Antimicrobial and antioxidant activities of a new metabolite from Quercus incana

Abstract Phytochemical investigations of Quercus incana led to the isolation of a new catechin derivative quercuschin (1), along with six known compounds: quercetin (2), methyl gallate (3), gallic acid (4), betulinic acid (5), (Z)-9-octadecenoic acid methyl ester (6) and β-sitosterol glucoside (7) from the ethyl acetate fraction of methanolic extract of the bark. Compound 1 was screened for its antibacterial, antifungal and antioxidant potential. Antibacterial and antifungal activities of the compound were tested against different bacterial and fungal strains, employing the agar well diffusion methods. The antibacterial activity was the highest against Streptococcus pyogenes with 80.0% inhibition, while the antifungal activity of the compound was the highest against Candida glabrata with 80.5% inhibition. The results of the antioxidant activity indicated that the compound exhibited antioxidant activity comparable to that of standard, butylated hydroxyanisole (51.2 μg/10 μl versus 45.9 μg/10 μl).

. Quercus incana (locally known as Serie or banj) a large evergreen tree is commonly known as blue jack oak or cinnamon oak. It represents an important genus of family Fagaceae commonly found in temperate regions of Pakistan (Nasir 1976;Sarwar et al. 2013). It is found in Balakot, Sangar, Kaghan, Swat, Dir and Kohistan. It is also found in Gadoon Gani Chatra and Swabi.
There are many ethnomedicinal uses of Q. incana. The acorns are given as diuretic and in gonorrhoea. These are also useful in indigestion and asthma (Jan et al. 2008). The decoction of the bark is used locally by the people in diarrhoea, pain, fever, inflammation and dysentery. It is also used as an astringent, diuretic and a santiasthmatic (Manan et al. 2007). The hydroalcoholic extract of plant showed marked antinociceptive activity and reduced gastrointestinal motility at various test doses (Sarwar et al. 2013). However, little work has been reported on its phytochemical and pharmacological activities.
Keeping in view the therapeutic potential of the plant, the current study deals with the isolation and charcterisation of secondary metabilties from Q. incana followed by screening for antibacterial, antifungal activity against various pathogens and free radical scavenging activity.
The COSY spectrum showed cross-peaks between methylene protons (δ 2.50, 2.85) and methine proton (δ 3.96). The methine protons in turn showed COSY correlation with the proton resonating at δ 4.55. Aromatic proton at δ 6.70 was COSY correlated with other aromatic protons at δ 6.75 and 6.82. The protons at δ 6.75 and 6.82 also showed COSY cross-peaks between them ( Figure S1). The correlation between δ 6.70 and 6.82 was observed to be weak and from the coupling constant value, it was assumed that they are placed meta to each other. The HMBC spectrum of 1 showed long-range correlations between -CH 2 -protons and quaternary carbons resonating at δ 100.9 and 157.9. HMBC correlations were also observed between methine proton (δ 6.82) and quaternary carbons (δ 157.9 and 132.2).
A symmetrical structure was proposed for the molecule in which a benzene ring is surrounded by three tetrahydropyran rings substituted at position 2 with a catechol moiety and a hydroxyl group at position 3. Absence of NOESY correlations between H-2 and H-3 suggested that the two protons are trans to each other. The trans-orientation of the two protons is also indicated by the large coupling constant (J 23 ). A similar class of compounds, epicatechins have cis-orientation of protons at these two positions, having a small J value (1.2 Hz), indicating the quasi-axial-quasi-equatorial (cis) orientation of the two protons (Wan et al. 1997). The large J value in compound 1 indicates a quasi-diaxial (trans) orientation for H-2 and H-3. The relative intensities of the signals in 13 C NMR spectrum were indicative of the repetitive structural features. The molecular formula of the proposed structure is C 33 H 30 O 12 , with a molecular weight of 618. The ESI-TOF spectrum gave a signal at m/z 600, which could be the M-H 2 O signal. The compound was characterised as a new compound. Structures of the other chemical compounds (2-7) ( Figure 2) were determined by comparing their data with the reported data (Watanabe et al. 1997;Núñez Sellés et al. 2002;Rahman et al. 2009;Asghar & Choudhry 2011;Eldahshan 2011;Haque et al. 2013). These were reported for the first time from Q. incana.
In the current study, we have reported significant data highlighting the antimicrobial potential of Q. incana as a source of new lead compound.
The new compound isolated from the plant exhibited significant antibacterial activity. The growth inhibitory potential of the tested compound was compared with that of standard drug, imipenem (10 μg/mL) and is presented in Table 1. Compound 1 (5 mg/mL) was found to inhibit pathogens: Bacillus subtilis, Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumonia and Escherichia coli. The compound showed the highest antibacterial activity against S. pyogenes (80.0% inhibition). It also revealed considerable activities against E. coli with 79.4% inhibition, S. aureus (77.5%), K. pneumonia (59.0%) and B. subtilis (52.8%), while the weakest activity was shown against P. aeruginosa (41.5%). The results showed that the plant exhibited a varied degree of antibacterial activity against various Gram-positive and Gram-negative bacteria. The results are supported by the antibacterial properties of related species of the genus Quercus like Q. dilatata (Jamil et al. 2012). The antifungal effect of the compound with mean zone of inhibition along with per cent inhibition observed with the test compound (5 mg/mL) against various strains is presented in (Table 2). It caused significant susceptibility against fungi; Candida glabrata (80.5%), Candida albicans (70.5%), Aspergillus niger (67.9%), Aspergillus flavus (65.9%), Fusarium solani (73.1%) and Microsporum canis (60.0%).
The emergence of resistance genes presents a great challenge for the practicing clinicians. This situation greatly intensifies the need for newer antimicrobial with novel mechanism of actions to combat with the challenges of bacterial resistance (Abdullah et al. 2014;Rauf et al. 2015). Natural organic healing material always proved to be the best alternative. The results of the isolated new compound as an antibacterial and antifungal agent were very attractive. Thus, it could be a new potential lead antimicrobial agent against infections caused by variety of microbes.
The result of the antioxidant activity against free radical, DPPH is presented in Table 3. The compound elicited considerable scavenging activity in the assay with IC 50 value of 51.2 μg/10 μL, which was comparable to that of the standard.
The DPPH free radical scavenging assay is the most commonly used test for the evaluation of test articles for antioxidant potential. The generation of free radical has generally been encountered during various infectious and inflammatory conditions , therefore, antioxidant potential of compounds are considered as an additional quality which obviously, augment the antimicrobial action of compounds. Thus, our newly isolated compound is a strong candidate for further detail studies in order to clinically estimate these characteristics.  Peshawar, identified the plant specimen which was then deposited in the herbarium having voucher number Bot.20052 (PUP). After botanical authentication, the plant material was air dried under shade and later on chopped and finely ground. The resulting powder material was subjected to extraction with methanol.

Fungal and bacterial strains
Tests were performed on six fungal and six bacterial strains. Bacterial strains included Gramnegative bacteria E. coli (ATCC-25922), P. aeruginosa (ATCC-27853) and K. pneumonia    . They were kept on an agar slant at 4°C and were stimulated/mobilised at 37°C for 24 h on nutrient agar or sabouraud glucose agar, respectively, for bacteria and fungi, before any screening.

Antibacterial activity
The isolated new compound was evaluated for its antibacterial potential, by agar well diffusion method (Naveed et al. 2013;Alam & Lee 2015) as presented in Table 1, with modifications according to the present experimental conditions. In order to prepare the stock solution, 5 mg/mL of the compound was dissolved in dimethyl sulfoxide (DMSO). Approximately 45 mL of molten nutrient agar was dispensed in sterilised Petri plates, and was allowed to harden. On these nutrient agar plates, bacterial culture was dispersed by making sterilised soft agar containing 100 μL of bacterial culture. A 6-mm-long clean decontaminated metallic borer was employed for well digging at appropriate gaps and marked for identification. After that 100 μL of sample was streamed into each well and left for incubation at 37°C for 24 h. The activity against bacteria was measured in the form of zone of inhibition (mm) and was compared with the standard antibacterial drug, imipenem (10 μg/mL), a broad spectrum antibacterial.

Antifungal activity
The isolated compound was evaluated for its antifungal potential, against different fungal strains as shown in Table 2, by agar well diffusion method (Quiroga et al. 2001;Nisar et al. 2013). Shortly, 5 mg/mL of the compound was mixed in dimethyl sulfoxide (DMSO) in order to prepare the stock solution. Approximately 50 mL of Sabouraud molten dextrose agar was poured in clean decontaminated Petri dishes, and allowed to solidify. On these nutrient agar plates, fungal culture was dispersed by making sterile soft agar containing about 100 μL of fungal culture. A sterilised metallic borer, 6-mm long, was employed for well digging at suitable spaces and spotted for illustration. After that, 100 μL of the sample was put into each well and left for incubation at 28°C for 48 h. The antifungal activity was measured in the form of zone of inhibition (mm) and then compared with the standard antifungal drug miconazole (broad spectrum antifungal) at 10 μg/mL.

DPPH free radical scavenging assay
The compound was tested for its antioxidant potential by the DPPH free radical scavenging activity method (Gulcin 2005(Gulcin , 2012Raziq et al. 2015). Briefly, 0.3 mM DPPH solution was prepared in ethanol. After which 5 μL of the test sample of various concentrations (62.5-500 μg) was mixed with 95 μL of DPPH solution. The mixture was then poured in 96-well plates and kept in incubator for 30 min at 37°C. The absorbance was measured at 517 nm using (microtitre plate reader). (%) radical scavenging activity was calculated by comparing with the dimethyl sulfoxide (DMSO)-treated control. The concentration of the compound that results 50% scavenging on DPPH was estimated as IC 50 . Butylhydroxy anisole was employed as standard.

Conclusion
This study furnished new scientific information about Q. incana regarding its biological potential and phytochemical assay that has never been recorded before, showing promising antimicrobial and antioxidant activities. The findings of the present study suggest the use of the plant in the discovery of drugs for the prevention of various common infectious diseases.