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Transcript of EVALUATION OF ANTI OSTEOARTHRITIC ACTIVITY OF CHIA … fileseeds was done by botanist Dr. Ramrao,...
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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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