mixing 05, 10, 15, 20, 30, 50, 60 and 70% w/v in dimethyl sulfoxide as solvent or blank. The 50 µl of extracts and blank was poured into each hole and incubated for 24 h at 37°C. All experiments were performed in triplicates and zone of inhibition were noted for MIC and MBC studies.

MIC and MBC determination:

MIC determination of plant extracts was done using macrodillution method. The extract, serially diluted two times to a concentration of 30%, 15%, 7.5%, 3.75%, and 1.87% w/v by using Mueller-Hinton broth for bacterial growth. The volume of 20 µL bacterial suspensions (1x10⁸ cfu/mL) was placed into each tested concentration and incubated for 20 h 37°C for 48 h. The MIC value has been calculated by looking for the least concentration associated with plant extract that will entirely inhibited the bacterial growth observed with the unaided eye. Minimum bactericidal concentration (MBC) was determined from the MIC range. Mueller-Hinton agar plates were sub-cultured from tubes and incubated at 37°C overnight. The petri dishes were observed macroscopically. The smallest concentration associated with plant extract that produced zero microbial growth on the solid medium was accepted as MBC.

Perseverance of minimum contact time:

The following study was targeted to find out the shortest time period needed by the extract to work as antiseptic agent. For this a series of concentrations of ethanolic extracts i.e. 40, 20, 10, and 5%w/v was used. Bacterial culture was transferred into each individual concentration associated with extract and incubated at room temperature by using defined time intervals i.e. 1, 2, 3, 4, 5, 6, 7 min. All tested microbial suspension inoculated into MHB and incubated at 37°C for overnight for any bacterial growth.

RESULTS:

Antibacterial activity:

In present study, the concentrated ethanolic extracts of Ocimum sanctum, Azadirachta indica, and Syzygium aromaticum were tested to screen the potential antibacterial properties against four human pathogens. The level of antimicrobial action was evaluated through the MIC value towards the bacterial strains. The well diffusion approach primarly applied to reveal the antibacterial ability associated with plant extracts against chosen four human pathogenic bacteria i.e. Staphylococcus aureus MTCC 96, Bacillus subtilis MTCC 441, Lactobacillus casei MTCC 1423, and Escherichia coli MTCC 1302.

The outcomes of the well diffusion approach suggested that crude ethanolic concentrates of Tulsi, Clove, and Neem showed different degrees of growth inhibition depending on the bacterial strains. The ethanolic concentrate of clove was shown the maximum antibacterial action by inhibiting the development of all bacterial strains examined in present study. Clove ethanolic extract shown the maximum antibacterial action as zone of inhibition (ZOI) towards all selected bacterial strains, ranges from 13mm to 17mm at 70%w/v extract concentration as displayed in Figure 1.

Figure 1: Antimicrobial activities of clove extract

Figure 2: Antimicrobial activities of Neem extract

Figure 3: Antimicrobial activities of Tulsi extract

Neem extract moreover shown considerable inhibition zone towards Bacillus subtilis MTCC 441, and Lactobacillus casei MTCC 1423 along with 13mm and 12mm of zone of inhibition with 70%w/v extract concentration, respectively (Figure 2). Tulsi recorded with maximum inhibition zone against Lactobacillus casei MTCC 1423 (ZOI 11mm) as displayed in Figure 3.

MIC and MBC findings:

Furthermore, two fold dilution approaches were employed regarding the determination associated with minimum inhibitory concentration (MIC) for the experimental plant extracts that are capable to generate area of inhibition in the course of screening procedure. MIC values of concentrated ethanolic extracts of Clove, Tulsi and Neem showed that clove possessed the highest antibacterial activity towards almost all bacterial strains examined (MIC 7. 5%w/v extract concentration) as displayed in Table 1, followed by Tulsi and Neem.

Table 1: MIC of ethanol extracts from three medicinal plants against human pathogens (%w/v).

Extract concentration (%w/v)	Tulsi Extract				Clove Extract
Extract concentration (%w/v)	*Staphylococcus aureus* MTCC 96	*Bacillus subtilis* MTCC 441	*Lactobacillus casei* MTCC 1423	Escherichia coli MTCC 1302	*Staphylococcus aureus* MTCC 96	*Bacillus subtilis* MTCC 441	*Lactobacillus casei* MTCC 1423	Escherichia coli MTCC 1302
30	+	+	+	+	+	+	+	+
20	+	+	+	+	+	+	+	+
15	+	+	+	+	+	+	+	+
7.5	-	-	+	-	+	-	+	-
3.75	-	-	-	-	-	-	-	-
1.87	-	-	-	-	-	-	-	-
MIC value	15	15	7.5	15	7.5	15	7.5	15

Continue Table 1

Neem Extract
*Staphylococcus aureus* MTCC 96	*Bacillus subtilis* MTCC 441	*Lactobacillus casei* MTCC 1423	*Escherichia coli* MTCC 1302
+	+	+	+
+	-	+	+
+	-	+	+
-	-	-	-
-	-	-	-
-	-	-	-
15	30	15	15

Note: + No microbial growth; - microbial growth

concentration in comparison to MIC (Table 2).

Table 2: MBC of ethanol extracts from three medicinal plants against human pathogens (%w/v).

Extract concentration (%w/v)	Tulsi Extract				Clove Extract
Extract concentration (%w/v)	*Staphylococcus aureus* MTCC 96	*Bacillus subtilis* MTCC 441	*Lactobacillus casei* *MTCC 1423*	Escherichia coli MTCC 1302	*Staphylococcus aureus* MTCC 96	*Bacillus subtilis* MTCC 441	*Lactobacillus casei* MTCC 1423	Escherichia coli MTCC 1302
30	+	+	+	+	+	+	+	+
20	+	+	+	+	+	+	+	+
15	-^*	+	+	+	+	+	+	+
7.5	-	+	+	-	+	-	+	-
3.75	-	-	-	-	-	-	-	-
1.87	-	-	-	-	-	-	-	-
MBC value	20^*	7.5	7.5	15	7.5	15	7.5	15

Continue Table 2

Neem Extract
*Staphylococcus aureus* MTCC 96	*Bacillus subtilis* MTCC 441	*Lactobacillus casei* MTCC 1423	*Escherichia coli* MTCC 1302
+	+	+	+
+	+^*	+	+
+	-	+	+
-	-	-	-
-	-	-	-
-	-	-	-
15	20^*	15	15

Note: + No microbial growth; - microbial growth, * Variation between MBC and MIC results

Table 3: Minimum contact time (min) results for different ethanolic %w/v plant extracts

Plant

%w/v extract conc.

Staphylococcus aureus

MTCC 96

Bacillus subtilis

MTCC 441

Lactobacillus casei

MTCC 1423

Escherichia coli

MTCC 1302

Time (min)

Tulsi

7.5

Clove

7.5

Neem

7.5

Note: + No microbial growth; - microbial growth

DISCUSSION:

Plant derived natural oils and components have been utilized from a long time, during food maintenance and storage, in pharmaceuticals as alternate choice of remedies and for natural treatments¹². It is needed to examine all those plants technically that have been practiced in conventional medication to enhance the level of quality of medical care. Plant extracts are possible solution concerning the novel anti-bacterial substances specifically towards bacterial pathogens. In vitro experiments within this study demonstrated how the plant extracts prevent bacterial growth^10,13. However, The antimicrobial action associated with various plant extracts was formerly evaluated and categorized as solid, medium or even weak^10,12. Typically the therapeutic plants such as cinnamon, timur, tulsi and origanum have been utilized commonly to treat inflammation, swelling, irritation, cough, toothache, antiseptics expectorant, and for various microbial treatment including fungi, yeast and bacterial^14,15. The antiseptic action of plant extracts has been linked to the existence of certain effective constituents in the extracts. Various Reports^16,17 mentioned about the fact that antibacterial action of clove was undoubtedly because of their key components, eugenol, eugenol acetate and gallic acid. These components are natural antioxidant and antimicrobial in nature. A significant feature associated with plant extracts together with their ingredients is usually their hydrophobicity¹⁸, that allow them to seperate the lipids in the microbial cell membrane and mitochondria, disturbing the particular cell constructions and even making them considerably more permeable which cause intensive leakage through bacterial cells and lead to bacterial death¹⁹. The inhibition developed due to herbal extracts on living bacteria will depend on different extrinsic and intrinsic constraints. Because of varied diffusability found in agar medium, the antiseptic attribute might not display because ZOI commensurate to the efficacy²⁰. For that reason MIC has additionally been calculated in this research. The MIC was explained for the least concentration of extract when the particular microorganism examined failed to show noticeable growth.

Based on the antibacterial assay carried out for the screenin gpurpose, each of the gram positive and even gram negative organisms i. e. Staphylococcus aureus, Bacillus subtilis, E. coli and Lactobacillus casei had been the most sensitive germs to any or all plant extracts. The efficiency concerning plant extracts fluctuate by organism to organism i. e. Clove extract indicate a good impact on almost all bacterial strains along with greater inhibition areas²⁰. The growth of E. coli has been mainly inhibited by clove however various other plants had slight impact on E. coli. All of these findings are most probably due to the effect of the variations within cell wall composition among Gram-positive and Gram-negative microorganisms, with Gram-negative external membrane behaving being an obstacle to a lot of external chemicals consisting antibiotics²¹. These types of studies promote the conventional understanding of localized people and it is an initial, methodical, validation for the usage of these types of plants pertaining to antibacterial action to enhance appropriate conservation and sustainable utilization of such type of plant resources.

CONCLUSION:

In summary, the outcomes from the current research encourage the application of the examined plants as antimicrobial agent that might be further more discovered to get antimicrobial action towards various other pathogenic microbes. Furthermore these types of plants, as remedies, shows that they symbolize an economical and harmless substitute in order to deal with contagious diseases. The outcomes associated with current analysis evidently suggest that the antibacterial activity differ along with the types of the plants. Hence, the research determines the significance of plants utilized in ayurveda, that could be of significant attention towards the progress of innovative medicines.

ACKNOWLEDGEMENT:

The authors are grateful to the authorities of Bhojia Institute of Life Sciences, Baddi, H.P. for the facilities.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 16.02.2018 Modified on 28.03.2018

Research J. Pharm. and Tech 2018; 11(7): 2895-2900.

DOI: 10.5958/0974-360X.2018.00533.4