Preliminarily Investigation on Antioxidant Phytochemical in Some Medicinal Plants of Kumaon Region
While screening Asparagus adscendens Roxb, Vitex negundo Linn. and Stephania glabra Miers in search of natural antioxidant, we first time developed HPLC method for the determination of α-carotene, β-carotene, xanthophyll and DL-α-tocopherol in the said spices. These antioxidants were extracted in light petroleum ether/methanol/ethyl acetate (1:1:1 ratio) and separated by phase separation method. Chromatogram of carotenoids and DL-α-tocopherol showed characteristic absorbance at 450 and 291 nm, respectively. This method was successfully applied to the analysis of antioxidants of these plants. The retention time of xanthophyll, α-carotene and β-carotene were found 2.045, 10.947 and 11.495 min, respectively. The retention time of DL-α-tocopherol was found 11.780 min. The maximum content of xanhophyll (8.08±0.93 mg/100 g), α-carotene (3.89±0.53 mg/100 g), β-carotene (243.53±0.66 mg/100 g) and total phenolics (324.55±0.47 mg/100 g) was found in V. negundo under the three investigated plants. The content of α-tocopherol (11.32±0.19 mg/100 g) and vitamin-c (311.45±0.47 mg/100 g) were maximum in A. adscendens. In the present study, the plants are found good source of antioxidants.
to cite this article:
Kundan Prasad, Kalpana Moulekhi and G. Bisht, 2014. Preliminarily Investigation on Antioxidant Phytochemical in Some Medicinal Plants of Kumaon Region. Research Journal of Phytochemistry, 8: 199-204.
Received: July 30, 2014;
Accepted: August 18, 2014;
Published: November 13, 2014
Asparagus adscendens Roxb (Liliaceae), occurs throughout the sub Himalayan
tract, the central and outer hill range up to 5000 feet. Roots have cooling,
demulcent and diaphoretic property. The herb is a nutritive tonic useful in
general debility, leucorrhea, sexual debility and spermatorrhea. This herb is
also used during pregnancy and postpartum. It helps to nourish the foetus and
increase the breast milk flow. Dried roots of A. adscendens Roxb are
used as drugs (Osmaston, 1976). Stephania glabra Miers.
(Menispermacae) occurs throughout the hills between 3000-6000 feet. Tubers are
used in pulmonary tuberculosis, asthma and intestinal complaints (Osmaston,
Vitex negundo Linn. (family Verbenaceae) commonly known as Nirgundi
or Shiwali occurs throughout the greater part of India up to an altitude of
1500 m in the outer western Himalayas (Anonymous, 1976;
Chopra et al., 1956). The leaves are green in
upper surface. Flowers are bluish purple, fruits are black when ripe. The plant
can grow on nutritionally poor soil (Prasad and Wahi, 1965).
Leaves are aromatic, bitter, acrid, astringent, anti-inflammatory, antipyretic
or febrifuge, tranquillizer, antihelmintic and vermifuge. Flowers are cool,
astringent, carminative, hepatoprotective, digestive, febrifuge, vermifuge and
are useful in haemorrhage and cardiac disorders. Fruits are nervine, cephalic,
aphrodisiac and vermifuge (Chopra et al., 1956).
Considering the importance of these plants in the traditional medicine and
pharmaceutical industries, it is important to screen the species for its antioxidant
contents. To the best of our knowledge on reference are available on the determination
of xanthophyll, α-carotene, β-carotene, α-tocopherol. The objective
of the present study is to provide database on HPLC estimation regarding the
natural compounds and their quantification.
MATERIALS AND METHODS
Chemical and reagents: Standard of xanthophyll, α-carotene, β-carotene
and DL-α-tocopherol were purchase from Sigma and each individual standard
were accurately weighted, developed and diluted with HPLC grade ethanol. Petroleum
ether, methanol, ethyl acetate and anhydrous sodium sulphate other chemicals
and reagents purchase form Merck.
Plant material: Asparagus adscendens Roxb, Vitex negundo
Linn and Stephania glabra Miers were collected in October 2006 from Harara
(Distt. Almora, Uttarakhand) identified by Prof. Y.P.S. Pangtey, Department
of Botany, Kumaun University, Nainital and also from Dr. H.C. Pandey, Botanical
Survey of India, Dehradun and herbarium deposited at phytochemistry lab D.S.B.
Campus, K.U. Nainital.
Isolation and extraction of carotenoids and tocopherol: The rhizomes
root and leaves of each were dried in shade and powdered using electrical grinder.
Dried plant material (1.0 g of each) was extracted with light petroleum ether/methanol/ethyl
acetate (1:1:1, V/V/V, 4x30 mL) until the extracts were colorless. The extracts
were mixed in a 250 mL separating funnel and shaken vigorously and allowed to
stand for phase separation. Upper layer was collected and lower layer was shaken
with 50 mL water and 50 mL petroleum ether for phase separation. Upper layers
were mixed with the first extracts. The organic extract was dried over anhydrous
sodium sulphate (10 g), filtered and evaporated to dryness in a Rotary Vacuum
Evaporator under reduced pressure. The residue was dissolved in light petroleum
ether (5 mL) and filtered by 0.2 μm membrane filter before HPLC analysis
(Bernstein et al., 2001).
HPLC analysis: The samples were analyzed by Shimadzu HPLC system, column
used was C18 phenomenex(R) (5 μ, 150x4.6 mm analytical
column) with solvent system 8:2:40:50 (methanol, ethyl acetate, acetonitrile
and acetone) flow rate 0.7 mL min-1, run time 20 min and detector
wavelength was 450 nm. The HPLC condition of the estimation DL-α-tocopherol
by as described by Kurilich et al. (1999) methods
at 291 nm.
Total phenolic compound analysis: The rhizomes root and leaves of each
were dried in shade and powdered using electrical grinder. The amount of total
phenolic content was estimated following Singleton et
al. (1999) with minor modification. The reaction mixture contained 100
dL of sample extract, 500 μL 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.
Vitamin-C (ascorbic acid) analysis: Ascorbic acid content was estimated
by (Ranganna, 1976) method with little modification. Dried
leaves powder (2.0 g) was extracted with 4% oxalic acid, made upto 100 mL and
centrifuged at 10,000 rpm for 10 min. Five milliliter supernatant liquid was
transferred in a conical flask, 10 mL of 4% oxalic acid was added and finally
titrated against standard dye solution (2, 6-dichlorophenol indophenol). The
procedure was repeated with a blank solution omitting the sample. Five milliliter
ascorbic acid with concentration (100 ppm) was used as standard.
RESULTS AND DISCUSSION
The aim of this study was to characterize the antioxidant value of these medicinal
plants with particular attention to carotenoids, vitamins and phenolics. In
this study, we have observed that xanthophyll, α-carotene, β-carotene
and DL-α-tocopherol contents are present in these plants. The composition
of these compounds is presented in Table 1. The retention
time of xanthophyll, α-carotene and β-carotene were found 2.045, 10.947
and 11.495 min, respectively. The retention time of DL-α-tocopherol was
found 11.780 min. The available percentage of compounds i.e., xanthophyll, α-carotene,
β-carotene and DL-a-tocopherol content is presented (Table
1). The chromatographic separation of xanthophylls, β-carotene, α-carotene
and DL-α-tocopherol standard is presented in Fig. 1-3.
In the present study we have observed that β-carotene in root Asparagus
adscendens Roxb were 15.47-15.54 mg/100 g and xanthophylls in roots varied
from 0.09-0.11 mg/100 g on dry weight basis. The range of α-tocopherol
was 11.19-11.46 mg/100 g, total phenolics were 234.12-234.9 and vitamin C was
310.63-311.81 mg/100 g but α-carotene was not determined in the roots of
plants. The Vitex negundo Linn. leaves contain the range of β-carotene
was 243.51-243.96 mg/100 g, α-carotene was 3.52-4.27 and xanthophyll was
8.08-8.73 mg/100 g on the dry weight basis. The range α-tocopherol were
9.84-10.31 mg/100 g, total phenolics were 324.21-324.88 mg/100 g and vitamin
C was 264.99±0.58 mg/100 g on dry weight basis. 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 xanthophylls are possibly linked
to a lower risk of cancer (Beecher and Khachik, 1984).
Studies have shown that carotenoids may play an important role in the prevention
of age related macular degeneration (AMD) (Landrum et
al., 1997). β-Carotene has proved to prevent peroxidation caused
by singlet oxygen and also by scavenging free radicals.
||Antioxidant content in the selected medicinal plants of Kunaon
|All values are mean of triplicate determinations expressed
on dry weight basis, ± is the standard error
||Chromatogram of standard peak of xanthophyll
||Chromatogram of standard peak of α-carotene and β-carotene
The rhizomes of Stephania glabra were contain 1.33-2.00 mg/100 g α-carotene,
15.51-15.54 mg/100 g β-carotene and 3.33-3.38 mg/100 g xanthophyll on dry
weight basis. The range of α-tocopherol in Stephania glabra were
10.09-10.98 mg/100 g on dry weight basis. Vitamin C in this plant was 264.99±0.58
mg/100 g and total phenolics were 183.19-183.95 mg/100 g on dry weight basis.
The result shows that this plant is very powerful antioxidant.
||Chromatogram of standard peak of DL-α-tocopherol
The maximum content of xanhophyll (8.08±0.93 mg/100 g), α-carotene
(3.89±0.53 mg/100 g), β-carotene (243.53±0.66 mg/100 g) and
total phenolics (324.55±0.47 mg/100 g) were found in V. negundo
under the three investigated plants. Phenolics, carotenoids and vitamins are
well known for its antioxidant activity (Kahkonen et
al., 1999; Javanmardi et al., 2002) 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 (Larson, 1988). Rosamarinic
acid, an important phytochemical has been found to be a potent active substances
against Human Immunodeficiency Virus type1 (HIV-1) (Mazumder
et al., 1997). The content of α-tocopherol (11.32±0.19
mg/100 g) and Vitamin C (311.45±0.47 mg/100 g) were maximum in A.
adscendens. In the present study, the plants are found good source of antioxidants.
Likewise, α-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 (Sun, 1990). The
major protective function of the vitamins against cancer is the lowering the
lipid oxidation in human body and counteract the prooxidative effect with other
compound like ascorbate and combination of ascorbate and β-carotene (Skibsted
et al., 2005).
This study concludes that the rhizomes and leaves of each are excellent source
of natural antioxidants. There is a great need to further study in which the
plants rhizomes and leaves easily available in the local markets for preparation
Authors are thankful to Dr Jagdish Singh, Head, Department of Biochemistry,
I.I.V.R., Varanasi for HPLC analysis.
Wealth of India-Raw Materials. Vol. 10, Council of Scientific and Industrial Research, New Delhi, India, Pages: 577
Beecher, G.R. and F. Khachik, 1984.
Evaluation of vitamin A and carotenoid data in food composition tables. J. Natl. Cancer Inst., 73: 1397-1404.PubMed |
Bernstein, P.S., F. Khachik, L.S. Carvalho, G.J. Muir, D.Y. Zhao and N.B. Katz, 2001.
Identification and quantitation of carotenoids and their metabolites in the tissues of the human eye. Exp. Eye Res., 72: 215-223.CrossRef | PubMed | Direct Link |
Chopra, R.N., S.L. Nayar and I.C. Chopra, 1956.
Glossary of Indian Medicinal Plants. CSIR, New Delhi, India, ISBN-13: 9788172360481, pp: 256-257
Javanmardi, J., A. Khalighi, A. Kashi, H.P. Bais and J.M. Vivanco, 2002.
Chemical characterization of basil (Ocimum basilicum
L.) found in local accessions and used in traditional medicines in Iran. J. Agric. Food. Chem., 50: 5878-5883.CrossRef | PubMed | Direct Link |
Kahkonen, M.P., A.I. Hopia, H.J. Vuorela, J.P. Rauha, K. Pihlaja, T.S. Kujala and M. Heinonen, 1999.
Antioxidant activity of plant extracts containing phenolic compounds. J. Agric. Food Chem., 47: 3954-3962.CrossRef | PubMed | Direct Link |
Kurilich, A.C., G.J. Tsau, A. Brown, L. Howard and B.P. Klein et al
Carotene, tocopherol and ascorbate contents in subspecies of Brassica oleracea
. J. Agric. Food Chem., 47: 1576-1581.CrossRef | PubMed | Direct Link |
Landrum, J.T., R.A. Bone, H. Joa, M.D. Kilburn, L.L. Moore and K.E. Sprague, 1997.
A one year study of the macular pigment: The effect of 140 days of a lutein supplement. Exp. Eye Res., 65: 57-62.CrossRef | Direct Link |
Larson, R.A., 1988.
The antioxidants of higher plants. Phytochemistry, 27: 969-978.CrossRef | Direct Link |
Mazumder, A., N. Neamati, S. Sunder, J. Schulz, H. Pertz, E. Eich and Y. Pommier, 1997.
Curcumin analogs with altered potencies against HIV-1 integrase as probes for biochemical mechanisms of drug action. J. Med. Chem., 40: 3057-3063.CrossRef | Direct Link |
Osmaston, A.E., 1976.
Forest Flora for Kumaon. International Book Distributors, New Delhi, India, ISBN-13: 9780785531265, pp: 540-541
Prasad, S. and S.P. Wahi, 1965.
Pharmacognostic study of leaf of Vitex negundo
Linn. (Nirgundi). J. Res. Indian Med., 72: 208-211.
Ranganna, S., 1976.
Handbook of Analysis of Quality Control for Fruits and Vegetable Product. 2nd Edn., Tata McGraw Hill Publication Company, New Delhi, India, Pages: 545
Singleton, V.L., R. Orthofer and R.M. Lamuela-Raventos, 1999.
Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Methods Enzymol., 299: 152-178.CrossRef | Direct Link |
Skibsted, L.H., U.C. Charlotte, V.K.O. Maiken, V.H. Rikke, B.M. Bibby and K. Dorthe, 2005.
Antioxidant and prooxidant in milk like emulsions: Effect of ascorbate, urate, tocopherol and carotenoids on early events in lipid oxidation. Milchwissenschaft, 60: 44-48.
Sun, Y., 1990.
Free radicals, antioxidant enzymes and carcinogenesis. Free Radical Biol. Med., 8: 583-599.CrossRef | Direct Link |