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Cornus macrophylla, the Antibacterial Activity of Organic Leaf Extracts and the Characterization of the More Lipophilic Components by GC/MS

In the present study, the antibacterial activity of Cornus macrophylla was examined. Organic solvent extracts of leaves were prepared using methanol, n-hexane, chloroform, and ethyl acetate. Antibacterial activity was examined by using a 100 mg/mL extract concentration. Penicillin was kept as a positive control while dimethyl sulfoxide was taken as a negative control. Methanolic extract exhibited a 21.5, 36.3, 25.3, and 23.7 mm inhibition zone diameter (IZD); n-hexane showed a 33, 40, 32.8, and 28.7 mm IZD; chloroform showed a 18.8, 29, 22.3, and 21.6 mm IZD; and ethyl acetate showed a 23.5, 30.2, 30, and 22.3 mm IZD against Erwinia carotovora, Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas axonopodis, respectively. The n-hexane extract revealed high antibacterial activity against all bacterial species as compared with methanolic, chloroform, and ethyl acetate extract. Gas Chromatography Mass Spectrometry (GC/MS) analysis of n-hexane extract depicted the presence of 55 compounds. Out of these compounds, one compound, identified as α-amyrin (Mol. wt = 426), exhibited the maximum peak area (32.64%), followed by A’-Neogammacer-22(29)-en-3-ol, acetate, (3.beta.,21.beta.)- (Mol. wt = 468) and β-amyrin (Mol. wt = 426) having peak areas of 25.97 and 6.77%, respectively. It was concluded that the antibacterial activity observed during the present investigation may be due to these compounds.


Introduction
Plants are a valuable source of bioactive compounds due to the production of secondary metabolites. Secondary metabolites of plants show antimicrobial activity against a number of pathogens [1,2]. The extracts of plants are also used for treatment of serious diseases [3]. For the maintenance of quality and quantity of food, there is a need to control plant diseases caused by various pathogens. At present, the most reliable method for controlling bacterial pathogens is the use of synthetic/chemical pesticides. Although pesticides are helpful to crops, they have negative impacts on biodiversity, pollute the environment [4,5], and cause health problems [6,7]. Also, bactericidal application kills microbes that help plants defend against pathogens [8]. Moreover, numerous pathogens have developed resistance responsible for cankers on Citrus maxima [34]. Additionally, Xanthomonas campestris pv. Mangiferae indicae is responsible for mango bacterial canker disease [35]. All of these plant pathogens have a broad host range and cause a number of diseases in many plants. Therefore, the present study was designed to assess the in vitro antibacterial activity of bioactive compounds of C. macrophylla separated through methanol, n-hexane, chloroform, and ethyl acetate. The metabolites in the most active organic fraction from C. macrophylla were identified with the help of GC/MS and have not been reported in earlier investigations. This study could help to further extend our knowledge of bioactive molecules that can be harnessed as natural eco-friendly antibacterial compounds. Figure 1 shows the antibacterial activity of C. macrophylla leaf extracts against E. carotovora, P. syringae, R. solanacearum, and X. axonopodis. In all of these experiments, DMSO kept as a negative control did not show any antibacterial activity while penicillin used as a positive control exhibited the maximum antibacterial activity in terms of IZD. 2.1. Antibacterial Activity of Methanolic, n-hexane, Chloroform, and Ethyl Acetate Extract of C. macrophylla Leaves on E. carotovora Methanolic extract significantly exhibited a 21.5 mm IZD against E. carotovora while penicillin showed a 46.3 mm IZD. The n-hexane extract revealed the maximum antibacterial activity as compared with methanolic, chloroform, and ethyl acetate extract. The n-hexane extract showed a 33 mm IZD against E. carotovora, whereas extract of chloroform showed an 18.8 mm IZD, which was less than all other extracts. The ethyl acetate extract also showed substantial results, forming an IZD of 23.5 mm. In the case of organic solvent fractions, a maximum 33 mm IZD was recorded. In the case of the n-hexane extract of C. macrophylla leaves, this IZD was less than the penicillin used as a positive control ( Figure 1). These results showed similarities to the findings of [36] in which researchers investigated the effect of Urospermum picroides against E. carotovora and recorded an inhibition zone of 7-8 mm. Inhibition caused by the organic solvent extract of C. macrophylla leaves on E. carotovora was greater than that caused by U. picroides. In a previous study, an ethyl acetate fraction of Amaranthus viridis leaf exhibited a 19 mm IZD against E. carotovora [37]. This higher efficacy can be attributed to a greater amount of antibacterial substances present in the leaves of C. macrophylla.

Results and Discussion
2.2. Antibacterial Activity of Methanolic, n-hexane, Chloroform, and Ethyl Acetate Extract of C. macrophylla Leaves on P. syringae Methanolic extract exhibited a 36.3 mm IZD against P. syringae whereas penicillin showed a 57.2 mm IZD. The n-hexane extract revealed the best antibacterial activity as compared with chloroform and ethyl acetate extract, exhibiting a 40 mm IZD against P. syringae. Extract of chloroform showed a 29 mm IZD, which was less than all other extracts. Ethyl acetate extract also showed significant results with an IZD of 30.2 mm (Figure 1). These results showed similarities to the findings of [38] in Polygonum cuspidatum roots against P. syringae and exhibited 100% inhibition after 24 hours at a 105.11 µg/mL concentration. In another study, an ethyl acetate fraction of A. viridis leaf caused a 21 mm IZD against P. syringae [37].
2.3. Antibacterial Activity of Methanolic, n-hexane, Chloroform, and Ethyl Acetate Extract of C. macrophylla Leaves on R. solanacearum The antibacterial activity of methanolic extract of C. macrophylla leaves is shown in Figure 1. Methanolic extract exhibited a 25.3 mm IZD against R. solanacearum whereas the corresponding value for penicillin was 54.7 mm. The n-hexane extract revealed more potent antibacterial activity than chloroform and ethyl acetate extracts. The n-hexane extract showed a 32.8 mm IZD against R. solanacearum. The extract of chloroform showed a 22.3 mm IZD, which was less than all other extracts. The ethyl acetate extract also showed significant results (a 30 mm IZD). In this experiment, n-hexane showed maximum antibacterial activity. In an earlier investigation, the methanolic extract of R. coriaria exhibited an 18 mm zone of inhibition against R. solanacearum [21]. Ethanolic extract of Ipomoea staphylina has antibacterial activity against Xanthomonas campestris, P. syringae, Klebsiella pneumonia, Escherichia coli, Salmonella typhi, P. aeruginosa and S. aureus. GC/MS analysis of the ethanolic extract revealed the presence of alkaloids, saponins, flavonoids, steroids, glycosides, phenols, and sterols [39].
2.4. Antibacterial Activity of Methanolic, n-hexane, Chloroform, and Ethyl Acetate Extract of C. macrophylla Leaves on X. axonopodis Figure 1 shows the data on the antibacterial activity of C. macrophylla extracts against X. axonopodis. Methanolic extract exhibited a 23.7 mm IZD against X. axonopodis whereas penicillin showed a 51.5 mm IZD. The n-hexane extract revealed substantial antibacterial activity as compared with methanol, chloroform, and ethyl acetate extract. The n-hexane extract showed a 28.7 mm IZD against X. axonopodis. On the other hand, the extract of chloroform showed a minimum (21.7 mm IZD) bactericidal activity. The ethyl acetate extract also showed significant results (a 22.3 mm IZD). A maximum IZD of 28.7 mm was recorded in the case of n-hexane extract, which was less than that of penicillin. These results are in agreement with the findings of [40] where Amaranthus tricolor showed 24%-62% antibacterial activity against X. axonopodis.

Gas Chromatography Mass Spectrometry (GC/MS) Analysis
In total, 55 compounds were identified in the n-hexane fraction of C. macrophylla. The retention time (RT), peak areas of component (%), molecular weight, and their molecular formulas are presented in Table 1. Of these compounds, only three compounds revealed >5% peak areas, viz., α-amyrin; A'-Neogammacer-22(29)-en-3-ol, acetate, (3.beta.,21.beta.)-; and β-amyrin (Figure 2A-C). The antibacterial activity of α-amyrin and β-amyrin was also reported against S. aureus, Bacillus subtilis, Enterococcus faecium and Staphylococcus saprophyticus [41,42]. Both αand β-amyrin triterpenes have also been isolated from Dorstenia arifolia and documented as having antimicrobial activities [43]. The compounds α-, β-amyrin, and α-amyrin phenylacetate reduced the bacterial viability to less than 20% [44]. S. aureus (MRSA) is an important human pathogen that has become resistant to antibiotics. The compound α-amyrin has been reported to exhibit antimicrobial activities against S. aureus. The compound α-amyrin regulates multiple desirable targets in cell division, the two-component system, ABC transporters, fatty acid biosynthesis, peptidoglycan biosynthesis, aminoacyl-tRNA synthetase, and ribosome and b-lactam resistance pathways [45], resulting in the destabilization of the bacterial cell membrane, a halt in protein synthesis, and inhibition of cell growth that eventually lead to cell death [46]. Furthermore, it causes disorganizing effects on cardiolipin-rich domains present in the membrane of E. coli [47]. The α-amyrin identified from Pyrus bretschneideri Rehd. also exhibited antibacterial activity [48]. Moreover, αand β-amyrin esters are also documented as antibacterial compounds [49]. In another investigation, β-amyrin isolated from leaves of Siraitia grosvenorii showed antibacterial activity against Streptococcus mutans, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum with minimum inhibitory concentrations of 48.80, >100, and 48.80 µg mL −1 , respectively [50]. On the other hand, there are no previous reports that describe the antibacterial activity of A'-Neogammacer-22(29)-en-3-ol, acetate. In the present study, a higher level antibacterial activity of the n-hexane extract of C. macrophylla leaves was recorded as compared with chloroform and ethyl acetate extracts; n-hexane is a non-polar solvent and has a greater ability to extract more lipophilic compounds like α-amyrin, as compared with chloroform and ethyl acetate. Since GC/MS of n-hexane extract of C. macrophylla leaves from Pakistan has shown the presence of α-amyrin having the highest peak area, more studies are required to isolate and characterize its bioactive constituents.

Collection and Identification of Plant Material
Fresh leaves of C. macrophylla were collected from the Bara Gali summer campus, University of Peshawar, Khyber Pakhtunkhwa (KPK), Galyat, Pakistan. The voucher specimen (UOG-000585) was deposited in the herbarium of the Department of Botany, University of Gujrat, Gujrat, Pakistan.

Preparation of C. macrophylla Leaves Extracts
After collection, leaves of C. macrophylla were sun dried for 1 week and dried leaves (1 kg) were ground with the help of a pestle and mortar to make a fine powder. The powder (400 g) was soaked in 1-L of methanol in a glass jar and incubated for 1 week at room temperature (25 • C) and frequently stirred with a glass rod. The filtration of the extract was performed by using four layered muslin cloth followed by a final filtration with Whatman filter paper No. 1. The filtrate was evaporated at 45 • C by using a rotary evaporator (Model: Laborata 4000/Gl, Heidolph, Schwabach, Germany). Extra methanol from this extract was evaporated under currents of clean air at room temperature to yield a viscous fluid termed as methanolic extract. This methanolic extract was reconstituted in double-distilled water (200 mL) and fractionated with three organic solvents, viz., n-hexane, chloroform, and ethyl acetate, first with 200 mL of n-hexane in a 500 mL separating funnel. This setup was left overnight until the n-hexane formed a layer in the upper portion of the separating funnel, which was then separated into a glass beaker. The process was repeated thrice by adding fresh solvent into the aqueous solution. A similar process was used for the extraction with chloroform and ethyl acetate. The organic solvent extracts thus obtained were evaporated by using a rotary evaporator, as discussed earlier, and stored at 4 • C until further use.

Culturing of Target Plant Pathogenic Bacterial Species
Plant pathogenic bacterial cultures were obtained from the Culture Bank of Pakistan, University of the Punjab, Lahore, Pakistan. The bacterial cultures with their accession numbers were E. carotovora (FCBP-PB-0421), P. syringae (FCBP-PB-0405), R. solanacearum (FCBP-PB-0407), and X. axonopodis (FCBP-PB-001). These cultures were sub-cultured on a Lysogeny broth (LB) medium in 9 cm diameter glass petri plates until colonies became visible and stored in a refrigerator at 4 • C for further use.

Preparation of Control and Stock Solutions, Culture Medium, and Antibacterial Assays
For antibacterial assays, a disk diffusion method was adopted according to the procedure described in our previous publication, with slight modifications [51]. For the preparation of the negative control solution, 166 µL of DMSO was mixed with 333 µL of autoclaved distilled water to make a final volume of 500 µL and for the preparation of the positive control solution, 50 mg of penicillin was dissolved in 166 µL DMSO and 333 µL of autoclaved distilled water was added to make a volume of 500 µL. Stock solutions of organic solvent extracts were prepared in a way similar to the preparation of the positive control solution. 50 mg of leaves extract in each organic solvent viz. methanol, n-hexane, chloroform and ethyl acetate were dissolved into 166 µL of DMSO and then added 333 µL of autoclaved distilled water to make volume up to 500 µL. In this way, the positive control, penicillin, and all organic solvent extracts of C. macrophylla leaves were tested for their antibacterial efficacy at a 100 mg mL −1 concentration. The LB medium was used for inoculation of bacterial species. For the preparation of the LB medium, 1000 mL of distilled water was added into the conical flask, then 5 g of yeast extract, 10 g of tryptone, 10 g of NaCl, and 15 g of agar powder were added and mixed well to dissolve all the nutrients. Afterwards, the flask opening was covered with aluminum foil and sterilized in autoclave for 20 min at 121 • C. After preparing the LB agar plates, bacterial inocula @ 1 × 10 5 cfu/mL were spread evenly onto these plates and, after spreading, filter paper discs (6 mm) were placed on these plates. Leaf extract (25 µL) for each solvent (methanol, n-hexane, chloroform, and ethyl acetate) was added onto these filter paper discs contained in Petriplates and incubated at 37 • C. Antibacterial activity was measured after 72 h in terms of inhibition zone diameter (IZD) with the help of a measuring scale [37]. All chemicals used were of Merck KGaA, Darmstadt, Germany.

Gas Chromatography Mass Spectrometry (GC/MS)
Constituents of n-hexane extract of C. macrophylla leaves showing higher bioactivity were analyzed by using GC/MS on a Clarus 500 Mass Spectrometer (PerkinElmer, Waltham, Massachusetts, USA) whose detectable mass range was set at 35-500 m/z. The ion source and interface temperatures were 200 • C and 250 • C, respectively. The start and end times were 2.50 min and 47.14 min, respectively. The column oven temp. was 40 • C whereas the injection temp. was 25 • C. Injection mode was split and flow control mode was set at a pressure of 100 kPa. Total flow was 13.9 mL/min while column flow was 1.78 mL/min with a linear velocity of 48.1 cm/sec. Purge flow was kept at 3.0 mL/min and a split ratio of 5.1. The oven temperature was programmed first at 40 • C for 5 min with an increase of 5 • C min −1 to 80 • C, then 5 • C min -1 to 300 • C for 5 min. The mass spectral library consulted for GC/MS analysis for the identification of components in our study was NIST14.lib. This part of the research was conducted at the Thermal Energy Research Lab., National University of Sciences and Technology, Islamabad, Pakistan.

Statistical Design and Analysis
The experiment was performed by adopting Completely Randomized Design (CRD). For statistical analysis, ANOVA was done followed by Fisher's least significant difference (LSD) Test using Minitab Statistical Software (Minitab 19, State College, Pennsylvania, USA).

Conclusions
The present study revealed the antibacterial efficacy of C. macrophylla leaf extracts. GC/MS analysis of n-hexane extract depicted the presence of α-amyrin having the highest peak area % age. It may be concluded that this compound, having the highest peak area % age, was responsible for the antibacterial activity recorded in the present study. The structure of this compound can be utilized further to develop eco-friendly bactericides in the future.