SUPPLEMENTARY MATERIAL
Identification and determination of flavonoids, carotenoids and chlorophyll
concentration in Cynodon dactylon (L.) by HPLC analysis
Saradha Devi Muthukrishnana, †, Ashokkumar Kaliyaperumalb, c,*, † and Annapoorani
Subramaniyana
a
Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University
for Women, Coimbatore-43, TN, India; bDepartment of Plant Biotechnology, Tamil Nadu
Agricultural University, Coimbatore-3, TN, India; cDepartment of Plant Sciences, University of
Saskatchewan, 51, Campus Drive, Saskatoon, Saskatchewan, S7N 5A8, Canada.
*
Corresponding author. Email: biotech.ashok@gmail.com
† Authors contributed equally to this article.
Abstract
Cynodon dactylon (L.) is a potent medicinal plant in the traditional and current Indian medicinal
systems. The objective of this research was to find out the levels of flavonoids, carotenoids and
chlorophyll b in C. dactylon leaves by high performance liquid chromatography (HPLC)
equipped with a diode array detector. HPLC analysis revealed that total carotenoid and total
flavonoid concentration were 62 mg/100g and 249.1 µg/g, respectively. The mean chlorophyll b
was 85.1 mg/100g in C. dactylon. Among the flavonoids, quercetin (164.7 µg/g) was the major
flavonoid followed by kaempferol (48.2 µg/g), rutin (18.4 µg/g), catechin (12.1 µg/g), and
myricetin (5.7 µg/g). Of the carotenoids, β-Carotene (35.2 mg/100g) was predominant followed
by lutein (17.0 mg/100g), violaxanthin (5.8 mg/100g) and zeaxanthin (4.2 mg/100g).
Chlorophyll b concentration was 85.1 mg/100g in C. dactylon. The results of this investigation
should be useful information for further pharmacological studies.
Key words: Cynodon dactylon; Carotenoids; Chlorophyll b; Flavonoids; HPLC
Experimental
Plant material and extraction
Cynodon dactlylon leaves were collected from the contaminant free area of the Avinashilingam
Deemed University for Women, Coimbatore. The plant was identified and authenticated from
Department of Botany; Avinashilingam University for Women (Coimbatore, Tamil Nadu, India).
Voucher specimen (HOD/BC/08-10-2012) is preserved in our laboratory for future reference. All
the chemicals and solvents used were of analytical grade. Fresh leaves of C. dactylon were
collected and washed with distilled water and allowed to dry using filter paper. The shade dried
leaves were cut into small pieces and ground for a few minutes in a domestic coffee grinder and
sieved. The 20 g ground sample were placed in a Soxhlet extractor and refluxed with 200ml of
petroleum ether at 60°C for 8 h to remove the chlorophyll, non-flavonoid components and lipid
dewaxing (Palanivel et al., 2008), and then petroleum ether extract were air dried for 12 hours.
After all the petroleum ether was evaporated, the dried sample was extracted with ethyl acetate
using Soxhlet apparatus (Ansari et al., 1976). Ethyl acetate extract was centrifuged at 10,000 g
for five minutes, filtered 0.2 µm PVDF membrane directly into a sample vial, and analyzed by
HPLC.
Carotenoids and chlorophyll b was extracted by Methanol (MeOH) : Dichloromethane
(DCM) solvent extraction method. 5 ml of extraction solvents were added in 1 g of sample tissue
in Pyrex tubes capped with Teflon liners. Samples were immediately vortexed followed by
shaking at 200 rpm for one hour and extracts were treated with the diluents (100% acetonitrile)
to remove proteins and some lipids, and centrifuged at 12,000 g for five minutes. For HPLC
separation, the centrifuged supernatant was filtered (0.2 µm PVDF), placed in 2-ml amber glass
HPLC vials and analyzed by HPLC.
Reagents and standard calibration
All the chemicals and organic solvents were obtained HPLC-grade Fluka (Sigma-Aldrich) and
unless otherwise noted. Reference compounds for authenticated standards (e.g., rutin, quercetin,
myrcetin, kaempferol) were obtained from Sigma-Aldrich used to construct linear standard
curves by injecting range of 20-200 µg. For carotenoid analysis, standards of lutein and
violaxanthin (ChromaDex), zeaxanthin and β-carotene, (Sigma-Aldrich) were used to construct
linear standard curves by injecting range of 4-100 µg. Flavonoids and carotenoids standards was
isolated and purified in our lab with individual purity not less than 98% (HPLC assay, UV
detection). Chemical reference compounds weighed to 0.1 mg, and all the reference stock
solutions were stored at -80°C. Diluted working solutions should be freshly prepared for each
HPLC analysis.
High performance liquid chromatographic separation of flavonoids, carotenoids and
chlorophyll b
Chromatography was performed using the HPLC system (Agilent 1100 serial), equipped with a
quaternary pump, online degasser, auto-sampler, column heater and variable wavelength
detector. Flavonoids separation was achieved on a reversed phase column (Phenomenex, Kinetex
XB-C18, 100A, 5 μm, 4.6 x 250 mm) preceded by a C18 guard column were used at 30°C and
methanol–acetonitrile–water (40:20:40, v/v/v isocratically) containing 0.5% acetic acid was
employed as the eluent. Ethyl acetate extracts were eluted for 20 min using elution solvents.
Spectrophotometric detection was achieved by means of a diode array detector in the range 190400 nm. The elution was kept constant at 0.5 ml/min and the peaks were simultaneously
identified using UV absorbance at 279 nm for quercetin and 254 nm for catechin, rutin, myrcetin
and kaempferol. The temperature of the column during analysis was maintained at 40°C. The
injection volume was 5µl for each technical repeat and total run time was 20 min. All the
injections were performed in triplicate. The chromatographic peaks of the analytes were
confirmed by comparing their retention time and UV spectra with those of the pure standards.
Quantification was carried out by the integration of the peak using the authentic standards. Each
biological sample was extracted in three technical repeats and analyzed by HPLC.
Carotenoids separation was done on a C30 carotenoid reverse phase column (3 µm, 4.6 x
250 mm), preceded by a C30 guard column were used at 24°C. MeOH-DCM extracts were
eluted with 40 min isocratic elution (58:22:20, CH3CN:CH3OH:CH2Cl2) at flow rate was 0.8
ml/min, to separate compounds in the extracts, and injection volume was 10 µl/sample. All
carotenoids and chlorophyll b peaks were detected at 450 nm. All individual carotenoids and
chlorophyll b were identified by ultraviolet-visible spectra analysis, and by comparing their
retention times with authentic standards.
Table S1. Molar mass, molecular formula and purity of individual flavonoids, carotenoids and
chlorophyll
Sl.No
Compounds
Molar Mass (g/mol)
Molecular formula
Purity (%)
Flavonoids
1.
2.
3.
Catechin
Rutin
Quercetin
290.27
610.52
302.24
C15H14O6
C27H30O16
C15H10O7
>98.0
>97.0
>98.0
4.
5.
Myricetin
Kaempferol
318.24
286.24
C15H10O8
C15H10O6
>98.0
>99.0
600.85
568.87
568.88
536.87
C40H56O4
C40H56O2
C40H56O2
C40H56
>97.0
>98.0
>98.0
>95.0
907.47
C55H70MgN4O6
>98.0
Carotenoids
1.
2.
3.
4.
Violaxanthin
Lutein
Zeaxanthin
β-Carotene
Chlorophyll
1.
Chlorophyll b
Figure S1. HPLC analysis detected individual carotenoids and chlorophyll b molecular structures
of Cynodon dactylon leaves.
Figure S2. HPLC chromatogram of flavonoid profile of Cynodon dactylon leaves. [(A), UV- Vis
detection at 254 nm; (B), UV-Vis detection at 279 nm]
Figure S3. Typical sample chromatogram of carotenoid profile of Cynodon dactylon leaves.
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