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JOURNAL OFFOOD COMPOSITION

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Journal of Food Composition and Analysis 20 (2007) 529–533

www.elsevier.com/locate/jfca

Original Article

Comparison of ascorbic acid content of Emblica officinalis fruitsdetermined by different analytical methods

V. Raghu, Kalpana Platel, K. Srinivasan�

Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore 570 020, India

Received 3 November 2006; received in revised form 31 January 2007; accepted 12 February 2007

Abstract

Emblica fruits have long been endowed with many pharmacological properties, which are attributable to their rich vitamin C content.

A recent report that Emblica fruits do not contain ascorbic acid, but contain two hydrolysable low molecular weight tannins with

antioxidant attributes, thus challenging the long-standing belief that amla fruits owe their potential pharmacological activity to their

vitamin C content. The present study examines the vitamin C content of Emblica fruits by employing a specific enzymatic method in

addition to the liquid chromatographic and the conventional spectrophotometric procedures to assess the extent of over-estimation, if

any. The specific enzymatic procedure employed here indicated that Emblica fruits do not totally lack vitamin C as claimed and

nevertheless, the conventional colorimetric method over-estimates the same in these fruits. A similar determination of vitamin C content

in a few other conventionally rich plant sources of this vitamin indicated that over-estimation of vitamin C by colorimetric method is also

evident in guava fruits (Psidium guajava) and amaranth leaves (Amaranthus gangeticus), but the extent was lower than that in the Emblica

fruits. Thus, the discrepancy in the reported value of this nutrient is not limited to Emblica fruits alone, but can be found in other plant

systems also. This study also examined the extent of retention of this vitamin after drying of the Emblica fruits and evidenced that shade

drying did not improve the same as compared to sun drying. However, the amount of vitamin C retained in the dried Emblica fruits is

comparable to the amounts present in several other fresh fruits.

r 2007 Elsevier Inc. All rights reserved.

Keywords: Ascorbic acid; Emblica fruits; Enzymatic determination

1. Introduction

Emblica (Emblica officinalis) fruits commonly grown intropical and subtropical parts of India, China, Indonesiaand Malaysia are an important dietary source of vitamin C.The fruits of Emblica are widely consumed raw, cooked orpickled, but they are also principal constituents of manymedicinal preparations in the indigenous system ofmedicine in India (as a potent rasayana, and for thetreatment of diarrhoea, jaundice and inflammations)(Scartezzini and Speroni, 2000). For many years, thetherapeutic potential of the Emblica fruits was attributed totheir ascorbic acid content (Kapoor, 1990), because of

e front matter r 2007 Elsevier Inc. All rights reserved.

a.2007.02.006

ing author. Tel.: +910821 2514876;

517233.

ess: ksri@sancharnet.in (K. Srinivasan).

tannins, the ascorbic acid does not oxidise even in the driedfruit, thus maintaining its anti-scurvy property unchanged.The fruit extract has been reported to have hypolipi-

demic, antidiabetic and anti-inflammatory activity, and toinhibit retroviruses such as HIV-1, tumour developmentand gastric ulcer (Sabu and Kuttan, 2002). Emblica fruitextract exhibits antioxidant properties, its aqueous extractbeing a potent inhibitor of lipoid peroxidation and ascavenger of hydroxyl and superoxide radicals in vitro(Scartezzini and Speroni, 2000).Contrary to the long-standing belief that Emblica fruits

owe their potential pharmacological activity to theirvitamin C content, Ghosal et al. (1996) employing liquidchromatography, asserted that Emblica fruits do notcontain ascorbic acid, either in the free or in the conjugatedform, but contain two hydrolysable tannins of lowmolecular weight, namely, Emblicanin A and EmblicaninB, and other tannins such as punigluconin and pedunclagin.

ARTICLE IN PRESSV. Raghu et al. / Journal of Food Composition and Analysis 20 (2007) 529–533530

The two Emblicanins are shown to exhibit a very strongantioxidant action (Ghosal et al., 1996). Whether this claimof the total absence of ascorbic acid in Emblica fruits basedon chromatographic methods is valid, needs to be examinedfurther by employing more specific methods.

Several methods are available for the determination ofascorbic acid in food samples. These analytical methodsemploy titrimetry (AOAC, 1990; Kabasakalis et al., 2000),spectrophotometry (Arya et al., 1998) and amperometry(Arya et al., 2000). Most of these methods may giveoverestimates due to the presence of oxidisable speciesother than ascorbic acid and/or exclude measurement ofdehydroascorbic acid. For example, the AOAC procedureemploying titration with 2,6-dichloroindophenol in acidicsolution is not applicable to all matrices. Substancesnaturally present in fruits such as tannins, sulphydrylcompounds, and metals such as copper, iron and cobalt areoxidised by the dye (Arya et al., 2000). One of thedifficulties of successful application of spectrophotometricmethods is that, beyond specified limits, the intensity ofabsorption is not directly proportional to concentration.Another disadvantage is that well-defined absorption bandin the ultraviolet region of the spectrum is subject tointerference from many other substances. The optimalmethods for ascorbic acid determination are separationtechniques, including capillary electrophoresis, gas chro-matography and liquid chromatography, which, however,cannot be used for routine analysis of a large number ofsamples (Versari et al., 2004; Silva, 2005). The enzymaticprocedure that employs ascorbic acid oxidase is highlyspecific to this compound and utilises the same as substratefor its reaction. Thus, determination of vitamin C in thesample before and after the ascorbic acid oxidase reactionusing an appropriate formazan method provides anaccurate estimate of the vitamin C content.

The present investigation was carried out to compare theascorbic acid content of Emblica fruits as determined byspectrophotometric, chromatographic and enzymatic meth-ods. In addition to Emblica, a few other rich sources ofascorbic acid such as guava fruits, amaranth leaves and greenchilli were also examined for their ascorbic acid content bymultiple procedures to ascertain if discrepancies in theestimates of ascorbic acid differ in different matrices. The useof the specific enzymatic method for ascorbic acid determi-nation would also confirm the presence of this vitamin inEmblica fruits. Since Emblica fruits are generally preserved intheir dehydrated form for use throughout the year, it wouldbe interesting to examine the vitamin C content retained inits dry form. In this context, vitamin C content wasdetermined in both sun-dried and shade-dried Emblica fruits.

2. Materials and methods

2.1. Materials

Fresh Emblica fruits (Emblica officinalis), guava fruits(Psidium guajava), amaranth leaves (Amaranthus gangeticus)

and green chilli (Capsicum annuum) were obtained fresh fromthe local orchards. 2,4-dinitrophenylhadrazine, 2,6-dichlor-ophenolindophenol, 3-(4,5-dimethyl thiozolyl-2)-2,5-dimethyltetrazolium bromide (MTT), 5-methyl phenazinium methylsulphate (PMS) and ascorbic acid oxidase (from Cucurbita-cae) were procured fromM/s. Sigma Chemical Co., St. Louis,USA. Xylene, methanol, o-phenylenediamine, norit and m-phosphoric acid were from SISCO Research Laboratories,Mumbai, India. Shimadzu UV-2450/2550 UV–visible spec-trophotometer (Shimadzu Corporation, Japan) was em-ployed for all spectrophotometric measurements.

2.2. Extraction of ascorbic acid

The fresh plant food materials were rinsed in distilledwater to remove adhering contaminants, and were used forextraction of ascorbic acid on the same day. Emblica,guava, chilli fruits and amaranth leaves were chopped, and5 g portions were immediately ground in 2% m-phosphoricacid solution using a pestle and mortar, filtered through aWhatman filter paper no. 1, and made up to 100mL. Thesame extract was divided and used fresh for the determina-tion of ascorbic acid by different methods on the same day.In the case of HPLC determination, the extract was storedfrozen overnight in an air-tight, dark container.

2.3. Drying of Emblica fruits

Two portions (approx. 200 g each) of fresh, de-seededEmblica fruits were dried under the sun (2 d) and in shade(4 d) in parallel, to a constant weight. Final constantweights of the fruits resulted at the end of 4 days of shadedrying or 2 d of sun drying. The dried samples were 22.2%of the original weight.

2.4. Analysis of vitamin C

Suitable aliquots of the sample extract of ascorbic acidprepared in m-phosphoric acid were used for the determi-nation of the same by various methods depending on theoptimum concentration range for the method involved.

2,4-Dinitrophenylhydrazine method: Vitamin C was de-termined based on the oxidation of ascorbic acid todehydroascorbic acid followed by coupling with 2, 4-dinitrophenylhydrazine under controlled conditions to givered coloured osazones (Roe and Osterling, 1943). Thecolour obtained was compared with standard solution ofascorbic acid (0–25 mg/mL).

Indophenol–xylene method: Vitamin C was determinedbased upon the quantitative decolouration of 2,6-dichlor-ophenol indophenol by ascorbic acid. The excess dye wasextracted with xylene and the colour intensity wasmeasured at 500 nm, and compared with standard ascorbicacid (0–80 mg/mL) (Robinson and Stotz, 1945).

Enzymatic method: Ascorbic acid in the sample wasdetermined after treatment with ascorbic acid oxidase inphosphate buffer (pH 5.6), as described by Bergmeyer

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(1984). One millilitre samples (in 2% m-phosphoric acid)were mixed with 1.5mL of 0.1M phosphate buffer (pH5.6). Fifty micro litres of ascorbic acid oxidase (6.25 units)were added and incubated at 37 1C for 15min. An aliquot(0.1mL) of this enzymatically treated sample was mixedwith 1mL of MTT solution (MTT, 7.5mmol/L; phosphate,0.2mol/L; citric acid, 0.2mol/L; EDTA, 2.0mmol/L, pH3.5), and 1.8mL of water, and incubated at 37 1C for 6min.The absorbance was read at 578 nm (A1). This sample wasmixed with 0.1mL PMS (15mmol/L) in dark, andincubated at 37 1C for 15min. The absorbance of theresultant formazan was read at 578 nm (A2). The differencebetween the two absorbencies was calculated, and sub-tracted from the similar OD value obtained for the samplethat had not undergone ascorbic acid oxidase reaction. Theworking range was 0.5–5 mg ascorbic acid.

Liquid chromatography and fluorescence detection: Thevitamin C samples were pre-derivatised as described byDodson et al. (1992). The ascorbic acid was oxidised todehydroascorbic acid (DHAA) in presence of Norit. Theoxidised form was then reacted with o-phenylene diamine(OPDA) to form a flourophore. Quantitation of the DHAA-OPDA derivative in the extracts previously cleaned up bypassing through 0.22mm membrane filter was made byHPLC in a Shimadzu HPLC-10AT system consisting of afluorescence detector, binary pump and manual sampleinjector. Chromatographic separation was accomplishedusing SGE 250� 4.6mm SS Excil C18 10mm column. Assayperformance and reproducibility were confirmed usinginternal standards. The DHAA-OPDA derivative wasanalysed by isocratic elution at the excitation wavelength350nm and emission wavelength 430nm. Sample (10ml) wasinjected on to a reverse-phase column and eluted with amobile phase containing methanol: water (55:45) at a flowrate of 1mL/min (Dodson et al., 1992). Quantitation of theDHAA-OPDA derivative was made from peak area ratio,which was based on a calibration curve generated fromstandard vitamin C derivative (20–100 ng).

2.5. Statistical analysis

All determinations were made in four replicates, usingfour different batches of samples and the average values are

Table 1

Ascorbic acid content (mg/100 g) of fresh plant foods as determined by variou

Sample Method of determination

2,4-DNPH Indopheno

Emblica fruits 468.8736.7 206.8732.0

Guava fruits 85.879.34 85.873.59

Amaranth leaves 62.374.39 60.474.79

Green chilli 55.074.00 71.177.94

Values are mean7SEM (Standard error of the mean) of four replicates of fou�Significantly lower than the values obtained by 2,4-DNPH method (Po0.��Significantly higher than the values obtained by 2,4-DNPH method (Po

reported. Statistical analysis of data was done employingStudent’s t-test according to Snedecor and Cochran (1976)and were considered significant when Po0.05.

3. Results and discussion

3.1. Ascorbic acid content of fresh fruits as determined by

different methods

Ascorbic acid content of Emblica fruits, guava fruits,green chilli and amaranth leaves as determined by 2,4-DNPH method, indophenol–xylene method, ascorbicacid oxidase method and HPLC method is presented inTable 1. The value in Emblica fruits ranged from 389 to560mg/100 g, with a mean of 468.8736.7 as obtained bythe conventional 2,4-DNPH method. This value however,was found to be much lower when determined by the otherthree methods. The values ranged from 110 to 287mg/100 gwith a mean of 206.8732.0mg/100 g in the indophenol–xylene method. Vitamin C values ranged from 121 to379mg/100 g with a mean of 213.6739.0 in the enzymaticmethod, while it ranged from 140.9 to 421mg/100 g with amean of 236.2734.2mg/100 g in the HPLC method.In order to verify if such a discrepancy in vitamin C

content arising out of the methods employed similarlyexists in other common sources of vitamin C, a few moreplant sources were examined in this context. Vitamin Ccontent of guava fruits was 85.879.34mg/100 g, rangingfrom 64.2 to 113.8mg/100 g in the individual fruits, asdetermined by the DNPH method. Indophenol–xylenemethod also provided a similar vitamin C value in guavafruits, namely, 85.873.59, ranging from 77.0 to 94.8mg/100 g. However, vitamin C content of guava fruits wasfound to be significantly lower, namely, 52.476.19(ranging from 29.7 to 79.0mg/100 g), as evidenced by theenzymatic method. HPLC method also provided similarlower vitamin C value in guava fruit and was 58.6711.9(ranging from 22.3 to 100.4mg/100 g). Thus, the spectro-photometric methods (DNPH and indophenol–xylene)provided an over-estimation of vitamin C content in guavafruits.Both DNPH method and indophenol–xylene method

provided a similar vitamin C value in the case of amaranth

s methods

l–xylene Enzymatic HPLC

� 213.5739.0� 236.2734.3�

52.476.19� 58.677.90�

36.874.86� 41.175.13��� 76.576.3�� 83.078.30��

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Method of Determination

2

Fig. 1. Vitamin C content of sun-dried and shade-dried Emblica fruits. 1:

Enzymatic method; 2: DNPH method; 3: Indophenol-xylene method.*Values significantly higher than the corresponding values obtained by

Enzymatic method.

V. Raghu et al. / Journal of Food Composition and Analysis 20 (2007) 529–533532

leaves (62.374.39mg/100 g by the DNPH method, and60.474.79mg/100 g by the indophenol–xylene method). Asin the case of guava fruits, both the enzymatic and HPLCmethod provided closer values, which were significantlylower than the values obtained with either DNPH orindophenol–xylene methods. The vitamin C values ofamaranth leaves were 36.874.86mg/100 g (enzymaticmethod) and 41.075.13mg/100 g (HPLC method). Thus,as in the case of guava fruits, the spectrophotometricmethods (DNPH and indophenol–xylene) provided anover-estimation of vitamin C content in amaranth leaves.

Unlike in the case of guava fruits or amaranth leaves, thevitamin C content of green chilli was similar whendetermined by the indophenol–xylene method (71.177.94mg/100 g), the enzymatic method (76.576.3mg/100 g),and the HPLC method (83.078.3mg/100 g). Whereas theconventional DNPH method gave a value which was lower(55.0710.0mg/100 g), than the other three methods. Thus,the situation in chilli resembled that of amla fruit in thatindophenol–xylene method provided values similar to thoseof enzymatic and HPLC methods. However, DNPHmethod has not over-estimated the vitamin C content inchilli, unlike in the other samples tested.

Thus, vitamin C content was lower than the valuesobtained by the DNPH method when determined by any ofthe other three methods employed. Although the enzymaticmethod and HPLC method are considered to be mostspecific and accurate, the indophenol–xylene method alsoprovided vitamin C values in these fruits comparable to theformer two methods, if the average of the five values wasviewed. However, values obtained with indophenol–xylenemethod were lower than those obtained with enzymaticand HPLC methods in the case of three fruits, while it wasthe opposite in the case of the rest. The values obtainedwith enzymatic and HPLC methods were reasonably closefor all the five fruits. It also emerges from these data that2,4-DNPH method has made an over-estimation ofvitamin C content in Emblica fruits.

Thus, the current investigation has indicated thatEmblica fruits do not totally lack ascorbic acid as claimedby Ghosal et al. (1996) , who drew their conclusions basedon the chromatographic method. The enzymatic procedureused in our investigation, which employs ascorbic acidoxidase is highly specific to ascorbic acid and uses the sameas substrate for its reaction. Thus, determination ofvitamin C in the sample before and after the ascorbic acidoxidase reaction using an appropriate formazan methodprovides an accurate estimate of the total ascorbic acidcontent. It is also true that the conventional DNPHmethod over-estimates the ascorbic acid content in Emblica

fruits. The actual ascorbic acid content would therefore be40–50% of what is reported by employing such conven-tional procedures. Similar over-estimation of ascorbic acidby DNPH method was evident also in guava fruits andamaranth leaves, but the extent was lower than that in theEmblica fruits, the actual ascorbic acid content beingaround 60% of the value obtained by DNPH method.

Therefore, such a discrepancy in the reported value of thisnutrient is not limited to Emblica fruits alone, but can befound in other plant systems also. Although this inferencecannot be sustained in the absence of any specificvalidation of the identity of the peak quantified in Emblica

as ascorbic acid, it still appears that based on theapplication of multiple procedures especially the morespecific enzymatic method, the results of Ghosal et al. maybe incorrect, and necessitates further investigation onEmblica.

3.2. Ascorbic acid content of dried amla fruits

Since Emblica fruits are generally preserved in theirdehydrated form for use throughout the year, the vitaminC content of sun-dried and shade-dried amla fruits wasdetermined. Vitamin C content of dried amla fruits asdetermined by enzymatic, DNPH and indophenol–xylenemethods are presented in Fig. 1. As in the case of ascorbicacid content in fresh Emblica fruits, the DNPH methodproduced an over-estimation of the ascorbic acid contentof both sun-dried and shade-dried Emblica fruits, followedby the indophenol–xylene and the enzymatic methods.Sun-drying of Emblica fruits for 2 d resulted in a moistureloss of 77.8%, while the same was achieved also by shadedrying for a duration of 4 d. Significant amounts of vitaminC were lost as a result of drying of Emblica fruits by eitherof the drying procedures. Only about 18% of the amountoriginally present in the fresh fruit was retained afterdrying under the sun for 2 d. Drying of these fruitsunder the shade did not improve the extent of retentionof this vitamin (Fig. 2). Thus, the methods of dryinghave not made any difference on the extent of vitamin Cretention.

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Fig. 2. Vitamin C retained in sun-dried and shade-dried Emblica fruits. 1:

Enzymatic method; 2: DNPH method; 3: Indophenol-xylene method.*Values significantly higher than the corresponding values obtained by

Enzymatic method.

V. Raghu et al. / Journal of Food Composition and Analysis 20 (2007) 529–533 533

It is a common belief that the vitamin C content is fairlywell conserved in Emblica during its drying/storage of thedried form, although there are no scientific evidences forthis. An early study has reported retention of vitamin C ofonly 2% in the preserves made out of Emblica fruits(Bhatia and Siddappa, 1956). The preparation of thesepreserves involved heat processing through blanching inboiling water for 5min. In another study, an extract ofEmblica fruits has been found to contain 2% vitamin C asdetermined by HPLC (Kim et al., 2005). This recent reporton the vitamin C content of Emblica fruit extractdetermined by a HPLC method confirms the presence ofvitamin C in amla fruits, thus contradicting the claim ofGhosal et al. (1996). In the present investigation, driedEmblica fruits contained 34–38mg vitamin C equivalent to100 g of fresh weight. This value is comparable to theamounts present in several other fresh fruits such as sweetlime, orange, guava, pineapple, etc., thus making driedamla still a good source of vitamin C.

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