- Research article
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Antiosteoporotic effect of Petroselinum crispum, Ocimum basilicum and Cichorium intybus L. in glucocorticoid-induced osteoporosis in rats
© The Author(s). 2016
- Received: 9 October 2015
- Accepted: 25 May 2016
- Published: 2 June 2016
Glucocorticoid-induced osteoporosis (GIO) is one of the serious side effects which have become the most common secondary osteoporosis. The purpose of this study is to evaluate the effect of aqueous extract of parsley, basil and chicory on glucocorticoid-induced osteoporosis in rats.
Fifty Female rats were divided into five groups and treated for 8 weeks as follow: group 1 served as control; group (2) subcutaneously injected with 0.1 mg/kg b. wt. dexamethasone dissolved in saline; group 3 received similar dose of dexamethasone together with aqueous parsley extract in a dose of 2 g/kg b. wt.; group 4 received similar dose of dexamethasone together with 400 mg/kg b. wt. aqueous basil extract and group 5 received similar dose of dexamethasone together with 100 mg/kg b. wt. aqueous chicory extract.
The dexamethasone group showed a significant decrease in serum E2, Ca, P levels and significant decrease in total BMD, BMC and a significant increase in serum PTH, ALP and ACP. Bone TBARs was significantly increased while GSH, antioxidant enzymes were significantly decreased. These changes were attenuated by parsley, basil and chicory extracts in the group 3, 4 and 5 respectively.
Aqueous extracts of parsley, basil and chicory showed bone protection against glucocorticoid-induced in rats. From our results, we concluded that chicory has a potent protective effect more than parsley and basil due to containing flavonoids and inulin.
- Bone biomarkers
- Histopathology and oxidative stress
Glucocorticoids (GCs) are widely used to treat various inflammatory, immunologic and allergic disorders that cause rheumatic, respiratory, bowel, hepatic, neurological, renal and skin diseases . Osteoporosis is one of the main complications of glucocorticoid application . GC therapy suppresses osteoblast function, increases bone resorption, decreases calcium gut absorption, and suppresses endogenous gonadal steroids, all of which lead to increase bone loss .
Aqueous parsley extract has been used for the treatment of diseases or conditions characterized by increased bone resorption . Parsley contains both calcium and vitamin c, as well as ergosterol, a precursor of vitamin D, which helps the body to absorb and utilize calcium . Phytochemical screening of parsley has revealed the presence of flavonoids (apiin, luteolin, and apigenin-glycosides), the methanolic extract from the aerial parts of parsley showed potent estrogenic activity which is equal to that of isoflavone glycosides from soybean [6, 7].
The profound medical effects of basil may be attributed to its antioxidant power due to flavonoids and polyphenols content . Flavonoids also called phytoestrogens because of their weak estrogenic activity with a chemical structure similar to 17β-estradiol, the most potent, naturally occurring estrogen as isoflavones bind to estrogen receptors, affecting estrogen-regulated processes [9, 10]. Phytoestrogens prevent bone resorption, and maintain or increase bone density and may inhibit osteoporosis to some degree in postmenopausal women, owing to their estrogenic activity because they are unlikely to cause the undesirable effects associated with steroid hormones [11, 12].
All parts of chicory plant possess medicinal importance as alkaloids, inulin, sesquiterpene lactones, coumarins, chlorophyll pigments, unsaturated sterols, flavonoids, saponins and tannins . Dietary supplementation with inulin-type fructans enhances the uptake of Ca, improves bone mineral content (BMC) in growing rats and alleviates the reduction in bone mineral content and bone mineral density (BMD) which follows ovariectomy or gastrectomy in rats .
The aim of this study was to investigate the anti-osteoporotic action of aqueous extract of Petroselinum crispum, Ocimum basilicum and Cichorium intybus L. in rats administered dexamethasone and the antioxidant capability of these extracts.
This work was conducted in the Chemistry Department, College of Science, Beni-Suef University.
Female albino rats (Rattus norvegicus) weighing about 120–150 gm. were used for the study and were kept in animal house at 26 ± 2 ° C with relative humidity 44 to 56 % along with light and dark cycles of 12 h, respectively. Animals were provided with standard diet and water ad libitum. All animal procedures were conducted in accordance with the standards set forth in the guidelines for the care and use of experimental animals by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the National Institutes of Health (NIH). The study protocol was approved by the Animal Ethics Committee of the Zoology Department in the College of Science at Beni-Suef University.
Dexamethasone [(Fortecortine® 8 mg – Mono ampoule) manufactured by Sigma – Tec Pharmaeutical industries – Egypt – S. A. E. under Licence of: Merck, Darmstadt, Germany]. Standard flavonoids (Luteo.6-arabinose8-glucose, Luteo.6-glucose8-arabinose, Apig.6-arabinose8-galactose, Apig.6-rhamnose8-glucose, Apig.6-glucose8-rhamnose, Naringin, Rutin, Hespirdin, Rosmarinic acid, Apig.7-o-neohespiroside, Apigenin-7-glucose, Quercetrin, Quercitin, Naringenin, Hespirtin, Kampferol, Apigenin) and saccharides (Glucuronic acid, Sucrose, Xylose, Rhaminose, Mannose, Arabinose, Manitol, Stachylose, Inulin, Fructose, Glucose) were purchased from Sigma Aldrich. Kit No. ES180S-100 purchased from Calbiotech. U.S. for E2 determination, kit No. MBS702121 purchased from My Biosource. U. S. A. for PTH determination, a commercial assay kit provided from Spinreact, Spain for Ca, P determination. Kit purchased from BioSystems Company, Spain for determination activity of alkaline phosophatase. Kit purchased from BIO Diagnostic Company, Egypt for determination activity of ACP and oxidative stress markers.
Parsley plant (P. crispum), Basil plant (O. basilicum), Chicory plant (C. intybus L.) leaves were collected from herbal medicine market (Cairo, Egypt) and identified by an ecologist in plant department, Faculty of Science, Beni-Suef University. A voucher specimen was deposited in the herbarium of the Botany Department, College of Science, Beni-Suef University, Egypt.
Preparation of aqueous parsley extract
The air dried parsley leaves (100 gm.) were extracted by adding 1000 ml of distilled water and boiled for 30 min. The extract was then filtered, and then filterate was evaporated, using rotary evaporator under reduced pressure to dryness (at 45 °C). The extract was dissolved in distilled water before the administration to rats .
Preparation of aqueous basil extract
The ground powder of dried basil leaves (300 gm.) was infused for 30 min in 200 ml of distilled water at 100 °C followed by filteration. The solution obtained was concentrated rotary evaporator under a vacuum at 65 °C. The resulting crude extract was suspended in 30 ml sterile distilled water and aliquots were stored at −20 °C till use .
Preparation of aqueous chicory extract
The powdered chicory leaves were added to the already boiling distilled water and infused for 15 min. Then, the infusion (2 % w/v) was filtered and the filtrate was freshly used .
Identification of flavonoids in extracts by HPLC analysis
The flavonoid compounds of the samples were extracted according to the method described . Three milliliters were collected in a vial for subsequent HPLC separation. HPLC instrument (Hewlett Packard, series 1050, country) equipped with stainless-steel column (Zorbax ODS 5 μ m 4.6 × 250 mm). Injection volume was 75 μl carried out with auto-sampling injector. The column temperature was maintained at 35 °C. Gradient separation was carried out with methanol and acetonitrile as a mobile phase at flow rate 1.0 ml/min. Elutes were monitored using UV detector set at 330 nm for flavonoid. Chromatographic peaks were identified by comparing the retention times with the respective retention times of known standard reference material.
Identification of saccharides in extracts by HPLC analysis
Sugar profiles were determined by the method described  high performance with modification that liquid chromatography coupled to a refraction index detector (HPLC-RI). Soluble sugar determined at 80 °C. The HPLC system was equipped with a Hewlett Packard 1050 HP1047A RI detector and with HPLC instrument (Hewlett Packard, series 1050, country) equipped with stainless-steel column (Zorbax ODS 5 μ m 4.6 × 250 mm). The mobile phase was isocratic elution system was used by deionized water at a flow rate of 1 ml/min. Sugar identification was made by comparing the relative retention times of samples peaks with standards.
Group 1: served as normal control.
Group 2: was given dexamethasone subcutaneously at 0.1 mg/kg b. wt./day dissolved in saline [ 19] and is considered as a control for groups 3, 4 & 5.
Group 3: received 0.1 mg/kg. b. wt. of dexamethasone together with 2 g/kg b. wt. of Petroselinum crispum leaves aqueous extract .
Group 4: received 0.1 mg/kg. b. wt. of dexamethasone together with 400 mg/kg b. wt. of Ocimum basilicum leaves aqueous extract .
Group 5: received 0.1 mg/kg. b. wt. of dexamethasone together with 100 mg/kg b. wt. of Cichorium intybus L. leaves aqueous extract .
All these groups were treated for three times per week for 8 consecutive weeks and the treatments with parsley, basil and chicory were performed orally between 7.00 and 9.00 a.m.
At the end of the experimental period (8 weeks), rats were sacrificed under diethyl ether anesthesia. Blood samples were collected from each rat, allowed to coagulate at room temperature then centrifuged at 3000 r.p.m. for 20 min. The clear, non hemolysed supernatant sera were quickly removed and kept at −20 °C till examined. For bone samples the left femurs were immediately removed, washed using chilled saline solution, weighed and minced in ice-cold 0.9 % saline solution using homogenizer. The homogenates were centrifuged, and the resultant supernatants were frozen at −20 °C .
The concentration of serum estradiol (E2) was determined by enzyme linked immunosorbent assay (ELISA) procedure . The concentration of serum parathyroid hormone (PTH) was determined by ELISA procedure . Serum and bone calcium and phosphorus concentrations were assayed according to the method of [25, 26] respectively. The activity of alkaline phosphatase in serum and bone was determined kinetically . Acid phosphatase activity in serum and bone was determined colorimetrically .
Bone mineral density and bone mineral content assay
The right femur of each animal was dissected and carefully cleaned for measuring bone mineral density (BMD) and bone mineral content (BMC) by dual energy x-ray absorptiometry (DEXA) using Norland XR 46, version 3.9.6/2.3.1 instrument equipped with dedicated software for small animal measurements in bone mineral density unit, Medical Service Unit, National research Center, Dokki, Egypt., this technique provides an software measure of right femur proximal, middle, distal and total areas.
Bone oxidative stress and antioxidant enzymes assay
The left femur of each animal was homogenized in cold 0.9 % NaCl to make up to 10 % homogenate (w/v). The homogenates were centrifuged, and the clear supernatants were used for estimation of malondialdehyde (MDA) , glutathione (GSH) , glutathione-S-transferase (GST) , glutathione peroxidase (GPx) , glutathione reductase (GR)  and catalase  levels.
Histopathological examination of bone
Right femur specimens were fixed in 10 % neutral buffered formalin for 24 h, decalcified in 10 % EDTA solution (pH = 7.4) and then processed till embedding in paraffin. Thin paraffin sections (4 μm) were stained with H&E .
The data were analyzed using the one-way analysis of variance (ANOVA)  followed by LSD test to compare various groups with each other. Results were expressed as mean ± standard deviation (SD) and values of P > 0.05 were considered non-significantly different, while those of P < 0.05, P < 0.01 and P < 0.001 were considered significant, highly and very highly significant, respectively.
Serum mineral parameters, enzyme activities in control and different treated groups
Dex. + parsley
Dex. + basil
Dex. + chicory
LSD at 5 %
LSD at 1 %
32.3 ± 6.45a
17.4 ± 0.86c
26.4 ± 2.31b
26.4 ± 0.38b
23.1 ± 0.73b
P < 0.001
8.36 ± 1.19d
17.4 ± 2.56a
14.0 ± 1.71b
15.6 ± 0.87ab
11.7 ± 0.91c
P < 0.001
9.94 ± 1.14a
5.49 ± 0.38c
7.05 ± 0.63b
6.69 ± 0.83b
6.66 ± 0.62b
P < 0.001
8.82 ± 0.39a
5.63 ± 0.46c
7.42 ± 1.07b
6.88 ± 0.69b
7.46 ± 0.81b
P < 0.001
199 ± 30.9b
393 ± 17.7a
202 ± 14.3b
203 ± 14.6b
210 ± 31.0b
P < 0.001
11.6 ± 2.16d
23.7 ± 1.62a
17.6 ± 2.93b
13.9 ± 1.62cd
16.5 ± 2.89bc
P < 0.001
Bone mineral parameters and bone enzyme activities in control and different treated groups
Dex. + parsley
Dex. + basil
Dex. + chicory
LSD at 5 %
LSD at 1 %
Ca (mg/g T)
15.9 ± 1.15a
10.2 ± 1.03d
15.5 ± 0.55ab
14.4 ± 0.93b
12.8 ± 0.75c
P < 0.001
P (mg/g T)
15.7 ± 0.49a
7.02 ± 0.16d
7.22 ± 0.47d
9.77 ± 0.91c
14.2 ± 0.72b
P < 0.001
ALP (IU/g T)
1186 ± 193d
2008 ± 89.2a
1396 ± 119b
1323 ± 94.9cd
1365 ± 142b
P < 0.001
ACP (IU/g T)
194 .1 ± 1.6b
213.1 ± 6.14a
197.6 ± 4.84b
195.9 ± 3.29b
195.1 ± 6.04b
P < 0.001
Bone mineral density and bone mineral contents in different areas in femur bone of control and different treated groups
Dex. + parsley
Dex. + basil
Dex. + chicory
LSD at 5 %
LSD at 1 %
Total BMD (mg/cm2)
60.5 ± 7.85a
38.7 ± 2.64c
50.9 ± 7.26b
51.5 ± 1.70b
55.5 ± 3.24bc
P < 0.001
Prox. BMD (mg/cm2)
55.8 ± 8.91a
43.9 ± 3.77c
52.2 ± 3.22ab
49.5 ± 2.12bc
52.7 ± 3.55ab
P < 0.001
Mid. BMD (mg/cm2)
69.0 ± 8.63a
38.2 ± 3.64d
61.7 ± 2.34b
54.7 ± 5.02c
54.9 ± 3.05c
P < 0.001
Dist. BMD (mg/cm2)
52.6 ± 6.63a
47.5 ± 6.61a
51.7 ± 4.79a
49.3 ± 3.86a
50.4 ± 4.13a
P > 0.05
Total BMC (mg)
83.0 ± 7.25a
28.5 ± 5.15d
50.4 ± 5.76c
60.8 ± 8.29b
55.0 ± 1.73bc
P < 0.001
Prox. BMC (mg)
43.6 ± 7.32a
12.4 ± 1.52c
27.3 ± 3.47b
24.5 ± 3.01b
25.0 ± 4.63d
P < 0.001
Mid. BMC (mg)
25.6 ± 4.17a
8.05 ± 1.33d
12.3 ± 1.56c
18.4 ± 3.19b
10.6 ± 0.78cd
P < 0.001
Dist. BMC (mg)
26.1 ± 2.87a
13.6 ± 2.49c
21.2 ± 1.03b
24.0 ± 2.47a
24.3 ± 0.87a
P < 0.001
Bone oxidative stress marker and antioxidant parameters in control and different treated groups
Dex. + parsley
Dex. + basil
Dex. + chicory
LSD at 5 %
LSD at 1 %
TBARS (nmol/g T)
18.1 ± 2.38c
56.3 ± 8.03a
32.1 ± 3.96b
30.4 ± 4.97b
23.5 ± 3.61c
P < 0.001
GSH (mg/g T)
6.76 ± 1.21a
2.37 ± 0.33c
2.96 ± 0.53bc
2.61 ± 0.54bc
3.49 ± 0.96b
P < 0.001
GST (U/g T)
2155 ± 254a
1277 ± 44.7d
1533 ± 59.2c
1648 ± 77.2bc
1741 ± 131b
P < 0.01
GPx (U/g T)
109 ± 21.7a
69.9 ± 7.58b
102 ± 11.5a
103 ± 16.5a
99.0 ± 13.8a
P < 0.01
GR (U/g T)
457 ± 67.9a
272 ± 70.7b
341 ± 88.6b
320 ± 55.9b
340 ± 91.2b
P < 0.01
Cat (U/g T)
0.97 ± 0.11a
0.32 ± 0.06c
0.37 ± 0.05c
0.59 ± 0.09b
0.88 ± 0.08a
P < 0.001
Dexamethasone decreased E2 levels associated with a significant decline in serum mineral concentrations, resulting in secondary hyperparathyroidism, which is consistent with the increase in PTH [37–39]. The enhanced bone turnover and fracture risk was reflected by the ALP activity . Also, lack of inhibiting activity of estrogen on osteoclasts caused an increase in ACP activity and in consequence increase in bone resorption . Glucocorticoids are known to alter the levels of TBARS and antioxidant enzymes in different tissue . Increased free radicals production overwhelms the natural antioxidants defense mechanisms, subjecting individuals to hyperoxidant stress and thus leading to osteoporosis . The decreased estrogen level in females increased the sensitivity of bones to the action of PTH leading to bone resorption with lower BMD  with concomitant decrease in bone matrix available for mineralization .
The ability of parsley extract to counteract the toxic effects may be attributed to the high nutritive value of parsley that concluded too high percent of vitamins (A, C, riboflavin and niacin) and minerals (Fe, Mg, P, K, Ca, Na and Zn). Essential trace elements are important parts of antioxidant enzymes as superoxide dismutase and glutathione peroxidase and may affect the antioxidant defense system . Parsley contains vitamin k which positively affects calcium balance, a key mineral in bone metabolism and vitamin K insufficiency might be involved in the pathogenesis of osteoporosis [46–48]. The most potent osteogenic chemicals ever discovered as petroselinum crispum is quercetin and diosmetin glycosides [49, 50]. Quercetin induces apoptosis in mature osteoclasts and inhibits bone resorption and diosmetin induce osteoblastic differentiation [51, 52].
Administration of O. basilicum leaf extracts in dexamethasone treated rats tends to bring the bone MDA back to normal. Rosmarinic acid in O. basilicum suggested that it might have a role in the scavenging of free radicals . Basil possessed good antioxidant properties attributed to free volatile aglycones in two different methods as the 2,2/-diphenyl-1-picrylhydrazyl radical scavenging method and ferric reducing/antioxidant power assay when compared with that of the essential oil and well known antioxidant butylatedhydroxytoluene . Antiradical activity of phenolic compounds seen in Ocimum species depend on their molecular structure; and the availability of phenolic hydrogens, which result in the formation of phenoxyl radicals due to hydrogen donation . An adequate supply of steroidal saponins of Anemarrhena asphodeloides prevented ovariectomized induced bone loss in rats through the promotion of bone formation . Volatile oil of basil has estragol, linalool, eugenol, methyl chavicol and small quantities of methyl cinnamate, cineole, and other terpenes, apigenin, luteolin, orientin and vicenin , also, apigenin induced apoptosis of mature osteoclasts obtained from rabbit long bone and inhibited bone resorption . The treatment with basil induced osteogenesis because O. bacilicum has a great number of compounds with oestrogenic activity  and phytoestrogens perform their antiosteoporotic effect by stimulating osteoblastic activity through an estrogen receptor mediated action, or by increasing the production of insulin 1 like growth factor-1 (IG-F) which is known to enhance osteoblastic activity .
In osteoporotic rats treated with chicory, it has been demonstrated that treatment with non-digestible fructans successfully increases Ca absorption and results in a corresponding increase in bone mineral  which is followed by a suppression of PTH, also non-digestible oligosaccharides from chicory roots on have the ability to reduce the elevation in the rate of bone turnover due to attributed to the ability to reduce the osteoclastic activity thus the rate of bone resorption decreases . The phytochemical screening of chicory extract confirmed the presence of such bioactive compounds, particularly total phenolic, which may contribute to protection of chicory extract against free radical generation . The treatment with chicory induced osteogenesis because C. intybus L. has osteoporosis preventive properties due to the protective effect of non-digestible oligosaccharides on bone in a rat model to mimic menopausal women was established through the following: 1) increased calcium absorption, 2) increased calcium balance, 3) increased bone mineralization, and 4) decreased bone turnover rate [63, 64].
Bone mineral density, bone mineral contents raising effects as well as the anti-oxidant properties of chicory make it has more ability to prevented bone loss and decreased resorption of bone in the dexamethasone treated group. This suggests that chicory represents a promising therapeutic option for the prevention glucocorticoids- induced osteoporosis.
%, percentage; ACP, acid phosphatase; ALP, alkaline phosphatase; ANOVA, one-way analysis of variance; b, bony matrix; b. wt, body weight; bm, bone marrow; BMC, bone mineral content; BMD, Bone mineral density; C, Cartilage; C. intybus L., Cichorium intybus L.; Ca, Calcium; Dex., dexamethasone; DEXA, Dual energy x-ray absorptiometry; dist., Distal; E2, Estradiol; ELISA, Enzyme linked immunosorbent assay; Fe, Iron; GCs, Glucocorticoids; GIO, glucocorticoid-induced osteoporosis; gm, gram; GPx, glutathione peroxidase; GR, glutathione reductase; GSH, glutathione reduced form; GST, glutathione-S-transferase; H&E, hematoxylin and eosin; HPLC, high-performance liquid chromatography; IG-F, insulin 1 like growth factor; IU/g T, international unit per gram tissue; K, potassium; LSD, least significant differences; MDA, malondialdehyde; Mg, magnesium; Mid., middle; Na, sodium; O. basilicum, Ocimum basilicum; P, phosphorus; P. crispum, Petroselinum crispum; Pg/ml, picograms per milliliter; Prox., proximal; PTH, parathyroid hormone; r.p.m, round per minute; RI, refraction index; SD, standard deviation; TBARs, thiobarbituric acid reactive substances; UV, ultra violet; w/v, weigh/volume; Zn, zinc; μg, microgramme; μL, microliter.
The authors are thankful to faculty of Science, Beni-Suef University for help in conducting this study and providing all required facilities. The authors are grateful to Professor Dr. Adel Mohamed Bakeer Kholoussy, Professor of Pathology, Faculty of Veterinary Medicine, Cairo University, for preparing histological sections.
The authors declare that they have received no funding for the research reported.
Availability of data and materials
The datasets supporting the conclusions of this article are presented in this main paper. Plant materials used in this study have been identified by an ecologist in plant department, Faculty of Science, Beni-Suef University. Animals were obtained from the animal house of the Research Institute of Ophthalmology, Giza, Egypt.
WH conceived of the study, was responsible for the conception of idea, design, interpreted the biochemical analysis and participated in revision of the manuscript. MD participated in the design of the study and participated in revision of the manuscript. HS participated in the design of the study, participated in the sequence alignment and drafted the manuscript. RA revised the histopathological sections, participated in revision of the manuscript. AK carried out biochemical analysis and performed the statistical studies. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All animal procedures were conducted in accordance with the standards set forth in the guidelines for the care and use of experimental animals by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and the National Institutes of Health (NIH). The study protocol was approved by the Animal Ethics Committee of the Zoology Department in the College of Science at Beni-Suef University.
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