Anti-oxidant activity of Hydro-alcoholic extract of some ...

Anti-oxidant activity of Hydro-alcoholic extract of some biologically

important medicinal plants

1*S.K.Gunavathy, 2H.Benita Sherine, 3N.Muruganantham and 4R.Govidharaju

1* Assistant Professor, Department of Chemistry, Srimad Andavan Arts and Science College (Autonomous),

(Affiliated to Bharathidasan University)Tiruchirappalli -620 005, Tamil Nadu, India. 2Assistant Professor, PG & Research Department Chemistry, Periyar E.V.R. College (Autonomous),

(Affiliated to Bharathidasan University) Tiruchirappalli -620 023, Tamil Nadu, India. 3,4 PG & Research Department of Chemistry, Thanthai Hans Roever College (Autonomous),

(Affiliated to Bharathidasan University), Perambalur - 621 220, Tamil Nadu, India.

Corresponding Author E.mail: [email protected]

ABSTRACT

The ethnomedicinal principles of plants form the basis of the herbal drug industry. India has

contributed its knowledge of traditional system medicines (Ayurveda and Siddha) to develop

herbal medicines with negligible side effects. The World Health Organization has also

recognized the benefits of drugs developed from natural products. Plectranthus mollis, Elaeagnus

conferta and Grewia tilaefolia leaf extracts, commonly used in Indian traditional system of

medicines. Traditionally these plants were used in the form of extracts/powder/paste by tribal

populations of India for treating common ailments like cough and cold, fever, stomach, kidney

and liver disorders, pains, inflammations, wounds, etc. Fresh leaves of Grewia tilaefolia were

extracted and evaluated for antioxidant activities by Reducing Power Assay, DPPH assay, ABTS

assay, Nitric oxide Assay. The results obtained showed that the compound extracted from

Grewia tilaefolia leaves can be considered as good sources of anti-oxidants can be incorporated

into the drug formulations. This study justifies the anti-oxidants activity of the compound

isolated from ethyl acetate fractions of Grewia tilaefolia leaf extracts. Further detailed analysis of

this sample is required to identify the presence of bioactive compounds responsible for anti-

oxidants activities. Studies are highly needed for future drug development.

Key words: Plectranthus mollis, Elaeagnus conferta and Grewia tilaefolia, leaf extracts, Anti-

oxidant activity, DPPH assay, ABTS assay, Total antioxidant activity etc.,

INTRODUCTION

Polyphenols are major secondary metabolites having important role in scavenging free

radicals in plant cells. Oxidative radicals are reported to be a potential cause of mutations,

Journal of Information and Computational Science

Volume 10 Issue 4 - 2020

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damage to lipids, DNA and proteins which in turn lead to certain disorders including cancer.

Higher antioxidant potency of polyphenolic compounds leads to growing interest in isolation and

use of natural products as antioxidants [1]. Plant extracts are rich sources of polyphenolic

compounds including phenolic acids, tannins and flavonoids [2, 3]. Flavonoids are mainly 15- C

compounds found generally throughout the plant kingdom [4]. Many natural compounds isolated

from plants having free radical scavenging potential are reported to be promising therapeutic

agents for several free radical pathologies [5, 6]. Tannins, high molecular weight polyphenols are

also found naturally in medicinal herbs and have a major role in free radical scavenging. Human

diets contain tannins most abundantly and various biologically important functions including

protection against oxidative damage and certain degenerative diseases, are reported to be

exhibited by tannins [7].

The family Lamiaceae is represented by about 236 genera and 7172 species in the world

[8]. Many members of this family are beneficial economically and are frequently useful for

several medicinal, ornamental, commercial and culinary purposes. Several previous studies

reported strong antioxidant potential of members of lamicea family [9- 11]. Thus, members of

the family are very important due to their medicinal and aromatic properties leading to

production of the herbal products and food supplements. Medicinal herbs belonging to family of

lamiacae are rich in essential oil content. Eseential oils are considered to be one of the potential

agents having strong antioxidant, antibacterial and anticancer activities [12- 14].

Dietary intake of such phytochemicals may be an important strategy for inhibiting or

delaying of pathological conditions caused by free radicals either formed by cellular metabolism,

exogenous chemicals or due to stress and is capable of oxidising biomolecules which may cause

many diseases [15]. The biomolecules obtained from the natural plants are in big demands

because they have no or less side effects, toxicity of food by synthetics and cosmetic

preservatives [16].

Hippophae rhamnoides belongs to the family Elaeagnaceae commonly known as Sea

buckthorn (SBT), a thorny nitrogen fixing actinomycetes deciduous shrub producing yellow

orange berries is native to Europe and Asia [17-18]. In India Hippophae rhamnoides is widely

distributed in cold Himalayan region at an altitude of 2500-4000 m. All parts of the plant are rich

source of bioactive substances which have been simultaneously investigated for their therapeutic



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potential and phytochemical contents. It has been used in the traditional system of medicine for

the treatment of cough, skin diseases, gastric ulcers, asthma and lung disorders for ages [19-21].

It was also reported that Hippophae rhamnoides leaf has no cytotoxicity or adverse effect after

oral administration [22-24].

Herbal drugs from ethnomedicinal plants have gained considerable importance in the

recent past not only in India but also around the world [25]. Traditional medicinal knowledge in

India has passed from one generation to the next, within specific geographical locations or tribal

groups [26]. This traditional knowledge finds its root in Indian traditional systems of medicine

i.e., Ayurveda and Siddha which is now gaining popularity in western world too.

Ethnomedicines/herbal medicines are much in demand as they are affordable and have much less

side effects [27]. Recently WHO has also recognized the importance of traditional medicine in

the healthcare sector [28-29]. In Ayurveda and Siddha systems, formulations from appropriate

parts of plants are made and used for treatment of various ailments. For almost past three

decades, many ethnomedicinal plants mentioned in Ayurveda and Siddha systems of medicines

are being scientifically evaluated [30].

Scientific evaluation of ethnomedicinal plants provides evidence-based alternative

medicines which form the basis of herbal drug industry and discovery of drug targets in the

pharmaceutical industry [31]. It may be emphasized here that usage of ethnomedicinal plants for

traditional medical treatment or for use in manufacture of Ayurvedic medicines/other herbal

drugs, when supported by scientific evidences can ensure safe and more effective utilization of

natural product drugs universally. Malvaceae family encompasses approximately 244 genera

with 4225 species of herbs, shrubs and trees [32]. Around 22 genera of the family are reported

from India, many of which have ethnomedicinal value e.g., Abutilon indicum, Gossypium

herbaceum, Hibiscus mutabilis, Hibiscus sabdariffa, Hibiscus rosa-sinensis, Sida acuta,

Sidacordifolia, Sida rhombifolia and several others [33].

Cucumbers are a valuable source of conventional antioxidant nutrients including vitamin

C, beta-carotene, and manganese. Fresh flowers of Cucumis sativus were extracted and evaluated

for antioxidant activities by 2,2-Diphenyl 1-picryl hydrazyl solution (DPPH), 2,2'-azino-bis(3-

ethylbenzthiazoline-6-sul- phonic acid (ABTS) assay and anti-inflammatory activites by human

blood cell (HRBC) membrane stabilization method and Inhibition of albumin denaturation



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method. The results obtained showed that the compound isolated from ethyl acetate fractions

of Cucumis sativus flowers can be considered as good sources of anti-oxidants, anti-

inflammatory and can be incorporated into the drug formulations [34].

Fresh flowers of Cassia auriculata were extracted with ethanol and evaluated for

antioxidant activities by 2,2-Diphenyl 1-picryl hydrazyl solution (DPPH), 2,2'-azino-bis(3-

ethylbenzthiazoline-6-sul- phonic acid (ABTS) assay and anti inflammatory activites by humen

blood cell (HRBC) membrane stabilization method and Inhibition of albumin denaturation

method [35].

The results obtained showed that the hydro alcoholic crude extract of Grewia tilaefolia

leaves can be considered as good sources of anti-oxidant activity can be incorporated into the

drug formulations.

MATERIALS AND METHODS

Collection of plant materials

Materials of Three plants (leaves) were harvested in March to May from Trichy and Perambalur

region. They were carefully washed, oven-dried for 1 h at 120°C and put in the shade in an

aerated place till complete drying, then were ground into a fine powder.

Preparation of plant extracts

The prepared powder was soaked in each of Hydroalcoholic extract solvents (plant material to

solvent ratio was 1:10, w/v) and extracted for 24 h at room temperature with shaking at 150 rpm.

Filtrates of the extracts were dried at 40°C. Then, samples were centrifuged (3000 rpm, 5

minutes) to eliminate the impurities and suspended solids. The supernatants were used as

aqueous crude extract in this study.

Hydro-Alcoholic Extraction

The plants powders were prepared as explained in previous section. For hydro-alcoholic

extraction, the powders were placed in flasks individually. Water and alcohol at a ratio of 50:50

in specified deal were added to the flasks and kept 24 hours in dark. Samples were poured in

rotary evaporator to remove as much as possible extra water and alcohol. Then, the concentrated

samples were centrifuged (3000 rpm, 5 minutes) and supernatants were used as hydro-alcoholic

crude extract in this study.

Reducing power assay

The sample together with Ascorbic acid solutions were spiked with 2.5ml of phosphate



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buffer (0.2 M, pH 6.6) and 2.5ml of 1% potassium ferricyanide. The mixture was kept in a 50°C

water-bath for 20min. The resulting solution was cooled rapidly, spiked with 2.5ml of 10%

trichloroacetic acid, and centrifuged at 3000rpm for 10 min. The supernatant (5ml) was mixed

with 5ml of distilled water and 1ml of 0.1% ferric chloride and incubated for 10min. The

absorbance was detected at 700nm on spectrophotometer. The extract concentration providing

the absorbance was calculated from the graph of absorbance at 700 nm against extract

concentration. Ascorbic acid was used as standard. Higher absorbance indicates higher reducing

power [36].

DPPH radical scavenging activity

DPPH radical scavenging activity was carried out by the method of Molyneux (2004). To

1.0 ml of 100.0 μM DPPH solution in methanol, equal volume of the test sample in methanol of

different concentration was added and incubated in dark for 30 minutes. The change in

coloration was observed in terms of absorbance using a spectrophotometer at 514 nm. 1.0 ml of

methanol instead of test sample was added to the control tube. The different concentration of

ascorbic acid was used as reference compound [37].

Percentage of inhibition was calculated from the equation

[(Absorbance of control - Absorbance of test)/ Absorbance of control] × 100.

IC50 value was calculated using Graph pad prism 5.0.

ABTS radical scavenging activity

ABTS radical-scavenging activity of the extract was determined according to Re et al.,

1999. The ABTS.+cation radical was produced by the reaction between 5 ml of 14 mM ABTS

solution and 5 ml of 4.9 mM potassium persulfate (K2S2O8) solution, stored in the dark at room

temperature for 16 hrs. Before use, this solution was diluted with ethanol to get an absorbance of

0.700 ± 0.020 at 734 nm. The plant extract at various concentrations with 1ml of ABTS solution

was homogenized and its absorbance was recorded at 734 nm. Ethanol blanks were run in each

assay, and all measurements were done after at least 6 min. Similarly, the reaction mixture of

standard group was obtained by mixing 950 µl of ABTS.+ solution and 50 µl of BHT. As for the

antiradical activity, ABTS scavenging ability was expressed as IC50 (µg/ml). The inhibition

percentage of ABTS radical was calculated using the following formula:

ABTS scavenging activity (%) = (A0 –A1) /A0 ×100

Where A0 is the absorbance of the control, and A1 is the absorbance of the sample [38].



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Nitric oxide scavenging activity

Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates

nitric oxide (NO), which interacts with oxygen to produce nitrite ions, which can be estimated

using Griess Illosvosy reaction (Garrat, 1964). Scavengers of NO compete with oxygen, leading

to reduced production of NO and a pink coloured chromophore is formed. The absorbance of

these solutions was measured at 540 nm against the corresponding blank solutions.

Percentage inhibition was calculated as NO scavenging activity (%)

= (A0 –A1) /A0 ×100

Where A0 is the absorbance of the control, and A1 is the absorbance of the sample [39].

Total Antioxidant capacity of the extract

An aliquot of 0.1ml of the sample solution containing a reducing species in DMSO was

combined in an Eppendorff tube with 1ml of reagent solution (0.6M Sulphuric acid, 28mM

sodium phosphate, and 4mM ammonium molybdate). The tubes were capped and incubated in

water bath at 95 °C for 90min. The samples were cooled to room temperature, and the

absorbance of each solution was measured at 695nm. The total antioxidant capacity was

expressed as mM equivalent of ascorbic acid [40].

RESULTS AND DISCUSSION

Oxidative stress is caused by an imbalance between the production of reactive oxygen

and a biological system's ability to readily detoxify the reactive intermediates or easily repair the

resulting damage. All forms of life maintain a reducing environment within their cells. This

reducing environment is preserved by enzymes that maintain the reduced state through a constant

input of metabolic energy. Disturbances in this normal redox state can cause toxic effects

through the production of peroxides and free radicals that damage all components of the cell,

including proteins, lipids, and DNA.

In humans, oxidative stress is involved in many diseases, such as atherosclerosis,

Parkinson's disease, Heart Failure, Myocardial Infarction, Alzheimer's disease and chronic

fatigue syndrome, but short-term oxidative stress may also be important in prevention of aging

by induction of a process named mitohormesis. Reactive oxygen species can be beneficial, as

they are used by the immune system as a way to attack and kill pathogens. Reactive oxygen

species are also used in cell signaling. This is dubbed redox signaling.



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In chemical terms, oxidative stress is a large rise (becoming less negative) in the cellular

reduction potential, or a large decrease in the reducing capacity of the cellular redox couples,

such as glutathione. The effects of oxidative stress depend upon the size of these changes, with a

cell being able to overcome small perturbations and regain its original state. However, more

severe oxidative stress can cause cell death and even moderate oxidation can trigger apoptosis,

while more intense stresses may cause necrosis.

A particularly destructive aspect of oxidative stress is the production of reactive oxygen

species, which include free radicals and peroxides. Some of the less reactive of these species

(such as superoxide) can be converted by redox reactions with transition metals or other redox

cycling compounds (including quinones) into more aggressive radical species that can cause

extensive cellular damage. The major portion of long term effects is inflicted by the damage on

DNA. Most of these oxygen-derived species are produced at a low level by normal aerobic

metabolism and the damage they cause to the cells is constantly repaired. However, under the

severe levels of oxidative stress that cause necrosis; the damage causes ATP depletion, and thus

preventing the controlled apoptotic death.

A plethora of previous studies indicate that certain plant derived compounds such as

vitamins, phenolic acids, flavonoids, dietary fibers and free radical scavengers play an important

role in the prevention of common diseases like cancer, inflammation, dibetes, certain

cardiovascular disorders and aging process [41-42]. Such natural compounds are considered to

be promising health promoting agents due to their antioxidant attibutes [43-45].

Alcohols extracts are the major plant compounds with antioxidant activity, which is

believed to be mainly due to their redox properties, that plays an important role in adsorbing and

neutralizing the free radicals, quenching singlet and triplet oxygen, or decomposition of the

peroxides.



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Reducing Power Assay

Reducing power assay activity for Hydroalcoholic extract of chosen plants.

Graphical representation of Reducing power assay activity for Hydroalcoholic extract of chosen plants.

0

10

20

30

40

50

60

70

80

50 250 500 750 1000

Sam

ple

Co

nce

ntr

atio

n (

µg/

ml)

% IC50 Cytotoxicity (µg/ml)

Reducing Power Assay

Ascorbic acid (STD)

P.mollis

E.conferta

G.tilifolia

Sample Ascorbic

acid

(STD) P.mollis E.conferta G.tilifolia

S.

No

Sample

Concentration

(µg/ml)


1. 50 44.90 27.21 25.85 32.65

2. 250 53.06 35.37 31.29 44.89

3. 500 61.90 48.97 38.09 60.54

4. 750 67.35 54.42 48.29 65.98

5 1000 72.79 66.66 51.70 70.74

IC50 165.49 µg/ml

594.70 µg/ml

893.49

µg/ml 386.76 µg/ml



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Graphical representation of IC50 Reducing power assay activity for Hydroalcoholic extract of chosen plants.

DPPH Assay

DPPH assay activity for Hydroalcoholic extract of chosen plants.

165.49

594.7

893.49

386.76

IC50- Reducing Power Assay

Ascorbic acid (STD) P.mollis E.conferta G.tilifolia

Sample Ascorbic

acid


S.

No

Sample

Concentration

(µg/ml)


1. 50 43.54 35.62 25.34 39.45

2. 250 51.02 43.15 33.56 42.85

3. 500 59.86 45.89 41.78 55.78

4. 750 63.95 53.42 50.00 61.90

5 1000 70.07 62.33 55.48 74.82

IC50 228.79

µg/ml

582.71 µg/ml

784.20 µg/ml

377.52

µg/ml



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Graphical representation of DPPH assay activity for Hydroalcoholic extract of chosen plants.

Graphical representation of IC50 DPPH assay activity for Hydroalcoholic extract of chosen plants.

0

10

20

30

40

50

60

70

80

50 250 500 750 1000

Sam

ple

Co

nce

ntr

atio

n (

µg/

ml)


DPPH Assay

Ascorbic acid (STD)

P.mollis

E.conferta

G.tilifolia

228.79

582.71

784.2

377.52

IC50 - DPPH Assay




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ABTS Assay

ABTS assay activity for Hydroalcoholic extract of chosen plants.

Graphical representation of ABTS assay activity for Hydroalcoholic extract of chosen plants.

0

10

20

30

40

50

60

70

80

90

50 250 500 750 1000

Sam

ple

Co

nce

ntr

atio

n (

µg/

ml)


ABTS Assay

Ascorbic acid (STD)

P.mollis

E.conferta

G.tilifolia

Sample Ascorbic

acid


S.

No

Sample

Concentration

(µg/ml)


1. 50 48.98 23.29 23.29 36.73

2. 250 55.10 32.19 32.19 49.65

3. 500 66.67 49.32 41.10 59.18

4. 750 73.47 60.96 48.63 72.10

5 1000 81.63 65.07 54.11 82.31

IC50 74.61

µg/ml

591.99 µg/ml

822.77 µg/ml

298.34 µg/ml



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Graphical representation of IC50 ABTS assay activity for Hydroalcoholic extract of chosen plants.

Nitric oxide Assay

Nitric oxide assay assay activity for Hydroalcoholic extract of chosen plants.

74.61

591.99

822.77

298.34

IC50 - ABTS Assay


Sample Ascorbic

acid


S.

No

Sample

Concentration

(µg/ml)

% IC50Cytotoxicity (µg/ml)

1. 50 38.10 25.34 20.55 33.33

2. 250 47.62 31.51 26.03 46.25

3. 500 53.06 47.26 32.19 55.10

4. 750 61.90 55.48 46.58 68.70

5 1000 64.63 58.22 52.74 69.38

IC50 399.96

µg/ml

682.57 µg/ml

915.07 µg/ml

393.93 µg/ml



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Graphical representation of Nitric oxide assay activity for Hydroalcoholic extract of chosen plants.

Graphical representation of IC50 Nitric oxide assay activity for Hydroalcoholic extract of chosen plants.

0

10

20

30

40

50

60

70

80

50 250 500 750 1000

Sam

ple

Co

nce

ntr

atio

n (

µg/

ml)


Nitric oxide Assay

Ascorbic acid (STD)

P.mollis

E.conferta

G.tilifolia

399.96

682.57

915.07

393.93

IC50- Nitric oxide Assay




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Total antioxidant activity

S.No Compound Total anti-

oxidant activity

(µg/ml)

1 P.mollis 124.83

2 E.conferta 106.11

3 G.tilifolia 152.07

4 Ascorbic acid (STD) 180.24

Total antioxidant activity for Hydroalcoholic extract of chosen plants.

Graphical representation of Total antioxidant activity for Hydroalcoholic extract of chosen plants.

The free radical scavenging activity of Grewia tilaefolia leaf extracts was studied by their

ability to decolourize the stable Reducing Power Assay, DPPH assay, ABTS assay and Nitric

oxide assay which provides information on the reactivity of compounds with a stable free radical

Table: 1,2,3,4,5and also indicates some graphical representations.

124.83

106.11

152.07

180.24

P.mollis

E.conferta

G.tilifolia

Ascorbic acid (STD)

0 20 40 60 80 100 120 140 160 180 200

Nam

e o

f Sa

mp

les

µg/ml

Total anti-oxidant activity (µg/ml)



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The results of this study showed that Grewia tilaefolia leaf extracts are effective in

scavenging Reducing Power Assay, DPPH assay, ABTS assay and Nitric oxide assay radical

scavenging abilities of the extracts were significantly less than those of ascorbic acid. This

indicates that the extracts have the proton-donating or scavenging ability and could serve as free

radical inhibitors or scavengers, acting possibly as primary antioxidants. Results obtained from

this assay further supported the validity of Reducing Power Assay, DPPH assay, ABTS assay

and Nitric oxide assay reconfirms the antioxidant potential of the Grewia tilaefolia leaf extracts.

Significant antioxidant activity showed by Grewia tilaefolia leaf extract provide a scientific

validation for the traditional use of these plants in traditional medicine system, however, work on

isolation and identification of active compounds and its efficacy needs further investigations

Table: 1,2,3,4,5and also indicates some graphical representations.

Antioxidant Activity Test of Plectranthus mollis, Elaeagnus conferta and Grewia

tilaefolia leaf extracts determined by the ability of a compound contained in extract to reduce the

purple color intensity of DPPH radical in its maximum wavelenght. The reduction of purple

colour intensity of DPPH radical is caused by the decrease of chromophore or conjugated double

bond in DPPH compound. It caused by potency of extract compound which scavenging the

radical by donating hydrogen atom to DPPH structure so that become reducted DPPH-H. DPPH-

H is a compound which have yellow colour. Finally we have demonstrated, that the hydro

alcohol extract of the leaves of Grewia tilaefolia leaf extracts showed potent activity when

compared to the Plectranthus mollis, Elaeagnus conferta leaves extracts, exhibiting considerable

activity (dose dependent) when compared with reference standard. The present research work

showed the validity and the clinical use of hydro alcohol extract of Grewia tilaefolia in the

control of anti-oxidant activity.

CONCLUSION

In conclusion, the present observations support that Grewia tilaefolia is a potential source

of antioxidants, their precursors and bioactive agents, which could be used as a natural

preservative and in the development of nutraceutical formulations to overcome the adverse

effects of synthetics. This study also provide a scientific support to the ongoing studies

exhibiting the effectiveness of sea buckthorn leaves in wound healing, inflammatory and free

radical mediated diseases. Further investigation on the isolation and identification of active

component(s) may lead to chemical entities with potential clinical uses. In the past few years



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interest in the search of new natural antioxidants has grown because reactive oxygen species

(ROS) production and oxidative stress is linked to many diseases. The use of synthetic

antioxidants generally leads to problems of toxicity. In this study, antioxidant potential of

nine extracts of four different plant Plectranthus mollis, Elaeagnus conferta and Grewia

tilaefolia leaf extracts were assayed by Reducing Power Assay, DPPH assay, ABTS assay and

Nitric oxide assay methods. The antioxidant activity of the extracts was also studied from

analysis and found strongly correlated with hydro alcoholic extracts. These correlations

suggested that hydro alcoholic extracts are mainly responsible for the antioxidant activity

displayed by plants.

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