Introduction

Secondary metabolism produced by many plants such as herbs and spices contain products like phenolics, phenolic acids, quinones, tannins, and flavonoids. Most studies in drug discovery have examined on antimicrobial potential of natural products and medicinal plants measured as either killing the microbial growth or inhibiting them. Medicinal plants are still being used as major therapeutic agents in treating many pathogenic human diseases. Some confirm inhibition of peptidoglycan synthesis by plant-derived compounds, and also they damage microbial membrane structures. They also influence Biofilm formation.

The anti-microbial activity of ginger has been demonstrated in many works. Ginger decreases joint pain from arthritis, lowers blood cholesterol. The gingerols have antibacterial and analgesic properties and increase the motility of the gastrointestinal tract. In Iranian traditional medicine, Ginger (Zingiber Officinale) is used for the treatment of fever, menstrual pain, nausea, indigestion, and vomiting. It has also been used for the treatment of chest diseases, cough, sore throat, skin, kidney and bladder infections, constipation, and other digestive problems like dysentery and intestinal inflammations (Azadpour et al., 2016).

Two important spices derived from nutmeg fruit are nutmeg and mace. Nutmeg has many health benefits and has been used for stomach cramps and to cure the plague. It detoxifies toxins in the body, lowers blood pressure and soothes stomach-ache. Joint and muscle pain are treated due to their anti-inflammatory properties. It helps in relieving infections and is found to reduce respiratory problems like a cough that arises from a common cold.

The antibacterial effect of nutmeg is only towards the pathogenic bacteria leaving behind the normal flora unharmed. The 157 E coli strain is sensitive to nutmeg extract, whereas the non-pathogenic strains of E. coli are not. In addition, Streptococcus mutans, the bacteria that cause cavities, are killed by nutmeg extract. In work done by (Shazia, Fatima, Farhan, & Mustafa, 2015), the study showed the potential antimicrobial, antioxidant, and cytotoxic activity of Myristica fragrans extracts. They also found that the methanolic extracts have maximum activity and can be used as a therapeutic agent for the treatment of many diseases.

Materials and Methods

Preparation of plant extracts

The two organic solvents used for the extraction were methanol and chloroform. Methanolic and chloroform extracts of ginger and nutmeg were prepared. Nutmeg extraction was done using the maceration method. Nutmeg shells were broken, and seeds were taken. The seeds were crushed into fine powder.100g of nutmeg was weighed and mixed with 500mL of the solvent (methanol and chloroform) in a conical flask and was sealed with aluminum foil and allowed to stand for about 72 hrs in room temperature. After this, it was filtered.

The filtrate was kept in a water bath to evaporate the solvent, and the pure extract was obtained. Ginger was taken and washed well. 720g of ginger was weighed and kept in a hot air oven for drying. After drying, the weight was 120g. The extracts were obtained by soxhlet extraction, 60g of dried ginger in 200ml of methanol at 65^oC and chloroform at 62^ᴼC using soxhlet apparatus. The extract was then concentrated by keeping in a water bath to obtain 100% methanolic and chloroform extracts of ginger (Ibrahim, Naem, & Abd-Sahib, 2013).

Phytochemical analysis

The methanolic and chloroform extracts of ginger and nutmeg were subjected to phytochemical studies (Terpenoids, Flavonoids, Saponins, Tannins, Reducing Sugars and Phenols) using the standard method described by (Trease & Evans, 1989).

Fourier Transform Infrared analysis

FTIR analysis was done for the extracts to identify the functional groups present. FTIR was analyzed using spectrum R1. Software version, Report builder, Rev.2.01. Then the obtained FTIR patterns were compared with the standards (Ragavendran, Ragavendran, Sophia, Raj, & Gopalakrishnan, 2011).

NMR Spectroscopy

Nuclear Magnetic Resonance (NMR) is an analytical technique used for determining the molecular structure of a sample. The ginger and nutmeg extracts were analysed by using NMR Spectroscopy (Moore & Dalrymple, 1997). All samples were dissolved in 600µl DMSO-d6, vortexed (1 min), sonicated (5 min), vortexed (1 min) again, and centrifuged (13,200 rpm). The supernatant was transferred to a 5mm NMR tube. NMR spectra were recorded at 400 MHz and 600 MHz. Spectra were acquired in 8 minutes per sample (32 scans). NMR analysis was done at the Sophisticated Analytical Instrument Facility (SAIF), IIT, Chennai.

Isolation of biofilm causing organisms

The test organisms causing biofilm used for the study were (Escherichia coli and Pseudomonas aeruginosa.) isolated. In the current study, Pseudomonas (isolate A) was isolated from soil samples and E.coli (isolate B) from sewage water samples using suitable selective media under optimized conditions. The organisms were subjected to various identification techniques. The identification was made using the differential staining technique, i.e., Gram's staining (Cappuccino & Sherman, 2014).

Bacterial Identification

The isolated bacterial strains from the samples were identified up to generic level by employing the standard morphological and biochemical characteristics described in Bergey’s manual of systematic bacteriology. Biochemical tests were performed as per standard procedure and are tabulated in Table 4 (Prauser et al., 1985).

Congo red assay

The production of Extra polymeric substances (EPS) for biofilm formation by the isolates were analysed by using the Congo Red assay. The overnight culture of P. aeruginosa and E. coli was spotted on to Congo red plates and incubated at 37◦ C for 48 hours (Kim & Park, 2013).

Anti-microbial activity

The antimicrobial activity of the extracts was studied using the Agar Plate well method. The antibacterial effect of the extract was assayed by using the extracts at 5 different concentrations, i.e., 20 %, 40%, 60%, 80%, and 100%. The procedure involved dilution of the pure extract to 20%, 40%, 60%, and 80% with the respective solvent. The crude extract was used as such for 100%.

The extract of varying concentrations was prepared and stored in sterile tubes. Fresh culture of P. aeruginosa and E.coli were plated in nutrient agar plates as swab culture. For the antibacterial effect study, wells were made with a uniform diameter on the agar plates using cork borer equidistantly, and 100 µL of the ginger and nutmeg extract of the 5 different concentrations were added to the wells. Triplicates were done, and plates were kept for incubation at 37^◦ C for 48 hrs, after which the zone of inhibition was measured.

Anti-biofilm activity

Air liquid interphase coverslip assay

Fresh culture of P. aeruginosa and E. coli was transferred to nutrient broth media poured to sterile Petri plates. Coverslips were added to the two bacterial cultures. It was then incubated at 37◦ C for 24 hours. After incubation, 2 coverslips that serve as control were taken out from both bacterial plates. It was then stained with crystal violet for 1 minute and observed under oil immersion objective to check the presence of biofilm formation. The biofilm obtained was treated with ginger and nutmeg extracts of different concentrations (60%, 80% &100%) and incubated at 37◦ C for overnight. After incubation, the coverslips were taken out and stained with crystal violet and observed under oil immersion objective to check the action of extracts on biofilm formation (O’toole et al., 1999).

Biofilm inhibition assay

Fresh culture of P. aeruginosa and E. coli was taken, and 100 µL of each culture, along with the equal volume of fresh media, were transferred separately to sterile microfuge tubes. To these tubes, 0.5 µL of ginger and nutmeg extracts were added, and 1 tube of each bacterial culture served as the control without extract. It was then incubated at 37^◦C for 48hours. After incubation, 0.1 mL of crystal violet was added to all tubes and kept for 5 minutes, after which the stain was removed by using distilled water. The microfuge tubes were then allowed to dry, and the activity of ginger and nutmeg extracts were observed based on the color intensity of crystal violet on the microfuge tubes.

Results and Discussion

The methanol and chloroform extracts of ginger and nutmeg were extracted using soxhlet apparatus. Four different extracts a) ginger methanol (GM), b) ginger chloroform (GC) c) nutmeg methanol (NM), and d) nutmeg chloroform (NC) extracts. All the four extracts obtained were subjected to phytochemical analysis, and results were tabulated (Table 1). The four different extracts obtained show positive for terpenoids and saponins. In addition to that, the nutmeg methanol extract alone revealed positive for the presence of flavonoids and reducing sugar.

Zingiberol is the principal aroma contributing component of ginger rhizome (Ali, Blunden, Tanira, & Nemmar, 2008). The species contains biologically active constituents, including the non-volatile pungent principles, such as the gingerols, shogaols, parasols, and zingerone that produce a hot sensation in the mouth. The following phytochemicals like Flavonoids, tannins, phenols, steroids were present in 27 ºC, 60ºC, 70ºC, and 80ºC temperatures. It has increased the quality of the medicinal plants. But free amino acid, anthraquinones, and phytosterols were absent in four temperatures.

The FTIR pattern of ginger methanol extract shows (Figure 1) that all the characteristic functional group frequencies of the isolated ginger extract samples were found to be matching with the absorption frequencies of the standard (Table 2). The nutmeg chloroform extract’s FTIR pattern (Figure 2) was observed. All the characteristic functional group frequencies for the isolated nutmeg chloroform extract samples were found to be matching with the absorption frequencies of the standard (Table 3). The H^1-NMR pattern of the nutmeg chloroform extract was confirmed for the presence of a trimyristin-a bioactive compound present in nutmeg (Figure 3). The H^1-NMR pattern of ginger methanol extract was found very closely relevant to the standard NMR pattern of gingerol (Figure 4).

The main constituents of the oils of M. fragrans are found to be alkyl benzene derivatives terpenes, alpha-pinene, beta-pinene, phenylpropanoids like myristicin, elemicin, safrole and fatty acids Trimyristin, myristic acid, tripalmitin, etc. Trimyristin proved to possess anxiogenic activity, the principle reason for using the herbal drug in various polyherbal formulations. Apart from this, it also has other activities like anti-inflammatory and anti-bacterial. (Baskaran, Ratha, & Kanimozhi, 2011), studied the effect solvent on the phytochemical composition of extracts of M. koenigii leaves. And reported that it is dependent on the nature of the solvent.

The broad peak at 3265 cm-¹ is because of the O-H stretching of a hydroxyl group, which indicates the presence of polyhydroxy compounds like flavonoids, non-flavonoids, and saponins (Rastogi & Arunachalam, 2011). The peak at 2926 cm^-1 is due to the asymmetric stretching of C-H groups of aromatic compounds. The peak at 1619 cm-¹ is corresponding to C=O stretching of peptide linkages C=O stretching of carbonyl and carboxylic groups. The peak at 1395 cm-¹ indicates the O-H bend of carboxylic acids, which in turn revealed the presence of flavonoids, tannins, saponins, and glycosides. The peak at 1036 cm-¹ indicates the S=O group revealed the presence of organosulfur compounds, including alliin, allicin, and diallyl disulfide.

The alcoholic solvents such as ethanol were more capable of the extraction of bioactive materials of mallow, but the presence of water was one of the reasons for the lack of antimicrobial effect of the hydro-alcoholic extract. Mallow contains phenolic compounds, anthocyanins, carotenoids, and vitamin E, which are antioxidant substances, are active ingredients of ginger (Zingiber Officinale) in introducing bioactive antibiotics for microbiology and pharmacology sciences. (Arani, Chaleshtori, & Rafieian-Kopaei, 2014) reported the mechanism actions of these plants or their components should be established and noted that phenolic compounds substantially possess antimicrobial activities.

The ethyl alcohol extract of nutmeg had antibacterial activity against the enteropathogenic E. coli. (Indu, Hatha, Abirosh, Harsha, & Vivekanandan, 2006) found that aqueous nutmeg extract had antibacterial activity against different serotypes of E. coli, Salmonella spp., Listeria, and Aeromonas hydrophila. The aqueous and ethanolic extracts of nutmeg were effective against both E. coli and Staphylococcus aureus.

Pseudomonas was obtained from soil samples and was isolated by plating on to cetrimide. Cetrimide agar is a selective medium used for the selective isolation of Pseudomonas aeruginosa. E.coli was obtained from sewage water samples. The isolation was done by plating onto Eosin methylene blue agar (EMB). It is a differential medium for the isolation of fecal coliforms. Escherichia coli and Pseudomonas aeruginosa, the strains used for the biofilm analysis, were isolated and identified on its physiological and biochemical characteristics (Table 5; Table 4).

After 48 hours of incubation, the colonies of the isolates causing biofilm on the Congo Red agar plate showed rugose colony morphology (Figure 5), which indicates the production and presence of extracellular polymeric substances (EPS) by the isolates. The anti-microbial activity of the plant extracts on Pseudomonas aeruginosa was carried out by the Agar Plate well method. It was found that the ginger chloroform extract showed a zone of inhibition in all five concentrations (20%, 40%, 60%, 80%, and 100%) (Table 6). 100% extract proved effective inhibition.

The methanolic ginger extract showed inhibition only in concentrations 60%, 80%, and 100%. The nutmeg chloroform extract showed inhibition in all concentrations except 20%. Here also 100% extract was found to be effective against bacteria. The methanolic nutmeg extract showed inhibition only in concentrations 60%, 80%, and 100%. Among these, 100% showed the maximum zone of inhibition. The anti-microbial activity of the plant extracts on Escherichia coli was also conducted by the Agar Plate well method. It was found all four extracts showed a zone of inhibition in 3 concentrations (60%, 80%, and 100%) and no inhibition in 20% and 40%. (Table 7).

All the four extracts showed maximum inhibition in 100% concentration. Among the four extracts, the nutmeg chloroform extract was found to be highly effective, showing the maximum zone of inhibition in 100%. From the results of antimicrobial activity, maximum inhibition on the growth of Pseudomonas aeruginosa was shown by the methanolic ginger extract. The air-liquid interphase coverslip assay was conducted in concentrations of 60%, 80%, and 100% of ginger methanolic extract (GM) and compared with control without extract. Biofilm reduction was observed in all 3 concentrations (60%, 80%, and 100%) and compared with the control (Figure 6).

From the results of the antimicrobial activity of extracts on Escherichia coli, the maximum zone of inhibition was shown by the nutmeg chloroform extract. The air-liquid interphase coverslip assay was conducted in concentrations of 60%, 80%, and 100% of a nutmeg chloroform extract (NC) and compared with control without extract. The reduction in biofilm was observed in all the 3 concentrations of the nutmeg chloroform extract with maximum biofilm reduction in 100% and was compared with the control (Figure 7). The biofilm inhibition assay was conducted with all the 4 extracts (GM, GC, NM, and NC) in 3 different concentrations (60%, 80%, and 100%) and compared with the control without extract. For both Pseudomonas aeruginosa and Escherichia coli, biofilm inhibition was observed in all 3 concentrations.

This study was done to check the inhibitory action of the ginger and nutmeg extracts on P.aeruginosa and E.coli biofilm formation. From this study, it was observed that as the extract concentration increases, the ability of organisms to form biofilm decreases.

The bacterial strains which created microcolonies on the glass surface were characterized by a different level of adhesion to polyurethane. The mechanism of biofilm formation may be connected to other features of the bac­terial strains. The relationship between biofilm formation and surface (polyurethane, glass) may be related to the hydrophobicity of the environment surface and outer membrane of a particular strain.

Probably, hydrophobic bacteria will attach better to hydrophobic polyurethane and vice versa. (Stoodley, Dodds, Boyle, & Scott, 1998), studied the biofilm formation of E.coli. (Ugurlu, Yagci, Ulusoy, Aksu, & Bosgelmez-Tinaz, 2016) analyzed, P. aeruginosa has the capacity to form a biofilm that works as a penetration barrier for antimicrobials. Significant reductions in biofilm formation in clinical P. aeruginosa isolates were observed in the presence of tested phenolic compounds. The addition of cinnamic acid, ferulic acid, and vanillic acid reduced biofilm production by 44%, 45%, and 46%, respectively, compared to the control. Methanolic extract of all the isolates was used to test antagonistic activity against pathogenic bacteria. (Nikolic, Vasic, Djurdjevic, Stefanovic, & Comic, 2014) observed that Z. officinale ethanolic extract has proved on a wide range of bacteria. It can be used in food.

The investigation of ginger has confirmed its significance, especially in the area of influence on tested Staphylococci, where the result achieved was much better in comparison with the previous investigations, and ginger extract had an effect against food spoilage organisms. The compound extracted and isolated from the methanol extract of the bark of M. cinnamomea also exhibited anti-QS. Reduction of bioluminescence in E. coli and Pseudomonas aeruginosa suggested anti-QS properties.

Conclusion

In the present study, two different plant species (Zingiber officinale, Myristica fragrans) were used for studying their antimicrobial and antibiofilm activity. Four different extracts were used, which includes the methanolic and chloroform extract of ginger and nutmeg, respectively. The ginger extracts were prepared using soxhlet extraction, and nutmeg extracts were prepared using the maceration method. The phytochemical analysis of the plant extracts was done by using standard methodology. FTIR analysis of the ginger and nutmeg extracts were carried out to study the functional groups present in the respective plant extracts. The extracts were also subjected to NMR spectroscopy.

Two different bacteria were used for the studies, which are good biofilm producers. Biofilms are produced by bacteria to survive in nature. Biofilm causing bacteria were isolated from a soil sample, and sewage samples wherein Pseudomonas aeruginosa were isolated on selective cetrimide agar media and Escherichia coli were isolated on EMB agar media. The bacterial isolates were subjected to standard morphological and biochemical characterization described in Bergey’s manual of systematic bacteriology. The antimicrobial activity of the methanolic and chloroform extracts of ginger and nutmeg were studied in the present work. It was found that the ginger methanol extract showed maximum zone of inhibition in Pseudomonas aeruginosa, while the nutmeg chloroform extract showed maximum zone of inhibition in Escherichia coli. The antibiofilm activities of the extracts on bacterial biofilm were studied using air-liquid interphase coverslip assay and biofilm assay. The results suggest that the ginger and nutmeg extracts have biofilm inhibition activity. This has a potential future application in various fields, which includes the pharmaceutical and food industries.