1.0 INTRODUCTION:

Drug resistance is a rapidly growing universal problem which is compounded by the short reproductive rates of microbes. Incomplete antibiotic treatment of patients and inappropriate usage of antimicrobials as food preservatives exacerbates this issue. Managing the use of medically important antibiotics in all kind of food products is very important to reduce risks from drug resistant pathogens. Frequent usage of antimicrobials in livestock may lead to the development of more virulent and multidrug resistant microbes which transmit from animals to humans¹. National Antimicrobial Resistance Monitoring System (NARMS) retail meat report 2010 revealed that meat and poultry products are contaminated with multidrug-resistant bacteria belonging to the species Campylobacter, Salmonella, Enterococcus and Escherichia coli². Staphylococcus aureus was found to be the most overriding multidrug resistant pathogen in 136 meat and poultry samples collected from five US cities³. A study conducted on the dissemination of drug resistant S.aureus in hospital wards, public places, both indoor and outdoor and poultry farms mainly revealed that air is the possible mode of transmission for these microbes ^4-6.

Future world would be most treacherous if the problem of drug resistance is neglected. Hence search for strong antimicrobial compound(s) active against drug resistant pathogens, may possibly be the key for disease control. The genus Penicillium is known for the production of secondary metabolites that include drugs against MRSA ⁷, cancer ⁸ and malarial parasite ⁹. Both bacteria and fungi from marine environments have been reported to produce metabolites with anti-MRSA activity ^10,11. In contrast, reports of anti-MRSA compounds isolated from soil environment, especially soil fungi, are very few. The present study involved the isolation and screening of soil fungal metabolites against clinical isolates of MRSA.

2.0 MATERIALS AND METHODS:

Media:

Media and solvents: Sabouraud Dextrose Broth (SDB), Czapek Yeast autolysate Broth (CYB) and other chemicals purchased from HiMedia. ManjuKori broth (MKB) (grams/liter): Glucose -20, yeast extract- 20, K₂HPO₄ – 4, CaCl₂.2H₂O-0.2, MgSO₄.7H₂O-1, Na₂SO₄-1, FeSO₄.7H₂O-0.010, MnSO₄.4H₂O-0.006, CuSO₄.5H₂O-0.015, ZnSO₄.H₂O-0.005g, pH-7.02.

Clinical isolates:

10 clinical isolates of MRSA obtained from Narayani Hospital, Vellore, India. All experiments were conducted in triplicate and mean of three values were used for the graphical presentation and tabulation.

2.1 Collection of soil sample and its analyses:

Soil sample was collected using sterile techniques, during the month of July-August 2010, Vellore, India. Top soil vegetation was cleared and sample was collected from 20 cm depth. Subsequently, a thermometer was inserted in deep soil for 10 minutes and temperature was recorded. pH was determined in slurry of 10 grams soil prepared in 50 ml of distilled water. Chemical and biological properties of soil were analysed by Shri AMM Murugappa Chettiar Research Centre (MCRC), Taramani, Chennai by following an alternative analytical indigenous technology developed by them and IIT (M). Chemical analyses and estimations carried out were the total organic carbon, NPK, Ca, Mg, Na, Fe, Mn, Cu, Zn, B, Mo, Sulphate and humus along with total minerals. Chromatogram of soil sample was prepared at MCRC using recommendations from Bio-Dynamic association of India¹², (http://www.biodynamics.in/ chrom.htm).

2.2 Isolation and identification of fungi and screening of their metabolites for anti-MRSA activity:

Soil fungi were isolated using the soil dilution method¹³. Colony characteristics were compared with data outlined in standard manuals¹⁴, literature reports¹⁵ and fungal database, available with International Mycological Association. Screening of metabolite’s antibacterial activity was carried out by growing fungi in Yeast Extract Sucrose broth (YESB) of pH 7.0 ± 0.2. In 5ml of sterile YESB, 8mm agar block of each fungus was inoculated and tubes were incubated at room temperature (28⁰C) for ten days. After incubation period, 100µl of each YESB was taken and checked for antibacterial activity by agar well diffusion method ¹⁶. Based on these results, the fungus which showed the highest activity i.e., Penicillium sp VIT-2012, was selected for further studies.

2.3 Selection of medium, temperature and pH for production of biomass and anti-MRSA metabolites of Penicillium sp VIT-2012:

In order to select the best medium that support for synthesis of anti-MRSA metabolites , Penicillium sp VIT-2012 was grown in five fungal media viz., SDB, Malt Extract Broth (MEB), YESB, CYB and MKB. MKB was an artificially designed medium based on the soil analyses report. The tubes were incubated for 10 days at room temperature (30⁰C) after which their antibacterial activity was assessed by agar well diffusion method. The medium in which highest anti-MRSA activity was found was considered as optimum medium. To determine the optimum temperature, 25ml of MKB was prepared in 100ml conical flasks and were inoculated with 1ml of 10⁶conidia/ml of Penicillium sp VIT-2012. Flasks were incubated at 5⁰C, 10⁰C, 15⁰C, 20⁰C, 25⁰C, 30⁰ C, 35⁰C, 40⁰C and 45⁰C for 10 days. Determination of optimum pH was carried out at the optimum temperature. All conditions like quantity of inoculum, medium and incubation time were maintained similar to the temperature experiments and the pH was adjusted from 3.0 to 9.0 at one pH unit intervals. On 10^th day, anti-MRSA activity, in terms of zone of inhibition and biomass (both wet and dry weights) were measured for temperature and pH experiments.

2.4 Mass production and determination of MIC of crude ethyl acetate extract of Penicillium sp VIT-2012:

With the optimum temperature and pH, the mass production of Penicillium sp VIT-2012 was carried out in three liters of MKB medium. After 10 days, broth and mat were separated and extracted crude metabolites with ethyl acetate thrice and concentrated to 10 ml using rotory evoparator. Since the yield and activity of mat extarct was higher then that of broth extract therefore crude extract of mat was used for further study. MIC of mat extract fraction was evaluated as per Clinical and Laboratory Standard Institute guidelines against 10 clinical isolates of MRSA obtained from Narayani Hospital, Vellore, India. MIC values of crude extract of mat were compared with that of standard vancomycin drug.

2.5 UV, FT-IR and GC-MS Analyses of crude extract:

A small amount of the mat extract was pelletized with KBr and the spectrum was recorded for functional group analysis. Extract was also analysed by GCMS, after filteration through membrane (0.45µm pore size) and data was interpreted through computer software TurboMass ver. 5.4.2 and matched with NISTO2 2008 version library.

2.6 Phylogeny of Penicillium sp VIT-2012 based on 28S rDNA sequencing

Penicillium sp VIT-2012 was sequenced for 28S rDNA gene for identification^17,18. Fungal DNA was isolated and amplified using the forward (LR0R-5’-ACCCGCTGAACTTAAGC-3’) and reverse (D1/D2-NL4–5’-GGTCCGTGTTTCAAGACGG-3’) universal primers of large subunit 28S rDNA gene. The resultant 718 nucleotide 28S rDNA sequence was matched with NCBI database in Blast and submitted to GenBank of NCBI.

RESULTS:

The temperature and pH of soil sample were found to be 30⁰C and 7.0 ±0.2 respectively. Its chromatogram revealed a prominent inner zone of diameter 3.1cm and was pinkish brown. The middle zone of diameter of 2.2 cm was dark brown in colour. No outer zone was observed but small protrusion of spikes into it was seen (Fig.1). Among macronutrients and micronutrients, the amount of nitrogen and phosphorus were found to be at low and boron and molybdenum were not detected. On the other hand, the concentration of calcium, magnesium, iron, manganese, copper and humus were high. Optimum concentrations of organic carbon, zinc, potassium and sulphur were found (Table 1).

Table 1: Chemical properties of soil sample:

Soil property	OC	HA	N	P	K	Ca	Mg	Na	Fe	Mn	Cu	Zn	Sulfate
Conc.	0.77	161	110.2	10.74	94.87	433	170	124	14	11.5	2.2	0.76	14.22
Rec. level MCRC	0.75-1.5	18 -31	113-182	18-36	60-138	>300	10-15	-	6-8	1.2-2.5	0.3-1	1.5-1	10-15

[Note: Organic Carbon (OC ) in %, humus (HA) to K in Kg/acre of soil, Ca to Sulfate in Mg/Kg of soil]

Table 2: Enzymatic and microbial properties of soil sample:

Soil property

Protease

(µgs Tyr/g/hr)

Cellulase

(µgs Tyr/g/hr)

Invertase

(µgs Tyr/g/hr)

Bacteria

(10⁹cfu/gm)

Actinomycetes

(10⁶cfu/gm)

Rhizobium

(10⁶cfu/gm)

Fungi

(10⁶cfu/gm)

Conc.

49.5

0.371

0.178

0.8

100

Bacteria were the dominant microbial flora in soil sample, especially those of Rhizobium species. This may be due to association of Rhizobium with roots of the vegetation. The second largest population was that of fungi. Among the soil enzymes, protease activity was found to be the maximum followed by that of cellulase and invertase (Table 2).

From the soil sample, sixty one fungal colonies, belonging to five genera viz., Penicillium, Fusarium, Aspergillus, Cladosporium and Trichoderma were isolated. Among these, ten species were identified, of which Penicillium sp and Fusarium sp were dominant followed by Aspergillus sp Cladosporium sp and Trichoderma sp. The metabolites from five isolates were found to be active. Genus Penicillium, comprising of 30%, showed highest activity, followed by Aspergillus (10%) and Trichoderma (10%) (Fig. 2).

Fig 1: Showing chromatogram of soil sample.

Fig 2: Total number of fungal species isolated from soil sample and anti-MRSA activity.

The zone of inhibition (in mm), of these metabolites against MRSA, for growth between 4^th to 20^th day, is shown in (Table 3).

Table 3: Anti-MRSA activity of soil fungi screened from 4^th day to 20^th day.

[^a Mean value of three, ^b Daysof incubation]

Soil sample	The organisms	Zone of inhibition (mm^a)
Soil sample	The organisms	4^b	8^b	12^b	16^b	20^b
02VITLB	Aspergillus niger	Nil	Nil	10	Nil	Nil
02VITLB	Penicillium sp1	Nil	Nil	Nil	05	08
02VITLB	Penicillium sp VIT-2012	Nil	08	10	16	18
02VITLB	Penicillium sp4	Nil	Nil	Nil	Nil	08
02VITLB	Trichoderma sp1	Nil	Nil	Nil	Nil	05

Among Penicillium isolates, only one species i.e., Penicillium sp VIT-2012 showed maximum and constant activity from 8^th to 20^th day of growth . Hence this isolate was selected for further studies. The metabolites of Penicillium sp VIT-2012 showed the maximum anti-MRSA activity on MKB compared to rest of the media (Fig. 3).

Fig 3: AntiMRSA activity and medium selection of Penicillium sp VIT-2012.

Penicillium sp VIT-2012 was identified by growing on different fungal media and also by SEM and 28S rDNA sequencing. Three point inoculation of Penicillium sp VIT-2012, on three fungal media, indicated colony morphology resembling that of its close relavtive Penicillium radicum¹⁹. On CYA , colony morphology showed cottony white mycelium with a yellow center. Yellowish green colour mycelia were observed on MEA surrounded by yellow colour vegetative mycelia. Light pigmentation was seen at the backside of the colony on CYA and MEA. However, the colony on SBD was yellow in colour, and contained constrictions radiating from its centre. SEM results of Penicillium sp VIT-2012 indicated the long stipe containing terverticillium metulae. Each matula contained conical shaped shrunken philides and was brush like in appearance. Conidia were arranged in chains which grew from each philide. The shape of the conidia were ellipsoidal, wrinkled and shruken with smooth surface (Fig. 4).

Phylogenetic tree analysis of experimental isolate constructed based on 28S rRNA gene sequence indicated its close relation with P. radicum. The Blast results showed 99% homology with P. radicum and a new isolate had emerged in lower clad as a separate branch, indicating new isolate¹⁹.The 718 nucleotide sequence was deposited in the GenBank database in NCBI with Accession No JX908779 (Fig. 5).

On this most favorable medium, i.e. MKB, neither growth nor activity were noticed between 0⁰C to 20⁰C and 40⁰C and 45⁰C. However, activity was observed at 25⁰C and 35⁰C in terms of zone of inhibition. The maximum activity was seen at 30⁰C. Growth of the organism commenced from 15⁰C with maximum growth being observed at 30⁰C, measured in terms of wet and dry biomass. The growth decreased beyond this temperature. Penicillium spVIT-2012 was found to grow at a wide range of pH, and growth, as well as anti-MRSA activity, of its metabolites was observed at almost all pH values, i.e., from 3.0 to 9.0. Activity was found to be comparable between pH 4 and 9 with significantly higher values being observed at pH 6.

However, maximum biomass was produced at pH 7.0 (Fig. 6).

Mass production of anti-MRSA metabolites on MKB, at optimum temperature 30^OC and pH 6 yielded 280 milligrams broth extract and 2.156 grams of mat extract. Both extracts showedstrong anti-MRSA activity against ten clinical isolates checked at mg/ml concentration. But the highest antiMRSA activity found in mat extract in contrast to broth extracts. Further the MIC values of mat extract were found to be 25µg against seven isolates and 12.5µg against three isolates in comparison to 1.6 µg MIC value of standard drug vancomycin (Table 4).

Fig 4: Colony morphology dentification of Penicillium sp VIT-2012 on different media. (A- CYA, B-MEA, C- SDB, D-SEM )

Table 5: Most probable compounds identified in crude ethyl acetate extract of Penicillium sp VIT-2012 by GC-MS analysis

REV	for	Compound Name	M.W	Formula
934	924	Dibutyl Phthalate	278	C₁₆H₂₂O₄
928	903	1,2-Benzenedicarboxylic Acid, Butyl 2-Methylpropyl Ester	278	C₁₆H₂₂O₄
916	892	Dibutyl Phthalate	278	C₁₆H₂₂O₄
915	871	Dibutyl Phthalate	278	C₁₆H₂₂O₄
901	871	1,2-Benzenedicarboxylic Acid, Bis(2-Methylpropyl) Ester	278	C₁₆H₂₂O₄
884	855	Phthalic Acid, Butyl Hexyl Ester	306	C₁₈H₂₆O₄
876	848	1,2-Benzenedicarboxylic Acid, Butyl Cyclohexyl Ester	304	C₁₈H₂₄O₄
870	838	Phthalic Acid, Isobutyl 2-Pentyl Ester	292	C₁₇H₂₄O₄
869	818	Phthalic Acid, Butyl Non-5-Yn-3-YL Ester	344	C₂₁H₂₈O₄
864	832	Phthalic Acid, Butyl 2-Pentyl Ester	292	C₁₇H₂₄O₄
858	830	1,2-Benzenedicarboxylic Acid, Bis(2-Methylpropyl) Ester	278	C₁₆H₂₂O₄
853	818	Phthalic Acid, Isobutyl 4-Octyl Ester	334	C₂₀H₃₀O₄
852	788	Phthalic Acid, Butyl 4-Nitrophenyl Ester	343	C₁₈H₁₇O₆N
850	820	1,2-Benzenedicarboxylic Acid, Bis(2-Methylpropyl) Ester	278	C₁₆H₂₂O₄
850	818	Phthalic Acid, Butyl Isoporpyl Ester	264	C₁₅H₂₀O₄
849	783	Phthalic Acid, Butyl Hex-2-YN-4-YL Ester	302	C₁₈H₂₂O₄
848	828	1,2-Benzenedicarboxylic Acid, Butyl 2-Ethylhexyl Ester	334	C₂₀H₃₀O₄
846	813	1,2-Benzenedicarboxylic Acid, Butyl Octyl Ester	334	C₂₀H₃₀O₄
842	812	Phthalic Acid, Isobutyl Non-5-YN-3-YL Ester	344	C₂₁H₂₈O₄
838	803	Phthalic Acid, Butyl Nonyl Ester	348	C₂₁H₃₂O₄

The pattern for the next abundant peak at 15.40 minutes (72%) revealed the probable presence of 1, 2-benzenedicarboxylic acid, butyl 2-methylpropyl ester and butyl 2-methylpropyl ester related compounds.

3.0 DISCUSSION:

Resistance to MRSA is emerging as a life threatening issue the world over, and the search for new compounds, to combat this problem, forms an important area of active research. These compounds may emerge from a wide variety of natural habitats, comprising of plant and microbial populations, and one such relatively under explored environs is the soil and its fungal population. Hence the current study was aimed to isolate and screen specific soil fungal metabolites for their activity against MRSA. Certain fungi, under favourable conditions of growth, may not produce an anti MRSA compound, but at times, due to fluctuations in physical and chemical nature of soil, they may present with altered physiology as a natural adaptation. This may lead to the production of specific secondary metabolites which otherwise may not be formed under regular conditions of growth. Temperature is a key environmental factor in the biosynthesis of secondary metabolites as reported for fumonisins and moniliformin by Fusarium oxysporum and ergosterol by Fusarium proliferatum²⁰. pH also plays an important role in their production as reported for Penicillium caseifulvum²¹. In our study, the fungus, Penicillium sp VIT-2012 was found to grow best at 30⁰C in laboratory conditions with the same temperature being observed in the area of sampling also, which is otherwise a high temperate region. However, the sampling temperature was 30⁰C as monsoon had set in during soil collection. The soil pH of 7.0, found during collection is probably due to even distribution of its acidic and basic constituents like humus, proteins, etc. The fungus was found to be thriving at this pH, although in general, fungi as known to be largely acidophilic. An adaptation may probably be the cause for the same.

Paper Chromatography was done here to detect the minerals, organic carbon, humus and protein which were separated into different layers or zones. The colour intensity and diameter of each zone indicates the concentration of respective chemical constituents in soil. Pinkish brown colour of inner zone and its diameter size revealed that the soil sample is rich in minerals (except for nitrogen and phosphorus) which are evenly distributed and this fact is supported by soil analysis report (Table 1). The dark brown colour of middle zone shows the presence of acidic humus and organic compound (starch) present in the sample. The improper protrusion of blunt spikes into the outer zone reveals the presence of low amount of proteins. The maximum population was seen for Rhizobium sp which is associated with grass roots and this may be because the soil was collected from grassland area.

In many fungi, macroelements and microelements were found to be gene regulators, not only for synthesis of bioactive compounds, but also for carrying out other cellular activities. In Aspergillus flavus and Fusarium graminearum, Zn²⁺, Cu⁺², Fe⁺² are known to regulate the growth, development and production of aflatoxin and zearalenone by metal modulated gene expression phenomenon²². The appropriate combination and concentration of elements seem to play an important role in secondary metabolite production. Combination of K₂HPO₄, KCl and MgSO₄and sodium nitrate-nitrogen (0.24-0.48 g/l) are ideal to enhance the sclerotium formation and production of carotenoid by Penicillium sp. PT95 ²³. Soil sample in the present study had metals like Mg, Fe, Mn, Cu in high concentrations along with humus and low amount of N and P. However their influence on growth and anti-MRSA activity of Penicillium sp VIT-2012 metabolites needs further analyses. Fungal diversity is reported to be different between seasons²⁴. Though different categories of secondary metabolites have been isolated from marine fungi, soil fungi were also found to be equally potent producers of secondary metabolites including that of anti-MRSA compounds. In the soil sample under study, Penicillium and Fusarium were the dominant organisms. Genus Penicillium is well known for the production of antibacterial secondary metabolites. Due to their versatility in nutrient requirements and production of a number of secondary metabolites active against many bacteria and fungi, they would have surpassed the remaining soil fungi in growth.

In the current study, maximum anti-MRSA activity was found for secondary metabolites synthesised from Penicillium sp VIT-2012 in MKB consisting of simple carbon source like dextrose which may easy to utilize, in comparison with other carbon sources. Though it has been reported that metals are inducers of secondary metabolites, the appropriate concentration of metals, for production of antibacterial compounds, in the medium is very essential²³. Improper ratio or lack of metals and nitrogen source may be the reason for reduced anti-MRSA activity on CYB, YESB and MEB. Presence of sucrose and improper nitrogen source, i.e., yeast extract may be one of the rationales for decreased anti-MRSA activity in YESB as compared to easy utilization of dextrose by microorganisms for production of secondary metabolites in MKB. On the whole, nitrogen source and metals concentration may act as inhibitors for production of secondary metabolites unless they are provided in optimum amounts.

The colony morphology on SDA is yellow in colour, colony constrictions radiating from the centre of colony clearly resembles the colony morphology of P. radicum²⁵. Few exceptional morphology that was observed under SEM were the structure of conidia in P. radicum which was rough (spikes) and bulged, in comparison to the smooth, shrunken and wrinkled conidia of Penicillium sp VIT-2012. The phialides of the Penicillium sp3 were thicker, wrinkled and shortened whereas in P.radicum they are straight and bulged²⁶. Further analysis by 28S rDNA sequencing clearly indiacated 99% similarity with P.radicum whose pure metabolite is known for its anti MRSA activity.

Physical parameters are vital for stimulating the production of anti-MRSA metabolites. The optimum temperature for growth and production of anti MRSA metabolites was found to be 30⁰C and other temperatures were totally unsuitable for both the conditions. In case of pH, both the growth and metabolite synthesis were flexible and seen at a range of 4.0-9.0 with maximum growth at pH 7.0 and activity at pH 6.0. This shows the versatile nature of this fungus and existence of difference physiological systems in it. The constant exposure of Penicillium sp VIT-2012, to conditions like high metal concentrations (Mg, Fe, Mn, Cu), humus and fluctuating temperature may be the responsible factors for this altered physiology.

A strong activity of crude medium extract was observed against clinical isolates of MRSA. Different types and concentration of antibacterial compounds present in the crude extract may be responsible for variation in MIC values i.e., 25 and 12.5 µg of crude extract. Another reason for this variation is heterogeneous nature of clinical MRSA that showed different levels of sensitivity to the particular antibiotic²⁷. The MIC of crude extract was found to be ten to twenty times higher than the standard drug vancomycin. MIC value of extract can be reduced to the lower level by further purification of crude extract. For its usage as a potential anti-MRSA formulation, its additional purification may be required along with its toxicity assessment.

The UV absorption peaks obtained for the components of the extract corresponds to the different components of secondary metabolites. Thirteen peaks are seen which infers that at least this many groups of compounds are present in the crude extract and any of these may have the antibacterial activity. The maximum OD is obtained at 207nm but this may or may not be the active compound.

The strong symmetrical stretching band of FTIR at 1633 cm and weaker peak at 1402cm corresponds to carboxylate anion. The moderately intense band at 744cm in the low frequency region indicates presence of carboxylic acid dimer and also indicates C-H bending of aromatic compounds. The peak at 1452 may indicate the presence of aromatic skeleton (1600-1300) and this is supported by the lack of strong band at a low frequency area (900-650). From these results it may be inferred that phthalic acid, benzenedicarboxylic acid and or its derivatives may be present as part of the metabolites. 1452 band also reveals the presence of cyclohexane which is evident for hexyl derivative of phthalic acid (Phthalic acid, Butyl Hex-2-YN-4-YL Ester) shown in Table 4. Presence of Butyl 2-Methylpropyl Ester may be supported by C-H bending for CH₃at 1452, C-C stretching vibration at 1080 and 933. The peak 1259 is due to C-H bending vibration (overtone) ²⁸. On the TLC sheet, four spots of crude extract designated the four groups of the metabolites. Each spot may have different types of components. The MS analysis of highest peak of GC i.e., 15.23 (100% relative abundance) corresponds to phthalate, 1, 2-benzenedicarboxylic acid, butyl 2-methylpropyl ester or butyl ester and their derivatives. No reports of their anti-MRSA activity are available in the literature. However the type, potentialilty and toxicity of the compound can be justified based on the futher purification of the crude extract.

However, it is expected from this study that a metabolite synthesised from Penicillium sp VIT-2012 may be a unknown compound as understood from the preliminary analysis of its crude extract by GC-MS studies. Further purification and structural elucidation of the pure compound from Penicillium sp VIT-2012, which is under progress, may confirm this inference.

4.0 ACKNOWLEDGEMENT:

The authors acknowledge VIT University, Vellore, India, for providing the infrastructure and financial support for carrying out this research work.

5.0 REFERENCES:

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Received on 20.09.2013 Modified on 21.10.2013

Research J. Pharm. and Tech. 6(12): Dec. 2013; Page 1340-1349

Clinical isolates	M1	M2	M3	M4	M5	M6	M7	M8	M9	M10
Crude extract (µg)	25	25	12.5	25	25	12.5	12.5	25	25	25
Vancomycin (µg)	1.6	1.6	1.6	1.6	1.6	1.6	1.6	1.6	1.6	1.6

2.0 MATERIALS AND METHODS:

Media:

The peak at 15.23 minutes showed maximum abundance (100%) and its MS fragmentation pattern revealed it probably as dibutyl phthalate (93.4%), 1, 2-benzenedicarboxylic acid and butyl 2-methylpropyl ester (92.8%) and their derivatives, from NIST02 library analysis (Table 5).

The pattern for the next abundant peak at 15.40 minutes (72%) revealed the probable presence of 1, 2-benzenedicarboxylic acid, butyl 2-methylpropyl ester and butyl 2-methylpropyl ester related compounds.