Introduction

Clerodendron inerme belongs to the Verbenaceae family in different species. It is found in tropical and subtropical regions of the world. The plant is widely distributed in southern and southeastern Asia, Australia, and the Pacific islands (Shrivastava & Patel, 2007). It is a constant flower that produces sweets and seeds (Rabiul et al., 2011). Common plant names are Sangkupi and Peechangu (Siripuram, Mondi, Gottumukkula, & Gowri, 2018; Wagner, Herbst, & Sohmer, 1999). The plant has therapeutic properties similar to neuroprotective effect. The leaves and roots of the plant cure rheumatism and skin diseases (Manoharan, Kavitha, Senthil, & Renju, 2006).

The plant has many bioactive compounds distributed throughout the leaves and roots, such as anthraquinones, flavonoids, phenolics, cardiac glycosides, proteins, carbohydrates, oils, and lignin (Al-Snafi, 2016; Manoharan et al., 2006). Anthraquinones were found in different parts of the plant (Prasad, Sushant, & Chikkaswamy, 2012; Wang, Luan, He, Wang, & Li, 2018). Among the compounds found in this plant are Aloe-emodin, Emodin, Chrysophanol, and 2,5- Dimethoxybenzoquinone (Nan, Yin, & Zhang, 2008; Shakeel, Shahid, Ahmad, & Ullah, 2017). They have antibacterial and other biological properties (Zahin, Aqil, Khan, & Ahmad, 2010).

These biological properties are evaluated for toxicity issues related to synthetic antioxidants and supplements.

This study is aimed to identify the antibacterial and antioxidant activities of the anthraquinones fraction from the leaves of C.inerme. In addition, GC-MS and FTIR analysis of anthraquinones are being investigated.

Materials and Methods

Collection of plant leaves

Fresh leaves of Clerodendrum inerme were collected from the university campus, University of Baghdad, Iraq in November 2018. The plant was identified by the Faculty of Science herbarium / University of Baghdad.

The plant leaves were washed under running tap water and for seven days dried and then put in an oven at 40^ºC for one day. The leaves were ground and stored until use.

Anthraquinones component of C. inerme leaves

This method is based on (Smita & Sushma, 2010). About 50 grams of dry powder of the leaves of this plant were added to methanol and deionized water and refluxed for 4 hours.

After that, the extract was stirred to 4 ml of concentrated HCL and mixed with the methanolic solution. Purification was performed with chloroform and filtered. The solution was then evaporated at 40^ºC by using a rotary evaporator until chloroform was released (Buchii- Switzerland). The product yield was 0.47%.

Test of bacteria

In this research, the antibacterial effect of C.inerme was tested against four types of bacteria: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa obtained from cultures maintained at the Department of Biological Science, University of Baghdad, Iraq.

Determination of antimicrobial activities

Well diffusion assay

An experiment was performed, based on the procedure (Radhika, Sastry, Harica, & Madhu, 2008) with some modifications. These strains of bacteria were uniformly dispersed on Mueller-Hinton agar (Oxoid, England) using sterile cotton swabs. In petri plates, wells of 6 mm diameter were arranged above the ground in the Mueller-Hinton agar. All samples were transferred to wells (50 µl) of different concentrations (100, 50, and 25 mg/ml) and incubated overnight at 37^ºC. The zones of inhibition were observed in three plates. Tetracycline as a concentration of 10 mg/ml using positive control and negative control was 5% Dimethyl sulfoxide (DMSO).

Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration (MIC) protocol used a standard protocol (Andrews, 2002) and the medium of culture bacteria used the Nutrient broth (Himedia, India). Anthraquinones fraction was divided by serially diluted two-fold with 5% (DMSO) of stock culture. One ml of broth was added to a tube containing 1-7. One ml of stock was added to all tubes without negative control and then incubated at 37^ºC for 24 hours. McFarland standard (0.5) was used to determine the number of units forming colony form (CFU) of bacteria in nutrient broth (1 x 10⁸ CFU / ml). MIC values ​​were determined as the lowest concentration of the bacteria kills bacteria after 24 hours.

Minimum bactericidal concentration (MBC)

The values of the minimal bacterial concentration (MBC) were determined by sub-culturing from the MIC assay tubes in Muller-Hinton agar (Oxoid, England) and then incubated at 37 °C for 24 hours. The disappearance of MBC has been identified as the lowest concentration of antimicrobial agents have killed 100% of the original pathogens (Swenson, 2005).

Determination of antioxidant activities

DPPH radical scavenging assay

The free radical scavenging activity was evaluated by 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) (Sigma Aldrich GmbdH, Germany) based on the method reported by (Bozin, Mimica-Dukic, Samojlik, Goran, & Igic, 2008) with some modifications involving the number of samples and conditions of incubation. Briefly, 950 µl of the reagent was added to 50 µl of the anthraquinones fraction (5, 10 µg/ml). The volume of the solution was increased up to 4 ml by adding 95% ethanol. The mixtures were shaken vigorously and allowed to stand at room temperature for two hours in the dark. Samples were calibrated by (Shimadzu spectrophotometer, Japan).

Ascorbic acid (10 µg/ ml) was used as positive control. The percentage of DPPH radical scavenging activity was calculated, using the following formula:

DPPH radical scavenging activity (% inhibition) = {(A0 – A1)/A0) x 100}

Where A0 is the detection of the control and A1 is the detection in the presence of all samples and positive control.

The IC₅₀ values representing sample concentrations depend on scavenging 50% of DPPH free radicals.

GC-MS and FTIR analysis

Anthraquinones isolated from C.inerme leaves were identified through the system of Agilent Technologies 7820A, USA. The electron ionization mode was detected by the mass spectrometer at 400 V and the range between 50-600 m/z in this way.

The column of the instrument was set (28 x 30.5 x 16.5 cm) and the temperature was increased from 50^°C to 300^°C at a rate of 1^°C/min. In this analysis, the gas flow rate was set at 1.2 ml/min and the sample volume was set at 1 µl. The concentrations of this fraction were analyzed for retention time and mass fragmentation patterns with the data in the NIST library provided by these institutions. The IR spectra of this fraction were recorded by the FTIR Spectrophotometer (infra Red Bruker Tensor 37, Austria) at room temperature from 375 to 7,500 cm-1 for direct scanning.

Statistical analysis

Data are presented as Mean ± SD. Statistical analysis was performed using SPSS version 20. A one-way ANOVA followed by Duncan's multiple comparisons was used to compare the values ​​of the samples compared with the control. A p-value < 0.05 was taken, indicating the samples' difference. Each treatment was performed three times and each experiment was repeated at least twice.

Results and Discussion

Antibacterial activity

Figure 1 shows the zones' inhibition of anthraquinone fraction from C.inerme leaves at 22.66 mm for S.aureus, 20.33 mm for E.coli, 18.33 mm for K.pneumoniae, and 13.66 mm for P.aeruginosa. The values of MIC and MBC for the leaves anthraquinones of this plant were 12.5, 12.5, 25, 25, 25, 25, and 25, 50 mg/ml for S.aureus, E.coli and K.pneumoniae, and P. aeruginosa, respectively (Table 1). According to numerous literature reviews, C.inerme has anthraquinones in the leaves. These substances have antibacterial, antifungal, and other biological properties (Al-Snafi, 2016; Florence, Joselin, & Jeeva, 2012; Shakeel, Sammia, Waqar, & Sami, 2017).

Antioxidant activity

The results of the percentage of DPPH radical scavenging activity of the leaves anthraquinones was a 91.40% (IC₅₀ 5.43 µg/l) compared to ascorbic acid at (5.16 µg/ml), as shown in Figure 2. The fraction has a high percentage of DPPH radical scavenging activity with significant IC₅₀ values. That said, C.inerme has good antioxidant activity (Chourasiya, Jain, Jain, Nayak, & Agrawal, 2010; Salahudeen & Bolaji, 2019). Previous studies indicate that the leaves of this plant contained anthraquinones so that this component of C.inerme have high antioxidant activity (Zengin, Locatelli, Ceylan, & Aktumsek, 2016) documented that anthraquinones have natural antioxidant fraction from plants.

GC-MS and FTIR analysis

The GC-MS results of the fraction of anthraquinones from the leaves of C.inerme identified nine compounds compared with a similar mass fragmentation in the NIST database library (Figure 3, Table 2). These compounds are Emodin, 1,4-Dihydroxyanthraquinone, 1-Methoxyanthraquinone, 2-Ethylanthraquinone, 9,10-Anthracenedione, 1,5-Dihydroxyanthrachinon, 1,4-Dimethoxybenzene, 1,4-Cyclohexanedione, and 2,3-Dimethylquinone. The results are different in the present study due to differences in the plant environment, GC-MS system, and isolation conditions. The results of the IR spectra (Figure 4) revealed O-H stretching as a strong band at 3347.84 cm-1 for anthraquinones extract from the leaves of C.inerme. The C-H stretch bands were found in bands 2924.15 and 2857.29 cm-1. The C-O group of C.inerme leaves were located in a band of 1733.18 cm-1, 1637.72 cm-1. The C-H bending groups were at 1375.64 cm-1 and the C-O groups were at 1236.81, 1050.16 cm-1. This is in line with the previously published research showing that the presence of main functional groups is anthraquinones in the studied plants (Lv, Chen, Ho, & Sang, 2011; Shami, 2015).

Conclusions

In conclusion, antibacterial activity and antioxidant capacity of anthraquinones fraction from C.inerme leaves grown in Iraq have been studied.This fraction of the leaves had antibacterial effects on S.aureus, E.coli, K.pneumoniae, and P.aeruginosa bacteria in well-diffusion. S.aureus was the most affected, while P.aeruginosa was the least affected. The results of MIC and MBC of S.aureus and E.coli were the smallest and P.aeruginosa was the highest. The anthraquinones fraction of the leaves of C.inerme showed a high level of antioxidant activity with significant values of IC₅₀.The GC-MS analysis of anthraquinones fraction from the green part of C.inerme had identified specific compounds that may be used to develop as antibacterial and antioxidant biopharmaceuticals in the future.