Cell Proliferative Potential of Ethnomedicinal plants for the management of Osteoporosis

 

Kunjal Vegad, Nimisha Kakadia, Niranjan Kanaki

Sharda School Pharmacy, Pethapur-Mahudi Road, Pethapur, Gandhinagar. K.B. Institute of Pharmaceutical Education and Research Centre, Gandhinagar, Gujarat, India.

 

ABSTRACT:

Osteoporosis is a silent disease leading to complications with the progression in the disease. Osteoporosis is characterized by a decrease in bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. During the early menopausal stage in women, there is reduction in circulating estrogen which leads to an increase in the rate of bone resorption. Hence, there is an imbalance between bone formation and resorption which lead to osteoporosis. Many traditional plants and phytoconstituents have been used as potential therapy for the treatment of the osteoporosis. However, the scientific approach for the validation of the use of phytoconstituents or herbs in the treatment of osteoporosis is the need of an hour. In present work five ethnomedicinal plants, Asparagus racemosus, Hemidesmus indicus, Berberis aristata, Emblica officinalis, and Nigella sativa were selected on the basis of their use in bone diseases in literature. The selected plants were studied for the positive effect on cell proliferation which is one of the important targets for the bone remodeling. Cell proliferative activity of aqueous and ethanolic extracts of selected plants was screened using MTT assay. Osteosarcoma cells (MG-63 cell line) and primary mesenchymal stem cells (MSCs) isolated from the rat femur were used for the MTT assay. The results of the above study showed significant (P < 0.001)cell proliferative activity of aqueous extract of seeds of Nigella sativaon MG-63 cells and MSCs and hence, supporting its ethnomedicinal use in the management of osteoporosis.

 

 

 

INTRODUCTION:

Osteoporosis is a major disease which has significant implications on public health. Osteoporosis is three times more common in women as compared to men⁽¹⁾. In aged women, pathogenesis of osteoporosis is complicated and depends on many exogenous and endogenous factors. Age-related changes lead to lesions of hormonal status, regenerative system, espsecially in combination with negative endogenous factors⁽²⁾. Osteoporosis means porous bone, and it is characterized by fragile bone and deteriorated bone tissues⁽³⁾. Osteoporosis causes progressive bone loss, which renders the bones susceptible to fractures with minimal trauma and is popularly known as “the silent disease” because early symptoms are absent⁽⁴⁾.

 

World Health Organization (WHO) defines osteoporosis as a bone mineral density of 2.5 standard deviations or more below the mean peak bone mass (average of young, healthy adults) as measured by dual-energy X-ray absorptiometry. Osteoporotic risk of fractures is associated with high mortality, morbidity and high medical expenses throughout the world⁽⁵⁾, The financial burden of osteoporotic fractures includes direct costs (hospital acute care, in-hospital rehabilitation, outpatient services, long term nursing care) and indirect costs (morbidity, loss of working days). Some costs are difficult to quantify, e.g. deterioration of quality of life, and time spent by the family on the care of the patient⁽⁶⁾.

 

The aim of the treatment is to prevent further bone loss in order to reduce initial and further risk of fractures. Many pharmacological agents, Vitamin D, calcium supplementation, biphosphonates, hormone replacement therapy, and selective estrogen receptor modulators are available for the treatment of osteoporosis⁽⁷⁾. The available treatments have several adverse effects; oral biphophonates cause gastrointestinal side effects such as abdominal pain, esophagitis, osteonecrosis of jaw and musculoskeletal pain. Estrogen therapy is associated with increased risk of cancer⁽⁸⁾, and prolonged use of calcium leads to deposition of calcium in blood vessels and increased risk of cardiovascular diseases⁽⁹⁾.

 

To overcome the wide range of side effects produced by these synthetic drugs, there is an increasing demand for ‘green medicines’ which are thought to be healthier and safer for the treatment of osteoporosis. There is a great demand of the herbsdue to their efficacy, safety and lesser side effects as compared to synthetic molecules. These herbs also have therapeutic role for age-related disorders like memory loss, immune disorders, etc. for which no modern medicine is available⁽¹⁰⁾. Various phytoconstituents such as flavonoids, alkaloids, carotenoids, terpenoids, sulphides, lignans, have been identified in various parts of these plants.

 

Since ancient time, many herbs are used in various bone related disorders like rheumatoid arthritis, osteodysplacia, osteoarthritis and osteomalacia. Based on the literature, roots of Asparagus racemosus (Ar), roots of Hemidesmus indicus (Hi), Bark of Berberis aristata (Ba), Fruits of Emblica officinalis (Eo), and Nigella sativa (Ns) were selected for their potential on bone cell proliferation. Two in vitro osteoblast model systems were used for the screening of selected plants viz., human osteoblast-like cells MG-63 and primary osteoblast cells isolated from rat femur. Cell viability was assessed by Trypan blue assay and cell proliferation assay was performed by MTT assay.

 

MATERIAL AND METHODS:

Collection and authentication of selected plants:

The fresh roots of Asparagus racemes and fresh fruits of Emblica officinalis were collected from the Botanical garden of Gandhinagar. The stem bark of Berberis aristata, roots of Hemidesmus indicus and seeds of Nigella sativa were purchased from Lallu Vrajlal Gandhi Herbal Store, Ahmedabad, Gujarat, India. The herbarium and voucher specimen of collected and procured crude drugs were prepared and submitted to the Pharmacognosy department of K.B. Institute of Pharmaceutical Education and Research, Gandhinagar. Freshly collected crude drugs were washed properly with water and dried. Dried drug materials were coarsely powdered and stored in a properly labeled airtight container until further use. All the crude drugs were authenticated by their morphological, microscopical and physicochemical parameters and compared with references.

 

Crude drug was powdered and evaluated for ash values (Total ash and acid insoluble ash), extractive values (alcohol and water extractive) and loss on drying for their authentication⁽¹¹⁾.

 

Preparation of Aqueous and ethanolic extracts:

Twenty gram of dried powdered drug was taken, and extracted with ethanol (100ml x 2) by heating under reflux on a water bath at 55˚C for 6 hours, and allowed to stand overnight and filtered. And for preparation of aqueous extract, 20 gram of dried powdered drug was extracted with water (100ml x 2) by heating under reflux on water bath for 6 hours, and allowed to stand overnight and filtered. Aqueous and ethanolic filtrates of each drug were concentrated to dryness on water bath temperature not exceeding 60˚C. Ethanolic and aqueous extract of each drug was labeled properly and stored in refrigerator till further use.

 

Procurement and maintenance of Cell line:

First the complete growth medium for the MG-63 cells and for primary mesenchymal stem cells (MSCs) was prepared using sterile liquid Dulbecco’s minimum essential medium DMEM, 10 % Fetal Bovine Serum and 1% Antibiotic solution (10000 U Penicillin and 10 mg Streptomycin/ml). The prepared complete growth media was labeled as basal media and stored at 8⁰C in a refrigerator for further use.

 

The human bone osteosarcoma cell line, MG-63 was used for the screening of extracts for their cell proliferation activity. MG-63 cell line was procured from NCCS, Pune prepared from 14 years human male bone with osteosarcoma. Cell line was maintained as per ATCC protocol throughout the work.

 

Sub culturing of Cells:

According to ATCC protocol cell line was allow to grow in DMEM supplemented with 1X antibiotic antimycotic solution and 10% FBS under standard growth conditions (temperature 37°C, 5% CO₂ and 95% humidity) in a CO₂ incubator. After confluent monolayer, cells were trypsinized with 0.25% trypsin–0.2% EDTA in Dulbecco’s phosphate buffered saline (DPBS) and subcultured to obtain enough number of cells for proliferation assay. For the proper growth of the cell media was changed every alternate day.

 

Cell viability by Trypan blue assay^(12-14)

The aliquot of 10µl of the trypsinized cell suspension was taken in a micro centrifuge tube and 1:1 mixture of the cell suspension was prepared by adding 10µl of the 0.4% trypan blue dye solution. It was gently mixed and kept at room temperature for about 5 min prior to use, the haemocytometer and the cover slip were sterilized with 70% (v/v) iso-propyl alcohol and were allowed to air-dry. Properly mixed cell suspension and trypan blue dye mixture was loaded on the chamber between the cover slip and the V-shaped groove in the chamber and allowed to set. The living cells are as clear form and dead cells are seen blue in colour under inverted microscope. Cell viability was calculated by counting living cells and non living cells using following formula,

 

^{%Cell Viability=(no.of living cells/ total cell count)*100}

 

Isolation of mesenchymal stem cells (MSCs) from rat femur^(15-18):

After the quarantine period, the animals were used for the experiment. The study was approved by the institutional animal ethics committee of K. B. Institute of Pharmaceutical Education and Research, Gandhinagar (KBIPER/ approval No: KBIPER/IAEC/ANIM/ 2016/577). The rats were euthanized by cervical dislocation under ether anesthesia, the femurs were removed aseptically and the soft tissue and muscles attached were cleaned-off from the femur. Femurs were cut from both ends and bone marrow was flushed with 2ml DMEM with help of a syringe with needle (27 gauge). Bone marrow cell suspension was filtered through sterile filter to remove cell debris and collected cell suspension was centrifuged at 1000rpm for 5 min. The MSCs were seeded in T-25cm² tissue culture flask. Primary cells (MSCs) were in were cultured DMEM supplemented with the 1X antibiotic-antimycotic solution and 10% FBS. MSCs were allowed to grow under standard growth conditions. The media was changed at every alternate day until the full confluence was achieved. Cells were trypsinized with 0.25% trypsin, 0.2% EDTA in Dulbecco’s phosphate buffered saline and either subcultured at a split ratio of 1:2 in 25 cm² volume tissue culture flask or seeded in microtitre plates (96 well and 24 well plates) for different cell based assays.

 

Cell Proliferation activity by MTT assay^(19-21):

Cell proliferation activity of extracts was screened by osteoblast cell line MG-63 and MSCs. Cells were plated at the concentration of 1×10⁴ cells/ml with DMEM in 96-well plate; Next day spent media was discarded carefully and the cells were treated with 3 different concentrations (1000μg/ml, 100μg/ml and 10μg/ml) of aqueous and ethanolic extracts of each drug in triplicate. After 48 h of incubation with drug extracts, the 20 µl of MTT (final concentration 0.5mg/ml) was added to each well and incubated for 4h at 37°C in dark place. Media was carefully pipetted out without disturbing the cell monolayer and 200 µl of DMSO was added to each well, the plate was shaken carefully and incubated for 1h in 37°C in dark place. Optical density (OD) of each well was measured at 570nm in microwell plate reader.

 

Statistical analysis:

Data was analyzed by applying one-way analysis of variance (ANOVA) followed by Tukey Test, using Graph Pad Prism software. The data was expressed as mean ± standard error of the mean (SEM). The results were considered statistically significant if the P<0.05.

 

RESULTS:

Authentication of selected plants:

Selected plants were authenticated by their physicochemical parameters and values were compared with the standard literature. Results of ash value, moisture content and extractive value are as shown in the Table 1.

 

Preparation of extracts:

Percentage yield of aqueous and ethanolic extracts of plant was as shown in Table 2.

 

Table 1: Physicochemical parameters of selected plants

Parameters Name of Plant	Ash value (%w/w)		Loss on Drying (%w/w)	Extractive value (%w/w)
	Total ash	Acid insoluble ash		Water soluble extractive	Alcohol soluble extractive
Roots of Asparagus racemosus	4.3 ± 0.19	0.45 ± 0.04	6.3 ± 0.31	33.7 ± 0.72	11.4 ± 0.42
Roots of Hemidesmus indicus	3.6 ± 0.31	0.28 ± 0.02	5.32 ± 0.47	9.6± 0.60	8± 0.42
Bark of Berberis aristata	12.1 ± 0.56	5.45 ± 0.32	5.98± 0.29	10± 0.54	5.5± 0.36
Fruits of Emblica officinalis	0.8 ± 0.35	0.04 ± 0.01	3.37 ± 0.63	10.2 ± 0.83	9.6 ± 0.64
Seeds of Nigella sativa	6.2 ± 0.65	0.4 ± 0.06	3.1 ± 0.42	7.5 ± 0.26	11.7 ± 0.67

 

Table 2 : Percentage yield of extracts

Name of Plant	Extract	% Yield of Extract (% w/w)
Asparagus racemosus (Root)	Aqueous (Ar A)	34.2
	Ethanolic (Ar E)	11.8
Hemidesmus indicus (Root)	Aqueous (Hi A)	9.4
	Ethanolic (Hi E)	8.3
Berberis aristata (Bark)	Aqueous (Ba A)	10
	Ethanolic (Ba E)	5.5
Emblica officinalis (Fruits)	Aqueous (Eo A)	10.5
	Ethanolic (Eo E)	9.1
Nigella sativa (Fruit)	Aqueous (Ns A)	8.1
	Ethanolic (Ns E)	12.0

Sub culturing of cells and Trypan blue assay:

The cells of the MG-63 cell line were sub-cultured at regular intervals when the cells in the tissue culture reached the confluency of 80-90%. Trypan blue is acid dye having two azo chomophore groups. Cell wall of the living cells is non permeable to trypan blue dye it only entre in the cell wall of nonliving cells. Total viable cell count and % cell viability by trypan blue was found to be 2.28*10⁶ cells/ ml and 98.21% respectively.

 

Cell proliferation assay:

Cell proliferative activity of the plant extracts was measured by MTT assay. Cell proliferation activity of each extract was evaluated both on osteoblast like cells of MG-63 cell line and normal cells of primary mesenchymal stem cells. Alendronate was taken as a positive control, and basal media was considered as a negative control. In our study we observed that, alendronate cause remarkably increase the proliferation of osteoblast like cells of MG 63 cell line as compare to the cells grown with basal media alone. Alendronate showed cell proliferation activity in dose dependent manner.

 

Aqueous and ethanolic extracts of roots of Hemidesmus indicus showed significant cell proliferation activity in dose dependent manner. Aqueous and ethanolic extract roots of Hemidesmus indicus showed significant cell proliferation potential (P < 0.01) at concentration 1000 µg/ml. Aqueous extract of seeds of Nigella sativa also showed highly significant proliferation potential (P < 0.001) at concentration 1000 µg/ml and signification proliferation (P < 0.01) at concentration 100 µg/ml and 10µg/ml. Aqueous and ethanolic extract of roots of Asparagus racemosus, bark of Berberis aristata and fruits of Emblica officinalis have no cell proliferation potential on cells of MG-63 cell line at any concentration (Shown in Figure 1).

 

 

Figure 1: Cell proliferation activity of selected plant extracts on MG-63 osteoblast like cells

The values depicted in the graph are from higher to lower concentrations (i.e. 1000 µg/ml, 100µg/ml, and 10µg/ml) for each drug extract. STD = Alendronate standard Ar A = Aqueous extract of Asparagus racemosus Ar E = Ethanolic extract of Asparagus racemosus Hi A = Aqueous extract of Hemidesmus indicus Hi E = Ethanolic extract of Hemidesmus indicus Ba A = Aqueous extract of Berberis aristate Ba E = Ethanolic extract of Berberis aristate Eo A = Aqueous extract of Emblica officinalis Eo E = Ethanolic extract of Emblica officinalis Ns A = Aqueous extract of Nigella sativa Ns E = Ethanolic extract of Nigella sativa Values are expressed in mean ± SEM. n=3. Significantly different from *P < 0.05 **P < 0.01 ***P < 0.001 Vs Control (Basal media)

Cell proliferation potential of extracts of selected plants was also evaluated on normal cells isolated from the bone marrow of rat femur. Results of the study indicated that aqueous and ethanolic extract of roots of Asparagus racemosus have highly significant (P < 0.001) cell proliferation property at the concentration of 1000µg/ml and low concentration 100 µg/ml and 10µg/ml have significant (P < 0.01) cell proliferation potential. Aqueous extract of seeds of Nigella sativa and also showed excellent (P < 0.001) cell proliferation potential but it was not in a dose dependent manner. Cell poliferative potential of ethanolic extract of seeds of Nigella sativa is also significant at concentration 10µg/ml while other concentration have non significant cell proliferation. Ethanolic extract of fruits of Emblica officinalis have highly significant (P < 0.001) cell proliferation compared to its aqueous extract. Aqueous and ethanolic extract of roots of Hemidesmus indicus showed cell proliferation activity in dose dependent manner but not as significant as aqueous extract of roots of Asparagus racemosus. Aqueous and ethanolic extract of bark of Berberis aristata have no significant cell proliferation potential compared to other extracts (Shown in Figure 2).

 

 

Figure 2: Cell proliferation activity of selected plant extracts on primary mesenchymal stem cells

The values depicted in the graph are from higher to lower concentrations (i.e. 1000 µg/ml, 100µg/ml, and 10µg/ml) for each drug extract. STD = Alendronate standard Ar A = Aqueous extract of Asparagus racemosus Ar E = Ethanolic extract of Asparagus racemosus Hi A = Aqueous extract of Hemidesmus indicus Hi E = Ethanolic extract of Hemidesmus indicus Ba A = Aqueous extract of Berberis aristate Ba E = Ethanolic extract of Berberis aristate Eo A = Aqueous extract of Emblica officinalis Eo E = Ethanolic extract of Emblica officinalis Ns A = Aqueous extract of Nigella sativa Ns E = Ethanolic extract of Nigella sativa Values are expressed in mean ± SEM. n=3. Significantly different from *P < 0.05 **P < 0.01 ***P < 0.001 Vs Control (Basal media)

 

DISCUSSION:

Osteoporosis constitutes a major public health problem, contributing significantly to morbidity, mortality and healthcare spending⁽²²⁾. Though osteoporosis is a senile disease, it will be started in young age and showed in adulthood⁽²³⁾. With increasing life expectancy the prevalence of osteoporosis is on rise and it poses a major public health issue⁽²⁴⁾.Natural menopause in women, making spontaneous cessation of estrogen has potent effect on the development and integrity of skeleton⁽²⁵⁾. Lifestyle changes, modifications in consumption of food, regular exercise have beneficial effects on bone health⁽²⁶⁾. The bone undergoes continuous turnover throughout life. Bone mass decreased due to activation of osteoclast which enhance bone resorption ⁽²⁷⁾, and osteoblast are bone forming cells. Bone metabolism depends upon these two types of cells. The state of our bones is always close to equilibrium between bone formation and bone resorption. In osteoporosis there is imbalance between bone formation and bone resorption⁽²⁸⁾. Currently available treatments for osteoporosis mostly include antiresorptive agents. Antiresorptive agents inhibit osteoclastic bone resorption and slow down loss of bone mass⁽²⁹⁾.

 

As ovarian hormone deficiency is a major risk factor for osteoporosis in the postmenopausal women, hormone replacement therapy (HRT), is the most effective treatment, but not preferred due to the risk of breast cancer and cardiovascular diseases. The other available therapeutic agents are also associated with adverse effects. So there is a search for the natural drugs in the treatment of osteoporosis. The advantages of the natural drugs are their easy availability, and negligible side effects.

 

Herbal drugs have been traditionally used in Ayurveda to accelerate the healing of bone fractures and to strengthen the bones. Five plants (roots of Hemidesmus indicus, roots of Asparagus racemosus, bark of Berberis aristata, fruits of Emblica officinalis) were selected on the basis of their use in bone disorder in literature.

 

For the treatment of osteoporosis, either there is increase in the activity of osteoblasts or there is reduction in the functioning of osteoclasts⁽³⁰⁾. As osteoblast proliferation is one of the important parameter of bone formation so cell proliferative potential of the extracts of selected plants was screened by MTT assay.

 

MTT assay is a colorimetric assay that measures the reduction of yellow 3-(4, 5-dimethythiazol2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, colored (dark purple) formazan product. Cell lysate was prepared by using an organic solvent and the released, solubilized formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the number of the viable cells.

 

Study suggests that the proliferative responses of normal cell or primary cell and human osteosarcoma cell lines are quite different^(31,32). So, in present work cell proliferative potential of each extract was evaluated by two in-vitro models using cells of MG-63 cell line and primary mesenchymal stem cells isolated from rat femur. Cells of MG-63 cell line are cancerous cells as it is obtained from 14 years old male with osteosarcoma and MSCs are normal cells. As bone marrow mesenchymal stem cells are the good source of bone forming cell- osteoblast, proliferation of MSCs is also important target for the treatment of osteoporosis ⁽³³⁾.

 

In present investigation, it was observed that aqueous extract of seeds of Nigella sativa showed good proliferation potential in the osteoblasts of MG-63 cell line as well as primary MSCs of bone marrow, whereas the ethanolic extracts of fruits of Emblica officinalis showed cell proliferative activity only in MSCs. Root of Hemidesmus indicus also have cell proliferative potential in dose dependent manner in both in-vitro models but have less significant in MSCs compared to the cells of MG-63 cell line. Thus, from the present study it can be concluded that aqueous extract of seeds of Nigella sativa causes significant proliferation stimulation amongst all five-selected drugs. Immunoprotective activity of aqueous extracts of seeds of Nigella sativa also support the results of the study⁽³⁴⁾. So it can be use for the treatment and management of osteoporosis. Further, the seeds of Nigella sativa can be used for the preparation of herbal formulation for the treatment of osteoporosis.

 

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Received on 13.02.2020           Modified on 14.03.2020

Accepted on 12.04.2020         © RJPT All right reserved