Pharmacognosy Magazine

ISSN: 0973-
0973-1296 PHCOG MAG.
Vol 4, Issue 16 (Suppl.), Oct-
Oct-Dec, 2008 An official Publication of Phcog.Net

PHCOG MAG.:Research Article

Evaluation of antioxidant phytochemical diversity in
Hedychium spicatum: a high value medicinal plant of
Himalaya
Indra D. Bhatt*, Kundan Prasad, Sandeep Rawat and Ranbeer S. Rawal
G. B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal Almora-263643.
*Author for correspondence: idbhatt@gbpihed.nic.in, id_bhatt@yahoo.com

ABSTRACT
While screening Hedychium spicatum in search of natural compounds, for the first time High Performance Liquid
Chromatography (HPLC) method for the determination of, -carotene, -carotene, xanthophyll and DL--
tocopherol was developed in the said species. All the three compounds were extracted in light petroleum
ether/methanol/ethyl acetate (1:1:1 ratio) and separated by phase separation method. Chromatogram of
carotenoids and DL--tocopherol showed characteristic absorbance at 450 nm and 291 nm respectively. This
method was successfully applied for the analysis of carotenoids and DL--tocopherol of H. spicatum rhizome
obtained from two different sources. Analysis of rhizome samples showed that phenolics and DL--tocopherol
contents was significantly (P<0.01) higher in one year old planted rhizomes as compared to the ones collected
from the wild.
KEY WORDS: Antioxidant, -carotene, -carotene, DL--tocopherol, HPLC, Hedychium spicatum, xanthophyll

INTRODUCTION
Recent researches on natural compounds have
highlighted the possibility of medicinal plants as a
source of functional food or food supplements (1).
Among other natural compounds, phenolics,
carotenoids and vitamins have found special place in
food by playing an important role as health protecting
factor and reducing the risk of oxidative stress (2-4).
Keeping the above in view, researchers are in
continuous search of new sources of such compounds
amongst wild plants (5).
Hedychium spicatum Ham. ex Sm. (Zingiberaceae),
commonly known as Kapurkachri, is a perennial
rhizomatous herb distributed in the Himalaya between
altitude of 800-3000 m asl. The species is frequently
used in Ayurvedic and Unani medicine (6). The rhizome
is considered as stomachic, carminative, stimulant and
emmenagogue and useful for liver complaints,
diarrhoea, food poisoning and various other diseases
(7). The crude extract of rhizome is used as a major
ingredient in making syrup and tablets with brand
name Vomicure and Vominil respectively and are being
sold in National and International market at the rate of
$ 40/bottle (www.suryaherbal.com). Recently the
crude extract of the rhizome has been used in the
preparation an anticancerous drug, PADMA 28 (8)
Considering the importance of species in traditional as
well as modern medicine, the present study was
undertaken to: (i) develop HPLC method for
determination of carotene, xanthophylls and
tocopherol, and (ii) assess range of variation in these
chemicals between wild and planted source. To the
best of our knowledge no reports are available on the
determination of total phenolics, xanthophyll, -
carotene, -carotene, DL -tocopherol in this species.
MATERIAL AND METHODS
Chemicals
Standard of xanthophyll, -carotene, -carotene and
DL--tocopherol were procured from Sigma Chemical
Co. St Louis, USA. Individual standard was accurately
weighed, developed and diluted with HPLC grade
ethanol. Petroleum ether, methanol, ethyl acetate and
anhydrous sodium sulphate and other chemicals and
reagents used in this study were purchased form Merck
Chemical Co. Mumbai, India.
Plant Material
Rhizomes of H. spicatum were collected from Kosi-
Katarmal, Almora forest (lat. 29 38 long. 79 36) at
an elevation of 1200 m asl and planted in the herbal
garden (year 2006) of G.B. Pant Institute of Himalayan
Environment and Development Kosi-Katarmal, Almora.

S 202
Pharmacognosy Magazine
ISSN: 0973-
0973-1296
Vol 4, Issue 16 (Suppl.), Oct-
Oct-Dec, 2008
Rhizomes were irrigated every alternate day and
harvested after one year of planting at senescence
stage. These rhizomes were regarded as planted
material. At the same time rhizomes were also
collected from the wild (Kosi - Katarmal forest) for
comparing the results and the material was termed as
wild. Three rhizomes in each case (planted and wild)
were processed for analysis.
Total phenolic content
The rhizomes of each source (wild and planted) were
dried in shade and powdered using electrical grinder
(Philip-HL1616). The amount of total phenolic content
was estimated following Singleton et. al. (9) with
minor modification. The reaction mixture contained
100 l of sample extract, 500 l Folins-Ciocalteus
reagent (freshly prepared), 2 ml of 20% Sodium
Carbonate and 5 ml of distilled water. After 15 min
reaction at 45 ,C the absorbance at 650 nm was
measured using spectrophotometer (HITACHI, Model U
2001). Results were expressed as mg of catechol
equivalent per 100 g of dry weight.
Extraction and Isolation of carotenoids and
tocopherol
Dried plant material (1.0 g of each) was extracted with
light petroleum ether/methanol/ethyl acetate (1:1:1,
V/V/V, 4 x 30 ml) until the extracts became colorless.
The extract was mixed in a 250 ml separating funnel,
shaken vigorously and allowed to stand for phase
separation. Upper layer was collected in a 100 ml flask
(Borosil India Co. Ltd.) and lower layer was shaken
with 50 ml water and 50 ml petroleum ether for phase
separation. Upper layer was mixed with the first
extract. The organic extract was dried over anhydrous
sodium sulphate (10 g), filtered and evaporated to
dryness in a Rotary Vacuum Evaporator (STEREOGLASS
SRL, Italy, Model 102/202) under reduced pressure.
The residue was dissolved in light petroleum ether (5
ml) and filtered by 0.2 m membrane filter prior to
HPLC analysis.
HPLC Analysis
All the samples were analyzed using MERCK HITACHI
HPLC system (Model L 7100, UV detector L-7400).
Column used was C18 phenomenex(R) (5 , 150 x 4.6 mm
analytical column) with solvent system 8:2:40:50
(methanol, ethyl acetate, acetonitrile and acetone),
flow rate 0.7 ml/min, run time 20 minutes and
detector wavelength was 450 nm. The HPLC condition
for the estimation DL--tocopherol was adopted as
described in Kurilich et al. (10).
Statistical analysis
PHCOG MAG.
An official Publication of Phcog.Net
Three replicates of each sample were used for
statistical analysis. Data obtained for each parameter
were analysed statistically using computer package
SYSTAT (11). Significant difference between means
(p<0.05) was tested using t test (12).
RESULTS AND DISCUSSION
The present study characterized and quantified the
total phenols, carotenoids and vitamin content in H.
spicatum. The data obtained for total phenolic content
varied considerably between two sources (Table 1).
The phenolic content (218 mg/100g) was found
significantly (p<0.05) higher in one year old planted
source as compared to wild ones (181.8 mg/100g). The
available percentage of compounds i.e. xanthophyll, -
carotene, -carotene and DL--tocopherol content is
presented (Table 1). The retention time of
xanthophyll, -carotene and -carotene were found to
be 2.045, 10.947 and 11.495 minute respectively (Fig.
1A&B). The retention time of DL--tocopherol was
found 11.780 minute. The content varied significantly
between two sources. While comparing with planted
rhizomes, xanthophyll, -carotene, -carotene content
were found significantly (P<0.01 and P<0.001) more in
the rhizomes of wild source. On the contrary, DL--
tocopherol was significantly (P<0.01) more in planted
source (Table 1).
Phenolics, carotenoids and vitamins are well known for
its antioxidant activity (13-14) and repeatedly been
used as natural antioxidants in fruits, vegetables and
other plants. For example, caffeic acid, ferulic acid,
and vanillic acid are widely distributed in the plant
kingdom (15). Rosamarinic acid, an important
phytochemical has been found to be a potent active
substances against human immunodeficiency virus
type1 (HIV-1) (16). Carotenoids are a group of natural
pigments, widely acceptable to consumers being
present in natural foods and are readily metabolized.
The hydrocarbon carotenoids have provitamin-A
activity and the oxygenated carotenoids or xanthophyll
are possibly linked to a lower risk of cancer (17).
Studies have shown that carotenoids may play an
important role in the prevention of age related
macular degeneration (AMD) (18). -Carotene has
proved to prevent peroxidation caused by singlet
oxygen and also by scavenging free radicals. Likewise,
-Tocopherol is known to have a number of biological
activities such as immune stimulation, inhibition of
nitrosamine formation and alteration of metabolic
activation of carcinogens (19). The major protective
function of the vitamins against cancer is the
S 203
Pharmacognosy Magazine
ISSN: 0973-
0973-1296 PHCOG MAG.
Vol 4, Issue 16 (Suppl.), Oct-
Oct-Dec, 2008 An official Publication of Phcog.Net

scavenging of reactive oxygen metabolites (ROMs) (20). Recent reports highlighted the role of -tocopherol in
Table 1. Carotenoids and vitamin content in Hedychium spicatum in planted and wild source.
Antioxidants Planted Wild Level of significance (t-test)
Total phenolics (mg/100g) 218 0.01 181.8 0.02 *
Xanthophyll (mg/100g) 0.23 0 1.65 0.1 **
-Carotene (mg/100g) 6.9 0.8 20.5 1.2 **
-Carotene (mg/100g) 19.3 2.4 61.8 2.8 ***
DL--tocopherol (mg/100g) 4.9 0.2 1.1 0.2 **
Level of significance p< *0.05, **0.01, ***0.001

Detector A (450nm)
Van-
Van-h-W-7-R1
Van-
Van-h-W-7-R1001

0.020 0.020

0.015 0.015

Volts Volts

0.010 0.010

1
0.005 0.005
3
2
0.000 0.000

0 2 4 6
Minutes 8 10 12 14

Figure 1A

Detector A (450nm)
sample8R1
sample8R1001
0.0125 0.0125

0.0100 0.0100

0.0075 0.0075

Volts Volts

0.0050 0.0050

1
0.0025
3 0.0025
2

0.0000 0.0000
0 2 4 6
Minutes 8 10 12 14

Figure 1B
Figure 1 : HPLC Chromatogram of H. spicatum 1- xanthophyll, 2- -carotene and 3- -carotene A- Wild, B- planted

S 204
Pharmacognosy Magazine
ISSN: 0973-
0973-1296
Vol 4, Issue 16 (Suppl.), Oct-
Oct-Dec, 2008
lowering the lipid oxidation in human body and
counteract the prooxidative effect with other
compound like ascorbate and combination of ascorbate
and -carotene (21).
The results of the present study are indicative that
mass scale plantation of the species may be adopted
for obtaining higher total phenols and -tocopherol
content. However, more details study is needed to
arrive at any conclusion. Studies on planted material of
the species for long time and periodic assessment for
these chemicals is urgently required prior to suggest
any options for alternate method of using synthetic
antioxidant compounds. This is a preliminary report on
the quantitative analysis of antioxidant content of the
species and perhaps can promote interest of
researchers on new sources of these compounds.
Moreover, the results of the present study may interest
pharmaceutical companies using rhizome in much
larger quantities for preparation of different drugs.
ACKNOWLEDGEMENT
Authors thanks to the Director of the Institute for
providing facilities and encouragement. Uttarakhand
Council for Science and Technology is greatly
acknowledged for funding the project (File No. UCS &
T/R&D/LS-21/06/775).
REFERENCES
1. I. Raskin, D.M. Ribnicky, S. Komarnytsky, N. Ilic, A. Poulev,
N. Borisjuk, A. Brinker, D.A. Moreno, C. Ripoll, N. Yakoby,
J.M. ONeal, T. Cornwell, I. Pastor, B. Fridlender. Plants and
human health in twenty first centaury. Trends in Biotechnology,
20(12): 522-531 (2002).
2. M. Kimura, D.B. Rodriguez-Amaya. A scheme for obtaining
standards and HPLC quantification of leafy carotenoids. Food
Chemistry 78:389-398 (2002).
3. P. Pedrelli, L.H. Skibsted. Antioxidant synergy and regeneration
effect of quercetin, (-)epicatechin and (+)catechin on -
tocopherol in homogenous solutions of peroxidating methyl
linoleate. J. Agr. Food Chem, 50:7138-7144 (2002).
4. J. Javanmardi, C. Stushnoff, E. Locke, J.M. Vivanco.
Antioxidant activity and total phenolic contents of Iranian
Ocimum accessions. Food Chemistry 83:547-550 (2003).
5. A.Z. Mercadante, D.B. Rodrigue-Amaya. Carotenoids
composition of a leafy vegetable in relation to some agriculture
variables. J. Agr. Food Chem., 39:1094-1097 (1991).
6. N.C. Shah. Need of systematic cultivation of medicinal herbs
used in indigenous systems traditional medicine. Indian drugs,
18(6): 210-217 (1981).
PHCOG MAG.
An official Publication of Phcog.Net
7. K.R. Kritikar, B.D. Basu. Indian Medicinal Plants. Vol I-II,
M/S Periodical Experts, New Dehli, (1988).
8. R. Nayab, H. Aingorn, L. Fallavollita, S. Sallon, R. Mechoulam,
I. Ginsburg, I. Vlodavsky, P. Brodt. PADMA-28, a traditional
Tibetan herbal preparation, blocks cellular responses to bFGF
and IGF-I. Inflammopharmacology, 12(4): 373-89 (2004).
9. V.L. Singleton, R. Orthofer, R.M. Lamula Raventos. Analysis
of total Phenols and other oxidation substrates and antioxidant
by means of folins ciocalteau reagent. Methods Enzymol.,
299:152-178 (1999).
10. A.C. Kurilich, G.J. Tsau, A. Brown, L. Howard, B.P. Klein,
E.H. Jeffery, M. Kushad, M.A. Walig, J.A. Javik. Carotene,
tocopherol and /ascorbate content in subspecies of Brassica
oleracea. J. Agr. Food Chem., 47:1576-1581 (1999).
11. L. Wilkinson. SYSTAT: A System for statististics. Systat-Inc.;
Evaston II (1986).
12. G.W. Snedechor, W.G. Cochran. Statistical methods. Oxfords
and IBH Publishing Co. Pvt. Ltd.; New Delhi, (1968).
13. M.P. Kahkonen, A.I. Hopia, H.J. Vuorela, J.P. Rauha, K.
Pihlaja, T.S. Kujala, M. Heinonen. Antioxidant activity of plant
extracts containing phenolic compounds. J. Agr. Food Chem.,
47(10): 39543962 (1999).
14. J. Javanmardi, A. Khalighi, A. Kashi, H.P. Bais, J.M. Vivanio,.
Chemical characterization of basil (Ocimum basiliam L.) found
in local accessions and used in traditional medicines in Iran. J.
Agr. Food Chem., 50: 58785883 (2002).
15. R.A. Larson. The antioxidants of higher plants. Phytochemistry,
27: 969978 (1988).
16. A. Mazumder, N. Neamati, S. Sunder, J. Schulz, H. Pertz, E.
Eich, V. Pommier. Curcumin analogues with altered potencies
against hiv-1 integrase as probes for biochemical mechanisms
of drug action. J. Med. Chem., 40: 30573063 (1997).
17. G. R. Beecher, F. Khachik. Evaluation of vitamin A and
carotenoid data in food composition tables. J. Natl. Cancer Inst.
73: 1397-1404 (1984).
18. J.T. Landrum, R.A. Bone, H. Joa, M.D. Kilburn, L.L. Moore,
K.E. Sprague. A one year study of the macular pigment: the
effect of 140 days of a lutein supplement. Exp Eye Res., 65 (1):
57-62 (1997).
19. Y. Sun. Free Radicals, antioxidant enzymes and carcinogenesis.
Free Radical Biol. Med., 8: 583-587 (1990)..
20. R.S. London, L. Murphy, K.E. Kitlowski. Breast cancer
prevention by supplemental vitamin E. J Am Coll. Nutr., 4
(5):559-64 (1985).
21. L.H. Skibsted, U.C. Charlotte, V.K.O. Maiken, V.H. Rikke,
B.M. Bibby, K. Dorthe. Antioxidant and prooxidant in milk like
emulsions: Effect of ascorbate, urate, -tocopherol and -
carotenoids on early events in lipid oxidation.
Milchwissenschaft, 60(1): 44-48 (2005).
S 205