Org.Chem.J. 2011, 1, 36-43 Antioxidant Activity and High Performance Liquid Chromatoghraphy

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Antioxidant Activity and High Performance Liquid

Chromatography Analyzation of Methanolic and

Aqueous Extract of Stachys Inflata

Sayyed Mehdy Hashemi Sohia, Anoosh eghdami*,b, Fathemeh sadeghib

aYoung researcher club, Saveh branch, Islamic Azad University, Saveh, Iran bBiology Department of Saveh branch, Islamic Azad University, Saveh, Iran

E-mail: eghdami_a@yahoo.com

Recevied date: January 19, 2011

Abstract

The objective of this paper was a screening of polyphnol and flavonoid by spectrophotometer. The type

of some polyphenols in Stachys inflata was determined by high performance liquid chromatography

(HPLC). The methanol extract of plant material was used for HPLC analysis. The HPLC system was

coupled with colorimetric detection of antioxidant activity. Additionally, the antioxidant activities were

determined by ferric reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picryl hydrazyl (DPPH)

radical scavenging activity. The flavonoid and total phenolic contents of methanolic extract was higher

than aqueous extract of Stachys inflata. A strong positive correlation of R2 = 0.90 between total

phenolic content and antioxidant activity was observed in this investigation. This study indicated that

Stachys inflata exhibite high antioxidant activity, flavonoid and phenolic content and can be used

potentially as a readily accessible source of natural antioxidant. Antioxidant activities of polyphenols

have been suggested to exert beneficial pharmacological effects on some diseases.

Keywords: Phenolic compound, DPPH, Antioxidants, PJs, Folin-Ciocalteou, HPLC

1. Introduction

The health promoting effect of antioxidants from plants is thought to arise from their potential effects

on the reactive oxygen/nitrogen species. In addition, antioxidants have been widely used in food

industry to prolong the shelf life. However, there is widespread agreement that some synthetic

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antioxidants such as butyl hydroxy anisole and butyl hydroxyl toluene need to be replaced with natural

antioxidants due to their potential health risks and toxicity.1 There is intense interest in plant

polyphenols as witnessed by the numerous papers devoted to various aspects of these compounds. The

use of plants, herbs as antioxidants in processed foods is becoming of increasing importance in the food

industry as an alternative to synthetic antioxidants.2 The genus Stachys (family Lamiaceae) is

represented by 270 species and distributed extensively in the tropical and subtropical countries. Thirty

four species of this genus are found in Iran of which 13 species are endemic.3 A resurgence of interest

has developed in wild species for their possible medicinal values in diets. Wild plant species provide

minerals, fiber, vitamins and essential fatty acids and enhance taste and colour in diets. In addition,

they have antibacterial, hepatoprotective and anti carcinogenic properties and therefore have medicinal

values extracts obtained from the aerial parts of (non-flowering stems of) Stachys inflata have been

used in Iranian folk medicine in infective, asthmatic, rheumatic and other inflammatory disorders.4,5

Polyphenols are a group of highly hydroxylated polyphenolic compounds present in the extractive

fractions Antioxidant activities of polyphenols have been suggested to exert beneficial pharmacological

effects on neurological disorders on the basis of in vitro observations.6,7 The use of plants, herbs as

antioxidants in prossesed foods is becoming of increasing importance in the food industry as an

alternative to synthetic antioxidants.8 Oxidative stress appears to play an important role in several

human chronic diseases, including atherosclerosis and cancer. Therefore, antioxidants may be useful in

the prevention and treatment of these conditions. Flavonoids are a group of polyphenolic substances

ubiquitous in foods of plant origin.9 In this research the antioxidant activity and total phenol contents of

Stachys inflata were investigated.

2. Results and Discussion

All the analyses were performed in triplicate. Results were expressed as means ± standard deviation.

Descriptive statistical analysis, Pearson correlation coefficients, one-way analysis of variance

(ANOVA) were performed using SPSS. Extract of Stachys inflata showed scavenging effects against

DPPH radical. Each value in the table was obtained by calculating average of three experiments ą

standard deviation.

The hierarchy for antioxidant capacity with respect to their EC50 values was methanolic

extract > aqueous extract. Correlation coefficient showed that total phenolic content was responsible

for antiradical efficiency in Stachys inflata, extracts. The antioxidant and total phenolic content levels

are also positively and significantly correlated. Our results strongly suggested Stachys inflata, extracts

can be promising sources of potential antioxidants. The methanol extracts of this plant had not

significantly difference in ferrous reducing antioxidant power (FRAP) relative to aqueous extract.

Many different detection methods

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Table 1: Some of the flavonoids structure.

Entry Compound name Compound structure

1 Coumric acid HO HO O

2 Vanilic acid HO

OO

OH

3 quercetin O

OHOOH

HO

OHOH

Table 2. Total phenolic ,flavonoids contents and antioxidant activities of methanol and aqueous extract of Stachys inflate.

variety Phenolic

content(mg

GAL/g)

Flavonoids

content((QE)/g)

Antioxidant

activity

By DPPH

(Inhibition%)

Antioxidant

(FRAP)

Methanolic

extract

92.2±0.04 36.7 ± 0.05 48.3±0.15 31.0787±

0.5319

Aqueous

extract

33.2±0.04 11.2±0.03 32.12 ±0.15 31.08 ± 0.53

are available in HPLC analysis. In general ultraviolet (UV) detectors are most popular and have been

extensively used in the detection of phenols. According to table of standards curve (Fig 1 and Fig 2)

and table of retention time of standards (Table 3), methanolic extract of Stachys inflata have

polyphenolic compounds (Fig 3) .

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Figure 1. HPLC chromatogram some of polypehnol standard (gallic acid, vanillic acid, coumaric acid,

benzoic acid respectively).

Table 3. Retention time of some poliphenols.

standards Retention time

Vanilic acid 20.10

quercetin 37.80

Gallic acid 6.25

Coumaric acid 30.02

catechin 27.3

Cafeic acid 20.28

3. Experimental

3.1. Plant collection

The plants were collected in July 2010 from nowbaran area of saveh city in the Mrkazy Province of

Iran. The area falls within the latitudes 35°.15' and longitudes 49°.45' and the altitude of area is 1680

m.

3.2. Chemicals

1,1-Diphenyl-2-picryl hydrazyl (DPPH) and quercetin were purchased from Sigma Chemical Co. (St.,

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Louis, USA). Gallic acid, Folin Ciocalteu reagent, trifluoroacetic acid (TFA) and methanol were

purchased from Merck Co. (Germany).

Figure 2. HPLC chromatogram some of polypehnol standard (quercetin).

Figure 3. HPLC chromatogram some of polyphenol from methanolic exteract.

3.3. Sample preparation

The samples were first ground to fine powder. For water extraction, 0.5 g of the fine powder was

extracted with 10 ml of ultra-filtered water at 100 °C for 30 min in a water bath. For methanol

extraction, 0.5 g of the powder was extracted with 10 ml of 80% methanol at 40 °C for 24 h. The

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samples were then cooled down to room temperature and centrifuged at 4500 rpm for 15 min. The

supernatant was recovered and used for the DPPH assay and total phenolic analysis. We used HPLC

analyzing for polyphenol types determination.

3.4. Determination of total phenolic content

Extracts was determined using the Folin- Ciocalteu reagent.10 The reaction mixture contained: 200 μl

of diluted Stachys inflata extract, 800 μl of freshly prepared diluted Folin Ciocalteu reagent and 2 ml of

7.5% sodium carbonate. The final mixture was diluted to 7 ml with deionized water. Mixtures were

kept in dark at ambient conditions for 2 h to complete the reaction. The absorbance at 765 nm was

measured. Gallic acid was used as standard and the results were expressed as mg gallic acid (GAE)/g

Stachys inflata .

3.5. Determination of total flavonoid content

Total flavonoid content was determined using aluminium chloride (AlCl3) according to a known

method11 using quercetin as a standard. The plant extract (0.1 ml) was added to 0.3 ml distilled water

followed by 5% NaNO2 (0.03 ml). After 5 min at 25°C, AlCl3 (0.03 ml, 10%) was added. After further

5 min, the reaction mixture was treated with 0.2 ml of 1 mM NaOH. Finally, the reaction mixture was

diluted to 1 ml with water and the absorbance was measured at 510 nm. The results were expressed as

mg quercetin (QE)/g Stachys inflata.

3.6. Antioxidant activity

3.6.1. DPPH (2,2'-diphenyl-1-picrylhydrazyl)

The antioxidant activity was tested by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) free radical

scavenging method. For each extract. Then, we also prepared a dilution 1 M of DPPH. The absorbance

of a mixture of 1 ml of the extract and 1 ml of the DPPH solution was measured at 517 nm. The radical

scavenging activity was calculated from the equation: Percentage of radical scavenging activity = (Abs

control - Abs sample)/Abs control × 100.12

3.6.1. Ferric reducing antioxidant power (FRAP) assay

FRAP assay is based on the ability of antioxidants to reduce Fe3+ to Fe2+ in the presence of 2,4,6-tri(2-

pyridyl)-s-triazine (TPTZ), forming an intense blue Fe2+-TPTZ complex with an absorption maximum

at 593 nm. This reaction is pH-dependent (optimum pH 3.6). The absorbance decrease is proportional

to the antioxidant content,13 0.2 ml of the extract is added to 3.8 ml of FRAP reagent (10 parts of 300

Mm sodium acetate buffer at pH 3.6, 1 part of 10.0 mM TPTZ solution and 1 part of 20.0 mM

FeCl3.6H2O solution) and the reaction mixture is incubated at 37 °C for 30 min and the increase in

absorbance at 593 nm is measured. FeSO4 is used for calibration. The antioxidant capacity based on

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the ability to reduce ferric ions of sample is calculated from the linear calibration curve and expressed

as mmol FeSO4 equivalents per gram of sample. ascorbic acid, quercetin, can be used as a positive

control.

3.7. HPLC analysis

Identification of individual phenolic compounds of the plant extracts was performed by HPLC. The

analysis was performed with a flow rate of 0.75 mL/min. using 0.2% trifluoroacetic acid (TFA) as

solvent A and Methanol as solvent B, with a linear gradient from 5 to 80% methanol in 50 min (Table

4).

Table 4. Gradient elution method performed with binary solvent system using as

mobile phase.

A B

0-5 5 95

5-8 10 90

8-11 15 85

11-14 20 80

14-17 25 75

17-20 30 70

20-23 35 65

23-26 40 60

26-29 45 55

29-32 50 50

32-35 55 45

35-38 60 40

38-41 65 35

41-44 70 30

44-47 75 25

47-50 80 20

AIl solvent used were filtered on Acetate Plus (0.22) before analysis. The selected flavonoid standards

required a greater concentration of Methanol (80%) and a longer HPLC run for their proper elution

than phenolic acids. Each standard was first injected individually to determine the exact retention time.

and chromatographic characteristics (λmax, absorbance ratio) followed by the analysis of the standard

mixture. The methanolic extract of Stachys inflata was filtered on qualitative circle, Whatman filter

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paper No. 1 (Whatman International Ltd., Maidstone U.K.) under vacuum and subsequently on Acetate

Plus filters (0.22 μm) to remove the undesirable contaminants. The limitations conceming the UV

detector are that the compound must absorbs ultraviolet light and any other contamination that also

absorbs UV radiation may interfere in the analysis.

4. Conclusion

The results from this study indicate that the leaves and stem extracts of Stachys inflata possess

antioxidant properties and could serve as free radical inhibitors or scavenger or, acting possibly as

primary antioxidants. The results of the current study showed that the methanol extract of Stachys

inflata had the highest scavenging activity. The amounts of phenol compounds of Stachys inflata

correlates to their scavenging effect. These results demonstrate that the antioxidant activities observed

can be ascribed both to mechanisms exerted by phenolic compounds and also to synergistic effects of

different phytocompounds. This study reveals that tested plant materials have moderate to significant

antioxidant activity and free radical scavenging activity. The result of the present study suggests that

selected plants can be used as a source of antioxidants for pharmacological preparations which is very

well evidenced by the present work.

References and Notes

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4. Ghahreman, A. Tehran, Research Institute of Forest and Rangelands and Tehran University Press, 1995.

5. Zargar, A. Tehran, Tehran University Press, 1992.

6. Moosmann, B.; Behl, C. Proc. Natl. Acad. Sci. USA 1999, 96, 8867.

7. Parr, A. J.; Bolwell, G. P. J. Sci. Food Agric. 2000, 80, 985.

8. Madsen, H. L.; Bertelsen, G. Trends in Food Science and Technology 1995, 6, 271.

9. Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Proc. Natl. Acad. Sci. USA 1995, 90, 7915.

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