Evaluation of ethanolic root extract of Daucus carota on steroid Induced Osteoporosis in rats.
Merlin Mary M*, Sudha KM
Institute of Pharmacology, Madras Medical College, Chennai-600 003.
*Corresponding Author E-mail: merlinjuliet325@gmail.com
ABSTRACT:
Objective: To evaluate the anti-osteoporotic activity of ethanolic extract of Daucus carota (DC) roots on steroid induced Osteoporosis in rats. Material and methods: Female wistar rats were divided into five groups of (n=6) each. Osteoporosis was induced by intramuscular administration of dexamethasone 7mg/kg once a week for four weeks to all groups of rats except normal control group. Group I served as normal control and received normal saline. Group II served as disease control injected with dexamethasone 7mg/kg (i.m). Group III served as standard control and treated with sodium alendronate 0.2mg/kg orally from 15th to 28th day. Group IV and V were treated with DC at doses of 200mg/kg and 400mg/kg (p.o) respectively from 15th to 28th day. Following treatment, antiosteoporotic effect of DC was evaluated by biochemical and biomechanical analysis and radiological observation along with histopathological examination among the experimental groups. Results: Rats treated with ethanolic extract of DC (200mg/kg and 400mg/kg) showed significant (P <0.01and P < 0.001) increase in level of serum calcium and phosphorus, whereas serum alkaline phosphatase was significantly (P < 0.05 and P < 0.001) decreased. A significant (P < 0.001) increase in bone weight, thickness, hardness was observed when compared to the disease control group. It was further confirmed by radiological and histopathological examination of femur bone. Conclusion: The present findings propose that ethanolic extract of Daucus carota possess potent antiosteoporotic activity in steroid induced osteoporotic rats.
KEYWORDS: Steroid induced osteoporosis, Daucus carota, Dexamethasone, Radiograph, Bone hardness.
INTRODUCTION:
It is also used in hypersensitivity reactions like atopic dermatitis, anaphylaxis, blood transfusion therapy, in drug allergies, asthma and auto immune disorders5.Glucocorticoids inhibits osteoblastogenesis. It lessens the lifespan of osteoblasts and osteocytes6. The antiresorptive drugs like Bisphosphonates lessens bone loss, improves BMD and reduces fracture rates. Alendronate, risedronate and zoledronic acid are recommended as first line therapy in men and women for primary and secondary prevention of vertebral, nonvertebral and hip fractures. Pharmacological agents are associated with severe adverse effects like epigastric distress, peptic ulcers and renal failure7,8. Hence the present research is focused at utilizing the nature’s gifts (Herbs) in prevention and treatment of osteoporosis with minimal or no adverse effects.
Daucus carota. Linn commonly known as “carrot” belong to the family Apiaceae and are cultivated all over the world as a vegetable9. The main constituents in the plant is alkaloids, carbohydrates, chlorogenic acid, phenols, essential oil, terpenoids and flavonoids including apigenin. Apigenin plays a major role in bone formation and resorption process in oxidative stress induced osteoporosis10. This plant exhibits nutritional and therapeutic benefits including antimicrobial, antioxidant, anticancer, hypolipidemic, cardioprotective, immunomodulatory, anti-inflammatory, analgesic antipyretic and many other pharmacological effects11. However so far, no scientific valid data has been made to establish it as an antiosteoporotic agent. Hence, on the basis of these evidences, it is assumed that apigenin of carrot can promote bone formation and may be useful in treatment of osteoporosis.
MATERIALS AND METHODS:
Plant material:
Roots of Daucus carota were purchased from Chennai, Tamil Nadu in the month of November 2018. The roots were authenticated by Dr. K. N. Sunil Kumar, Research Officer and HOD of Pharmacognosy, Siddha Central Research Institute, Arumbakkam, Chennai.
Plant Extraction:
The freshly collected roots of this plant were chopped, shade dried. The dried material was powdered and passed through a 40-mesh sieve. The powder was then extracted with ethanol (99.9%) using a Soxhlet extractor. The extract was then concentrated by using a rotary evaporator. The percentage yield was found to be 11.42%w/w.
Experimental animals:
The present study was conducted after obtaining approval from the Institutional Animal Ethics Committee and this protocol met the requirements of national guidelines of CPCSEA/ IAEC approval no: 1917/ReBi/S/16/CPCSEA/25.10.2016 and 31/AEL/IAEC/MMC, Date:16.11.2018. Female Wistar albino rats used for this study were procured from Animal house, Madras Medical College, Chennai, India. Acclimatization was done as per CPCSEA guidelines before the study was carried out. The animals were housed in spacious polypropylene cages and fed with standard pellet diet and water ad libitum throughout the study.
Experimental procedure:
Thirty healthy female albino wistar rats were weighed and grouped randomly into six groups (n=6). GroupI kept as normal control received normal saline. Group II considered as disease control group was given dexamethasone i.m at a dose of 7mg/kg once a week for 4 weeks. Group III considered as standard control was treated with dexamethasone 7mg/kgonce a week for 4 weeks intramuscularly and standard drug sodium alendronate 0.2mg/kg/dayorally from 15th to 28th day. Group IV and Group V were considered as treatment groups that received Daucus carota at doses of 200mg/kg and 400mg/kg/day/orally administered for 14 days along with dexamethasone 7mg/kg/once a week, intramuscularly.
Evaluation of antiosteoporotic activity:
Changes in body weight:
The weight of the animals was noted every week till the end of the study period and changes in body weight was observed.
Biochemical Analysis:
The blood was collected by cardiac puncture after anaesthetizing the animals with isoflurane 2-3%, using a fine capillary tube. The collected blood was then processed by centrifuging at 3000rpm for 15minutes in order to separate serum from cells and stored at temperature of -20℃. Serum was collected in rapid clot activator tubes and subjected for biochemical analysis of serum calcium, serum phosphorus, serum alkaline phosphatase which was measured spectrophotometrically using automatic analyser.
Biomechanical Analysis:
After euthanizing the animal by overdose of isoflurane, the animal was placed on a dissecting board and the hip ball and socket joint was reached by invasion and retraction. The bone was dislocated and cleansed by removing the tissues around it and femoral bone was stored in 10% neutralized buffer formalin solution. The femur was then subjected to biomechanical evaluation. The weight of each bone was measured using digital balance. The length is measured by using ruler and the thickness was measured at the epiphyseal growth plate region by using Vernier Caliper. The hardness of the bone was measured in order to determine the periosteal and endosteal arrangement of cortical bone by determining the fracture point. The fracture point is the measure of the point at which the bone breaks when weight was applied. In this study hardness tester was used to determine bone hardness.
Radiological observation:
At the end of the study, radiographs were taken using table top procedure (44kVp and 2 mAs) with exposure time of 10ms and working distance of 1m in all animals.
Histopathological examination:
The isolated femur bone was defatted by treating the bone with 5% nitric acid for a period of 24 hours. Then the bone samples were dehydrated using automated vacuum tissue processor. Dehydrated samples were embedded in paraffin wax and then sectioned. The sectioned bone was stained with haematoxylin and eosin (H&E) and observed under light microscope.
Statistical analysis:
The results were expressed as mean ± SEM. The data were statistically analysed by means of one-way ANOVA followed by Dunnett’s multiple comparison test using Graph Pad Prism Software version 8.0.2. One-way ANOVA was used to correlate the statistical difference between the variables. P values (P<0.05, P<0.01, P<0.001) were considered as statistically significant.
RESULTS AND DISCUSSION:
Long term exogenous glucocorticoid therapy causes profound reduction of bone mineral density, bone quality, bone formation and bone mechanical parameters, which leads to fracture. Glucocorticoids inhibits osteoblastogenesis and reduces the lifespan of osteoblasts and osteocytes. They impair the replication, differentiation and function of osteoblasts and induce the apoptosis of mature osteoblasts and osteocytes. For assessing the anti-osteoporotic activity, glucocorticoid induced rat model was selected. Female wistar albino rats were chosen for this study. Duration of the study was four weeks. Dexamethasone was injected at the dose of 7mg/kg intramuscularly to all group of animals except control group of rats. Ethanolic root extract of Daucus carota was given from 15th to 28th day at the dose of 200mg/kg and 400mg/kg orally.
Body weight changes:
There was an increase of body weight in normal rats throughout the experiment. The disease control rats showed decrease in body weight. There was a slight increase in body weight in rats treated with low dose (200mg/kg) and high dose(400mg/kg) of Daucus carota. However, the above changes were not statistically significant.
Effect of Daucus carota on biochemical parameters:
Glucocorticoids in high doses interfere with active transmembrane calcium transport. They also inhibit the expression of genes dependent on vitamin D3. They decrease calcium absorption in the gastrointestinal tract and increase urine calcium loss, which causes a negative calcium balance. This, in turn, leads to hyperstimulation of the parathyroid glands and secondary hyperparathyroidism. As a result of negative calcium balance and inhibited osteoblast activity, bone tissue demineralization occurs.
In this study dexamethasone injected group significantly (P < 0.001) decreased serum levels of calcium and phosphorus when compared to the normal control rats. Treatment of standard group showed significantly (P < 0.001) increased levels of calcium. A significant(P < 0.01 and P < 0.001)increase in level of serum calcium was also found in low and high dose extract treated group when compared with disease control rats.There was significant (P < 0.001, P < 0.01 and P < 0.001) increase in serum phosphorus in groups III, IV and V rats when compared to disease control rats.
Alkaline phosphatase is an important biochemical marker of bone turnover.ALP is indicator of bone activity and is used to monitor metabolic bone disease. The serum ALP level was found to be significantly (P < 0.001) increased in osteoporotic rats compared with the normal control rats. Also, from the results alkaline phosphatase was seen to be significantly (P < 0.05 and P < 0.001) reduced in rats treated with ethanolic extract of Daucus carota at both the doses (200mg/kg and 400 mg/kg respectively) when compared to the disease control rats. Similar effect was seen in sodium alendronate treated rats. (Table 1)
Table 1: Effect of ethanolic root extract of Daucus carota on biochemical parameters.
|
Group |
Treatment |
Serum calcium (mg/dl) |
Serum phosphorus (mg/dl) |
Serum alkaline phosphatase (IU/L) |
|
I |
Normal control |
9.15±0.26 |
8.01±0.28 |
126.04±5.94 |
|
II |
Disease control |
3.84±0.38### |
4.89±0.38### |
266.72±22.1### |
|
III |
Standard control |
7.54±0.53*** |
7.53±0.32*** |
140.72±4.3*** |
|
IV |
Low dose (200mg/kg) |
5.90±0.34** |
6.53±0.19** |
212.25±16.3* |
|
V |
High dose (400mg/kg) |
7.01±0.52*** |
7.37±0.28*** |
158.30±6.7*** |
All the values are expressed as mean± SEM (n=6). *P < 0.05, **P < 0.01and ***P < 0.001 compared with disease control; ###P < 0.001 compared with normal control.
Effect of Daucus carota on biomechanical parameters:
Weight and length of femoral bone was decreased (P < 0.001 and P < 0.01) in disease control group compared with normal control group. Administration of sodium alendronate (0.2mg/kg) showed significant (P < 0.001) increase in bone weight compared with disease control group of rats. Ethanolic extract of Daucus carota at the dose of 200mg/kg and 400mg/kg showed significant (P < 0.05 and P < 0.001) increase in the bone weight when compared to the disease control group of rats, whereas there were no significant changes in length of the femoral bone observedwhen treated with sodium alendronate (0.2mg/kg) and ethanolic extract of Daucus carota at the dose of 200mg/kg and 400mg/kg.
Thickness of femoral bone was decreased (P < 0.001) significantly in disease control group when compared with normal control group. Administration of ethanolic extract (200mg/kg and 400mg/kg) and alendronate treated group showed improvement in the thickness when compared to the disease control group.
The major impact of glucocorticoid induced osteoporosis is an unequivocal increase in fracture risk at both trabecular and cortical sites. Disease control group showed significant (P<0.001) decrease in hardness when compared to the normal control rats. Daucus carota (low dose 200mg/kg and high dose 400 mg/kg) treated group showed an increase in hardness of bone when compared to disease control group. Alendronate treated rats also shows a similar effect in osteoporotic rats.
A. Bone weight
B. Bone length
CBone Thickness
D. Bone thickness
Figure 1: Effect of ethanolic root extract of Daucus carota on biomechanical parameters. (A) Bone weight changes;(B) Bone length changes;(C) Bone thickness;(D) Bone hardness. All the values are expressed as mean± SEM (n=6). *P < 0.05, **P < 0.01and ***P < 0.001 compared with disease control; ##P < 0.001,###P < 0.001 compared with normal control.
Radiographic observation:
Radiographic observation showed features of osteoporosis in disease control group. Radiological findings confirmed osteoporosis as evidenced by radiolucent view of thinning of cortical bone. Treatment with alendronate and test drug showed marked improvement in bone formation, thickening of cortical bone which confirmed that test drug has effect in reducing porous bone and improving the bone formation by radiological observation. (Figure 3)
A. Normal control
B. Disease control
C. Standard control
D. Low dose (200mg/kg)
E. High dose (400mg/kg)
Figure 2: X-ray imaging of femur bones. (A) Normal control(untreated rats); (B) Disease control rats (dexamethasone 7mg/kg intramuscularly); (C) standard control rats (sodium alendronate 0.2mg/kg orally); (D) low dose of Daucus carota (200mg/kg orally); (E) high dose of Daucus carota (400mg/kg)
Histopathological examination:
Femur bone section was examined for any histological changes. Control group animals showed normal architecture and normal bone compactness. The rats in steroid induced model group showed thinning of trabeculae, increase in number of osteoclasts and decreased osteoblasts. Alendronate and Daucus carota group exhibited significant restorative process with increased osteoblast and trabecular thickness. Treatment with high dose of Daucus carota showed resemblance to alendronate group with more osteoblastic number. Therefore, from the above histopathological examination, it is proved that the ethanolic extract of Daucus carota is active in bone formation and has beneficial effects in the treatment of osteoporosis. (Figure 3)
|
|
|
|
|
|
A. Normal control |
B. Disease control |
C. Standard control |
|
|
|
|
||
|
D. Low dose (200mg/kg) |
E. High dose (400mg/kg) |
||
Figure 3: H and E staining of femur bone.(A) Normal control (untreated rats);
(B) Disease control rats (dexamethasone 7mg/kg intramuscularly); (C) standard control rats (sodium alendronate 0.2mg/kg orally); (D) low dose of Daucus carota (200mg/kg orally); (E) high dose of Daucus carota (400mg/kg)
From the results and discussion, it was clear that the ethanolic root extract of Daucus carota has an ameliorative effect on steroid induced osteoporosis.
On reviewing literatures on the beneficial effects of apigenin on bone it was found to inhibit osteoclast differentiation by reducing receptor activator of nuclear factor kappa ligand (RANKL)and calcitonin receptor resulting in the inhibition of multinucleated osteoclast formation12. Present work suggests that Daucus carota containing apigenin may have a potential role against bone loss induced by steroids.
CONCLUSION:
From the study it is concluded that the ethanolic extract of Daucus carota root possesses beneficial effect against steroid induced osteoporosis which has been proved by the valid data obtained from the in-vivo evaluation which includes serum markers, biomechanical properties, radiological observation and histopathological examination.
The present study provided basic evidence that the Daucus carota has beneficial effect for treatment of osteoporosis. Further studies are required to elucidate the molecular mechanism of action and its therapeutic potential in the treatment of osteoporosis.
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Received on 26.04.2019 Modified on 30.05.2019
Accepted on 18.06.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(11):5461-5466.
DOI: 10.5958/0974-360X.2019.00947.8