Introduction

Nowadays, upper respiratory tract infection (URTI) is one of the common and acute problems among the common population and causes significant morbidity (Adeshina et al. 2014). Mostly the upper respiratory tract infections are self-limited and mild. A variety of bacteria can cause URTI (upper respiratory tract infections) which refer to infections such as laryngitis, tonsillitis, sinusitis, pharyngitis, otitis media, and common cold (Passioti et al. 2014). Some common symptoms of respiratory tract infections (RTIs) include sore throat, sneezing, coughing, headaches, fever, and nasal congestion.

Respiratory tract infections are responsible for more than 50 million deaths worldwide. The main reasons behind the high death rate are malnutrition, clinic visits, and poor immunity. Though the antibiotics treatment brings relief from bacterial infection but, over some decades, the increasing number of antibiotic-resistant bacteria is becoming a global problem (Kotwani and Holloway 2014).

To overcome these issues, researches have been focused on natural herbal supplements and medicines. More focused is on those natural antimicrobial agents who show the least adverse effects on human health (Mandal et al. 2010). Treatment by using traditional herbal medicine or plant extracts is increasingly acknowledged and accepted by the common population of both developing as well as developed countries (Bardia et al. 2007). However, the uses of herbs for the treatment of respiratory infections are common in some countries (Chen et al. 2007; Said et al. 2002). Therefore in this study, five different extracts, including Glycyrrhiza glabra, Vitis vinifera, Terminalia chebula, Camellia sinensis, and Terminalia arjuna, were screened for their possible antibacterial activity against some upper respiratory infection causing bacteria.

Materials and methods

Plant extract collection

Standardized powdered extract of Terminalia chebula, Camellia sinensis, Glycyrrhiza glabra, Terminalia arjuna, and Vitis Vinifera were obtained from Saamir international Pvt. Ltd. (Delhi, India) and K. Patel Phyto Extractions Pvt. Ltd. (Gujarat, India). The powdered extracts were properly dissolved in an aqueous solvent and kept at -4 ºC for further use.

Bacteria and growth condition

The bacterial strains of Streptococcus pyogenes (MTCC 1927), Haemophilus influenza (MTCC 3826), Staphylococcus aureus (MTCC 96) were procured from MTCC and were grown in brain heart infusion broth and nutrient broth media at 37 °C, pH 7.0. Media, including NB, BHI was procured from HI media.

MIC (Minimum Inhibitory Concentration)

The MIC (Minimum Inhibitory Concentration) of five different extracts against Streptococcus pyogenes, Haemophilus influenza, and Staphylococcus aureus was investigated by using the broth dilution method as per NCCLS, USA, 2006 guidelines. Streptococcus pyogenes, Haemophilus influenza, and Staphylococcus aureus bacterial culture (OD 600 nm = 1) were grown at 37 °C for 24 h in the Mueller-Hinton broth with or without extracts in 96 well plates. The concentration at which the bacterial growth was found completely inhibited was noted as the MIC for the extracts (Kalia et al. 2020).

Antibacterial activity (cut-well diffusion method) of extracts

In brief, the bacterial inoculums were spread on the prepared agar plate surface by using a sterile glass spreader. Then, aseptically a well is cut with the help of a sterile tip, and extracts sample (100 µL) is placed into their respective wells. Further, these agar plates are incubated at 37 °C for 24 h. The clear effective inhibition halo zone around the well was measured, showing the extracts antimicrobial activity against different strains. The diameter of the clear zone was noted in mm (Kalia et al. 2020).

Animals

Healthy adult male rats were kept in the animal house of the United Institute of Pharmacy. Standard feed and water were provided ad libitum throughout the experimental period. The experiments were performed following the Institutional Animal Ethics Committee (IAEC) formed as per the direction of the CPCSEA Government of India, New Delhi. The research protocol was approved by the Institutional Animal Ethics Committee (IAEC) (UIP/IAEC/Nov.-2019/06) dated 15/12/2019, United Institute of Pharmacy (Allahabad). All the tests were performed during the light period (0800:1600 h).

Acute oral toxicity studies

The extracts acute oral toxicity was conducted as per the OECD (Organization for Economic Co-operation and Development) test guidelines 423 (OECD 2001). Single oral doses of aqueous extracts 5 mg/kg, 50 mg/kg, 300 mg/kg, and 2000 mg/kg body weight were administered to the overnight fasting rats (three animals per dose) except the control group rats. During 14 days of observation, the different wellness parameters, including mortality, skin and fur, body weight, food and water intake, diarrhea, hair loss, mortality, and behavioral pattern changes, were observed (Lipnick et al. 1995).

Statistical analysis

The analysis was performed in triplicate and expressed as average ± standard error. All the statistical analysis was performed by following ANOVA using Prism5. At p ≤ 0.05, the significance value of the data was determined.

Results

The description related to the extracts are presented in Table 1.

Table 1 Showing the detailed description of the selected extracts used in the study
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Antibacterial activity (cut-well diffusion method) of extracts

The antibacterial activity of the extracts against the bacteria was calculated by measuring the clear halo zone around the cut wells. The antimicrobial activity of the different extracts was examined against Streptococcus pyogenes, Haemophilus influenza, Staphylococcus aureus, and a maximum clear halo inhibitory zone of 22.6 mm of Terminalia arjuna extract were observed against Staphylococcus aureus. The minimum inhibition zone of 10.3 mm of Terminalia chebula extract was observed against Haemophilus influenza (Fig. 1).

Fig. 1
figure 1

Antibacterial activity by cut-well diffusion method

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MICs (minimum inhibitory concentrations)

The MICs (minimum inhibitory concentrations) were calculated by using the microdilution method in 96 well microplates. According to the obtained result, the lowest MIC (0.8 mg/ml) was found for Camellia sinensis against the Streptococcus pyogenes and Staphylococcus aureus (Table 2).

Table 2 Minimum inhibitory concentration value of different extracts
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Acute oral toxicity

When the single oral dose of herbal extracts including Terminalia chebula, Camellia sinensis, Glycyrrhiza glabra, Terminalia arjuna, and Vitis Vinifera was orally administered to rats (n = 5), no significant changes were observed during 14 days of observations (Table 3).

Table 3 The effect of doses on the animal’s physical parameters during ld50 analysis
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Discussion

Upper respiratory tract infections (URTIs) are the most common problem among the general population (Goossens et al. 2005). Bacteria, including Streptococcus pyogenes, Haemophilus influenza, Staphylococcus aureus, are usually responsible for causing upper respiratory tract infections. Many times antibiotics are prescribed for the treatment of respiratory tract infections. However, bacteria developing resistance against antibiotics are the major threats for public health worldwide (Vervloet et al. 2016). Plant extracts contain the phytochemical compounds that employ the therapeutic effects on health and pose antimicrobial activity (Heinonen 2007). Herbal remedies have started gaining greater attention in this modern era than ever before, just because of their easy availability, least side effects, efficacy, and low cost when compared with the market available drugs (Mohan et al. 2011).

Respiratory tract infection was selected in this study because it is a common disease, especially among children, and is directly correlated with air pollution and industrialization. S. aureus causes severe bacterial pneumonia, secondary disease, to viral infections, which causes widespread mortality and morbidity (Kumar et al. 2009). H influenza infection causes swelling of the epiglottis to ensue and progressive erythema. S. pyogenes infections normally take the form of fever (rash), pharyngitis, scarlet, and impetigo (Wong and Yuen 2012). This study aims to screen the antimicrobial activity of five different herbal extracts: Terminalia chebula, Camellia sinensis, Glycyrrhiza glabra, Terminalia arjuna, and Vitis Vinifera against some upper respiratory infection causing bacteria.

MIC determination is a simple and easy method for calculating the inhibitory doses of herbal extracts (an antibacterial agent) against the particular bacteria. All five extracts showed successful bacterial growth inhibition. On the other side zone of inhibition was calculated to determine the antibacterial activity of the different extracts. All the extracts showed antibacterial activity (Kalia et al. 2020; Chan et al. 2011; Pradhan and Dubey 2020). The maximum zone of inhibition was shown by T. arjuna against S. aureus (22 mm) and H. influenza (21 mm), followed by V. vinifera against S. pyogenes (20 mm), H. influenza (21 mm), and S. aureus (18 mm) and T. chebula against S. pyogenes (21 mm). Simultaneously, a minimum inhibitory zone of T. chebula was observed against H. influenza (10 mm) and S. aureus (14 mm). The zone of inhibition result suggests that the extract contains bioactive compounds that will directly affect the S. pyogenes, H. influenza, and S. aureus destruction (Adeshina et al. 2014; Bardia et al. 2007; Kaushik 2019; Pradhan and Dubey 2020).

Further, the in vivo acute oral toxicity of the five extracts was analyzed following the OECD 423 guidelines. The results showed that all the five extracts are found non-toxic when given up to the dose of 2000 mg/kg. During 14 days of acute oral toxicity study, no mortality, no signs of toxicity, and no behavioral changes were observed (Lipnick et al. 1995; Limsuwan et al. 2020).

Conclusions

Herbal extracts are the potent antibacterial and can be used to decrease the antibiotic resistance problem. However, the use of CAM (complementary and alternative medicine) therapies, including herbal extracts, herbal formulations, could reduce prescribing different antibiotics in the condition when there is no clinical indication, like viral infections. The five different extracts screened in the study have the potential and can be used as an alternative remedy for treating upper respiratory infection. In the future, these results may be considered for in vivo and clinical analysis.