GC-MS profile of antimicrobial and antioxidant fractions from Cordia rothii roots

Abstract

Context: An ethnobotanical survey of Cordia rothii Roem. & Schult. (Boraginaceae) reveals it as a medicinal plant.

Objective: Antimicrobial and antioxidant potential evaluation and identification of chemical constituents via GC-MS of C. rothii roots fractions. To the best of our knowledge, this is the first systematic investigation of the roots exploiting GC-MS.

Materials and methods: Extraction and fractionation of C. rothii roots furnished various fractions using solvents of varying polarity, i.e., n-hexane, chloroform, ethyl acetate, acetone and methanol. In vitro antimicrobial and antioxidant screening was performed using disk diffusion and DPPH methods, respectively. MIC of active fractions was also determined using disk diffusion method. GC-MS was used to identify constituents which may be responsible for these activities.

Results: Among various fractions from C. rothii roots, fraction KA-C showed strong antibacterial activity against 17 microorganisms tested, with MIC ranging from 250–31.25 μg/mL. Fractions KA-A, KM and KM-A exhibited significant antioxidant potential with EC₅₀ 46.875 μg/mL, while fractions KEA-PE, KM-PE and KM-M were good with EC₅₀ 93.750 μg/mL. Forty-five phytochemicals were identified in GC-MS studies including eight hydrocarbons, six free fatty acids, 11 fatty acids esters, two phenylpropanoids, four aromatics, four terpenoid quinones/hydroquinones, three triterpenes, four phytosterols, two hexose metabolites and a DNA base. Of these, 32 constituents have been reported for the first time from C. rothii, 24 from genus Cordia and 15 from Boraginaceae.

Discussion and conclusion: Strong antibacterial and antioxidant potential of C. rothii roots may be due to the contribution of phytoconstituents identified through GC-MS studies.

Keywords:

• Boraginaceae
• free radical scavenging
• minimum inhibitory concentrations
• phytochemicals

Introduction

Cordia (Boraginaceae) is well known for its phytochemical and pharmacological diversity (Matias et al. 2015). Cordia rothii Roem. & Schult., locally called gondani, is phytochemically and pharmacologically a less investigated species and has been selected for this study due to its immense medicinal properties such as antidote, antiseptic, antidiarrhoeal, astringent, abortifacient, anti-inflammatory, antileprotic (Chauhan & Chavan 2009), wound healer (Hajabhai et al. 2012) and immunomodulation (Firdous et al. 2014).

Increased antimicrobial resistance is the main cause of spread of many infectious diseases. Efforts have been made to overcome the pathological effects of these etiological agents (Doğan et al. 2013). Since plant-derived drugs offer minimum or no side effects, search for such therapeutics has become the goal of phytochemists and microbiologists to reduce the suffering of mankind. According to an analysis, 50% of the marketed drugs during 1981 to 2006 originated from natural products e.g., morphine, quinine, artemisinin, etc. (Kingston 2011). Free radical species are responsible for various diseases including cardiovascular, diabetes mellitus and neurodegenerative disorders (Valko et al. 2007). Literature survey of C. rothii reveals its use in heart ailments, diabetes (Chauhan & Chavan 2009) and CNS depression (Asolkar et al. 1992). In this study, antimicrobial potential of C. rothii roots fractions against some pathogenic microbes and antioxidant activity against free radical species is tested. Chemical composition of these fractions is also determined by GC-MS.

Materials and methods

Collection of plant material

C. rothii roots collected in July 2010 from the University of Karachi Campus were identified by Prof. Dr. Surraya Khatoon, Department of Botany, University of Karachi. A voucher specimen (No. 85853) has been deposited in the herbarium of the same department.

Extraction procedure

Classical extraction procedure was adopted to obtain various fractions using solvents of varying polarity. Freshly collected, powdered roots (4.5 kg) from C. rothii were defatted by soaking in n-hexane (2 × 10 L) at room temperature for two days furnishing fraction 2 A. The marc left was then sequentially percolated in solvents of increasing polarity; chloroform, ethyl acetate, acetone and methanol to afford chloroform (4A), ethyl acetate (6A), acetone (KA) and methanol soluble (KM) fractions, respectively. Detailed fractionation is elaborated in Schemes 1 and 2. A total of 19 fractions thus obtained were screened for their antimicrobial and antioxidant potential. Out of 19, 12 non- to moderate polar fractions (2A, 4A, KC-PE, KC-C, 6A, KEA-PE, KEA-C, KA-PE, KA-C, KM-PE, KM-C and KM-EA) were subjected to GC-FID and GC-MS analyses on the basis of their resolved TLC profile. The protocol already reported in earlier communications (Azmat et al. 2010; Muhammad et al. 2015) was used with little variations.

Scheme 1. Extraction, and fractionation and identified compounds f rom C. rothii roots. *Continued in Scheme 2.

Scheme 2. Extraction and fractionation and identified compounds from C. rothii roots. *Continued from Scheme 1.

Gas chromatography-mass spectrometric (GC-MS) analyses of C. rothii roots

Initial profiling of fractions was performed using gas chromatography-flame ionization detection (GC-FID) on a Shimadzu GC-17 A (Shimadzu Corp., Kyoto, Japan) equipped with a SPB-5^® capillary column (30 m ×0.25 mm ID and 0.25 μm df). Carrier gas was helium with a flow rate of 1 mL/min. The oven was kept initially at 70 °C for 5 min, programmed to 260 °C at a rate of 5 °C/min and kept isothermal for 20 min. Temperature and splitting ratio of injector was set to 280 °C and 1:30, respectively. For GC-MS, Hewlett-Packard 5890 (Bunker Lake Blvd, Ramsey, MN) gas chromatograph coupled to Jeol JMS-600 mass spectrometer and equipped with ZB-5MS^® capillary column (30 m × 0.25 mm ID and 0.25 μm df) was used, keeping analytical conditions similar as described for GC-FID. EI ion source was kept at 250 °C and 70 eV.

For the identification of compounds, electronic mass spectral survey (NIST 2005) and EIMS data reported in literature were compared (Moir & Thomson 1973; Manners & Jurd 1977) with the experimental mass spectra. The order of elution of compounds was confirmed through reported retention indices (RI) data (Kováts 1958; Van den dool & Kratz 1963). Area normalization method was used to quantify constituents.

Bacterial and fungal strains

Pure bacterial cultures (15 Gram-positive and 13 Gram-negative, Table 1) and 14 fungal strains were obtained from Department of Microbiology, University of Karachi. The fungal strains included six saprophytic fungi; Aspergillus flavus, Aspergillus niger, Fusarium sp., Penicillium sp., Rhizopus sp., Helminthosporium, three non-filamentous fungi; Candida albicans, Candida albicans ATCC 0383, Saccharomyces cerevisiae and five dermatophytes; Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton mentagrophytes and Trichophyton tonsurans.

Table 1. In vitro antibacterial activity of C. rothii roots against Gram-positive and Gram-negative bacteria.

Name of bacteria	Zone of inhibition (mm)
	2A	4A	KC-PE	KC-C	6A	KEA-PE	KEA-C	KA	KA-PE	KA-C	KA-EA	KM-PE	KM-C	KM-EA	KM-M
Gram-positive bacteria
Bacillus cereus	–	–	–	–	9	10	10	–	11	12	9	–	11	9	–
Bacillus subtilis	11	9	10	10	9	11	10	9	–	11	–	–	9	9	9
Bacillus thuringiensis	–	–	–	10	–	–	–	–	11	15	–	–	11	11	–
Corynebacterium diphtheriae	–	–	–	9	–	–	9	–	12	16	9	10	13	10	–
Corynebacterium hofmannii	–	–	–	–	–	13	11	–	–	15	10	–	15	–	–
Corynebacterium xerosis	9	–	9	–	9	10	–	10	13	18	–	12	16	13	–
Micrococcus luteus	10	–	9	9	10	10	9	9	–	15	9	–	10	13	–
Micrococcus luteus ATCC 9341	10	9	10	–	10	12	13	–	14	23	10	11	20	14	–
Methicillin resistant Staphylococcus aureus	–	–	–	–	–	10	9	–	–	15	9	9	12	10	–
Staphylococcus aureus	11	9	9	–	–	9	11	9	9	12	–	–	–	–	–
Staphylococcus aureus AB188	9	–	9	9	10	13	9	–	12	15	10	–	12	–	11
Staphylococcus epidermidis	–	9	11	10	9	–	–	10	17	18	–	11	13	15	–
Streptococcus faecalis	10	–	9	–	–	9	–	–	15	20	–	–	13	14	–
Streptococcus saprophyticus	14	9	11	11	12	10	10	11	10	17	10	–	15	9	–
Streptococcus pyogenes	–	–	–	–	–	–	–	–	12	16	–	9	10	11	–
Gram-negative bacteria
Enterobacter specie	11	–	10	10	10	12	12	10	12	13	11	10	12	10	–
Escherichia coli ATCC 8739	–	–	–	–	–	14	12	9	11	15	10	–	10	11	–
Escherichia coli	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
E. coli MDR	–	–	–	10	–	13	9	–	–	–	9	–	–	–	–
Klebsiella pneumoniae	12	10	13	12	10	11	10	12	11	15	–	11	11	10	–
Proteus mirabilis	–	–	–	–	–	–	–	–	–	17	–	11	14	12	–
Pseudomonas aeruginosa	–	–	–	–	–	–	–	–	10	15	–	10	12	10	–
Pseudomonas aeruginosa ATCC 9027	13	9	11	11	11	–	–	11	10	13	–	–	10	10	–
Salmonella typhi	12	–	10	13	13	9	–	10	–	–	–	–	–	–	–
Salmonella paratyphi A	–	–	–	–	–	13	12	–	–	12	10	–	–	–	–
Salmonella paratyphi B	–	–	–	–	–	–	–	–	–	–	11	9	–	–	9
Shigella dysenteriae	14	11	10	11	14	–	–	12	–	–	–	–	–	–	–
Shigella flexneri	14	–	10	13	14	–	–	–	–	–	–	–	–	–	–

– : Inactive; Negative control = DMSO; KA-A, KA-M, KM and KM-A did not exhibit antibacterial activity against any of the Gram-positive or -negative bacteria.

Antimicrobial assay

Roots extracts were screened for in vitro antibacterial and antifungal (data not shown as extracts were weakly antifungal) bioassays using disk diffusion method (Bauer et al. 1966). Then 50 mg/mL stock solution was used to prepare sterile disk of extracts, and 10 μL containing 500 μg sample was loaded onto the disks. Finally prepared disks were incubated for 24 h at 37 °C. Dimethylsulfoxide (DMSO) was used as control. The samples exhibiting strong antibacterial activity ≥15 mm (zone of inhibition/concentration in μg) in preliminary screening were evaluated for their MIC. Samples were serially diluted to get different concentrations (31.25–250 μg/disk) for determining MIC (Table 2).

Table 2. Minimum inhibitory concentration (μg/disc) of C. rothii roots.

Name of bacteria	KA-C	KM-C	Name of bacteria	KA-C	KM-C
Bacillus thuringiensis	31.25	–	Staphylococcus epidermidis	250	–
Corynebacterium diphtheriae	250	–	Streptococcus saprophyticus	62.50	62.50
Corynebacterium hofmannii	125	250	Streptococcus pyogenes	250	–
Corynebacterium xerosis	250	125	Streptococcus faecalis	125	–
Micrococcus luteus	31.25	–	Proteus mirabilis	250	–
Micrococcus luteus ATCC 9341	250	31.25	Pseudomonas aeruginosa	125	–
Methicillin resistant Staphylococcus aureus	62.50	–	Escherichia coli ATCC 8739	62.50	–
Staphylococcus aureus AB188	<31.25	–	Klebsiella pneumoniae	31.25	–

Antioxidant assay using DPPH protocol

The method for antioxidant bioassay was adopted from Lee et al. (1998). Ethanol was used to make 300 μM solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH). Reaction mixture containing 10 μL test sample in DMSO and 90 μL DPPH was incubated for 30 min at 37 °C, and absorbance was measured at 515 nm. Comparison between the DMSO-treated control (ascorbic acid) and test sample provided the % inhibition. The EC₅₀ concentration of test sample was calculated in μg/mL (Table 3).

Table 3. Antioxidant potential of C. rothii roots.

Sample code	% Inhibition ± SD	EC50 μg/mL	Sample code	% Inhibition ± SD	EC50 μg/mL
2A	61.11 ± 0.02	187.5	KA-EA	33.45 ± 0.01	–
4A	51.67 ± 0.02	375	KA-A	71.76 ± 0.01	46.875
KC-PE	43.88 ± 0.01	–	KA-M	53.95 ± 0.05	375
KC-C	46.58 ± 0.01	–	KM	76.79 ± 0.01	46.875
6A	58.63 ± 0.03	375	KM-PE	62.94 ± 0.07	93.750
KEA-PE	67.65 ± 0.01	93.750	KM-C	58.27 ± 0.02	375
KEA-C	56.11 ± 0.02	375	KM-EA	57.0 ± 0.01	375
KA	59.89 ± 0.01	187.5	KM-A	69.24 ± 0.02	46.875
KA-PE	23.56 ± 0.03	–	KM-M	72.58 ± 0.02	93.750
KA-C	63.84 ± 0.02	187.5	–	–	–

Results

C. rothii roots were extracted sequentially with organic solvents of increasing polarity furnishing 19 fractions. All these fractions (Schemes 1 and 2) were screened for their antimicrobial potential against 15 Gram-positive, 13 Gram-negative bacteria and 14 fungal strains. KA-C exhibited broad spectrum antibacterial activity against all the Gram-positive and Gram-negative bacteria tested. Strong antibacterial activity was observed against B. thuringiensis, M. luteus, MRSA, S. aureus AB188, S. saprophyticus, E. coli ATCC 8739 and K. pneumoniae (MIC 31.25–62.50 μg/disk). Good antibacterial activity was observed against C. diphtheriae, C. hofmannii, C. xerosis, M. luteus ATCC 9341, S. epidermidis, S. pyogenes, S. faecalis, P. mirabilis and P. aeruginosa (MIC 125–250 μg/disk) (Tables 2). Similarly, KM-C exhibited significant antibacterial activity against M. luteus ATCC 9341 and S. saprophyticus (MIC 31.25–62.50 μg/disk) and good antibacterial activity against C. hofmannii and C. xerosis (MIC 125–250 μg/disk) (Tables 2). KA-PE and KM-EA showed good antibacterial activity against S. epidermidis (MIC 250 μg/disk) while KA-PE strongly inhibited S. faecalis (MIC 31.25 μg/disk). With the exception of KA-A, KA-M, KM and KM-A, all samples exhibited little to moderate antibacterial activity. KA-PE and KM-EA exhibited no activity (Table 1). No antifungal activity was observed in these fractions, except KEA-PE, which exhibited some activity against Fusarium specie and S. cerevisiae.

The above-mentioned fractions were also screened for their antioxidant potential. KA-A, KM and KM-A exhibited significant antioxidant activity (EC₅₀ 46.875 μg/mL), whereas KEA-PE, KM-PE and KM-M showed good antioxidant potential (EC₅₀ 93.750 μg/mL). Most of the samples exhibited antioxidant activity with inhibitory effect above 50%. Fractions 2A, 4A, 6A, KEA-C, KA, KA-C, KA-M, KM-C and KM-EA exhibited little antioxidant activity (EC₅₀ 187.5–375 μg/mL) (Table 3).

Analyses of the 12 non-polar to moderately polar fractions of C. rothii roots (Schemes 1, 2 and Table 4) through GC-MS resulted in identification of 45 out of 95 metabolites (constituting ∼44%). To the best of our knowledge 32 of these constituents (comprising 71.11% of composition) are reported for the first time from C. rothii, 24 from genus Cordia and 15 from Boraginaceae. Non-polar fraction 2 A provided the most promising results. Out of 25, 19 phytoconstituents, quantifying 71.91% of the sample have been identified. These included seven hydrocarbons (HC); 1–4, 6, 8 and 10; two free fatty acids (FFA); 13 and 18; eight fatty acid esters (FAE); 5, 7, 11, 12 and 14–17; a terpenoid quinone (TQ); 9; and a phytosterol (PS); 19. Twelve of these 1, 2, 4, 5, 7, 11, 12, 14–17 and 19 are being reported for the first time from C. rothii. KM-PE also furnished promising results in GC-MS analyses, with 17 identifications out of 22 constituents and 57.08% concentration. These metabolites included a terpenoid hydroquinone (THQ); 21, two PS; 25 and 26, three triterpenoids (TT); 31, 32 and 43, two hexose metabolites; 36 and 37; three FFA; 38, 40 and 41; a FAE; 42, a phenylpropanoid (PP); 39 and a DNA base; 35. Eleven of these constituents (21, 25, 31, 32, 35–39, 42 and 43) are being reported for the first time from C. rothii. GC-MS studies on fractions KC-PE and KM-EA resulted in identification of 15 and 11 metabolites from a total of 37 and 24 metabolites, constituting 52.93 and 33.92% of fractions, respectively. These fractions resulted in identification of a HC; 20, two FAE; 22 and 23, a THQ; 24; a PS; 27, a PP; 28 and two aromatics (Ar); 44 and 45. The rest of the identified metabolites reappeared. This is the first report of phytoconstituents 22, 23, 24 and 28 from C. rothii. A THQ; 29 and a FFA; 30 were identified in KEA-PE while two aromatics; 33 and 34 in KEA-C along with other constituents. Three of these metabolites, 29, 33 and 34 are being reported for the first time from C. rothii.

Table 4. Composition (%) of phytochemicals in GC-MS studies on C. rothii roots.

Identified compounds/trivial and IUPAC name	2 A	4 A	KC-PE	KC-C	6 A	KEA-PE	KEA-C	KA-PE	KA-C	KM-PE	KM-C	KM-EA	Sub-total	% overall	R.I.
n-Tridecane^a (1)	1.29	–	–	–	–	–	–	–	–	–	–	–	1.29	0.11	1300
n-Tetradecane^a,b (2)	1.51	–	–	–	–	–		–	–	–	–	–	1.51	0.13	1400
n-Pentadecane^b (3)	1.45	–	–	–	–	–	–	–	–	–	–	–	1.45	0.12	1500
iso-Hexadecane^a,b (4)	1.86	–	–	–	–	–	–	–	–	–	–	–	1.86	0.16	–
Lauric acid ethyl ester^a,b,c (n-dodecanoic acid ethyl ester) (5), co-eluted with 4	–	–	–	–	–	–	–	–	–	–	–	–	0.00	–	1554
n-Heptadecane (6)	1.33	–	–	–	0.20	–	–	–	–	–	–	–	1.53	0.13	1700
Myristic acid ethyl ester^a,b (n-tetradecanoic acid ethyl ester) (7)	1.80	–	–	–	0.35	–	–	–	–	–	–	–	2.15	0.18	1798
n-Octadecane (8)	2.40	1.90	1.17	–	–	–	–	–	–	–	–	–	5.47	0.46	1800
Cordiachrome C (5,6,7,8-tetrahydro-7-isopropenyl-6-methyl-6-vinyl naphthalene-1,4-dione) (9)	8.51	–	14.75	0.75	–	32.61	–	–	13.0	–	–	–	69.62	5.86	n.a.
n-Nonadecane (10)	2.26	–	–	–	–	–	–	–	–	–	–	–	2.26	0.19	1900
Palmitic acid methyl ester^a,b (n-hexadecanoic acid methyl ester) (11)	3.34	8.84	5.80	–	3.65	1.44	–	7.93	–	2.17	1.02	0.92	35.11	2.96	1938
Palmitic acid ethyl ester^a (n-hexadecanoic acid ethyl ester) (12)	7.35	–	–	–	–	–	–	–	–	–	–	–	7.35	0.62	1975
Palmitic acid (n-hexadecanoic acid) (13)	7.99	28.68	5.55	5.49	–	3.75	–	15.03	4.17	6.67	3.05	4.19	84.57	7.12	1983
Oleic acid methyl ester^a,b (octadec-9Z-enoic acid methyl ester) (14)	3.47	–	1.95	–	–	1.21	–	9.08	–	–	–	–	15.71	1.32	2107
Linoleic acid ethyl ester^a,b (octadec-9Z,12Z-dienoic acid ethyl ester) (15)	3.21	–	1.93	–	–	–	–	–	–	–	–	–	5.14	0.43	2162
Elaidic acid ethyl ester^a,b (octadec-9E-enoic acid ethyl ester) (16)	5.24	–	4.62	–	–	–	–	–	–	–	–	–	9.86	0.83	2168
Oleic acid ethyl ester^a,b (octadec-9Z-enoic acid ethyl ester) (17)	9.88	–	–	–	–	–	–	–	–	–	–	–	9.88	0.83	2171
Stearic acid (n-octadecanoic acid) (18)	3.63	15.35	2.83	3.35	–	2.05	–	2.80	–	4.38	2.75	–	37.14	3.13	2177
Stigmasta-3,5-diene^a,b (19)	5.39	–	–	1.25	–	–	–	11.80	–	–	–	–	18.44	1.55	2525^d
n-Hexadecane (20)	–	1.10	–	–	0.82	–	–	–	–	–	–	–	1.92	0.16	1600
Cordiachromene A^a (2-methyl-2-(4-methylpent-3-enyl)-2H-chromen-6-ol) (21)	–	–	5.48	13.00	–	3.13	–	11.31	8.67	7.70	4.20	6.78	60.27	5.07	n.a.
Linoleic acid methyl ester^a,b (octadec-9Z,12Z-dienoic acid methyl ester) (22)	–	–	1.59	0.99	–	–	–	–	–	–	–	–	2.58	0.22	2098
Stearic acid methyl ester^a,b (octadecanoic acid methyl ester) (23)	–	–	1.18	–	–	–	–	–	–	–	–	–	1.18	0.10	2134
Cordiaquinol C^a (5,6,7,8-tetrahydro-7-isopropenyl-6-methyl-6-vinylnaphthalene-1,4-diol) (24)	–	–	2.24	–	–	–	–	–	–	–	–	4.37	6.61	0.56	n.a.
Stigmasta-4,22-diene-3β-ol^a,b (25)	–	–	1.21	2.24	–	1.75	–	–	–	3.58	4.00	2.84	15.62	1.32	2739^d
β-Sitosterol (stigmast-5-en-3β-ol) (26)	–	–	2.17	3.91	–	4.13	–	4.90	3.32	9.59	9.01	6.19	43.22	3.64	2731^d
Stigmasterol (stigmasta-5,22-diene-3β-ol) (27)	–	–	0.46	–	–	–	–	–	–	–	–	–	0.46	0.04	2739^d
Coniferyl alcohol^a (((1E)-4-(3-hydroxy-1-propenyl)-2-methoxyphenol) (28)	–	–	–	5.03	–	–	–	–	–	–	–	4.29	9.32	0.78	1727
Cordiol A^a (5,6,7,8,8a,9,10,10a-octahydro-5,5-dimethyl-anthracene-1,4,8a-triol) (29)	–	–	–	3.41	–	1.20	–	–	2.84	–	–	–	7.45	0.63	2274
Linoleic acid (octadec-9Z, 12Z-dienoic acid) (30)	–	–	–	–	–	3.13	–	–	–	–	–	–	3.13	0.26	2157
Cycloartenol^a,b,c (9,19-cyclolanost-24-en-3β-ol) (31)	–	–	–	–	–	0.66	–	–	–	2.00	–	–	2.66	0.22	2816^d
24-Methylene cycloartenol^a,b (24-methylene-9,19-cyclolanostan-3β-ol) (32)	–	–	–	–	–	0.85	–	–	–	2.00	3.10	–	5.95	0.50	2834^d
Phenyl ethylene^a,b (phenyl ethene) (33)	–	–	–	–	–	–	18.81	–	–	–	–	–	18.81	1.58	895
Benzyl acetate^a,b,c (acetic acid phenylmethyl ester) (34)	–	–	–	–	–	–	4.39	–	–	–	–	–	4.39	0.37	1184
Thymine^a,b,c (2,4-dihydroxy-5-methylpyrimidine) (35)	–	–	–	–	–	–	–	–	–	0.48	–	–	0.48	0.04	1118^d
2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one^a,b,c (36)	–	–	–	–	–	–	–	–	–	0.60	–	–	0.60	0.05	1167
Hydroxymethyl furfuraldehyde^a ((5-(hydroxymethyl)-2-furan-carboxaldehyde) (37)	–	–	–	–	–	–	–	–	–	0.53	–	–	0.53	0.04	1256
Lauric acid^a,b (n-dodecanoic acid) (38)	–	–	–	–	–	–	–	–	–	0.72	–	–	0.72	0.06	1566
Hydrocaffeic acid^a,b (3,4-dihydroxy-benzenepropanoic acid) (39)	–	–	–	–	–	–	–	–	–	0.62	–	1.74	2.36	0.20	1790^d
Myristic acid (n-tetradecanoic acid (40)	–	–	–	–	–	–	–	–	–	0.99	–	0.87	1.86	0.16	1774
Oleic acid (octadec-9Z-enoic acid) (41)	–	–	–	–	–	–	–	–	–	8.98	6.54	–	15.52	1.31	2161
2-Monopalmitin^a,b (hexadecanoic acid 2-hydroxy-1-(hydroxymethyl)-ethyl ester) (42)	–	–	–	–	–	–	–	–	–	4.73	3.72	–	8.45	0.71	2498^d
Cycloeucalenol^a,b (4α,14α-di-methyl-9,19-cycloergost-24(28)-en-3β-ol) (43)	–	–	–	–	–	–	–	–	–	1.34	–	–	1.34	0.11	2760^d
p-Salicylic acid^a (4-hydroxy-benzoic acid) (44)	–	–	–	–	–	–	–	–	–	–	–	0.79	0.79	0.07	1522
Vanillic acid^a (4-hydroxy-3-methoxy-benzoic acid)(45)	–	–	–	–	–	–	–	–	–	–	–	0.94	0.94	0.08	1608

*Constituents arranged and numbered in the order of their appearance in Schemes 1 and 2.

^aFirst report from C. rothii; ^bFirst report from genus Cordia; ^cFirst report from family Boraginaceae;

^dEstimated values as mentioned in NIST.

Figure 1 summarizes number of total and identified phytoconstituents along with the % composition while Figure 2 shows the diversity in the compounds identified. The structures of chemical constituents identified through GC-MS studies are given in Figure 3. A table in supplementary data justifies their natural occurrence.

Figure 1. Identified chemical constituents from C. rothii roots through GC-MS studies.

Figure 2. Diverse classes of natural products identified through GC-MS studies.

Figure 3. Structures of chemical constituents identified through GC-MS studies.

Discussion

Plants of genus Cordia are highly reputed for their diverse pharmacology and phytochemistry (Matias et al. 2015). However, C. rothii is relatively less explored specie. Fractionation using organic solvents in increasing order of polarity affects antimicrobial property since change in solvents result in the extraction of different metabolites. Plant based antimicrobials not only show promising results but are also reported to have reduced side effects. The synergism arises due to the complex structural variety of natural products as individual or in mixture whose interaction(s) with pathogens or toxins do not affect other healthy cells or molecules (Di Pasqua et al. 2005; Negi et al. 2012). It is also true for antagonism. Studies showed that lipophilic or hydrophilic properties of compounds are associated with antimicrobial properties, e.g., the proposed order of antimicrobial activity against various phytochemicals is phenols > aldehydes > ketones > alcohols > esters > hydrocarbons. Increasing carbon chain length also imparts good effect on antibacterial activity (Bendiabdellah et al. 2013).

Hydrocarbons are also reported to exhibit antimicrobial property (Bassolé & Juliani 2012). Essential oil from Minuartia meyeri containing hydrocarbons as major constituents has exhibited antibacterial activity (Yayli et al. 2006). Role of fatty acids as antimicrobial agents in food additives (Freese et al. 1973) and the antifungal and antibacterial properties of their esters are also well known (Zhang et al. 2013). All the FFAs identified in this study are reported to exhibit antimicrobial properties (Agoramoorthy et al. 2007). Although the exact mechanism of antibacterial activity of FFA is unknown, but it may be suggested that hydrophobic part is responsible for the activity by interacting with the hydrophobic parts of bacterial cell membrane. Moreover, elongated carbon chain length, presence of double bond and its position also affects antibacterial activity (Furtado et al. 2014). Organic compounds having aromatic and phenolic moiety may attribute increased antimicrobial activity to the compound (Patnaik et al. 2008). Essential oils, having phenols or quinones as major constituents, exhibited potent antimicrobial activity (Shigeharu et al. 2006). Sesquiterpenoid quinones and hydroquinones also exhibited antimicrobial potential (Cariello et al. 1982; Takahashi et al. 2008). Structure–activity relationship (SAR) studies indicate that side chain of PS is responsible for its antibacterial activity (Tanaka et al. 2013).

Fractions from this study did not exhibit antifungal potential. In general, samples exhibiting antibacterial activity usually do not show antifungal behaviour or it may be due to the antagonistic effect of the other components present in the samples.

Antimicrobial and antioxidant activities of phenolic compounds have been evaluated in many earlier studies (Osoro et al. 2013). It is now a well-established fact that antioxidant and antimicrobial potential of plant extracts is characterized by their phenolic content (Proestos et al. 2005). Although several citations are available for the correlation between antioxidant potential and phenolic content of plants (Guruvaiah et al. 2012) but this might not always be true. Antioxidant potential is correlated with the applied method, concentration, nature and physicochemical properties of the sample under investigation (Stamenic et al. 2014).

Antioxidant behaviour of FFA has been evaluated earlier (Zhao et al. 2013). Fatty acid content and antioxidant property can be associated with many biological benefits. An increased amount of unsaturated FFA imparts positive effect on antioxidant potential of seaweeds (Huang & Wang 2004). Antioxidant activity of benzoic and cinnamic acid derivatives is in the order of p-hydroxy < p-hydroxymethoxy < dihydroxy < p-hydroxydimethoxy (Natella et al. 1999). p-Hydroxybenzoic acid and its derivatives exhibit diverse range of biological activities including antimicrobial and antioxidant (Manuja et al. 2013). Aromatic compounds with methoxy group exhibits high antioxidant activity (Narayanan et al. 2012). Such compounds have been identified in this study.

Various environmental factors generate oxidative stress in plants. Consequently, formation of phenolic metabolites with antioxidant properties is induced to overcome this stress (Grace 2005). Free radical species are reduced by hydroxyl group of an aromatic system as a result of which resonance stabilized phenoxyl radical is produced (Calil et al. 2012). Phenolic acids and their derivatives scavenge reactive oxygen species showing antioxidant behaviour. Compounds having one, two or three free hydroxyl groups on aromatic ring exhibit low, medium and high antioxidant activity, respectively (Katti & Ranjekar 2014). In another contrast study maximum antioxidant activity was exhibited by monohydroxybenzoic acid derivatives and position of carboxylic acid and hydroxyl group is found responsible for the antioxidant activity of a compound. Highest antioxidant activity was observed in compounds having -OH group oriented ortho- or para- to the -COOH group. The antioxidant activity decreases if the hydroxyl group is blocked whereas no pronounced effect was observed in the case of blocked carboxylic acid group (Velika & Kron 2012). Structures of metabolites 39, 44 and 45 are noteworthy for their probable activity. Phenylpropanoids play important role as antioxidants (Kurkin 2013), which is related to the hydroxyl moiety of aromatic ring (Park et al. 2003). SAR study shows that number of -OH groups and the nature of alkyl spacer between aromatic ring and carboxylic group play important role in antioxidant behaviour of a compound (Siquet et al. 2006). Sesquiterpenoid hydroquinones and sesquiterpenoid quinones are also reported as antioxidant agents with hydroquinones being stronger as compared to quinones (Belisario et al. 1992). Thus, 21, 24 and 29 can also be the potential contributors in antioxidant potential of C. rothii. Phytosterols are reported as the antioxidant constituents of plants (Conforti et al. 2007).

Conclusion

C. rothii roots showed significant antimicrobial and antioxidant potential. Thirty-two out of 45 phytoconstituents identified through GC-MS studies are here reported for the first time. These fractions were evaluated in mixture, therefore, it is suggested that these activities could be due to the synergistic effect of the components identified. Further, inhibitory effects against a broad range of bacterial strains reveal the diversified antimicrobial potential of the plant due to the extraction of secondary metabolites of varying nature, in different organic solvents.

Compounds 9 (Shigeharu et al. 2006), 13 (Agoramoorthy et al. 2007), 26 (Yadav et al. 2014) and 21 (Benslimane et al. 1988) being antimicrobial agents may be the potential contributors of fraction KA-C. Similarly 13 and 18 (Agoramoorthy et al. 2007), 21 (Benslimane et al. 1988), 26 (Yadav et al. 2014), 41 (Agoramoorthy et al. 2007) and 42 (Zheng et al. 2012), reported as antimicrobial agents may be responsible for the significant antimicrobial potential of fraction KM-C.

The most active antibacterial fractions (KA-C and KM-C), exhibited little or no antioxidant activity while fractions exhibiting significant antioxidant activity; KA-A, KA-M, KM and KM-A did not exhibit antibacterial activity. Fractions exhibiting good antioxidant activity KEA-PE, KM-PE, KM-M, showed variable activity against Gram-positive and Gram-negative bacteria tested and fractions exhibiting good antibacterial activity (KA-PE and KM-EA) showed little or no antioxidant activity at all (Table 1–3).

Polar fractions (KA-PE, KA-C, KA-A, KM, KM-PE, KM-C, KM-A and KM-M) having varying classes of natural products with different polar functionalities were found to be more active, which is in accordance with various earlier reports. However, reports where chloroform extract was proved to be more active than methanol extract are also available. Further studies on identification and characterization of natural constituents from the bioactive fractions are in progress. These may lead to unveil templates for bioactive lead compounds.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.