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HPTLC Analysis of Vitamin C from Pithecellobium Dulce, Benth (Fabaceae) Nilakshi V

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HPTLC Analysis of Vitamin C from Pithecellobium Dulce, Benth (Fabaceae) Nilakshi V Research Article Nilakshi V. Gambhir et al. / Journal of Pharmacy Research 2011,4(4),1197-1198 Available online through ISSN: 0974-6943 http://jprsolutions.info HPTLC analysis of vitamin C from Pithecellobium dulce, Benth (Fabaceae) Nilakshi V. Gambhir* and V. V. Bhaskar. Department of Botany, GDM Arts, KRN Commerce and MD Science College, Jamner, Dist. Jalgaon, Pin. 424 206, India Received on: 04-01-2011; Revised on: 17-02-2011; Accepted on:16-03-2011 ABSTRACT This work describes the simplest HPTLC method developed for quantification of vitamin C from methanolic extract of Pithecellobium dulce pods, a traditional medicinal plant widely used in Asia. A good separation was achieved with mobile phase ethanol: water (2:1) on precoated silica gel 60 F 254 HPTLC plates. Quantitative analysis was carried out in the absorbance at 254nm. A good linear relationship, r2= 0.992 with respect to peak area was observed between the concentration ranges of 1.0 -6.0 µg. Pithecellobium dulce pods are a rich source of vitamin C and can be considered as a functional food. The method developed is reproducible and can be used for routine analysis of vitamin C in crude drugs and in herbal and traditional dosage containing Pithecellobium dulce as an ingredient. Key words: Pithecellobium dulce pod, HPTLC analysis, Vitamin C, traditional medicine. INTRODUCTION The family Fabaceae with 730 genera and over 19,400 species is an economically One ml of sample extract was further diluted to 10 ml with 50% Methanol important family found throughout the world [1]. Pithecellobium dulce benth is which was used for quantitative estimation. one of the common road side fabaceae member found in Asia. The plant is extensively used in number of traditional medicine treatments and is reported to Preparation of Standard Vitamin C and calibration curve: be a folk remedy for ear ache, leprosy, peptic ulcer and tooth ache. Infusions of Ten mg of std. vitamin C sample dissolved in 10 ml of 50% of Methanol for the different plant parts have been used traditionally to treat dysentry, intestinal preparation of Standard Curve. The six standard levels (1, 2, 3, 4, 5 and 6 µg) of disorders, ulcer, and indigestion etc. Pithecellobium dulce pods are customarily Std. vitamin C were used for the calibration curve for which 1, 2, 3, 4, 5 and 6 µl sold on roadside stands in tropical countries, contain a thick sweetish, but also of standard solution was applied in triplicate on a TLC Plate. acidic pulp, eaten raw or made into a drink by tribal peoples and also used in traditional medicines but still most of the chemical constituents of the pods are The chromatogram was developed for 15 mts, dried at room temperature and remain unexplored and underutilized [2,3,4]. scanned at 254 nm; average peak areas of 3 standards were calculated. The calibration curve of the standard drug concentration (X-axis) over the average Vitamine C is an effective anti-oxidant and is necessary for treatment of scurvy, peak area (Y-axis) was prepared to get a regression equation by Win Cats in humans. It performs numerous physiological functions in human body. The richest natural sources of vitamin C are fruits and vegetabels [5,7]. The present investigation has therefore been made to find out Vitamin C con- tent of the fresh Pithecellobium dulce pods by simple and reproducible HPTLC method. MATERIALS AND METHODS : Plant Material: Fresh and healthy Pithecellobium dulce pods were collected from the forests of Thane district, Maharashtra, India. The plant was authenticated at Department of Botany, MD Science College, Jamner, Maharashtra. A herbarium specimen, number MDSC/PS/20 was also preserved in the same college. Fresh pod pulp was then separated from the seeds and husk. Figure 1. HPTLC Chromatogram of vitamin C Extraction of Plant Material: Fresh pulp weighing 100gm were macerated with 150 ml of 50% methanol for 24 hrs with occasional shaking. The extract was separated and mark was again extracted twice with 50 ml of 50% methanol (fresh). The extract was pooled (mixed) and concentrated in a rotary vaccum evaporator to get 100 ml of sample [6]. *Corresponding author. Nilakshi V. Gambhir Department of Botany, GDM Arts, KRN Commerce and MD Science College, Jamner, Dist. Jalgaon, Pin. 424 206, India Figure 2. Standard curve of vitamin C with respect to area Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1197-1198 Nilakshi V. Gambhir et al. / Journal of Pharmacy Research 2011,4(4),1197-1198 Figure 3. Superimposed UV spectra of vitamin C in standard and sample Figure4. 3D View of all tracks software, which was used for the estimation of vitamin C in Pithecellobium Pithecellobium dulce consumption will contribute to redox homeostasis of the dulce [6,8]. human body and consequently, this fruit could be considered as a functional food. Chromatographic analysis : Sample solution of 20µl was spotted on TLC aluminum sheets silica gel 60 F ACKNOWLEDGEMENTS: 254 pre coated layer, thickness 0.2 mm using Linomat V spotting device of The authors are grateful to Head, Department of Botany, MD Science College, Camag HPTLC system, consisting scanner III with Win Cats 4 software. The Jamner for authentification and preservation of plant herbarium and Dr. Rekha mobile phase and development chamber used were Ethanol : Water (2:1) and Vartak, Head, Department of Biology, Homi Bhabha Centre for Science Educa- Twin through chamber of (20 x 10). Slit dimension was 6.00 X 0.45 mm, Micro. tion, TIFR, Mumbai for providing laboratory facilities. Also grateful to Dr. R. Sets were prepared in Triplicates. The chromatograms were scanned at 254 nm. Shukla from P.S.G.V.P. M’s Arts, Science and Commerce College, Shahada for The mean peak area of triplicate samples was calculated and the content of the valuable guidance. vitamin C was quantified using the regression equation obtained from the stan- dard curve [6,8]. REFERENCES: 1. Allen O.N., Allen E.K., The Leguminosae: A Source Book of Charac- RESULTS AND DISCUSSION: teristics, Uses and Nodulation, The University of Wisconsin Press, The standard vitamin C has an Rf value of 0.88 [fig.1]. A good linear relationship Madison, Wisconsin, 1981. (r2= 0.992 with respect to peak area) was observed between the concentration 2. Anonymous, The Wealth of India, 11 volumes, Council for Scientific ranges of 1.0 -6.0 µg [fig. 2]. The regression equation was found to be Y= 5.017x and Industrial Research (CSIR) Publications, New Delhi, 1948–1976. + 957.3 with respect to area, where Y is the peak area and X is the concentration 3. Duke J.A., Wain K.K., Medicinal plants of the world. [Computer of standard Vitamin C [fig. 2]. The UV spectrum of test sample is super imposable index with more than 85,000 entries], 3 vols, Longman group UK with that of standard ascorbic acid [Fig. 3] indicating purity of peak. Simplicity, Limited, 1981. specificity and sensitivity of the newly developed method makes it the appro- 4. Murugesansugumaran, Pithecellobium dulce Benth: a review, Phar- priate choice for monitoring the vitamin C content at the time of drug prepara- maceutical reviews, 6, 2008. tions and for quality control of finished product. 5. Padayatty, Katz A., Wang Y., Eck P., Kwon O., Lee J., Chen S., Corpe C., Vitamin C as an antioxidant : evaluation of its role in The Vitamin C content found in the Pithecellobium dulce fruit was 140mg/100 disease prevention, Journal of the American College of Nutrition, 22, gm of fresh pulp, using the present HPTLC method. Thus the vitamin C 2003, p. 18–35. content of fresh Pithecellobium dulce pod pulp is much higher than that re- 6. Sethi P. D., Quantitative analysis of drugs in Pharmaceutical formu- ported in apple (6-8mg/100gm), plum (10mg/100gm), mango (28mg/100gm), lations, CBS Publisher and distributors, New Delhi, 1997, p. 589. orange (50mg/100gm), litchy (71mg/100gm), pea (24-54mg/100gm), and beans 7. Vaughan J. G., Geissler C. A., The New Oxford Book of Food Plants, (27mg/100gm) [7]. Vitamin C consumption has been associated with antioxidant Oxford University Press, USA, 2009. and neuroprotective effects, lower risk of cardiovascular diseases and 8. Weins C., Hauck H. H., Advances and developments in thin layer with prevention/treatment of scurvy, cancer [5, 7]. Thus, it is expected that chromatography, LC-GC International, 9, 1996, p. 710-7. Source of support: Nil, Conflict of interest: None Declared Journal of Pharmacy Research Vol.4.Issue 4. April 2011 1197-1198.
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