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ANTIMICROBIAL AND ANTIOXIDANT SCREENING OF Gardenia tetrasperma.

Byline: M. Ajaib, M. Latif, S. A. Mirza, Aziz-Ul-Hassan, M. A. Iqbal and Z. Khan

Keywords: Gardenia tetrasperma, Antimicrobial, Aspergillus niger, Antioxidant activity.

INTRODUCTION

Plant constituents differ widely in terms of their structure and biological properties and become a source of active natural products. Fresh fruits, vegetables and plant beverages are rich in natural antioxidants that prevents humans from many disorders such as cancer and cardiovascular diseases. The antioxidant and antimicrobial potential of plant products is due to the presence of several compounds in them which have distinct mechanisms of action in which some are enzymes and proteins while others are low molecular weight compounds such as vitamins, carotenoids, flavonoids, anthocyanins and other phenolic compounds (Ajaib et al., 2016).

According to the recent research,WHO has assessed that almost 80% world's population depend on herbal medicines for health care system (Amir et al., 2018). Compounds include antioxidants not only lack the reactive free radicals especially ROS (reactive species of oxygen) but reduce or stop the improvement of deteriorating diseases including cancer, cardiovascular, inflammatory and other chronic diseases (Sreejayan and Rao, 1996; Ajaib et al., 2015). Many plants of family Rubiaceae demonstrated antioxidant, antimicrobial, antimalarial activities due to presence of certain bioactive compounds. Nauclea latifolia, Crossopteryx febrifuga and Mitragyna inermis are important medicinal plants of family Rubiaceae. Many potent compounds are awaiting exploration in this family (Karou et al., 2011).

G. tetrasperma Roxb. (Figure 1) of family Rubiaceae commonly known as "Guggle" in District Kotli, Azad Jammu and Kashmir is fairly a common species of open slopes, in the middle of rocks and dry river beds; arising upto 2000 m. It is a shrubby plant height about 2 m. Flowers are greenish-white, sweet scented; fruit is upto 8 mm in diameter, rounded dark purple or black (Nazimuddin and Qaiser, 1989).

MATERIALS AND METHODS

Plant material: The plant material such as bark, leaves and seeds of G. tetrasperma was collected from Tehsil Khuiratta, District Kotli Azad Kashmir at N. 33Adeg18A', E. 74Adeg01.5A' with an elevation 850 m. The plant material was identified from Dr. Sultan A. Chaudhry Herbarium (SAH), Botany Department of GCU Lahore with a voucher no. BOT. 2747.

Plant Extracts: 250 g powdered plant material was used for extraction, following maceration technique (Singh, 2008) using petroleum ether, chloroform, methanol and distilled water as sovents.

Antimicrobial activity

Test Organisms: For appraisal of antibacterial and antifungal potential of bark and leaf of G. tetrasperma, two gram-negative, two gram positive bacteria and two fungal strains were used following Cruick-shank et al. (1975).

Antimicrobial bioassay: Agar well diffusion method according to Ferriera et al. (1996) and Ortega et al. (1996) was employed to investigate the antimicrobial activity. Minimum Inhibitory Concentration (MIC) of G. tetrasperma was evaluated using Broth-dilution method following Murray et al. (1999).

Antioxidant assays: DPPH free Radical Scavenging Activity of G. tetrasperma was evaluated using Lee and Shibamoto (2001) method.Total Antioxidant Activity (TAA) of selected plant was studied by using phosphomolybdenum complex formation technique following Prieto et al. (1999). Total Phenolic Contents (TPC) of G.tetrasperma was assessed using the methodology applied by Makkar et. al. (1993). Ferric reducing antioxidant power (FRAP) assessment of the plant extracts was completed by employing methodology adopted by Benzie and Strain (1996) with slight amendments.

RESULTS AND DISCUSSION

Verification that the antibacterial and antifungal potential was displayed decently by crude extract of G. tetrasperma plant only and not by solvents, negative response was observed.

All plant parts possesses satisfactory results against bacterial and antifungal strains (Table 3). Methanolic extract of bark showed 44+-0.5 mm against Escherichia coli as compared to standard disc Cephradine 26+-0.3 mm. Methanolic extract of bark against S.aureus showed 36+-2.2 mm as compared to standard disc Azithromycin, i.e. 15+-0.8 mm where as methanolic extract of bark against P.aeruginosa showed 49.66+-5.1 mm zone of inhibition. Methanolic extract of bark showed 45.67+-2.9 mm zone of inhibition against B.subtilis. It was noticed that methanolic extracts of bark showed best results as compared to the standard disc. Methanolic extracts of seeds also showed best results, i.e. 36.66+-1.73 mm against E.coli, 54.34+-2.6 mm against S.aureus, 23+-4.2 mm against P.aeruginosa, 20.66+-1.7 mm zone of inhibiton against B.subtilis. Leaves extract of chloroform showed best result against E.coli 91.66+-0.4 mm and against S. aureus 62+-1.1mm.

Seed extracts of methanol showed poor results as compared to methanolic extracts of leaf and bark.

Methanolic extracts of G. tetrasperma during MIC, displayed substantial confrontation against both types of bacteria. Furthermore significant results of seed extracts, i.e. 0.10+-0.01 against E.coli and 0.29+-0.04 against P. aeruginosa was recorded. E.coli showed susceptibility against leaf extracts which was 0.02+-0.1. P. aeruginosa showed susceptibility against leaf extracts with a value 0.04+-0.01. All part of plants showed good results against bacterial strains (Table 4). Methanolic extract of leaf have supreme results in contrast to F.solani and A.niger with the values 42.42+-1.5 and 36.22+-1.11 respectively. On the other hand, lowest value showed by aqueous extract of the leaf against F. solani, i.e., 16.43+-1.37. The seed methanolic extract also displayed satisfactory results against F. solani 42+-1.75. Whereas, lowest value is exposed by the water extract of the seeds, i.e., 12.33+-0.8. Maximum value showed by seed methanolic extract, i.e. 42.42+-1.5 against A.niger.

Minimum zone displayed by aqueous extract, i.e. 15+-0.62 against A. niger (Table 5). The MIC assay was performed for further study which presented the significant MIC value, i.e. 0.02+-0.1 at 0.8 mg/mL of leaves extract against E.coli (Table 4) where as 0.02+-0. 9 at 0.2 mg/mL concentration of leaves extract against A. niger (Table 6).

Best results of DPPH free radical scavenging was shown by bark extract in petroleum ether (PE) at absorbance value 95.51+-1.7 at 500 uL/ml, then by chloroform extract with absorbance of 87.19+-0.79 at 250 uL/ml, on the other side, a smaller amount of proficiency is revealed by the bark aqueous extract (43.19+-0.99 at 125uL/mL). In all extracts, petroleum ether and chloroform extracts of leaf showed uppermost value, i.e., 91.36+-0.77 and 81.6+-0.77 respectively at 1000 uL/mL. Whereas, least value was shown by the aqueous extract of leaf, i.e. 31.11+-0.89 at 125 uL/mL. Petroleum ether seed seed extracts of G. tetrasperma showed maximum absorbance at 1000 uL/mL with a value of 89.37+-0.01.

Alike, methanolic and chloroform extract exhibited the significant value at 1000 uL/mL, i.e., 84.3+-19.1and 72.12+-0.53 respectively. Overall least value is displayed by the water extract, i.e. 32.43+-1.2 at 125 uL/mL (Table 7).

The total phenolic components (TPC) present in the leaf, bark and seed of the G.tetrasperma were predictable in relevance to the Gallic acid, attained by linking the subsequent values with the standard curve of Gallic acid in GAE mg/mL (Table. 10). To determine TPC the plant extract was allowed to react with FC reagent and absorbance value was taken at 726 nm. The petroleum ether bark extract of plant exhibited maximum absorbance, i.e. 1.53+-0.27 GAE mg/mL. Maximum value of absorbance exhibited by chloroform extracts of leaves 1.11+-0.45 GAE mg/mL. Although the aqueous extracts of the seed showed absorbance of 1.8+-1.26 GAE mg/ml which is the maximum value amongst all aqueous extracts of this assay. During whole experiment bark extracts of G. tetrasperma were found to have maximum phenolic substances as associated to the extracts of other parts (Table 10).

Ferric reducing antioxidant power assay is beneficial over all other extracts of G.tetrasperma. The assay was performed following Benzie and Strain (1996) and the results were measured in TE (uM/mL). Maximum reduction potential was recorded by the bark methanolic extract, i.e., 65.66+-0.01 uM/mL (Table 12) whereas the minimum value was shown by the leaf water extract, i.e., 13.44+-0.37 uM/mL.

The antioxidant potential of G. tetrasperma was assessed using Phoshomolybdenum method (Table-11). The results were paralleled with the known value of BHT. Greatest antioxidant power was displayed by the methanolic extract of bark, i.e., 1.29+-0.11 mm whereas the minimal effectiveness was presented by the aqueous extract of seed, i.e., 1.01+-0.77 mm.

Almost all plant parts of G. tetrasperma showed good antimicrobial results against bacterial as well as fungal strains. Methanolic extract of bark showed 44+-0.5 against E.coli as compared to standard disc Cephradine 26+-0.3mmmay be due antimicrobial compounds collected in the bark and the results were more significant than standard antibiotic discs, such findings are also testified by (Ajaib et al., 2015) throughout the determination of antimicrobial compounds in Clerodendrum splendens. Antioxidant potential, i.e. Total phenolic contents, DPPH, Total antioxidant assay FTC was evaluated. In whole process bark extract of Petroleum ether presented good results (95.51+-1.7%) at 500 uL/ml (Table 7) followed by leaf extracts of Chloroform (91.44+-0.6%) at 1000 uL/mL (Table 8) while less competency was disclosed by water extracts of Seed (32.43+-1.2%) at 125 uL/mL (Table 9).

Similar situations were also reported by Ebrahimzadeh et al. (2008) while investigating oxidative stress on Iranian Corn Silk.

The total phenolic contents were determined in comparison to the Gallic acid. Petroleum ether extract of bark of G. tetrasperma showed highest absorbance, i.e. 1.53+-0.27GAEmg/mL. Maximum value shown by leaf extract in chloroform was 1.11+-0.45 GAEmg/mL as compared to other solvents. Bark macerates of plant have significant phenolic contents as compared to other parts of G. tetrasperma (Table 10). Somewhat similar results were noticed by Ajaib et al. (2016) while working on Chenopodium ambrosioides for antimicrobial and antioxidant screening.

The significant TPC was possessed by methanolic and petroleum ether extracts of bark and leaf, i.e., 1.29+-0.11 mm and 1.19+-0.5 mm respectively very close to BHT standard with a value 1.2+-0.1 (Table 11). The antioxidant potential was found in order Methanol >Petroleum ether >Chloroform > Water. Aqueous extracts presented minimum antioxidant activity as reported by Siddiqui et al. (2015) and Riaz et al. (2012) during investigations on Cotinus coggyria and Pyrus pashia.

Table-1: Inhibitory Zone formation by standard antibacterial discs (Positive control).

###Antibacterial Standard Disc###Conc. (ug)###Bacterial Strains###Zone of Inhibition

###(mm)

###Azithromycin###15###S. aureus###15+-0.8

###Amikacin###30###B. subtilis###17+-0.41

###Ampicillin###10###P. aeruginosa###22+-0.2

###Cephradine###30###E. coli###26+-0.3

Table-2: Inhibitory Zone formation by standard antifungal discs (Positive control).

###Conc.

###Antifungal standard disc###Fungal strains###Zone of inhibition (mm)

###(ug)

###Itraconale###100###F. solani###9+-0.97

###Voriconazole###100###A. niger###39+-1.00

Table-3: Zone of Inhibition produced by bark, leaf and seed extracts of G. tetrasperma against bacterial strains.

Plant Parts###Extract###Zone of Inhibition (mm)

###E.coli###S.aureus###P.aeruginosa###B.subtils

###Bark###Petroleum ether###30.34+-1.7###53+-2.5###20+-2.1###20.34+-1.8

###Chloroform###50+-3.2###38.67+-3.2###43+-0.5###30+-2.0

###Methanol###44 +-0.5###36+-2.2###49.66+-5.1###45.67+-2.9

###Aqueous###37.66+-0.11###39+-0.4###22.66+-1.3###20.6+-1.1

###Leaf###Petroleum ether###33.66+-0.3###52+-1.4###75+-0.3###15+-1.1

###Chloroform###91.66+-0.4###62+-1.1###49+-1.5###43.6+-1.4

###Methanol###25.33+-0.2###71.66+-1.1###44.67+-1.1###29+-0.3

###Aqueous###51.33+-2.6###49+-2.6###34+-1.4###27.67+-4.1

###Seed###Petroleum ether###35+-1.7###43+-1.2###32+-0.2###24+-4.2

###Chloroform###32.34+-0.5###40+-0.3###35+-0.8###36.6+-1.6

###Methanol###36.66+-1.73###54.34+-2.6###23+-4.2###20.66+-1.7

###Aqueous###32+-4.2###46.3+-1.5###26+-3.2###26.33+-0.2

Table-4: MIC values of bark , leaf and seed extracts of G. tetrasperma against bacterial strains.

Plant###Bacterial strains

parts

###E.coli###P.aregenosa###B.subtilis###S.areus

###Conc.###MIC###Conc.###MIC###Conc.###MIC###Conc.###MIC

###mg/mL###mg/mL

Bark###1###0.06+-0.37###0.7###0.04+-0.01###0.3###0.21+-0.37###0.7###0.96+-0.02

Leaf###0.8###0.02+-0.1###0.9###0.53+-0.37###0.7###0.25+-0.21###0.9###1.08+-0.1

Seed###0.9###0.10+-0.01###0.8###0.29+-0.04###0.8###1.22+-0.01###0.4###0.09+-0.03

Table-5: Zone of Inhibition produced by leaf, bark and seed extracts of G. tetrasperma against fungal strains.

###Plant part###Solvent###Zone of inhibition (mm)

###Fusarium solani###Aspergilus niger

###Bark###Petroleum Ether###22.33+-0.2###20+-0.5

###Chloroform###37.3+-0.16###21+-0.5

###Methanol###49+-0.6###42.3+-1.4

###Aqueous###26.3+-0.3###43+-0.5

###Leaf###Petroleum Ether###15.34+-1.4###19+-0.5

###Chloroform###21.33+-1.8###16+-1.5

###Methanol###42.42+-1.5###36.22+-1.11

###Aqueous###16.43+-1.37###18.66+-2.4

###Seed###Petroleum Ether###18+-0.5###28+-1.5

###Chloroform###16+-0.8###22+-0.5

###Methanol###42+-1.75###42.42+-1.5

###Aqueous###12.33+-0.8###15+-0.62

Table-6: MIC values of bark, leaf and seed extracts of G. tetrasperma against fungal strains.

###Plant parts###Fungal Strains

###F.solani###A.niger

###Conc. mg/mL###MIC###Conc. mg/mL###MIC

###Bark###0.9###0.05+-0.1###0.8###0.25+-0.37

###Leaf###1.00###0.30+-0.04###0.2###0.02+-0. 9

###Seed###0.4###0.70+-0.03###0.7###0.177+-0.2

Table-7: % Free radical scavenging activity of bark of G. tetrasperma.

Plant Part###Extract###Concentrations###Absorbance

###(uL)###In %

###Petroleum###1000###67.66+-1.5

###ether###500###95.51+-1.7

###250###89.37+-0.5

###125###51.56+-0.4

###Chloroform###1000###80.22+-1.04

###500###70.11+-0.3

###250###90.21+-0.5

###125###66.13+-0.5

Bark

###Methanol###1000###59.86+-0.9

###500###62.67+-0.4

###250###70.24+-0.4

###125###40.25+-0.7

###Aqueous###1000###79.40+-1.1

###500###68.29+-0.4

###250###49.8+-2.7

###125###43.19+-0.99

BHT###91.35

Standard

Table-8: % Free radical scavenging activity of leaf of G. tetrasperma.

Plant###Extract###Concentrations(u###Absorbance

Part###L)###In %

###Petroleum###1000###81.6+-0.77

###ether###500###75.5+-1.1

###250###58.566+-1.3

###125###64.13+-0.6

###Chlorofor###1000###91.44+-0.6

###m###500###82.84+-1.2

###250###73.9+-1.86

###125###43.45+-0.5

Leaf

###Methanol###1000###87.34+-0.6

###500###61.64+-0.67

###250###58.76+-0.18

###125###53.34+-1.5

###Aqueous###1000###78.14+-1.12

###500###42.34+-1.4

###250###67.76+-1.3

###125###31.11+-0.89

BHT###91.35

Standard

Table-9: % Free radical scavenging activity of seeds of G. tetrasperma.

Plant###Extracts###Concentrations###Absorbance

Part###In %

Seeds###Petroleum###1000###89.37+-0.01

###ether###500###85.73+-0.45

###250###76.66+-1.45

###125###54.5+-1.4

###Chloroform###1000###72.12+-0.53

###500###39.54+-0.9

###250###45.47+-0.45

###125###44.1+-0.2

###Methanol###1000###86.5+-2.1

###500###52.6+-1.2

###250###58.41+-0.7

###125###44.1+-0.7

###Aqueous###1000###51.64+-0.9

###500###42.45+-1.4

###250###45.87+-1.3

###125###32.43+-1.2

BHT###77.3+-0.7

Standard

Table-10: Total phenolic content of G. tetrasperma.

Plant Part###Extract###Absorbance

Bark###Petroleum ether###1.702+-0.3

###Chloroform###0.63+-0.1

###Methanol###1.43+-.6

###Aqueous###0.4+-1.4

Leaf###Petroleum ether###0.71+-0.7

###Chloroform###1.11+-0.45

###Methanol###0.84+-2.4

###Aqueous###0.56+-0.9

Seed###Petroleum ether###0.8+-1.7

###Chloroform###0.95+-2.3

###Methanol###1.032+-0.1

###Aqueous###1.07+-3.7

Table-11: Total antioxidant activity of G. tetrasperma.

Plant part###Extract###Absorbance

###Petroleum ether###1.07+-0.1

###Chloroform###1.03+-0.6

###Bark

###Methanol###1.29+-0.11

###Aqueous###1.17+-0.3

###Petroleum ether###1.19+-0.5

###Chloroform###1.13+-0.6

###Leaf

###Methanol###1.14+-0.1

###Aqueous###1.175+-0.4

###Petroleum ether###1.09+-0.1

###Chloroform###1.03+-0.2

###Seed

###Methanol###1.04+-0.7

###Aqueous###1.01+-0.77

Standard###1.2+-0.1

Table-12: FRAP assay of G. tetrasperma.

###Plant part###Extract###Absorbance

###Bark###Petroleum ether###45.66+-0.03

###Chloroform###24.33+-0.09

###Methanol###65.66+-0.01

###Aqueous###39.33+-0.4

###Leaf###Petroleum ether###45.23+-0.02

###Chloroform###20.43+-0.04

###Methanol###53.66+-0.2

###Aqueous###13.44+-0.37

###Seed###Petroleum ether###41.2+-0.3

###Chloroform###50.44+-0.2

###Methanol###42.22+-0.01

###Aqueous###18.63+-0.05

Conclusion: it was concluded that G. tetrasperma contains bioactive compounds which are potent antimicrobial and antioxidants.

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