Glycyrrhiza glabra HPLC fractions: identification of Aldehydo Isoophiopogonone and Liquirtigenin having activity against multidrug resistant bacteria

Background Medicinal plants have been founded as traditional herbal medicine worldwide. Most of the plant’s therapeutic properties are due to the presence of secondary metabolites such as alkaloids, glycosides, tannins and volatile oil. Methods The present investigation analyzed the High-Pressure Liquid Chromatography (HPLC) fractions of Glycyrrhiza glabra (Aqueous, Chloroform, Ethanol and Hexane) against multidrug resistant human bacterial pathogens (Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus and Pseudomonas aeruginosa). All the fractions showed antibacterial activity, were subjected to LC MS/MS analysis for identification of bioactive compounds. Results Among total HPLC fractions of G. glabra (n = 20), three HPLC fractions showed potential activity against multidrug resistant (MDR) bacterial isolates. Fraction 1 (F1) of aqueous extracts, showed activity against A. baumannii (15 ± 0.5 mm). F4 from hexane extract of G. glabra showed activity against S. aureus (10 ± 0.2 mm). However, F2 from ethanol extract exhibited activity against S. aureus (10 ± 0.3 mm). These active fractions were further processed by LC MS/MS analysis for the identification of compounds. Ellagic acid was identified in the F1 of aqueous extract while 6-aldehydo-isoophiopogonone was present in F4 of hexane extract. Similarly, Liquirtigenin was identified in F2 of ethanol. Conclusions Glycyrrhiza glabra extracts HPLC fractions showed anti-MDR activity. Three bioactive compounds were identified in the study. 6-aldehydo-isoophiopogonone and Liquirtigenin were for the first time reported in G. glabra. Further characterization of the identified compounds will be helpful for possible therapeutic uses against infectious diseases caused by multidrug resistant bacteria.


Background
Medicinal plants are used for the treatment of various infections [1,2]. These plants contributed as a source of inspiration for novel therapeutic compounds [3]. The medicinal value of plants is due to the presence of a wide variety of secondary metabolites including alkaloids, glycosides, tannins, volatile oil and terpenoids [4]. The distribution of G. glabra plant is worldwide, the plant is reported from different regions e.g. Central Asia, Spain, Italy, China, Turkey, Iran, India [5] and Pakistan.
Glycyrrhiza glabra has a sweet wood and usually employed medicinally as an expectorant and carminative. Moreover, since long time G. glabra is used as a potential therapeutic herb found in various parts of the world. The herb is also used for the treatment of resistant microbes involved in the infections of skin, respiratory tract, urinary system and as anti-ulcer activity. The use of G. glabra is also reported in Unani an Ayurveda medicines [6,7]. Besides, Oil from G. glabra is used as a flavoring agent and natural sweetener. The compounds which add these features are glycyrrhizin and some volatile compounds, saponins and flavonoids. Due to these characteristic G. glabra oil extract is also used in confectioneries, personal care products, food items, beverages and cosmetics [8]. Phenolics compounds from the root of G. glabra are isolated which protect low density lipoproteins and from oxidative damage [9].
Literature data is available on the antibacterial activity of G. glabra crude extract; however, no data exist on the biological activity of HPLC fractions from G. glabra extracts. In the present study, gradient HPLC fractions were collected, and only these fractions with antibacterial activity were further investigated by LC MS/MS analysis for the identification of bioactive compounds. Findings of the study will be helpful for elucidation of lead molecules for possible therapeutic intervention.

Methods
The current study was performed to check the antibacterial activity of G. glabra's chromatographic fractions against multidrug resistant (MDR) pathogenic bacterial isolates at the Department of Microbiology, Kohat University of Science and Technology (KUST), Kohat.
Ethical approval was waived by the Departmental Review Board (DRB) with a reference number MIC/KUST/2118.

Processing of G. glabra
G. glabra (Mulaithi) plant was collected from Khyber Pakhtunkhwa (KPK), Pakistan, and was morphologically identified by plant taxonomist Prof. Dr. Waheed Murad at the Herbarium of Botany department, Kohat University of Science & Technology, where the voucher sample (10,052/GG) was deposited. After identification, G. glabra was washed, air dried and chopped. The plant pieces were dried and mashed into powder form for further processing [10]. The powdered plant was subjected to extraction process as described earlier [11].

Gradient HPLC fractionation
Solidified extracts of plants were processed for HPLC fractionation by dissolving in 60% methanol as described [12].

Collection of multi drug resistant (MDR) bacteria
Pure cultures of the MDR human were obtained from the Department of Microbiology, KUST and confirmed on the basis of culture, microscopy and biochemical characteristics [13].

Antibacterial activity of plant extracts
Bacterial cultures were inoculated on Muller Hinton agar. Three HPLC fractions of G. glabra plant were poured in three wells of each plate while DMSO was placed as a negative control. Results were interpreted by using standard guidelines [14].

LC MS/MS analysis
Those fractions which showed antibacterial activity were processed bioactive compounds identification using LC MS/MS (LTQ XL, Thermo Electron Corporation, USA) method as described earlier [15].

Bioinformatics and data analysis
Structure parameters for each compound were obtained by using online database software (www.chemspider.com).

Results
A total twenty (n = 20) gradient HPLC fractions (Five fraction for each extract) were collected from extracts of G. glabra plant and processed for antibacterial activity.
The zones of inhibition showed by different fractions of G. glabra extracts were measured against known MDR bacterial isolates. Among total twenty HPLC fractions, three fractions of aqueous, hexane and ethanol extract exhibited activity against MDR bacteria. Among the total HPLC fractions of G. glabra, three fractions showed activity against multidrug resistant (MDR) bacterial isolates. When five HPLC fractions from aqueous extracts were checked, only fraction 1 (F1) exhibited a zone of inhibition (15 ± 0.5 mm) against A. baumannii (Table 1).
A zone of inhibition (10 ± 0.2 mm) was observed against S. aureus (MRSA) by fraction 4 (F4) of hexan extract of G. glabra. When F2 of ethanol extract was processed for antimicrobial activity, a zone of inhibition (10 ± 0.5 mm) was observed against S. aureus. All the active fractions were subjected to mass spectrometric analysis for compounds identification (Table 1).
After LC MS/MS and bioinformatics analysis (Table 2) , Ellagic acid was identified in the F1 of aqueous extract of G. glabra (Fig. 1). In the F4 of hexane extract, 6aldehydo-isoophiopogonone was identified (Fig. 2).  Lastly Liquirtigenin was identified in the F2 of ethanol extract (Fig. 3).

Discussion
In the present study G. glabra was selected for HPLC fractionation and to check the antibacterial activity of these fractions. These plant HPLC fractions were screened against selected multi-drug resistant bacteria. Fraction of chloroform extracts showed considerable activity against A. baumannii and E. coli. Previously the compounds responsible for their antimicrobial activity were identified by HPLC fractionating plant extract and determining the antimicrobial activity of each fraction against A. baumannii [16]. Earlier study has been conducted to check the antimicrobial activity of G. glabra extracts prepared in different solvents. In a study the chloroform and acetone extracts of G. glabra showed good activity against two Gram positive and two Gram negative bacteria [17]. Among ethanol extract HPLC fractions, only fraction 2 (F2) have antibacterial effect against S. aureus. Potential antimicrobial activity of G. glabra extracts was reported against S. aureus and E. coli respectively [18,19]. Aparajita Gupta (2013) also evaluated the promising activity of methanol and acetone extract of G. glabra against E. coli and S. aureus [20]. The F4 fraction of hexane extract showed considerable antibacterial activity. In a study six medicinal plants including G. glabra were investigated for pharmaceuticals and their role in antimicrobial activity against antibiotic resistant bacteria isolated from pharmaceuticals and hospital [21]. Likewise F1 of aqueous showed anti-MDR activity. The above results of a study suggested that the ethanol fraction contained maximum soluble bioactive compounds which may be responsible for the highest antibacterial activity.
Fractions exhibited potential antimicrobial activity was further subjected to LC MS/MS analysis for the identification of bioactive compounds. Fraction 2 from ethanol was identified as Liquirtigenin. 6-aldehydo-isoophiopogonone was identified in fraction 4 of Hexane extract. There is no data reported on 6-aldehydo-isoophiopogonone from G. glabra; however, this compound was isolated from Ophiopogon japonicas plant as homoisoflavonoids which showed antioxidant activities [22]. Ellagic Acid was identified in fraction 4 of hexane extract. This compound is for the first time reported in G. glabra; however other studies reported this compound for its anti-mutagenic, antimicrobial, antioxidant properties [23].

Conclusions
Among the total HPLC fractions of plant G. glabra, three fractions showed activity against multidrug resistant (MDR) bacterial isolates. 6-aldehydo-isoophiopogonone and Liquirtigenin were for the first time reported in G. glabra. Further studies on these compounds may lead to the development of new potential antibacterial compounds.

Availability of data and materials
The data and materials are available from the corresponding author on reasonable request.
Authors' contributions HR and IK designed the study, carried out the experiments, analyzed the data and drafted the manuscript. AH, AAS and AT helped in the experiments, interpreted the results and drafted the manuscript. MQ analyzed the data and drafted the manuscript. MA, MSA, RU and SNK contributed in plant selection, results interpretation and discussion. All authors read and approved the final manuscript.
Ethics approval and consent to participate A local ethics committee ruled that no formal ethics approval was required in this particular case.