Evaluation of Antimicrobial behaviour of Tylophora indica leaf extracts against selected food microbial pathogens

 

Charu Khanna^1,2, Shalini Singh^1*, Manish Vyas², Sujata Das¹

¹School of Bioengineering and Biosciences, Lovely Professional University, Punjab, India -144411.

²School of Pharmaceutical Sciences, Lovely Professional University, Punjab, India -144411.

 

ABSTRACT:

In the last few decades, tremendous rise in food trade along with, consumer awareness for hygienic food stuffs has been witnessed. The loss and wastage of food throughout the food supply chain along with serious incidences of food illnesses, owing to consumptions of contaminated foods, has opened the scope for development of newer and effective preservation technology involving natural substances, for Food Microbiologists. To add on, the emergence of multi drug resistant strains due to excessive and inappropriate use of antibiotics have created an immediate need to look in for natural anti-microbial for control of microbial infections and thus, extensive efforts are being made globally to replace chemical based food preservatives & antibiotics with natural ones. Plants based products are one of the most important alternatives as these are natural, relatively lesser toxic, economical along with higher inclination towards their acceptance in general population. Decades back, herbs such as Neem and Haridra were used to preserve foods, which were gradually overtaken by the synthetic preservatives. Owing to the health issues with chemical preservatives and additives such as allergies, asthma, dermatitis and seizures, plants have attracted the focus of the scientific community and are being explored for their preservative potential. In view of the above, different extracts viz. methanolic, ethanolic, aqueous and ethyl acetate extracts leaves of Tylophora indica were explored for their antimicrobial potential against selected food pathogens (E. coli, P. aeruginosa, S. aureus, B. subtilis and L. monocytogenes). The efficiency of different extracts was found in the order methanolic> aqueous> ethyl acetate> ethanolic, thus confirming methanolic to be most effective antimicrobial crude extract. Thereafter, the authors are looking forward to analyse the effects of selected extracts towards shelf life of food products. The plant extracts tested offer scope for developing potential safe and natural food preservatives that alone or in combinations with other antimicrobial agents, can be used in food formulations for enhancing shelf life of different food materials.

 

 

 

INTRODUCTION:

In estimation, nearly one-third of the food is wasted or lost from moment of its production up till it is consumed by the humans. This loss and wastage is comparatively higher and even throughout the food supply chain in the developing countries especially with the livestock because of diseases such as pneumonia, parasite infections and digestive infections at the rearing stage^[1].

 

As per a news release, WHO estimated that one in a ten people fall sick due to consumption of contaminated food every year resulting nearly 4,20,00 deaths, annually. The major food borne illness is contributed by African and South-east Asian regions^[2]. Additionally, extensive use of antibiotics in such conditions has caused the emergence of multidrug resistant pathogenic strains (MDR) including food pathogens, leading to worsening the situation. To combat the growth of health threatening food pathogens in food along with enhancement of shelf life and to check the generation of MDR strains, use of natural substances are being looked upon by health and food scientists nowadays^[3]. More recently, orientation of research towards the antimicrobial potential of natural plant extracts can be well witnessed. Plants have always been a part of health and food community since ancient times. The different bioactive components such as alkaloids, essential oils, glycosides and resins have been responsible for their antimicrobial potential^[4]. Few studies has been reported where the biologically active ingredient of the plants (garlic, thymol, Mentha pulegium oil and  grape fruit seed extract ) alone or in combination with other natural agents such as nisin has been studied against different microbes^[5,6]. Tylophora indica is a semi shrub climber and is the member of Asclepiadiaceae family. The leaves of this plant are the potential as anti-asthmatic agent. It has various phenanthroindolizidine alkaloids along with other non-alkaloid substances which are responsible for the pharmacological and antimicrobial potential of this plant. Authors have already reported a review for pharmacological and antimicrobial potentials of this plant in other chapters along with the standardisation of this herb^[7,8].

 

In the present work, the behaviour of different extracts of T. indica leaves towards selected food pathogens has been studied. Studies on different plants such as, Mangifera indica for their antimicrobial potential against few food pathogens such as S. aureus and E. coli have been reported but their role as food preservative is still not extensively evaluated. Also, the antimicrobial activity of T. indica has not been evaluated against L. monocytogenes which may be potential against this noxious food pathogen.

 

MATERIALS AND METHODS:

Materials:

T. indica plant was collected from the Ayushya Vatika, Lovely Professional University, and authenticated from the Department of Botanical and Environmental Sciences, GNDU, Amritsar with voucher reference no. 2350 dated 23/6/2017. Leaves were collected, dried, powdered, stored and characterised for the presence of different phytoconstituents along with their standardization as already discussed in the other chapter^[7,8].

 

Procurement and Maintenance of cultures:

Five indicator strains (three Gram positive and two Gram negative) used in this study were E. coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Listeria monocytogenes. The lyophilised culture strains of E. coli (MTCC1687) and Listeria monocytogenes (MTCC1143) were purchased from MTCC, Chandigarh and revived in Nutrient broth and Brain Heart Infusion (BHI) broth, respectively. S. aureus (MTCC 96), B. subtilis (MTCC 121) and P. aeruginosa (MTCC 4673) were obtained from the glycerol stocks maintained at -20^oC in the laboratory of Microbiology Department, Lovely Professional University, Punjab, India. Subsequently, they were revived in Nutrient Broth at 37^oC for 24 hrs. The working cultures of all the indicator bacterial strains were sub cultured from the broth by streaking on appropriate agar media and incubated at 37^oC for 24 hours. The cultured slants/plates were stored at 4±2^oC during the experimental trials and were refreshed periodically. To ensure long term preservation, 1 ml of the bacterial broth culture was added aseptically to 1 ml of 30% sterilised glycerol, in a 2ml cryo vial and thereafter stored at -20^o C.

 

Preparation of crude T. indica leaf extracts:

To each 15g of leaves powder, 200ml of each solvent (methanol, ethanol, double distilled water and ethyl acetate) was added in sterile 500ml Erlenmeyer flasks, appropriately covered with aluminium foil, and were kept for 48 h with 6 h of continuous shaking over a magnetic stirrer at 700rpm. It was then followed by filtration, decanting and evaporation of filtrate in a pre-weighed dried china dish at 45^oC. The thick syrupy mass was cooled and weighed with reconstitution of crude extracts in 100% dimethyl sulphoxide (DMSO) to prepare a stock solution of 200mg/ml. This prepared solution was stored in sterilised bottles at 4±2^oC for future use. Seven dilutions: 100mg/ ml, 50mg/ml, 25 mg/ml, 20mg/ml, 15mg/ml, 10mg/ml and 5mg/ml, were prepared using sterilised 5% DMSO from the stock solution (200 mg/ml) aseptically for the study^[9,10,11].

 

Evaluation of antimicrobial activity (Agar well diffusion assay) of leave extracts of T. indica against select test organisms:

25-30ml of sterilised Muller Hinton agar was poured in 90mm sterilised petri plates aseptically, and allowed to cool and solidify. A lawn of 18-24 h old indicator strains was prepared with sterile cotton swabs aseptically, followed by cutting of 8 mm wells with sterilised borer. 100μl of the test sample (extracts dissolved in DMSO) was added in each well and the plates were kept at 4^oC in the refrigerator to allow the diffusion of the samples in the media. Sterilised 5% DMSO was taken as the control. Thereafter, the plates were incubated at 37^oC overnight and zone of inhibition measured. The test has been performed in triplicates^[12].  

 

Table 1: Bacterial cultures used as indicator strains

Incubation temp: 37^oC; Incubation time: 24 h

MTCC: Microbial Type Culture Collection, Chandigarh, Punjab, India

ML, LPU*: Microbiology Laboratory, Department of Microbiology, Lovely Professional University, Punjab, India.

 

RESULTS AND DISCUSSION:

Procurement and Maintenance of cultures:

The five indicator strains were properly cultured on nutrient media and maintained for their use as indicator strains for anti-bacterial study (Table 1).

 

Preparation of crude T. indica leaf extracts:

Methanolic, ethanolic, aqueous and ethyl acetate crude extracts of T. indica leaves were prepared to evaluate their antimicrobial potential and the extractive values have been reported in Table 2. The prepared extracts were dissolved in dimethyl sulphoxide (DMSO) as the solvent to obtain a stock solution of 200mg/ml and stored in capped plastic bottles at 4^oC. Maximum extraction was determined with methanol (28%) as compared to ethyl acetate which was found only to be 5%. Subsequently, seven test concentrations were prepared aseptically from the stock solution, using sterilised 5% DMSO as the solvent in the sterilised test tube (Table 2). DMSO is a polar aprotic solvent and capacitates the dissolution of both non-polar and polar compounds. It is miscible in wide range of organic solvents and water. DMSO is one of the extensively applied extracting solvent and does not exhibit anti-microbial activity against organisms such as, S. aureus, B. subtilis and E. coli^[13,14].  Hence, DMSO has been used as the solvent for the obtained extracts, as also reported in other studies^[13,14,15].   

 

Table 2: Preparation of crude extractives of T. indica leaves

Crude powder drug (g): Solvent (ml) =15: 200

Concentration of stock solution used: 200mg/ml

Solvent used: Stock preparation: 100% DMSO; Working samples: 5% sterilized DMSO

 

Evaluation of antimicrobial activity (Agar well diffusion assay) of leave extracts of T. indica against select test organisms:

Four different extracts (methanolic, ethanolic, aqueous and ethyl acetate crude extracts) of T. indica leaves, prepared as above, were studied for their anti-bacterial potential against selected food pathogens (Table 3). With 5mg/ml concentration, the methanolic and aqueous extracts could inhibit S. aureus only with ZOI (mm) 9.3±0.5 and 9.6±0.4, respectively. Other two extracts were ineffective at this concentration towards any test strain. With 10mg/ml concentration, methanolic extracts were found to inhibit all indicator strains except, L. monocytogenes. E. coli was most sensitive strain to methanolic, aqueous and ethyl acetate extract with maximum ZOI was determined to be 11.3±0.5mm with aqueous extract. With 15mg/ml as the test concentration, most sensitive Gram-negative strain was E. coli where ZOI with ethyl acetate extracts was found to be 12±0.8 mm, while S. aureus was most sensitive amongst Gram positive with methanolic extracts where ZOI was determined to be 11.7±0.9mm. The ethanolic extracts were ineffective against any indicator organism up to 15mg/ml. With 20mg/ml as test concentration, maximum sensitivity was exhibited by E. coli (ZOI: 13.3±1.2mm) with ethyl acetate extracts. With 25mg/ml as the test concentration, maximum ZOI (13.7±0.4mm) was exhibited by E. coli (aqueous extracts) followed by S. aureus (12.7±0.5mm) with methanolic extracts. This concentration of ethanolic extract was a potential to inhibit L. monocytogenes (11.3±0.5mm) which other extracts were not able to exhibit. With 50mg/ml as the test concentration, most sensitive Gram-negative strain was E. coli where ZOI with aqueous extracts was found to be 16±0.8 mm, while S. aureus was most sensitive amongst Gram positive with methanolic extracts where ZOI was determined to be 15±1.4mm. With 100mg/ml test concentration, methanolic and aqueous extracts could inhibit all indicator strains with ZOI (mm) against E. coli, P. aeruginosa, S. aureus, B. subtilis and L. monocytogenes was exhibited to be 15.7±1.7, 16.7±0.9, 16±1.4, 14.3±0.5,  9.7±0.4 and 16.7±1.2, 13.3±0.4, 12.3±0.5, 12.7±1.2, 13.3±0.5 respectively. The only resistant strain to the ethanolic extract and ethyl acetate extract was S. aureus and P. aeruginosa, respectively. The sensitivity for Gram negative indicator organism was E. coli > P. aeruginosa; while for Gram positive organisms, it was S. aureus > B. subtilis > L. monocytogenes.

 

The methanolic crude extracts of T. indica leaves were reported to be effective against P. aeruginosa, S. aureus and B. subtilis whereas E. coli was found to be resistant using 50 to 1000μg/ml concentration^[16]. With similar extracts, E. coli was again found to be resistant with 1mg/ml concentration while inhibition was found against E. coli, P. aeruginosa and S. aureus with 10mg/ml and 50mg/ml.

 

In the same study, ethyl acetate extracts also exhibited same results^[17]. In another study, the aqueous and ethanolic extracts were reported to exhibit no activity against E. coli, P. aeruginosa and S. aureus while methanolic extracts were effective against only E. coli^[18]. Some studies have reported that the alcoholic extracts of T. indica leaves did not possess any activity against E. coli and S. aureus^[19,20]. The current study, does not comply with some of these result findings as our methanolic extracts showed biological activity against E. coli, P. aeruginosa, S. aureus and B. subtilis at 10mg/ml concentration. Interestingly, aqueous extracts were also found to be effective selectively at different test concentrations as cited earlier.

 

Table 3: Antimicrobial potential of different concentration of Tylophora indica leaf extracts against test organisms

Sample	Conc. (mg/ml)	Organism	Methanolic Extract	Ethanolic Extract	Aqueous Extract	Ethyl acetate Extract
			ZOI (mm)	ZOI (mm)	ZOI (mm)	ZOI (mm)
1	5	E. coli	Nil	Nil	Nil	Nil
		P.  aeruginosa	Nil	Nil	Nil	Nil
		S. aureus	9.3±0.5	Nil	9.6±0.4	Nil
		Bacillus subtilis	Nil	Nil	Nil	Nil
		L. monocytogenes	Nil	Nil	Nil	Nil
2	10	E. coli	10.7±0.9	Nil	9.7±0.9	10.6±0.9
		P.  aeruginosa	9.6±0.4	Nil	Nil	Nil
		S. aureus	10.3±0.5	Nil	10.3±0.5	9.3±0.4
		Bacillus subtilis	9.3±0.5	Nil	Nil	9±0
		L. monocytogenes	Nil	Nil	Nil	Nil
3	15	E. coli	11±1.6	Nil	11.7±0.9	12±0.8
		P.  aeruginosa	10±0.8	Nil	Nil	Nil
		S. aureus	11.7±0.9	Nil	10.7±0.4	10±0.8
		Bacillus subtilis	10±0.8	Nil	9.3±0.5	9.3±0.4
		L. monocytogenes	Nil	Nil	Nil	Nil
4	20	E. coli	11.7±0.9	Nil	12±0	13.3±1.2
		P.  aeruginosa	10.7±0.5	Nil	Nil	Nil
		S. aureus	12.3±0.5	Nil	11.3±0.5	10.3±0.5
		Bacillus subtilis	10.3±0.5	9±0	9.7±0.4	9.7±0.9
		L. monocytogenes	Nil	Nil	Nil	Nil
5	25	E. coli	11.3±0.5	Nil	13.7±0.4	13.3±0.5
		P.  aeruginosa	11.7±0.4	Nil	Nil	Nil
		S. aureus	12.7±0.5	Nil	11.3±0.5	10.3±0.4
		Bacillus subtilis	10.7±0.9	10.3±0.5	9.7±0.4	10.3±0.5
		L. monocytogenes	Nil	11.3±0.5	Nil	Nil
6	50	E. coli	14±1.4	Nil	16±0.8	13.7±0.5
		P.  aeruginosa	14.7±0.4	Nil	12.3±0.5	Nil
		S. aureus	15±1.4	Nil	11.7±0.5	11.7±0.9
		Bacillus subtilis	12.7±0.9	13.3±0.4	10.7±0.4	10.3±0.5
		L. monocytogenes	Nil	13±0.8	Nil	Nil
7	100	E. coli	15.7±1.7	14.7±0.5	16.7±1.2	15.3±0.4
		P.  aeruginosa	16.7±0.9	11±0.8	13.3±0.4	Nil
		S. aureus	16±1.4	Nil	12.3±0.5	14.7±0.9
		Bacillus subtilis	14.3±0.5	14±0.8	12.7±1.2	11.3±0.5
		L. monocytogenes	9.7±0.4	18.3±0.5	13.3±0.5	13.7±0.9
8	DMSO (Control)	E. coli	Nil	Nil	Nil	Nil
		P.  aeruginosa	Nil	Nil	Nil	Nil
		S. aureus	Nil	Nil	Nil	Nil
		Bacillus subtilis	Nil	Nil	Nil	Nil
		L. monocytogenes	Nil	Nil	Nil	Nil

·       Dilutions of plant extracts have been prepared in 5% Dimethyl sulphoxide

For antibacterial analysis: Well- Diffusion Method (Well size:8mm); Amount of sample used: 100 μl; Incubation period: 24h, Incubation temp: 37°C; Media used: Muller Hinton Agar (Himedia); pH: 7.3±0.1, for  L. monocytogenes: Listeria selective agar; pH: 7.3±0.2

 

No study has been conducted by T. indica leaf extracts against L. monocytogenes. This is the first report of such findings. The plant extracts exhibited low potential against L. monocytogenes. 100mg/ml of ethanolic extracts showed maximum zone (18.3±0.5) against this organism.

 

The crude alkaloid extract of T. indica leaves (which possess phenanthroindolizidine alkaloids such as tylophorine and tylophorinine) inhibit protein synthesis, cause leakage of proteins and sugars from the bacterial cell membranes and cause cell death^[21]. The performance of different extracts with same concentration, exhibited quite variation against tested strains. This may be because of the difference in solubility of active compounds with solvents of different polarity^[22]. The dose up to 200mg/kg was tolerated by male wistar rats during toxicity evaluation for methanolic extracts of T. asthmatica (synonym of T. indica) leaves. The toxic effects of the extracts were exhibited from 500mg/kg dose. Thereafter, the methanolic extracts were concluded to be mildly toxic in this study^[23]. Correspondingly, the human equivalent dose (HED) has been determined to be approximately 32.4mg/kg for methanolic T. indica leaves crude extracts ^[24]. Hence, the toxicity of T. indica leaves must be taken into consideration while applying this part of the plant.

 

CONCLUSION:

Immense international attention is being focussed on improvement of food hygiene, with global rise in food business and rapid increase in consumer awareness for food safety and hygiene. The control of food borne infections/illnesses owing to presence of microbial pathogens such as E. coli, P. aeruginosa, Campylobacter jejuni, Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus sp. and Listeria monocytogenes, in foods, play a key role in maintaining health of common population and needs urgent attention. The antimicrobial potential of leaves extracts of T. indica against common food borne pathogens open up great scope to add to the search of safer and effective natural food preservatives. Further studies on using T. indica leaves extracts in combinations with other efficient antimicrobials and subsequent development of food formulation packaging films need to be thoroughly investigated to explore full potential of the T. indica as a natural preservative. The authors are already investigating the extracts of T. indica leaves for their potential as preservatives. These potentials, as determined in this study, can be explored by not only the food industry for the bio preservation of food products, such as by their incorporation in packaging films, but also the health industry even as therapeutic agents against studied food pathogens. The authors may be looking forward to analyse the shelf life analysis of food using the extracts of T. indica leaves.

 

ACKNOWLEDGEMENT:

Authors are thankful to second International Conference of Pharmacy, held by School of Pharmaceutical Sciences, Lovely Professional University on September 13-14, 2019 to fund the publication of this manuscript.

 

CONFLICT OF INTERESTS:

The authors declare that there is no conflict of interests regarding the publication of this paper.

 

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Received on 19.11.2019           Modified on 06.02.2020

Accepted on 01.04.2020         © RJPT All right reserved