The funder of this project is Cotton Research Development Corporation (CRDC), Australia. CRDC website is
Cotton gin trash (CGT), a waste product of cotton gins, make up about 10% of each bale of cotton bolls ginned. The current study investigates high value volatile compounds in CGT to add value to this by-product. The volatile compounds in CGT and different parts of the cotton plant were extracted using various methods, identified by gas chromatography-mass spectrometry (GC-MS) or nuclear magnetic resonance (NMR) spectroscopy, and then quantified by gas chromatography-flame ionisation detection (GC-FID) against available standards. Terpenoids including monoterpenoids and sesquiterpenoids were found to be the most abundant, making up 64.66% (area under peak) of total volatiles extracted by hydro-distillation. The major extractable terpenoids in CGT were α-pinene (13.69–23.05 μg/g), β-caryophyllene (3.99–74.32 μg/g), α-humulene (2.00–25.71 μg/g), caryophyllene oxide (41.50–102.08 μg/g) and β-bisabolol (40.05–137.32 μg/g). Recoveries varied between different extraction methods. The terpenoids were found to be more abundant in the calyx (659.12 μg/g) and leaves (627.72 μg/g) than in stalks (112.97 μg/g) and stems (24.24 μg/g) of the cotton plant, indicating the possible biological origin of CGT volatiles. This study is the first to identify and quantify the different terpenoids present in CGT and significantly, β-bisabolol, an abundant compound (sesquiterpene alcohol) which may have valuable biological prospects. These findings therefore contribute to identifying alternative management strategies and uses of CGT.
Cotton gin trash (CGT) is the waste product generated from cotton ginning, a process whereby mature cotton bolls are separated into different components such as seeds and fibres, which are used as raw materials for production of end products such as cotton seed oil and fabric [
Volatile chemicals are typical secondary metabolites of many plants including cotton [
Generally, determination of the composition of plant volatiles can be achieved by extracting the compounds from various parts of the plant. Extraction procedures are dependent on the nature (matrix properties) of the plant and its parts, and differ by temperature, extraction solvent, time and pressure [
Although phytochemical study of cotton suggests the plant is a reservoir of extractives, particularly terpenoids, information on the complete volatile composition of CGT is still unclear. In this study, different extraction and analytical methods were implemented to achieve recovery, identification and quantification of terpenoids present in the trash. Methods implemented for extraction of compounds from CGT are well established for extracting a wide range of chemicals from plant materials [
Analytical standards were procured from Sigma Aldrich, Australia and included (+)-α-pinene (#80605), (-)-β-pinene (#80609), α-humulene (#12448), β-humulene (#53676), (-)-α-bisabolol (#95426), (-)-caryophyllene oxide (#91034) and (-)-
CGT was kindly supplied by Namoi Cotton Co-operative (Yarraman Gin, NSW). All material was air-dried at 40 °C for 48 h. To achieve better uniformity and further size reduction, samples were subject to a 60 sec pulse in a pulveriser (Labtechnics Pulveriser, WA, Australia). Samples were stored at room temperature prior to extraction. Dried post–harvest trash (PHT) samples were first separated into the different components of stalks, stems, leaves and calyx (burr) prior to grinding using a Mixer mill 301 (Retsch GmbH, Germany).
Preliminary hydro-distillation were performed to determine an optimised duration for the extraction procedure. CGT sample (300 g) was distilled in 2,700 mL milli-Q water sequentially for 6 h, 12 h and 18 h and extracted oil collected for the three time durations. Recovered oil was extracted with hexane and the extract analysed for terpenoids by gas chromatography-mass spectroscopy. The methods finally used for extraction, identification and semi-quantification of the volatiles from the trash samples are explained in the following sections.
For all hydro-distillation, 300 g of CGT sample was submerged in 2700 mL milli-Q water and boiled for six hours after mixing in a 5,000 mL round bottom flask connected to a chiller and essential oil collected in a graduated collection tube. Following distillation, the volume of oil was measured using a graduated collection tube. The collection tube was washed with acetone and combined with collected oil was dried in a fume hood overnight. The recovered extracted oil was mixed with 3 mL milli-Q water and 5 mL hexane, shaken vigorously and centrifuged at 3,000 rpm for 3 mins. The hexane fraction was collected in a 25 mL volumetric flask and the extraction procedure repeated twice with equal volumes of hexane. Total extract in the volumetric flasks were made up to 25 mL and transferred to clean, pre-weighed 40 mL vials. Extracts were stored at 4 °C until further analysis.
Identification and semi-quantification was performed on an Agilent 6890A GC instrument equipped with a ZB-5 capillary column (Phenomenex) of dimensions 300 mm length, × 0.53 mm internal diameter (I.D) × 1.50 μm film thickness. Samples were injected in the split mode (split ratio of 1:25) under the following conditions: injection port set at 280 °C, oven temperature held at 50 °C for 1 min, then programmed at a rate of 8 °C/min to 300 °C, helium carrier gas at a flow rate of 1.2 mL/min. An Agilent 5973 network mass selective detector was used as the detector and operated in scan mode, with a scanning mass range of 35 to 300 atomic mass unit (amu) at 5.19 scans/sec. Electron ionisation (EI) for the mass selective detector was 70eV and a solvent delay time of 6 mins was maintained when no spectra was collected. Volatile compounds in CGT extracts were identified by comparing mass spectrum with GC-MS library Wiley 275.L database and their relative abundance were compared using the peak area in the total ion chromatogram (TIC).
CGT oil obtained by hydro-distillation was fractionated using high-performance liquid chromatography (prep-HPLC, Agilent Technologies 1260) equipped with an ultraviolet (UV) detector and fraction collector. The CGT oil (400 μl) was mixed with methanol (600μl) before injection. Separation of fractions was performed using a Luna C18 column (150 mm x 21.20 mm, 55 μm, Phenomenex Co., USA) at a flow rate of 20 mL/min of mobile phase with methanol + 0.05% trifluoroacetic acid (TFA) and water +0.05% TFA. A linear gradient elution mode was maintained with mobile phase set at 0 min for 60% methanol, 2 min for 60% methanol, 8 min for 100% methanol, 22 min for 100% methanol, 25 min for 60% methanol and 30 min for 60% methanol. The UV detection was at 210, 280 and 360 nm. A total of four injections of 250 μl each were done with fractions collected between 4 and 18 min at an interval of 0.24 min.
The purity of each fraction was checked using the GC-MS method described above and liquid chromatography-mass spectrometry (LC-MS, Agilent Technologies 1260), equipped with a vacuum degasser, binary pump and auto injector, diode array detector (DAD, 1260) and quadrupole mass detector (MSD, 6120). An Agilent eclipse plus C18 RRHD column with specifications of 1.8 μm, 2.1 x 50 mm was used. The mobile phase was composed of water and 0.005% TFA and acetonitrile and 0.005% TFA and the flow rate was at 0.3 mL/min. Samples were analysed using a linear elution gradient with acetonitrile, 10% at 0 min, 99% at 10 min, 99% at 11.5 min, 10% at 13 min and 10% at 15 min. Fractions collected from prep-HPLC were directly injected for LC-MS analysis and injection volume was 0.1μl. The mass selective detection (MSD) was carried out in electrospray ionisation (ESI) mode using the following parameters: drying gas flow, 12.0 L/min; nebulizer pressure, 35 psig; drying gas temperature, 350 °C; capillary voltage, 3000 V (positive) and a scan mass range of 100
Fractions containing unknown sesquiterpenoids were dried under a stream of nitrogen gas, dissolved in methanol and further purified using the same prep-HPLC described above fitted with a semi-preparative HPLC column (Luna C18 column, 250 x 10mm, i.d 55μm, Phenomenex Co., USA). The flow rate was at 4 mL/min with a linear gradient of methanol 0 mins-85%, 2 mins-85%, 20 mins-93%, 20.5 mins-100%, 21.1 mins-100%, 22 mins-85% and 30 mins-85% methanol. The fractions were collected between 10.70 and 15.00 mins at time slice of 0.10 min and their purity was checked using the GC-MS and LC-MS method described above. The confirmed pure fractions were dried under a stream of nitrogen gas and subjected to nuclear magnetic resonance (NMR) spectroscopy analysis.
The chemical structure and identity of the isolated compound was confirmed by 1H-NMR and 13C-NMR and 2D (heteronuclear multiple bond correlations (HMBC), homonuclear correlation spectroscopy (COSY) and rotating-frame overhauser spectroscopy (ROESY)) NMR using a Bruker 800MHz NMR with the isolated compound dissolved in deuterated chloroform (CDCl3). Optical rotation measurement was conducted on a JASCO P-1020 Polarimeter to confirm the stereochemistry of the compound isolated.
The organic solvents hexane, methanol (MeOH), dicholoromethane (DCM), diethyl ether (DEE), ethanol and ethyl acetate (EA) were used to extract volatile compounds from 1 g of CGT samples. Parallel extraction of compounds using each organic solvent was done in triplicate. One gram of CGT was weighed into 22 mL vials and 3 mL of each respective extraction solvent added. The mixture was sonicated for 15 min in a sonication bath (Soniclean) and centrifuged using a benchtop centrifuge (Sigma 2–5 10134 centrifuge) at 3,000 rpm for 3 mins. The supernatant (extraction solvent) containing extracts from the CGT sample was transferred into a clean 10 mL volumetric flask. The extraction procedure using 3 mL of solvent was repeated twice and the supernatant collected in a volumetric flask and the volume made up to 10 mL with addition of the respective solvent. Each type of solvent extract was transferred into clean, pre-weighed 22 mL vials and stored at 4 °C until further analysis. One mL aliquots were collected from all extracts into clear 2 mL screw-cap HPLC vials and kept at 4 °C with bulk extracts for further analysis.
PHT components were weighed (1 g) into clean 22 mL vials and 10 mL s of extraction solvent, methanol and hexane added to the samples. Extraction was done in duplicate. Samples were sonicated for 30 mins and left overnight to allow maceration of samples. The following day, samples were vigorously shaken by hand and centrifuged at 3,000 rpm for 3 mins. The supernatant (extraction solvent) was collected into clean, pre-weighed 22 mL vials and 1 mL aliquots collected for further analysis. All extracts were stored at 4 °C until further analysis.
Volatiles were extracted from a larger CGT sample using methanol and hexane. In each 500 mL conical flask, 40 g of CGT was mixed with 200 mL extraction solvent and sonicated for 1 h. After sonication, the respective CGT and extraction solvent mixtures were filtered through a Whatmann Grade 4 filter paper. At this stage, 1 mL aliquots were collected from the respective extraction solvents for further analysis.
A Hewlett Packard 6890 series GC instrument equipped with a BPX-5 capillary column (SGE Analytical Science) (50 mm x 0.22 mm x 1 μm film thickness) fitted with a flame ionisation detector was used. Samples were injected in split mode (split ratio of 1:25) under the following conditions: injection port set at 280 °C, oven temperature held at 50 °C for 1 min, then programmed at a rate of 8 °C/min to 300 °C, helium carrier gas at a flow rate of 1.2 mL /min. Samples were analysed alongside analytical standards of known concentration (β-caryophyllene 4.5 mg/g, α-bisabolol 3.6 mg/g, caryophyllene oxide 1.0 mg/g, α-pinene 4.4 mg/g, β-pinene 4.5 mg/g and α–humulene 2.1 mg/g) and concentration of volatiles calculated using equations derived from standard calibration curve plotted for dilutions of analytical standards.
Data generated from each experiment was analysed using MSD chemstation data analysis software for GC-MS and LC-MS. Microsoft Excel, 2013 was used to calculate concentration, mean values and standard deviation and percentage composition of identified chemical volatiles. GenStat 64-bit Release 18.1 (18th edition), was used to calculate analysis of variance (ANOVA) and determine significant difference (P≤0.05) using the Duncan’s multiple range test. NMR spectroscopy data were analysed using Bruker’s TopSpin™ software.
Gas chromatography-mass spectrometry analysis of extracts obtained from hydro-distilled CGT oil showed different volatile chemicals. Preliminary experiments to determine the appropriate duration (6 h, 12 h or 18 h) for maximal extraction of volatile compounds from CGT by hydro-distillation indicated that most of the terpenoids in CGT were extracted after 6 h of distillation (
Detection of the major terpenoids (
Peak labelled with numbers on top are terpenoids (% area of total area under peaks): (1) α-pinene (0.04), (2) myrcene (0.02), (3) β-pinene (0.01), (4) safranal (0.03), (5) α-copaene (0.1), (6) β-caryophyllene (2.4), (7) β-santalene (0.5), (8) α-curcumene (0.4), (9) α-humulene (1.2), (10) β-farnesene (0.7), (11) nerolidol (1.2), (12) caryophyllene oxide (15.5), (13) gossonorol (5.7), (14) humulene epoxide II (5.3) and (15) β-bisabolol (28.9).
Terpenoids identified in this study from the oil extracts of hydro-distilled CGT samples comprised of 64.7% (area under peaks) total terpenoids, consisting of about 0.1% and 64.5% monoterpenoids and sesquiterpenoids, respectively. The other non-terpenoid volatiles constituted 35.3% of total volatiles identified in the CGT samples.
In order to confirm the identity of the most abundant volatile in the extract of CGT which was tentatively identified as β-bisabolol by GC-MS analysis, isolation by prep-HPLC and semi-prep HPLC (
Data obtained from LC-MS analysis of the isolated compound represented in
C | Carbon shift (δC) | Proton shift (δH) |
---|---|---|
1 | 34.4 | (1.86, 2.17) |
2 | 118.6 | 5.30 (s) |
3 | 134.2 | |
4 | 27.2 | (1.94, 2.17) m |
5 | 31.2 | (1.59, 1.62) m |
6 | 72.4 | 1.62 (s) |
7 | 42.2 | 1.45 (m) |
8 | 31.1 | (1.05, 1.69) m |
9 | 26.8 | (1.91, 2.10) m |
10 | 125.0 | 5.12 (t) |
11 | 131.6 | |
12 | 25.9 | 1.69 (s) |
13 | 17.7 | 1.61 (s) |
14 | 13.8 | 0.92 (d) |
15 | 23.5 | 1.67 (s) |
The total ion chromatogram (TIC) in
Hydro-distillation (HD), hexane (Hex), methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), ethyl acetate (EtOAc) and diethyl ether (DE). Error bars represent standard deviation of terpenoids concentration in replicate samples.
Terpenoid yield by hydro-distillation was compared to yields from use of different organic solvents, to ascertain that the target compounds have been effectively recovered. Extraction of terpenoids from 1 g of CGT samples by use of ethanol (EtOH), methanol (MeOH), diethyl ether (DE), ethyl acetate (EtOAc), hexane (Hex) and dichloromethane (DCM) resulted in the recovery of similar terpenoids extracted by hydro-distillation with the exception of α-bisabolol (
The percentage composition of terpenoids in individual organic solvent extracts (
Total major terpenoids identified were calculated for CGT hydro-distilled and organic solvents extracts and a significant difference (P< 0.001) was observed across the different extracts.
Data generated from solvent extraction of volatile compounds from a larger CGT sample size (40 g) show that recovery of volatile terpenoids was higher in hexane extracts compared to methanol extracts (
Error bars represent standard deviation of terpenoids concentration in replicate samples. Different superscript letters and numerals indicate significant differences (P< 0.05) between terpenes quantified in methanol and hexane extracts of CGT respectively.
Volatiles quantified in hexane extracts from 1 g of different components of the post-harvest trash (PHT) (
Terpenoids | Concentration (μg/g) in PHT components | |||
---|---|---|---|---|
Calyx | Leaves | Stalks | Stems | |
α-pinene | 2.9 ± 0.1b | 1.2 ± 0.3ab | 7.0 ± 1.4c | 0.0 |
β-pinene | 1.6 ± 0.1c | 0.3 ± 0.0a | 0.9 ± 0.1b | 0.0 |
β-caryophyllene | 16.9 ± 2.8b | 23.7 ± 2.3c | 16.0 ± 2.3b | 3.3 ± 0.1a |
α-humulene | 5.0 ± 2.1b | 8.9 ± 1.1c | 5.7 ± 0.5b | 1.2 ± 0.0a |
caryophyllene oxide | 50.8 ± 8.8c | 28.8 ± 2.8b | 10.8 ± 1.4a | 1.4 ± 0.3a |
β-bisabolol | 582.0 ± 73.6b | 564.8 ± 57.5b | 72.7 ± 10.5a | 18.3 ± 1.6a |
Values presented as mean concentration ± standard deviation (S.D). Different superscript letters indicate significant differences (P< 0.05) of terpenes between the different components of post-harvest trash.
Crop residues can serve as an alternative and cheaper source of biologically active natural products. In this study, the volatile chemical composition of waste materials from cotton ginning was investigated following the hypothesis that bioactive chemicals distributed in parts of the cotton plant could be carried over into cotton gin trash (CGT) during the ginning process. Extraction of bioactive chemicals from plants or plant-based materials is achieved by means of different methods including distillation, use of solvent, maceration, Soxhlet extraction, headspace etc. [
The hydro-distillation process involves temperature and time, which are two important factors [
Notwithstanding, the different concentration of individual terpenoids identified in CGT extracts from hydro-distillation and solvent extraction, the recovery abundance for all the extracts with the exception of dichloromethane (DCM) was β-bisabolol > caryophyllene oxide > β-caryophyllene > α-humulene > α-pinene or α-bisabolol. In this study, CGT samples were from mature cotton plants and therefore, the composition of volatile terpenoids identified not only gives a clear indication of the chemical profile of the waste material but also provides added knowledge of chemical composition of the cotton plant at full maturity. Terpenoids such as β-caryophyllene, caryophyllene oxide, humulene and β-bisabolol which are constitutive and inducible terpenoids in cotton plant parts including leaves and bracts, are known to play defensive roles against insect attack on the plant. Hence, their continuous synthesis in cotton plant tissues catalysed by the activity of terpene synthases during the period of plant growth, results in carryover of the volatiles into the waste material. The results further reveal that at full maturity, cotton plant terpenoids are mostly comprised of sesquiterpenoids, with the most abundant found to be β-bisabolol, β-caryophyllene and caryophyllene oxide.
The relatively high amount of β-bisabolol (confirmed by NMR spectroscopy) observed in CGT samples is in agreement with Thompson, Baker,
Specific terpenoids are concentrated in different parts of the cotton plant at different proportions. Therefore, the concentration of terpenoids identified in the different components of PHT in this study, can be linked to the original distribution of the volatiles in the cotton plant. Terpenoids found in parts of the cotton plant are either stored in that part of the plant or synthesised at the time of need indicating the special functions of these terpenoids in their locations in the plant [
Herbivoral activity, which is one of the external factors, is known to target mostly the aerial parts of the cotton plant including leaves, fruits, bolls and flowers, and less frequently other parts such as the stalks and stems [
β-Bisabolol, identified as the most abundant terpenoid in CGT, is a sesquiterpenoid alcohol and an isomer of α-bisabolol, a major sesquiterpenoid found in chamomile flowers [
In this study, we demonstrate that bio-extractives like terpenoids are present in CGT. Most notably is the high concentration of the sesquiterpenoid alcohol β-bisabolol in extracts from hydro-distillation and organic solvent extraction. Hydro and steam distillations have been commonly used to extract volatiles from plant materials on an industrial scale and they should be suitable to produce essential oil rich in β-bisabolol from CGT. Although not investigated in this study, pH of the extracting solvents maybe exploited to further optimise the recovery of volatile compounds from CGT. The distribution of terpenoids in different components of PHT suggests that these volatile compounds are from specific plant parts and carried over to CGT occurs from harvesting cotton fibres. The presence of the volatiles in CGT and PHT suggests these by-products from cotton industry could be exploited as a source of potentially valuable bioactive compounds.
Isolated fraction containing suspected β-bisabolol (black arrow) at UV of 280nm (A) and 210 nm (B) performed by preparative HPLC.
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Hexane (Hex), methanol (MeOH), ethanol (EtOH), dichloromethane (DCM), ethyl acetate (EtOAc) and diethyl ether (DE). Error bars represent standard deviation of terpenoids concentration in replicate samples. Different superscript letters indicate significant differences (P < 0.05).
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