Tecomella undulata G. Don: A rich source of flavonoids

Industrial Crops and Products 43 (2013) 213–217

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Tecomella undulata G. Don: A rich source of flavonoids Abdul Qayoom Laghari a , Shahabuddin Memon a,∗ , Aisha Nelofar b , Abdul Hafeez Laghari b a b

National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080, Pakistan Pakistan Council of Scientific and Industrial Research, Karachi 75280, Pakistan

a r t i c l e

i n f o

Article history: Received 19 March 2012 Received in revised form 11 July 2012 Accepted 13 July 2012 Keywords: Tecomella undulata Flavonoids Antioxidant activity Microwave extraction

a b s t r a c t The present study describes identification of flavonoids extracted from leaves and flowers of Tecomella undulata and their antioxidant potential. The total flavonoid contents (TFC) were determined by UV–vis spectrophotometry. Identification and quantification of individual flavonoids in the extracts were done by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS). The aqueous ethanol (70%) fractions of leaves and flowers are highly rich in flavonoids. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity of the extract was also determined. Of all the methods microwave extract has highest antioxidant activity as compared to other extracts obtained by sonication, marination, reflux and soxhlet extraction methods. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

2. Experimental

Flavonoids belong to polyphenolic phytochemicals with variable phenolic structures; they are antioxidants (Laghari et al., 2010, 2011a,b; Memon et al., 2010) and play a protective role in carcinogenesis (Hertog et al., 1992). Tecomella undulata G. Don (Bignoniaceae) commonly known as Rohira in Sindh is used in traditional medications. It is widely distributed in Thar Desert of Pakistan and India. This plant contains flavonoids, (Taneja et al., 1975; Azam and Ghanim, 2000) iridoid glucoside (Joshi et al., 1975) ferulic ester (Joshi et al., 1986) and Chromone glucoside (Gujral et al., 1979). The plant has many pharmacological activities such as hepatoprotective activity against thioacetamide-induced hepatotoxicity (Khatri et al., 2009), antiinflammatory and analgesic potential (Ahmad et al., 1994; Chal et al., 2011). In the indigenous systems of medicine, it has been used for liver, spleen and abdominal complaints (Parekh and Chanda, 2007). In the present study, total flavonoid contents and identification of different flavonoids as well as their antioxidant activity were determined in the extracts from leaves and flowers of T. undulata. Herein, we report six flavonoids for the first time in leaves and flowers extracts of T. undulata. Among all these six flavonoids only rutin (a well known flavonoid and antioxidant) was reported elsewhere from flowers (Taneja et al., 1975).

2.1. Chemicals

∗ Corresponding author. Tel.: +92 22 9213430; fax: +92 22 9213431. E-mail address: [email protected] (S. Memon). 0926-6690/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.indcrop.2012.07.025

In this study, butylated hydroxyanisole (BHA) (BDH, Poole, England), methanol, ethanol, acetonitrile, petroleum ether (40–60 Bp) (HPLC grade) (Lab-Scan Analytical Sciences, Bangkok, Thailand), formic acid, aluminum chloride, sodium nitrite and sodium hydroxide (Merck, Darmstadt, Germany) were used. 2.2. Plant material The flowers and leaves of T. undulata were collected in March 2010 from a small village Rana Bajeer district Mithi at Tharparkar of Sindh province in Pakistan. The plant species were identified by a plant taxonomist in the Institute of Plant Sciences, University of Sindh, Jamshoro/Pakistan and identified plant sample is submitted with the voucher specimen number 15176 in herbarium of this institute. The plant material was shade dried, coarsely powdered and used for further studies. 2.3. Extraction of flavonoids Leaves/flowers (coarsely powdered and shade dried) were defatted and flavonoids extraction was made by a reported method (Zhu et al., 2010) with slight modification. 2.3.1. Microwave extraction One gram of defatted leaves/flowers was taken in 50 mL aqueous ethanol (70%) and placed into microwave decomposition

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system (Start E Microwave Extraction system, Mile stone, ModelAct 38-Revol-03106) at 50 ◦ C for 9 min. The sample was filtered and made up to 50 mL in a volumetric flask with aqueous ethanol. 2.3.2. Soxhlet extraction One gram of defatted leaves/flowers were taken in 160 mL of aqueous ethanol (70%) on soxhlet assembly on water bath at 100 ◦ C. Extraction time was 5 h. Solvent was then evaporated under vacuum and remaining extract was made up to 50 mL with aqueous ethanol (70%). 2.3.3. Sonication extraction One gram of defatted leaves/flowers were taken in 50 mL of aqueous ethanol (70%). It was sonicated in ultrasonic bath (Supersonic X-3, Model DSD80A5QS, power 80 W) for 1 h at 30 ◦ C. It was filtered and made up to 50 mL with aqueous ethanol (70%). 2.3.4. Reflux condensation extraction One gram of defatted leaves/flowers were taken in 50 mL aqueous ethanol (70%). It was refluxed for two and half hours at 80 ◦ C. Extract was filtered and diluted to 50 mL with aqueous ethanol (70%).

Table 1 Total flavonoids identified in present study as well as in previous studies from Tecomella undulata. Parts of the plant

Flavonoids identified

References

Flowers

Rutin, quercetin, luteolin 7-glucoside Cirsimaritin

Taneja et al., 1975

Leaves

Azam and Ghanim (2000)

Cirsilineol Flowers and leaves

Rutin Tiliroside 5,6-Dimethyoxy-3 ,4 dioxymethylene-7-O-(6 ’-ˇ-dglucopyranosyl-ˇ-dlucopyranosyl) flavanone Genistein 4 ,7-O-diglucoside methylmalonylated Luteone 4 ,7-O-diglucoside Luteolin 3 ,4 -dimethylether-7O-ˇ-d-glucoside

Present work

2.3.5. Marinated extraction One gram of defatted leaves/flowers was taken in 50 mL of aqueous ethanol (70%). After standing for 48 h at room temperature (25 ◦ C) it was filtered and made up to 50 mL with aqueous ethanol (70%).

software was used for data acquisition. Analytical conditions were: positive ion mass spectra of the column elute were recorded in the range m/z 200–700. Capillary temperature was kept at 200 ◦ C at 4.5 kV. Sheath gas flow rate was kept at 70 (arbitrary unit). Identification of individual flavonoids in extracts was carried out by LC–MS-ESI by comparing their masses and max with the reported values (Lin and Harnly, 2007; Jung and Park, 2007; Sultana et al., 2010; Stobiecki et al., 2010; El-Desoky et al., 2007).

2.4. Determination of total flavonoid content

2.7. DPPH antioxidant activity

Total flavonoids were calculated by a reported method (Zhu et al., 2010). The reaction mixture containing 2 mL plant extracts, 0.6 mL sodium nitrite (5%, w/v), 0.5 mL aluminum chloride (10%, w/v), 3 mL sodium hydroxide (4.3%, w/v) was made up to 10 mL with water. In each step, 6 min time period was given for shaking to complete reaction at room temperature (25 ◦ C). The solution was allowed to stand for 15 min and absorbance was measured at 500 nm on a Specord 200 spectrophotometer. Sample extract in aqueous ethanol (70%) without any other reagent was used as reference, rutin was used as a standard and results were calculated as rutin equivalents (Rutin eq., mg/mL) of T. undulata.

DPPH scavenging ability of T. undulata leaves or flowers extracts was measured by their bleaching capability toward purple DPPH methanol solution. This assay was performed according to the reported method (Yu et al., 2008; Laghari et al., 2011a,b) by mixing 3 mL of 0.1 mM DPPH in methanol and 1 mL of sample solution in aqueous ethanol (70%). The absorbance was measured at 517 nm. Decreasing amplitude of signal at the selected wavelength confirmed a high radical scavenging activity. The antioxidant activity of BHA as standard reference was assayed. Methanol was used as blank and the measurement of solutions without sample were used as the control. The inhibition of DPPH radicals by the samples was calculated as follows:

2.5. Chromatographic analysis of flavonoids

DPPH inhibition (%) = The liquid chromatograph system was equipped with the photo diode array detector (PDA) and a vacuum degasser. A Hypersil Gold C18 (250 mm × 4.6 mm, 5 ␮m) (Thermo electron corporation USA) column was used for all separations and analytical data was evaluated by using X-Caliber data processing system (2.0 SR2). The mobile phase was composed of methanol–acetonitrile (7:3) (A) and 0.1% (v/v) formic acid in water (B). The flow rate was 1 mL/min. The gradient programming was as follows; starting from concentration of A at 5% for 5 min and then gradual increase from 5% to 30% in 10 min. Isocratic step of 5 min and then brought back to 5% in 5 min followed by 5 min for column equilibration; total run time was 25 min The eluent was monitored by PDA detector set at three different wavelengths 270, 320 and 360 nm. 2.6. Identification and qualitative analysis of constituents by LC–MS Identification of the constituents was performed with a Thermo Finnigan LCQ Advantage ion trap mass spectrometer (Vernon Hills, Illinois, USA) fitted with the liquid chromatograph system coupled on-line with electro-spray ionization (ESI) source and X-Caliber

A−B × 100. A

(1)

where A is the absorbance without extract and B is the absorbance with the extract. IC50 values were calculated by considering the 50% inhibition of DPPH free radical in 5 min by leaves or flowers samples at minimum concentration and comparing with same response shown by BHA. 3. Results and discussion 3.1. Identification and quantification of flavonoids As described above the T. undulata has largely been used medicinal plant in folk medication system and rich in bioactive compounds responsible for that property. Previously Taneja et al. (1975) and Azam and Ghanim (2000) have reported flavonoids from this plant. A comparative profile of flavonoids already reported in the literature from T. undulata and those discussed in the present study are given in Table 1. Identification of the individual flavonoids was done by HPLCPDA–ESI-MS/MS, by comparing their masses and max with the

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Table 2 Flavonoid glycosides identified by HPLC-PDA–ESI-MS/MS from the leaves and flowers extracts of Tecomella undulata. Compound No.

max (nm) reported

Identity

1 2 3

Rutin Tiliroside 5,6-Dimethyoxy-3 ,4 dioxymethylene-7-O-(6  -ˇ-dglucopyranosyl-ˇ-d-glucopyranosyl) Flavanone Genistein 4 ,7-O-diglucoside methylmalonylated Luteone 4 ,7-O-diglucoside Luteolin 3 ,4 -dimethylether-7-O-ˇ-d-glucoside

4 5 6

max (nm) samples

Calculated molecular mass

Mass found in samples M+1

MS2

256, 352 266, 310 275,324

255, 349 266, 320 277,316

610 594 668

611 595 669

465, 303 287 345

–

277, 316

694

695

533, 271

– 255, 267, 352

287, 317 252, 266, 349

678 546

679 547

517, 461, 355, 299 329, 219

reported value (Table 2) (Lin and Harnly, 2007; Jung and Park, 2007; Sultana et al., 2010; Stobiecki et al., 2010; El-Desoky et al., 2007). Rutin (1), Tiliroside (2), 5,6-dimethyoxy-3 ,4 -dioxymethylene7-O-(6 -ˇ-d-glucopyranosyl-ˇ-d-lucopyranosyl)flavanone (3), Genistein 4 ,7-O-diglucoside methylmalonylated (4), luteone 4 ,7O-diglucoside (5), luteolin 3 ,4 -dimethylether-7-O-ˇ-d-glucoside (6) were identified from the extracts (Fig. 1).

Quantification was performed in relative percentage by taking area of all constituents from HPLC-PDA chromatogram (Fig. 2) and taking it as denominator to calculate each constituent by putting area of that content in numerator and multiplying by 100. Relative percentage of individual flavonoids (1–6) was also found to be higher in microwave extraction technique as compared to other extraction techniques (Table 3).

OH O

HO

OH OH

HO O OH

O

O

OH O

H 3C HO HO

OH

O

O

HO HO

OH

O

O

OH OH

Rutin (1)

HO O

O

OH

OH O

O CO-CH2-COOCH3

O

HO

Genistein 4’,7-O-diglucoside methylmalonylated (4)

O

O

OH

O

OH O

O OH

O

HO HO

OH

HO

O

O

OH

OH (CH3)2C=CH-CH 2

H OH

HO O O

OH O

OH

Tiliroside (2)

HOH2C HO HO

HO

OH OH

Luteone 4ʹ,7-O-diglucoside (5)

O

OH

O

O

HO HO

O HO

O

O

OMe

OH

O

HO HO

OMe O

O

O

OH

MeO OMe

O

5,6-dimethyoxy-3’,4’-dioxymethylene-7-O-(6’’-β -D-glucopyranosyl-β-Dglucopyranosyl) Flavanone (3)

OH O Luteolin 3’,4’-dimethylether -7-O- β-D-glucoside (6)

Fig. 1. Chemical structures of flavonoids (1–6) identified from the leaves and flowers.

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Fig. 2. HPLC-PDA chromatogram comprised of (1) Rutin, (2) Tiliroside, (3) 5,6-dimethyoxy-3 ,4 -dioxymethylene-7-O-(6  -ˇ-d-glucopyranosyl-ˇ-d-lucopyranosyl) Flavanone, (4) Genistein 4 ,7-O-diglucoside methylmalonylated, (5) luteone 4 ,7-O-diglucoside, (6) luteolin 3 ,4 -dimethylether-7-O-ˇ-d-glucoside.

Table 3 Relative percentage of each flavanoid in flowers and leaves extracts with different extraction techniques. Compound

Extraction techniques Microwave

1 2 3 4 5 6

Soxhlet

Marination

Sonication

Reflux

Flowers

Leaves

Flowers

Leaves

Flowers

Leaves

Flowers

Leaves

Flowers

Leaves

28.1 17.8 21.9 23.8 19.3 32.9

28.2 18.0 22.1 24.2 19.6 33.8

20.7 16.7 20.3 18.5 18.7 20.1

21.2 17.2 18.4 19.2 19.4 21.0

16.5 23.3 23.0 21.4 18.2 10.4

19.0 25.3 21.7 22.9 19.5 11.3

16.5 20.6 23.9 18.1 13.5 20.3

16.8 21.1 21.6 18.6 13.9 21.1

14.2 17.5 17.3 14.3 26.1 11.9

14.7 18.2 16.0 14.8 27.3 12.6

All values are in relative %.

3.2. Total flavonoid contents Total flavonoid contents were found higher, i.e. 5.9 mg/g and 3.2 mg/g in microwave extractions of leaves and flowers of T. undulata, respectively (Table 4). 3.3. Assay of DPPH Leaves or flowers extract shows higher DPPH scavenging ability than the reference Butylated hydroxyanisole (BHA) (Table 5). Microwave extracts show highest antioxidant activity than rest of the extracts obtained by other techniques. Leaves show higher antioxidant activity as compared to flowers. It can be concluded

Table 4 Total flavonoids content in the leaves and flowers and comparison in the extraction methods. Extraction methods

Total flavonoids contents in leaves (mg/g)

Total flavonoids contents in flowers (mg/g)

Microwave Soxhlet Reflux Sonication Marinated

5.9 5.5 4.1 4.4 4.0

3.2 2.1 2.0 1.7 1.0

A.Q. Laghari et al. / Industrial Crops and Products 43 (2013) 213–217 Table 5 DPPH % inhibition activity of reference BHA standard and various extracts of Tecomella undulata from leaves (a) and flowers (b). Extraction sample/Reference

IC50 (a)a , b

IC50 (b)a , b

BHA Microwave Soxhlet Marinated Sonication Reflux

347.2 ␮M 1.7 mg/L 2.4 mg/L 3.2 mg/L 3.0 mg/L 5.2 mg/L

347.2 ␮M 2.2 mg/L 3.6 mg/L 4.7 mg/L 4.3 mg/L 7.8 mg/L

a Concentration (in mg/L) at which 50% inhibition of DPPH is observed by samples in the same time period as the reference. b Concentration (in moles) at which 50% inhibition of DPPH is observed by reference.

that the contribution to antioxidant ability of T. undulata extracts is mainly due to the presence of all identified flavonoids, which were extracted in highest quantity by microwave extraction. 4. Conclusion Of all the techniques, microwave extraction process is most appropriate as it gives highest yield of flavonoids in minimum possible time. T. undulata is a rich source of flavonoids and all extracts show noticeable antioxidant activity. The importance of the plant is due to its high antioxidant activity and total flavonoids contents. It is a better alternative to the synthetic antioxidant agents and flavonoids. This data may also help to pharmaceutical industry as well as to folk medication practitioners who use this plant as a source of curing diseases. Acknowledgements Support during the present work made by the National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro/Pakistan and Pakistan Council of Scientific and Industrial Research, Karachi/Pakistan is gratefully acknowledged. References Ahmad, F., Khan, R.A., Rasheed, S., 1994. Preliminary screening of methanolic extracts of Celastrus paniculatus and Tecomella undulata for analgesic and antiinflammatory activities. J. Ethnopharmacol. 42, 193–198.

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