1.1 Anemia: Modern Institute of Pharmaceutical Sciences Indore

Anti-anemic activity of hydroalcholic extracts of Tagetes erecta
flower on phenylhydrazine- induced anaemic rats
1. INTRODUCTION
1.1 ANEMIA
Anaemia is a blood disorder. Blood is a vital liquid that your heart constantly pumps
through your veins and arteries and all throughout your body. When something goes
wrong in your blood, it can affect your health and quality of life. Many types of anaemia
exist, such as iron-deficiency anaemia, pernicious anaemia, aplastic anaemia, and
haemolytic anaemia. The different types of anaemia are linked to various diseases and
conditions. Anaemia can affect people of all ages, races, and ethnicities. Some types of
anaemia are very common, and some are very rare. Some are very mild, and others are
severe or even life-threatening if not treated aggressively. The good news is that anaemia
often can be successfully treated and even prevented.
Signs of Anaemia
 Weakness
 Shortness of breath
 Feeling tired
 Tiredness or weakness
 Faintness or dizziness
 Increased thirst
 Sweating
 Lower leg cramps
1.1.1 Type of anaemia

 Iron-Deficiency Anemia
 Aplastic Anemia
 Pernicious Anemia
 Hemolytic Anemia
 Megaloblastic Anemia
 Hemorrhagic Anemia
Modern Institute of Pharmaceutical Sciences Indore Page 1

a) Iron-Deficiency Anemia
Inadequate absorption of iron, excessive loss of iron, increased iron requirement,
or insufficient intake of iron causes iron deficiency anemia, the most common
type of anemia. Women are at greater risk for iron-deficiency anemia due to
menstrual blood losses and increased iron demands of the growing fetus during
pregnancy. Gastrointestinal losses, such as those that occur with malignancy or
ulceration, also contribute to this type of anemia.
b) Aplastic Anemia
Destruction of red bone marrow results in aplastic anemia. It is caused by toxins,
gamma radiation, and certain medications that inhibit enzymes needed for
hemopoiesis.
c) Pernicious Anemia
Insufficient hemopoiesis resulting from an inability of the stomach to produce
intrinsic factor, which is needed for absorption of vitamin B12 in the small
intestine, causes pernicious anemia.
d) Hemolytic Anemia
RBC plasma membranes rupture prematurely in hemolytic anemia. The released
hemoglobin pours into the plasma and may damage the filtering units (glomeruli)
in the kidneys. The condition may result from inherited defects such as abnormal
red blood cell enzymes, or from outside agents such as parasites, toxins, or
antibodies from incompatible transfused blood.
e) Megaloblastic Anemia
Inadequate intake of vitamin B12 or folic acid causes Megaloblastic anemia in
which red bone marrow produces large, abnormal red blood cells (megaloblasts).
It may also be caused by drugs that alter gastric secretion or are used to treat
cancer.
f) Hemorrhagic Anemia

Excessive loss of RBCs through bleeding resulting from large wounds, stomach
ulcers, or especially heavy menstruation leads to hemorrhagic anemia. (Tortora)
g) Thalassemia
An inherited form of hemolytic anemia, Thalassemia comes from the production
of abnormal hemoglobin. It is characterized by low hemoglobin and unusually
small and fragile RBCs (microcytosis), although the RBC count may be normal.
Thalassemia has several types that involve imbalances in the four chains of amino
acids that comprise hemoglobin (alpha- and beta-globins). In Thalassemia minor
or Thalassemia trait (heterozygous Thalassemia), also called alpha-Thalassemia,
there is an imbalance in the production of the alpha chain of amino acids. In
Thalassemia minor, fetal hemoglobin (HbF), the hemoglobin form that circulates
in the fetus, does not decrease normally after birth and may remain high in later
life. A child may inherit Thalassemia trait when only one parent has the genes
responsible for it. It is usually not treated and does not have serious consequences.
Thalassemia major (homozygous Thalassemia or Cooley's anemia) occurs in
children in whom both parents pass on the genes responsible. It is known as beta-
Thalassemia, because of an imbalance in the beta chain amino acids of
hemoglobin. It also involves the persistence of HbF with larger than normal
amounts appearing in the child's circulation. Alpha-Thalassemia occur most
commonly in African Americans; beta-Thalassemia most commonly affect people
of Mediterranean or middle-Eastern ancestry and Southeast Asians. Hemoglobin
H disease is another form of Thalassemia in which three of the four beta-globin
genes are missing.
h) Sickle cell anemia

Sickle cell anemia is an inherited, chronic, incurable blood disorder that causes
the body to produce defective hemoglobin, the abnormal HgbS, which occurs
primarily in African Americans. The condition is characterized by abnormal,
crescent-shaped RBCs. Unlike normal oval cells, fragile sickle cells cannot hold
enough hemoglobin to nourish body tissues. The deformed shape makes it hard

for sickle cells to pass through narrow blood vessels. When capillaries become
obstructed, a life-threatening condition called sickle cell crisis is likely to occur. A
child who inherits the sickle cell gene from each parent will have the disease. A
child who inherits the sickle cell gene from only one parent carries the sickle cell
trait but does not have the disease.
Synthetic Drug Used For the Treatment Of Anemia

Iron Preparation
1. Ferrous sulfate: (hydrated salt 20% iron, dried salt 32% iron) is the cheapest;
may be preferred on this account. It often leaves a metallic taste in mouth
2. Ferrous gluconate (12% iron)
3. Ferrous fumarate (33% iron): is less water soluble than ferrous sulfate and
tasteless
4. Colloidal ferric hydroxide (50% iron):
5. Ferrous succinate (35% iron)
6. Iron choline citrate
7. Iron calcium complex (5% iron)
8. Ferric ammonium citrate (scale iron)
9. Ferrous aminoate (10% iron)
10. Ferric glycerophosphate
11. Iron hydroxy polymaltose
Vitamin B12
1. Cyanocobalamin
2. Hydroxocobalamin
3. Methylcobalamin
Other
Erythropoietin

Herbal drugs used in the treatment of anemia
A. Punarnava Mandoor
Punarnava is a wonderful Ayurvedic remedy with diuretic It is an excellent kidney

and heart tonic. This medicine is useful in maintaining the health of the skin. These
capsules can clear excess Kapha from the stomach and chest. They act as a
rejuvenator and can be used in treating impaired urinary conditions. They are very
useful in the reduction of excess body fluids, congestion and edema due to excess
Kapha. Punarnava has properties to clear toxins from the blood and increase red
blood cells in the body. The hemoglobin content increases and oxygen carrying
capacity increases. They are useful in treating swollen joints due to fluid retention
caused by imbalance in Kapha. This herbal medicine is useful in eliminating toxins
accumulated in the joints through kidneys and urine. It is effective in the treatment of
arthritis and gout. It is helpful in the breakdown of the 'fibrin', a blood clotting
protein. Punarnava Mandoor is very effective in treating anemia. Punarnava Mandoor
is 100% natural tablet without any preservatives or chemicals. It is available in pack
of 120 tablets.
Dosage -1-2 tablets twice or thrice daily, after meals.
B Arogyavardhini vati
An Ancient Ayurvedic formulation that has been useful for the Liver and
especially for cases of Fatty liver and Jaundice. It is useful in clearing
accumulated bile. It is enriched with kutki a herb which is excellent liver cleanser.
Arogayavardhini vati is 100% natural tablet without any preservatives or
chemicals. It is available in pack of 120 tablets.
Dosage - 1-2 tablets twice or thrice daily, after meals.

C Chanderprabha vati
It is a classical Ayurvedic formulation for weakness due to vata disorders and

general debilities. It is enriched with Loh Bhasma and Shilajit along with many
vata pacifying herbs. It is very effective in treating anemia. Chanderprabha vati is
100% natural tablet without any preservatives or chemicals. It is available in pack
of 120 tablets.
Dosage -1-2 tablets twice or thrice daily, after meals.
D. Wheat Grass powder
Planet Ayurvedic Wheat Grass powder is made from organic fresh small leaves of
baby wheat plants. It helps to detoxify Liver, Improves digestion, balances body
pH, restores alkalinity, boosts formation of Red Blood cells and treats Anaemia.

2. LITERATURE REVIEW
2.1 Review on biological activity of the plant
 Marigold and its derivatives and characterized secondary metabolites are widely used for
medicinal purposes, are becoming popular all over the world as a natural alternative to
synthetically produced chemicals both in traditional and allopathic system of medicines.
(Lokesh J Shetty et.al in 2015)
 This article has given a well discussion medicinal value of Tagetes erecta with
pharmacological and photochemistry of Tagetes erecta and its application in the
treatment of various ailments like the flower parts of plants are used as a
hepatoprotective, insecticide, anti-oxidants and analgesic.( R. Islam1, S.M.H. Rashid
et. al. in 2004)
 Effects of Haematinics on Body Weight and Some Haematological Values in animals

The results revealed that the body weight gain, total erythrocyte count, haemoglobin
concentration, packed cell volume, mean corpuscular volume, mean corpuscular
haemoglobin and mean corpuscular haemoglobin concentration were increased
significantly in animals administered with haematinics compared to those of the
control.( G. Gopi1, A. Elumalai et. al. in 2012)
 The extracts of roots, foliage & flowers of Tagetes erecta having a vast usage, the
review has indicated that the major bioactive components in Tagetes are polytheinyls,
terpenoids & majorly the caretenoids there are various beneficial effect of the
flavanoids and caretenoids in pharmaceutical & other applications. They also have
nematocidal & biocidal value.( Helen Fennimore, , et al 2013)
 The review enlightens that the Marigolds are easy to grow, bloom reliably all summer
and have few insect and disease problems. They have understandably been a favourite
annual in this country for many years. (Abdul-Wasea a. Asrar et al., 2010)

 The genus has been recognized as a potential source of very interesting biologically
active products i.e. caretenoids that are used as food colorants, feed additives This
genus has been investigated for various biological activities like antimicrobial,
antiplasmodial, The Tagetes oil has been mainly used for the compounding of high-
grade perfumes and also acts as antihaemorrhagic, anti-inflammatory, antiseptic,
antispasmodic, astringent, diaphoretic and immenagogue. Antioxidant, insecticidal
etc. The present review summarizes the biological activities and phytoconstituents of
this genus. And possess anticancer and antiageing effects, essential oil known for
antimicrobial and insecticidal properties, thiophenes with a marked biocidal activity
and flavanoids having pharmacological properties. (Tanveer hussain et al, 2013)
 The genus Tagetes (Asteraceae) contains 56 species, T.patula L. which have been
widely used in the world nowadays; the species widely used throughout the world
were T. erecta L., T.patula L. and T.tenuifolia (Soule, 1996). In China, T.erecta L.
and T. patula L. were introduced and widely cultivated as important garden plants. In
addition, the inflorescence of pigment T. erecta L. flowers were also ideal materials
for extracting lutein. Therefore, it was very important to study Tagetes plant with
their great economic value..(V.b. Pratheesh, Nify Benny,et al., 2007)
 Marigold is a widely cultivated plant and found plenty. Farmers cultivate marigold
(Tagetes erecta and Tagetes patula) as a major crop in a multi crop system by rotating
it with other agricultural and horticultural crops like tomatoes and other vegetables.
Currently, the cropping area covers around 1600 hectares of land in the Farrukhnagar
block. The flowers are generally sold in the nearby markets in Delhi and are being
used for decorative, ornamental, social and religious purposes with several other
medicinal values. (Pin zhang, li Zeng, et al., 2011)
 The bioactive components (secondary metabolites) of medicinal plants Tagetes

erecta have high therapeutic values in the medical field These bioactive
compounds are widely used as an alternative source in the modern medicine
which has taken an exponential growth in terms of natural origin and lesser side

effects [5]. The effective therapeutic value occurs by the combinations of plant
metabolites like tannins, flavanoids, resins, gums, steroids, terpenes, alkaloids
caretenoids etc. (P.ghosh and P.pal et al. 2008)
.
 The bioactive components (secondary metabolites) of medicinal plants have high
therapeutic values in the medical field These bioactive compounds are widely
used as an alternative source in the modern medicine which has taken an
exponential growth in terms of natural origin and lesser side effects. The effective
therapeutic value occurs by the combinations of plant metabolites like. They used
as an alternative source in the modern medicine which has taken an exponential
growth in terms of natural origin and lesser side effects . The effective therapeutic
value occurs by the combinations of plant metabolites like tannins, flavanoids,
resins, gums, steroids, terpenes, alkaloids caretenoids etc. Sonja Nicholson,
(Llorna Cox, et al 2008) .
2.2 Review on chemical constituents of the plant
 the effects of lutein, zeaxanthin and meso-zeaxanthin on macular pigment optical

density (MPOD) in randomized controlled trials (RCTs) Pearson correlation
analysis was used to determine the relationship between the changes in MPOD
and blood xanthophylls caretenoids or baseline MPOD levels Xanthophylls
caretenoids supplementation was associated with significant increase in MPOD in
AMD patients and healthy subject. Stratified analysis showed a greater increase in
MPOD among trials supplemented and combined with meso-zeaxanthin. (Luís
cláudio Paterno silveira1 et al. 2009)
 The present paper deals with the chemistry, isolation, separation, characterisation
and stabilisation of the Marigold oleoresin and its application as a natural
component. Marigold (Tagetes Erecta L) has as a rich source of natural
antioxidant-Lutein Chromatographic separations of saponified and unsaponified

oleoresin were performed and Trans-Lutein identified as the major constituent.

Well-preserved flowers exhibit a high yield of Xanthophylls content in contrast to
the unpreserved flower sample emphasizing the significance of flower
preservation in the extraction of xanthophylls. (Devika regaswami, Justin
koilpillai et al., 2014)
 Lutein is oxycarotenoid/xanthophylls containing two cyclic end groups and the

basic C40 isoprenoid structure. It is a non-vitamin A caretenoids that cannot be
synthesized by humans and lutein and zeaxanthin are the only dietary caretenoids
present Marigold flower petals are excellent sources of lutein as they contain
high levels of lutein (of the order 4500 mg/lb) and no significant levels of other
caretenoids. (Nagaraj b1, Krishnmurti nbl et al. 2012)
2.3 Review on hematinics
 Samples of coagulated and ethylenediaminetetraacetate (EDTA) anticoagulated

blood were sent directly by post to the Department of Haematology and
Department of Clinical Biochemistry and Immunology, Addenbrooke’s Hospital,
Cambridge (Addenbrooke’s) after their collection. Serum samples were
obtained by centrifugation of the coagulated blood sample.for the several
examinations of blood contents. (Sayema aefin, Md towhidul et al., 2015)
 Hematopoiesis (formation of blood) is a complex process of proliferation,

differentiation, and maturation of cellular components of blood (erythrocytes,
leucocytes and platelets) from the bone marrow stem cells. It is regulated by
balanced interaction between endogenously derived hematopoietic growth factors
and exogenously supplied essential nutrients (haematinics). Inadequate supply of
either the growth factors or the haematinics results in deficiency of normal blood
cells which is manifested as anaemia, thrombocytopenia or neutropenia. D.
(Vidhya sagar swamy et al. 2002)

3. REASEARCH ENVISAGED
The aim of this study is to find the “Anti anemic activity of hydroalcoholic extract of
Tagetes erecta on phenyl hydrazine induced anemic rats.”
The reasons for choosing Tagetes erecta are:-

Easy availability and wide geographical distribution of the plant globally in almost every
climatic condition. Presence of an important medicinally active phytoconstituents Lutine
in abundant amount.
The outcome of the study would be

1) Establishment of Lutine as an anti anemic agent.
2) The drug dose efficiency.

4. PLAN OF WORK
1) Collection, identification and drying of drug
2) Extraction of drug by soxhlet extraction method (Hydroalcoholic)
3) Phytochemical studies
 Qualitative chemical evaluation
 Determination of percentage yield
4) Pharmacological studies
 Acute toxicity study (fixed dose)
 Anti-Anemic Activity
4) Compilation of work

5. MATERIALS AND METHODS

5.1 DESCRIPTION OF PLANT
T.erecta is a perennial subscandent shrub found wild in the southern parts of peninsular
India, Tripura, and West Bengal and cultivated in garden throughout India and Ceylon.
(Veluri, 1999)
Family: Asteraceae
Genus: Tagetes
Species: Tagetes erecta
Hindi: genda
English: Marigold (Indian medicinal plants 1468)
Synonym: Calendula officinalis, pot marigold
Origin: India, Africa.
Category: herbs
Height: 6 inches to 3 feet
Spacing: 18-24 in. (45-60 cm)
Sun Exposure: Light Shade
Bloom color: yellow and gold to orange, red and mahogany.
Bloom Time: Late Winter/Early Spring

Fig no.1 Tagetes erecta plant
Foliage: Rich dark green and scented
Other details: Average Water Needs
Distribution: Cultivated throughout India, perhaps a native of Bengal, southern India
and Africa.
Leaves: 3.8 -7.5 by 2.2 -3.8 cm ovate, or elliptic, dark green.
Flower: several striped, bicoloured, finely cut, fernlike. 2-2.5 cm long.
Soil PH: 6.2- 6.5
Humidity: 40 - 70

5.2 COLLECTION AND IDENTIFICATION
 Collection Time: August, 2016
 From: Herbal garden MIPS
 Identified by: Dr. S.B.Singh Assistant College of agriculture, Indore

 Voucher number- HRB/IAC/2016/0555
 Specimen was submitted in Department of Pharmacognosy, MIPS,Indore , (M.P.)
5.3 EXTRACTION AND PRELIMINARY PHYTOCHEMICAL SCREENING
a) Extraction
Fruits was collected from herbal garden Modern institute of Pharmaceutical Sciences,
Indore Madhya Pradesh, during August 2016. It was dried in shade. The dried fruits were
powdered, weighed (100 g) and filled in soxhlet apparatus for extraction. The powdered
drug was extracted with hydroalcoholic solvent (70:30) until drug was completely
extracted. % yield was calculated for each extract after drying under vacuum. (Kokate,
2000)
b) Determination of percentage yield: -

The percentage yield of each extract was calculated by using following formul
Weight of Extract
Percentage yield = -------------------------------------------------- x 100
Weight of powder drug Taken
% percentage yield was found to be = 20% w/w
c) Qualitative Chemical Evaluation (Mukherjee, 2002 and Kokate, 1996)

Alkaloid test
o The acidic solution of the extracts were made by dissolving them in 1% solution
of Sulphuric acid and subjected for test.
o 0.1ml of extract soln + 1 drop of Dragendorff’s reagent

o Inference: Orange ppt. shows presence of alkaloid.
o 0.1ml of extract soln + 1 drop of Mayer’s reagent

o Inference: Cream ppt shows presence of alkaloid
o 0.1ml of extract soln + 1 drop of Wagner’s reagent

o Inference: Orange ppt shows presence of alkaloid
o 0.1ml of extract soln + 1 drop of Hager’s reagent

o Inference: Yellow ppt shows presence of alkaloid
Carbohydrate tests
Molish’s test
o Extracts were treated with ά napthol and conc. H2SO4, Purple colour if produced
indicates +ve test
o Inference: Purple colour shows presence of Carbohydrates.
Reduction of Fehling solution

o Extract was treated with equal quantity of Fehling’s soln A and Fehling’s soln B
and heated.
o Inference: Brick red ppt shows presence of reducing sugar.
Glycoside test
Test 1

o Extract + 5ml of dilute (10%) H2SO4 and heated on water bath for 2min., and
filtered.
o Acid extract is neutralized with a 5% soln of NaOH 1 ml of Fehling’s solution A
and then Fehling’s soln B were added until alkaline and heated on water bath for 2
min. Quantity of red ppt formed is noted and compared with that of formed in test
2.
Test 2
o Extract +volume of water equivalent to the volume of NaOH used in test 1, step 2
above. 1ml of Fehling’s soln A and then Fehling’s soln B is added until the
solution became alkaline and heated on water bath for 2 min. Quantity of red ppt
formed is noted.
o Inference: If ppt was not same in both test 1 and test 2, shows presence of
Glycoside.
Borntrager’s test
o Ethereal extract soln was treated with caustic soda or NH4 (made alkaline)
o Inference: - Aqueous layer pink, red, or violet colour shows presence of
Glycoside.
Legal’s test
o Extract dissolved in pyridine + Sodium nitropruside soln and soln was made
alkaline
o Inference: Pink or red colour shows presence of Glycoside.
Fixed oils and fat

o 1ml of 1% CuSo4 soln + 5 drops of 10% NaOH soln.
o Inference: Clear-blue solution shows presence of glycerol.

o 5 drops of extract + a pinch of sodium hydrogen sulphate

o Inference: Pungent odour shows presence of Fixed oil.
Phenolics and tannins

o Extract +Ferric chloride soln
o Inference: Green or violet colour shows presence of Phenolics and tannins
o Extract + 1% gelatine Soln (10% NaCl)

o Inference: - Buff colour ppt shows presence of Phenolics and tannins
o Extract + Lead acetate

o Inference: - ppt shows presence of Phenolics and tannins
Flavanoids test
o Extract + Zinc dust + Conc. HCl
o Inference: Magenta colour (purplish pink) shows presence of flavanoids.
o Extract + pinch of magnesium + Conc. HCl

o Inference: - Pink colour shows presence of flavanoids.
o Extract was treated with NaOH

o Inference: Pale yellow colour shows presence of flavanoids.
Saponin
o Extract + Water to be shaken
o Inference: - Foam formation shows presence of Saponin.
Naphthaquinone
o Extract + Alkali
o Inference - Change in colour shows presence of Naphthaquinon

5.4 PHARMACOLOGICAL INVESTIGATION
5.4.1 Selection of animals
Wistar Male Albino rats weighing between 150 – 180 g were obtained from
animal house, Department of Pharmacology Modern institute of Pharmaceutical
Sciences, Indore (M.P). These animals were used for the acute toxicity, anti-anemic
activity. The animals were stabilized for 1 week. They were maintained in standard
condition at room temperature 60 ± 5% relative humidity and 12 h light dark cycle. They
had been given standard pellet diet supplied by Hindustan Lever Co. Mumbai and water
ad-libitum throughout the course of the study. The animals were handled gently to avoid
giving them too much stress, which could result in an increased adrenal output.
5.4.2 Acute toxicity studies
The preliminary pharmacological studies were conducted to assess the acute
pharmacological effects and LD50 of the hydroalcoholic drug extracts.
The acute toxicity study was carried out in adult female albino rats by “Fixed Dose
Procedure” method (OECD guidelines 420)
The animals were fasted overnight and next day extract of the flowers of the plant
Tagetes erecta (suspended in 0.5 % w/v sodium CMC). Then the animals were observed
continuously for three hour for general behavioural, neurological, autonomic profiles and
then every 30 min for next three hour and finally death after 24 hour. The observations
were tabulated according to ‘Irwin’s Table’. Sighting studies result show dose level of
2000 mg/kg of Tagetes erecta shows death. So, for the main study cut off 1000 mg/kg
was selected. In main study the, 5 animals in each group were taken. Group I Tagetes
erecta then animal were observed for 24 hour for the signs of any toxicity. Animal were
kept in the separated cage were observed for 14 days and on the 14th day animal blood
was collected and animal was sacrificed for the histopathological studies of liver and

kidney. The organs for histopathological studies were sent to College of Veterinary
Science and Animal Husbandry, Mhow, Madhya Pradesh.
5.4.3 Selection of doses
For the assessment of analgesic and anti-inflammatory activity, dose level were
chosen in such a way that, the dose was approximately one tenth and one fifth of the
maximum dose during acute toxicity therefore dose for the Tagetes erecta extract
100mg/kg and 200 mg/kg respectively.
5.4.4 Anti Anemic Activity
Induction of anaemia: Anaemia was induced in rats by intraperitoneal administration of

40 mg / kg / day of phenylhydrazine (PHZ) for two days (D0 and D1) [16, 17]. The
treated rats with phenylhydrazine whose haemoglobin concentration <13 g / dl were
considered as anemic and included for the study.
Treatment of animals: Seven groups of 5 rats were formed and treated daily for 4 weeks
as follows:
-Group I (G1) - Normal control received 10 ml / kg of 0.5% CMC (carboxy methyl

cellulose) from day D2 to D28.
-Group II (G2) -Anaemic control received 0.5% CMC (10 ml / kg) from day D2 to D28.
-Group III (G3) - Treated with Vitamin B12 (Vit B12) syrup (1 ml /day) from day D2 to
D28.
-Group IV (G4) - Treated with the ethanolic extract of T.erecta (100 mg / kg) from day
D2 to D28.

-Group V (G5) - Treated with the ethanolic extract of T.erecta (200 mg / kg) from day D0
to D28.
5.4.8 Statistical Analysis
The data were expressed as mean ± SEM. The data of anti-inflammatory and
analgesic activity were analyzed by one way analysis of variance (ANOVA) followed by
Dunnet’s-‘t’ test. A p value less than 0.05 was considered as statistically significant.

6. RESULTS
Table 1. Various chemical compounds identified in T.erecta flower.
Extracts Aqueous Ethanolic

Chemical Groups
Alkaloids + +
Polyphenols + +
Sterols and Terpenes + +
Catechin Tannins + +
Gallic tannins - -
Flavonoids + +
Quinones - +
Cardiac Glycosides + +
Saponins + -
Leucoanthocyanins + +
Signs: + = Present; - = Absent

Body Weight and Percentage Weight Variation of rats before and

after induction of anemia with phenylhydrazine.
Groups Body Weight (g)

st
D0 D2 1 week 2nd week 3rd week 4th week
Normal Control
(10ml/kg of 0.5% CMC) 209.5 212.4 221.2 220.4 222.8 224.3
Anemic Control 222.3 235.0 226.5 235.3 239.0
233.0  b b b b
(10ml/kg of 0.5% CMC) a
Vit B12 syrup 179.9 193.3 200.9 202.5 203.3

(1ml/day) 185.5 
a
b b b b
HAE T erecta 182.3 188.1 189.9 191.0 192.4
190.6  b b b b
(100mg/kg) from D2 a
HAE T erecta 181.2 187.5 190.1 193.8 197.1

193.9  b b b b
(200mg/kg) from D2 a

Effect of Hydroalcoholic extracts of Tagetes erecta flower

on the number of red blood cells during and after induction
of anaemia with phenylhydrazine in rats.
Drug treatment RBC (106cells/ /µl)
D0 D2 1st week 2nd week 3rd week 4th week
Normal Control     7.46   7.39  
(10 ml/kg of 0.5% CMC)
Anemic Control       5.77   6.69  
(10 ml/kg of 0.5% CMC) a** b b +61.34b b**
Vit B12 syrup       5.02   5.92  
(1ml/day) a** b b +71.20b b***
HAE T erecta (100mg/kg)       5.23   5.90  
from D2 a** b b +67.30b b***
HAE T erecta (200mg/kg) 7.43   4.38   5.53   6.81  
from D2 a** b b +63.69b* b**
Values are expressed as Mean  SEM (n = 5). *P <0.05, **P <0.01 & ***P<0.001
a: Percentage variation compared to day D0, b: Percentage variation compared to day D2. ; HAE: hydroalcoholic extract:

Effect of hydroalcoholic extracts of Tagetes erecta flower on Haemoglobin (Hb) of rats after
induction of anemia with phenylhydrazine.
Drug treatment Hemoglobin (g/dl)

Normal Control      13.37   13.20  
Anemic Control       12.33   12.80  
(10 ml/kg of 0.5% CMC) a** b b +57.83b* b**
Vit B12 syrup       13.00   13.40  
(1ml/day) a** b** b** +73.80b*** b***
HAE T erecta       12.60   12.93  
(100mg/kg) from D2 a*** b* b** +69.71b** b**
HAE T erecta    8.16   12.70   13.70  
(200mg/kg) from D2 a** b b** +67.89b** b***
Values are expressed as Mean  SEM (n = 5). *P <0.05, **P <0.01 & ***P<0.001
a: Percentage variation compared to day D0, b: Percentage variation compared to day D2. HAE: hydroalcoholic
extract:.

Effect of Hydroalcoholic Extracts of Tagetes erecta flower on Haematocrit

during and after induction of anaemia with phenylhydrazine in rats.
Drug treatment Haematocrit (%)

Normal Control     43.65    
Anemic Control       41.17  41.84  42.25 
(10 ml/kg of 0.5% CMC) a*** b*** b*** b*** b***
Vit B12 syrup       41.93  42.35  44.05 
(1ml/day) a*** b*** b*** b*** b***
HAE T erecta       41.60 42.00  42.67 
(100mg/kg) from D2 a*** b*** b*** b*** b***
HAE T erecta       41.47  42.35  42.90 
(200mg/kg) from D2 a*** b*** b*** b*** b***
Values are expressed as Mean  SEM (n = 5). ***P<0.001
a: Percentage variation compared to day D0, b: Percentage variation compared to day D2. HAE: hydroalcoholic
extract.

7. DISCUSSION:
Plants are a rich source of drugs because they produce a host of bioactive molecules, most
likely acts as chemical defence against predators or infectious agents [20]. Phytochemical
analysis revealed the presence of large chemical groups that are: alkaloids, tannins,
flavanoids, polyphenols, quinones, sterols, terpenes, cardiac glycosides, saponins and
leucoanthocyanins. They have antioxidant power, promote regeneration of tissue, reduce
the permeability of blood capillaries and increase their resistance to haemolysis [21]. The
presence of these chemicals by their properties justifies the resistance of red blood cells of
treated rats with the extract. Indeed saponins and alkaloids have shown anti-anaemic
properties [22]. Alkaloid inhibits cyclic adenosine monophosphate (cAMP)
phosphodiesterase thereby accumulating cAMP. This effect stimulates phosphorylation of
proteins and synthesis of proteins, which improves erythropoietin [23]. Saponins are also
known to inhibit platelet aggregation and thrombosis. Saponin containing in herbs have
been successfully used in the management of liver inflammation, as tonic sedative
formulas, to promote and vitalize blood circulation [24, 25]. Since saponins are active
agents which lyse the membrane of red blood cells or other wall, it is likely that red blood
cells were first lysed by the plant. Then the cells have overcome this inhibition by
producing a glycoside enzyme which cleaves some of the terminal sugars from the
saponin, which causes its detoxification [26]. This detoxification of saponins has
reinforced the proper use of iron contained in the ethanolic extract of T.erecta flower
allowing to synthesize heme / haemoglobin for new red blood cells, thus leading to an
improvement of Hb, RBC and PCV. Saponins especially terpenes glycosides enhance the
natural resistance and have the recovery powers of body [27]. Also, flavanoids have an
anti-anaemic potential and veinotonic property, which protects the blood capillaries [21].
The anti-anemia potential and haemoglobin restoring effect of ethanolic extract of Tagetes
erecta flower as suggested by the data in this study could be attributed in part to its
phytochemical constituents.
As regards the weight of the rats, there was a reduction in body weight after induction of
anaemia by phenylhydrazine [Table 2]. This observation is in agreement with the previous
report of Saimak [28]. The loss of body weight is one of the symptoms of anaemia. This

would be due to lack of appetite in anaemic rats. During treatment these rats resumed
appetite thus promoting body weight gain. This decrease in body weight in anemic rats
could be explained by a reduction of the activities of disaccharidases (enzymes that
catalyze the last stage of carbohydrate digestion) in anemic rats [29, 30]. The ethanolic
extract of Tagetes erecta flower has better improved the percentage of weight gain in
treated rats during the study period. This improvement in the percentage of weight gain of
rats treated of the ethanolic extract is in line with that of the anti-anemic rats that received
antianemic of reference Vit B12.
The intra peritoneal administration of 40 mg / kg / day of phenylhydrazine for 2 days (D0

and D1) in Wistar rats caused a significant mean decrease of the concentration of
haemoglobin, red blood cells and the packed cell volume (PCV). Our results are similar to
those of Ryu and Yook [31] who observed a decrease of number of blood cells and
haematocrit (50% and 55% respectively) with a phénylhydrazine administration at 40 mg /
kg / day for 4 days in Sprague Drawley rats. The rats of groups IV and V received the
extracts of the plant at the same time as the phenylhydrazine administration (from day D0).
The treatment from the day D0 allowed red blood cells from the beginning to develop a
resistance vis a vis of PHZ and the extract containing the saponin. Rats of Groups IV and
V were treated after the administration of PHZ (from day D2), only the ethanolic extract
administered to rats of group IV showed a significant increase (P <0.01) haemoglobin
content after the first week of treatment. Considering the results of the groups IV and V,
the (200mg) ethanolic extract of T.erecta flower has a higher anti-anaemic potential than
that of ethanolic extract (100mg). In addition, Vit B12 reference drug showed a significant
increase (P <0.01) of the content in haemoglobin after the first week of treatment. The
anti-anemic effect of the aqueous extract of T.erecta flower was comparable to that of
Vit B12.

8. CONCLUSION:
The injection of phenylhydrazine to rats caused a haemolytic anemia characterized by
reducing haematological parameters. The oral administration of ethanolic extract of
Tagetes erecta in the dose of 100 mg/kg/day and 200 mg / kg / day significantly increased
haemoglobin level in the first week of treatment. The anti-anaemic effect of the 200
mg/kg/day ethanolic extract was more pronounced than that of the 100 mg/kg/day
ethanolic extract (haemoglobin content against at the fourth week). The
anti-anaemic potential of the plant could come from Phytochemical and also the possible
vitamin and mineral constituents.

REFERENCES:
1. Lokesh J Shetty, 2015, et. al. worked on pharmacological screening, of marigold.
2. R. Islam1, S.M.H. Rashid, 2004, et. al. worked on effects of hematinics on body
weight and some hematological values in sheep and goats.
3. G. Gopi1, A. Elumalai, 2012, et. al. worked on a concise review on Tagetes erecta.
4. G. Gopi1, A. Elumalai, 2012, et. al. Worked on hematopoietic drugs.
5. Helen Fennimore, 2013, et. al. Worked on hemorrhage-adjusted iron requirements,

hematinics and Hepcidin define hereditary hemorrhagic Telangiectasia as a model
of hemorrhagic iron deficiency
6. Abdul-Wasea a. Asrar et al., 2010, worked on alleviation of drought stress of
marigold (Tagetes erecta) plants by using arbuscular mycorrhizal fungi.
7. Tanveer hussain, 2013 et al., worked on proximate analysis, phytochemical
screening and antioxidant activity of Tagetes erecta flower.
8. V.b. Pratheesh, Nify Benny, 2007, et. al. worked on isolation, stabilization and
characterization of xanthophylls from marigold flower- Tagetes erecta-l.
9. Pin zhang, li Zeng, 2011, et al., worked on Karyotype studies on Tagetes erecta
l. And Tagetes patula l.
10. P.ghosh and P.pal. 2008, et al., worked on performance of tagesta erecta under
various influences.
11. Sonja Nicholson, Llorna Cox, 2008 et al., worked on appendix n methods of blood
analysis and quality control.
12. Luís cláudio Paterno silveira1 2009 et al., worked on marigold (Tagetes erecta l.)
As an attractive crop to natural enemies in onion fields
13. Devika regaswamy and Justin koilpillai 2014 et al., worked on physicochemical
screening of Tagetes erecta Linn.
14. Nagaraj b1, Krishnmurti nbl 2012 et al., worked on green synthesis of gold
nanoparticles using Tagetes erecta l. (Mari gold) flower extract & evaluation of
their antimicrobial activities.
15. Home and garden information center marigold 2015.
16. Sayema aefin, Md towhidul 2015 et al., worked on proximal analysis, phytochemical screening and
antioxidant activity of Tagetes erecta flower grown in coastal areas of Bangladesh.

17. D. Vidhya sagar swamy 2002 et al., worked on bioactive phytochemical screening from marigold
18. Pankaj gupta and Neeru vasudeva 2012 et al., worked on Marigold a potential ornamental plant dru
19. Pasch, J. H. and von Elbe, J. H. 1979 et al.,Betanine stability in buffered solutions
containing organic acids, metal cations, antioxidants, or sequestrates, Journal of
Food Science.
20. Philip, T 1997 et al., Purification of lutein-fatty acid esters from plant materials.
US Patent 4.
21. Gregory, G.K., Chen, T.S. 1986 et al.,. Quantitative analysis of lutein esters in
marigold flowers by high performance liquid chromatography. Journal of Food
Science,
22. Mabry, T.J; Drieiding. A.S. 1968 et al., the Betalaines. In Recent Advances in
Phytochemistry,
23. Madhavi, DL, Kagan DI. 2002 et a., Process for the isolation of mixed caretenoids
from plants. US Patent
24. Verghese, J. 1998 et al., Focus on xanthophylls from Tagetes Erecta L the giant
natural complex-I. Indian Spices.
25. Khachik, F. 1995 et al., Process for isolation, purification, and recrystallization of
lutein from saponified marigold oleoresin and uses thereof. US Patent
26. Ali, M.H., 1992 et al., A comparative study of hematinics in goat. M. Sc. Thesis,
Bangladesh Agricultural University, Mymensing, Bangladesh Bostedt, H., 1979.
Effect of iron dextran on the development of hemogram in artificially rared lambs.
Berliner and Munchener Tierazlliche.
27. Tauson, A.H. and M. Neil, 1993 et al., Vitamin B12 supplementation to Mink
(Mistela vison) in the prevention of feed induced iron deficiency anemia. 1. Effect
on growth performance and fur quality characteristics. Acta Agric. Scandinavica.
Section A, Anim. Sci.
28. Shetty LJ. Harikiran H, Fernandes J. 2009 et al., Pharmacological evaluation of
ethanolic extract of flowers of Tagetes erecta on epilepsy. Journal of Pharmacy
Research.

29. Shinde NV, Kanase KG, 2009, et al., Antinociceptive and anti-inflammatory
effects of solvent extracts of Tagetes erecta Linn (Asteraceae). Trop. J. Pharm. Res

1.1 Anemia: Modern Institute of Pharmaceutical Sciences Indore

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Anti-anemic activity of hydroalcholic extracts of Tagetes erecta

flower on phenylhydrazine- induced anaemic rats

1.1.1 Type of anaemia

Modern Institute of Pharmaceutical Sciences Indore Page 1

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h) Sickle cell anemia

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Synthetic Drug Used For the Treatment Of Anemia

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Herbal drugs used in the treatment of anemia

Punarnava is a wonderful Ayurvedic remedy with diuretic It is an excellent kidney

Dosage -1-2 tablets twice or thrice daily, after meals.

Dosage - 1-2 tablets twice or thrice daily, after meals.

Modern Institute of Pharmaceutical Sciences Indore Page 5

It is a classical Ayurvedic formulation for weakness due to vata disorders and

Dosage -1-2 tablets twice or thrice daily, after meals.

D. Wheat Grass powder

Modern Institute of Pharmaceutical Sciences Indore Page 6

2.1 Review on biological activity of the plant

 Effects of Haematinics on Body Weight and Some Haematological Values in animals

Modern Institute of Pharmaceutical Sciences Indore Page 7

 The bioactive components (secondary metabolites) of medicinal plants Tagetes

Modern Institute of Pharmaceutical Sciences Indore Page 8

2.2 Review on chemical constituents of the plant

 the effects of lutein, zeaxanthin and meso-zeaxanthin on macular pigment optical

Modern Institute of Pharmaceutical Sciences Indore Page 9

oleoresin were performed and Trans-Lutein identified as the major constituent.

 Lutein is oxycarotenoid/xanthophylls containing two cyclic end groups and the

2.3 Review on hematinics

 Samples of coagulated and ethylenediaminetetraacetate (EDTA) anticoagulated

 Hematopoiesis (formation of blood) is a complex process of proliferation,

Modern Institute of Pharmaceutical Sciences Indore Page 10

The reasons for choosing Tagetes erecta are:-

The outcome of the study would be

Modern Institute of Pharmaceutical Sciences Indore Page 11

1) Collection, identification and drying of drug

2) Extraction of drug by soxhlet extraction method (Hydroalcoholic)

 Qualitative chemical evaluation

 Determination of percentage yield

 Acute toxicity study (fixed dose)

Modern Institute of Pharmaceutical Sciences Indore Page 12

5. MATERIALS AND METHODS

Species: Tagetes erecta

English: Marigold (Indian medicinal plants 1468)

Synonym: Calendula officinalis, pot marigold

Origin: India, Africa.

Height: 6 inches to 3 feet

Spacing: 18-24 in. (45-60 cm)

Sun Exposure: Light Shade

Bloom color: yellow and gold to orange, red and mahogany.

Bloom Time: Late Winter/Early Spring

Modern Institute of Pharmaceutical Sciences Indore Page 13

Fig no.1 Tagetes erecta plant

Foliage: Rich dark green and scented

Other details: Average Water Needs

Distribution: Cultivated throughout India, perhaps a native of Bengal, southern India

Leaves: 3.8 -7.5 by 2.2 -3.8 cm ovate, or elliptic, dark green.

Flower: several striped, bicoloured, finely cut, fernlike. 2-2.5 cm long.

Soil PH: 6.2- 6.5

Modern Institute of Pharmaceutical Sciences Indore Page 14

5.2 COLLECTION AND IDENTIFICATION

 Collection Time: August, 2016

 From: Herbal garden MIPS