Journal of Saudi Chemical Society (2012) 16, 125129

King Saud University

Journal of Saudi Chemical Society

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Variation in fat soluble vitamins (A, D, E, K) in in vitro and ex vitro germinated chickpea (Cicer arietinum L.) seedlings, as revealed by high performance liquid chromatography
Junaid Aslam
a b c

a,c,*

, Sheba Haque Khan b, Saeed Ahmad Khan

Dubai Pharmacy College, Al Muhasinah 1, P.O. Box 19099, Dubai, United Arab Emirates G.F. College, Department of Chemistry, M.J.P. Rohilkhand University, Bareilly, Uttar Pradesh, India Department of Biotechnology, Jamia Hamdard (Hamdard University), New Delhi 110062, India

Received 15 November 2010; accepted 10 December 2010 Available online 15 December 2010

KEYWORDS Cicer arietinum L.; Fat soluble vitamins; In vitro and ex vitro seed germination; High performance liquid chromatography

Abstract In present communication, effect of in vitro and ex vitro culture conditions was investigated on the yield of fat soluble vitamins in chickpea (Cicer arietinum L.) seedlings analyzed by high performance liquid chromatography (HPLC). Amongst the tested culture conditions (in vitro and ex vitro); in vitro condition proved to be highly effective compared to ex vitro. We noticed a significant difference in vitamins D (ergocalciferol), E (a-tocopherol) and K (phylloquinone) yields in chickpea seedlings grown in two different conditions. Maximum yield with a linear increase in vitamins D and E was noticed upto 9 days old in vitro grown seedlings. Vitamin K yield was also high in in vitro grown seedlings with a linear increase upto 11 days. Although, vitamin A was not detected, the vitamin production in germinating seeds was, therefore, age and culture condition

* Corresponding author at: Dubai Pharmacy College, Al Muhasinah 1, P.O. Box 19099, Dubai, United Arab Emirates. Tel.: +91 9990706557. E-mail address: Junaidg1@gmail.com (J. Aslam). 1319-6103 2010 King Saud University. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2010.12.001

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dependent. The study revealed that, in in vitro condition, the level of fat soluble vitamins enhanced in seedlings, which could be used for human consumption with value addition in the diet of vegetarians.
2010 King Saud University. Production and hosting by Elsevier B.V. All rights reserved.

1. Introduction Chickpea (Cicer arietinum L.) is the third important food legume of the world grown in 40 countries over an area of about 11.2 million hectares, with a production of 9.2 million tons and an average yield of 818 kg/ha. It is commonly known as, gram, bengal gram, chana, chole, garbanzo etc.; is an important pulse which is utilized for human consumption in various forms and recipes viz., unripe green grain boiled in water, roasted pods with developed soft grains, ripe grain boiled in water, sprouted grain after soaking in water, split graindal without seed coat, whole grain powder and split grain dal powder without seed coat. It is grown and consumed in different countries of Asia, Africa, Europe, Middle East, North and South America (Asthana et al., 1996; ICRISAT, 2002). Its grain is rich of protein (1920%) and is utilized in preparation of several tasteful recipes alone or in combination with other food materials like cereals, pulses, vegetables, meat and sh. Besides nutrition, chickpea also has medicinal properties as hypocholesteremic and aphrodisiacs agent. It is used to provide succor and ameliorate in the treatment of bronchitis, catarrh, cutamenia, cholera, constipation, diarrhea, dyspepsia, atulence, snakebite, sunstroke and warts (Duke, 1981; Geervani, 1991). The vitamin content in pulses may increase in germinating grains or sprouts and may lead to value addition in food before cooking (Gopalan et al., 2007). Vitamins are reported to reduce the damage by free radicals and check degenerative disease (Jacab and Sotoudeh, 2005). Vitamin supplements have ergogenic and performance enhancing effect (Clarkson, 1993; Zhao et al., 2004). The grains of pulses lack vitamins A and C, however, recently the water soluble B group of vitamins are found to be present in germinating or sprouted grains (Gopalan et al., 2007). Fat soluble vitamins are accumulated in the body and help in carrying out important functions, such as utilization of proteins and carbohydrates in the body. The information on the occurrence of fat soluble vitamins in chickpea grain was lacking. The present investigation was, therefore, undertaken to determine the fat soluble vitamins in germinating chickpea seed for possible value addition in the diet for human consumption. 2. Experimental 2.1. Plant material Chickpea seeds were purchased from a public supermarket at Qusais in Dubai, United Arab Emirates (U.A.E.). 2.2. In vitro seed germination and culture conditions

double-distilled water. Surface-disinfected seeds (1525 in number) were placed in GA-7 Magenta vessels (Sigma, St. Louis, MO, USA) containing 50 ml MS (Murashige and Skoog, 1962) medium without organic compounds and plant growth regulators, incubated at 25 2 C under 16 h photoperiods with cool white uorescent illumination (100 lmol m2 s1 PFD). Germinating seedlings were removed from the culture vessels and used for the analysis of fat soluble vitamins. 2.3. Ex vitro seed germination In ex vitro conditions, seeds were sown in well prepared seed bed with appropriate moisture. The temperature at the time of sowing was 41 C. The data for fat soluble vitamins were analyzed after three days of sowing and continued upto 11 days. 2.4. Chemicals and reagents All the chemicals and reagents used were of analytical grade viz., HPLC water (Merck), Methanol (Merck), Acetonitrile (Merck), Retinol acetate (SigmaAldrich), Ergocalciferol (SigmaAldrich), a-tocopherol and Phylloquinone (Sigma Aldrich). Water was distilled and deionized by using millipore direct q system. 2.5. Mobile phase The mobile phase was used for the analysis that contained methanol, acetonitrile and water mixed in the ratio (84:14:2) and was ltered through 0.45 micron membrane using millipore ltration unit. 2.6. Standard stock solution Standard of vitamins A, D, E and K was prepared as follows: 11.8 mg of vitamin A (retinol acetate), 16.6 mg of vitamin D (ergocalciferol), 13.3 mg of vitamin E (a-tocopherol) and 11.13 mg of vitamin K (phylloquinone) were dissolved separately in 10 ml of volumetric ask. The nal volume (10 ml) was made up using mobile phase. 2.7. Working standard The working standard was prepared as follows: 562 ll (1.068 mg/ml) of vitamin A; 60.5 ll (1.66 mg/ml) of vitamin E, 510 ll (0.069 mg/ml) of vitamin D and 292 ll (0.856 mg/ ml) of vitamin K were taken from the stock solution and the nal volume was made up to 10 ml using mobile phase. 2.8. Calibration curve

The seeds were washed in running water for 15 min and surface-sterilized in 70% ethanol for 10 min and rinsed three times with sterilized double-distilled water, treated with 0.5% mercuric chloride for 2 min followed by H2O2 (1%). After every treatment, the seeds were rinsed four times in sterilized

Calibration curve was made by using mix standard in mobile phase with ve point calibration, analyzed independently by HPLC and a standard curve was plotted between concentration and peak area. The injected quantities showed good

Variation in fat soluble vitamins (A,D,E,K) in in vitro and ex vitro germinated chickpea linearity. The data of peak area vs. used standard vitamin concentration were treated by linear least square regression from the regression equation. Standard curve from different vitamins standard concentrations was used to estimate fat soluble vitamins in different samples. 2.9. Sample preparation Five grams of each sample was homogenized, weighed and transferred into 100 ml conical ask. 0.5 g ascorbic acid and 25 ml ethyl alcohol was added, shaken for 5 min and kept. Thereafter, 5 ml of KOH (12 N) was added and then reuxed for 3040 min at 80 C, cooled down for 30 min and then sample was extracted three times. For quick separation 23 drops of sodium chloride salt was added and nally concentrated to obtain 1 ml of sample, respectively. 2.10. High performance liquid chromatography (HPLC) For HPLC analysis, a waters symmetry C18 column (4.6 250 mm 5 lm) was used with a linear gradient methanol, acetonitrile and water mixed in the ratio (84:14:2) at a constant ow rate of 1 ml/min with 2300 pressure by using waters pump (1515 isocratic) and a UV (2487) detector was employed for the detection of peaks, using two channels simultaneously at a wavelength of 292 nm, a bandwidth of 5 nm and another wavelength of 280 nm. All the analyses were performed with 3 replicates. 2.11. Recovery study The accuracy of the present method was checked in the sample by recovery study. Pre-analyzed samples were spiked with extra 50,100 and 150% of the standard vitamins; the mixtures were reanalyzed by adapting the proposed method. The experiment was repeated 3 times, average recovered vitamins content was quantied using regression equation and the % recovery was calculated accordingly. 2.12. Precision The precision of the used method was obtained by determining inter and intra-day variations in the three replicates of fat soluble vitamins at two concentration levels (200400 ng per spot) and the Percent Standard Relative Deviation (RSD) was calculated. 2.13. Quantication of fat soluble vitamins 60 ll of each sample was used in triplicates and the vitamin yield was quantied using regression equation of calibration curve of each standard. 2.14. Statistic analysis

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The data on the effect of culture conditions (in vitro and ex vitro) on fat soluble vitamins yield were analyzed by one way analysis of variance (ANOVA). Values are mean of four replicates from two experiments, and the presented mean values were separated using Duncans Multiple Range Test (DMRT) at p 6 0.05. 3. Results and discussion The quantitative analysis of fat soluble vitamins was carried out at 292 and 280 nm absorbance modes. The equation obtained from the various standard concentrations showed a good linear relationship (Table 1). The method used for vitamin extraction and subsequent estimation afforded recovery of 97.18100.88%. The inter and intra-day variations of vitamins at two different concentration levels showed a low % Relative standard Deviation (0.941.57%). The content of fat soluble vitamins in in vitro and in ex vitro raised samples was analyzed from the regression equation using the value of area obtained from win-cat software. The quantitative HPLC analysis of fat soluble vitamins was carried out in in vitro and in ex vitro germinated chickpea seedlings. The vitamins D and K contents were maximum in in vitro grown seedlings, whereas, a linear increase in vitamin D content was noticed upto 9 days old in in vitro germinated seedlings. However, in the case of vitamin K, a signicant increase in contents was noticed upto 11 days. Vitamin A in both the conditions was not detected. In addition a signicant increase in vitamin E content was noticed upto 5 days, thereafter, increase continued with a marginal difference upto 9 days old seedlings. The mean values of the various fat soluble vitamins analyzed from ex vitro and in vitro germinated seedlings are presented in Fig. 1. Effect of the two culture conditions was studied on fat soluble vitamins yield in chick pea seedlings. We have found a signicant variation in the fat soluble vitamins content analyzed from in vitro and from ex vitro germinated seedlings by high performance liquid chromatography. The variation in vitamins content might be due to the light intensity, which regulates the metabolic pathways of particular type of fat soluble vitamins. These results agreed with previous report, where the vitamin yield was directly inuenced by the light intensity and culture conditions (Gopalan et al., 2007; Junaid et al., 2008). The present study revealed that fat soluble vitamin production was age and culture condition dependent. These results corroborate the earlier nding in some other plant system including medicinal and food plants, where increased accumulation of active principle was noted as the seedlings grew, reached a steady state when the plants became older (Elliot and McPherson, 1971; Kundu and Das, 1986; Datta and Srivastava, 1997; Yohe,

Table 1
A D E K

The important parameters of calibration curve.

y = ax + b Y = 7.09e + 004x 5.59e + 005 Y = 9.80e + 004x 1.13e + 005 Y = 1.6e + 005x 2.37e + 004 Y = 1.57e + 005x + 1.2e + 004 r 0.9964 0.9977 0.9932 0.9953 Concentration range (mg/ml) 1.7320.8 1.67420.01 4.3452.0 4.1950.4 Retinol acetate Ergocalciferol a-Tocopherol Phylloquinone

Fat soluble vitamins

a slope; b intercept; r correlation coefcient.

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J. Aslam et al.

Fat soluble vitamins contents (ug g-1)

A
Fat soluble vitamins Yield (ug g -1)

0.25

B
a a
Vitamin A VitaminE Vitamin D Vitamin K

0.6

a
0.5 0.4 0.3 0.2 0.1 0

0.2 0.15 0.1 0.05 0

In vitro

b a b b

b a b c c d
Ex vitro

d
In vitro

c
Ex vitro

Fat soluble vitamins contents (ug g -1 )

1.2 1 0.8 0.6

b a

Fat soluble vitamins contents (ug g -1 )

3 Days old germinated seeds

D
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 In vitro

5 Days old germinated seeds

c a b c d d

b c c d b a

0.4 0.2 0 In vitro 7 Days old germinated seeds Ex vitro

d
Ex vitro 9 Days old germinated seeds

E
Fat soluble vitamins contents (ug g -1 )

1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 In vitro 11 Days old germinated seeds Ex vitro

a a b b

Figure 1 Variation in fat soluble contents at different days interval in in vitro and in ex vitro culture conditions (A) 3 days old sample (B) 5 days old sample (C) 7 days old sample (D and E) 9 and 11 days old sample. Values represented in terms of bar diagrams are the mean of three replicates. Bar diagrams in each condition (in vitro and ex vitro) with same alphabets are not signicantly different according to the Duncan Multiple Range Test (DMRT) at p P 0.05.

2002; Pande et al., 2002; Paul and Shaha, 2004; Bhat et al., 2008; Junaid et al., 2008). It has also been established in other plants that the metabolic pathways was not only restricted to certain tissues, but it was also modulated by different developmental and environmental conditions (Pasquali et al., 1992; Adebiyi et al., 2005). The increase in the level of vitamins content in in vitro germinated seedlings might be due to the photoperiod and the xed culture temperature. From the present observation it is apparent that the differentiation and development of the tissues are clearly the preconditions for the biosynthesis of more complex fat soluble vitamins. As there is no report on systemic monitoring of fat soluble vitamins in in vitro and in ex vitro germinated seedlings, this study will be of signicant value for synthesizing and improving the level of fat soluble vitamins using in vitro methodology. 4. Conclusion Fat soluble vitamins yield was age and culture condition dependent, as maximum fat soluble vitamins were isolated from in vitro germinating chickpea seedlings; that too in older tissues. The present investigation determined the suitable age

and the condition for the production of fat soluble vitamins in chickpea. Based on these results it can be concluded that germinated chickpea could be used for human consumption in order to supply fat soluble vitamins in the diet of vegetarians, and this chickpea culture is a potential source of fat soluble vitamins that can be enriched to a level of acceptability, even industrially, if in vitro mutagenesis, other biotechnological techniques and semi-synthetic method are integrated. Acknowledgments The rst author (Dr. Junaid Aslam), gratefully acknowledges the Chairman, Board of Trustees of Dubai Pharmacy College for providing all facilities to carry out the present research work. References
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Variation in fat soluble vitamins (A,D,E,K) in in vitro and ex vitro germinated chickpea
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Junaid, A., Muhajir, M.S., Khan, S.A., Khan, A.Q., 2008. Afr. J. Biotechnol. 7, 23102314. Kundu, A.S.K., Das, S.M., 1986. Appl. Microbiol. 19, 598603. Murashige, T., Skoog, F., 1962. Physiol. Planta 15, 473497. Pande, D., Purohit, M., Srivastava, P.S., 2002. Plant Sci. 162, 583587. Pasquali, G., Goddijin, O.J.M., De Wall, A., Verpoorte, R., Schilperoort, R.A., Hoge, J.H.C., Memelink, J., 1992. Plant Mol. Biol. 18, 11211131. Paul, D.K., Shaha, R.K., 2004. Pak. J. Biol. Sci. 7, 238242. Yohe, J., 2002. Science 57, 214. Zhao, B., Tham, S.Y., Lu, J., Lai, M.H., Lee, L.K.H., Moochhala, S.M., 2004. J. Pharm. Pharmaceut. Sci. 7 (2), 200204.