Stability Studies of Garcinia indica by Determination of Anthocyanins

 

 

Mandar B Mulik1, Ganesh S Deshmukh1 and Mitesh D Phale2*

1Oriental College of Pharmacy, University of Mumbai, Sandpada, Navi Mumbai 400705, India.

2Divakar Chemicals Ltd., Analytical Research and Development, 203-204, Sant Bhavan, Sharma Ind. Estate, Walbhat Road, Mumbai 400063, India.

 

 

ABSTRACT:

Stability testing is to provide evidence on how the quality of plant products varies with the time under influence of a variety of environmental factors such as temperature and humidity. Hence, kokum juice was subjected to accelerated stability studies and evaluated by anthocyanins content. Anthocyanins are present in the fruit of Garcinia indica, which is responsible for colour of kokum juice. The anthocyanins in juice were studied during six months period by subjecting it to accelerated conditions. The total acidity, brix and pH of the formulation were also monitored as the parameter of stability testing. The study was carried out as per the ICH guidelines.

 

The method used for total anthocyanins determination involved UV spectroscopy. Measurement of the absorbance was done at 510 nm on sample diluted with pH 1.0 and 4.5 buffers. The pigment content was calculated in absolute quantities with the aid of extinction coefficients. 30 ml of juice was withdrawn from four samples of capacity 200 ml each on 0, 1 and 3 month intervals. The results suggest that an increase in temperature decreases the anthocyanins stability. The degradation of anthocyanins in kokum juice samples was observed which is out of the limits of acceptance criteria according ICH guidelines.

 

KEYWORDS: Anthocyanins, cyanidin‑3‑glucoside, Garcinia indica, kokum juice.

 

 

INTRODUCTION:

Anthocyanins are a group of naturally occurring phenolic compounds responsible for the color of many plants, flowers and fruits. Anthocyanins are the best known natural red colorant in food industries. Anthocyanins are commercially derived from various sources which includes grapes, blackcurrant, raspberry, red cabbage, black carrot, etc. At pH 1.0, anthocyanins exist in the highly coloured oxonium or flavilium form, while at pH 4.5 they are present in the colourless carbinol form.1, 2

 

Fruit rinds of Garcinia indica (G. indica) belonging to family Guttiferae contain 10–30% (-)-hydroxycitric acid (1, 2 dihydroxypropane- 1, 2, 3-tricarboxylic acid; and it is valued for health benefits.3 The kokum juice is extracted from the rinds of G. indica which is rich source of anthocyanins. Cyanidin‑3‑glucoside (Fig.1) is the major anthocyanin present in kokum rind. Thus kokum juice with its rich source of anthocyanin has reported to show antioxidant activity, anti carcinogenic, anti-inflammatory, and useful in reducing risk of coronary heart disease and stroke. 4, 5

 

Literature survey revealed the presence of various techniques for the extraction and analysis of anthocyanins from fruits and berries 6, 7; however there is no reported method to evaluate the stability of anthocyanins in kokum juice. Stability is considered one of the most important criteria in pharmaceutical quality control.

 

But now a day’s beverage industry also considers; that only stable preparation would promise precise delivery of the content to the customer. Expiration dating on any product is based upon scientific studies at normal and stressed conditions. 8

 

 

Considering this objective an attempt has been made to conduct a stability study and to prove   how the quality of natural product varies with the time under influence of a variety of environmental factors such as temperature and humidity. The shelf life of the kokum juice was also determined.

 

MATERIAL AND METHODS:

Chemicals and reagents:

Hydrochloric acid, Sodium acetate, Potassium chloride, Sodium hydroxide and Phenolphthalein indicator were obtained from S. D. Fine Chemicals Limited, Mumbai (India). Normal sugar was used as excipient in the preparation of juice, which was procured from local general store.

 

Plant material:

The fruit of kokum plant were procured from local market of Mumbai and was authenticated. The fruit thus obtained was authenticated as Garcinia indica Guttiferae fruits.

 

Equipment:

Stability of samples was done as per ICH guidelines in stability chamber of Thermo Lab humidity cum photo stability chamber. The centrifugation operation carried out in Remi high speed centrifuge. pH of the extracts was checked on a pH/ion analyzer (Lab India PHAN, India). The UV system employed in the identification was Jasco V-550. And the Abbeys portable hand-refractometer was used for measurement of brix value.

 

Preparation of sample:

A kokum juice was prepared by from 20 g of G. indica rinds which were spilt open in halves and soaked in 40 gm of sugar for 8 hours. Soaked rind then extracted with 200 ml of water at 15 lbs/in pressure for 20 min and filtered.3 The extraction and filtration was repeated twice for complete extraction of the juice. The extract was filtered trough muslin cloth and centrifuged to remove vegetative parts from juice. The supernatant was then diluted to 2000 ml with purified water and stabilized with addition of more 240 gm of sugar. The diluted stabilized juice was packed in sealed 10 polybags and stored at 4°C until further use. Each polybag contained 200 ml of clear kokum juice. This procedure was very crude and time consuming as well, but juice obtained by this method was more stable. Taking into consideration stability of the colouring and flovouring ingredients of juice i.e., flavonoids obtained by this method, the sample thus extracted was stored at 4°C and used as a standard for further study. 4 polybags labeled as Standard (S1 to S4)

 

Remaining 4 polybags subjected to accelerated stability testing in Thermo Lab humidity cum photo-stability chamber and labeled as sample (C1 to C4). These samples thus obtained were compared with the standard juice prepared to compare the content of glycoside cyanidin‑3‑glucoside along with total acidity, pH and brix value. Stability study protocol is summarized in Table 1.

 

Table 1: Stability studies protocol of kokum juice

Study

Storage condition

Minimum time period covered by data at submission

Cold storage

4 ºC ± 2 º C

3 months (0,1M, 2 M, 3 M)

Accelerated

 

40 ºC ± 2 º C / 75 % RH ± 5 %RH

3 months (0,1M, 2 M, 3 M)

 

Analysis of the kokum juice:

The G. indica juice so obtained by all the sample preparation procedures was evaluated for following parameters:

 

Organoleptic evaluation:

Appeal, taste, and odour form a very important part of the products. Added ingredients may modify the original taste of any product. The organoleptic characteristics include appearance; colour, odour/flavor, and taste of the S1 to S4 and C1 to C4 preparations were evaluated.

 

Physical evaluation:9, 10

Determination of pH: The measurement of pH was done with a suitable calibrated potentiometer know as pH meter fitted with two electrodes, one constructed of glass and sensitive to hydrogen-ion and the other is calomel reference electrode. The determination was carried out at a temperature of 30°C (Room temperature).

 

Determination of total titratable acidity: The juice was allowed to settle down for 5 min. to obtain a clear sample. An 50 ml burette on a retort stand was filled with standardized 0.1 M sodium hydroxide (NaOH

) solution. 100 ml of distilled water (H2O) was neutralized with phenolphthalein as indicator. 10 ml of prepared sample was pipetted accurately into the 250 ml container, containing the distilled water. The sample was titrated until a pink colour end point was achieved that persisted.

 

Thus the titratable acidity was calculated.

 

Titratable Acidity (gm/l as per tartaric acid)   

=    ml (titre) * M (NaOH) * 0.75

10 ml * 0.1 M (NaOH)

 

Determination of brix value: The samples were placed between the two surfaces of prisms of Abbes portable hand refractometer. Light was adjusted with the help of mirror for maximum reflection and the knob was rotated to bring the boundary (between bright and dark halves of light) to the centre of the cross wire. Reading was noted which was the brix value of the sample.

 

Table 2: Organoleptic, physical and chemical Evaluation of kokum juice subjected to accelerated stability studies

Juice

Day

Appearance

Taste

pH at 30°C

Total titratable acidity (g/100 ml)

Brix value

(%solid content)

Anthocyanin content (mg/L)

Sample

0

Red liquid

Sweet and sour

2.54 ±0.0503

0.1402 ±0.0036

14.76 ±0.1527

1.3042 ±0.2585

30

Red liquid

Sweet and sour

2.57 ±0.0251

0.1429 ±0.0036

14.70 ±0.1000

1.2653 ±0.2641

60

Red liquid

Slightly Sour

2.62 ±0.0230

0.1452 ±0.0064

14.93 ±0.1154

0.6365 ±0.0597

90

Red liquid

Slightly Sour

2.59 ±0.0281

0.1376 ±0.0050

14.80 ±0.2000

0.2547 ±0.0625

Standard

0

Red liquid

Sweet and sour

2.55 ±0.0218

0.1444 ±0.0042

14.43 ±0.2357

1.4432 ±0.1213

30

Red liquid

Sweet and sour

2.56 ±0.0401

0.1472 ±0.0045

14.26 ±0.1941

1.4101 ±0.0693

60

Red liquid

Sweet and sour

2.54 ±0.0101

0.1472 ±0.0023

14.39 ±0.1253

1.3938 ±0.0785

90

Red liquid

Sweet and sour

2.56 ±0.0193

0.1437 ±0.0073

14.60 ±0.2164

1.3806 ±0.1024

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Chemical evaluation:

Quantitative estimation of anthocyanin by pH differential method: 11-13

 

Principle: The structural conformation of cyanidin‑3‑glucoside (the major anthocyanin pigment of G. indica) undergoes changes with changes in pH. Anthocyanin pigments can be described as being indicators, that is, their hue (shade of colour) and intensity (depth of colour) change with pH. At pH 1.0, anthocyanins exist in the highly coloured oxonium or flavilium form, while at pH 4.5 they are present in the colourless carbinol form. The quantitative procedure for determining anthocyanin content described here is based on these facts.

 

One aliquot of an aqueous anthocyanin solution is adjusted to pH l.0 and another to pH 4.5. The difference in absorbance at the wavelength of maximum absorption (510 nm) will be proportional to the anthocyanin content.

Determination of anthocyanin content is based on Lambert‑Beer's Law:

A = eCL

A      is the absorbance; L is the path length in cm and e is the molar absorbance, a physical constant for a molecular species in a given solvent system at a given wavelength. Molar absorbance values for purified pigments taken from the literature can be used, cyanidin‑3‑glucoside is the major anthocyanin of G. Indica and its molar absorbance, e is 29.600 l / (mol cm) and its molecular weight, M is 445 g/mol. C is the molar concentration and by rearranging the Lambert‑Beer's Law equation and multiplying by the molecular weight (M) of the pigment, the concentration in milligrams per liter is determined by:

C (mg/l) = DA/eL x M x 103 x D

Where, D is the dilution factor, and DA is the difference in absorbance of the sample at maximum absorption (510 nm) in the pH 1 and pH 4.5 buffers.

 

Dilution of the sample: The sample must be diluted, so that the sample at pH 1.0 has an absorbance of less than 1.0 and preferably in the range of 0.4‑0.6. The sample must be diluted the same amount at pH 1 and pH 4.5.

 

Corrections for haze or sediment: The diluted samples should be clear and contain no haze or sediment. Sediment should be removed by centrifugation or filtration of the sample. If the sample is free of haze, the absorbance at 700 nm should be zero. To correct for turbidity (haze) the absorbance at 700 nm is subtracted from the absorbance at 510 ‑ 540 nm (the wavelength of maximum absorption).

 

Preparation of buffers for determination of anthocyanin content: pH 1.0 buffer: 125 ml of 0.2M KCl (14.9 g/l) + 335 ml of 0.2M HCl

pH 4.5 buffer:        400 ml of 1M sodium acetate (136 g/l) + 240 ml of 1M HCl (83.0 ml concentrated HCl /l) + 360 ml distilled water

Procedure: Two 10 ml aliquots were each diluted to 50 ml with pH 1.0 and 4.5 buffers. The wavelength of maximum absorption was 510 nm. The absorbance at 510 nm of the pH 1.0 solution and that of the pH 4.5 solution was taken. The samples appeared to be free from haze and the absorbance for both samples at 700 nm was zero.

 

RESULTS:

The kokum juice (S1 to S4 & C1 to C4) so prepared was evaluated for parameters like organoleptic, physical, and chemical parameters, which included determination of pH, total titratable acidity, brix value and quantitative estimation of anthocyanin. The results are summarized in Table 2.

 

DISCUSSION:

The anthocyanin content has decrease variably in all samples. It has been observed that samples in accelerated conditions have anthocyanin percent degradation at 1, 2 and 3 month interval was 2.98, 351.19 and 80.47 % respectively. While samples in cold storage showed a degradation of 2.29, 3.42 and 4.33 %. Other physical parameters (including pH) were fairly constant through out the sampling time except, the colour has changed markedly. The comparison is shown in the Fig.2.

 

The physical parameters such as pH, brix value and total titratable acidity of sample initially and analyzed after 1, 2 and 3 months of storage at accelerated conditions were found similar while the color of samples has changed compare to samples stored at cold condition. The assay for anthocyanin indicates its unstable nature in all samples subjected to accelerated studies, while there was a limit decrease in anthocyanin content in samples subjected to cold condition. The results suggest that an increase in temperature decreases the anthocyanin stability.

 

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Received on 21.12.2009

Accepted on 05.03.2010   

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(2): March -April 2010, 144-147