Accepted Manuscript Title: Determination of phytochemicals, antioxidant activity and total phenolic content in Andrographis paniculata using chromatographic methods Author: Marzanna Kurzawa Anna Filipiak-Szok Ewa Kłodzi´nska Edward Szłyk PII: DOI: Reference:

S1570-0232(15)30022-2 http://dx.doi.org/doi:10.1016/j.jchromb.2015.05.021 CHROMB 19453

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

2-2-2015 11-5-2015 14-5-2015

Please cite this article as: Marzanna Kurzawa, Anna Filipiak-Szok, Ewa Klodzi´nska, Edward Szlyk, Determination of phytochemicals, antioxidant activity and total phenolic content in Andrographis paniculata using chromatographic methods, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2015.05.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Determination of phytochemicals, antioxidant activity and total phenolic content in Andrographis paniculata using chromatographic methods Marzanna Kurzawa1 , Anna Filipiak-Szok1*, Ewa Kłodzińska2, Edward Szłyk1 1

Nicolaus Copernicus University in Toruń, Faculty of Chemistry, Gagarin 7 St., 87-100 Toruń, Poland 2

Institute for Engineering of Polymer Materials and Dyes, M. Skłodowskiej-Curie 55 St., 87-100 Toruń, Poland

Corresponding

author:

Anna

Filipiak-Szok*,

e-mail:

[email protected],

phone number +48 0056 611 4369

Antioxidant activity, total phenolics content and selected phytochemicals (alkaloids and andrographolides) were determined in Andrographis paniculata and in dietary supplements containing this plant. Antioxidant activity was measured by FRAP, CUPRAC and DPPH procedures and ranged from 503.36 to 6164.09µmol TE/100g d.m. depending on methods, part of plant and kind of dietary supplement. The total phenolics (175.13 – 1723.79 mg GAE/100g) and andrographolides content (19.44 – 85.13 mg/g) in the studied samples were correlated with antioxidant activities determined by CUPRAC, FRAP and DPPH (r>0.95, p98%) indicates good accuracy, acceptable for the pharmacopoeial methods. Precision of analysis (measured six times on the same day and once in five consecutive days) was determined for standard solutions of the studied analytes. The intra-day precision was between 0.94 – 1.08%, while the inter-day parameter was estimated from 0.99 to 1.15%. Total andrographolides content (TAC) was calculated as the sum of every analyzed andrographolide.

Andrographolides in AP were identified by GC-MS technique using temperature program : 100°C hold for 1 min then ramped with a rate of 8°C/min up to 290°C, hold for 10 min. Each analysis was performed using 10:1 split ratio and sample volume 10µl. The detection unit was a 5975C VL MSD with triple-axis Mass Selective Detector (Agilent) in TIC mode, with a full scan range 20-200 amu).

2.5.

Determination of antioxidant activity

2.5.1. Cupric ion reducing antioxidant activity - CUPRAC method The CUPRAC procedure is based on the reduction of Cu(II) (0.01M), in ammonium acetate (1.0 M) in the presence of 0.0075M neocuproine (2,9-dimethyl-1,10-phenanthroline), by polyphenols, yielding a Cu(I) complexes. Absorbance of Cu(I) complex was measured at λmax = 455 nm after 30 min of incubation at 22°C. The calibration curves were prepared using 0.02–0.10 µmol/mL working methanolic solutions of Trolox (TE) (y = (9.804 ±0.141) x (0.069 ± 0.009), R2 = 0.9998, RSDslope = 0.71%, detection limit DL= 0.0018 µmol/mL and quantification limit QL= 0.0045 µmol/mL). Detection limit was calculated as DL=(3.3Sx/y )/a; quantification limit as QL=(10Sx/y )/a; where Sx/y is standard deviation of y-residuals and a is slope, whereas mean recovery was 99.10% ± 0.32 (± SD).

2.5.2. DPPH method The DPPH assay was performed according to Filipiak-Szok et al. [17] procedure. Freshly prepared DPPH (0.5 mL) methanolic solution (330 µmol/mL),

and 2.0 mL of

methanol was added to Trolox solution (0.5 mL) or studied extracts (0.10-0.50 mL), stirred for 1 min, incubated in the dark for 15 min and measured at 517 nm against methanol (blank). Calibration curves (concentration range of Trolox 0.01–0.10 µmol/mL) were calculated by the least-squares method. Obtained equation: y = (714.02±15.32)x + (4.253 ± 0.9505) revealed R2 = 0.9993, RSDslope = 1.54 %, DL= 0.00298 µmol/mL and QL= 0.00729 µmol/mL). The mean recovery was 98.71% ± 0.55 (± SD).

2.5.3. Ferric ion reducing antioxidant parameter – FRAP method Freshly prepared FRAP reagent: 2.5 mL TPTZ solution (10 mmol/L) in 2.5 mL FeCl3 (20 mmol/L) and 25 mL acetate buffer (0.3 mol/L, pH 3.6) was incubated at 37°C 15 min. The aliquots of AP extracts (0.2 mL) were mixed with 2.0 mL of FRAP reagent and made up 10 mL in a volumetric flask. Obtained blue solutions were kept at room temperature for 20 min and absorbance measured at 593 nm. The calibration curve was calculated using standard

methanolic solutions of Trolox (0.001–0.020 µmol/mL), resulting in equation: y = (47.858 ±1.451)x - (0.0315 ± 0.0174); R2 = 0.9991, RSDslope = 2.23%, DL= 0.00081 µmol/mL and QL= 0.00198 µmol/mL, whereas mean recovery was 99.15% ± 0.14 (± SD).

2.6.

Determination of total phenolic compounds An appropriate amount of the Folin-Ciocalteau reagent and saturated sodium

carbonate solution (0.20 g/mL) were added to the extract, mixed, filtrated, if necessary, incubated for 1h at 22°C and absorbance measured (λmax=725 nm). Calibration solutions of gallic acid (GA) (0.50-10.00 mg/L) were prepared, and calculated calibration curve equation was: y= (0.0789±0.0013)x + (0.08722±0.0080), whereas

R2=0.9997, RSDslope = 1.32%,

DL=0.24 mg/L and QL=0.57 mg/L. Results were expressed as gallic acid equivalent (GAE) in mg/100 gram dry mass (d.m.). Obtained mean recovery was 99.14% ± 0.35 (± SD). 2.7.

Determination of alkaloids by different chromatographic methods Presence of purine and isoquinolic alkaloids in AP was confirmed by GC-MS

technique set at the same condition as for andrographolides analysis. LC-MS/MS analysis with ESI ionization was performed using a reverse phase column (Kinetex, 2.6 u C18 100A, 100 × 3.0 mm, Phenomenex) at the following parameters: nebulizing gas: 1.5 L/min; dryging gas: 15 L/min; desolvation line temperature: 250 °C; and heat block temperature: 400 °C. Positive ionization MRM was executed. The sample injection volume was 1µL. The mobile phase was mixture of methanol - 2% acetic acid at a total flow rate of 0.4 mL/min in gradient conditions (0 – 5 min from 2 to 45% B, 5 – 8 min from 45 to 55% B, 8 – 12 min from 55 – 100% B and maintain 100% B for the next 3 minutes. Analysis was carried out in less than 20 minutes.. Calibration curves were calculated by standard solution analysis (concentration range 0.002 – 0.10 mg/L and 0.1 – 1.2 mg/L for every alkaloids) in 5 repetitions, resulting in DL 0.04 mg/L and QL were and 0.15 mg/L for the concentration range

0.1 – 1.2 mg/L.

Determination coefficient was > 0.9992. Developed procedure was used for selected alkaloids determination.

2.8.

Statistical analysis The Pearson correlation test was used for correlations between variables, AC, TPC and

TAC in different plants samples. The mean differences were considered significant at the p < 0.05 level. One-way ANOVA, followed by Duncan test, were preformed to analyse the significant differences between data (p < 0.05).

Principal component analysis (PCA) was performed for AC, TPC and TAC in the AP samples data using Statistica (Windows software package, version 10.0 PL). PCA score plot and cluster analysis were used to determine whether AP at various sources could be grouped into different classes.

3. Result and discussion 3.1.

Andrographolides Chromatograms of the A. paniculata (APs-leaves, APs-roots, Pn) ethanolic extracts

are presented at Figure 1. The andrographolides were determined at the isocratic condition (methanol:acetic acid 70:30 v/v) and eluted within 20 min. The separation of peaks (I, II, III) is acceptable for identification of APe, NAP and DIAP. The purity of each peak was checked by a PDA software. Due to the complexity of the natural samples extracts, lack of standards and applied detector, the precise identification of the every peak on chromatograms was not performed.

140000

120000

Absorbance [a.u.]

100000

80000

60000

Pn

40000

AP roo

AP lea

20000

0 0.0

5.0

10.0 Retention time [min]

15.0

Fig. 1. HPLC chromatograms of A. paniculata extracts (APs-leaves, APs-roots, Pn, I – APe, II – DIAP, III – NAP).

Obtained results for studied andrographolides (APe, DIAP, NAP) and TAC in AP samples are listed in Table 1 and Table 2. One-way ANOVA, followed by the Duncan test (p < 0.05), revealed that APe concentration in APb-leaves and APs-leaves does not differ significantly, whereas for another andrographolides significant differences were noted. It worth noting that the APe concentration in every AP samples was higher than DIAP and NAP, moreover, DIAP content exceeds NAP.

Different letters (a-e) within the same column indicate significant differences (one-way ANOVA and Duncan test, p < 0.05)

Different letters (a-e) within the same column and (x and y) for the same line indicate significant differences (one-way ANOVA and Duncan test, p < 0.05).

The TAC in APs-roots and APs-leaves varies from 50.71 ± 0.36 mg/g d.m. to 78.71 ± 0.48 mg/g d.m., respectively, whereas for dietary supplements (Pn and DK) TAC was found between 19.52±0.15 mg/g and 22.18±0.15 mg/g d.m.. Higher concentration of andrographolides was found in extracts from leaves than roots (Table 2). In addition, one-way ANOVA followed by Duncan test, p < 0.05, indicated that TAC for APs-leaves and APbleaves do not differ significantly. Results for other analytes listed in Table 2 indicate significant differences which can be related to differences in genetic and environmental factors, as well as growing conditions.

GC-MS method has been used for identification of the of selected andrographolides. GC chromatogram and MS spectrum of AP are presented at Figure 2.

Fig. 2. GC chromatogram and MS spectrum of andrographolide in AP

Our obtained results of andographolides are in accordance with those obtained by other authors. Akowuah et al. [9] used the HPLC with UV detection for determination of APe and DIAP in leaves of the A. paniculata cultivated in different locations of Malaysia (APe varied from 21.50 to 29.50 mg/g d.m. and DIAP from 12.70 to 19.60 mg/g d.m. in methanol extracts, while in water extracts from 0.7 to 1.4 mg/g d.m. and 0.4 to 0.8 mg/g d.m., respectively). Moreover Bhan et al. [18] determined TAC from 5.17 to 10.17 g for dry herbage of plants harvested in September, and from 32.50 to 49.50 g in October and 37.00 to 85.67g in November. APe content determined by Cheung et al. [12] varied from 0.61 ± 0.04 to 3.54 ± 0.25 (% w/w) for roots and a whole plant, respectively, while DIAP concentration was 0.61 ± 0.01 and 1.27 ± 0.09 (% w/w) for leaves and stems, respectively. The smaller percentage content of NAP was noted in leaves (0.26 ± 0.01 %w/w), while the highest in stems (1.37 ± 0.00 % w/w). The content of APe determined by Zhao et al. [19] by microemulsion electrokinetic capillary chromatography (MEEKC) was 0.97 - 1.23%, while DIAP 0.73 – 1.06%.

3.2.

Alkaloids Determined alkaloids concentrations are listed in Table 3. Alkaloids content in most

cases reveal significant differences (one-way ANOVA followed by Duncan test, p < 0.05).

However, for APb-leaves and APs-leaves, similar concentrations for theophylline, harmane and brucine are evident. Strychnine was observed only in APs-leaves and DK, while harmol and theobromine were detected only in DK. Because DK supplement contains besides AP other plants, hence the richest contents of alkaloids were found. Moreover caffeine, theophylline, harmane, harmine and brucine were determined in all tested samples. In APsroots, in comparison to AP-leaves revealed the smaller concentration of the studied alkaloids, and lower diversification is evident. Alkaloids are classified as stimulants (caffeine, theobromine, theophylline, nicotine, harmine), many of them are used in medicine (yohimbine, strychnine, harmine, harmol), however some are toxic (brucine, harmine, strychnine). Many alkaloids in appropriate doses can be effective medicine for many diseases and ailments, however when overdosed they become poisonous (e.g. strychnine). It is well known, that some plants naturally produce an excess of toxic alkaloids (strychnine, brucine, harmine, narcotine, etc.). Hence, it is necessary to analyse herbs and medicinal plants as well as ingredients of dietary supplements. To the best of our knowledge, there are no reports on the alkaloids content in AP hence the results can be useful for dietary supplements formulations.

Different letters (a-e) within the same column and line indicate significant differences (one-way ANOVA and Duncan test, p < 0.05); udl – under detection limit, n.d. – not detected.

3.3.

Antioxidant activity and total phenolic content Results of antioxidant activity (AA) and total phenolic content determination are listed

in Table 2. It can be noted that AA, TPC and TAC concentration in the studied plant extracts differ significantly from each other (Duncan test, Table 2). This variability can be related to the impact of genetics, agronomic, environmental and different cultivation regions. APsleaves reveals the highest AA (CUPRAC = 6164.09±5.93 µmol TE/100 g d.m.), while the lowest was for Pn (769.32±2.46 µmol TE/100g d.m.). Moreover, in every samples AA determined for leaves was higher than for roots samples. It is noteworthy that FRAP values (503.36 - 4975.57 µmol TE/100g) and DPPH results (515.64 - 4737.61 µmol TE/100g) seems to be similar, however the Duncan test (Table

2) indicates significant differences between them. For studied plant extracts AA’s obtained by CUPRAC method were higher than the one determined by FRAP and DPPH. Probably, phytochemicals in the studied samples significantly contribute to the antioxidant activities. Dietary supplements revealed smaller TPC, TAC and AA values than AP extracts (Table 2). However, the studied extracts of AP and dietary supplements can be ranked as rich sources of total phenolics (Table 2). As reported the F-C reagent is not selective against antioxidants and responds to the total phenolic content, as well as to other reducing substances (sugars, proteins, vitamins etc.). Therefore F-C reagent measures the total reducing activity of a sample, however can be used for comparison of samples containing various antioxidants. The obtained results were compared to the ones reported by others. Akowuah et al. [9] studied free radical scavenging activity of A. paniculata extracts using DPPH assay. Analyzed methanol extracts exhibited free radical scavenging activity ranging from 45.67% to 53.82%, however water extracts revealed lower activity (25.29% to 28.77%) [9]. Antioxidant action of the aqueous extract of AP against mice’s lymphoma was analyzed and compared with doxorubicin (DOX) by Verma and Vinayak [20] and results confirm significant effect on lymphoma level in mice. Moreover, the antioxidant activity of AP has been found greater than DOX.

3.4.

Principal components analysis and cluster analysis The scores of the first two principal components - PC1 and PC2 and cluster analysis

for the studied A. paniculata extracts from various parts of plants, of different origin and from dietary supplements are presented in Fig. 3 and Fig. 4, respectively. A set of five orthogonal variables by PCA (PCs) was generated. The first two principal components took into account 99.91% (PC1 = 99.77% and PC2 = 0.14%) of the total variation. The first principal component (PC1) revealed the highest eigenvalue (4.988), and accounted for 99.77% of the variability in the data set (Table 3). The remaining four PCs (PC2, PC3, PC4 and PC5) yielded progressively lower eigenvalues ( 91.5%) and precision (RSD0.94, p < 0.05 level). TPC and TAC results correlate with each other and with AA values. The proposed procedures are relatively simple, precise and convenient for AA determination in various parts of AP plants. The studied AP samples appear to be good sources of antioxidants. Presented results on phytochemicals, antioxidants, alkaloids and andrographolides content in Andrographis paniculata suggest possible usefulness of this plant in dietary supplements and other formulations. Acknowledgement

Author (Anna Filipiak-Szok) wishes to thank National Science Centre (Poland) for the financial support grant No. DEC-2012/07/N/NZ9/00965.

References [1] K.R. Kirtikar, B.D. Basu, In: Indian Medicinal Plants, vol. 3. Periodical Experts, New Delhi. (1975) pp. 1884–1886. [2] T. Matsuda, M. Kuroyanagi, S. Sugiyama, K. Umehara, A. Ueno, K. Nishi, Cell differentiation-inducing diterpenes from Andrographis paniculata Nees. Chemical and Pharmaceutical Bulletin, 42 (1994) 1216–1225. [3] Y.K. Rao, G. Vimalamma, C.V. Rao, Y. Tzeng, Flavonoids and andrographolides from Andrographis paniculata. Phytochemistry, 65 (2004) 2317-2321. [4] P. Radhika, Y.R. Prasad, K.R. Lakshmi, Flavones from the stem of Andrographis paniculata, Nees. Nat Prod Commun. 5(1) (2010) 59-60. [5] R.Y. Koteswara, G. Vimalamma, R.C. Venkata, Y.M. Tzeng, Flavonoids and andrographolides from Andrographis paniculata. Phytochemistry, 65 (2004) 2317. [6] L.X. Chen, F. Qiu, H. Wei, G.X. Qu, X.S. Yao, Nine New ent-Labdane Diterpenoids from the Aerial Parts of Andrographis paniculata, Helvetica Chimica Acta, 89 (2006) 2654-2664. [7] S. Saxena, D.C. Jain, M.M. Gupta, R.S. Bhakuni, H.O. Mishra, R.P. Sharma, High performance thin layer chromatographic analysis of hepatoprotective diterpenoids from Andrographis paniculata. Phytochem. Anal.11 (2000) 34–36. [8] A. Srivastava, H. Misra, R.K. Verma, Chemical fingerprinting of Andrographis paniculata using HPLC, HPTLC and densitometry. Phytochem Anal. 15 (2004) 280–285. [9] G.A. Akowouah, I. Zhari, I. Norhayati, A. Mariam, HPLC and HPTLC densitometric determination of andrographolides and antioxidant potential of Andrographis paniculata. Journal of Food Composition and Analysis, 19 (2006) 118-126. [10] D.C. Jain, M.M. Gupta, S. Saxena, S. Kumar, LC analysis of hepatoprotective diterpenoids from Andrographis paniculata. J Pharm Biomed Anal. 22 (2000) 705–709. [11] W.K. Li, J.F. Fitzloff, HPLC-PDA determination of bioactive diterpenoids from plant materials and commercial products of Andrographis paniculata. J Liq Chromatogr Related Technol. 27 (2004) 2407–2420. [12] H.Y. Cheung, C.S. Cheung, C.K. Kong, Determination of bioactive diterpenoids from Andrographis paniculata by micellar electrokinetic chromatography. J Chromatogr A., 930 (2001) 171–176.

[13] A.Sharma, L. Krishan, S.S. Handa, Standardization of the Indian crude drug Kalmegh by high pressure liquid chromatographic determination of andrographolide. Phytochem. Anal. 3 (1992) 129-131. [14] B.Q. Wang, Z.G. Pang, C.Y. Wang, Indirect chemiluminescence to quantify andrographolide. Huaxue Tongbao 7 (1994) 50. [15] Q. Liang, J. Qu, G. Luo, Y. Wang, Rapid and reliable determination of illegal adulterant in herbal medicines and dietary supplements by LC/MS/MS, Journal of Pharmaceutical and Biomedical Analysis, 40 (2005) 305-311. [16] J. Wang, D. Yang, Z. Wang, B. Chen, S. Yao, Simultaneous of illegal additives in dietary supplements and traditional medicines by high performance liquid chromatography– electrospray ionization mass spectrometry, Food Chemistry 113 (2009) 227–232. [17] A. Filipiak-Szok, M. Kurzaw, E. Szłyk, Determination of anti-oxidant activity and content of phenols, phenolic acids, and flavonols in Indian and European gooseberry. Chemical Papers, 66 (4) (2012) 259–268. [18] M.K. Bhan, A.K. Dhar, S. Khan, S.K. Lattoo, K.K. Gupta, D.K. Choudhary, Screening and optimization of Andrographis paniculata (Burm.f.) Nees for total andrographolide content, yield and its components. Scientia Horticulturae, 107 (2006) 386–391. [19] Y. Zhao, Y. Ming, H. Zhang, X. Luo, L. Chen, Y. Li, Rapid determination of diterpenoids an Andrographis paniculata by microemulsion electrokinetic capillary chromatography with short-end injection. Chromatographia 62 (2005) 611-615. [20] N. Verma, M. Vinayak, Antioxidant action of Andrographis paniculata on lymphoma. Mol. Biol Rep. 35 (2008) 535-540.

Table 1. Andrographolides (APe, DIAP, NAP) concentration in Andrographis paniculata samples determined by HPLC-DAD analysis

Samples

Andrographolides content [mg/g d.m. ± SD (n=3), p =0.05] APe

DIAP

NAP

APb-leaves

37.29±0.30d

26.49±0.14d

10.37±0.14d

APs-roots

26.53±0.54c

17.47±0.20c

6.70±0.23c

APs-leaves

38.83±0.35d

28.42±0.64e

11.45±0.39e

DK

12.91±0.13b

6.92±0.19a

2.34±0.11b

Pn

10.08±0.18a

7.33±0.17b

2.12±0.07a

Table 2. Antioxidant activities (determined by the CUPRAC, FRAP and DPPH methods), total phenolic content and total andrographolides concentration in Andrographis paniculata samples

Samples

Antioxidant activity

TPC

TAC

[µmol TE/100g d.m. ± SD (n=3), p = 0.05]

[mg GAE/100g

[mg/g d.m.

d.m. ± SD

± SD (n=3),

CUPRAC

DPPH

FRAP

(n=3), p = 0.05] d

d,,x

4135.91±3.14

d,y

1537.02±4.45

d

p = 0.05] 74.14±0.21d

APb-leaves

5204.82±7.03

4019.16±4.05

APs-roots

3317.67±2.27c

2514.39±4.90c,x

2658.59±2.38c,y

620.07±1.98c

50.71±0.36c

APs-leaves

6164.09±5.93e

4737.61±7.17e,x

4975.57±8.17e,y

1723.79±7.51e

78.71±0.48d

DK

817.66±2.62b

618.23±2.61b,y

610.31±1.75b,x

235.47±4.06b

22.18±0.15 b

Pn

769.32±2.46a

515.64±1.73a,y

503.36±1.68a,x

175.13±2.88a

19.52±0.15a

Table 3. Alkaloids content in Andrographis paniculata plants and dietary supplements by LC-MS/MS Concentration of alkaloids [mg/g d.m. ± SD (n=3), p = 0.05 Alkaloids

APb-leaves

APs-roots

APs-leaves

DK

Pn

0.18±0.02c

0.07±0.00a

0.22±0.02d

0.31±0.02e

0.13±0.01b

Theobromine

n.d.

n.d.

n.d.

0.08±0.01

n.d.

Theophylline

0.11±0.01c

0.03±0.00a

0.12±0.01c

0.15±0.02d

0.08±0.00b

Harmine

0.13±0.01d

0.04±0.00a

0.11±0.01c

0.15±0.02e

0.08±0.01b

Harmane

0.22±0.03c

0.08±0.01a

0.25±0.02c

0.33±0.04d

0.11±0.02b

Harmol

n.d.

n.d.

n.d.

0.13±0.01

n.d.

Strychnine

udl

n.d.

0.01±0.00a

0.01±0.00a

n.d.

Brucine

0.11±0.01c

0.03±0.00a

0.10±0.01c

0.14±0.02d

0.04±0.00b

Yohimbine

0.04±0.00a

n.d.

0.04±0.00a

0.07±0.01b

udl

Caffeine

Determination of phytochemicals, antioxidant activity and total phenolic content in Andrographis paniculata using chromatographic methods.

Antioxidant activity, total phenolics content and selected phytochemicals (alkaloids and andrographolides) were determined in Andrographis paniculata ...
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