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Anti-inflammatory Effect of Bioactive Compounds of Tagetes erecta (Linn.)

Flower Extract

Article  in  Journal of Pure and Applied Microbiology · October 2015

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Rengaswamy Devika Yesudason Justin Koilpillai

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JOURNAL OF PURE AND APPLIED MICROBIOLOGY, Sept. 2015. Vol. 9(3), p. 2547-2550

Anti-inflammatory Effect of Bioactive Compounds of

Tagetes erecta (Linn.) Flower Extract
R. Devika* and Justin Koilpillai

*Department of Biotechnology, Sathyabama University, Chennai- 600 119, India.

Department of Botany, St. Joseph’s College, Trichirappalli, India.

(Received: 02 April 2015; accepted: 09 June 2015)

Today, plant based drugs has taken its great attention in modern pharmaceutical
industries for its less cost and side effects. Phytomedicines utilization has exhibited
effective treatment in curing many aliments in Human. The present investigation
emphasize on the compounds (Flavonoid and salicylic acid) extracted from Tagetes
erecta Linn. flower sample. Tagetes erecta Linn. belong to the Asterceae family and it is
proved to exhibit potent anti-inflammatory effect. Flavonoid showed significant effect
than the salicylic acid during the period of study.

Key words: Therapeutic, bioactive compounds, phytomedicine,

anti-inflammatory, macrophage, vero cells.

Inflammation in the tissue is known for The plant sources had became a valuable resource
its complexicity and pathophysiological for new drug preparation because of its easy
processes1 conducted by a variety of response availability, low cost and with less side effect 8,9.
actions by leucocytes, macrophytes, mast cells etc.2 Literature indicate that the plants are the source
at the point of target site with symptoms of redness, for therapeutic values and it is old as 4000-5000
swelling, pain etc. 3,4 . The introduction of B.C and appears in Rigveda (1600-3500 B.C) 10. It
inflammation was started by the Greek Physician, was also referred that over 7800 pharmaceutical
Hippocrates as the beginning of a healing process industries consume 2000 tonnes of herbs
with edema and erysiplelas5. A comprehensive annually11,12 and they have proved that the phenolic
report on inflammation was put forward by Aulus compounds of plants have high anti oxidant
Celsus in his De Medicina which described four properties13,14 and it was revealed that over 1.5
symptoms such as rubor, humor, color and dolor million practitioners use herbal therapeutics as
which means redness, swelling, heat and pain, preventive medicine or curative applicable
respectively 1. Later, after 100 years, the fifth medicine 15 either as raw, crude drugs or as
symptoms was introduced as functiolaesa formulation of pharmaceuticals forms16.
(impaired function) by Galen of Pergaman 5. Reasearch evidences proved that the
Inflammation can be classified into two types as berries of Solanum nigrumare highly effective
acute inflammation and chronic inflammation against inflammation, tuberculosis17 and highly
associated with infilteration, activation, active for acute and chronic inflammations 18.
proliferation etc., respectively during curing6. Achilllea millefolium L. of Asterceae family
God’s creation forms a complete store- contained three different flavonoids (rutin,
house of therapeutics remedies for many diseases7. aspigenin 7-0-glucoside and luteolin-7-0-
glucoside)19 which inhibited the human neutrophil
* To whom all correspondence should be addressed. elastase and proved to be anti-inflammatory during
E-mail: [email protected] in vitro studies20. The target plant (Tagetes erecta
Linn.) of this investigation, has been thoroughly
2548 DEVIKA & KOILPILLAI: ANTI-INFLAMMATORY EFFECT OF Tagetes erecta

studied in terms of qualitative analysis of in RPMI-1640 medium supplemented with 10% Fetal
phytochemical 21 , and the constituents were Bovine Serum (FBS). From the suspension about
separated by column chromatographic methods 20 µL of cells (1 x 108 cells/ mL) were added to 20 µL
and the evidence showed the presence of of the floral bioactive fraction sample (each isolated
flavonoids and salicylic acid22. In the present compound; flavonoid and salicylic acid) along with
investigation, an attempt has been made to evaluate 40 µL of RPMI1640. The plates were incubated at
the anti inflammatory action and the percentage of 37ºC in 5% CO2 atmosphere. After 24 hours, 20 µL
toxicity of the separated compounds (Flavonoid of Candida albicans (5 x 107 particles/ mL), 20 µL
and salicylic acid). of Nitroblue tetrazolium (NBT) and 1.5 mg/mL in
Phosphate Buffer Solution (PBS) were added and
MATERIALS AND METHODS incubated for 10 minutes. The medium was
removed and the cells were rinsed with the fresh
Healthy, disease free plants of Tagetes medium. Then the cells were washed with 200 µL
erecta (Linn.) was collected, taxonomically of methanol to remove unreduced NBT. Finally,
identified and authenticated in the Herbal Science, 120 µL of 2M KOH and 140 µL of DMSO were
Plant Anatomy Research Centre, Chennai. The added to each well and the absorbance was read at
plant parts were segregated separately as stem, 570 nm. The percentage of NBT reduction was
leaf, flower and roots. The collected plant parts calculated by the following formula.
were washed properly in the tap water and rinsed OD control – OD sample
NBT reduction = x 100
with distilled water carefully (damaged plant parts
were removed). The fresh plants parts were OD control
separately air dried in a closed room (25-28oC) for Ttoxicity studyvero Cell Preparation
10-15 days. After assuring the full dryness of the MTT assay24 is a calorimetric technique
plant parts, they were pulverized to obtain as which is based on the ability of the live cells to
powder form by using sterile electrical blender. The reduce yellow tetrazolium dye to a purple formazon.
powdered samples (leaf, stem, flower and root) Vero cells were maintained in DMEM medium
were stored in air tight containers and protected supplemented with 10% Fetal Bovine Serum at 37oC
from sunlight for further investigation. in humidified atmosphere with 5% CO2.
After various investigation and analysis, MTT Assay
it was confirmed that the flower extract of Tagetes The vero cells (1.2 x 104 cell/well) were
erecta (Linn.) had higher therapeutic value plated in 96 well microtiter plates and was placed
compounds such as flavonoids and salicylic acid. undisturbed at 37 o C overnight (For cell
These compounds were extracted by column attachment). Later, the developed cells were
chromatography 22 and the compounds were incubated with different concentrations (100µg,
separated by Thin Layer Chromatography (TLC) 200µg, 300µg) of flavonoid and salicylic acid for 24
and the purified compounds was authenticated by hours. After incubation, fresh 10 µl of MTT (5mg/
High Performance Liquid Chromatography (HPLC) ml) was added. After 4 hours, the medium was
with their respective standards. The extracted discarded and 100µl of DMSO was added to
compounds (Flavonoids and salicylic acid) were dissolve the formazan crystals. Then the
subjected to anti inflammatory analysis with RAW absorbance was read at 570nm in a microtitre plate
264.7 macrophage cells and the toxicity study (MTT reader. Cyclophosphamide was used as a positive
Assay) was conducted as per standard control.
procedures. Cell viability % = (Test OD/control OD) x 100
Macrophage scavanging assay Cytotoxicity % = 100 - viability%
The effect of the column fractions on the
phagocytic activity of RAW 264.7 macrophages RESULTS AND DISCUSSION
was studied according to the standard method23.
Fresh heparinised blood from healthy volunteers The effect of isolated bioactive
was used for the isolation of Polymorphonuclear compounds (Flavonoid and salicylic acid) from
cells (PMNs). PMNs were isolated and suspended Tagetes erecta Linn. was subjected to macrophage

J PURE APPL MICROBIO, 9(3), SEPTEMBER 2015.

 DEVIKA & KOILPILLAI: ANTI-INFLAMMATORY EFFECT OF Tagetes erecta 2549

scavenging assay23. Various concentrations (50 µg/ inhibitory effect against a-glucosidase enzyme with
ml, 100 µg/ml, 150 µg/ml) of flavonoid and salicylic RAW 264.7 cells activated with lipopolysaccharide
25
acid were prepared and they were analysed for .
NBT reduction at 570 nm. Duplicate analysis were Toxicity study was conducted in a 96 well
made for all the concentrations and the average microtitre plate with vero cells. After incubation
readings were used for calculation of percentage period, the absorbance was observed at 570 nm
NBT reduction. The results showed an increase of and the viability percentage was calculated and
NBT reduction as the concentration of bioactive recorded in the Table 2.Interestingly, the lower
compounds increased. The maximum reduction was concentration of both flavonoid and salicylic acid
observed at 150 µg/ml and the minimum reduction (50 µg) recorded the maximum viability percentage
was observed at 50 µg/ml with flavonoid sample with 92.59 and 88.81, respectively. Percentage of
were 25.26% and 19.6%, respectively. Interestingly, toxicity was maximum at 150 µg flavonoid and
salicylic acid also showed equivalent percentage salicylic acid with 18% and 21%, respectively.
reduction of NBT during the period of study (Table Cancer cell lines MTT assay proved to have an 30-
1).Macrophages are scavengers which are 50% inhibition on the mitochondrial activity26.
responsible to remove the body of worn out cells, Evidences also proved that the patients with
debris and create a vital immune response. Hot hematological malignancies showed significant
water extract of Actinidia arguta stem showed viability with multidrug resistant malignancies 27.

Table 1. Percentage NBT reduction by flavonoid and salicylic acid of Tagetes erecta (Linn.)

Parameter Control Flavonoid (µg/ml) Salicylic acid (µg/ml)

50 100 150 50 100 150

Absorbance at 570 nm 0.927 0.751 0.724 0.698 0.742 0.726 0.684

0.914 0.749 0.717 0.701 0.738 0.712 0.692
Average absorbance 0.920 0.750 0.720 0.699 0.740 0.719 0.688
% NBT reduction 0 18.52 21.73 24.00 19.61 21.89 25.26

Table 2. Toxicity studies of flavonoid and salicylic acid against vero cells

Parameter Control Vero cell line with Flavonoid Vero cell line with Salicylic Acid
50µg 100µg 150µg 50µg 100µg 150µg

% of Viability 100 92.58 86.15 81.81 88.81 83.07 78.74

% of Toxicity 0 7.41 13.84 18.18 11.18 16.92 21.25

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