International Journal of Pharmacognosy and Phytochemical Research 2016; 8(3); 519-523
*Author for Correspondence
Evaluation of Antioxidant and Anti-inflammatory Potentials of
Selected Siddha Herbal Drugs – An In vitro Study
Rajalakshmi P*, Vadivel V, Abirami K, Brindha P
Herbal drugs such as cardamom (Elletaria cardamomum L.), ginger (Zingiber officinale Roscoe), arrow root (Maranta
formulations. The afore-said herbal drugs exhibited therapeutic effect against the loss of appetite, indigestion, gastric reflex,
hiccup, flatulence, itching, scabies, cold, cough, head-ache and joint pains. Even though these herbal drugs have been used
to manage arthritis, there is no scientific evidence regarding mechanism of action. Hence, the present study dealt with the
evaluation of antioxidant and anti-inflammatory potentials of selected herbal drugs through in vitro studies so as to provide
scientific evidences for their anti-arthritic effect.
Key words: Herbal drugs, phytochemistry, polyphenols, antioxidant, anti-inflammatory.
Arthritis is a joint disorder accompanied with pain, joint
stiffness, inflammation, swelling and frequent changes in
body structure due to the age, trauma or infection in the
joints. According to Siddha system arthritis is called as
Santhu vaatham, Kizh vayu, Moottu vali, Aamavatham,
Megasulai and Mudakkuvayu
. Vatham is the causative
joint pain is called Santhu vaatham
. The affected person
stiffness, difficult to move, muscle weakness, increase the
pain after flex the affected joint and tenderness. Siddha
literature describes that this disease has the symptoms like
join swelling, pricking pain, disable to flex and fold the
joint, body tiredness, giddiness, tongue dryness, or
excessive salivary secretion
. Causes of arthritis are
dryness of synovial fluid, fracture, oxidative stress and
. Vatham-induced arthritis was stimulated
cold foods, venereal diseases, genetic disorders, exposure
to chill weather and moist conditions
Diclofenac sodium and Ibrufen, but they have various side
effects. In Siddha system Amukkara chooranam,
Thirikaduku chooranam, Panchadeepakini chooranam,
Astathi chooranam Elathi choornam and Thiribala
chooranam are used for arthritis problem
, but they do not
. Siddha System of medicine
medicine. The crude drugs are derived from different
sources such as plants, animals, metals and minerals
in Siddha system of medicine could be investigated
scientifically to act against arthritis
. Therefore, the present
inflammatory activities of selected herbal drugs such as
cardamom (Elletaria cardamomum L.), ginger (Zingiber
officinale Roscoe), arrow root (Maranta arundinacea L.),
yew leaves (Abies webbiana (D. Don) Spach), Indian rose
chestnut (Mesua ferrea L.), pepper (Piper nigrum L) and
clove (Syzygium aromaticum (L.) Merrill & Perry)
The afore-said herbal drugs exhibited therapeutic effect
against the loss of appetite, indigestion, gastric reflex,
hiccup, flatulence, itching, scabies, cold, cough, head-ache
and joint pains
Raw drugs (Elletaria cardamomum L., Zingiber officinale
Roscoe, Maranta arundinacea L., Abies webbiana (D.
Don) Spach, Mesua ferrea L., Piper nigrum L., Syzygium
herbal market, Thanjavur, Tamilnadu, India and identified
in the NABL accredited lab of CARISM, SASTRA
University and authenticated using macroscopic and
(particle size 1 mm) individually using a lab mill and used
for further analysis.
Ten grams of powdered raw drugs were taken separately
in a conical flask and extracted with 100 ml of ethanol and
kept for 24 h. Then the extract was filtered through filter
Table 2: Total phenolic content of selected herbal drugs
S. No. Sample Name
Total Phenol content
(mg GAE/100 g)
Elletaria cardamomum 104.42 ± 1.06
179.42 ± 4.36
35.00 ± 1.65
1314.17 ± 17.68
2194.17 ± 515.01
226.75 ± 19.21
460.00 ± 49.50
Table 3: Antioxidant activity of selected herbal drugs
(DPPH assay, %)
37.53 ± 5.274
80.30 ± 3.461
26.69 ± 2.967
80.94 ± 1.071
82.52 ± 0.330
83.33 ± 0.494
Table 4: Anti-inflammatory activity of selected herbal
89.97 ± 0.964
34.34 ± 0.857
83.56 ± 1.750
85.15 ± 3.857
paper and used for the analysis.
Phytochemical profile of herbal drugs extract was
of concentrated sulphuric acid, shake well and kept away
some time and noted for colour change. Terpenoids were
analyzed by heating the extract in mild flame with tin and
thionylchloride and noted the colour change. For alkaloid
test, 1 ml of sample was added with 1 ml of diluted acetic
acid and few drops of Dragendorff’s reagent and noted the
precipitate. For the phenol test 0.5 ml of extract was taken
with 1 ml of alcoholic ferric chloride and noted for colour
change. For flavonoids test, 0.5 ml of extract was taken and
1 mg of magnesium turning and few drops of concentrated
hydrochloric acid were added, boiled for 5 min and noted
the colour change. For the tannins experiment, few drops
of ferric chloride solution was added with extract and
noted the colour change. Presence of saponins was
examined by taking the extract with water and shaken
hardly and noted for froth. For the quinone, 0.5 ml of
extract was added with 1 ml of sodium hydroxide (10%)
and observed for colour change. For the coumarin
assessment 0.5 ml of extract was taken in a test tube with
1 ml NaOH and shaken well and noted the colour change.
To check the presence of sugars, extract was treated with
Fehlings solution A and B and boiled and noted for
The total phenolic concentration of ethanolic extract of all
the drugs was estimated according to the modified Folin-
Ciocaltue reagent method
. Extract (10 μl) was taken in a
of 4.4% of Na
for 30 min
nm in the ELISA plate reader (Make: Biotek, Model:
A calibration curve was prepared using standard
= 0.9978) and used to
express the results as gallic acid equivalents (GAE).
The antioxidant activity of ethanolic extracts was analyzed
using DPPH free radical scavenging assay
. Extracts (10
DPPH solution (2.5 mg/100 ml) and incubated
Epoch). The radical scavenging activity of tested samples
was calculated using the formula (Antioxidant activity =
Abs control – Abs test / Abs control x 100) and expressed
on percentage basis.
The anti-inflammatory activity of the ethanolic extracts
was evaluated using RBC membrane stabilization
. Blood sample (2 ml) was collected from
volunteer in a heparinized tube and washed with PBS twice
and centifuged at 3000 rpm for 10 min (Centrifuge Make:
Eppendorf, Model 5810-R). Then RBC was suspended in
Table 1: Phytochemical screening results of herbal drugs
normal saline and taken in a tube (0.5 ml) with 0.5 ml of
extract and 0.5 ml hypotonic solution and incubated for 30
min at room temperature. Then the contents were
centrifuged at 1500 rpm for 10 min and the supernatant
was collected and the absorbance was read at 560 nm using
Micro plate reader (Make: Biotek, Model: Epoch). Based
on the absorbance of extract and control, the membrane
stabilization effect was calculated and expressed on
RESULTS AND DISCUSSION
Data obtained in the preliminary phytochemical screening
of herbal drugs was shown in Table 1. Red colour was
formed when extract was treated with few drops of
concentrated sulphuric acid, which shows the presence of
sterols in cardomomum, ginger, yew, Mesua and clove.
This result was similar to earlier reports in cardomomum
Extract given pink colour upon reaction with tin and
same result was noted in previous work
. For alkaloid test,
and clove when treated with acetic acid and few drops of
Dragendorff’s reagent. Bluish black colour was appeared
in phenol test in yew and clove extracts. Presence of
flavonoids was noted in cardomomum, ginger, yew, mesua
and pepper, based on formation of red colour during
extract was reacted with magnesium turning and
concentrated hydrochloric acid. Similarly the presence of
flavonoid was reported in pepper
. Extracts of
ginger shows the positive result of bluish black colour
when few drops of ferric chloride solution was added.
Presence of saponins was noted based on appearance of
froth in ginger, mesua, pepper and clove. Similarly the
presence of saponins was reported in clove
. Quinone was
of end point colour when extract added with sodium
hydroxide. Yellow colour was formed when extract was
treated with NaOH, which indicated the presence of
coumarin in cardomomum, ginger and mesua. This is in
agreement with previous result in cardomomum
Presence of sugars was recorded in the extracts of
confirmed by the formation of brick red coloured
precipitation upon treatment with Fehlings solution A and
B. This result was similar to earlier reports in mesua
mg GAE/100 g), which is followed by Abies webbiana
(1314.17 mg GAE/100 g). Previous, higher level of total
phenol content was noted in M. ferrea 98.15mg GAE/g
and A. webbiana. Phenolic compounds represent the
synthesized by higher plants, probably as a result of
antioxidative strategies. Polyphenols can scavenge the free
radicals such as .OH, O2.-, and ROO. and suppress the free
radical-mediated oxidation. The formation of free radicals
may be inhibited by reducing hydroperoxides and
hydrogen peroxide and by sequestering metal ions through
complexation / chelation reactions. The antioxidant and
anti-inflammatory role of polyphenols were proven in past
Antioxidant activity was determined using 1,1-diphenyl-2-
on the measurement of the loss of DPPH colour at 517 nm
after reaction with test compounds and the reaction is
monitored by a spectrophotometer. DPPH is a stable free
radical of purple colour and in the presence of an
antioxidant its colour changes to yellow based on the
efficiency of the antioxidant. The change in the absorbance
with respect to control (DPPH solution, 100 % free radical)
is calculated as per cent scavenging power. Radical
scavenging action is dependent on both reactivity and
concentration of the antioxidant. The antioxidant activity
of herbal drugs was measured in terms of DPPH radical
scavenging potential and the results are shown in Table 3.
Among the drugs, clove (83.33%), Mesua (82.52%),
ginger (83.33%) and Abies (80.94%) exhibited maximum
level of antioxidant activities. The higher level of
antioxidant activity recorded in Mesua and Abies is
correlated with their high content polyphenols (Table 2).
Similar level of antioxidant activity was reported in
previous studies on Zingiber officinale (79.0%)
and clove (93%)
Some free radicals such as nitric oxide, superoxide radical
reactive nitrogen species (RNS) mediate cells in signalling
where excessive quantities of ROS/RNS overcome
endogenous antioxidant capacity, leading to oxidation of
enzymes, proteins, DNA and lipids. Oxidative stress is the
contributing feature in the development of chronic
degenerative diseases including coronary heart disease,
Table 5: Anti-arthritic and related activities reported in various herbal drugs and their major phytoconstituents.
Analgesic and Anti-inflammation
Energy producer and Increase bone strength
cancer, arthritis, and aging
. The DPPH can be used as a
that involved in oxidative stress condition.
Typical inflammatory diseases such as rheumatoid
arthritis, asthma, colitis and hepatitis are the leading cause
of disability and death. The excessive production of
reactive oxygen metabolites by phagocytic leucocytes
during the inflammatory process, as part of host defence,
disregulates cellular function causing tissue injury which
in turn augments the state of inflammation leading to
chronic inflammatory diseases. Antioxidants, which
scavenge these reactive oxygen metabolites, have been
found to complement the anti-inflammatory process and
promote tissue repair
. In the present study, we have used
inflammatory activity of herbal drugs. A possible
explanation for the membrane stabilization effect could be
an increase in the surface area / volume ration of the cell.
The membrane denaturation of RBC was brought about by
expansion of cell membrane by incubating the cells in
Among the presently studies herbal drugs of cardamomum
(89.97%), pepper (85.15%) and Abies (83.56%) were
exhibited higher level of anti-inflammatory activities in
terms of RBC membrane stabilization. The notable anti-
inflammatory activity of Abies could be due to the high
amount of polyphenols recorded in the same sample of the
present study (Table 1) These results are supported by the
previous in vivo studies on anti-inflammatory activity of
cardomomum, pepper and Abies
Based on literature survey, we have found the presence of
drugs and their anti-arthritic and related activities (Table
5). So, only the Mesua ferrea exhibits anti-arthritic effect
based on literature reference, while ginger, pepper and
clove were showed analgesic properties. Both ginger and
Abies revealed anti-inflammatory activity whereas
cardamom displayed immune-modulatory property. So,
consumption of these herbal drugs could relieve the
arthritis pain and also strengthen the bone joints in addition
improve the immune system and prevent the cellular
damages through antioxidant and anti-inflammatory
In the present work, the total phenolic content, antioxidant
and anti-inflammatory activities of ethanolic extract of
different herbal drugs were analyzed. Even though these
drugs have been used to treat arthritis in folklore medicine,
the mechanism of action is not yet proved and hence the
present investigation provides scientific evidence for the
action of this drug against arthritis through antioxidant and
anti-inflammation pathways. Among the drugs, ginger,
Abies, Mesua and clove are responsible for the high
antioxidant power while anti-inflammatory property was
contributed by cardamom, Abies, Maratha and pepper. So,
combination of these drugs could exhibit both antioxidant
as well anti-inflammatory activities and thus provides
therapeutic effect against arthritis as indicated by the in
vitro studies. Further in vivo models are necessary to prove
the efficacy and mechanism of action of this drug against
arthritis, which is a major burden for aged people
throughout the world.
Authors are thankful to the Hon’ble Vice Chancellor of
SASTRA University for their constant encouragement and
support to carry out this research work.
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