3.1 Extraction of A. sativum clove juice
The A. sativum clove juice was prepared using different solvents (aqueous, methanol and ethanol) and filtered using Whatman filter paper (Figure 1a, 1b, 1c and 1d).
3.2 Phytochemical analysis
The preliminary qualitative phytochemical analysis of A. sativum clove juice with and without skin was performed. The preliminary screening tests may be useful in detection of bioactive principles and subsequently may lead to drug discovery and development.
The phytochemical analysis of the current study revealed the presence or absence of phenol, alkaloids, steroids, cardiac glycosides, flavonoids, saponins, phlobatannins, volatile oil and glycosides (Table 2; Figure 2 – 10)).
3.3 Gomutra analysis to confirm the absence of microorganisms
The gomutra culture showed no presence of any bacteria or fungi. Also the urine analysis showed the absence of bile salts and pigments (Figure 11).
3.4 Agar well diffusion assay
3.4.1 Antifungal assay
The results of the antifungal assay reveal that the extracts possessed antifungal properties in various degrees on test organisms (Graph 1 and 2).
220.127.116.11 A. sativum extract without skin:
Zone of inhibition was observed in all solvent extracts but the aqueous A. sativum extract showed the maximum zone of inhibition against both A. niger (17mm) and Penicillium sp (14mm) (Table 3a and 3b). The aqueous, methanolic and ethanolic A. sativum extract without skin when mixed with FCU showed 15, 15 and 16 mm zone of inhibition against A. niger, respectively (Figure 9a). The aqueous, methanolic and ethanolic A. sativum extract without skin when mixed with FCU showed 16, 1 and 1 mm zone of inhibition against Penicillium sp respectively (Figure 10a and 11a). There was no zone of inhibition with FCU against both the tested fungi.
18.104.22.168 A. sativum extract with skin:
Zone of inhibition was observed in all solvent extracts but the aqueous A. sativum extract showed the maximum zone of inhibition against both A. niger (18mm) and Penicillium sp (17mm) (Table 3a and 3b). The aqueous, methanolic and ethanolic A. sativum extract with skin when mixed with FCU showed 16, 14 and 1 mm zone of inhibition against A. niger respectively (Figure 9b). The aqueous, methanolic and ethanolic A. sativum extract with skin when mixed with FCU showed 16, 1 and 1 mm zone of inhibition against Penicillium sp respectively (Figure 10b and 11b). There was no zone of inhibition with FCU against both the tested fungi.
3.4.2 Antibacterial assay
The results of the antibacterial assay reveal that the extracts possessed antibacterial properties in various degrees on test organisms (Graph 3 and 4).
22.214.171.124 A. sativum extract without skin
Zone of inhibition was observed in all solvent extracts but the aqueous extract without skin showed the maximum zone of inhibition against both Pseudomonas sp (17mm) and S. aureus (14mm) (Table 4a and 4b). When the aqueous, methanolic and ethanolic A. sativum extract without skin was mixed with FCU, it showed 18, 1 and 4 mm zone of inhibition against Pseudomonas sp respectively (Figure 15a). Only the aqueous A. sativum extract without skin mixed with FCU showed 13 mm zone of inhibition against S. aureus (Figure 16a). There was no zone of inhibition with FCU against both the bacteria.
126.96.36.199 A. sativum extract with skin
The aqueous A. sativum extract with skin showed the maximum zone of inhibition against both Pseudomonas sp (16mm) and S. aureus (16mm) (Table 4a and 4b). When the aqueous and methanolic A. sativum extract with skin was mixed with FCU it showed 12 and 2 mm zone of inhibition against Pseudomonas sp respectively (Figure 15b). Whereas the ethanolic and methanolic A. sativum extract when mixed with FCU didn’t show any zone of inhibition against Pseudomonas sp and S. aureus respectively. Whereas the aqueous and ethanolic A. sativum extract when mixed with FCU showed 14 and 2 mm zone of inhibition against S. aureus respectively (Figure 16b). There was no zone of inhibition with FCU against both the bacteria.
Graph (5, 6, 7 and 8) shows the typical FTIR spectrum of A. sativum extracts and Graph 9 shows the FTIR analysis of gomutra. The FTIR analysis of A. sativum aqueous extract without skin (GJA) is shown in Table 5a and Graph 5. The FTIR analysis of A. sativum aqueous extract with skin (GJWSA) is shown in Table 5b and Graph 6. The FTIR analysis of A. sativum ethanolic extract without skin (GJE) is shown in Table 5c and Graph 7. The FTIR analysis of A. sativum ethanolic extract with skin (GJWSE) is shown in Table 5d and Graph 8. The FTIR analysis of FCU is shown in Table 5e and Graph 9.
3.6 HPLC Analysis
The HPLC analysis of various solvent extracts of A. sativum for allicin were analyzed using UV detector and shown in Graph 11 – 14. The retention time for allicin is between 2.5 to 4 minutes. The HPLC analysis of the FCU was also recorded as shown in Graph 10. The chromatogram obtained by analyzing a solution of standard allicin is also shown in Graph 11.
3.7 GC/MS analysis
For identification of bioactive compounds in ethanolic solvent extract of A. sativum without skin, GC-MS analysis was performed (Graph 15). The ethanol extract showed presence of N-hexadecanoic acid as main compound with other 8 compounds in trace quantity (Table 6). Other minor compounds included alpha-D-glucopyranoside – O-alpha D-Glucopyranosyl, and Oleic acid. From the results it can be interpreted that the identified compound may be responsible for the antimicrobial activity of garlic.
3.8 Calculation of MIC of various solvent extracts of A. sativum
MIC value of the various solvent extracts of A. sativum was determined. The MIC value for GJA, GJE, GJWSA and GJWSE against Pseudomonas sp is 1, 10, 10 and 1 mg/ml respectively (Graph 16). The MIC for GJA, GJE, GJWSA and GJWSE against S. aureus is 100, 100, 100 and 1 mg/ml respectively (Graph 17).
A medicinal plant is any plant in which, one or more of its organs, contains substances that can be used for therapeutic purpose of which are precursors for the synthesis of useful drugs. The Phytochemical screening of A. sativum extract in this study revealed the presence of phenols, alkaloids, flavonoids, glycosides, saponins, volatile oil, cardiac glycosides, phlobatannins, and steroids. The presence of some of these bioactive components confirms similar research conducted by (Dahiru et al., 2006). The presence of alkaloids, flavonoids, saponin, and cardiac glycosides is in agreement with the work done by other researchers (Idowu et al., 2008). These classes of compounds especially alkaloids, saponins and flavonoids are known to have curative activity against several pathogens (Usman et al., 2009). Saponins possess both beneficial (cholesterol-lowering) and deleterious (cytotoxic permeabilization of the intestine) properties and also exhibit structure dependent biological activities (Osagie and Eka, 1998). Plants produce saponins to fight infections by parasites and in humans saponins help the immune system and also protect against viruses and bacteria.
Flavonoids are water soluble polyphenolic molecules and therefore belong to the polyphenol family. Together with carotenes, flavonoids are also responsible for the coloring of fruits, vegetables and herbs. Some flavonoid containing plants are diuretics (e.g. buchu), antispasmodic (e.g. liquorice) and others have antimicrobial properties (Trease and Evans, 2002). Flavonoids detected in A. sativum bulbs could be used in the treatment of various disease conditions like edema, toothache, fever, common cold, diarrhea and dental caries. These could be possible as the root extracts contains some antibacterial activities. The flavonoids are acting on bacteria by inhibiting its protein synthesis (Hong-xi and Song, 2001).
Since 1940s, the development of effective and safe drugs to deal with bacterial infections has revolutionized medical treatment, and the morbidity and mortality from microbial disease have been dramatically reduced. Unfortunately, the development of effective antibacterial drugs has been accompanied by the emergence of drug-resistant organisms. The phenomenon of resistance imposes serious constraints on the options available for the medical treatment of many bacterial infections.
The present study has demonstrated that A. sativum juice extract effectively inhibited the growth of both Pseudomonas sp and S. aureus though their sensitivity to the extract varied based on the presence or absence of skin and the different solvents used for extraction. The MIC values were found to be in the range of 1 to 10 mg/ml in case of Pseudomonas sp and 1 to 100 mg/ml in the case of S. aureus (Graph 16 and 17).
In previous studies, it has been reported that a component named allicin that is present in A. sativum exhibits its antimicrobial activity mainly by immediate and total inhibition of RNA synthesis, although DNA and protein syntheses are also partially inhibited, suggesting that RNA is the primary target of allicin action (Feldberg et al., 1988). The structural differences of the bacterial strains may also play a role in the bacterial susceptibility to A. sativum constituents.
The present study showed that A. sativum extracts also have antifungal activity which is in accordance with Davis et al., 2003. It is clear that A. sativum juice may be useful as an antimicrobial agent against the E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa, and S. aureus (Yadav et al., 2015). The present study suggests that A. sativum is active against organisms that cause wound infection. It has been previously reported that A. sativum is active against organisms that are found to be resistant to conventional antibiotics (Jezowa et al., 1966; Li et al., 2015). Moreover, studies also indicate that combination of A. sativum extracts with conventional antimicrobials leads to partial or total synergism (Didry et al., 1992; Gaekwad and Trivedi, 2013)
The susceptibility of some bacterial strains to the extract A. sativum may be a pointer to its potential as a drug that can be used against these susceptible bacterial strains. Furthermore, antibacterial resistance, especially, among Gram-negative bacteria is an important issue that has created problems in the treatment of infectious diseases and necessitates the search for alternative drugs or natural antibacterial remedies (Khosravi and Behzadi, 2006).
The antibacterial activity of aqueous, ethanolic and methanolic extracts of A. sativum was determined using Pseudomonas sp and S. aureus. From the result of the zone of inhibition in the microbial study, it was seen that all the extracts demonstrated antibacterial activity. The aqueous extract demonstrated the higher activity followed by ethanolic and methanol extracts.
Findings from this work reveal that FCU doesn’t show any antimicrobial activity. Also the combination of A. sativum juice with FCU didn’t show any synergistic effect. This study also reveals that A. sativum had both antibacterial and antifungal activity. This indicates that the extracts could also be used in the treatment of some wound infections caused by gram negative and gram positive bacteria.
Further research should be conducted to impregnate the bandages with active compounds of A. sativum and analyze whether it is active against infections in a real wound setting.
The phytochemical screening and investigation into the antibacterial potential of the extract of A. sativum showed or highlighted the antibiotic spectra of the A. sativum extract under assay, suggesting a promising lead as an alternative antibiotic and it yielded scientific support to their use in traditional ayurvedic medicine. The A. sativum extract was found to exhibit slight synergistic activity with gomutra against some tested organism.
From the entire experiment, it can be concluded that A. sativum juice extract with skin have the potential natural antibacterial and anti fungal compounds and can be used against organism which cause wound infection. The activity was influenced by gomutra and it was more effective in aqueous extract. However, if plant extracts are to be used for medicinal purposes, issues of safety and toxicity will always need to be considered. Hence I conclude that further research should be carried out on the dosage, in vivo evaluation of the garlic extracts in bandages against infections in a real wound setting.