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Reverse phase-high performance liquid chromatography-diode array detector

(RP-HPLC-DAD) analysis of flavonoids profile from curry leaf (Murraya koenigii.
L)

Article in Journal of Medicinal Plant Research · December 2013

DOI: 10.5897/JMPR2013.5150

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 Vol. 7(47), pp. 3393-3399, 17 December, 2013
DOI: 10.5897/JMPR2013.5150
ISSN 1996-0875 ©2013 Academic Journals Journal of Medicinal Plants Research
[Link]

Full Length Research Paper

Reverse phase-high performance liquid

chromatography-diode array detector (RP-HPLC-DAD)
analysis of flavonoids profile from curry leaf (Murraya
koenigii. L)
Kaliyaperumal Ashokkumar1,2*, Kumarakurubaran Selvaraj3 and Saradha Devi K. M.4
1
Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India.
2
Department of Plant Sciences, University of Saskatchewan, Saskatoon, S7N 5A8, SK, Canada.
3
Department of Biology, University of Saskatchewan, Saskatoon, S7N 5E2, SK, Canada.
4
Department of Biochemistry, Biotechnology and Bioinformatics, Avinashilingam University for Women, Coimbatore-43,
Tamil Nadu, India.
Accepted 1 December, 2013

Murraya koenigii L. Spreng, a medicinally important herb of Indian origin, has been used for centuries
in the Ayurvedic system of traditional Indian medicine and popularly used in Indian cuisine on a daily
basis. To evaluate the quality of M. koenigii, L. leaves, a sensitive, simple and precise reversed-phase
high performance liquid chromatography (RP-HPLC) method was developed for assessment of four
major bioactive flavonoids: rutin, quercetin, myricetin and kaempferol. Separation was achieved on a
reversed phase column (ZORBAX, Eclipse plus-C18, 5 μm, 4.6 × 150 mm, Agilent, USA) and
methanol:acetonitrile:water:acetic acid ([Link], v/v/v/v) was employed as the eluent. Sample was
eluted at 0.8 ml/min and peaks were simultaneously identified using UV absorbance at 350 nm for
kaempferol and 254 nm for rutin, myrcetin, and quercetin. The authenticated four analytes were used to
construct linear standard curves by injecting range of 20 to 200 ng. The correlation coefficients of the
calibration curve for the analytes existed higher than 0.999. Isolation of four compounds in M. koenigii,
leaves was achieved by the RP-HPLC method and the results showed that mean value of rutin (924.25
mg/kg) and quercetin (85.88 mg/kg) accumulated greater concentration. Lowest flavonoid concentration
5.88 and 0.20 mg/kg were found to possess in myricetin and kaempferol, respectively. Total flavonoids
concentration was observed to be 1415.50 mg/kg. This study suggested that accumulation of the
greater amount of bioactive components: rutin and quercetin in M. koenigii leaves will be more useful
information for further pharmacological investigations.

Key words: Flavonols, polyphenol, rutin, Murraya koenigii, reversed-phase high performance liquid
chromatography (RP-HPLC).

INTRODUCTION

Flavonoids belong to a group of natural substances with vegetables, grains, bark, roots, stems, flowers, tea, wine
variable phenolic structures and abundance in fruits, and medicinal plants (Pekkarinen et al., 1999). Flavonoids

*Corresponding author. E-mail: [Link]@[Link].

3394 J. Med. Plants Res.

are the most ubiquitous groups of plant phenolics. Due to Earlier studies of M. koenigii, the phytochemical
their importance in food organoleptic properties and analysis was done mostly using thin layer chromato-
human health, a better understanding of their structures graphy (TLC), HP-TLC, and Spectrophotometer. The
and biological activities indicates their potentials as detailed data for the extraction of the individual flavonoids
therapeutic agents and also for predicting and controlling from M. koenigii leaves were limited, and until none of
food quality (Tapas et al., 2008). Researchers have them was characterized by all the components. There-
attracted the greatest attention, and have been fore, the present study was carried out to find appropriate
extensively studied, because they are highly effective precise extraction method and quantification of flavonoids
antioxidants with lower toxicity than synthetic antioxidants from M. koenigii leaves extracts using reversed phase-
such as butylated hydroxyanisole (BHA) and Butylated high performance liquid chromatography-diode array
hydroxytoluene (BHT) (Pekkarinen et al., 1999). detector (RP-HPLC-DAD) analysis. In addition, validate
Every group of flavonoids has their capacity to act the leaf flavonoid components, thus to obtain an
potential as antioxidants. Among them, flavones and informative profile which may serve as a basis for further
catechin components predominately acted as the most utilization of M. koenigii leaves. Furthermore, the results
powerful flavonoids in protecting the body against will be important as an indication of M. koenigii leaves
reactive oxidative species damage (De Groot, 1994). flavonoids as a new source of bioactive flavonoids.
Several studies reported that quercetin had the ability to
inhibit low-density lipoproteins (LDL) oxidation (Graf et MATERIALS AND METHODS
al., 2005), and the oxidation of LDL can result in the
formation of atherosclerotic plaques, leading to cardio- Plant
vascular disease (Hollman and Katan, 1997). Quercetin,
Freshly harvested M. koenigii L. Spreng, leaves were collected from
acting as free radical scavengers was shown to exert a local cultivar grown in Jayankondam (Ariyalur District, Tamil Nadu,
protective effect in liver reperfusion ischemic tissue India). The leafy portions were manually separated, and shade
damage. In addition, quercetin can also reduce dried under room temperature condition. The moisture content of
inflammation by scavenging free radicals. Free radicals dried samples was estimated by toluene distillation method (ASTA,
can activate the transcription factors that generate pro- 1985) and further used for the extraction of flavonoids.
inflammatory cytokines, which are frequently found,
elevated in patients that suffer from chronic inflammatory Sample extractions
diseases (Boots et al., 2008). Quercetin, rutin, myricetin,
and kaempferol, acted as antioxidants, exhibited Dried leaves (100 g) were coarse ground in a blender for about 10
s, and ground powder was weighed 1 g in a 50 ml polypropylene
beneficial effects such as anti-inflammatory, antiviral, and tube and extracted with 10 ml methanol (Romero et al., 2010).
antiallergic, in addition to anticancer activity (Hillwell, Chlorophyll was removed with ice cold acetone (1:20) and mixed
1994; Fraga et al., 1987). thoroughly, and then chlorophyll layer was removed. The mixture
Curry leaf Murraya koenigii (Rutaceae), is a perennial was sonicated for 1 h at room temperature. After centrifugation, the
plant commonly known as Curry Veppilai (Tamil) and supernatant was filtered through a 0.45 µm pore size cellulose
membrane filter and the precipitate was re-extracted twice more
“Curry Patta” (Hindi) is widely used as a spice throughout
following the same steps. The three filtrate supernatants were
India and other tropical countries (Joseph and Peter, combined, from which 3ml was evaporated to dryness in a vacuum
1985). M. koenigii, a medicinally important herb of Indian and the residue was re-dissolved in 500 μl of methanol, placed in 2-
origin, has been used for centuries in the Ayurvedic ml amber glass HPLC vials and analyzed by RP-HPLC coupled with
system of medicine, and very popularly used in Indian DAD.
cuisine on daily basis. Several parts of M. koenigii have
been used in traditional medicine for the treatment of Reagents
rheumatism, traumatic injury and snake bite, because of
it has been reported to have antioxidant (Ninjappa et al., HPLC-grade chemicals and reference compounds for authenticated
standards (e.g. (±) rutin, quercetin, myrcetin, kaempferol) were
2008), antimicrobial (Goutam and Purohit, 1974), anti- obtained from Fluka (Sigma-Aldrich; Bangalore, India), and were
diabetic and anti-dysenteric activities (Kong et al., 1986; used to construct linear standard curves by injecting range of 20 to
Kesari et al., 2007; Mishra et al., 2009; Yankuzo et al., 200 ng. Flavonoids standards (Figure 1) were isolated and purified
2011). It has also been used as anti-inflammatory in lab with individual purity not less than 98% (HPLC assay, UV
(Muthumani et al., 2009), anticancer (Kok et al., 2012; detection). Acids and bases were obtained by ACS quality. Filtered
Muthumani et al., 2009), antinociceptive (Patil et al., water was obtained from a Milli-Q Ultrapure water purification
system (Millipore Pvt. Ltd, Mumbai, India). All the reference stock
2012), anthelmintic (Sharma et al., 2010), solutions were stored at -80°C. Diluted working solutions were
anticholinesterase and antiamnesic activity (Tembhurne freshly prepared for each HPLC analysis.
and Sakarkar, 2011). The recent research reported urry
leaves to have a rich source of polyphenols, inhibit the
HPLC analysis for flavonoids
proteolytic activity of the cancer cell proteasome, and
cause cell death (Noolu et al., 2013). The HPLC system was equipped with an Agilent 1100 serial
 Ashokkumar et al. 3395

Figure 1. Chemical structure of Rutin, Quercetin, Myricetin, and Kaempferol.

system, consisting of a quaternary pump, online degasser, auto- 20 to 200 ng, all the four standards were injected and
sampler, column heater and variable wavelength detector. good correlation of linearity has been achieved. The
Separation was achieved on a reversed phase column (ZORBAX,
Eclipse plus-C18, 5 μm, 4.6 × 150 mm, Agilent, USA) preceded by a
regression equation and correlation coefficient deter-
C18 guard column at 40°C with a diode array detector (DAD) set at mined from the reference were presented in Table 2.
190 to 600 nm and methanol:acetonitrile:water:acetic acid
([Link], v/v/v/v isocratically) employed as the eluent. The
elution was kept constant at 0.8 ml/min and the peaks were Reproducibility and accuracy
simultaneously identified using UV absorbance at 350 nm for
kaempferol and 254 nm for rutin, myricetin and quercetin. The The study of reproducibility was performed on three
injection volume was 10 µl for each technical repeat. All the consecutive days (n = 9) indicating a relative standard
injections were performed in triplicate. The chromatographic peaks
deviation of 3.50% for rutin, 3.25% for quercetin, 3.00%
of the analytes were confirmed by comparing their retention time
and UV spectra with those of the pure standards. Quantification for myricetin and 3.36% for kaempferol. The accuracy of
was carried out by the integration of the peak using the external the analytical method was determined by assaying at
standard method. least triplicate applications of each sample. The recovery
test was used to evaluate the accuracy of the method.
The average recovery (n=5) of quercetin and myricetin
RESULTS AND DISCUSSION were both 99 and 98.2% for rutin and 99.2% for
kaempferol. Relative standard deviation (RSD) was 2.77,
Range of linearity 0.71, 1.58 and 2.17% for in rutin, quercetin, myricetin and
kaempferol, respectively (Table 2). Analytes peaks were
The retention time of all the four individual analytes were simultaneously identified using UV/Vis diode array
presented in Table 1. The linearity was investigated for detection at 350 nm for kaempferol and 254 nm for rutin,
the authenticated four flavonoids standard by plotting myricetin and quercetin (Figure 2). Typical chromato-
peak area against the injected amounts. In the range of grams showed comparison between the kaempferol 40
3396 J. Med. Plants Res.

Table 1. Identification of flavonoids by RP-HPLC analysis.

a b
S/N Compounds Molar mass (g/mol) Formula RT (min) Lambda Max
1 Rutin 610.52 C27H30O16 0.89 254
2 Quercetin 302.24 C15H10O7 1.29 254
3 Myricetin 318.23 C15H10O8 1.66 254
4 Kaempferol 286.23 C15H10O6 2.23 350
a b
RT: Retention time; Lambda max, absorbance spectrum wavelength (nanometer).

Table 2. Standard calibration curve and average recovery percentage.

2 a b
S/N Compound Standard purity (%) Regression equation Correlation (R ) Average recovery (%) RSD (%)
1 Rutin >98.0 4.4582 × -0.7012 0.9999 98.2 2.77
2 Quercetin >98.0 5.6667 × -1.3192 0.9995 99.0 0.71
3 Myricetin >97.0 5.2132 × -1.0153 0.9998 99.0 1.58
4 Kaempferol >98.0 5.4721 × +0.2124 0.9999 99.2 2.17
a
Average recovery: Observations (n=9); RSD (%): relative standard deviation (%).
Absorbance (mAU)

Time (min)
Figure 2. Chromatogram of external standards (catechin, quercetin, myricetin and kaempferol) concentration in
40 ng and absorbance at 254 nm.

ng concentration detected at 350 nm with highly repeats) were extracted following the earlier mentioned
significant greater peak area than 254 nm (Figures 2 and procedure, and all injections were performed in triplicate
3). analyzed in the Agilent HPLC system. The concentration
of flavonoid analytes was determined by the regression
equation. Total flavonoid concentrations were calculated
RP-HPLC analysis and quantitative determination of by the sum of four individual flavonoids along with
flavonoids unknown flavonoids. The unknown flavonoids presented
in the same chromatogram profile were calculated by
Three biologically replicated samples (nine technical using regression equation of rutin. HPLC assay was
 Ashokkumar et al. 3397

Absorbance (mAU)

Time (min)
Figure 3. Typical chromatogram of external standard kaempferol (40ng) absorbance at 350 nm.

reproducible, sensitive and validated. Hence, it was In this study, the RP-HPLC analysis also showed good
successfully applied for the determination of rutin, resolution of peak separation of myricetin and kaempferol
quercetin, myricetin, and kaempferol in M. koenigii leaf from M. koenigii leaves. The average value of methanolic
samples. The results showed that three biologically extracts of curry leaf of myricetin and kaempferol are
replicated samples had similar retention time in HPLC shown in Table 3. In addition, total flavonoids concen-
chromatogram for all four individual analytes. Of the four tration was 1415.50 mg/kg (Figure 4). Kaempferol and
analytes, mean value of rutin (924.25 mg/kg or 92.42 myricetin are acting as antioxidants exhibited beneficial
mg/100 g) and quercetin (85.88 mg/kg or 8.6 mg/100 g) effects such as anti-inflammatory, antiallergic, antiviral as
accumulated greater in methanol extracts of M. koenigii well as anticancer activity (Hillwell, 1994; Fraga et al.,
leaves (Table 3 and Figure 4). Earlier study by Vijayand 1987; Ningappa et al., 2008). Dasgupta et al. (2003)
and Wesely (2011) reported M. koenigii leaves to contain observed that M. Koenigii leaves had significant reduction
quercetin and rutin and it confirmed our results. in tumor cells from Swiss albino mice and the blood-
In recent studies, it was reported that quercetin could glucose levels of diabetic rats was treated with methanol
inhibit LDL oxidation. In addition, the intake of quercetin extracts of curry leaf which was observed to have
greater than 4 mg/day has been found to have 21% significant reduction as compared to diabetic control
reduction in cardiovascular disease mortality (Graf et al., groups (Math and Balasubramaniam, 2005). Therefore,
2005). Nevertheless, quercetin can also reduce our study results observed that M. koenigii leaves have
inflammation by scavenging free radicals (Boots et al., the natural abundance of rutin and flavonols, and it may
2008). Based on this report, our present study results to exhibit several health benefits.
intake of 50 g/day, curry leaf was expected to produce
approximately more than 4.0 mg of quercetin, and it will
be helpful in reducing cardiovascular disease in human Conclusion
being. Flavonoid components act as the most powerful
flavonoids for protecting the body against reactive Methanol extracts of curry leaf contains rutin, quercetin,
oxidative species (ROS) damage (De Groot, 1994). Our myricetin and kaempferol identified by RP-HPLC-DAD
study results showed that methanolic extract of curry leaf analysis. The individual flavonoid concentration was
leaves have the huge amount of rutin. Hence, intake of determined by the authenticated reference analytes
antioxidants rich curry leaf will be more useful in regression equation, and assay was reproducible, sensi-
protecting the human body against the oxidative stress tive and validated. The analysis revealed that rutin and
damage by scavenging free radicals. quercetin are subjected to the presence predominantly,
3398 J. Med. Plants Res.

Table 3. Quantification of flavonoid concentration in Murraya koenigii leaves.

a b c
S/N Flavonoid mg/kg SD SE
1 Rutin 924.25 3.77 1.26
2 Myricetin 5.17 0.15 0.05
3 Quercetin 85.88 4.84 1.61
4 Kaempferol 0.19 0.04 0.01
5 Total flavonoids 1415.50 7.01 2.34
a b c
mg/kg: Milligram per kilogram; SD: standard deviation (n=9); SE, Standard error.
Concentration (mg/kg)

Total flavonoid

Figure 4. Flavonoid concentration in curry leaf (Murraya koenigii) leaves with 5% error bar.

whereas myricetin and kaempferol concentrations were Boots AW, Willms LC, Swennen EL, Kleinjans JC, Bast A, Haenen GR
accumulated with significant amount in curry leaf leaves. (2008). In vitro and ex vivo anti-inflammatory activity of quercetin in
healthy volunteers. Nutrition 24(7-8):703-710.
This study suggests that accumulation of greater amount Dasgupta T, Rao AR, Yadava PK (2003). Chemomodulatory action of
of bioactive components like rutin and quercetin in M. curry leaf (murraya koenigii) extract on hepatic and extrahepatic
koenigii leaves will provide more useful information for xenobiotic metabolising enzymes, antioxidant levels, lipid
peroxidation, skin and forestomach papillomagenesis. Nutr. Res.
further pharmacological investigations.
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