Effectiveness of Aqueous extract of Green, Black and Red Tea Leaves against some types of Gram positive and negative bacteria

 

Dr. Nada Khazal Kadhim Hindi¹*, Iman Fadhil Abdul-Husin², Dr. Zainab Khudhur Ahmad     Al-Mahdi¹, Ruqaya M. J. Ewadh³, Ahmed Obaid Hossain⁴, Mohanad Jawad Kadhim⁴,

Ali Hussein F. Alnasraui⁴, Amean A. Al-Yaseri⁵

¹Basic and Medical Science Department, College of Nursing, Babylon University, Babylon Province, Iraq

²College of Biotechnology, Al-Qasim Green University, Babylon Province, Iraq

³DNA Research Center, Babylon University/ Iraq

⁴College of Biotechnology, Al-Qasim Green University, Babylon Province, Iraq

⁵College of Nursing, University of Babylon, Babylon Province, Iraq

 

ABSTRACT:

Objective: An evaluation for Antibacterial activity of aqueous extract of Green, Black, Red, and Tea Leaves against gram positive bacteria Staphylococcus aureus, Staphylococcu sepidermidis, Streptococcus pneumonia, Streptococcus feacalis, Streptococcus mutanus, Streptococcus feacalis and gram negative bacteria Escherichiacoli, Salmonella typhi, Moraxalliacatarralis, Pseudomonas aeroginosa, Proteus mirabilis, Klebsiella pneumonia, Enterobacter spp. Acinetobacter, Serratia spp.

Methods: Three types of aquatic extract of green, black and red tea leaves were used to determine the antibacterial activity. Agar well diffusion method was used in this study.

Results: All three types of tea aquatic extract exhibited excellent and best antibacterial activity against both gram positive and gram negative bacteria and show good  inhibition zone and better than the  effect of ciprofloxacin against different types of bacteria.

Conclusion:  aqueous extract of Green, Black, Red, and Tea Leaves show high antibacterial activity against both gram positive and gram negative bacteria, therefore most types of tee can provide protection to a certain extent against our natural enemies like bacterial pathogens.

 

 

 

 

INTRODUCTION:

The most popular drink world-wide “tea” is produced from Camellia sinensis plant[1] with four varieties; white, green, Oolong, and black, Oolong tea from partially fermented leaves; while black tea from fully fermented leaves.

 

From buds or very young tea leaves; white tea is produce; while green tea is made from mature unfermented leaves [2]. Red tea or Rooibos tea is naturally caffeine free making it a good choice for breastfeeding or pregnant women, red tea contain high number of antioxidants[3]. Camellia sinensis plant is a safe, nontoxic, cheap and available traditionally in Asian countries. These properties make it a very good alternative antimicrobial agent [4]. Due to rich content of phenolic compounds in Camellia sinensis leave has the preventive potential for esophageal, colon, lung cancers, urinary infections and dental curie. These polyphenols may account for as much as 30% of the dry weight of fresh tea leaves in green tea, while black tea contains between 3 and 10 percent[5]. There are many compounds in green tea have broad spectrum of biological activities such as antifungal, and antitumor activity and antioxidant functions[5]. Four polyphenol compounds, are present in tea extract according to the previous studies, Epigallocatechingallate (EGCG), Epicatechingallate (ECG), Epigallocatechin (EGC) and Epicatechin (EC) are significant antioxidants constituents. EGCG is the most luxuriant component in tea extract and the most potent chemical tested for biological activity[7,34]. Aim to determined antimicrobial activity of tea extracts leaves against commonly isolated pathogens(Gram positive and negative bacteria) from hospitalized patient with intestinal ailments, blood and skin infections. These bacterial isolates include Gram negative bacteria: Salmonellatyphi, Salmonella typhimurum, Pseudomonas aeroginosa, Pseudomonas fluroscences, Proteus merabilis, Proteus vuligaris, Klebsiella pneumonia, Enterobacter spp. Acinetobacter, Escherichia coli, Serratiaspp and Gram positive bacteria: Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pyogenes, Streptococcus pneumonia, Streptococcus mutanus, Streptococcus fecalis, Streptococcus agalagtia

 

MATERIALS AND METHODS:

Preparation of Aqueous extracts:

Tea leaves were collected from a retail food store (Al- Hilla) 2016. Then aqueous extract (Green, Black and Red Tea Leaves) was soaked in fifty gram of powder by 100 ml distillated water, and allowed to stand for 72 hr and sterilized by filtration (using Millipore 0.45 filter paper). This extract was considered as fifty percent (50%) of the extract concentration[8].

 

Bacterial Isolates :

A total of 11 Gram negative, and 8 Gram positive (isolated from clinical samples) were used in this study. The bacterial isolates represented by; S. aureus, S. epidermidis, S. pyogenes, E. feacalis, S.pneumoniae, P. aeruginosa, P. fluresence, E. coli, S. typhi, E. aerugenes, K. pneumoniae, Proteus mirabilis, P. vulgaris, Acinetobacter. These bacteria were activated and cloned three successive times in nutrient agar and stored on nutrient agar slant at 4ºC. Conventional biochemical tests were used for identification of these organisms[9].

 

Antibacterial activity assay:

Agar disc diffusion method was used for determination antibacterial activity[9]. Agar plates were inoculated with 0.1 ml broth culture of tested organisms, the bacterial inoculum was spread bya sterile an L-shaped rod glass spreader. Ciprofloxacin were disk was used as a positive control and was plated in the center of agar plate. (The plates were performed in triplicates). All plate of the tested organisms were allowed for overnight incubation at 37°C. After one day (24 h)incubation, each extract was screened their inhibition zone for all isolates. The zone of inhibitions was measured by measuring the diameters of scale in millimeter (mm).

 

In vitro Antimicrobial activity testing using Agar well diffusion assay:

Loopfull bacterial isolates growths were inoculated into nutrient broth tube and incubated at 37°C for 18 hours. Normal saline was used for preparation of bacterial suspensions and adjust the turbidity and compare with standard tube (McFarland number 0.5) to yield a uniform suspension containing 1.5×10⁸ CFU / ml. Dip cotton swab into adjustment suspension and streak the entire Mueller-Hinton agar, all plates were left for one 5 -15 minutes at room temperature to dry. Media were cut into four wells (5mm diameter) by cork borer and add 0.1ml of the extracts. The plates were incubated at 37°C for overnight. Zone of inhibition was measured from the edge of well to the edge of inhibition of growth [10, 35.36, 37].

 

RESULT AND DISCUSSION:

The inhibition zone diameter of tested bacteria were illustrated in Fig 1 -3, which reveals that the aqueous extract of (Green, Black and Red Tea Leaves) have been observed that they have been showed potential antimicrobial activity which was varied based on its effects because their activity affect by their concentration and may depending on the species of bacterial isolates. This activity may due to its content of alkaloids, and flavonoids [11].

 

Concerning aqueous extract of black tea antimicrobial activity has been reveals in figure(1);

 

The highest antimicrobial activity of black tea were against Streptococcus species; in Streptococcus agalagtia was with inhibition zone (33)mm then Streptococcus pneumonia, Streptococcus mutanus with (30)mm inhibition zone and Streptococcus fecalis was (28)mm.

 

This may related to the high content of phenolic compounds in black tea[13].These compounds cause cell death by degrading the cell wall (disrupt the cytoplasmic membrane and interfere with membrane-integrated enzymes) or by damaging the proteins of cell wall[14].

 

Figure (1): Antibacterial activity of black tea against bacterial isolates\

 

Hamdi et al’s found that black tea has a preventive effects on biofilm formation of Streptococcus mutans bacteria [15]. Other study showed that the antibacterial activity of black tea is not extensive [16]. Aqueous extract of black tea aqueous extract was also exhibits high activity against tested staphylococcus species: Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus with inhibition zone (28, 30, 30)mm respectively. The most sensitive bacteria was Staphylococcus aureus to the addition of tea extracts to the media[16]. The aqueous extract has less activity against P. aeruginosa and E. coli. Proteusmerabilis Proteus vuligaris, Klebsiella pneumonia, Enterobacter spp, Acinetobacter and Serratia spp. These are Gram-negative bacteria which typically resistant to antimicrobial agents than Gram-positive bacteria. This has been explained by the structure of the outer membrane permeability barrier in Gram-negative bacteria, which limits access of the antimicrobial agents to their targets in the bacterial cells. About of 42.19% of S. typhi was inhibited by this extract [18];Other study reported that black extract had a good activity against P. aeruginosa and E. coli.[19]. Concerning aqueous extract of red tea antimicrobial activity has been reveals in figure(2);

 

Figure (2): Antibacterial activity of red tea against bacterial isolates

 

The highest antibacterial activity of aqueous extract of red tea was against Streptococcus fecalis, Staphylococcus saprophyticus with (30 mm) inhibition zone; followed by Streptococcus agalagtia, Acinetobacter and Streptococcus mutanus (28mm) inhibition zone. This finding was compatible with other study reported by (20) that Rooibos extracts possess antimicrobial activity against Escherichia coli, Staphylococcus aureus and Streptococcus mutans in liquid culture.

 

Red tea is wealthy with polyphenols and flavonoids which useful resource inside the discount of cellular damage due to loose radicals that may weaken herbal defenses and subsequently result in ageing and the onset of sickness; Pholyphenols as catechin act on a few bacterial lines belonging through producing hydrogen peroxide [21] and through changing the permeability of the microbial membrane [22]. Microbes careworn by way of publicity to polyphenols upregulate proteins related to protecting mechanisms, which protect cells whilst concurrently down regulating diverse metabolic and biosynthetic proteins involved, for instance, in amino acid and protein synthesis in addition to phospholipid, carbon, and energy metabolism [23]. Furthermore, polyphenols were interfere with bacterial quorum sensing, the production of small signal molecules by way of bacterial cells of Escherichia coli, Pseudomonas putida and Burkholderiacepacia that cause the exponential increase of a bacterial population[24]. However, unclear whether or not the antimicrobial pastime of the complicated rooibos extract is due to the polyphenols alone or to a few different element of the extracts.

 

Many studies conducted the antimicrobial abilities of green tea; Chou et al.[25], proven that green tea, specifically, are capable of killing bacteria since green tea demonstrated higher overall performance against Escherichia coli and Salmonella. Inexperienced tea’s effectiveness as an antimicrobial agent can in component be attributed to its low degree of fermentation. At some point of the fermentation manner, catechins, which includes EGCG are destroyed, decreasing the tea’s antimicrobial activity. This may be definitely seen inside the consequences of the take a look at, as the noticeably fermented black tea killed the least microorganism in almost every case[25].

 

Figure (3): Antibacterial activity of green tea against bacterial isolates

 

The properties of inexperienced green tea which inhibit bacterial boom are in particular associated with their polyphenolic components which include epicatechin, epicatechingallate, epigallocatechin, and epigallocatechingallate against numerous Gram-positive and Gram-negative bacteria[27]. Similarly to the antimicrobial effects of mentioned catechins (damage to the bacterial mobile membrane, inhibition of fatty acid synthesis, inhibition of enzyme activity, etc.), there are some consequences that could make contributions to the full antimicrobial impact in infected individuals. These outcomes consist of inhibition of irritation (mainly infection caused by oxidative pressure, which include vascular), extra specifically, with the aid of growing the synthesis of nitric oxide [28]. Inhibiting angiotensin II and IL-6 brought on c-reactive protein expression [29]., suppression of IL-6 and RANK manufacturing in infected osteoblast-like cells [30]., inhibiting IL-8 manufacturing [31], and inhibiting hyaluronidase hobby (activated by continual irritation) by inhibition of IL-12 [32].

 

In the present work we also compare between the result obtained by tested extract (Green, Black, Red, Tea Leaves and Mentha) and results obtained by ciprofloxacinantibiotic (as most traditional treatment for infection caused by tested bacteria) concerning their antimicrobial activity against tested bacteria;

 

Figure 4: effect of ciprofloxacin against different types of bacteria.

 

All three types of tea aqueous extracts showed more activity as antimicrobial agent against tested bacterial isolates compare to ciprofloxacin according to inhibition zone diameter and even antibiotic resistant strains(Pseudomonas aeroginosa, Pseudomonas fluroscences, Klebsiella pneumoniae) and less sensitivity bacteria to ciprofloxacin (Enterobacter spp., Proteus mirabilis Proteus vuligaris, Staphylococcus aureus, Staphylococcus epidermidis). These bacterial strain is a mainly multi-drug resistant bacteria that are most commonly found, especially through nosocomial infections [33].

 

Present study concludes that the tea extract can be controlled growth of ciprofloxacin resistance bacterial strains which involved in this study within vitro condition.

 

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Accepted on 29.05.2017        © RJPT All right reserved