Gas Chromatographic Analysis of the Phyto-Constituents and the Assessment of the Anti-Microbial Properties of the Leave Extracts of Nigeria-Grown Gingko biloba

1 Department of Applied Biochemistry, NnamdiAzikiwe University, Awka, Nigeria. 2 National Biosafety Management Agency, Abuja, Nigeria Department of Applied Microbiology, Nnamdi Azikiwe University, Awka, Nigeria. 4 Department of Biochemistry, Federal University of Gusau, Nigeria. 5 Department of Medical Biotechnology, National Biotechnology Development Agency, Abuja. 6 Department of Applied Biochemistry, NnamdiAzikiwe University, Awka, Nigeria.


Introduction
For many years, plants have been used as medicine because they are an important source of many biologically and therapeutically active products. These plants have been

Collection and Extraction of Plant Leaves
Healthy fresh leaves of Ginkgo biloba were collected from Ginkgo biloba tree at Park Avenue, GRA, Enugu, Enugu State, Nigeria in a large quantity. The plant was identified and authenticated by a plant taxonomist in the Botany Department of Nnamdi Azikiwe University, Awka. The voucher specimen was deposited in the Hebarium of the Department. The leaves were dried at room temperature for three weeks. The dried leaves were later ground into a fine powder using a local steel grinder. To obtain an aqueous extract, 30g of the ground leaves was mixed with 300ml of distilled water. Then 30g of the ground leaves was mixed with 80% (v/v) ethanol to obtain an ethanol extract. Both mixtures were refluxed in a water bath at 65 0 C for 1 hour and filtered using Whitman filter paper No. 1 followed by the evaporation of the filtrate to slurry form using a rotary evaporator.

Phytochemical Quantification
Quantification of the phytochemical constituents in the aqueous and ethanol extracts was done using gas chromatography, fitted with flame ionization detector (GC-FID) employing Martin and Synge (1941) method and modified by Ujowundu et al. (2015).

Procedure:
For each extract, 0.5ml was introduced into separating funnels containing 0.5g of sodium sulphate. Then 20ml of N-hexane was added to each column and was left to stand for a few minutes after which the N-hexane layer was extracted and used for the GC-FID analysis.

Conditions for the Quantification:
The quantification of the phytochemicals was performed on a BUCK M910 Gas Chromatograph equipped with a flame ionization detector. A RESTEK 15 meter MXT-1 column (15 x 250 x 0.15µm) was used. The injector temperature was 280 0 C with a splitless injection of 2µl of the sample and a linear velocity of 30cms -1 . Helium 5.0pa.s was the carrier gas with a flow rate of 40mlmin -1 . The oven operated initially at 200 0 C, it was heated to 330 0 C at a rate of 3 0 C min -1 and was kept at this temperature for 5 minutes. The detector operated at a temperature of 320 0 C.
The phytochemicals were then determined by the ratio between the area and mass of internal standards and the area of the identified phytochemicals. The concentration of the different phytochemicals was expressed in µg/ml.

Test Organisms:
A total of three pathogenic bacteria namely: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and three pathogenic fungi, Candida albicansAspergillusfumigatus, Penicilliumcyclopium were obtained from the Reference Laboratory section of the Department of Applied Microbiology and Brewing, NnamdiAzikiwe University Awka, Anambra State, Nigeria. These organisms were maintained on Nutrient Broth for Bacteria and Sabouraud Dextrose Broth for fungi.

Antimicrobial Susceptibility Test:
This was determined by the modified method described by Agu et al. (2013). Plates that had confluent and/or semi-confluent growth were selected for the antimicrobial susceptibility tests. The disk diffusion method was used to assay the effect of the extracts on the various microorganisms. Mueller-Hinton Agar was used for the bacteria and Sabouraud Dextrose Agar (SDA) for the fungus. Twenty four hours broth cultures of the test organisms were serially diluted, then 10 -1 and 10 -2 dilutions were used to seed the fungal isolates, while 10 -2 was used to seed the bacterial plates. Then 0.1ml of the appropriate dilution of the broth culture of each microorganism was uniformly spread using a sterile glass spreader on the surface of the media, and sterile filter papers were soaked in the neat Ginkgo biloba extracts (aqueous and ethanol) that is in slurry form and placed on two points on each petri dish. Incubation was done at room temperature for 24 hours for bacteria and 48 hours for fungi. Clear zones of inhibition around the wells indicated antimicrobial activities of the extracts against the test organisms. The diameters of the zones of inhibition were measured and recorded in millimetres. Negative controls were set up with sterile water and positive controls were set up using 0.5% Nystatin for fungi and 0.5% Ciprofloxacin for bacteria.

Determination of Minimum Inhibitory Concentration (MIC):
The Modified method of Pallotaet al. (2007) was used for this study. The Minimum Inhibitory Concentration (MIC) was determined by the broth dilution method. Different test tubes containing Nutrient Broth (bacteria) and Sabouraud Dextrose Broth (fungi), which contained 20%, 40%, 60% and 80%, of the extracts were inoculated with known amounts of the bacterial and fungal isolates, with a dilution factor of 10 -2 for bacteria and 10 -1 and 10 -2 for fungi and controls (negative and positive) were set up also. The plates were incubated at room temperature for 24 hours for both bacteria and fungi in a metabolic rotary shaker (220 revs/min). Thereafter, the incubated test tubes were then sub-cultured onto sterile freshly prepared plates and incubated for 24 hours for bacteria and 48 hours for fungi. At the end of the incubation period, the plates were counted and the total microbial count recorded in CFU/ml. The plate with the lowest count was recorded as the MIC. The MIC is defined as the lowest concentration required to arrest the growth of the micro-organism at the end of 24 h or 48 h of incubation.

Results and Discussion
Some data obtained in this study were evaluated using the Statistical Package for Social Sciences (SPSS). The values were expressed as a mean of duplicate determination + standard deviation. Analysis of variance (ANOVA) was done at 0.05 level of significance, if P<0.05, there is a significant difference.

Discussion
In this study, gas chromatography linked with flame ionization detector (GC-FID) showed the quantities of 12 and 13 specific phytochemicals in the aqueous and ethanol extracts respectively, as seen in tables 1 and 2. The identification of these specific phytochemicals was done by comparison of the GC-retention time of the standard phytochemicals which was saved in the data system library. Flavonoids content was observed to be in the highest quantity in both extracts considering the concentration levels of the different components of flavonoids like epicatechin, kaempferol, anthocyanin, catechin and rutin. Catechins have shown to possess antibiotic properties due to their role in disrupting a specific stage of the bacterial DNA replication process (Gradisar et al., 2007). It may have contributed immensely to the many reported antibacterial effects of the leaf. Rutin has been reported to have clinically relevant functions that include anti-inflammatory, antihaemorrhagic activity and strengthening of capillary permeability and stabilization of platelets (Guo and Wei, 2008).Kaempferol and anthocyanin are antioxidant components and could have aided in the massive reports of the antioxidant properties of the leaves. Anthocyanin has been reported to be involved in the improvement of vision, decreased platelet aggregation and neuroprotective effects (Tyagi et al., 2010). The specific alkaloid; lunamarine has shown to be immunomodulatory and anti-amoebic activity, particularly against Entamoeba histolytica. Alghabban (2014) in his study confirmed that the Ginkgo biloba leaf extract inhibited and treated Entamoeba histolytica infection. Tannin was also identified as shown in Table 1 and 2 for aqueous and ethanol extracts. Tannin has been reported to be an essential antioxidant, antimicrobial and anti-cancer activities (Dillard and German, 2000). Saponin content in aqueous and ethanol extracts is quite significant with concentrations of 21.3164µg/ml and 21.3336µg/ml respectively. The saponins are generally known to exert hypoglycaemic and anticholesterol effect through intra-lumenal physiochemical interactions (Price et al., 1987). Saponin has also been implicated to act as antibacterial agents (Lacaille-Dubois & Wagner, 2000).
This study as with many other studies on Ginkgo biloba had indicated the high content of phenols in the leaf and had contributed immensely with steroids and terpenes (sapogenin) and flavonoids to its highly recognized antioxidant effect. The study also identified very low content of anti-nutrients (oxalate and phytate) in both extracts which are very good for consumption. The study has shown that the extracts contain appreciable phytochemicals that exact many therapeutic functions, it can be said that the extracts may be safe for human consumption for medicinal purposes. However, it is pertinent to state that the percentage of each constituent in the extracts can vary according to the country where it was grown and the season in which the plant leaves were harvested (Sati et al., 2013). Table 3 and 4 showed that the aqueous extract did not show any antibacterial and antifungal activity on the three bacteria and fungi. Ethanol may be a better solvent for the extraction of the secondary metabolites from the leaves especially catechin, as shown in comparison between table 1 and 2. Catechin may have being the leaf's constituent that easily diffused into the bacterial and fungal cell wall. The reason for the reduced effect of the extracts on gram-negative bacteria and mould may have been due to the complexity of the cell wall of gram-negative bacteria and the central core of the mould cell wall that is a branched β1,3,1,6 glucan linked to chitin via β 1, 4 linkage (Latge, 2007) respectively. Majorly, β 1, 3 glucan synthase inhibitors is used clinically to target Aspergillusfumigatus and other mould fungi (Latge, 2007). This result is in consonance with Sati and Joshi (2011) who confirmed the antibacterial effect of Ginkgo biloba ethanol extract of Indian origin on many bacteria including Escherichia coli that showed low inhibition. The result is also in consonance to those of Sati et al., (2012) who confirmed that the aqueous leaf extract of Ginkgo biloba from India did not show any activity on gram-negative and gram-positive bacteria, and mould and yeast whereas other extracts including ethanol showed antibacterial and antifungal effect.
In table 5, the colony count was high in 20% dilution of the Ginkgo biloba ethanol extract for Staphylococcus aureus and Escherichia coli. This is due to the low concentration of the extract, however 40%, 60% and 80% concentrations for Staphylococcus aureus showed no colony count indicating that 40% dilution (0.4ml of extract in 0.6ml of broth) extract may have been able to inhibit and kill Staphylococcus aureus. As for the E.coli, 40%, 60% and 80% dilution concentration of the extract showed a decrease in the colony count in that sequential order. Therefore it can be said that at 40% dilution concentration, the extract may inhibit E.coli to the greatest minimum, and at 80% dilution concentration, the extract may kill E.coli.
In table 6, the colony count was high in 20% of 10 -1 dilution factor with 34 + 1.41 count and 20% of 10 -2 dilution factor with 15 + 2.83 count of Candida albicans. This is due to the low concentration of the extract while 40% and 60% concentration for 10 -1 dilution factor; the count decreased showing greater inhibition on Candida albicans. The 80% concentration of the extract may be able to kill Candida albicans. It was the same for 10 -2 dilution factor with 60% concentration that may be able to inhibit and kill Candida albicans.
The same inhibition based on concentration was observed in Penicilliumcyclopiumas in Candida albicans. However, at 60% concentration of the ethanol extract, it was able to inhibit and kill Penicilliumcyclopium at 10 -1 and 10 -2 dilution factors.
The inhibition activity of the ethanol extract of Ginkgo biloba leaf signifies the leaf antimicrobial effect and this might be due to presence of variety of active compounds in the extract such as tannin, saponin, alkaloids and flavonoids as suggested by Abo et al., (2000). This study had established the potential of using Ginkgo biloba ethanol leaf extract of Nigerian origin for microbial treatment.

Conclusion and Suggestion
In conclusion, this study revealed that aqueous and ethanol leaf extracts of Ginkgo biloba from Nigeria possess phyto-constituents of health importance and have shown to possess antibacterial and antifungal properties. The importance of natural products with phyto-constituents of health implication and antimicrobial properties cannot be overemphasized; therefore greater awareness should be vigorously pursued to effectively appropriate the rich chemical constituents and anti-microbial properties of Ginkgo biloba found in Nigeria.