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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 12  |  Issue : 1  |  Page : 11-19

Effect of cephalandra indica against advanced glycation end products, sorbitol accumulation and aldose reductase activity in homoeopathic formulation


M. M. College of Pharmacy, M. M. University, Ambala, Haryana, India

Date of Web Publication3-Apr-2018

Correspondence Address:
Dr. Randhir Singh
M. M. College of Pharmacy, M. M. University, Mullana, Ambala - 133 207, Haryana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijrh.ijrh_88_16

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  Abstract 


Background: Extreme generation of free radicals leads to oxidative stress which has been apprehensive in several disease processes such as diabetic complications and vascular and neurodegenerative diseases. Objective: The present study was designed to evaluate the potential of homoeopathic preparations of Cephalandra indica L. against oxidative stress. Materials and Methods: Potencies of Cephalandra indica (mother tincture, 6C and 30C) were procured from Dr. Willmar Schwabe India Pvt. Ltd. The antioxidant activity of Cephalandra indica was evaluated by employing various in vitro antioxidant methods. Results: The total phenol content was found to be 1905, 849 and 495 mg/g gallic acid equivalents in mother tincture, 6C and 30C of Cephalandra indica and total antioxidant capacity was found to be 2710, 759 and 510 μM/g ascorbic acid equivalents, respectively. Mother tincture, 6C and 30C of Cephalandra indica was found to have strong reducing power, 2,2-diphenyl-1-picrylhydrazyl radical, hydrogen peroxide, nitric oxide and superoxide radical scavenging activity. Percentage inhibition of AGEs formation by mother tincture, 6C and 30C of Cephalandra indica (10–50 μl) was found to be 30.34%–91.77%, 29.98%–65.71% and 33.05%–57.75%, respectively. Mother tincture, 6C and 30C of Cephalandra indica showed inhibitory effect against sorbitol accumulation with IC50value of 26.12 μl, 203.10 μl and 897.3 μl, respectively, whereas, in aldose reductase inhibition assay, the IC50value was 32.54 μl, 175.02 μl and 834.34 μl, respectively. Conclusion: The results revealed that homoeopathic preparations of Cephalandra indica exhibit protective effect against oxidative stress.

Keywords: Advanced glycation end products, Aldose reductase inhibition, Cephalandra indica, diabetic complications, Homoeopathic preparations, Oxidative stress, Sorbitol accumulation


How to cite this article:
Kishore L, Singh R. Effect of cephalandra indica against advanced glycation end products, sorbitol accumulation and aldose reductase activity in homoeopathic formulation. Indian J Res Homoeopathy 2018;12:11-9

How to cite this URL:
Kishore L, Singh R. Effect of cephalandra indica against advanced glycation end products, sorbitol accumulation and aldose reductase activity in homoeopathic formulation. Indian J Res Homoeopathy [serial online] 2018 [cited 2018 Jun 18];12:11-9. Available from: http://www.ijrh.org/text.asp?2018/12/1/11/229072




  Introduction Top


Generation of free radicals and reactive oxygen species (ROS) leads to the oxidative damage to cellular biomolecules such as proteins, lipids and DNA which is considered to play an important role in the prevalence of several chronic diseases.[1],[2]

Cephalandra indica belongs to family Cucurbitaceae, also known as Kundru in Hindi and Ivy Gourd in English, is a creeper vegetable that grows wild and in abundance in major parts of India. It is a perennial climbing herb with tuberous roots, fruiting throughout the year. The plant has been used since ancient times for treating Diabetes Mellitus in the Indian system of medicine known as Ayurveda. As it was significantly effective in diabetes treatment, Cephalandra indica was described by some as 'Indian Substitute for Insulin'.[3] The plant also gained many reported scientific values as antidiabetic medicine.[4],[5] Fresh juice of roots is used to treat diabetes; tincture of leaves is used to treat gonorrhoea and paste of leaves is applied to the skin diseases. Dried bark is a good cathartic. Leaves and stem are antispasmodic and expectorant. The fleshy green fruit is very bitter. Green fruit is chewed to cure sores on the tongue.[6],[7]

In the present study,in vitro antioxidative potential of mother tincture, 6C and 30C potencies of Cephalandra indica was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, nitric oxide (NO) scavenging assay, hydrogen peroxide scavenging assay, reducing power, superoxide radical scavenging activity, total phenolic content by Folin-Ciocalteu method and total antioxidant activity by phosphomolybdenum method.


  Materials and Methods Top


Materials and reagents

Potencies (Mother tincture, 6C and 30C) of Cephalandra indica L. were procured from Dr. Willmar Schwabe India Pvt. Ltd. DPPH and NADPH were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Gallic acid, bovine serum albumin (BSA), nitroblue tetrazolium (NBT), Folin–Ciocalteu reagent and N-(1-Naphthyl) ethylenediamine dihydrochloride were purchased from Molychem Pvt. Ltd., India. All other chemicals and reagents used were of analytical grade. Study protocol was approved and conducted in MMCP, Maharishi Markandeshwar University, Mullana, Ambala, Haryana, India.

In vitro antioxidant activity

Determination of total phenol content

Total phenolic content in the mother tincture, 6C and 30C of Cephalandra indica was determined with Folin–Ciocalteu reagent using gallic acid as a standard phenolic compound. Sample was diluted appropriately to obtain absorbance in the range of calibration curve. An aliquot of 1 ml of sample solution was mixed with 1 ml of Folin–Ciocalteu reagent. Three minutes later, 3.0 ml of 2% sodium carbonate was added, and the mixture was allowed to stand for 3 h with intermittent shaking. The absorbance of the blue colour that developed was measured at 760 nm (UV-VIS spectrophotometer - 1800, Shimadzu, Japan). The concentration of total phenolic compounds in the sample was obtained as milligrams of gallic acid equivalent (GAE) per gram dry weight.[8]

Total antioxidant capacity

An aliquot of 0.3 ml of mother tincture, 6C and 30C of Cephalandra indica was mixed with 3 ml of the reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). In case of blank, 0.3 ml of water was used instead of sample. The tubes were capped with aluminium foil and incubated in boiling water bath at 95°C for 90 min. After the samples had cooled to room temperature, the absorbance was measured at 695 nm against a blank. Ascorbic acid was used as a standard. Total antioxidant capacity was expressed as equivalents of ascorbic acid (μmol/g).[9]

2,2-diphenyl-1-picrylhydrazyl scavenging activity

The DPPH radical scavenging ability of mother tincture, 6C and 30C of Cephalandra indica was evaluated according to the method given in literature with slight modification.[10] The different concentrations in each reaction set were mixed with 1.0 ml of 0.1 mM of DPPH in ethanol. The mixture was incubated in the dark for 30 min at room temperature. Degree of inhibition of DPPH by monitoring the decrease in absorbance measured at 517 nm. Ascorbic acid was used as positive control. Radical scavenging activity was expressed as inhibition percentage of free radical by the sample and was calculated using the following formula:



Where A0 was the absorbance of control (blank without sample) and At was the absorbance in presence of sample. All the tests were performed in triplicate and graph was plotted with mean values.

Hydrogen peroxide scavenging activity

The hydrogen peroxide scavenging activity was evaluated as described previously.[11] An aliquot of 40 mM H2O2 solution (0.6 ml) was mixed with various concentrations of mother tincture, 6C and 30C of Cephalandra indica. To the mixture, 2.4 ml of phosphate buffer (0.1 M, pH 7.4) was added and the mixture was shaken vigorously and incubated at room temperature for 10 min. Then, the absorbance of the reaction mixture was determined at 230 nm. Ascorbic acid was used as positive control. The H2O2 scavenging activity was calculated as follows:



Where A0 is the absorbance of the control (water instead of sample), A1 is the absorbance of the sample and A2 is the absorbance of the sample only (phosphate buffer instead of H2O2 solution). The IC50 value represented the concentration of the compounds that caused 50% inhibition of H2O2.

Reducing power assay

The Fe3+-reducing power of mother tincture, 6C and 30C of Cephalandra indica was determined according to the method described in literature.[12] Different concentrations of samples (2.5 ml) were mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide and incubated at 50°C for 20 min. After incubation, 2.5 ml of 10% trichloroacetic acid (w/v) was added and the mixture centrifuged at 1000 rpm for 8 min. The supernatant (5 ml) was mixed with 5 ml of distilled water and 1 ml of 0.1% of ferric chloride, and the absorbance was measured spectrophotometrically at 700 nm. The assay was carried out in triplicate and the results expressed as mean values ± standard deviations. Ascorbic acid was used as positive control. The sample concentration providing 0.5 of absorbance (EC50) was calculated from the graph plotted between absorbance at 700 nm against sample concentration.

Nitric oxide scavenging activity

At physiological pH, aqueous solution of sodium nitroprusside spontaneously generates NO [13] which interacts with oxygen to produce nitric ions that can be estimated using Griess reagent. Scavengers of NO compete with oxygen, leading to reduce the production of NO. The reaction mixture of 5 mM sodium nitroprusside in phosphate buffer saline (PBS) and 3.0 ml of different concentrations of the mother tincture, 6C and 30C of Cephalandra indica was incubated at 25°C for 150 min. After incubation, the samples were added to Greiss reagent (1% sulphanilamide, 2% H3 PO4 and 0.1% naphthyl ethylenediamine dihydrochloride). The pink chromophore generated during the diazotisation of nitrite with sulphanilamide and subsequent coupling with naphthyl ethylenediamine was measured at 546 nm. Ascorbic acid was used as positive control. The percentage of inhibition was measured by the following formula:



Where A0 was the absorbance of the control (blank, without sample) and At was the absorbance in the presence of the sample. All the tests were performed in triplicate and the graph was plotted with the mean values.

Superoxide radical scavenging activity

The activity was measured by the reduction of NBT reagent method as described by Shukla et al., 2009.[14] The method is based on the generation of superoxide radical (O2−) by auto-oxidation of hydroxylamine hydrochloride in the presence of NBT, which gets reduced to nitrite. Nitrite in the presence of ethylenediaminetetraacetic acid (EDTA) gives a colour that was measured at 560 nm. Different concentrations of mother tincture, 6C and 30C of Cephalandra indica were taken in a test tube. To this, reaction mixture consisting of 1 ml of (50 mM) sodium carbonate, 0.4 ml of (24 mM) NBT and 0.2 ml of 0.1 mM EDTA solutions were added to the test tube and immediate reading was taken at 560 nm. After incubating the reaction mixture at 25°C for 15 min, about 0.4 ml of (1 mM) of hydroxylamine hydrochloride was added to initiate the reaction and reduction of NBT was measured at 560 nm. Ascorbic acid was used as the positive control. Decreased absorbance of the reaction mixture indicates increased superoxide anion scavenging activity. The percentage of inhibition was calculated according to the following equation:



Where A0 was the absorbance of the control (blank, without sample) and At was the absorbance in the presence of the samples. All the tests were performed in triplicate and the graph was plotted with the mean values.

Antiglycation activity

In vitro antiglycation activity of mother tincture, 6C and 30C of Cephalandra indica was examined by testing their ability to inhibit the fluorescence of BSA in accordance with a previous method.[15] The reaction mixture of BSA (10 mg/ml), 1.1 M fructose in 0.1 M PBS and pH 7.4 containing 0.02% sodium azide with or without sample (Mother tincture, 6C and 30C of Cephalandra indica dissolved in PBS) was incubated in darkness at 37°C for 1, 2, 3 and 4 weeks. AGE formation was measured by fluorescent intensity at an excitation wavelength 355 nm and emission wavelength 460 nm using Elico-SLI74 spectrofluorometer fitted with Xenon Lamp (Elico, India). Aminoguanidine (AG; 500 μg/ml) was used as a positive control for this study.

Erythrocyte sorbitol accumulation inhibition

Five millilitres of heparinised blood was collected from overnight fasted healthy male Wistar rats and erythrocytes were separated from the plasma by centrifugation at 3000 g for 30 min. The cells were washed three times with isotonic saline at 4°C, and in the final washing, the cells were centrifuged at 1500 g for 15 min to obtain a consistently packed cell preparation. The packed cells (1 mL) were then incubated in Krebs-Ringer bicarbonate buffer (pH 7.4) (4 mL) containing 55 mM glucose in the presence or absence of samples (Mother tincture, 6C and 30C of Cephalandra indica) at 37°C for 3 h. The erythrocytes were washed with cold saline by centrifugation at 2000 g for 5 min, precipitated by adding 6% of cold perchloric acid (3 mL) and centrifuged again at 2000 g for 10 min. The supernatant was neutralised with 2.5 M K2 CO3 at 4°C and used for sorbitol determination.[16] The relative fluorescence due to NADH was measured by a fluorescence spectrometer (Elico-SLI74 spectrofluorometer fitted with Xenon Lamp, Elico, India) at an excitation wavelength of 366 nm and an emission wavelength of 452 nm. The experiments were performed in triplicates.

Aldose reductase enzyme inhibition

Partial purification of aldose reductase (ALR1) from rat kidney (IAEC Protocol No: MMCP/IAEC/13/07) was carried out following the previously described methods.[17] Isolated kidney was homogenised in 3 volumes of 10 mM sodium phosphate buffer, pH 7.2 containing 0.25 M sucrose, 2.0 mM EDTA and 2.5 mM 2-mercaptoethanol. The homogenate was centrifuged at 10,000 g for 20 min and the supernatant was subjected to ammonium sulphate precipitation. Precipitate obtained between 45% and 75% saturation was dissolved in the above buffer. The supernatant was used as the source of ALR1. The activity of ALR1 was measured spectrophotometrically by monitoring the oxidation of NADPH at 340 nm as a function of time at 37°C using glyceraldehyde as substrate. The assay mixture in 1 ml contained 50 mM sodium phosphate buffer of pH 7.2, 0.2 M ammonium sulphate, 10 mM DL-glyceraldehyde, 5 mM β-mercaptoethanol and 0.1 mM NADPH. Various concentrations of mother tincture, 6C and 30C of Cephalandra indica were added to assay mixtures of ALR1 and incubated for 5 min before initiating the reaction by NADPH as described above. The percentage inhibition was calculated considering the activity in the absence of mother tincture, 6C and 30C of Cephalandra indica as 100%. The IC50 values were determined by linear regression analysis of the plot of percentage inhibition versus inhibitor concentration.


  Results and Discussion Top


Total phenol content

Phenols and polyphenols are the main class of natural antioxidants found in plants whose functions is to strengthen the oxidative stability of foods and human systems due to their redox properties, which plays significant role in neutralising free radicals, quenching singlet oxygen or decomposing hydroperoxides.[18],[19] Total phenol content in homoeopathic preparations of Cephalandra indica was determined by Folin–Ciocalteu reagent. The total phenol content was found to be 1905, 849 and 495 mg/g GAEs in mother tincture, 6C and 30C of Cephalandra indica, respectively.

Total antioxidant capacity

The total antioxidant capacity is used for the evaluation of both water- and fat-soluble antioxidants. The phosphomolybdenum method used for measuring total antioxidant capacity is based on the formation of green phosphate/reduced Mo (V) complex at acidic pH.[20] Total antioxidant capacity of mother tincture, 6C and 30C of Cephalandra indica was found to be 2710, 759 and 501 μM/g ascorbic acid equivalent, respectively.

2,2-diphenyl-1-picrylhydrazyl scavenging activity

Assay based on the use of DPPH radicals is the most popular spectrophotometric methods for the determination of the antioxidant capacity of plant extracts, foods, beverages and vegetable extracts because the radical compounds can directly react with antioxidants.[21] The present results suggest that mother tincture, 6C and 30C of Cephalandra indica is apparently good free radical scavengers. The IC50 value of mother tincture, 6C and 30C of Cephalandra indica was 28.23, 155.14 and 923.02 μl respectively [Figure 1] and that of ascorbic acid was found to be 1.43 μg/ml.
Figure 1: Effect of Cephalandra indica on 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities in homoeopathic formulation. Values are mean ± standard deviation for n = 3

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Hydrogen peroxide scavenging activity

H2O2 is considered as one of the main inducers of cellular ageing and could strike many cellular energy-producing systems.[22] H2O2 is an uncharged species and is not very reactive itself. It penetrates in cellular membrane and leads to formation of hydroxyl radical which the capability of damaging almost every molecule found in the living cells.[23] The IC50 value of mother tincture, 6C and 30C of Cephalandra indica was found to be 32.52 μl, 185.14 μl and 939.05 μl ml, respectively [Figure 2]. IC50 value of ascorbic was found to be 80 μg/ml.
Figure 2: Effect of Cephalandra indica on H2O2radical scavenging activities in homoeopathic formulation. Values are mean ± standard deviation for n = 3

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Reducing power assay

The electron donating capacity reflects the reducing power of bioactive compounds and is associated with antioxidant activity. Antioxidants can be reductants, and inactivation of oxidants by reductants can be described as redox reactions in which one reaction species is reduced at the expense of the oxidation of the other. The presence of reductants, such as antioxidant substances in the samples, causes the reduction of the Fe3+/ferricyanide complex to the ferrous form.[24] The formation of ferrous ion complex was observed and EC50(effective concentration at which the absorbance is 0.5) was calculated [Figure 3]. EC50 was found to be 37 μl, 250 μl and 989 μl for mother tincture, 6C and 30C of Cephalandra indica, respectively, and 21.42 μg/ml for ascorbic acid.
Figure 3: Effect of Cephalandra indica on reducing power in homoeopathic formulation. Values are mean ± standard deviation for n = 3

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Nitric oxide scavenging activity

NO is generated from amino acid L-arginine by vascular endothelial cells, phagocytes and certain cells of the brain. NO is classified as a free radical because of its unpaired electron and displays important reactivity with certain types of proteins and other free radicals. The toxicity of NO becomes adverse when it reacts with superoxide radical, forming a highly reactive peroxynitrite anion.[25] The various concentrations of mother tincture, 6C and 30C of Cephalandra indica showed significant inhibition against NO radical in a dose-dependent manner. The concentration of mother tincture, 6C and 30C of Cephalandra indica required for 50% inhibition (IC50) was found to be 42.13 μl, 207.05 μl and 999 μl, respectively [Figure 4]. IC50 of ascorbic acid was found to be 63.55 μg/ml.
Figure 4: Effect of Cephalandra indica on NO radical scavenging activities in homoeopathic formulation. Values are mean ± standard deviation for n = 3

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Superoxide scavenging activity

Superoxide anion radicals are formed due to electron leakage from electron transport chain in aerobic cells and acts as a precursor for ROS that contributes to tissue damage and various other pathological conditions.[26] Mother tincture, 6C and 30C of Cephalandra indica, had significant activity against superoxide radicals in a dose-dependent manner [Figure 5]. IC50 of ascorbic acid was found to be 27.96 μg/ml and that of mother tincture, 6C and 30C of Cephalandra indica was found to be 33.65 μl, 223 μl and 987 μl, respectively.
Figure 5: Effect of Cephalandra indica on SOD radical scavenging activities in homoeopathic formulation. Values are mean ± standard deviation for n = 3

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AGEs inhibition activity

Hyperglycaemia leads to the production of AGEs and their receptors. AGEs are responsible for production of ROS and thus results to oxidative stress, the major element for onset of diabetic complications.[27] In the present study, the formation of AGEs was monitored weekly by measuring fluorescence intensity of the BSA-fructose solutions for 4 weeks and mother tincture, 6C and 30C, potencies of Cephalandra indica were found to have inhibitory effect against the formation of AGEs. A significant inhibition of AGEs formation (93.37%) was observed in fructose-induced glycated BSA plus AG (500 μg/ml). At 4th week of incubation, the percentage inhibitions of AGEs formation by Cephalandra indica mother tincture (10–50 μl) were 30.34%–91.77%, respectively; 6C (50–250 μl) was 29.98%–65.71% and for 30C (200–1000 μl) was found to be 33.05%–57.75%, respectively [Figure 6], [Figure 7], [Figure 8].
Figure 6: The effects of Cephalandra indica mother tincture on the formation of fluorescent advanced glycation end products in bovine serum albumin incubated with fructose. Values are mean ± standard deviation for n = 3. AG: Aminoguanidine

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Figure 7: The effects of Cephalandra indica 6C on formation of fluorescent advanced glycation end products in bovine serum albumin incubated with fructose. Values are mean ± standard deviation for n = 3. AG: Aminoguanidine

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Figure 8: The effects of Cephalandra indica 30C on the formation of fluorescent advanced glycation end products inbovine serum albumin incubated with fructose. Values are mean ± standard deviation for n = 3. AG: Aminoguanidine

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Erythrocyte sorbitol accumulation inhibition

The excessive formation and accumulation of sorbitol during chronic hyperglycaemia lead to increased oxidative stress and ultimately to diabetic complications.[28] The different potencies of Cephalandra indica in the present study were found to exhibit significant inhibitory effect on accumulation of sorbitol in red blood cells [Figure 9]. IC50 of ascorbic acid was found to be 183.08 μg/ml and that of mother tincture, 6C and 30C of Cephalandra indica was found to be 26.23 μl, 203.14 μl and 897.54 μl, respectively.
Figure 9: Effect of Cephalandra indica on erythrocyte sorbitol accumulation inhibition assay in homoeopathic formulation. Values are mean ± standard deviation for n = 3

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Aldose reductase inhibitory activity

Chronic hyperglycaemia is responsible for the activation of polyol pathway through the enzyme aldose reductase. The increased activity of this pathway leads to excess formation of sorbitol.[28] The enzyme ALR1 was obtained from kidney of Wistar rat and the activity of ALR1was measured spectrophotometrically by monitoring the oxidation of NADPH at 340 nm. IC50 of quercetin (standard) was found to be 5.30 μg/ml and that of mother tincture, 6C and 30C of Cephalandra indica was found to be 32.19 μl, 175.01 μl and 834 μl respectively [Figure 10].
Figure 10: Effect of Cephalandra indica on aldose reductase inhibitory assay in homoeopathic formulation. Values are mean ± standard deviation for n = 3

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  Conclusion Top


Free radicals produced from different physiological and non-physiological processes are one of the bases of several diseases. Therefore, there is an urge of safe and potent antioxidants to overthrow the free radicals. The results from the present study revealed the presence of phenols in homoeopathic preparation of Cephalandra indica which plays a significant role in the reduction of oxidative stress. Cephalandra indica was found to scavenge different radicals such as in DPPH, H2O2, NO and SOD. Cephalandra indica was also found to inhibit the formation of AGEs and sorbitol accumulation, major culprits for onset of diabetic complications. These defensive effects of homoeopathic preparation of Cephalandra indica may suggest its use for the attenuation of oxidative stress and diabetic complications.

Acknowledgement

Financial assistance from CCRH, Ministry of AYUSH, Government of India is highly acknowledged.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]



 

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