Abstract
Context:
D. macrostachyum is an epiphytic orchid abundant in Southern India and is reported for pain relief in folklore.
Aims:
The objective of the present study was to determine in vitro free radical scavenging and anti-inflammatory activity of D. macrostachyum and to perform LCMS based metabolic profiling of the plant.
Settings and Design:
Sequential stem and leaf extracts were assessed for its antioxidant and anti-inflammatory activity by in vitro methods.
Materials and Methods:
The
antioxidant activity determined by assays based on the decolourization
of the radical monocation of DPPH, ABTS and reducing power. Total amount
of phenolics for quantitative analysis of antioxidative components was
estimated. In vitro anti-inflammatory activity was evaluated
using protein denaturation assay, membrane stabilization assay and
proteinase inhibitory activity. Methanolic extract of plant was
subjected to LCMS.
Results:
The stem ethanolic extracts exhibited significant IC50
value of 10.21, 31.54 and 142.97 μg/ml respectively for DPPH, ABTS
radical scavenging and reducing power activity. The ethanol and water
extract was highly effective as albumin denaturation inhibitors (IC50 = 114.13 and 135.818 μg/ml respectively) and proteinase inhibitors (IC50
= 72.49 and 129.681 μg/ml respectively). Membrane stabilization was
also noticeably inhibited by the stem ethanolic extract among other
extracts (IC50 = 89.33 μg/ml) but comparatively lower to aspirin standard (IC50
= 83.926 μg/ml). The highest total phenol content was exhibited by
ethanolic stem and leaf extracts respectively at 20 and 16 mg of gallic
acid equivalents of dry extract. On LCMS analysis 20 constituents were
identified and it included chemotaxonomic marker for Dendrobium species.
Conclusions:
The
results showed a relatively high concentration of phenolics, high
scavenger activity and high anti-inflammatory activity of the stem
extract compared to the leaf extract. The results indicate that the
plant can be a potential source of bioactive compounds.
KEYWORDS: Anti-inflammatoryactivity, antioxidant activity, dendrobium, Dendrobium macrostachyum, general unknown screening, in vitro bioassays, marathilotti, orchidaceae, radam, reducing power, total phenol
INTRODUCTION
The
genus Dendrobium with 1190 species is one of the most important genera
in the orchid family. Orchidaceae, as being the source of wide variety
of biologically active compounds are used extensively as crude material
or pure compounds for treating various disease conditions.[1] Among them, Dendrobium macrostachyum Lindl (Common name: Radam/Marathilotti)
is one of the widespread Dendrobium orchid species found in the plains
of Southern India. It is an epiphytic herb, stem tufted, leaves
membranous and deciduous during flowering season with small green
flowers and narrow petals.[2,3,4] Presence of alkaloids, leaf flavonoids, sterols, glycosides, tannins and phenols were reported in a study.[4] A detailed study on ethylacetate fraction of ethanolic stem extract was found to possess good anti-lipoxygenase activity in-vitro.[5] Traditional use of tender tip juice for earache is reported.[6] Rural folk of Northern Andaman islands use whole plant to relieve pain.[7]
The
utilization of liquid chromatography coupled to single (LC – MS) and
tandem (LC – MS/MS) mass spectrometry has grown rapidly and is now
widely recognized as powerful tool for a comprehensive General Unknown
Screening (GUS) of organic molecules in a multiple component mixture.[8] In the present investigation, an effort is made to phytochemically profile Dendrobium macrostachyum Lindl. using these advanced techniques. The determination of these characters along with their in-vitro
anti-oxidant and anti-inflammatory activity screening will help
establish the pharmaceutical values of this pharmacologically unscreened
plant in future studies.
MATERIALS AND METHODS
Collection and authentication of plant
The
plant materials collected at the flowering state in June from Melattur
(North Kerala, India), was identified and a voucher specimen (5/23/2011 –
12/Tech. 785) was deposited in the Herbarium, Southern Regional Centre,
Botanical Survey of India.
Preparation of extract and bioactivity studies
The
fresh plant materials were collected, washed, stems and leaves
separated, blended into small fragments and shade dried. The dried plant
materials were disintegrated using mixture grinder, powdered well and
then powder was passed through sieve No: 60 and stored. Dried ground
stem and leaves (50 g) were extracted in Soxhlet extractor sequentially
in 150 ml using increasing polarity (Petroleum Ether, Ethanol, Methanol,
Water) to give eight extracts of the same nature (Solid). The extract
yields were noted and tabulated [Table 1].
Total phenolic contents (TPC) were determined spectrophotometrically
using Folin – Ciocalteu reagent and results were expressed as (+)
milligrams of gallic acid equivalents (GAE).[9]
Total phenolic content and yields of Dendrobium macrostachyum extracts
Antioxidant activity was determined by spectrophotometric methods based on the reduction of an ethanol solution of DPPH[10] and by measuring ABTS+ radical cation formation induced by metmyoglobin and hydrogen peroxide.[11]
We also used FRAP assay which uses anti-oxidants as reductants in a
redox linked colorimetric method to test the total antioxidant power
directly.[12]
In-vitro
anti-inflammatory activity was evaluated using inhibition of protein
denaturation which was calculated with bovine albumin fraction after
heating (37°C for 20 min and then at 57°C for 20 min) and read at 600
nm.[13] Red blood cell membrane stabilization method (RBC method)[14] and anti-proteinase activity[15] at different concentrations, was also used for the confirmation of anti-inflammatory activity in vitro.
Ascorbic
acid was used as positive control for free radical scavenging and
antioxidant activities whereas aspirin for anti-inflammatory activity
studies. The tests were done in triplicates for each sample and the
means were used to determine the percentage radical scavenging activity
by (A – B/A) ×100, where A is the activity without test material and B
is the activity with test material. The activities studied were
expressed as IC50.
Sample and Buffer preparation for LC – MS
Extract
was reconstituted in 1 ml of a 10% methanol solution. Standards were
prepared by spiking stock solutions of drug mixtures to make
concentrations ranging from 0.500 to 5 mg/ml, resulting in a set of
standards with the following concentrations: 0.500, 1.000, 1.500, 2.000
and 5.000 mg/ml. A buffer (pH 2.2) was prepared with a 20 mmol/l K2 HPO4 solution.
Chromatographic and mass spectral conditions
Extract
was subjected to chromatographic separation on Phenomenex Reverse phase
18 column (dimension 25 cm × 2.5 cm) and operated at a column
temperature of 25°C with a flow rate of 2 ml/min. Injection volume was
10 µl. Electronic spray ionization (ESI) mode was used with m/z range of
50–1000 for negative and 50–980 for positive. Class VP integrated
software was used and identification of isolated compounds was based on
the comparison of the mass spectral data with METWIN 2.0.
RESULTS AND DISCUSSION
The
amounts of TPC were found to vary over a wide range (3 to 20 mg/g) with
it being higher in stem extract as compared to leaf extract [Table 1]. The phenolic substances are known to possess the ability to reduce oxidative damage and acts as antioxidants.[16]
The
results exhibited a relatively high concentration of antioxidants, high
scavenger activity and high anti-inflammatory activity in stem compared
to leaves. On comparing various stem extracts, ethanolic extract was a
more effective radical scavenger and anti-inflammatory as compared to
petroleum ether, methanol, water extracts and positive standards
(ascorbic acid and aspirin respectively). IC50 (µg/ml) values
of the scavenging and anti-inflammatory activity were determined from
the concentration–effect regression lines is tabulated in Tables Tables22 and and33.
Antioxidant activities of Dendrobium macrostachyum extracts
Anti-inflammatory activity of Dendrobium macrostachyum extracts at IC50
GUS analysis for exploring metabolites present in Dendrobium macrostachyum
Lindl with LC – MS identified 20 compounds including chemotaxonomic
marker Flavidin (a phenantherene class of compound), of the genus
Dendrobium. The compounds are listed below in Table 4
with their molecular mass. Liquid Chromatography – Mass Spectrometry
(LC – MS) is proved to be a useful technique for plant metabolite
profiling which allows the quantification of a large variety of plant
metabolites in a single chromatogram.[17] The LCMS spectra of both positive mode and negative mode are included as Figures Figures11 and and22
respectively. Identification is done with the aid of Computer assisted
evaluation of the resulting data by searching against the spectral
library. Some of the identified compounds are reported to have great
pharmacological importance, while some compounds are novel and yet to be
studied.
Identified compounds from LC-MS Spectrum
Liquid chromatography-mass spectrometry spectrum (positive mode)
Liquid chromatography-mass spectrometry spectrum (negative mode)
Present investigation constitutes the first screening of Dendrobium macrostachyum
Lindl and the resultant scavenging and anti-inflammatory activities
gives insight to the strong presence of bioactive constituents. Recent
research centers on various strategies to protect crucial tissues and
organs against oxidative damage induced by free radicals. Hence,
possible identification and isolation of biologically active compounds
will be valuable.
CONCLUSIONS
This study suggests that the ethanolic stem extract of Dendrobium macrostachyum
Lindl possesses high phenolic content, antioxidant and
anti-inflammatory activity that might be helpful in preventing or
slowing the progress of various oxidative stress related diseases.
Metabolic profiling provided an evidence of the presence of promising
compounds for the abovesaid activity. Further investigation on the
isolation, identification and mode of action may lead to chemical
entities with potential for clinical use.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
REFERENCES
1. Bulpitt CJ, Li Y, Bulpitt PF, Wang J. The use of orchids in Chinese medicine. J R Soc Med. 2007;100:558–63. [PMC free article] [PubMed]
2. Andre S. The strange case of Dendrobium aphyllum. Orchid Rev. 2011;119:104–10.
3. Abraham A, Vatsala P. Introduction to Orchids with Illustration and Descriptions of 150 South Indian Orchids. 1st ed. Trivandrum: Tropical Botanical Garden and Research Institute Press; 1981.
4. Nimisha PS, Hiranmai YR. Proximate and physicochemical analysis of Dendrobium macrostachyum Lindl. Int J Pharm Pharm Sci. 2011;4:385–6.
5. Nimisha PS, Hiranmai YR. Anti-lipoxygenase (LOX) activity of Dendrobium macrostachyum Lindl. Int J Res Pharm Sci. 2012;3:601–3.
6. Hossain MM. Therapeutic orchids: Traditional uses and recent advances – An overview. Fitoterapia. 2011;82:102–40. [PubMed]
7. Prasad PR, Reddy CS, Raza SH, Dutt CB. Folklore medicinal plants of North Andaman Islands, India. Fitoterapia. 2008;79:458–64. [PubMed]
8. Sauvage
FL, Picard N, Saint-Marcoux F, Gaulier JM, Lachâtre G, Marquet P.
General unknown screening procedure for the characterization of human
drug metabolites in forensic toxicology: Applications and constraints. J Sep Sci. 2009;32:3074–83. [PubMed]
9. Singelton
VR, Orthifer R, Lamuela-Raventos RM. Analysis of total phenols and
other oxidation substrates and antioxidants by means of Folin-Ciocalteu
reagent. Methods Enzymol. 1999;299:152–78.
10. Piao
XL, Park IH, Baek SH, Kim HY, Park MK, Park JH. Antioxidative activity
of furanocoumarins isolated from Angelicae dahuricae. J Ethnopharmacol. 2004;93:243–6. [PubMed]
11. Mathew S, Abraham TE. In vitro antioxidant activity and scavenging effects of Cinnamomum verum leaf extract assayed by different methodologies. Food Chem Toxicol. 2006;44:198–206. [PubMed]
12. Selvakumar K, Madhan R, Srinivasan G, Baskar V. Antioxidant assays in pharmacological research. Asian J Pharm Technol. 2011;1:99–103.
13. Mizushima
Y, Kobayashi M. Interaction of anti-inflammatory drugs with serum
proteins, especially with some biologically active proteins. J Pharm Pharmacol. 1968;20:169–73. [PubMed]
14. Azeem AK, Dilip C, Prasanth SS, Junise V, Shahima H. Antiinflammatory activity of the glandular extracts of Thunnu salalunga. Asia Pac J Med. 2010;3:412–20.
15. Oyedepo OO, Femurewa AJ. Anti-protease and membrane stabilizing activities of extracts of Fagra zanthoxiloides, Olax subscorpioides and Tetrapleura tetraptera. Int J Pharmacongn. 1995;33:65–9.
16. Halliwell B. Antioxidants and human disease: A general introduction. Nutr Rev. 1997;55:S44–9. [PubMed]
17. Payal SS, Tribhuwan S, Rekha V, Jayabaskaran C. Liquid chromatography-mass spectrometry based profile of bioactive compounds of Cucumiscallosus. Eur J Exp Biol. 2013;3:316–26.
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