Sandalwood, an Indian medicinal plant attenuates the microbial growth and influence up/down regulation of the metabolites

The methanolic extract of sandalwood (SwME) was prepared by soxhlet apparatus and the antibacterial assay was performed. Further, the metabolite profiling of SwME and lysates of E. coli and E. coli grown in the presence of SwME was generated. SwME showed maximum inhibition against E. coli (MTCC 443) i.e. 82.71%, and minimal against B. subtilis (MTCC 736) i.e. 26.82%. The metabolome profiles of E. coli and SwME were generated using gas chromatography-mass spectrometry (GC-MS) technique. Comparative studies were done to understand to what extent metabolite modifications differ between SwME, E. coli lysate and the E. coli strain grown in presence of extract. Result revealed 23 peaks with major compounds present in E. coli were 9-Octadecenoic Acid (Z)-, Methyl Ester (26.85%), Hexadecanoic Acid, methyl ester (20.5%) and Hexadecanoic acid, trimethylsilyl ester (15.79%). When E. coli was grown in the presence of SwME, GC-MS analysis showed 25 peaks with major compounds such as 9-Octadecenoic Acid, Methyl Ester (21.97%), Hexadecanoic Acid, Methyl Ester (17.03%), and Hexadecanoic Acid, Trimethylsilyl Ester (14.96%). Correlating the metabolic profiles with the changes occurring is essential to progression their comprehension and in the development of new approaches to identify the metabolomics regulation in E. coli in response to SwME.


INTRODUCTION
With the advancement in human livings affected by technology and such innovations, disease causing organisms have also developed themselves.This is currently the most focused aspect of humans, the new or improved ways of defence and cure from the life threatening disease.The present chemical based pharmaceutical is looking at new hopes in traditional or natural origin medicines which would be efficiently effective against disease causing micro-organisms which are now said to immune themselves from the chemical based medicine available in the market (Baker et al., 2007;Beghyn et al., 2008;Briskin, 2000).Nature has all the solutions to mankind problems; such is the natural or ayurvedic medicine derived from the plants, animals or marine.Therefore, several plants have been selected based on their use in traditional systems of medicine (Ji et al., 2009).
Among the thousands of such therapeutic plants, Sandalwood is one of the well known plants from the Indian origin that has been used in cosmetics and therapeutics and is used by Ayurvedic medicinal practitioners in India for almost 3000 years.It is known for its multi-therapeutic properties and is being used since a long time in this part of world as a cosmetic and therapeutic agent.
The Indian plant, Santalum album may serve as a dietary antioxidant with several modes of action to protect the body organs against oxidative damage and age-related cognitive decline.Aqueous extract of S. album showed good activity against Staphylococcus aureus, Bacillus subtiliss, Pseudomonas aeruginosa and Escherichia coli and the aqueous extract showed maximum activity against S. aureus (Shamshi et al., 2014).
Plants are known to possess the various complex phytochemical compounds that may be used in the prevention and treatment of several human diseases (Hall et al., 2002).Identification of phytochemicals or bioactive compounds and their characterization are required to explore the medicinal value of the plant which is an ongoing challenge in this era.This process needs several extraction methods for the metabolites extraction and the advanced techniques that can identify, detect and characterize these bioactive compounds (Wolfender et al., 2015).
Metabolomics is a field where various analytical strategies and platform in application of natural extracts develop the chemical profiles.The information obtained from this approach might be useful in the determination of various biological aspects of plants such as defense, growth, development, productivity and responses to external stresses etc (Desai and Alexander, 2013;Misra et al., 2014).Gas chromatography (GC) coupled with mass spectrometry (MS) is the analytical platform and strategy that can be employed for the analysis of metabolites found in the plant extracts qualitatively and quantitatively (Wong et al., 2015).GC-MS analysis can identify up to approximately 100 metabolites in the simple plant extracts and can generate a metabolite profile (Benina et al., 2013;Obata et al., 2013;Sauter et al., 1991;Lee et al., 2014).The effective and better identification of the obtained metabolites can be easily done by comparing the results with the database stored in the large mass spectral library based on GC-MS electron impact (EI) (Musharraf et al., 2013).The sample preparation for the extraction of metabolites also plays a key role in this analytical platform of metabolomics and is known to be a major factor that affects the profiling of metabolite (Ying et al., 2009).Metabolites extraction can be done in various organic solvents such as ethanol, water, methanol and hexane and can be carried out by using various conventional extraction methods followed by lyophilization and then monitoring and generating the profiles of extracted metabolite using several analytical platforms (Lapornik et al., 2009;Amzad and Shah, 2015).Various factors such as type of solvent, extraction method and temperature etc affect the metabolic content of crude extracts of plants (Kindt et al., 2009).For the study of the aforementioned properties of S. album, antibacterial assay and GC-MS analysis was performed to know the metabolite composition of S. album methanolic extract, E. coli (the most susceptible bacterial strain) lysate and the E. coli grown in the presence of S. album methanolic extract.

Plant material/ Chemicals
The medicinal plant sandalwood (S. album) was purchased from Local Ayurvedic Clinic, Rampur, Uttar Pradesh, India.The chemicals of analytical grade were purchased from Merck and Himedia.

Preparation of methanolic crude extract of sandalwood
The sandalwood was finely ground in a grinder mixer and then 10 g of powdered sandalwood placed in a Soxhlet apparatus.Extraction was carried out with 250 ml of methanol for 36 to 48 h.The temperature was maintained below the boiling point of the solvent.Further, the extract was filtered through a 0.45 μm filter.The filtrate was then concentrated by Rotary Evaporator and it served as the methanol extract of sandalwood (SwME) for further exploration and activities.The extract was stored in a refrigerator at 4°C for further use.

Determination of antibacterial potential of sandalwood methanolic extract (SwME)
SwME was evaluated for its antibacterial potential against several bacterial strains using the method as described by Shamsi et al. (2014) with some modifications.A single colony form agar plates of each bacterial strain was inoculated in autoclaved Luria broth (LB) media for bacterial growth.The cultures were incubated at 37°C for 12 to 24 h.Subsequently, the LB broth was added to the bacterial culture to dilute it upto 1 × 10 4 colony-forming units (CFU).Approximately, 50 μl of 5 mg/ml SwME (250 μg SwME) was mixed with diluted bacterial cultures in 96well plate.The final volume in each well was maintained up to 300 μl using LB media.Ampicillin (Stock 10 mg/ml) was used as positive control and 25 μl of it (250 μg ampicillin) was applied in each well containing LB media and bacterial broth culture.The results were compared with negative controls which contained the LB media and bacterial culture.Blanks contained LB media only and the LB media with SwME.96well plate was further incubated at 37°C for 14 to 16 h.After incubation, absorption measurements were determined using ELISA microplate reader at 600 nm.The percentage mean growth inhibition (MGI%) was calculated by % MGI = [(Ac -At)/Ac] × 100.Where, Ac and At are the absorbance of negative control and SwME treated strains respectively.The EC50 value (concentration causing 50% reduction in the bacterial growth) was calculated using Microsoft office Excel 2007, based on the readings.

Homogenization and extraction
Homogenization and extraction were the preliminary step to quench biomass for the release of metabolites.Homogenization is carried out using a grinder.The solvent methanol was degassed and cooled before the extraction procedure.

Biomass quenching
SwME, and the lysates of E. coli and E. coli grown in the presence of SwME were taken and the biomass centrifuged for 20 to 30 min to pellet down the biomass at 5000 rpm at 4°C.Thereafter, the supernatant was discarded and an approximately 1 ml aliquot preserved for the determination of leakage of internal metabolites.Further, the pellets and supernatants were frozen in liquid nitrogen followed by storing them at -80°C and then further analysis done.

Extraction of metabolites
The biomass in the form of pellets were mixed with 500 μl of 100% chilled methanol (-48°C), followed by freezing in liquid nitrogen and then allowed to thaw on dry ice.The process of freezing followed by thawing was performed several times which helped in the permeabilization of the cell membrane, which resulted in the metabolite leakage from the cells.The samples were then centrifuged at 10000 g for 5 to 10 min at 4°C.Thereafter, the supernatant was obtained and preserved on dry ice.Approximately, 500 μl aliquot of 100% methanol (-48°C) was further mixed with the pellet and then stored on dry ice to perform further analysis.

Mass spectrometric analysis
GC-MS analysis of lysate of E. coli, SwME and E. coli grown in presence of SwME was performed on a GC-MS equipment.The dimensions of GC-MS column (non-polar column) used were about 30 m × 0.25 mm with a film thickness of 0.25 mm based on capillary standard.The flow rate was maintained at 0.5 ml/min and approximately 1 µl of the concentrated sample was utilized.The methanolic samples were run fully at a range of 50 to 650 m/z and the inlet temperature was maintained at 250°C.The samples were run for approximately 90 min and GC-MS examined using electron impactionization at 70eV.The obtained spectrums of the samples were compared with the database of spectrum of known compounds which are stored in the GC-MS NIST library to evaluate the total ion count for compound identification and robust quantification.Further, the peak areas and retention time etc were measured and data processing performed by Turbo-Mass software.The raw GC-MS chromatograms were transferred to a server station by recording two scans per second in full scan mode.

Data acquisition by GC-MS
Peak identifications were carried out by matching retention indices and mass spectral similarity against a user-defined metabolite library.Metabolite peaks were quantified by area of target ion traces.The obtained number of metabolites was counted per chromatogram.

Identification of components
Identification of the obtained compounds were done by comparing their GC-MS mass spectrum using the database of National Institute Standard and Technology (NIST) with software Turbomas 5.2 which stores approximately 62,000 patterns based on their molecular mass, molecular structure and calculated fragments.The database then generated a table which included the name, molecular weight and structure of the tested components by comparing the database.The percentage relative of each compound was calculated by comparing the average peak area in the chromatogram to the total areas.The detection of compounds helped in the determination of the current commercial, industrial and traditional use of crude extracts as an herbal medicine.This method also helped in the determination of the best extraction methods for these components.The identified compound can now further be explored for their putative biological or therapeutic relevance.GC-MS software that was able to perform multi-target analysis also included quantitation by area and height of the peak in the chromatogram.

Statistical analysis
The experiments were performed in triplicates (n=3).Graph Pad Prism Software was used to prepare Graphs and calculate the statistical parameters such as mean and standard error of mean (SEM) etc.The data obtained were expressed as mean ± SEM.

Determination of antibacterial activity of SwME
Antibacterial potential of the SwME was evaluated by  Generally, B. subtilis was the only strain which was less susceptible to the antibacterial effect of the SwME and ampicillin as compared to the other strains.SwME exhibited different degree of growth percentage inhibition among tested bacterial strains which were presented in the form of EC50 values (Table 1).

GC-MS analysis
Identification and quantification up to 100 metabolites can be performed using an important and critical technique, that is, GC-MS based metabolite profiling.These metabolites may incorporate sugars, hydrocarbons, phenolic compounds, carboxylic acids, sugar alcohols, alcohol acids, esters, amino acids and polyamines etc and provide a far reaching scope of the primary metabolism central pathways.The present study emphasizes on use of the GC-MS analysis for identification and quantification of the metabolites or compounds present in the methanolic extract of sandalwood, E. coli and the E. coli grown in presence of SwME.

Identification of compounds in E. coli grown in the presence of SwME
When E. coli was grown in the presence of SwME to find the impact of bioactive compounds of the SWME extract on the metabolites present in the E. coli, the methanolic lysate was explored to find the up or down-regulated metabolites using GC-MS technique.The results obtained showed 25 peaks indicating the presence of twenty one compounds.This time, some different compounds were observed which was neither present in SwME nor E. coli.
The results obtained here suggested that, sandalwood up regulated few metabolites in E. coli which may be present in the negligible amount and hence, this up regulation might be responsible for the bacterial death.The different metabolites observed were 4).0( 6 [1.alpha.,5.alpha.,6.alpha

Time (min)
Figure 4: A typical chromatogram of the metabolites present the methanolic lysate of E. coli grown in presence of sandalwood methanolic extract.
The present investigation is only a preliminary characterization of the presence of some particular properties in sandalwood extract.Further investigation and in-depth knowledge is required to identify the biochemical and phytochemical functions of the herbs.The metabolites which were identified here by GC-MS analysis revealed that they possess many biochemical and pharmacological potential and can be based on Dr. Duke's Phytochemical and Ethnobotanical Databases created by Dr. Jim Duke of the Agricultural Research Service/USDA.

DISCUSSION
Medicinal plants are considered new as new assets for producing agents that go about as alternatives to antibiotics against antibiotic-resistant bacteria.It is very important for an antimicrobial agent to be eco-friendly and biologically safe.This study revealed that sandalwood could be a promising antibacterial agent as it showed inhibition against various bacterial strains, that is, the maximum and minimum inhibition of SwME was found to be against E. coli and B. subtilis, respectively.In addition, the compounds which get up/down regulated might be responsible for this activity.Shamsi et al. (2014) in their study investigated that the aqueous extract of S. album showed effective inhibitory activity against S. aureus (MTCC 902).The results from antibacterial activity of SwME revealed that E. coli was the most sensitive strain with highest percentage inhibition towards methanolic extract.Stenz et al. (2008) in their study showed when 9-octadecenoic acid was added after primary adhesion was developed; it stimulated biofilm formation and hence, bacterial growth.Through our study, it is confirmed that when E. coli was grown in the presence of SwME, 9octadecenoic acid was down-regulated which confirms the result of the aforementioned study.
The presence of different compounds in SwME through GC-MS analysis showed sandalwood to be the promising therapeutic agents as the compounds present in methanolic extract possess anti-microbial, anti-oxidant, anti-ulcer and anti-asthamic activities etc. Hexadecanoic acid present in methanolic extract of sandalwood posses antiinflammatory, anti-oxidant, hypocholesterolemic nematicide, pesticide, anti androgenic flavor, hemolytic, 5-Alpha reductase inhibitor and potent mosquito larvicide (Aparna et al., 2012;Kumar et al., 2010;Rahuman et al., 2000;Mustapha et al., 2016), 9-12 Octadecenoic Acid (Z)-, Methyl Ester shows anti-cancer and antimicrobial activity (Yu et al., 2005;Rahman et al., 2014).Piperidine has characteristic anti-asthamic activity (Matsushita et al., 1998).The literature survey also confirmed the presence of 9-Octadecenoic Acid as a plant metabolite in the hexane soluble extract of the Croton tiglium seeds (Saputera et al., 2006).Sreevidya and Chandradhar (2015) in their study revealed the presence of α-santalol as chemopreventive agent appears to be very promising in skin cancer control.The present study also confirms the anti-cancerous activity of S. album through the presence of 9 Octadecenoic acid and Sentlyl acetate in the methanolic extract.
The results pertaining to GC-MS analysis of the methanolic extract of S. album has led to the identification of various compounds which have manifold therapeutic benefits.The identified metabolites were known to possess various bioactivities such as anti-fungal, anti-inflammatory, skin disorders and anti-bacterial etc.The extract showed the presence of a variety of fatty acids and antiinflammatory compounds such as hexadecanoic acid, fragrance and flavoring agents such as tetradecanoic acid are found in the plant extract (Burdock and Carabin, 2007).This study explored the therapeutic significance of the sandalwood which has great effect on bacteria causing the metabolite regulation and can be proposed as a herb of phytopharmacological importance.Various chemical compounds which are indentified in methanolic extract of sandalwood, E. coli lysate and E. coli grown in methanolic extract of sandalwood help in the determination of the possible synergistic benefits of the plant and their potential as anti-microbial agent.This also opens up ways to investigate it further by isolating the single bioactive compounds leading to the further investigation of specific biological and pharmacological potentials (Sermakkani and Thangapandian, 2012).

Conclusions
This study evaluated the anti-bacterial potential of SwME and the profiling of bioactive compounds generated by GC-MS analysis in SwME extract and in the lysate of E. coli.The results concluded that SwME was a strong anti-microbial agent showing fair anti-bacterial activity against all the bacterial strains.GC-MS chromatogram also showed various compounds in SwME with maximum peak intensity which could contribute to the medicinal value of the sandalwood.This study was performed to analyze the effect of sandalwood methanolic extract on the bacterial metabolites when grown in the presence of the extract.The result showed the presence of the compound in the extract such as hexadecanoic acid and 9 Octadecenoic acids which exerts antimicrobial activity.Anti-bacterial activity of methanolic extract found to be impressive against E. coli proved that sandalwood has great potential for development of therapeutic agents against microbial ailments and it can also be used as alternative or antibiotic.However, isolation and purification of single bioactive compound and their biological activity will be certainly giving productive outcomes and open a new area of investigation of individual components and their pharmacological potency.From these results, it could be inferred that S. album contains diverse bio-active compounds.Evaluation of pharmacological activity is under progress.Along these lines, it is suggested as a plant of phytopharmaceutical importance.

Figure 1 :
Figure 1: Bar diagrammatic representations of in vitro antibacterial activity of sandalwood methanolic extract.The bars represent the percentage mean growth inhibition by methanolic extract of sandalwood when tested against 4 bacterial strains.

Figure 2 :
Figure 2: A typical chromatogram of the bioactive compounds present in methanol crude extract of sandalwood plant showing different peaks.
Figure 3: A typical chromatogram of the metabolites found in the methanolic lysate of E. coli.

Table 1 :
Determination of EC50 values of SwME and ampicillin and represented in mg/ml.
Bacterial strains EC50 (mg/ml)E.coli(MTCC443)0.50 S. aureus(MTCC 902)0.95P.aeruginosa(MTCC245)0.57B.subtilis(MTCC736)1.54assessing the percentage mean growth inhibition in the presence of SwME and the results were compared to the proper positive control.The results exhibited fair antibacterial property in-vitro against all the tested strains, that is, the bacterial growth was diminished in the presence of SwME as shown in Figure1.Results showed that SwME had strongest inhibitory activity against E. coli (MTCC

Table 2 :
Biologically active chemical compounds of methanol extract from sandalwood, showing retention time, molecular formula and peak area (%).

Table 2
Contd: Biologically active chemical compounds of methanol extract from sandalwood, showing retention time, molecular formula and peak area (%).

Table 4 :
GC-MS analysis revealed the presence of compounds in the methanolic lysate of E.coli grown in presence of sandalwood methanolic extract showing retention time, molecular formula and peak area (%).