evaluation of in vivo and in vitro antiarthritic potential

Human Journals

Research Article

October 2021 Vol.:13, Issue:1

© All rights are reserved by Nilesh Desai et al.

Evaluation of In Vivo and In Vitro Antiarthritic Potential of

Annona squamosa Leaves

www.jcpr.humanjournals.com

Keywords: Arthritis, CFA, Annona Squamosa, SGOT, SGPT.

ABSTRACT

In India there are several herbs in different forms are used

in the traditional systems of medicine. For several years the

herbs are used for the treatment of several diseases such as

arthritis and also other inflammatory diseases. The herbs

are considered as the safe and effective treatment for many

diseases. By utilizing the ethnobotanical knowledge we

described the benefits of Annona Squamosa in different

conditions of arthritis. The antiarthritic potential of

different extracts of Annona Squamosa was tested by using

In vivo & In vitro arthritic models. In vivo study of Aqueous

Extract of Annona Squamosa (AEAS) against CFA induced

arthritis showed significant changes in different arthritic

parameters like paw edema, paw diameter,

biochemical/serum parameters like SGOT & SGPT. In vitro

study of Ethanolic Extract of Annona Squamosa (EEAS)

showed potential antiarthritic effect against protein

denaturation assay.The findings from the present study

justify the traditional use of Annona Squamosa in the

treatment of arthritis. However, further fractionation and

isolation of extract is required to observe the safety,

efficacy, and potency of Annona Squamosa against arthritis.

Nilesh Desai1*, Kishorkumar Burade2, Ritesh

Agrawal3

1 Research Scholar, Pacific University Udaipur

Rajasthan, India.

2 Principal, Government College of pharmacy Karad

Maharashtra, India.

3 Head IPR Cell Pacific University, Professor Pacific

College of pharmacy Rajasthan. India.

Submitted: 23 September 2021

Accepted: 28 September 2021

Published: 30 October 2021


Citation: Nilesh Desai et al. Jcpr.Human, 2021; Vol. 13 (1): 27-44. 28

1. INTRODUCTION:

Arthritis is a chronic inflammatory disease that affects several parts of the joints including

cartilage, synovium tendons, and muscles. Joints can be inflamed for many reasons which

result in pain, swelling, and limited movement cartilage damage, and erosions of the

underlying bone which is characteristic of chronic arthritis. RA is a specific type of arthritis

and is a complex chronic inflammatory disease dependent on multiple interacting

environments and genetic factors making it difficult to understand pathogenesis. The main

causes of arthritis are osteoarthritis (OA), rheumatoid arthritis (RA) & gout [1].

Rheumatoid arthritis is a common form of inflammatory arthritis and has a substantial social

effect in terms of cost, disability, and lost productivity. Although the pathogenesis of RA

remains incompletely understood, much insight into the cellular and molecular mechanism

has been involved has been gained in the past decade. The exact cause of rheumatoid arthritis

is not known but is considered to be an autoimmune disorder in which joints usually

including those of hands and feet are symmetrically inflamed resulting in swelling, pain, and

often the eventual destruction of the joints interior. The immune system attacks the soft tissue

that lines the joint and also attacks the connective tissue in other parts of the body. The joints

deteriorate at a highly variable rate [2,3].

The current arthritis therapy is strongly established it is associated with numerous side effects

like NSAID-induced peptic ulcer, heartburn, abdominal cramps, bone marrow suppression,

idiosyncratic allergic-like pulmonary injury, severe hepatic damage, associated with

lymphomas and occasionally with severe skin reactions, stomatitis, teratogenic cataracts,

glaucoma, dysfunction of the adrenal glands, etc. Since these antiarthritic agents are usually

recommended for prolonged use, the risk of significant dose-dependent side effects is a major

drawback of this therapy. It is widely accepted that there is increasing use of natural remedies

to the general public to replace or complement conventional medicine [5,6].

The demand for antiarthritic drugs is constantly increasing, our life expectancy is also

increasing so systemic study on an anti-arthritic effect of Indian medicinal plants was done by

many investigating workers. Hence the present work aims to evaluate the anti-arthritic

efficiency of a well-known Indian herb Annona squamosa using the Complete Freund’s

Adjuvant (CFA) induced arthritis model in rats.



1.1. Introduction to Annona Squamosa:

Annona Squamosa is commonly known as custard apple in English, sitaphal in Hindi. It is a

small well branched evergreen tree is cultivated throughout India for its fruits, various parts

of Annona squamosa are used for their medicinal value in the treatment of many disorders.

Annona Squamosa plant with fruit.

Annona Squamosa leaves Annona Squamosa Flower

1.2. Plant Profile:

Botanical Name: Annona squamosa Linnaeus.

Family: Annonaceae

Synonyms: Annona cinerea Dunhal, Annona forskahlii DC, Annona asiatica L

1.3. Scientific Classification:

Kingdom- Plantae



Order- Mangnoliales

Family- Annonaceae

Genus- Annona

Species- A. squamosa

1.4. Chemical Constituents:

Pulp contains moisture (62-64%) and reducing sugars 6.55%. Seeds yield oil and resin; seeds,

leaves, and immature fruit contain an acrid principle that is insecticidal. Fruit is rich in

vitamin C. The aporphone alkaloids anonain roemerine, noreorydine, corydine,

norisocorydine, and glaucine have been isolated from Annona Squamosa. The seed contains a

saponin. The bark contains tannin (2.02%), camphor, borneol, β-sitosterol. The green leaves,

on steam distillation yield a yellow olive-green, pleasant smelling bitter essential oil (0.08%).

The oil primarily consists of β- caryophyllen (50%). Other components are α-pinene (7.0%),

a monocyclic terpene (4.04%) & two bicyclic sesquiterpenes.

1.5. Ethno Botanical Uses:

Fruits are normally eaten fresh. The pulp can be used as a flavouring in ice cream. Between

50-80% of the fruit is edible. The vitamin C content is appreciable (35-42 mg/100 g) and

slightly higher than in grapefruit. The nutrient value of thiamine, potassium and dietary fiber

is also significant. Fruit enriches the blood, increases muscular strength, relieves vomiting,

sedative to the heart. The root is cathartic, drastic purgative, administered in acute dysentery.

The tree is a good source of firewood. Green fruits, seeds, and leaves have effective

vermicidal and insecticidal properties. Leaves, shoots, bark, and roots have been reported to

have medicinal properties. The unripe fruit is astringent, and the root is a drastic purgative.

The plant is traditionally used for the treatment of epilepsy, dysentery, cardiac problems,

worm infestation, constipation, hemorrhage, antibacterial infection, dysuria, fever,

inflammation & ulcer. It also has antifertility, antitumor, and abortifacient properties.

Ethanolic extracts of leaves and stems are reported to have anticancer activity. The aqueous

leaf extract has also been reported to ameliorate hyperthyroidism, which is often considered a

causative factor of Diabetes mellitus. The ripe fruit is medicinally considered as maturant and

when bruised and mixed with salt, is applied to malignant tumors to hasten suppuration. The



seeds contain an acrid principle fatal to insects, and the dried unripe fruit, powdered and

mixed with gram flour is used to destroy vermin. [7,8]

1.6. Reported Activities:

1) Antibacterial activity:

The antibacterial screening by agar cup method indicates that the highest zone of inhibition

was shown by the methanol extract followed by petroleum ether and aqueous extracts for

Annona squamosa leaf. Extracts of Annona Squamosa inhibited the growth of all test strains

except Salmonella typhimurium. Annona Squamosa had strong antibacterial activity against

these bacterial strains Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus

& Vibrio alginolyticus.

2) Anti Diabetic Activity:

Annona Squamosa root extract indicates the antidiabetic activity in STZ induced

hyperglycemia in rats. The antihyperglycemic activity of the aqueous extract of Annona

squamosa roots was comparable with Glibenclamide, a standard hypoglycaemic drug. The

ethanolic extract of Annona squamosa leaves possesses considerable hypoglycaemic activity

in normal rats.

3) Anti Genotoxic Agent:

The aqueous and ethanolic bark extracts of Annona Squamosa significantly reduced the

frequency of MnPCEs (micro-nucleated polychromatic erythrocytes) and chromosomal

aberration in DMBA treated hamsters.

4) Antihyperlipidemic Activity:

Aqueous extract of Polyherbal formulation of Annona Squamosa indicates effects on blood

glucose, plasma insulin, tissue lipid profile, and lipid peroxidation in streptozotocin induced

diabetic rats. The polyherbal formulation also resulted in a significant decrease in tissue lipids

and lipid peroxide formation. The decreased lipid peroxides and tissue lipids clearly showed

the antihyperlipidemic and antiperoxidative effect of polyherbal formulation apart from its

antidiabetic effect.



5) Antioxidant Activity:

The free radical scavenging potential of the leaves of Annona Squamosa was studied by using

different antioxidant models of screening. The extract showed only moderate scavenging

activity of superoxide radicals and anti-lipid peroxidation potential, which was performed

using rat-brain homogenate.

6) Antitumour Activity:

The effect of aqueous and organic extracts from defatted seeds of Annona Squamosa was

studied on a rat histolytic tumour cell line AK-5. Both the extracts caused significant

apoptotic tumour cell death with enhanced caspase-3 activity down regulation of

antiapoptotic genes Bcl-2 and Bclxi and enhance the generation of intracellular ROS, which

correlated well with the decreased levels of intracellular GSH. In addition, DNA

fragmentation and annexin V staining confirmed that the extracts induced apoptosis in

tumour cells through oxidative stress.

7) Cytotoxic Activity:

Annonaceous acetogenins are a new class of compounds that have been reported to have

potent pesticide, parasiticidal, antimicrobial, cell growth inhibitory activities. In this study,

organic and aqueous extracts from the defatted seeds of Annona squamosa (custard apple)

were tested on different human tumour cell lines for antiumoural activity. Treatment of MCF-

7 and K-562 cells with organic and aqueous extracts resulted in nuclear condensation, DNA

fragmentation, induction of reactive oxygen species (ROS) generation & reduced intracellular

glutathione levels. In addition, down regulation of Bcl-2 and PS externalization by Annexin -

V staining suggested induction of apoptosis in MCF-7 and K-562 cells by both the extracts

through oxidative stress. These observations suggest that the induction of apoptosis by

Annona Squamosa extracts can be selective for certain types of cancerous cells.

8) Chemopreventive & Antilipid peroxidative:

The chemopreventive and antilpidperoxidative potential of Annona squamosa bark extracts in

DMBA induced hamster buccal pouch carcinogenesis. Oral administration of aqueous &

ethanolic bark extracts of Annona Squamosa at a dose of 500 mg/kg & 300 mg/kg prevented

the tumor formation as well as decreased the levels of lipid peroxidation byproducts &

enhanced the antioxidants defense mechanism in DMBA painted hamsters.



9) Hepatoprotective Activity:

The extracts of Annona Squamosa (300 & 350 mg/kg bw) were used to study the

hepatoprotective effect in isoniazid + rifampicin induced hepatotoxic model in albino Wistar

rats. There was a significant decrease in total bilirubin accompanied by a significant increase

in the level of total protein and also a significant decrease in ALP, AST, and ALT in the

treatment group as compared to the hepatotoxic group. In the histopathological study, the

hepatotoxic group showed hepatocytic necrosis and inflammation in the centrilobular region

with portal triaditis. The treatment group showed minimal inflammation with moderate portal

triaditis and their lobular architecture was normal. In another study, the protective effect was

evaluated in diethyl nitrosamine induced hepatotoxicity. This study revealed that the extracts

of Annona Squamosa exerted a hepatoprotective effect.

10) Insecticidal Activity:

The ethanolic Annona squamosa extract showed potent activity against Sitophilus oryzae

pest. [9,10]

2. MATERIALS & METHODS:

2.1. Animals:

Male Wistar rats (150-250 g) or female Swiss albino mice (20-25 gm) were used for study.

2.2. Collection of Plant Material:

The leaves of Annona Squamosa were collected from Kolhapur region of Maharashtra.

2.3. Preparation of Aqueous Extract of Annona squamosa leaves:

Leaves of Annona Squamosa were shade dried and coarsely powdered by using a grinder

mixer. The powdered material was macerated in a sufficient quantity of distilled water with a

small quantity of chloroform to prevent fungal growth and kept for 3 days. During

maceration, it was shaken twice daily. On the 3rd day, it was filtered and dried at 60 °C on a

water bath [14]. The extract was then preserved in the desiccator and then used for

phytochemical and pharmacological studies. Ethanolic extract of Annona Squamosa was

prepared by the soxhlet extraction method [15].



2.4. Acute Oral Toxicity Study

The oral toxicity study of ATG was carried out as per OECD guidelines No. 423 (Annexure-2

d) for acute oral toxicity.

2.5. Experimental Designs:

Prophylactic effect of Aqueous Extract of Annona Squamosa leaves (AEAS) on

Complete Freund’s Adjuvant (CFA) induced arthritic rats.

CFA induced arthritis model was used to assess the anti-arthritic activity in albino wistar rats.

Animals were randomly divided into six groups of six animal each (n=6). Wistar rats were

made arthritic by a single sub plantar injection of 0.1 ml of CFA. Drug treatment was started

from the initial day i.e. from the day adjuvant injection (0day) and continued till the 21st day.

Paw volume and Paw diameter were measured on 0th,4th, 8th, 14th, 21st day by using a

plethysmometer and vernier caliper respectively [15,16].

Table 1: Treatment Schedule in Prophylactic model of arthritis.

Group No. Treatment Dose Route of

Administration

I Normal Distilled water 5 ml/kg Per Oral.

II Control Complete Freund’s adjuvant

(CFA) 0.1ml Sub Planter.

III Diclofenac 10 mg/kg Intraperitoneally.

CFA 0.1ml Sub Planter.

IV AEAS 100mg/kg Per Oral.


V AEAS 200 mg/kg Per Oral.


VI AEAS 400 mg/kg Per Oral.


2.6. In Vitro Anti-arthritic activity of Annona Squamosa.

The reaction mixture (10 mL) consisted of 0.4 mL of egg albumin (from fresh hen’s egg), 5.6

mL of phosphate-buffered saline (PBS, pH 6.4), and 4 mL of synthetic derivatives (1000,



800, 600. 400, 200μg/ml). A similar volume of double-distilled water served as a control.

Then the mixtures were incubated at (370c ±2) in an incubator for 15 min and then heated at

700c for 5 min. After cooling, their absorbance was measured at 660 nm by using the vehicle

as blank. Diclofenac sodium at a concentration (1000, 800, 600. 400, 200μg/ml) was used as

a reference drug and treated similarly for determination of absorbance. The percentage

inhibition of protein denaturation was calculated by using the following formula [17].

% inhibition = absorbance of control - absorbance of test / absorbance of control x 100

2.7. Statistical analysis:

The values were expressed as mean ± SEM (n=6). The statistical significance was assessed

using student t-test or one-way analysis of variance (ANOVA) followed by Dunnet’s test and

P<0.05 and P<0.01 were considered to be statistically significant.

3. RESULTS AND DISCUSSION:

3.1. Phytochemical & Pharmacological evaluation of Aqueous Extract Annona

Squamosa (AEAS).

Table 2: Phytochemical screening of AEAS.

Plant Constituents Test performed Aqueous extract of

Annona Squamosa

Test for Steroids Salkowaski Reaction +

Liebermann-Buchard Reaction +

Test for Glycosides

Baljet’s Test +

Keller-Killiani Test +

Legal Test --

Borntrager’s Test --

Tests for Saponins Froth Test +

Tests for Carbohydrates

Molisch’s Test +

Fehling’s Test +

Benedict’s Test +

Test for Alkaloids Mayer’s Test +

Dragendroff's Reagent +

Tests for Flavonoids Lead acetate Test +

Shinoda Test +

Test for Tannins Ferric chloride Test +

Gelatin Test --

Test for Proteins

Millon’s Test --

Xanthoproteic Test --

Biuret Test --

Ninhydrin Test --



The phytochemical analysis of the aqueous extract of Annona Squamosa confirms the

presence of flavonoids, alkaloids, saponins, carbohydrates, tannins, steroids etc.

3.2. Acute Oral Toxicity Records for AEAS.

According to the OECD guidelines for acute oral toxicity animals were tested at the dose of

2000mg/kg and 5000mg/kg. Animal treated with AEAS 5000mg/kg showed symptoms of

toxicity like salivation, convulsions, and excitement, and motility is reported within 48 hours.

Animal treated with AEAS 2000mg/kg did not show any symptoms of toxicity. Hence,

according to the guidelines the dose 2000mg/kg was found to be safe, and hence the different

doses of 100 mg/kg, 200 mg/kg, and 400 mg/kg of AEAS were selected for the present study.

3.3. Prophylactic effect of Aqueous Extract of Annona Squamosa (AEAS) on Complete

Freund’s Adjuvant (CFA) induced arthritis in rats.

Table 3: Prophylactic effect of AEAS on CFA induced Paw Volume (ml).

Groups Days

0 4 8 14 21

Normal 0.90 ±

0.02

0.90 ±

0.02

0.90 ±

0.03

0.91 ±

0.02

0.92 ±

0.04

Control 0.93 ±

0.04

1.30 ±

0.05##

1.64 ±

0.10##

2.14 ±

0.07##

2.80 ±

0.07##

Diclofenac 10 mg/kg 0.92 ±

0.05

1.02 ±

0.02**

1.07 ±

0.03**

1.23 ±

0.05**

1.18 ±

0.04**

AEAS 100 mg/kg 0.96 ±

0.03

1.27 ±

0.09

1.62 ±

0.11

2.04 ±

0.06

2.80 ±

0.04

AEAS 200 mg/kg 0.95 ±

0.04

1.20 ±

0.04

1.59 ±

0.12

1.91 ±

0.12

2.79 ±

0.07

AEAS 400 mg/kg 0.97 ±

0.02

1.20 ±

0.06

1.54 ±

0.10

1.82 ±

0.10*

2.48 ±

0.10*

Values are expressed as mean ± SEM (n=6). *P<0.05, **P<0.01 as compared with control

(One-way ANOVA followed by Dunnet’s test). ## indicates significant induction when

compared with the normal group.

Sub planter injection of CFA in the rat hind paw led to the development of arthritis, which

reached peak edema on the 21st day of injection. There was a significant decrease in paw

edema in Diclofenac (10mg/kg) treated group on day 4th, 8th, 14th & 21st with P<0.01. The

result indicates that AEAS (100 mg/kg) & AEAS (200 mg/kg) did not show any significant



change in paw edema, whereas the AEAS (400 mg/kg) treated group suppressed paw edema

significantly on day 14th & 21st with P<0.05.

Figure 1: Prophylactic effect of AEAS on CFA induced Paw Volume (ml).

Table 4: Prophylactic effect of AEAS on CFA induced Paw Diameter (mm).

Groups Days

0 4 8 14 21

Normal 9.16 ±

0.30

9.16 ±

0.47

9.16 ±

0.47

9.33±

0.42

9.33±

0.21

Control 9.33 ±

0.61

11.50±

0.56

14.33±

0.84##

20.66±

0.84##

22.33±

0.71##

Diclofenac 10 mg/kg 9.00 ±

0.57

9.66±

0.66

11.50±

0.67

12.50±

0.67**

10.00±

0.25**

AEAS 100 mg/kg 9.33±

0.66

11.30±

0.7

13.66±

0.88

19.33±

0.98

22.16±

0.7

AEAS 200 mg/kg 9.66 ±

0.88

11.33±

0.88

13.33±

0.91

17.16±

1.24

19.83±

0.87

AEAS 400 mg/kg 8.83±

0.47

10.83±

0.94

12.83±

0.60

16.33±

1.62*

19.00±

0.85*

Values are expressed as mean ± SEM (n=6). *P<0.05, **P<0.01. as compared with control


compared with the normal group. Increased paw diameter, due to inflammation and edema

was also observed. In the control group, paw diameter showed changes up to the 21st day.

There was a significant decrease in paw diameter in Diclofenac (10 mg/kg) treated group on



day 14th and 21st with P<0.01. AEAS (100 mg/kg) & AEAS (200 mg/kg) treated group did

not show any significant change in paw diameter, whereas AEAS (400 mg/kg) treated group

suppressed paw diameter significantly on day 14th & 21st with P<0.05.

Figure 2: Prophylactic effect of AEAS on CFA induced Paw Diameter (mm).

Table 5: Prophylactic effect of AEAS on various Biochemical Parameters.

Groups SGPT (IU/L) SGOT (IU/L)

Normal 38.88 ± 2.99 48.56 ±3.12

Control 77.11 ± 3.10## 127.27 ± 8.55##

Diclofenac 10 mg/kg 58.01 ± 3.25** 64.26 ± 5.03**

AEAS 100 mg/kg 72.99 ± 3.84 123.38 ± 3.25

AEAS 200 mg/kg 70.93 ± 4.71 114.35 ± 4.45

AEAS 400 mg/kg 63.64 ± 2.93* 106.43 ± 6.60*

Values are expressed as mean ± SEM (n=6). *P<0.05, **P<0.01. as compared with control


compared with the normal group.

The biochemical parameters such as SGOT & SGPT were estimated for assessing the effect

on liver function. There was a significant increase in the level of SGPT & SGOT is observed



in the control group when compared with normal. Diclofenac (10 mg/kg) treated group

significantly decreased the level of SGPT & SGOT with P<0.01. Only AEAS (400 mg/kg)

showed significant changes in the level of SGPT & SGOT with P<0.05.

Figure 3: Prophylactic effect of AEAS on various Biochemical Parameters.

3.4. In vitro Antiarthritic activity of Annona squamosa.

Table 6: Phytochemical Analysis of Ethanolic Extract of Annona Squamosa (EEAS).

Sr. No. Test Observation

1 Test for glycosides +

2 Test for saponins +

3 Test for flavonoids +

4 Test for Alkaloids +

The phytochemical analysis of Ethanolic Extract of Annona squamosa (EEAS) confirms the

presence of glycosides, saponins, flavonoids, alkaloids etc.



Table 7: Anti-arthritic activity of Ethanolic Extract of Annona Squamosa (EEAS) by

Protein denaturation assay.

Groups Concentration

(mg/ml)

Absorbance at

660nm % Inhibition

Control - 0.89 -

Ethanolic extract of

Annona squamosa

0.2 0.84 5.60

0.4 0.79 11.23

0.6 0.71 20.22

0.8 0.60 32.58

1 0.56 37.07

Diclofenac Sodium 1 0.28 68.53

Figure 4: Anti-arthritic activity of Ethanolic Extract of Annona Squamosa (EEAS) by

Protein denaturation assay.

In the present study, rats were selected to induce arthritis because rats develop a chronic

swelling in multiple joints with an influence of inflammatory cells, erosion of joint cartilage,

and bone destruction. CFA induced models are extensively used to study the pathogenesis of

rheumatoid arthritis for testing therapeutics. One of the reasons for the wide utilization of this

model is due to the strong correlation between the efficacy of the therapeutic agents in this

model and rheumatic conditions in humans. In CFA induced arthritis, bacterial peptidoglycan



and muramyl dipeptide are responsible for arthritic induction. It occurs through cell mediated

autoimmunity by structural mimicry between mycobacteria and cartilage proteoglycans in

rats [18,19]. In the present investigation the arthritic rats showed a soft tissue swelling that

was noticeable around the ankle joints during the acute phase of arthritis and was due to be

edema of periarticular tissues such as ligaments and joint capsules. The paw swelling and

paw diameter is increasing in the initial phase of inflammation and then become constant for

two weeks. In the Prophylactic model AEAS (400 mg/kg) significantly suppressed paw

volume & paw diameter. Significant suppression of paw volume & paw diameter by AEAS

(400 mg/kg) may be due to the suppression of different cytokines such as IL‐1, IL‐6,

interferon‐γ (IFN‐γ) & TNF‐α which are responsible for joint swelling & paw inflammation

[20]. Assessment of the serum levels of SGOT & SGPT provides an excellent and simple

tool to measure the anti-arthritic activity of the target drug. The activities of SGOT & SGPT

increase significantly in arthritic rats since these are good indices of liver and kidney

impairment which is also considered a feature of CFA induced arthritis. Earlier findings

suggest that CFA administration in rats immunologically alters the hepatic biochemistry

which is responsible for changes in the level of Serum SGOT & SGPT. Serum SGOT and

SGPT have been reported to play a vital role in the formation of biologically active chemical

mediators such as bradykinins in the inflammatory process. In the present study, the

challenge with CFA has significantly elevated the serum SGOT & SGPT levels. However,

administration of AEAS (400 mg/kg) showed significant changes in the SGOT & SGPT

levels. This effect may be related to the significant immunological alteration in the hepatic

biochemistry by AEAS [21]. In the present study the protein denaturation bioassay was

selected for in vitro assessment of antiarthritic activity of ethanolic extract of Annona

Squamosa (EEAS). Denaturation of tissue proteins is one of the well-documented cases of

inflammatory and arthritic diseases. Production of autoantigens in certain arthritic diseases

may be due to the denaturation of proteins in vivo [22]. Agents that can prevent protein

denaturation therefore, would be worthwhile for antiarthritic drug development. The

significant change in % inhibition of test samples concerning control indicated stabilization

of protein i.e. inhibition of heat induced protein (albumin) denaturation by EEAS and

reference drug diclofenac sodium [23]. EEAS contains alkaloids, flavonoids, saponin, and

glycosides that are known to promote antiarthritic activity [24]. The result of the present

study reveals the anti-arthritic effect of AEAS at 400mg/kg as evidenced by (i) a significant

reduction in paw volume (ii) a significant reduction in paw diameter (iii) a significant change

in serum parameters (SGOT & SGPT) (iv) In vitro inhibition of protein denaturation. Our



photochemical investigation revealed that the presence of Saponins, flavonoids, steroid

glycoside, and alkaloids in the aqueous extract. The presence of a wide range of constituents

indicates the good efficacy of this plant in various pathological disorders. Saponin, steroids,

alkaloids are known to inhibit articular swelling, decrease arthritic index and regulate down

the content of IL-IB and TNF-α in the inflammatory tissues of arthritic rats [18,19]. Besides

this flavonoid has been reported to inhibit the cyclooxygenase enzyme thereby inhibiting

prostaglandin synthesis which interrupt might result in increased lysosomal membrane

stability. Pharmacological studies indicate that flavonoids and steroidal saponin have anti-

inflammatory, antiarthritic, antiviral, antimicrobial, and antitumor activity. Saponin may have

antiarthritic effect properties by suppressing intestinal protozoa which may have a role in

joint inflammation. Dietary saponin reduces blood cholesterol level these effects result in

saponin binding with cholesterol and excreted in bile [25].

4. CONCLUSION:

Thus, in the light of the above facts, it can be demonstrated that the different extracts of

Annona Squamosa leaves may serve as an effective anti-arthritic drug and the effect might be

speculated due to phytochemicals such as alkaloids, saponins & flavonoids. This study

warrants the investigation to isolate and identify the active principles and to investigate the

exact mechanism of action against arthritis.

5. ETHICAL STATEMENT:

The experimental design and research plan along with animal handling and disposal

procedure were approved by the Institutional Animal Ethics Committee of Tatyasaheb Kore

College of Pharmacy, Warnanagar Kolhapur. IAEC approval No: TKCP/2017/01/03.

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

7. ACKNOWLEDGMENT:

As said “where there is a will there is a way”, I had the will but the way to accomplish my

goal was shown only and only by my esteemed research Guide Dr. K.B.Burade, Principal

Government College of Pharmacy Karad. I express my deep sense of gratitude and am

indebted towards him who always guided me to work and added new dimensions to my

thinking in a scientific way. I express my sincere thanks to another research guide Dr. Ritesh



Agrawal, Head IRP Cell Pacific University, Professor Pacific College of pharmacy Rajasthan

for inspiring me, timely attention & kind interest, since the beginning of my course.

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Mr. Nilesh V. Desai1 (M Pharm Pharmacology)

Research Scholar,

Pacific University Udaipur Rajasthan, India.

Dr. Kishorkumar B. Burade2

Principal, Government College of pharmacy Karad

Maharashtra, India.

Dr. Ritesh Agrawal3

Head IPR Cell Pacific University,

Professor Pacific College of pharmacy Rajasthan.

evaluation of in vivo and in vitro antiarthritic potential

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