PHYTOCHEMICAL INVESTIGATION AND INVITRO

ANTI-OXIDANT POTENTIAL OF ACHYRANTHES ASPERA LINN

Thesis submitted to

FACULTY OF PHARMACY
OSMANIA UNIVERSITY, HYDERABAD

Submitted by:

J. SHRIYA KUMARI(256221881037)
K. SHIVANANDU(256221881038)
K. SURESH(256221881039)
K. VARDHAN REDDY (256221881040)
K. AKSHITHA (256221881041)
In partial fulfillment for the award of the degree of

BACHELOR OF PHARMACY
Under the guidance of

Dr. DIVYA YADA, M. Pharm, Ph. D

Department of Pharmaceutics

MALLA REDDY COLLEGE OF PHARMACY, SECUNDRABAD-500100 (T.S.)

FEBRUARY-2025

1
 DECLARATION BY THE CANDIDATE

I hereby declare that this thesis entitled PHYTOCHEMICAL INVESTIGATION AND INVITRO
ANTI OXIDANT POTENTIAL OF ACHYRANTHES ASPERA LINN is the result of original work
carried out by [Link] KUMARI, [Link], [Link], [Link],
[Link] in Malla Reddy College of Pharmacy under the guidance of Dr. DIVYA YADA, M.
Pharm, Ph.D. Professor, Department of Pharmaceutics for submission to Osmania University for
the award of the degree of Bachelor of Pharmacy. This work has not been submitted earlier in part or
full to any other university or college for the award of any other degree.

Place: MAISAMMAGUDA

Date:

(J. SHRIYA KUMARI)

(K. SHIVANANDU)

(K. SURESH)

(K. VARDHAN REDDY)

(K. AKSHITHA)

2
 CERTIFICATE

This is to certify that the thesis entitled PHYTOCHEMICAL INVESTIGATION AND INVITRO
ANTI OXIDANT POTENTIAL OF ACHYRANTHES ASPERA LINN has been carried out by
J. SHRIYA KUMARI, [Link], [Link], [Link] REDDY, [Link]
Under my supervision for partial fulfilment for the award of degree BACHELOR OF PHARMACY.
This work has not been submitted earlier to any university either in part or in fulfilment of the award
of any degree.

Signature of the Guide

Dr. Divya yada, M. Pharm, Ph.D.

Associate Professor

Secunderabad-500010

3
 CERTIFICATE

This is to certify that the thesis entitled PHYTOCHEMICAL INVESTIGATION AND INVITRO
ANTI OXIDANT POTENTIAL OF ACHYRANTHES ASPERA LINN has been carried out by
[Link] KUMARI,[Link],[Link],[Link] REDDY,[Link] in
Malla Reddy College of Pharmacy for the partial fulfillment of Practice school in BACHELOR OF
PHARMACY.

Signature of HOD

Dr. Aneesa Fathima, M, Pharm, Ph.D.

Professor

Malla Reddy College of Pharmacy

Secundrabad-500100

4
 CERTIFICATE

This is to certify that the thesis entitled PHYTOCHEMICAL INVESTIGATION AND INVITRO
ANTI OXIDANT POTENTIAL OF ACHYRANTHES ASPERA LINN has been carried out by
[Link] KUMARI,[Link],[Link],[Link] REDDY,[Link] in
Malla Reddy College of Pharmacy for the partial fulfillment for the award of degree BACHELOR
OF PHARMACY.

Signature of Principal

Dr. M. Sudhakar, M. Pharm, Ph.D.

Professor & Principal
Malla Reddy College of pharmacy
Secunderabad- 500100

5
 ACKNOWLEDGEMENT

I consider this as an opportunity to express my gratitude to all the dignitaries who have been involved
directly or indirectly with the successful completion of this dissertation. The satisfaction that
accompanies the successful completion of any task would be incomplete without mention of the people
who made It possible with constant guidance, and encouragement that crows all efforts with success. I
would like to express my appreciation for all our efforts to my beloved family who has always supported
my ideas wholeheartedly.
Many thanks to almighty Parents, for if they began this work in me and carried it to completion. It is He
who was blesses me with the people whose names I feel privileged to mention here first, I consider it a
real privilege to express my heartfelt gratitude and sincere thanks to my esteemed guides [Link]
YADA, [Link], Ph.D., Assoc. Professor, Department of Pharmaceutical chemistry, Malla Reddy
College of Pharmacy, Hyderabad for their valuable suggestion, encouragement, motivation, guidance
and cooperation during my thesis work.
I consider myself to be very fortunate to have Dr. M. SUDHAKAR, M. Pharm, Ph.D., Principal,
Malla Reddy College of Pharmacy, who with his dynamic approach boosted my morale, which helped
me to a very great extent in the completion of this dissertation. I express my deep gratitude to other
faculty of Malla Reddy College of Pharmacy for valuable guidance during the course of study.
I take this opportunity to thank the Librarian MR. VIJAYA RAJ (MRCP), for his support and help. I
also express my special thanks to Ms. Mamatha for her help to get chemicals and non-teaching staff
and office staff of MRCP, for their direct and indirect contributions during the dissertation.

6
 TABLE OF CONTENTS

SNO. NAME OF THE CHAPTER PAGE NO.

1. 11-21
INTRODUCTION

2. 22-24
LITERATURE REVIEW

3. AIM & OBJECTIVES 25-26

4. EXPERIMENTAL SECTION 27-33

5. RESULTS AND DISCUSSION 34-39

6. CONCLUSION AND SUMMARY 40-41

7. REFERENCES 42-44

8. ANNEXURE 45

7
 LIST OF TABLES

SNO. NAME OF THE TABLE PAGE NO.

Table 1 Percentage yield of various extracts of 35

Achyranthes aspera

Table 2 Phytochemical analysis of various extracts of 35

Achyranthes aspera

Table 3
Ferric reducing power activity of Ethanolic 39
extract of Achyranthes aspera Linn

8
 LIST OF FIGURES

SNO. NAME OF THE FIGURE PAGE NO.

Figure 1 Achyranthes aspera 13

Figure 2 Calibration Curve Of Gallic Acid 36

37
Figure 3 Calibration Curve Of Quercetin

Figure 4 The Reaction of Alkaloids With BCG 37

Figure 5 calibration curve of atropine 38

Figure 6
DPPH free radical scavenging activity 38

Figure 7 Total Antioxidant Activity Of Ethanolic 39

Extract

9
 ABSTRACT

Antioxidants are renowned protective molecules that are significant in the management of many
chronic diseases. They can support the free radical mechanism to quench the cellular stress to
prevent oxidative damage and improve existing therapy by playing a vital role in protection
through antioxidant activities. The family Amaranthaceae is a well-known family for various
health benefits. The selected plant Achyranthes aspera was also cited in the modern and
traditional literature for their diversified health applications and pharmacological properties. So,
the current work aims to screen the total phenolic, flavonoid, and alkaloid content of fractions
made with solvents with various polarities and to screen their detailed antioxidant potential
through DPPH assay, reducing power assay, and total antioxidant activity. It is inferred from the
results that the ethanolic extract was rich in antioxidant components that are statistically
significant with the reference standards.

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 CHAPTER 1
INTRODUCTION

11
 INTRODUCTION

Knowledge of herbs has been handed down from generation to generation for thousands of years [1].
Herbal drugs constitute a major part in all traditional systems of medicines. Herbal medicine is a triumph
of popular therapeutic diversity. Plants above all other agents have been used for medicine from time
immemorial because they have fitted the immediate personal need, are easily accessible and inexpensive
[2]. In the recent past there has been a tremendous increase in the use of plant based health products in
developing as well as developed countries resulting in an exponential growth of herbal products
globally. An upward trend has been observed in the research on herbals. Herbal medicines have a strong
traditional or conceptual base and the potential to be useful as drugs in terms of safety and effectiveness
leads for treating different diseases. World Health Organization has made an attempt to identify all
medicinal plants used globally and listed more than 20,000 species [3].

According to the WHO more than 80 % of the world’s population relies on traditional herbal medicine
for their primary health care [4]. Plants continue to serve as possible sources for new drugs and
chemicals derived from various parts of plants [5]. In recent time there has been a marked shift towards
herbal cures because of the pronounced cumulative and irreversible reactions of modern drugs.
However, due to over population, urbanization and continuous exploitation of these herbal reserves, the
natural resources along with their related traditional knowledge are depleting day by day [6].

In the present era of drug development and discovery of newer drug molecules many plant products are
evaluated on the basis of their traditional uses. One of the many plants which are being evaluated for
their therapeutic efficacies is Achyranthes aspera which is commonly known as Latjeera (Hindi) &
Rough Chaff tree (English). It is an erect or procumbent, annual or perennial herb, 1-2m in height, often
with a woody base, commonly found as a weed of waysides, on roadsides [6] [7] [8]. Although it has
many medicinal properties, it is particularly used spermicidal [10], antipyretic [11] & as a cardiovascular
agent [9].

12
 Figure 1: Achyranthes aspera

TRADITIONAL USES:

Traditionally, the plant is used in asthma and cough. It is pungent, antiphlegmatic, antiperiodic, diuretic,
purgative and laxative, useful in oedema, dropsy and piles, boils and eruptions of skin etc. Crushed plant
is boiled in water and is used in pneumonia. Infusion of the root is a mild astringent in bowel complaints.
The flowering spikes or seeds, ground and made into a paste with water, are used as external application
for bites of poisonous snakes and reptiles, used in night blindness and cutaneous diseases [13]. For snake
bites the ground root is given with water until the patient vomits and regains consciousness. Inhaling
the fume of Achyranthes aspera mixed with Smilax ovalifolia roots is suggested to improve appetite
and to cure various types of gastric disorders [14]. It is useful in haemorrhoids, leaves and seeds are
emetic, hydrophobia, carminative, resolve swelling, digestive and expel phlegm. Ash of the plant is
applied externally for ulcers and warts. The crushed leaves rubbed on aching back to cure strained back
[15]. A fresh piece of root is used as tooth brush. Paste of the roots in water is used in ophthalmia and
opacities of the cornea. Paste of fresh leaves is used for allaying pain from bite of wasps [12]. The plant
is useful in liver complaints, rheumatism, scabies and other skin diseases. It also possesses tranquillizing
properties [16] [17]

Traditional methods:

Achyranthes aspera (prickly chaff flower) vary based on regional practices, but the plant's parts leaves,
roots, seeds, and stems are commonly utilized in different forms for therapeutic purposes. Here are some
traditional methods:
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1. Leaf Paste for External Application:

For Wounds and Inflammation: The fresh leaves are crushed to make a paste, which is then applied
directly to cuts, bruises, or inflamed areas of the skin. The paste helps reduce pain, swelling, and speeds
up the healing process.

For Skin Problems: A paste made from the leaves is also used for conditions like eczema, rashes, or
itching. The anti-inflammatory and antimicrobial properties of the leaves are believed to provide relief.

2. Root Decoction for Internal Use:

For Fever and Digestive Issues: A decoction made from the roots is commonly consumed to treat
fever, indigestion, or diarrhea. To prepare, the roots are boiled in water until reduced, then the liquid is
strained and consumed. This decoction can also be used to treat dysentery.

For Urinary Disorders: A decoction of the root is traditionally used to treat kidney stones, urinary tract
infections, and as a diuretic to increase urine flow.

3. Powdered Seeds for Digestive Aid:

As a Laxative: The seeds of Achyranthes aspera are dried and ground into a fine powder. This powder
is sometimes mixed with water or honey and consumed to relieve constipation, as it has mild laxative
effects.

For Stomach Upset: The powdered seeds may also be mixed with other herbs to treat digestive
disturbances like bloating or indigestion.

4. Leaf Juice for Respiratory Health:

For Cough and Asthma: Fresh leaf juice is traditionally taken to help treat respiratory issues like
asthma, bronchitis, or persistent cough. The juice is typically consumed in small quantities, often
combined with honey or other herbs to enhance its efficacy.

As an Expectorant: The juice is also believed to help loosen mucus from the lungs, making it easier to
expel

5. Leaf and Root Infusion for Fever and Pain Relief:

As a Fever Reducer: An infusion or tea made from the leaves and roots is a traditional remedy for
fever. The mixture is boiled and strained, then drunk for its cooling and fever-reducing effects.

Pain Relief: The infusion is also used to relieve body aches and pains associated with conditions like
arthritis or general inflammation.

14
6. External Application for Insect Bites and Stings:

For Pain and Swelling: The leaves or root are crushed and applied to the affected area for relief from
insect bites, stings, or mild skin irritation. This is often used in rural areas where access to modern
treatments may be limited.

7. Traditional Tonic for General Health:

For Energy and Vitality: In some cultures, an infusion of Achyranthes aspera (especially the leaves
or roots) is consumed as a tonic to improve energy levels, boost immunity, and enhance overall vitality.

8. Seed Paste for Joint Pain:

For Arthritis: The seeds are sometimes ground into a paste and applied to affected joints to reduce
inflammation and provide relief from pain caused by conditions like arthritis.

15
 ACHYRANTHES ASPERA:

INTRODUCTION:

Herbal medicine has a long history in treatment of several diseases [18]. Medicinal herbs are used to
treat illness, maintain and promote health[19]and are the only available and inexpensive source of
primary health care, especially in the absence of access to modern medical facilities. Various plant parts
(leaves, stems, root and bark) are used for medicine preparation, because they contain biologically active
ingredients, for treating mild or chronic ailments. Alkaloids, tannins, flavonoids, and phenolic
compounds are the most important bioactive constituents of plant. Sometimes they are also added to
foods of pregnant and nursing mothers for medicinal purposes [20]. Now-a-days pills, capsules, powders
or in more concentrated liquid form called extracts and tincture forms of herbal medicines are also
available. These can also be used topically in creams or applied directly to the skin [21].

16
Achyranthes aspera Linn. (family Amaranthaceae) is commonly known as Latjira in Hindi. It is an erect
stiff medicinal plant. The plant is used by traditional healers for the treatment of dysentery, fever and
diabetes [22]. A. aspera is available as weed in whole India, Asia and many parts of the world such as
Mexico, Central America and Africa [23]. It is described as bitter, pungent, purgative, heating, laxative,
stomachic, carminative and digestive and is useful for the treatment of bronchitis, heart maladies, piles,
itching abdominal problems, ascites, rheumatism, abdominal enlargement, rabies and for enlarged
cervical gland [24] [25]. The aim of present review is to provide entire available information regarding
pharmacology and ethnobotanical uses of A. aspera.

History and Origin:

Two varieties of A. aspera, red and white are mentioned in Ayurvedic and Chinese medicines.
Achyranthes aspera as a rough flowered stalk is described as in Sanskrit synonyms. It is described in
'Nighantas' as pungent, purgative, digestive, and a remedy for inflammation of the internal organs, itch,
piles, abdominal enlargements and enlarged cervical glands. The diuretic property of the plant was well

17
known to the natives of India and European physicians. Various plant parts form ingredients in many
native prescriptions were used in combination with more active remedies. The plant is globally available
as a medicinal weed in Baluchistan, Ceylon, Tropical Asia, Africa, Australia and America. It is reported
as an invasive alien species in northern Bangladesh. It is also found to be the most prevalent weed in
Shivbari, Himachal Pradesh and an exotic medicinal herb of district, Lalitpur (Uttar Pradesh), India .
Throughout India A. aspera is found in field boundaries, road sides and waste places as a medicinal
herb.

Scientific taxonomic classification:

Kingdom - Plantae

Subkingdom - Tracheobinota

Super Division - Spermatophyta

Division - Mangoliophyta

Class - Mangoliophsida

Subclass - Caryophyllidae

Order - Caryophyllales

Family - Amaranthaceae

Genus - Achyranthes

Species – aspera

Common names of Achyranthes aspera:

African vernacular names: Swahili- Turura, Sotho-Bohomane, Bohome-bo-bolek, Mo-tswarak-gano,

Belgian Congo-Denge, Gnegna, Kalambata, Lenge, Lenamo.

Arabian - Atkumah, Na´eem, No´eim, Mahout, Wazer (Yemen)

Ayurvedic - Apamarga, Chirchita, Shikhari, Shaikharika

Bengali - Apaang.

English - Prickly chaff flower, Hawai chaff flower, Devil´s horse whip, Prickly chaff flower

French - Achyranth a feuilles rudes, Collant, Gendarme

Gujarati - Safad Aghedo, Anghadi, Andhedi, Agado.

18
Hindi - Latjira, Chirchira, Lamchichra, Sonpur, Onga.

Kannada - Uttaranee, Utame.

Latin - A. aspera

Malayalam - Kadaladi, Vankadaladi, Katalati.

Marathi - Aghada, Pandhara-aghada.

Punjabi - Kutri

Sanskrit - Aghata, Apamargah, Mayoorah, Markatapippalee, Durgrahan, Khara-manjari

Tamil - Shiru-kadaladi, Nayuruvi

Telugu– Uttaraene, Utareni, Aduchinnike, Antisha, Pamargamu, Uttaraene.

Unani – Chirchitaa.

Geographical Distribution:

It is found on road sides, field boundaries and waste places as a weed throughout India up to an altitude
of 2100 m and in South Andaman Islands. The plant is also widespread in Baluchistan, Ceylon, Tropical
Asia, Africa, Australia and America. It was reported as an invasive alien species in northern Bangladesh
It was found to be the most prevalent herb in Shivbari sacred grove of Himachal Pradesh, India and an
exotic medicinal plant of district Lalitpur, Uttar Pradesh, India.

[Link] and Soil Requirements:

Climate: Achyranthes aspera thrives in warm, tropical climates but can also grow in subtropical
regions. It requires full sun to partial shade.

Soil: It prefers well-draining, loamy to sandy soil. The plant is quite adaptable and can grow in poor
soils, including those with slightly acidic to neutral pH levels.

[Link]:

Seeds: This plant is most commonly propagated by seeds. The seeds are small and should be sown
directly into the soil.

Sowing: Sow the seeds in shallow furrows or scatter them on the surface and lightly cover with soil.
Seeds should be spaced 20-30 cm apart.

19
Germination: Achyranthes aspera seeds typically germinate in 7-10 days under ideal conditions
(warm temperatures and consistent moisture).

Cuttings: In some cases, the plant can also be propagated via cuttings. Select a healthy stem, cut it to a
few inches long, and plant it in the soil.

[Link]:

Regular watering is essential, especially during the dry season. However, the plant is somewhat
drought-tolerant once established. Avoid waterlogging, as it can harm the roots.

[Link]:

Achyranthes aspera grows well in fertile soil, but over-fertilizing may lead to excessive foliage growth
at the expense of flowering.

Use organic compost or a balanced fertilizer to improve soil health during planting.

[Link] and Maintenance:

Weeding: Regular weeding is necessary to prevent competition for nutrients and space.

Pruning: Prune back any leggy or overgrown stems to encourage bushier growth.

Pest Control: While not typically prone to serious pest issues, aphids, and caterpillars may occasionally
affect the plant. Use organic insecticides if necessary.

6. Harvesting:

Achyranthes aspera flowers bloom from late summer to fall. The leaves, stems, and roots are often
harvested for medicinal uses.

Leaves and Stems: Can be harvested once the plant is well-established (around 3-4 months after sowing).
For medicinal purposes, harvest leaves and stems by cutting them gently with pruning shears.

Roots: If using the roots, dig them out carefully to avoid damaging the plant too much.

[Link] and Diseases:

The plant is relatively resilient, but common pests like aphids or whiteflies may appear. In general,
Achyranthes aspera is not highly susceptible to diseases.

20
[Link]:

This plant is often used in traditional medicine for its anti-inflammatory, diuretic, and wound-healing
properties. It can be used as a decoction, poultice, or in herbal remedies for a variety of ailments.

Pharmacological actions:

Spermicidal Activity:

Extracts from roots of Achyranthes aspera have been reported to possess spermicidal activity in human
and rat sperm, as studied by Paul et al. (2010). Study was made on hydroethanolic, n-hexane and
chloroform extracts, which were found to be most effective for sperm immobilization, sperm viability,
acrosome status, 5’-nucleotidase activity and nuclear chromatin decondensation. Vasudeva and Sharma
(2006) reported the ethanolic extract of the root of Achyranthes aspera shows post coital antifertility
activity in female albino rats. According to their study, the extract exhibited 83.3% anti-implantation
activity when given orally at 200 mg/kg body weight.

Antiparasitic Activity:

Ethyl acetate extracts of A. Aspera have been proved to contain anti parasitic activity by Zahir et al.
(2009). It has been studied that dried leaf, flower and seed extract of A. Aspera are active against the
larvae of cattle tick Rhipicephalus (Boophilus)microplus (Acari:lxodidae),sheep internal parasite
Paramphistomum cervi.

Hypoglyceamic and Cancer Chemo preventive Activity:

Aqueous methanolic extract of the whole plant have been shown to possess hypoglycaemic activity by
Akhtar and Iqbal (1991). Methanolic extracts from leaves of Achyranthes aspera have been proved to
have cancer preventive action on Epstein- Barr virus early antigen activation induced by tumor promoter
12-O-tetradecanoylphorbol-13-acetate in Raji cells, as reported by Chakraborty et al. (2002).

Hepatoprotective Activity:

Bafna and Mishra (2004) reported that the methanolic extract of the aerial parts of Achyranthes aspera
shows hepatoprotective activity on rifampicin induced hepatotoxicity in albino rats. Methanolic extract
showed dose dependent decrease in the levels of SGPT, SGOT, ALKP and total bilirubin

21
 CHAPTER 2
LITERATURE REVIEW

22
 LITERATURE REVIEW

Priyamvada et al.,(2021) estimated investigation to screen the phytochemicals in the methanolic and
petroleum ether leaf extracts of Achyranthes aspera and evaluate their antidiabetic and antioxidant
activities by using an in vitro model. Phytochemical screening of the extracts revealed the presence of
alkaloids, tannins, saponins, flavonoids, terpenoids, phenols and steroids. The results of α–amylase and
α–glucosidase enzymes inhibition activities were found in a dose-dependent manner. The strongest
activity was showed by methanolic fraction (55.0±0.50% for α–amylase and 53.06±0.23% for α–
glucosidase inhibition at 160 μg/ml) compared to petroleum ether extract (51.87±00% for α–amylase
and 46.0±0.22% for α–glucosidase inhibition at 160 μg/ml). The plant extracts were also examined for
its antioxidant activities by using DPPH scavenging method. The DPPH assay exhibited significant
antioxidant activity of isolated phytochemical compounds’. The DPPH radical scavenging activity of
the methanolic extract (68±0.44% at 250 μg/ml) was higher than that of petroleum ether extract
(63.06±0.56% at 250 μg/ml). Thus, in conclusion this study can recommend this plant due to the
presence of antioxidant components which have potential prospective for the control of diabetes and the
related condition of oxidative stress. This knowledge will be helpful in exploring more potent
antidiabetic principle from the natural resources for the clinical development of antidiabetic
therapeutics.

Sandeep Gupta et al (2013) investigated antimicrobial properties of methanolic and aqueous extracts
of leaf, stem, root and flower of Achyranthes aspera against bacteria namely Pseudomonas aeruginosa
(MTCC -7296), Staphylococcus aureus (MTCC- 7443), Salmonella typhi (MTCC- 733),
Mycobacterium tuberculosis (MTCC-300) and fungi namely Microsporum canis (MTCC- 2820),
Epidermophyton floccossum (MTCC-613), Trichophyton rubrum (MTCC-296) and Aspergillus
candidus (MTCC-1989).The methanolic extract of flower of Achyranthes aspera, showed zone of
inhibition against Salmonella typhi, Pseudomonas aeruginosa and Staphylococcus [Link] highest
zone of inhibition (08.99mm) was found against Salmonella typhi. Both the extract of root (methanolic
and aqueous) and methanolic extract of stem was found effective against Epidermophyton floccossum,
whereas only aqueous extract of root and stem was found effective against Microsporum canis. The
aqueous extract of leaf and flower and methanolic extract of stem was found with potential antifungal
property against Aspergillus candidus. The highest zone of inhibition (17.19 mm) was found in the
aqueous extract of flower against Aspergillus candidus. Present finding pave the path for further
investigation for identification of specific molecule against specific organism

23
.

Hemanta Mukherjee et al., ( 2013) aimed this study to evaluate the antiviral potential of methanolic
extract (ME) of Achyranthes aspera, an Indian folk medicine and one of its pure compound oleanolic
acid (OA) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The ME possessed weak
anti-herpes virus activity (EC50 64.4 μg/ml for HSV-1 and 72.8 μg/ml for HSV-2). While OA exhibited
potent antiherpesvirus activity against both HSV-1 (EC50 6.8 μg/ml) and HSV-2 (EC50 7.8 μg/ml). The
time response study revealed that the antiviral activity of ME and OA is highest at 2–6 h post infection.
The infected and drug-treated peritoneal macrophage at specific time showed increased level of pro-
inflammatory cytokines (IL6 and IL12). Further, the PCR of DNA from infected cultures treated with
ME and OA, at various time intervals, failed to show amplification at 48–72 h, similar to that of HSV
infected cells treated with acyclovir, indicating that the ME and OA probably inhibit the early stage of
multiplication (post infection of 2–6 h). Thus, our study demonstrated that ME and OA have good anti-
HSV activity, with SI values of 12, suggesting the potential use of this plant.

Edwin, S. et al., (2008) evaluated wound healing and antioxidant activity of ethanol and aqueous
extracts of leaves of Achyranthes aspera. The wound healing activity was studied using two wound
models, excision wound model and incision wound model. The free radical scavenging activity of the
extracts was also assessed using two methods, DPPH radical scavenging activity, and superoxide
scavenging activity. The extracts responded significantly in both the wound models tested. Also, the
plant exhibited good antioxidant effect by preventing the formation of free radicals in the two models
studied. Comparatively, the aqueous extract was found to be more effective. These findings could justify
the inclusion of this plant in the management of cuts by the local people.

Naidu, et al., (2006) investigated In-vitro antimicrobial activity on successive extracts of Achyranthes
aspera herb parts has been investigated for against Staphylococcus aureus, Bacillus subtilis, Escherichia
coli and Salmonella typhi by disc diffusion method. Different solvents such as methanol, acetonitrile,
chloroform and n-hexane were selected for extraction. Ciprofloxacin and Cotrimoxazole 1000 µg/ ml
were used as standard drugs. Methanol extracts of Achyranthes aspera powder inhibited the growth of
Staphylococcus aureus, Salmonella typhi and Bacillus subtilis with inhibition zone diameter of 9 mm,
7 mm and 9 mm respectively. Chloroform extract of Achyranthes aspera plant parts was found to inhibit
S. aureus, S. typhi with zone diameter of 6 mm (leaves), and 6 mm (inflorescence) respectively. N-
hexane extract showed the growth inhibition against S. aureus, E. coli and B. subtilis with 6 mm (stem),
8 mm (inflorescence) and 6 mm (whole plant) respectively.

24
 CHAPTER 3

AIM & OBJECTIVE

25
 AIM & OBJECTIVE
AIM:

To screen the phytoconstituents, In-Vitro studies on In-Vitro antioxidant potential of ethanolic extract of
Achyranthes aspera

OBJECTIVE:

 To prepare various extracts of Achyranthes aspera using microwave irradiation.

 To screen phytoconstituents by using the methods.

 To study Total Flavonoid Content, Total Phenolic Content, Total Alkaloid Content of prepared extracts of
Achyranthes aspera

 To study In vitro anti-oxidant potential of ethanolic extract of Achyranthes aspera

 by DPPH, iron reducing power, Total antioxidant capacity method.

26
 CHAPTER 4

EXPERIMENTAL SECTION

27
 EXPERIMENTAL SECTION
COLLECTION OF PLANT MATERIAL: Whole plant of Achyranthes aspera Linn was collected
from the Hyderabad rural areas and authenticated by Dr. P.V. Prasanna, Scientist “G”,Botanical survey
of India, Hyderabad, Telangana

EXTRACTION:
Microwave Assisted Extraction: Microwave-assisted extraction (MAE) is a technique based on the
use of the microwave energy to help the transfer of the solutes from the matrix into the solvent. As
consequence, extraction yield is increased, whereas time and solvent consumption are decreased.
Procedure of Microwave Assisted Extraction: Preparation of Plant Material: Grind the dried
Achyranthes aspera plant material into a fine powder using a mill or grinder. 
Selection of Solvent: From nonpolar to polar nature.
Preparation of Extraction Vessel: Place the powdered plant material in the extraction vessel and add
the solvent.
Microwave Irradiation: Place the extraction vessel in the microwave oven and irradiate with
microwave energy at a controlled power level (e.g., 300-600 W) and time (e.g., 30-90 seconds).
Cooling and Filtration: Remove the extraction vessel from the microwave oven and allow it to cool.
Filter the extract using filter paper or cloth to separate the solvent from the plant material. Evaporate to
get crude extracts.
PHYTOCHEMICAL SCREENING: The preliminary phytochemical screening of all the extracts of
Achyranthes aspera Linn were performed according to the standard procedures
Qualitative analysis for identification of phytoconstituents:
A. Test for Alkaloids
Extract of each plant sample (0.5gm) was separately stirred with few ml of dilute hydrochloric acid and
filtered. The filtrate was tested with various alkaloidal reagents as
follows:
Dragendorff’s test: To a few ml of filtrate, add 1 or 2ml of Dragendorff’s reagent (Potassium bismuth
iodide solution). A prominent yellow precipitate indicates the presence of alkaloids.
Mayer’s test: To a few ml of filtrate, add 1ml of Mayer’s reagent (potassium mercuric iodide
solution). White or cream-coloured precipitate indicates the presence of alkaloids.
Hager’s test: To a few ml of filtrate, add 2ml of Hager’s reagent (saturated aqueous solution of picric
acid); yellow coloured precipitate indicates the presence of alkaloids.
Wagner’s test: To a few ml of filtrate, add 2ml of Wagner’s reagent (iodine in potassium iodide),
formation of reddish brown precipitate indicates the presence of alkaloids.

28
B. Test for Saponins
Stir each of the plant extract (0.5gm) separately with water in a test tube, foaming which persist on
warming will be taken as an evidence for the presence of saponins.
C. Test for Glycosides
Test A: Extract 200mg of drug with 5ml of dilute sulphuric acid by warming on a water bath. Filter it.
Neutralise the acid extract with 5% solution of sodium hydroxide. Add 0.1ml of Fehling’s solution A
and B until it becomes alkaline (test with pH paper) and heat on a water bath for 2min. Note the quantity
of red precipitate formed and compare with that of red precipitate Test B.
Test B: Extract 200mg of the drug using 5ml of water. After boiling add equal amount of water. Add
0.1ml Fehling’s solution A and B until alkaline and heat on water bath for 2min. Note the quantity of
red precipitate formed. Compare the quantity of precipitate formed in Test B with that of formed in Test
A. If the precipitate in Test A is greater than in Test B, then glycoside may be present. Test B represents
the amount of free reducing sugars already present in the crude drug, where as Test A represents free
reducing sugar plus those related on acid hydrolysis of any glycoside in the crude drug.
D. Test for Carbohydrates
Extract of each sample (100mg) was separately dissolved in 5 ml of water and filtered. The filtrate was
subjected to the following test.
Molisch’s Test: To 2ml of filtrate, add two drops of alcoholic solution of α-naphthol the nightmixture
shake well and add 1ml of concentrated sulphuric acid slowly along the sides of the test tube and allow
to stand. Purple or reddish violet colour ring at the junction of the two liquids reveal the presence of
carbohydrates.
Fehling’s test: Boil one ml of filtrate on a water bath with 1ml each of Fehling solutions A and B.
Formation of a red precipitate indicates the presence of sugars.
Benedict’s test: To 0.5ml of filtrate, add 0.5ml of Benedict’s reagent. Heat the mixture on a boiling
water bath for 2min. A characteristic red coloured precipitate indicates the presence of sugars.
E. Test for Tannins and Phenolic compounds
Ferric chloride test: Dissolve the extract (50mg) in 5ml of distilled water. To this, add few drops of
neutral 5% ferric chloride solution. Formation of a dark green coloured indicates the presence of
phenolic compounds.
Gelatine test: Dissolve the extract (50mg) in 5ml of distilled water and add 2ml of 1% solution of
gelatine containing 10% sodium chloride to it. Formation of white precipitate indicates the presence of
phenolic compounds.
Lead acetate test: Dissolve the extract (50mg) in distilled water and to this add 3ml of 10% lead acetate
solution. Formation of a bulky white precipitate indicates the presences of phenolic compounds.

29
F. Test for Flavonoids
Treat the extract of each sample with a small piece of magnesium ribbon; and add by drop wise
concentrated hydrochloric acid. Formation of colours ranging from orange to red indicates the presence
of flavones, red to crimson indicates flavanols, crimson to magenta indicates flavanones.
G. Test for Steroids
Libbermann-Burchard test: Dissolve 50mg of the extract in a few drops of chloroform, and add 2ml
of acetic anhydride warm and cool and add one or two drops of concentrated sulphuric acid along the
sides of the test tube. Appearance of bluish green colour shows the presence of sterols.
Salkowski test: Dissolve the extract in chloroform and add equal volume of conc. H2SO4. Formation
of bluish red to cherry colour in chloroform layer and green fluorescence in the acid layer indicates the
steroidal components in the tested extract.
H. Test for Proteins and Amino Acids
The extract of each sample (100mg) was dissolved in 10ml of distilled water and filtered through
Whatmann No.1 filter paper and the filtrate was subjected to the following test.
Biuret Test: Treat an aliquot of 2 ml of filtrate with one drop of 2% copper sulphate solution. To this,
add 1ml of ethanol (95%), followed by excess of potassium hydroxide pellets. Pink colour in the
ethanolic layer indicates the presence of proteins
Ninhydrin Test: Add two drops of ninhydrin solution (10mg ninhydrin in 200ml of acetone) to 2ml of
aqueous filtrate. A characteristic purple colour indicates the presences of amino acids.
Millon’s Test: Add few drops of Millon’s reagent to 2ml of filtrate. Formation of white a white
precipitate indicates the presence of proteins.
I. Test for Fixed Oils and Fats
Spot Test: Press a small quantity of each extract between two filter papers. Oil stain on the paper
indicates the presence of fixed oils.
Saponification Test: To a few drops of 0.5N alcoholic potassium hydroxide solution add a small
quantity of extract along with a drop of phenolphthalein. Heat the mixture on a water bath for 2h. The
formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats.

ESTIMATION OF TOTAL PHENOLIC, FLAVONOID AND ALKALOID CONTENT:

Estimation of Total Phenolic Content:
The total phenolic content (TPC) for the Ethanolic extract of Achyranthes aspera (EEAA) was
determined by using Foline- Ciocalteau phenol reagent method [27]
Principle:
The principle of the Folin-Ciocalteu assay is the reduction of the Folin-Ciocalteu reagent in the presence
of phenolics which results in the production of molybdenum-tungsten blue that is measured

30
spectrophotometrically at 760nm and the intensity increases linearly with the concentration of the
phenolics.
Chemicals used:
 Foline -Ciocalteu reagent (FCR)
 Gallic acid Ethanol
 7% Sodium Carbonate
 Distilled water
Briefly, 1.0 mL of the extract at various concentrations was mixed with 2.5 mL of 10% Foline-
Ciocalteau reagent and 2.5 mL of 7.5% sodium carbonate. The contents were thoroughly mixed and
allowed to stand for about half an hour (30 minutes). The absorbance was read at 750 nm in a
spectrophotometer. The total phenol content was expressed as gallic acid equivalents in milligram per
gram of the extract.
Estimation of Total Flavonoid Content:
The flavonoid content (TFC) for Ethanolic extract of Achyranthes aspera was screened using
aluminium chloride colorimetric method[27] . Quercetin was used as a reference standard for plotting a
calibration curve.
Principle:
The basic principle of the Aluminium Chloride colorimetric method is that Aluminium Chloride forms
acid-stable complexes with the C-4 keto groups and either with the C-3 or C-5 hydroxyl groups of
flavones and flavanols.
Chemicals used:
 Aluminium Chloride
 Quercetin
 1M Potassium acetate
 Ethanol
 Distilled water
TFC is determined by the following procedure 0.5mL of the extract at various concentrations was mixed
with 3mL of 95% methanol, 0.1mL of 10% (weight/volume) aluminium chloride, 0.1mL of 1M
potassium acetate, and 2.8mL of distilled water. Let the reaction mixture to stand at room temperature
for about 30 minutes and absorbance was read at 415 nm against the blank sample. A calibration curve
was generated using quercetin in methanol. The flavonoid content was expressed as Quercetin
equivalents in milligram per gram of the extract.
Estimation of Total Alkaloid Content:
The total alkaloid content for Ethanolic extract of Achyranthes aspera was determined by a slightly
modified Bromocresol green (BCG) method. Atropine was used as the reference standard for plotting

31
the calibration curve.
Principle:
A simple spectrophotometric method based on the reaction with Bromocresol Green (BCG) was
developed for the determination of total alkaloids in medicinal plants. A yellow complex forms and is
easily extractable by chloroform at pH 4.7.
Chemicals used:
 Atropine Sulphate
 Bromocresol green (BCG)
 Dimethyl Sulfoxide (DMSO)
 Phosphate buffer (pH- 4.7)
 Chloroform
Procedure:
Sample preparation: Dissolve 1mg of Ethanolic extract of Achyranthes aspera in 1ml of 2N HCl and
add 1ml of Dimethyl sulfoxide then filter it. From the above solution take 1 ml and transfer it into
separating funnel.a5 Add of bromo cresol green solution and add 5 ml of phosphate buffer then the
mixture was shaken with 1ml,2ml,3ml,4ml of chloroform .Collect the chloroform layer after every
shaking then make up the volume up to 10ml with chloroform in 10 ml volumetric flask. Measure the
absorbance at 470 nm
Standard preparation:
Standard drug : Atropine sulphate
Dissolve 100mg of atropine sulphate in 100ml of distilled water then concentration 1mg/ ml is aliquots
of 0.4ml ,0.6ml,0.8ml and 1.2ml are transferred into a separating funnel. Add 0.4ml of concentrated
solution in 5ml of phosphate buffer and then add 5ml of bromocresol green solution, then extract with
1ml ,2ml,3ml,and 4ml of chloroform and make up the volume to 10ml with chloroform. Measure the
absorbance at 470nm.
In-vitro Antioxidant Activity:
DPPH Antiradical capacity:
The free radical potential of Ethanolic extract of Achyranthes aspera’s was determined
spectrophotometrically[28]. A stock solution of DPPH was prepared by dissolving 33mg of DPPH in 1
liter of methanol. Five different concentrations of Ethanolic extract of Achyranthes aspera (20, 40,60,
80 and 100 µg/ml) were mixed with 100 µL of DPPH radical solution in a 96-well microplate and
incubated for 20 min at room temperature. The resultant mixture was read spectrophotometrically at 517
nm against a methanol blank and the following equation was used to calculate the % inhibition of each
extract:
% inhibition= (Ao−As)/ (Ao) ×100

32
Where, A o is the absorbance of the control, and As is the absorbance of the test sample. The IC 50
represented the concentration of the extract that inhibited 50% of radical
Fe Reducing power Determination:
The reducing ability of a compound generally depends on the presence of reductants which exhibits
antioxidant potential by breaking the free radical chain, donating a hydrogen atom. The Fe 2+ reducing
power of plant extract was determined by the method [29]
Principle:
The reducing power assay method is based on the principle that substances that have reduction potential
react with potassium ferricyanide (Fe 3+) converted to potassium ferrocyanide (Fe 2+) which in turn reacts
with ferric chloride to form ferric–ferrous complex ions, which are identified by a change in color from
yellow to bluish-green.
Ethanolic extract of Achyranthes aspera (0.75mL) at various concentrations was mixed with 0.75 ml of
phosphate buffer (0.2 mole, pH 6.6) and 0.75 mL of potassium ferricyanide K 3 Fe(CN) 6 (1%w/v),
followed by incubating at 50°C for 20 mins. The reaction was stopped by adding 2.5mL of 10% (w/v)
trichloroacetic acid followed by centrifugation at 3000 rpm for 10min. Finally, 1.5mL of the upper layer
was mixed with 1.5mL of distilled water and 0.5mL of FeCl 3 (0.1%) and the absorbance was measured
at 700 nm. Higher the absorbance of reaction mixture indicated greater the reducing power. Ascorbic
acid is used as reference compound.
Total Antioxidant Activity:
Total antioxidant activity was estimated by phosphomolybdenum assay [30]
Principle:
This method is based on the reduction of phosphomolybdic acid to phosphomolybdenum blue complex
by sodium sulfide. The obtained phosphomolybdenum blue complex is oxidized by the addition of nitrite
and this causes a reduction in the intensity of the blue colour.
Reagent preparation:
1ml each of 0.6M Sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybate were added
in 20ml of distilled water and made up volume to 50ml by adding distilled water.
Procedure:
Ethanolic extract of Achyranthes aspera in different concentration ranging from 20µl to 100µl were
added to each test tube individually containing 3ml of distilled water and 1ml of molybdate reagent
solution. These tubes were kept incubated at 95ºC for 90 min. After incubation, these tubes were
normalized to room temperature for 20- 30 min and the absorbance of the reaction mixture was measured
at [Link] values from independent samples were calculated for each extract ascorbic acid was
used as positive reference standard.
Total antioxidant=OD of test X concentration of standard in µg X made up volume of sample

33
 CHAPTER 5

RESULTS AND DISCUSSION

34
 RESULTS AND DISCUSSION
Percentage Yield: The percentage yield with all the extracts was calculated and depicted in Table 1.
The Ethanol yielded more quantity, followed by extraction with water.
Table 1: Percentage yield of various extracts of Achyranthes aspera

Solvent % yield

Petroleum ether 3.17

Chloroform 3.35

Ethyl acetate 0.57

Ethanol 5.15

Aqueous 4.05

Phytochemical screening was carried out for all extract of Achyranthes aspera by preliminary tests.
From the results it was evident that, carbohydrates, Alkaloids, steroids, Glycosides, Flavonoids,
Saponins, Phenols, Tannins. Results were shown in Table 2. ‘+’ indicates the presence and ‘-’ indicates
the absence of phytochemical constituents.
Table 2: Phytochemical analysis of various extracts of Achyranthes aspera
Phyto Constituent Pet Ether Chloroform Ethyl Ethanol Aqueous
(PEAA) (CEAA) Acetate (EAEE) (AEAA)
(EAAA)

Alkaloids - + + + -

Saponins - - + + +

Carbohydrates - - - + +

Phenols - - + + -

Flavonoids - - + + -

Steroids + + - - -

Glycosides + + - - -

Terpenoids + + - - -

Quinones - - - + +

Tannins - + - + -

35
Estimation of Total Phenolic Content:
The total phenolic content in various fractions of the whole plant of Achyranthes aspera was determined
using the Folin-Ciocalteau method. The results were expressed in terms of milligram per gram
equivalents of gallic acid .A calibration curve was plotted by the absorbance and concentrations using
prepared dilutions. The regression analysis was performed and the resulting equation was Abs(y) =
0.0041x + 0.028. The coefficient of determination for standard curves was greater than 0.99 (R² =
0.9999). Thus, the calculated straight line could explain more than 99% of the experimental data Fig. 1.
The total phenolic content of the extracts was calculated and expressed as mg gallic acid equivalents
(GAE) per gram of sample in dry weight (mg/g). Where y is the absorbance at 760 nm and x is the total
phenolic content in the extracts. Among the five extracts, ethanolic extract (101.22 ± 0.22) and ethyl
acetate extract (15.87 ± 0.25) has shown the higher and lower amount of phenolic compounds for
Achyranthes aspera Linn.
The concentration of the phenolic compounds was increased with an increase in the dose.

Calibration Curve of Gallic acid

2.5

2 y = 0.0041x + 0.028
R² = 0.9999
Absorbance

1.5

0.5

0
0 100 200 300 400 500 600
Conc(µg/mL)

Figure 2: Calibration Curve Of Gallic Acid

Estimation of Total Flavonoid Content:

Flavonoids scavenge the excess free radicals that cause cellular stress. Flavonoids are polyphenolic
substances obtained from a large number of plants. They exhibit various biological activities such as
antioxidants, antimicrobials, anti-inflammatory, antiallergic and antiviral. The total flavonoid content
profile of the plant extracts was established through the colorimetric method using AlCl 3. A calibration
curve (y = 0.0039x + 0.1428, R² = 0.9987) was plotted using various concentrations of standard
quercetin (15–480 µg/mL) and expressed in quercetin equivalents (QE) per gram.

36
 Calibration curve of Quercetin
2.5
2 y = 0.0039x + 0.1428
R² = 0.9987

Absorbance
1.5
1
0.5
0
0 100 200 300 400 500 600
Conc(µg/mL)

Figure 3: Calibration Curve Of Quercetin

Estimation of total alkaloid content:

The determination of total alkaloids using a visible spectrophotometric method with Bromocresol Green
(BCG) is a simple and sensitive technique that requires no special equipment. BCG can react with certain
alkaloids, i.e., the ones that have nitrogen inside their structure, but not with amine and amide alkaloids.
The reaction of alkaloids with BCG forms a yellow-coloured product. Figure 3

Figure 4: The Reaction Of Alkaloids With BCG

Among the five extracts, ethanolic extract (81.22 ± 0.22) , Chloroform( 57.27 ± 0.3) and ethyl acetate
extract (11.47 ± 0.35). Ethanolic extract has shown the higher and ethyl acetate extract has shown lower
amount of alkaloidal compounds for Achyranthes aspera Linn Figure 4.

37
 Calibration Curve of Atropine
2.5 y = 0.0038x + 0.0859
R² = 0.9938
2
1.89

Absorbance
1.5

1 1.03
0.64
0.5
0.31
0.171
0.09
0
0 100 200 300 400 500 600
Conc(µg/mL)

Series1 Linear (Series1)

Figure 5: calibration curve of atropine

In-VITRO ANTI_OXIDANT POTENTIAL:

DPPH Assay:

DPPH is a relatively stable free radical and the assay determines the ability of Ethanolic extract of
Achyranthes aspera Linn reduced DPPH free radicals to the corresponding hydrazine by converting the
unpaired electrons to paired ones. Antioxidant can act by converting the unpaired electron to paired one.
When DPPH accepts an electron donated by an antioxidant compound, the DPPH is decolorized which
can be quantitatively measured from the changes in absorbance at 517nm and also for visible deep purple
colour.(Figure 6) and the IC50 value was calculated as 187.09 μg/mL using GraphPad Prism 8.3.1 in
Figure.5

DPPH free radical scavenging activity of

Ethanolic extract of Achyranthes aspera Linn
100

80
% of inhibition

60

40

20

0
100 200 400 600 800
Conc(µg/mL)

EEAA Ascorbic acid

Figure 6: DPPH free radical scavenging activity

38
Fe Reducing power assay:

Reducing power experiment is a good reflector of antioxidant activity of the plant. The plant having
high reducing power generally reported to carry high antioxidant potential too. In this experiment, Ferric
ions are reduced to ferrous ions, identified by colour change from yellow to bluish green. The results
for ferric reducing power activity of Ethanolic extract of Achyranthes aspera Linn in comparison with
ascorbic acid are reported in Figure 6 and Table 3. Reducing power potential of extracts increase with
the dose, however the extract exhibited low reducing power than that of ascorbic acid.

Table 3: Ferric reducing power activity of Ethanolic extract of Achyranthes aspera Linn

CONCENTRATIONS ABSORBANCE at 700nm

100μg 1.051
200μg 1.262
400μg 1.880
600μg 2.217
800μg 4.000
Total Antioxidant Activity:

The Ethanolic extract of Achyranthes aspera Linn at various concentrations were assayed for their
antioxidant potency by the formation of green phosphomolybdenum complex. The total anti-oxidant
capacity was measured by taking ascorbic acid as standard. IC50 was calculated by using GraphPad
Prism 8.3.1 and was 160.98µg/ml

Total anti-oxidant capacity of ethanolic extract of

Achyranthes aspera Linn
250
Anti oxidant capacity

200

150

100

50

0
100 200 400 600 800
Conc(µg/mL)

Figure 7: Total Antioxidant Activity Of Ethanolic Extract

39
 CHAPTER 6

SUMMARY & CONCLUSION

40
 SUMMARY & CONCLUSION

To conclude the results, A. aspera was screened for various phytochemicals and proved to cherish with
various phytochemicals such as alkaloids, glycosides, saponins, tannins, steroids and carbohydrates.
Various fractionation of the plants is studied for detailed phytochemical profiling and reported to possess
a good amount of phenolic content, flavonoid content and total alkaloidal content. The antioxidant
activity of ethanolic extract of A. aspera and was screened in various in-vitro methods such as DPPH
assay, reducing power assay, and total antioxidant assay. The results suggest that the polyphenolic
contents present in the whole plant of A. aspera are responsible for the reported antioxidant activity

41
CHAPTER 7

REFERENCES

42
 REFERENCES

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44
CHAPTER 8

ANNEXURE

45
 World Journal of Pharmaceutical Sciences
ISSN (Print): 2321-3310; ISSN (Online): 2321-3086
Available online at: [Link]
Review Article

EVALUATION OF ANTI-INFLAMMATORY ACTIVITY THROUGH IN VITRO AND IN VIVO

MODELS
Akshitha kalwarala2, Shriya Kumari.J2,ShivaNandu.K2,Suresh.K2,Vardhan Reddy.K2, [Link] Yada1
1
Associate Professor, Department of Pharmaceutical Chemistry, Malla Reddy College of Pharmacy,
Secunderabad. Affiliated to Osmania University, Hyderabad-500100.
2
[Link] Student, Malla Reddy College of Pharmacy, Secunderabad. Affiliated to Osmania University,
Hyderabad-500100.

Received: 28-01-2025 / Revised Accepted: 02-02-2025 / Published: 07-02-2025

ABSTRACT:
Inflammation is a complex biological response of the immune system to harmful stimuli such
as pathogens, damaged cells, or irritants. Chronic inflammation is associated with various
diseases, including autoimmune disorders, cardiovascular diseases, and cancer. Anti-
inflammatory agents aim to modulate or suppress inflammation, thus providing therapeutic
benefits. This paper explores the in vivo and in vitro models used to study anti-inflammatory
mechanisms and evaluate the efficacy of potential anti-inflammatory agents. In vitro models,
such as cell cultures and cytokine assays, provide controlled environments to study specific
molecular and cellular pathways. In contrast, in vivo models, including animal studies, offer
insights into systemic responses and pharmacokinetics. A comprehensive understanding of
these models is critical for developing effective anti-inflammatory therapies. This review
highlights the advantages, limitations, and applications of both in vivo and in vitro
approaches, providing a framework for selecting appropriate models in preclinical research.

Key words: Inflammation, cytokine assays, in vitro and in vivo models.

INTRODUCTION
An important physiological reaction that shields the body from wounds and infections is inflammation. On the
other hand, chronic disorders can result from dysregulated or protracted inflammation, which presents serious
global health issues. The goal of anti-inflammatory treatments is to lessen related harm and bring the
inflammatory response back into equilibrium.
The development and evaluation of anti-inflammatory agents rely on robust experimental models that replicate
the complexity of inflammatory processes. In vitro models offer precise control over experimental conditions,
enabling detailed analysis of molecular and cellular mechanisms. Common in vitro techniques include cytokine
profiling, cell viability assays, and co-culture systems. These models are invaluable for high-throughput
screening of potential anti-inflammatory compounds.
Conversely, in vivo models replicate the dynamic and systemic character of inflammation in a living thing. The
pharmacodynamic and pharmacokinetic characteristics of anti-inflammatory drugs are frequently evaluated
using animal models, such as paw edema caused by carrageenan and systemic inflammation caused by
lipopolysaccharide (LPS). An overview of the main in vitro and in vivo models used in anti-inflammatory
research is given in this study, with a focus on their applicability in preclinical investigations. By contrasting
these models, we hope to help researchers choose the best approaches to further the development of anti-
inflammatory drugs.
WHAT IS INFLAMMATION:-
Inflammation is a biological function that is caused after a mechanical tissue disorder or from the reactions by
the occurrence of a physical, chemical, or biological mediator in the body.

Address for Correspondence: Dr. Divya Yada, Associate Professor, Department of Pharmaceutical Chemistry, Malla Reddy
College of Pharmacy, Secunderabad. Affiliated to Osmania University, Hyderabad-500100.; E-Mail: [Link]@[Link].

How to Cite this Article: Dr. Divya Yada, Evaluation of Anti-Inflammatory Activity through In Vitro and In Vivo Models.
World J Pharm Sci 2025; 13(01): 95-111; [Link]

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 Dr. Divya Yada et al, World J Pharm Sci 2025; 13(01): 95-111

Figure1:-Inflammation picture

TYPES OF INFLAMMATION:-
There are two types of inflammations:-
 Acute inflammation
 Chronic inflammation

1. Acute inflammation
Acute inflammation is an immediate, beneficial response to injury or infection that helps the body fight off
harmful microorganisms and speed up healing. It is a protective process designed to eliminate the cause of tissue
damage, remove damaged cells, and initiate tissue repair. This response typically occurs quickly and lasts for a
short period, ranging from a few hours to a few days.

2. Chronic Inflammation
Chronic inflammation is when the body continues to send inflammatory cells even when there's no danger. This
can damage healthy cells, tissues, and organs, and lead to diseases like cancer, heart disease, and diabetes.
CAUSES OF INFLAMMATION:-
Inflammation is the body’s natural response to injury, infection, or harmful stimuli. The causes of inflammation
can be divided into two main categories: acute and chronic.
Acute Inflammation Causes
Acute inflammation is a short-term response that typically resolves after the harmful stimulus is removed.
Common causes include:
[Link]:
 Bacterial (e.g., strep throat, urinary tract infections)
 Viral (e.g., influenza, COVID-19)
 Fungal or parasitic infections
[Link]:
 Cuts, scrapes, or wounds
 Sprains or fractures
 Burns or frostbite
[Link]:
 Chemicals (e.g., acids, alkalis)
 Allergens (e.g., pollen, dust)
[Link]:
 Environmental pollutants
 Bacterial toxins

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Chronic Inflammation Causes

Chronic inflammation occurs when the immune response persists, potentially leading to tissue damage. Causes
include:
1. Chronic Infections:
 Untreated infections like hepatitis, tuberculosis
[Link] Disorders:
 Rheumatoid arthritis
 Lupus
 Multiple sclerosis
[Link] Exposure to Irritants:
 Smoking
 Pollution
 Alcohol abuse
[Link] and Metabolic Disorders:
• Excess adipose tissue can release inflammatory cytokines.
[Link] Acute Inflammation:
 Inadequate treatment of infections or injuries.
[Link] Diseases:
 Diabetes
 Cancer
 Cardiovascular diseases
SYMPTOMS OF INFLAMMATION:-
Inflammation presents with a range of symptoms that depend on whether it is acute or chronic. The
classic signs of acute inflammation are localized and often appear suddenly, while chronic inflammation
may cause more subtle, systemic symptoms over time.
Classic Symptoms of Acute Inflammation (Localized)
1. Redness : Due to increased blood flow to the affected area.
2. Swelling : Caused by fluid accumulation in the tissue.
3. Heat : The inflamed area feels warm due to increased blood supply.
4. Pain :Resulting from pressure on nerves and the release of inflammatory chemicals.
5. Loss of Function : The affected area may become difficult to use due to pain or swelling.
Systemic Symptoms of Acute Inflammation
In severe cases, inflammation may affect the whole body, leading to:
• Fever
• Fatigue
• Malaise (general discomfort)
• Elevated white blood cell count
Symptoms of Chronic Inflammation (Systemic)
Chronic inflammation is more subtle and may cause symptoms such as:
1. Fatigue: Persistent tiredness or lack of energy.
2. Aches and Pains: Muscle or joint pain, often diffuse.
3. Digestive Issues: Bloating, diarrhoea, or constipation in cases like inflammatory bowel disease.
4. Weight Changes: Unexplained weight loss or gain.
5. Low-Grade Fever: Often unnoticed but persistent.
6. Frequent Infections: Weakened immune function due to prolonged inflammation.

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MODELS USED TO STUDY ANTI -INFLAMMATORY ACTIVITY:-

Figure 2:-invivo and Invitro models

WHAT ARE INVIVO AND INVITRO MODELS:

In vivo models:
Definition: In vivo refers to experiments or studies performed within a living organism, such as animals or
humans.
Examples:
• Testing drug effects in mice or humans.
• Observing disease progression in a live animal model.
Advantages:
• Provides a realistic environment, reflecting complex biological interactions.
• Enables the study of systemic effects, such as metabolism and immune responses.
Disadvantages:
• Ethical concerns, especially in animal studies.
• Higher costs and longer timelines.
• Variability between species may limit direct applicability to humans.
In Vitro Models
Definition: In vitro refers to experiments conducted outside a living organism, typically in a controlled
environment like test tubes, petri dishes, or cell cultures.
Examples:
• Studying cell behavior in culture dishes.
• Testing chemical effects on isolated tissues or enzymes.
Advantages:
• High level of control over experimental conditions.
• Cost-effective and faster than in vivo studies.
• Avoids ethical concerns related to animal use.
Disadvantages:
• Limited ability to replicate the complexity of a whole organism.
• Results may not always translate directly to in vivo conditions
ABOUT ANIMAL MODELS:-
Animal models are non-human species used in scientific research to study biological processes, diseases, and
treatments in ways that mimic human systems. They serve as a bridge between in vitro (test-tube or cell-based)

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studies and human clinical trials, helping scientists understand complex physiological and pathological
mechanisms.
Purpose of Animal Models:
• Understanding Diseases: To study disease mechanisms and progression.
1. Testing Treatments: Evaluating the safety and efficacy of new drugs, therapies, or medical devices.
2. Biological Insights: Exploring basic physiological processes, such as reproduction, immunity, and
metabolism.
3. Genetic Studies: Investigating the role of specific genes in health and disease.
• Types of Animal Models
1. Natural models: Animals that naturally develop diseases similar to human conditions (e.g., dogs with
diabetes).
2. Induced Models: Conditions are experimentally induced using chemicals, surgery, or environmental
factors (e.g., rodents exposed to carcinogens).
3. Genetically Modified Models: Animals genetically engineered to express or suppress specific genes to
mimic human diseases (e.g., transgenic mice for Alzheimer’s research).
4. Homologous Models: Animals that closely replicate human diseases with similar symptoms and
mechanisms (e.g., monkeys for Parkinson’s disease).
5. Orphan Models: Used for studying rare diseases or conditions unique to animals that may have human
applications.
• Commonly Used Animals
1. Rodents (Mice and Rats): Most widely used due to their genetic similarity to humans, short lifespans,
and ease of handling.
2. Zebrafish: Ideal for genetic and developmental biology studies.
3. Non-Human Primates: Used for studying neurological and infectious diseases.
4. Pigs: Common in cardiovascular and organ transplantation research.
5. Dogs and Cats: Used in veterinary research and for studying specific conditions like heart disease.

IN VIVO MODELS TO STUDY ANTI-INFLAMMATION:-

1. Carrageenan induced paw edema:-
The carrageenan-induced paw edema model is a widely utilized experimental method for studying acute
inflammation and evaluating the efficacy of anti-inflammatory agents in rodents, particularly rats and mice. This
model involves the subcutaneous injection of carrageenan, a sulfated polysaccharide, into the hind paw, leading
to a measurable inflammatory response characterized by edema (swelling).
• Principle:
The underlying principle of this model is that carrageenan injection induces a biphasic inflammatory response:
1. Early Phase (0–2 hours post-injection): This phase is primarily mediated by the release of histamine,
serotonin, and bradykinin, leading to increased vascular permeability and fluid exudation.
2. Late Phase (3–6 hours post-injection): Dominated by the production of prostaglandins, particularly PGE2, and
the infiltration of neutrophils, resulting in sustained edema and inflammation.
The extent of paw swelling serves as an indicator of the inflammatory response, allowing researchers to assess
the anti-inflammatory potential of test compounds by measuring their ability to reduce this edema.
• Procedure:
1. Animal Preparation: Select healthy adult rodents and acclimate them to the laboratory environment.
Ensure ethical approval and adherence to animal welfare guidelines.
2. Baseline Measurement: Measure the initial paw volume or thickness using a plethysmometer or calipers
to establish a baseline.
3. Carrageenan Injection: Prepare a 1% carrageenan solution in sterile saline. Inject 0.1 mL of this solution
subcutaneously into the plantar surface of the hind paw.
4. Administration of Test Compounds: Administer the test substance or standard anti-inflammatory drug
(e.g., indomethacin) at the desired dose and route (oral or intraperitoneal), typically 30–60 minutes before
carrageenan injection.
5. Post-Injection Measurements: At predetermined time intervals (e.g., 1, 2, 3, 4, and 5 hours post-
injection), measure the paw volume or thickness to monitor the development of edema.
6. Data Analysis: Calculate the increase in paw volume or thickness relative to baseline. Assess the
percentage inhibition of edema in treated groups compared to the control group to determine the anti-
inflammatory efficacy of the test compound.

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Figure .3:-carrageenan induced paw edema

Mechanism of action:-
The inflammatory response induced by carrageenan involves multiple mediators and cellular events:
Early Phase: Carrageenan triggers the release of histamine and serotonin from mast cells, and bradykinin from
kininogen, leading to vasodilation and increased vascular permeability.
Late Phase: The initial mediator release stimulates the production of prostaglandins through the
cyclooxygenase pathway, particularly COX-2, resulting in sustained inflammation. Additionally, neutrophil
infiltration occurs, contributing to the release of reactive oxygen species and further propagating the
inflammatory response.
This biphasic response provides a comprehensive model for evaluating both early and late anti-inflammatory
effects of pharmacological agents.1,2,3
2) Cotton pellet induced granuloma:-
The cotton pellet-induced granuloma model is a widely used experimental method for evaluating the efficacy of
anti-inflammatory agents, particularly in the context of chronic inflammation. This model involves the
subcutaneous implantation of sterile cotton pellets in rodents, leading to granuloma formation, which serves as
an indicator of the proliferative phase of inflammation.
Principle:
The principle behind the cotton pellet-induced granuloma model involves the body’s response to a foreign
material (cotton pellet), which leads to the formation of a granuloma. The process can be summarized as
follows:
1. Foreign Body Reaction: When a cotton pellet is implanted into the subcutaneous tissue, it is recognized as a
foreign body by the immune system, triggering an inflammatory response.
2. Acute Inflammation: Initially, there is acute inflammation characterized by the recruitment of immune cells
such as neutrophils and macrophages, and the release of inflammatory mediators like prostaglandins,
cytokines, and histamines.
3. Chronic Inflammation and Granuloma Formation: Over time, the persistent presence of the foreign
material leads to chronic inflammation. Macrophages aggregate and transform into epithelioid cells, forming
a granuloma around the cotton pellet. This granuloma serves to “contain” the irritant.
4. Fibrosis: Fibroblasts may also contribute to fibrosis the granuloma, further encapsulating the foreign
material.
This model is used to simulate chronic inflammatory responses, allowing researchers to study mechanisms of
granuloma formation and evaluate potential anti-inflammatory treatments.

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Procedure:
1. Animal Preparation: Select healthy adult rodents (rats or mice) and acclimate them to laboratory
conditions.
2. Cotton Pellet Preparation: Sterilize cotton pellets weighing approximately 50 mg each by autoclaving.
3. Implantation: Under appropriate anaesthesia, make a small incision in the dorsal region of the animal
and implant the sterile cotton pellet subcutaneously. Close the incision with sutures.
4. Treatment Administration: Administer the test compound or standard anti-inflammatory drug at the
desired dose and route, typically starting on the day of implantation and continuing for a specified period
(e.g., 7 days).
5. Granuloma Extraction: At the end of the treatment period, euthanize the animals, carefully extract the
cotton pellets along with the surrounding granulomatous tissue, and dry them at 60°C for 24 hours.
6. Weight Measurement: Weigh the dried granulomas to determine the dry weight, which reflects the
amount of granulomatous tissue formed.
7. Data Analysis: Compare the granuloma weights between treated and control groups to assess the anti-
inflammatory efficacy of the test compounds.

Figure 4:-cotton pellet induced granuloma

Mechanism of Action:
The cotton pellet-induced granuloma model involves a triphasic inflammatory response:
1. Transudative hase: Characterized by the transudation of fluid from the blood vessels into the surrounding
tissue.
2. Exudative Phase: Involves the escape of fluid and blood cells into the tissue, leading to edema.
3. Proliferative Phase: Marked by the proliferation of fibroblasts and the formation of collagen, resulting in
granuloma formation.
Anti-inflammatory agents may exert their effects by inhibiting one or more of these phases, thereby reducing
granuloma formation.4,5,6,7

3) Formalin induced paw edema:-

The formalin-induced paw edema model is an established experimental method for studying inflammatory
responses and evaluating the efficacy of anti-inflammatory agents in rodents, particularly rats and mice. This
model involves the injection of formalin into the paw, leading to localized inflammation characterized by edema
(swelling) and nociceptive behaviors.

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Figure.5:-formalin induced paw edema

Principle:
Formalin-induced paw edema is a widely used experimental model to study acute inflammation, nociception,
and the efficacy of anti-inflammatory or analgesic agents in rodents. Its mechanism of action involves two
distinct phases:
1. Early Phase (Neurogenic Pain Phase)
• Duration: Occurs within the first 0–10 minutes after formalin injection.
• Mechanism:
• Formalin directly activates nociceptors by stimulating TRPA1 (Transient Receptor Potential Ankyrin
1) ion channels on sensory neurones.
• This leads to the release of substance P and calcitonin gene-related peptide (CGRP), which contribute
to pain perception and vasodilation.
• The early phase primarily reflects neurogenic pain and the activation of peripheral nociceptive fibers.
2. Late Phase (Inflammatory Pain Phase)
• Duration: Begins around 10–15 minutes after injection and lasts up to 60 minutes or longer.
• Mechanism:
• Involves an inflammatory response mediated by:
• Recruitment of immune cells (e.g., neutrophils, macrophages).
• Release of inflammatory mediators such as histamine, serotonin, prostaglandins (PGE2), bradykinin,
and cytokines (e.g., TNF-α, IL-1β).
• These mediators increase vascular permeability, leading to edema and sustained pain.
• This phase reflects inflammatory pain and involves both peripheral and central sensitization.
Procedure:
1. Animal Preparation: Select healthy adult rodents and acclimate them to the laboratory environment.
Ensure ethical approval and adherence to animal welfare guidelines.
2. Baseline Measurement: Measure the initial paw volume or thickness using a plethysmometer or calipers
to establish a baseline.
3. Formalin Injection: Inject a specific concentration of formalin (commonly 1–5% formaldehyde solution)
subcutaneously into the plantar surface of the hind paw, typically in a volume of 50–100 µL.
4. Observation Period: Monitor the animal for nociceptive behaviors (e.g., licking, biting, or shaking of the
injected paw) and measure paw edema at predetermined time intervals to assess the inflammatory
response.
5. Administration of Test Compounds: Administer the test substance or standard anti-inflammatory drug at
the desired dose and route (oral or intraperitoneal), either before or after formalin injection, depending on
the study design.
6. Data Analysis: Evaluate the extent of paw edema and nociceptive behaviors in treated and control groups
to determine the anti-inflammatory and analgesic efficacy of the test compounds.
Mechanism of Action:-
Formalin induces inflammation and pain through several mechanisms:
• Direct Nociceptor Activation: Formalin directly stimulates sensory neurons , leading to immediate pain
responses. This effect is mediated, in part, by the activation of the TRPA1 cation channel, which plays a
significant role in inflammatory pain.

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• Release of Inflammatory Mediators: Formalin injection leads to the release of various inflammatory
mediators, including substance P, prostanoids, serotonin, and histamine, which contribute to vasodilation,
increased vascular permeability, and edema formation.
• Neurogenic Inflammation: At higher doses, formalin induces neurogenic inflammation mediated by
neuropeptides such as substance P, resulting in increased vascular permeability and edema.
• This model is valuable for assessing both the inflammatory and nociceptive components of pain and for
evaluating the efficacy of pharmacological agents targeting these processes. 8,9,10,11
4) TPA induced ear edema:-
The 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced ear edema model is a widely used experimental
method for evaluating the anti-inflammatory properties of compounds in vivo. This model involves the topical
application of TPA to the mouse ear, leading to acute skin inflammation characterized by edema, erythema, and
leukocyte infiltration.
Principle:
TPA, a potent activator of protein kinase C (PKC), induces a rapid inflammatory response when applied to the
skin. This response is marked by increased production of pro-inflammatory mediators, resulting in edema and
other signs of inflammation. The extent of ear swelling serves as a quantitative measure of the inflammatory
response, allowing for the assessment of anti-inflammatory agents.
Procedure:
1. Animal Preparation: Select healthy adult mice and acclimate them to laboratory conditions. Ensure
ethical approval and adherence to animal welfare guidelines.
2. Baseline Measurement: Measure the initial thickness of both ears using a micrometer to establish a
baseline
3. TPA Application: Dissolve TPA in a suitable solvent, typically acetone, to achieve the desired
concentration (e.g., 2.5 µg/ear). Apply the TPA solution topically to the inner and outer surfaces of one
ear (usually the right ear) using a micropipette. The contralateral ear may serve as a control.

Figure6:-TPA induced ear edema

4. Treatment Administration: Administer the test compound or standard anti-inflammatory drug either
topically or systemically at the desired dose and route, prior to or after TPA application, depending on
the study design.
5. Observation Period: Allow the inflammatory response to develop over a specified period, typically 4 to
6 hours post-TPA application. During this time, monitor the animals for any adverse reactions.
6. Measurement of Edema: At the end of the observation period, measure the thickness of both ears again
using the micrometer. Calculate the increase in ear thickness as an indicator of edema.
7. Data Analysis: Compare the ear thickness measurements between treated and control group to assess the
anti-inflammatory efficacy of the test compounds.

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Mechanism of Action:
TPA induces inflammation through several mechanisms:
• Activation of Protein Kinase C (PKC): TPA directly activates PKC, leading to the phosphorylation of
various substrates involved in inflammatory pathways.
• Production of Pro-inflammatory Mediators: Activation of PKC results in the upregulation of enzymes
such as cyclooxygenase (COX) and lipoxygenase (LOX), leading to increased synthesis of eicosanoids like
prostaglandins and leukotrienes, which contribute to edema and pain.
• Induction of Cytokines and Chemokines: TPA application stimulates the release of pro-inflammatory
cytokines (e.g., TNF-α, IL-1β) and chemokines, promoting leukocyte infiltration into the affected tissue.
• Oxidative Stress: TPA-induced activation of inflammatory cells leads to the production of reactive
oxygen species (ROS), contributing to tissue damage and further propagating the inflammatory response.
• The TPA-induced ear edema model is particularly useful for evaluating the efficacy of anti-inflammatory
agents targeting these pathways.12,13,14

5) Xylene induced ear edema:-

The xylene-induced ear edema model is a widely used experimental method for evaluating the anti-
inflammatory properties of compounds in vivo. This model involves the topical application of xylene to the
mouse ear, leading to acute inflammation characterized by edema (swelling), erythema (redness), and increased
vascular permeability.
Principle:
Xylene, an organic solvent, acts as an irritant when applied to the skin, inducing an acute inflammatory
response. This response is marked by increased capillary permeability, fluid accumulation, and leukocyte
infiltration into the affected tissue. The degree of ear swelling serves as a quantitative measure of the
inflammatory response, allowing for the assessment of anti-inflammatory agents.
Procedure:
1. Animal Preparation: Select healthy adult mice, typically weighing between 18 to 21 grams, and acclimate
them to laboratory conditions. Ensure ethical approval and adherence to animal welfare guidelines.
2. Baseline Measurement: Measure the initial thickness or weight of both ears to establish a baseline.
3. Xylene Application: Apply a specific volume of xylene (commonly 30 µL) to the inner surface of the right
ear using a micropipette. The left ear serves as a control.
4. Treatment Administration: Administer the test compound or standard anti-inflammatory drug orally or via
another appropriate route, either before or after xylene application, depending on the study design.

Figure .7:-xylene induced ear edema

[Link] Period: Allow the inflammatory response to develop over a specified period, typically 30
minutes post-xylene application. Monitor the animals for any adverse reactions during this time.
[Link] of Edema: After the observation period, euthanize the animals humanely. Remove both ears
and weigh them separately. The difference in weight between the treated and control ears indicates the extent of
edema
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7. Data Analysis: Compare the ear weight differences between treated and control groups to assess the anti-
inflammatory efficacy of the test compounds.
Mechanism of Action:
Xylene induces inflammation through several mechanisms:
• Increased Capillary Permeability: Xylene application leads to the release of inflammatory mediators
that increase capillary permeability, resulting in plasma exudation and tissue swelling.
• Leukocyte Infiltration: The irritant effect of xylene promotes the infiltration of leukocytes,
particularly neutrophils, into the affected tissue, contributing to the inflammatory response.
• Neurogenic Inflammation: Xylene exposure may stimulate sensory nerve endings, leading to the
release of neuropeptides such as substance P, which further enhance vascular permeability and
inflammation.
The xylene-induced ear edema model is particularly useful for evaluating the efficacy of anti-inflammatory
agents that target these acute inflammatory pathways.15,16,17
IN VITRO MODELS TO STUDY ANTI INFLAMMATION:-
1) cox inhibition assay:-
The COX inhibition assay is used to evaluate the effectiveness of compounds in inhibiting cyclooxygenase
enzymes (COX-1 and COX-2), which are involved in the synthesis of prostaglandins, mediators of
inflammation and pain.
Principle:
Cyclooxygenase (COX) enzymes catalyze the conversion of arachidonic acid to prostaglandins, which are
critical mediators of inflammation. COX-1 is constitutively expressed in many tissues and plays a role in
maintaining normal physiological functions, while COX-2 is induced in response to inflammation. Inhibition of
these enzymes, particularly COX-2, is a target for anti-inflammatory drugs.
The COX inhibition assay typically measures the reduction in prostaglandin production after exposure to a test
compound, using either COX-1 or COX-2 as the enzyme source.
Procedure:
1. Preparation of Reagents:
COX enzyme solution: Purified COX-1 or COX-2 enzyme.
Arachidonic acid: Substrate for COX enzymes.
Test compound: Compound to be tested for COX inhibition, prepared at various concentrations.
Detection reagent: Prostaglandin E2 (PGE2) or other prostaglandins, detected by colorimetric or fluorometric
methods.
2. Assay Setup:
• Mix the COX enzyme solution with the test compound in a micro plate well.
• Add arachidonic acid to start the enzyme reaction.
• Incubate the mixture at 37°C for a specified duration (e.g., 10-15 minutes).
3. Detection:
• After the incubation, stop the reaction and add the detection reagent that reacts with the prostaglandin
product.
• Measure the absorbance or fluorescence of the reaction using a micro-plate reader. The higher the
signal, the more prostaglandin was produced.
4. Data Analysis:
• Calculate the percentage inhibition of COX activity by comparing the signal from treated wells to a
control (no inhibitor).

Figure 8:-cox inhibition assay

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• Determine the IC₅₀ value (concentration of the test compound that inhibits 50% of COX activity).
Mechanism of Action:
COX enzymes have two functional sites:
1. Cyclooxygenase site: This site catalyzes the conversion of arachidonic acid to prostaglandin G2 (PGG2).
2. Peroxidase site: This site catalyzes the conversion of PGG2 to prostaglandin H2 (PGH2), which is the
precursor for other prostaglandins.
Inhibitors can bind to either of these sites, preventing the conversion of arachidonic acid to prostaglandins. Non-
selective COX inhibitors block both COX-1 and COX-2, while selective COX-2 inhibitors primarily block the
inducible form of the enzyme associated with inflammation. 18,19,20
2) LPS stimulated macrophages assay:-
The LPS-stimulated macrophage assay is a commonly used in vitro model to study inflammation and test the
efficacy of anti-inflammatory agents. LPS (lipopolysaccharide), a component of the outer membrane of Gram-
negative bacteria, is a potent inducer of inflammation, primarily through activation of macrophages and the
release of pro-inflammatory cytokines.
Principle:
LPS activates macrophages via the toll-like receptor 4 (TLR4), leading to the production of inflammatory
mediators like cytokines (e.g., TNF-α, IL-6, IL-1β) and prostaglandins. These molecules play crucial roles in the
immune response to infection, but excessive production can lead to chronic inflammation and tissue damage.
The LPS-stimulated macrophage assay helps assess the anti-inflammatory effects of compounds by measuring
their ability to inhibit LPS-induced cytokine production and other markers of inflammation.
Procedure:
1. Cell Culture Preparation:
• Macrophage Cells: Cultivate macrophage cell lines (e.g., RAW 264.7, J774A.1) or primary
macrophages (from mouse or human sources) in appropriate culture conditions (e.g., RPMI-1640 or
DMEM media with 10% FBS, incubated at 37°C with 5% CO2).
2. Treatment Preparation:
• Prepare the test compound (e.g., potential anti-inflammatory drug) at various concentrations (e.g., 1,
10, 100 µM).
• Dilute LPS to the desired concentration, typically 1 µg/mL for stimulation.
3. Incubation with LPS:
• Seed macrophages in a 96-well plate and allow them to adhere for several hours (usually 2–4 hours).
• Add the test compound to the cells and incubate for a pre-determined period (e.g., 1 hour) to allow any
effects on macrophage function.
• Stimulate the cells with LPS (1 µg/mL) to activate the macrophages.
4. Cytokine Measurement:
• After a specified period of LPS stimulation (usually 24 hours), collect the culture supernatant.
• Measure the levels of cytokines such as TNF-α, IL-6, and IL-1β using enzyme-linked immunosorbent
assays (ELISA), cytokine bead arrays (CBA), or other detection methods.

Figure 9:-LPS stimulated macrophages

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5. Cell Viability (Optional):

• Perform a cell viability assay (e.g., MTT or MTS assay) to ensure that the compound does not induce
cytotoxicity.
6. Data Analysis:
• Analyze the inhibition of cytokine production by comparing the levels of cytokines in LPS-stimulated
cells with and without the test compound. A reduction in cytokine release indicates anti-inflammatory
activity.
Mechanism of Action:
1. Activation of TLR4
LPS binds to TLR4 on the surface of macrophages, triggering a downstream signaling cascade through
adaptor proteins (e.g., MyD88, TRIF) and activation of NF-κB (nuclear factor kappa-light-chain-
enhancer of activated B cells) and MAPK (mitogen-activated protein kinase) pathways.
2. Inflammatory Mediators Release:
Activation of NF-κB and MAPKs leads to the expression of pro-inflammatory genes, including
cytokines (e.g., TNF-α, IL-6, IL-1β) and enzymes like COX-2 (cyclooxygenase-2), which produce
prostaglandins (e.g., PGE2), a key inflammatory mediator.
3. Effect of Anti-inflammatory Compounds:
Anti-inflammatory compounds can inhibit the activation of TLR4 or block the downstream signaling
pathways (NF-κB or MAPK), thereby reducing the production of pro-inflammatory cytokines and
mediators.21,22,23
3)Mast cell degranulation:-
The mast cell degranulation assay is used to study the activation and degranulation of mast cells, which are
immune cells involved in allergic reactions and inflammation. Mast cells release histamine and other pro-
inflammatory mediators upon activation, making them a key target in the study of allergic diseases and
inflammation.
Principle:
Mast cells contain granules filled with histamine, proteases, and other inflammatory mediators. Upon activation
(e.g., by antigen binding to IgE or by other stimuli), mast cells undergo degranulation, releasing these mediators
into the surrounding environment. The mast cell degranulation assay evaluates the extent of this degranulation,
often by measuring the release of histamine or other substances, and helps assess the potential effects of
compounds in modulating this process.
Procedure:
• Preparation of Mast Cells:
• Cell Source: Use mast cell lines (e.g., RBL-2H3 or MC/9 cells) or primary mast cells derived from
animal models (e.g., mice or rats).
• Cell Culture: Cultivate cells in appropriate media (e.g., RPMI-1640 or DMEM) supplemented with
fetal bovine serum (FBS) and maintain under standard cell culture conditions (37°C, 5% CO2).
• Priming Mast Cells (if needed):
• IgE Sensitization: For allergic reactions, mast cells are often “sensitised” by incubating them with anti-
IgE antibodies (e.g., DNP-IgE) that bind to the high-affinity IgE receptor (FcεRI) on mast cells. This
primes the mast cells to respond to specific antigens (e.g., dinitrophenylated bovine serum albumin,
DNP-BSA).
• Treatment with Test Compound:
• Incubate the mast cells with the test compound at various concentrations for a specified period (e.g., 1–
2 hours). This allows the compound to potentially influence mast cell function, either by inhibiting or
enhancing degranulation.
• Induction of Degranulation:
• Stimulation: After pre-treatment with the test compound, stimulate mast cells by adding a triggering
agent. For IgE-mediated degranulation, an antigen (e.g., DNP-BSA) is added, which cross-links the IgE
receptors on the mast cells, causing degranulation.
• Alternative Stimuli: Some assays use non-IgE-dependent stimuli, such as compound 48/80, calcium
ionophore A23187, or other chemical agents that directly induce degranulation.
• Measurement of Degranulation:
• Histamine Release: The most common method of assessing degranulation is by measuring the release
of histamine into the supernatant. Histamine can be quantified using specific assays like the
fluorometric or colorimetric assay.
• Other Markers: In some cases, other markers of degranulation, such as β-hexosaminidase or tryptase
activity, can also be measured.

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• Data Analysis:
• Calculate the percentage of mast cell degranulation by comparing the histamine release in the
stimulated cells (with and without the test compound) to baseline (unstimulated) cells.

Figure .10:- mast cell degranulation

Mechanism of Action:
1. Mast Cell Activation:
• Mast cells are activated when an antigen (e.g., DNP-BSA) binds to the IgE antibodies already bound to
FcεRI receptors on the surface of mast cells. This cross-linking leads to the activation of intracellular
signaling pathways.

2. Signal Transduction:
• The cross-linking of IgE receptors triggers the activation of various intracellular signaling pathways,
including the PLC/IP3 pathway, leading to the release of intracellular calcium and the activation of protein
kinases (e.g., PKC, MAPK). This cascade ultimately causes mast cell degranulation.
3. Degranulation and Mediator Release:
• The intracellular signaling pathways lead to the fusion of mast cell granules with the cell membrane,
releasing pre-stored inflammatory mediators like histamine, cytokines, proteases, and lipid mediators
(prostaglandins and leukotrienes).
4. Effect of Inhibitors:
• Anti-inflammatory compounds or inhibitors can modulate these pathways by blocking the activation of
receptors (e.g., blocking IgE-FcεRI interaction), inhibiting signaling molecules (e.g., PLC, PKC, or
MAPKs), or stabilizing the mast cell membrane to prevent granule release.24,25,26
4) Mono layer cell culture model:-
The monolayer cell culture model is a widely used in vitro system where cells are cultured in a single layer
on a flat surface, typically a culture dish or well plate. This model allows for the study of cellular behavior,
including proliferation, differentiation, migration, and response to external stimuli, such as drugs, toxins, or
growth factors.

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Principle:
The monolayer cell culture model is based on the ability of adherent cells (such as fibroblasts, epithelial cells, or
endothelial cells) to grow in a single layer when provided with a suitable surface (e.g., tissue culture-treated
plastic) and appropriate culture conditions (temperature, CO₂ concentration, and nutrient-rich medium). Cells
are typically grown in a nutrient medium, and their responses to various treatments can be studied through
various assays, such as cell viability, migration, and gene expression analysis.
Procedure:
1. Cell Preparation:
• Select an appropriate cell line (e.g., HeLa cells, NIH-3T3 cells, or RAW 264.7 macrophages).
• Culture cells in appropriate growth medium (e.g., DMEM or RPMI-1640) supplemented with 10% fetal
bovine serum (FBS) and antibiotics (e.g., penicillin-streptomycin) to prevent contamination.
• Maintain cells in a humidified incubator at 37°C with 5% CO₂.
2. Subculture and Seeding:
• Subculture the cells to prevent over-confluence by trypsinizing them and transferring them to fresh
culture vessels.
• Seed a known number of cells (e.g., 1 x 10⁴ cells per well) onto a flat surface of a multi-well plate or a
petri dish.
• Allow cells to adhere and spread out to form a monolayer, typically taking 12–24 hours, depending on
the cell type and seeding density.
3. Treatment and Exposure:
• Once the monolayer is formed, treat the cells with the experimental compounds, drugs, or stimuli.
Treatment concentrations and durations will depend on the specific experiment being conducted (e.g.,
24–48 hours for drug exposure).
• For migration studies, induce stress conditions or use stimuli like growth factors or chemical agents
(e.g., EGF for cell migration).

Figure11:-monolayer cell culture

4. Assays and Measurements:
• Cell Viability: Assess cell viability using colorimetric assays such as MTT, MTS, or Alamar Blue,
which measure metabolic activity or cell proliferation.
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• Cell Migration/Invasion: Perform scratch wound healing assays or transwell assays to evaluate the
ability of cells to migrate or invade.
• Gene/Protein Expression: Use PCR, Western blotting, or immunohistochemistry to assess changes in
gene or protein expression due to treatment.
• Morphological Changes: Observe changes in cell morphology under a microscope, often used to
assess differentiation or response to stimuli.
5. Data Analysis:
• Quantify results (e.g., percentage of wound closure, cell proliferation rates, or relative expression
levels) using appropriate software.
• Analyze the effect of the treatment by comparing experimental groups with control groups

Mechanism of Action:
1. Cell Attachment and Spreading:
• When seeded onto a surface, adherent cells bind to the extracellular matrix (ECM) proteins (e.g.,
fibronectin, collagen) via integrins. This interaction triggers intracellular signaling pathways that
facilitate cell spreading and migration across the surface to form a monolayer.
2. Cell Proliferation and Growth:
• Once the monolayer is established, cell growth and division are regulated by growth factors and nutrients
in the medium. Cell proliferation is controlled by signals through receptors, such as the epidermal growth
factor receptor (EGFR) and fibroblast growth factor receptor (FGFR).
3. Response to External Stimuli:
• Cells in the monolayer respond to external stimuli, such as cytokines, drugs, or mechanical stress, by
activating signaling pathways like MAPK, PI3K-AKT, or NF-κB. These pathways can lead to various
outcomes, including cell proliferation, apoptosis, differentiation, or changes in gene expression.
4. Cell-Matrix Interaction and Mechanical Forces:
• The integrity of the monolayer is maintained by adhesion molecules like E-cadherin and β-catenin, which
form adherent junctions. Mechanical forces such as tension and compression, often applied during assays
like migration or invasion, activate pathways such as Rho-GTPase signaling, which regulates cell shape,
motility, and cytoskeletal organization.27,28,29

CONCLUSION:-
In conclusion, the diverse experimental models used to study inflammation and related biological processes,
such as the carrageenan-induced paw edema model, cotton pellet-induced granuloma model, formalin-induced
paw edema model, TPA- and xylene-induced ear edema models, COX inhibition assay, LPS-stimulated
macrophage assay, mast cell degranulation assay, and monolayer cell culture model, provide invaluable tools for
preclinical research.
Each model offers distinct insights into specific aspects of acute and chronic inflammation, pain, immune
activation, and cellular responses. Together, they enable a comprehensive evaluation of the mechanisms
underlying inflammation and the pharmacological effects of potential therapeutic agents. Their robustness,
reproducibility, and relevance to human pathophysiology make them critical in the discovery and development
of novel anti-inflammatory and analgesic drugs, as well as in understanding fundamental biological processes.
These models continue to play a pivotal role in bridging the gap between basic research and clinical application,
advancing the field of biomedical research and the development of effective treatments for inflammatory and
immune-mediated diseases.

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