Analytical & Bioanalytical Shah and Lele, J Anal Bioanal Techniques 2012, 3:4

http://dx.doi.org/10.4172/2155-9872.1000141
Techniques

Research Article Open Access

Extraction of Diosgenin, a Bioactive Compound from Natural Source Dioscorea

alata Var purpurae
Heena J Shah and Lele SS*
Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai, India

Abstract
Diosgenin, a commercially important bioactive sapogenin, extracted from tubers belonging to Dioscorea species,
is used as a precursor in the manufacturing of sex hormones, oral contraceptives and other pharmaceutically
important steroidal drugs. Diosgenin content of two Indian variety viz., D. deltoidea and D. prazeri ranges from 0.32
- 1%. With the view to commercially exploit a new source of diosgenin, a lesser explored tuber D. alata Var purpurae
(purple yam), from Western Ghats, India was studied. The diosgenin content was determined from tuber (fresh and
dried) and callus of D. alata by acidic hydrolysis of the glycosides and extraction with an organic solvent, followed by
the HPTLC analysis. HPTLC mobile phase was optimized to toluene: ethyl acetate 7:3 v/v using the PRISMA model
and diosgenin content was found to be 0.078, 0.133 and 0.048% respectively after extraction for 8 hrs in alcohol.

Keywords: D. alata var purpurae; Acid hydrolysis; Solvent extraction; Hydrolysis

HPTLC; PRISMA model; Diosgenin
Introduction
Diosgenin (Figure 1) is a bioactive steroidal sapogenin belonging
to the triterpene group and is of great interest to the pharmaceutical
industry [1]. It is the aglycone formed by the hydrolysis of saponin
dioscin (Figure 1), a compound found in Dioscorea spp. [2]. It serves
as an important starting material for the production of corticosteroids,
sexual hormones, oral contraceptives as well as other steroidal drugs
via hemisynthesis [3]. Different chemical and biological protocols are
reported to extract diosgenin [4]. Past research shows various methods
analyzing Diosgenin such as microscopy [5], spectrophotometeric [6],
GLC [7], HPLC [8] and HPTLC [9].
In the present work, we studied extraction and estimation of
Diosgenin content of the lesser explored tuber Diosocrea alata Var
purpurae (purple yam), from Western Ghats, India [3]. We tried
extracting diosgenin from fresh and dried tuber and callus developed
by inoculating tuber as explant and optimizing the HPTLC method to
analyze it.
Materials and Methods
Raw material
Dioscorea alata Var purpurae (purple yam) was procured from
a fixed vendor in the local market of Mumbai. Tuber was dried in
horizontal tray dryer and was pulverized to mesh size 40. Fresh tuber
cortex was inoculated on Murashige and Skoog media to get callus by
8-10 days of inoculation.
Fresh tuber paste (5 gms), dried tuber powder (5 gms) and fresh
callus powder (0.91 gms) were treated with slightly modified method
described by Drapeau et al. [10]. Samples were hydrolyzed in 150 ml
of refluxing 20% H2SO4 in 70% isopropanol for 8 h. The extract was
filtered and extracted with hexane (50 ml×3). The three hexane extracts
were combined and rinsed thrice times with 5% alkali and then rinsed
thrice with distilled water. The extract was then passed through a
column of Na2SO4 to eliminate any remaining water. The samples
were concentrated to dryness by evaporating the solvent at 40°C in a
Rotary evaporator. The dried crude extract was solubilized in 0.5 ml
of chloroform prior to the quantitative determination of diosgenin by
Thin layer chromatography. All the chemicals were of analytical grade
from Himedia.
HPTLC analysis
Instrument used for HPTLC analysis was A CAMAG TLC system
comprising of a Linomat-5 applicator and CAMAG TLC densitometer
scanner of ANCHROM. Stationary phase used was silica gel 60 F254,
20×10 cm TLC plate obtained from Merck. Solvents Toluene (T), Ethyl
Acetate (EA), Formic Acid (FA) and Glacial Acetic Acid (GAA) were of
analytical grade from Himedia.
To begin the analysis, mobile phase T:EA:FA:GAA in the ratio
2:1:1:0.75 v/v reported by Kshirsagar et al. [9] was preferred. From
the preliminary runs we came to the conclusion that GAA is not a
significant component affecting the separation of Diosgenin. Remaining
three mobile phase components where optimized by following the runs
derived from PRISMA model (Figure 2).

*Corresponding author: Prof. S.S. Lele, Professor of Biochemical Engineering,

O Dioscin O Food Engineering and Technology Department, Institute of Chemical Technology,
Diosgenin HO
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Matunga, Mumbai, 400 019, India, Tel: +91-22-3361 1111; Fax: +91-22-3361 1020;
H O O H E-mail: shah.heena146@gmail.com, dr.smita.lele@gmail.com
H
O
H O H H
Received May 26, 2012; Accepted August 21, 2012; Published August 27, 2012
H HO OHO O
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Citation: Shah HJ, Lele SS (2012) Extraction of Diosgenin, a Bioactive Compound
H H from Natural Source Dioscorea alata Var purpurae. J Anal Bioanal Techniques
HO HO OH 3:141. doi:10.4172/2155-9872.1000141
OH
Copyright: © 2012 Shah HJ, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
Figure 1: Bioactive molecules of Dioscorea spp. use, distribution, and reproduction in any medium, provided the original author and
source are credited.

J Anal Bioanal Techniques

ISSN:2155-9872 JABT, an open access journal Volume 3 • Issue 4 • 1000141
Citation: Shah HJ, Lele SS (2012) Extraction of Diosgenin, a Bioactive Compound from Natural Source Dioscorea alata Var purpurae. J Anal Bioanal
Techniques 3:141. doi:10.4172/2155-9872.1000141

Page 2 of 3

Sr. No. Toluene E A FA Mean Rf SD Comment

FA
(0:0:10) 1 3 2 5 0.69 0.00
2 2 3 5 0.00 0.00 Smudgy
3 4 2 4 0.67 0.02
4 3 3 4 0.85 0.00
(2:0:8) (0:2:8)
5 2 4 4 0.93 0.54 Smudgy
6 4 3 3 0.74 0.00
7 3 4 3 0.92 0.01
(4:0:6) (3:1:6) (0:4:6)
8 3 1 6 0.49 0.03 Spread
(4:1:5) (3:2:5) (2:3:5) 9 4 1 5 0.46 0.04
10 5 1 4 0.43 0.01
(6:0:4) (5:1:4) (4:3:4) (3:3:4)(2:4:4) (0:6:4) 11 6 1 3 0.44 0.00
(6:1:3) (5:2:3)(4:3:3)(3:4:3) 12 5 2 3 0.59 0.00
13 6 2 2 0.59 0.00
(8:0:2) (7:1:2) (6:2:2)(5:3:2) (0:8:2) 14 7 1 2 0.43 0.01
(8:1:1) (7:2:1)(6:3:1) 15 5 3 2 0.67 0.01 Sharp
16 8 1 1 0.44 0.08
T EA 17 7 2 1 0.56 0.01 Sharp
(10:0:0) (8:2:0) (7:3:0)(6:4:0) (4:6:0) (2:8:0) (0:10:0)
18 6 3 1 0.62 0.00
19 8 2 0 0.45 0.00
Figure 2: PRISMA model for mobile phase optimization.
20 7 3 0 0.56 0.01 Sharp
21 6 4 0 0.60 0.00
Table 1: PRISMA model runs.

Track Volume applied (μl) Conc. (ng) Area (AU) X (calc., ng) Remark
1 4 200 5719
2 8 400 8060
3 12 600 9533
4 16 800 11063
5 20 1000 12745
6 24 1200 14241
7 28 1400 14727
8 32 1600 14733
9 36 1800 17186
10 5 ** 37600 36813.57 Fresh tuber
pwd
11 5 ** 64113 63326.49 Dried tuber
pwd
Figure 3: TLC plate of std Diosgenin and callus, fresh and dried tuber. 12 5 ** 4963 4176.67 Fresh callus
Table 2: Densitometer scanning results of std Diosgenin and samples.

For each run the factors kept constant were 1) Solvent front: 8 cm, Sample Diosgenin STD added Total Conc. Actual % Recovery
2) Time of run: 12-15 mins, 3) Developing reagent: Anisaldehyde in EA (ng) (ng) Conc.
and conc. H2SO4, 4) Time of development: 15 min in oven at 60°C with Fresh tuber pwd 36813.57 100 36913.57 36909.75 96.18
air circulation, 5) Densitometry scan: 430 nm. Dried tuber pwd 63326.49 100 63426.49 63425 98.51
Fresh callus 4176.67 100 4276.67 4274.38 97.71
50 μgml-1 of working standard of Diosgenin (sigma-aldrich,
Table 3: Recovery study of Diosgenin.
95% pure) prepared in chloroform was taken for the study. The
calibration curve from 200-1800 ng/spot was prepared and checked for nm after development with the anisaldehyde reagent. Figure 4 is the
reproducibility, linearity and validating the method. chromatogram of standard and callus sample. The linear regression data
Validation (n=6) showed a good linear relationship over a concentration range of
200-1800 ng/spot with regression coefficient of 0.98315 and standard
The developed method was validated in terms of linearity range, deviation of 6.28%. The calibration curve was prepared by plotting the
accuracy, limit of detection (LOD) and limit of quantification (LOQ). concentration of Diosgenin versus average area of the peak (Figure 5).
Results and Discussion The amount of Diosgenin in fresh and dried tuber as well as callus
A solvent system giving clear sharp spots with Rf value in the range was computed from calibration curve and back calculated to infer
0.45-0.65 was desired for quantification of Diosgenin (Figure 3). Table the diosgenin content to be 0.078, 0.133 and 0.048% respectively. The
1 shows that of all the mobile phase runs derived from PRISMA model, developed method was validated as per ICH guidelines [11]. In Table 3
Toluene: Ethyl acetate (7:3 v/v) gave Rf value of 0.56 ± 0.01 for standard the percentage recovery was observed in the range from 96-98%. Other
Diosgenin. Table 2 shows the densitometer scanning results at 430 parameters of the validation study were 1) linearity range: 200-1800

J Anal Bioanal Techniques

ISSN:2155-9872 JABT, an open access journal Volume 3 • Issue 4 • 1000141
Citation: Shah HJ, Lele SS (2012) Extraction of Diosgenin, a Bioactive Compound from Natural Source Dioscorea alata Var purpurae. J Anal Bioanal
Techniques 3:141. doi:10.4172/2155-9872.1000141

Page 3 of 3

3. Burkill IH (1960) The organography and the evolution of Dioscoreaceae, the

600 500 family of the Yams. J Linn Soc (Bot) 56: 319-412.
AU
AU
500 4. Zhang YQ, Liang JH, Fu EH, Li BX (2007) Effect of Modified Enzymatic
Diosgenin Std 400 Catalysis on the Extraction of Diosgenin from Dioscorea zingiberensis C. H.
400 Wright. Chem Eng Technol 30: 1488-1494.
300
5. Ravishankar GA, Grewal S (1991) A simple method for selection of high
300
diosgenin yielding clones of Dioscorea deltoidea callus. Biotechnology
200 Techniques 5: 281-282.
200
6. Sánchez GL, Medina Acevedo JC, Soto RR (1972) Spectrophotometric
Diosgenin in Callus
100 100 determination of diosgenin in Dioscorea composita following thin-layer
] ]
chromatography. Analyst 97: 973-976.
] ]
0
0.09 0.29 0.49 0.69 0.89 Rs 0
0.09 0.29 0.49 0.69 0.89 Rs
7. Cooke BK (1970) Determination of diosgenin in Dioscorea deltoidea and
Dioscorea sylvatica by using gas-liquid chromatography. Analyst 95: 95-97.
Figure 4: Chromatogram of std Diosgenin and callus.
8. Niño J, Jiménez DA, Mosquera OM, Correa YM (2007) Diosgenin quantification
by HPLC in Dioscorea polygonoides tuber collection from Colombian flora. J
Braz Chem Soc 18: 1073-1076.
20000 Diosgenin Std curve @ 430nm 9. Kshirsagar VB, Deokate UA, Bharkad VB, Khadabadi SS (2008) HPTLC method
development and validation for the simultaneous estimation of Diosgenin and
AU Levodopa in marketed formulation. Asian J. Research Chem 1: 36-39.

15000
10. Drapeau D, Sauvaire Y, Blanch HW, Wilke CR (1986) Improvement of diosgenin
yield from Dioscorea deltoidea plant cell culture by use of a non-traditional
hydrolysis method. Planta Med 6: 474-478.

11. ICH guidelines Q2A (1994) Text on Validation of Analytical Procedure.

International Conference of Harmonization, Geneva, 1-5.
10000

Y = 5268.950 + 6.698 + X
r = 0.98315
5000
sdv = 6.28%
Linear regression model

0
500 1000 1500 ng

Figure 5: Std Curve of Diosgenin (range: 200-1800 ng).

ng, 2) accuracy: ~97%, 3) limit of detection (LOD): 10 ng, 4) limit of

quantification (LOQ): 20 ng.

Conclusion
he proposed extraction and HPTLC method resulted in a simple,
faster and cost effective quantitative method for routine analysis of
Diosgenin. From validation studies we concluded that the method
is showing good performance. Dioscorea alata Var Purpurae, a lesser
explored tuber from Western Ghats of India was confirmed as a
probable source of Diosgenin. Diosgenin was quantified from the fresh
tuber, dried tuber powder and its callus. Further increase in diosgenin Submit your next manuscript and get advantages of OMICS
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References • 200 Open Access Journals

1. Oncina R, Botia JM, Del Rio JA, Ortuno A (2000) Bioproduction of diosgenin in
callus cultures of Trigonella foenum-graecum L. Food Chemistry 70: 489-492.
2. Olayemi JO, Ajaiyeoba EO (2007) Anti-inflammatory studies of yam (Dioscorea
esculenta) extract on wistar rats. African Journal of Biotechnology 6: 1913-
1915.
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J Anal Bioanal Techniques

ISSN:2155-9872 JABT, an open access journal Volume 3 • Issue 4 • 1000141