EVALUATION OF ANTI OSTEOARTHRITIC ACTIVITY OF CHIA … fileseeds was done by botanist Dr. Ramrao, Central drug testing laboratory, Bengaluru. Extraction: 200g of dried seeds were coarsely

www.wjpps.com Vol 8, Issue 6, 2019.

745

Arpitha et al. World Journal of Pharmacy and Pharmaceutical Sciences

EVALUATION OF ANTI OSTEOARTHRITIC ACTIVITY OF CHIA

SEEDS (SALVIA HISPANICA) IN RATS

Arpitha M.*, Dr. M. S. Rajesh, Devika Rani K. and Dr. S. Ramachandra Setty

Department of Pharmacology, Government College of Pharmacy, Subbaiah Circle Bengaluru,

Karnataka, India.

ABSTRACT

Osteoarthritis(OA) is a classic age-related chronic degenerative

disorder. In OA degradation and loss of the articular cartilage is a

central feature that is sometimes attributed to “wear and tear”. The

present study was designed to evaluate the anti-osteoarthritic activity

of Salvia hispanica. Seeds of Salvia hispanica were extracted with

mixture of water and ethanol (30:70). Invivo anti-inflammatory

activity and anti-osteoarthritic activity were evaluated. Acute toxicity

studies were carried out as per OECD guideline 420. The effect of

hydro alcoholic extract of seeds of Salvia hispanica were screened

using models like Corticosteroid induced OA for anti-osteoarthritis,

Carrageenan induced paw edema (1% carrageenan) and Cotton pellet

method, by implanting 10mg of cotton subcutaneously for anti-inflammatory activity.

Diclofenac sodium (11mg/kg b.w) and alandronate sodium (1mg/kg b.w) were used as

reference standard, the degree of protection was determined by bone density, bone width,

serum calcium, and phosphorous for anti-osteoarthritis and granuloma tissue formation and

paw volume were evaluated for anti-inflammatory effect. Treatment with hydro alcoholic

extract of chia seeds significantly increased serum calcium, phosphorous and bone density

favouring anti-osteoarthritic activity, and it also exhibited inhibition in granuloma tissue

formation and decrease in paw volume facilitating anti-inflammatory activity. Further the

histopathological evaluation revealed decrease in parameters like bone resorption, cyst

formation and depth surface induced by corticosteroids which may be helpful in preventing

osteoarthritis and inflammation respectively.

KEYWORDS: Osteoarthritis, Salvia Hispanica, Anti-Inflammatory.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 7.421

Volume 8, Issue 6, 745-756 Research Article ISSN 2278 – 4357

*Corresponding Author

Dr. Arpitha M.

Department of

Pharmacology, Government

College of Pharmacy,

Subbaiah Circle Bengaluru,

Karnataka, India.

Article Received on

02 April 2019,

Revised on 23 April 2019, Accepted on 14 May 2019

DOI: 10.20959/wjpps20196-13779


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INTRODUCTION

Arthritis means pain in joints, due to inflammatory/immune mediated injury of the joints.

There are three types of arthritis, mainly rheumatoid arthritis, osteoarthritis, and psoriatic

arthritis. Among them osteoarthritis is a major type seen all over the world. Osteoarthritis

(OA) is a classic age-related disorder. It is often described as a chronic degenerative disease.

In OA degradation and loss of the articular cartilage is a central feature that is sometimes

attributed to “wear and tear”. OA can be viewed as the clinical and pathological outcome of a

range of disorders that result in structural and functional failure of synovial joints with loss

and erosion of articular cartilage, subchondral bone alteration, meniscal degeneration, a

synovial inflammatory response, and bone and cartilage overgrowth. OA occurs when the

dynamic equilibrium between the breakdown and repair of joint tissues becomes unbalanced.

OA can occur in synovial joint in the body but is most common in the knees, hips and hands.

In Ayurvedic system of medicine, numerous plants have been used to treat osteo arthritis, as a

part of this study a field survey was conducted and found a plant by name chia (Salvia

hispanica) (Family: Laminacae ) upon literature survey it was found that the plant contain

fatty acids such as n-3 and n-6 alpha-lenolenic acid conjugated linoleic acid (CLA) these

helps to prevent the inflammation, CLA helps in reducing body weight.[4]

The plant also

contain Minerals such as calcium(18%), magnesium, phosphorous, vitamins like A, D, E, K,

C in that Vitamins A, C, and D are responsible for the stimulation of cartilage regeneration. It

also contain amino acids such as lysine, valin, alanin, glycine and several other in that the

lysine is responsible for absorption of calcium.

But there are no reports regarding the utility of this plant in treating osteoarthritis. Since the

plant is reported to possess minerals that are required to strengthen bones, vitamins to

stimulate cartilage regeneration and amino acids to enhance calcium absorption; “It is

hypothesized that this plant may be helpful in treating osteoarthritis”.

MATERIALS AND METHOD

The plant material (seeds) were procured from registered suppliers and authentication of

seeds was done by botanist Dr. Ramrao, Central drug testing laboratory, Bengaluru.

Extraction: 200g of dried seeds were coarsely powdered and extracted with petroleum ether

using Soxhlet apparatus for defatting and the defatted marc was further used for

hydroalcoholic extraction. The solvent was completely removed and the exract further was

subjected to phytochemical evaluation and pharmacological screening.


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Materials used: Alandronate sodium, Prednisolone, Diclofenac sodium, Carrageenan, Pet.

Ether, Methanol, Hydroalcoholic extract of Salvia hispanica.Anaesthetic ether, Formalin,

Xylazine, Hydrochloric acid, Lignocaine.

Animals used: 8-12 week old wistar albino rats of either sex weighing between 150-200g

were housed in polypropylene cages and were maintained at standard laboratory conditions

under 12h light and 12h dark cycle, with free access to food and water ad libitum and the

animals were allowed to acclimatize to laboratory conditions for about 2 weeks former the

study. The experimental protocol was approved by Institutional Animal Ethical Committee.

Phytochemical screening: Proximate analysis of the powdered Chia seeds was performed

for Moisture content, water and alcohol soluble extractive value, Ash value and phyto

chemical screening.

Screening Models

Anti Inflammatory models

1. Cotton pellet induced rat paw edema[15]

Method described by D’Arcy et al, 1960 was followed, female wistar rats were divided into 4

groups with 6 animals in each group.

Group I: Disease control: Vehicle

Group II: Std control: Diclofenac sodium

Group III: Treated (low dose): HAE of Chia seeds(200mg/kg)

Group IV: Treated (high dose): HAE of Chia seeds(400mg/kg)

Anti-inflammatory activity will be evaluated by implanting sub-cutaneously pre-weighed

sterile cotton pellets (10+_mg) in ventral region of rat under light ether anesthesia. All drug

doses and vehicle are administered as mentioned for seven consecutive days after the

implantation of cotton pellet. On the 8th

day the animals are anesthetised and the pellets with

granuloma tissues are carefully removed and freed from extraneous tissuses, dried at 60O

c for

24h and weighed.

2. Carrageenan induced rat paw edema :Female wistar rats were treated according to

the method described by winter et al[15]

Group I: Normal control: Vehicle

Group II: disease control: Vehicle + 1% of carrageenan solution(0.1ml).


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Group III: standard control: Diclofenac 11mg/kg bodyweight.

Group IV: Treated (low doses): HAE of Chia seeds (200mg/kg bw).

Group V: Treated (high doses): HAE of chia seeds (400mg/kg bw).

In this method inflammation was produced by injecting 0.ml of 1% 0.1ml carrageenan

suspension into sub plantar region of hind paw of rat subcutaneously, after 1hr of

administration of test substance or standard samples. The raw volume was measured using

Plethsmometer from 0-6hr, at an hourly interval. Percentage inhibition of edema was

calculated for each group with respect to its control group.

Corticosteroid induced Osteoarthritis in rats

Female wistar rats were treated according to the method described by

Group I: Normal control: vehicle

Group II: Disease control: Prednisolone (6mg/kg)+ Vehicle

Group III: Treated (low dose): Prednisolone (6mg/kg) + HAE of Chia seeds

(200mg/kg bw)

Group IV: Treated (high dose) : Prednisolone (6mg/kfg bw)+Alendronate.

Animals are randomised into 5 group with 8 rats in each group. Prednisolone 6mg/kg bw is

orally administered daily for 80days to all the groups except normal control. On 81st day all

animals sacrificed and Blood is collected by carotid bleeding and serum is separated for

estimation of Ca2+ and phosphorous, femur and tibia are removed, cleaned and evaluated to

obtain ca2+,phosphorous content of ash, bone strength, bone density and histopath.

RESULTS

Phytochemical evaluation Table No.1.

Sl. No. Chemical constituents

Tests Observations Inference

1. Protiens and

amino acids

Millon’s test Formation of white precipitate

Proteins and amino

acids present Biuret test

Appearance of pink colour in the

ethanolic layer

Ninhydrin test Appearance of characteristic purple

colour

2. Carbohydrates

Fehling’s test Formation of red precipitate

Carbohydrates present Barfoed’s test Formation of red precipitate

Benidict’s test Formation of characteristic colour

A. Cotton pellet induced granuloma in rats


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Table No. 2: Effect of chia seeds extract on inflammation in cotton pellet induced

Granuloma.

Groups Mean Granuloma tissue

formation in mg % of inhibition

Normal control 12.25± 1.31 -

Standard control 3.67 ± 0.26**

70.o4%

High dose(400mg/kg) 3.92 ± 0.47**

68%

Lowdose(200mg/kg) 4.67 ± 0.22**

61.87%

All values are expressed as mean± SEM. p<0.05*, p<0.01

**, p< 0.001

***,against to Disease

control. Compare to control group there will be a significant decrease in the granuloma

formation the group treated with higher and lower dose of chia seeds extract.

Cotton pellet induced granuloma in rats

B. Carrageenan induced rat paw edema.

Table No. 3: Effect of chia seeds extract on carrageenan induced rat paw edema.

Mean paw volume in ml

Groups 0 hr 1 hr 2 hr 3 hr 4 hr 5 hr

Disease control 1.52±0.08 1.58±0.10 1.65±0.14 1.82±0.13 95±0.11 2.10±0.10

Standr d

control 97±0.06** 1.01±0.06** 63±0.06 37±0.07** 1.25±0.07** 0.91±0.09**

High dose 1.04±0.06** 1.23±0.07** 1.34±0.09* 1.29±0.06** 1.21±0.06** 1.13±0.05**

Low dose 1.18±0.03** 1.40±0.03 1.52±0.03 1.44±0.02** 1.39±0.02** 1.35±0.03

All values are expressed as mean ±SEM. p<0.05*, p<0.01

**, p<0.001

*** against to Disease

control.

Carrageenan induced rat paw edema.

Carrageenan induced rat paw edema


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Disease control

Standard control

High dose

Low dos

C. Corticosteroid induced osteoarthritis in rats

Compared with disease control group very significantly increased in ash calcium and

phosphorous, femur density, femur weight, tibia weight, and serum calcium & phosphorous

in standard group. Treatment with high dose of chia seeds extract and low dose of chia seeds

extract significantly increased in ash calcium & phosphorous, femur & tibia weight, femur

density, serum calcium & phosphorous compare to disease control group.

Effect of chia seeds extract on bone density

Effect of chia seeds extract on length of femur and tibia

Effect of chia seeds extract on serum calcium and phosphorous


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Effect of chia seeds on bone ash calcium and phosphorous.

Effect of chia seeds extract on carticosteroind induced osteoarthritis in rats

Table No. 5 All groups are comparing to normal control.

Sl No Parameters Normal

control Disease control

Standard

control High dose Low dose

1 Bone density in g/cc 1.49±0.02 1.15±0.01**

1.38± 0.02* 1.32±0.02

** 1.25±0.01

**

2 Femur Bone

weight in g 0.425±0.01 0.34 ±0.02 0.365±0.01 0.360±0.01 0.345±0.01

3 Tibia Bone

weight in G 0.36±0.03 0.28±0.03 0.335±0.02 0.345±0.01 0.32±0.0

4 Length of

Femur in mm 32.5±0.5 30±1.00 30.5±0.05 30.5±0.5 31±1.00

5 Length of

Tibia in mm 36±1.00 33.5±3.15 33.5±0.5 33.5±2.5 34±1.00

6 Serum calcium 8.085±0.07 7.925±0.1** 7.925±0.1**

8.95±0.1**

8.53±0.1**

7 Serum

Phosphorous 4.515±0.5 1.705±0.1** 3.755±0.06 2.975±0.08

* 2.45±0.4

*

8 Body weight 135.00±1.89 123.75±2.63* 127.50±2.50 122.50±4.11

* 133.75±2.63

All values are expressed in terms of mean ± SEM. p<0.05*, p<0.01

**, p< 0.001

***, against to

Normal control.


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Table No. 6 Effect of chia seeds extract on corticosteroid induced osteoarthritis in rats.

All groups are comparing to disease control group.

Sl No Parameters Normal

control

Disease

control

Standard

control High dose Low dose

1 Bone density

in g/cc 1.49±0.02

** 1.15±0.01 1.38± 0.02

** 1.32±0.02

** 1.25±0.01

*

2 Femur Bone

weight in g 0.425 ±0.01 0.34 ±0.02 0.365±0.01 0.360±0.01 0.345±0.01

3 Tibia Bone

weight in G 0.36±0.03 0.28±0.03 0.335±0.02 0.345±0.01 0.32±0.0

4 Length of

Femur in mm 32.5±0.5 30±1.00 30.5±0.05 30.5±0.5 31±1.00

5 Length of

Tibia in mm 36±1.00 33.5±3.15 33.5±0.5 33.5±2.5 34±1.00

6 Serum

calcium 10±0.1

** 7.925 ± 0.1 7.925±0.1

** 8.95±0.1

** 8.53±0.1

7 Serum

Phosphorous 4.515±0.5

** 1.705±0.1 3.755±0.06

* 2.975±0.08

* 2.45±0.4

*

All values are expressed in terms of mean ± SEM. p<0.05*, p<0.01

**, against to Disease

control.

Histopathology of femurs


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Report: The microscopic observation shows

The disease control group shows that the formation of resorption, cysts, depth surfaces, and

wide range of lesions. Compare to disease control group there will be significant

improvement in the standard group (Alandronate) and the group treated with high dose (400

mg of chia extract) in decreasing the resorption, lesions, cysts formations. The group treated

with low dose (200mg of chia extract) shows less effect in decreasing bone resorption, cysts,

depth surfaces and lesions.

DISCUSSION

The extract was screened for acute oral toxicity as per OECD guidelines 420. The extract was

found safe till 2000mg/kg bw did not observed any mortality. Hence 1/10th

(200mg/kg bw

low dose) of 2000mg/kg bw was selected as therapeutic dose and 2TD dose for further

studies. The anti-osteoarthritic activity of hydro alcoholic extract of chia seeds was evaluated

by using three animal models. The results of anti-inflammatory activity of hydro alcoholic

extract of chia seeds were recorded.

The results shows increased granuloma tissue formation 12.25± 1.31 in disease control.

Compared to disease control there were be significant decrease in the granuloma tissue

formation in the group treated with diclofenac (standard control) 3.67 ± 0.26 (70.04%), the

group treated with higher dose (400mg/kg bw) of hydro alcoholic extract of chia seeds

showed 3.92 ± 0.47 (68%) decrease in the granuloma tissue formation and the group treated

with lower dose (200mg/kg bw) showed 4.67 ± 0.22 (61.87%) decrease in granuloma tissue

formation. The effect of HAE of chia seeds on anti-inflammatory activity was evaluated in

carrageenan induced rat paw edema. The group treated with carrageenan and vehicle (disease

control) showed increased paw volume 2.10 ± 0.10 at fifth hour. Compare to disease control


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group there were be a significant decrease in the paw volume 0.91 ±0.09 in the group treated

with diclofenac (11mg/kg bw) at fifth hour and the group treated with higher dose (400mg/kg

bw) of hydro alcoholic extract of chia seeds shows no significant effect at 2nd

hour 1.34 ±

0.09 but it shows significant effect at fifth hour 1.13± 0.05 in decreasing the paw edema

volume and the group treated with lower dose (200mg/kg bw) of hydro alcoholic extract of

chia seeds not shows significant decrease in the paw edema volume in 2nd & 3rd hour (1.40±

0.03, 1.52 ± 0.03) but it showed significant effect in fifth hour in decreasing the paw volume

1.35 ± 0.03, but the effect is lesser than 2TD or at higher dose (400mg/kg bw ). The results

were recorded in the table no 9. The anti-osteoarthritic activity of HAE of chia seeds was

studied against corticosteroid induced osteoarthritis in rats for 80 days. Egras MA, Hamilton

RW, lenz LT, monaghan SM et al., reported that Decrease in the body weight was related to

the effect of chia seeds (salvia hispanica) on muscle mass and boost metabolism20,32. In this

study the groups treated with chia seeds extract shows marginal effect on reducing body

weight of animals in group treated with higher dose of HAE of chia seeds. The table no10

&11showed that group treated with standard drug alaldronate (1mg/kg bw) showed

significant increase in serum and ash calcium, phosphorous(7.925±0.1 & 3.755±0.06), the

group treated with high dose (400mg/kg bw) of hydro alcoholic extract of chia seeds showed

that significant improvement in the serum and ash calcium, phosphorous,( 8.95±0.1,

2.975±0.08 ) and the group treated with lower dose (200m/kg bw) showed increased level of

serum and ash calcium, phosphorous compare to disease control.The group treated with

higher and lower dose (200 and 400mg/kg bw) of HAE of chia seeds shows significant

increase in the bone density when compare to disease control the results were recorded in the.

The results were further supported by histopathological studies. The observation of slide A

(disease control) showed that the formation of resorption, cysts, depth surfaces, and wide

range of lesions. Compare to disease control group there will be significant improvement in

the slide B (standard control Alandronate) and in slide D (the group treated with high dose

400 mg/kg bw of chia seeds extract) in decreasing the resorption, lesions, cysts formations.

And in slide E (the group treated with low dose 200mg/kg bw) showed less effect in

decreasing bone resorption, cysts, depth surfaces and lesions.

CONCLUSION

The present study was made to evaluate the anti-osteoarthritic activity of HAE of chia Seeds

against cotton pellet induced granuloma, carrageenan induced paw edema and corticosteroid

induced osteoarthritis in rats. The phytochemical investigation showed that the plant contains


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minerals, carbohydrates, proteins and amino acids. The hydro alcoholic extract of chia seeds

prevented bone loss and resorption in corticosteroid (prednisolone) induced osteoarthritis and

prevented inflammation in cotton pellet induced granuloma and carrageenan induced

inflammation in wistar rats. Maira Rubi Segur-Campos et al., reported that the plant contain

Minerals such as calcium(18%), magnesium, phosphorous, vitamins like A, D, E, K, C in that

Vitamins A, C, and D Are responsible for the stimulation of cartilage regeneration19. Sona

Nitrayova, Matej Brestensky et al., reported that the plant contain amino acids such as lysine,

valin, alanin, glycine and several other in that the lysine is responsible for absorption of

calcium23 and Dr.Tajudeen Abdul Hameed et al., reported that n-3 and n-6 alpha-lenolenic

acid conjugated linoleic acid (CLA) these are helps to prevent the inflammation17, These

findings suggest that chia has significant remineralisation and anti-inflammatory activity in

osteoarthritis in wistar rats. The beneficial effects of HAE of chia seeds indicate its potential

in treating osteoarthritis in natural way through herbal resources. Thus chia could be

employed in managing osteoarthritis. This has potential implication in further development of

herbs and mineral formulations as anti-osteoarthritis agents.

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Documents

EVALUATION OF ANTI OSTEOARTHRITIC ACTIVITY OF CHIA … fileseeds was done by botanist Dr. Ramrao, Central drug testing laboratory, Bengaluru. Extraction: 200g of dried seeds were coarsely