Volatile components of fruits of Ligustrum lucidum Ait . stimulate proliferation and differentiation of rat calvarial osteoblasts

The fruits of Ligustrum lucidum Ait., (FLL), which contain rich volatile components, are commonly used as tonic for kidney and liver in the traditional Chinese medicine prescriptions. This study aimed to investigate the effects of volatile components of FLL on the proliferation and differentiation of rat calvarial osteoblasts by the MTT method and measuring the activity of alkaline phosphatase (ALP). Results showed that volatile components (1 to 100 μg/mL) of FLL significantly (p<0.01) stimulated the proliferation and increased the ALP activity of rat calvarial osteoblasts which indicated that volatile components of FLL played an important role in osteoblastic bone formation just as non-volatile components in FLL. Such finding accredited the FLL as a potential candidate that might be useful in bone engineering and in treating bone defects including osteoporosis. The volatile components were analyzed by GC-MS. A total of 67 compounds were identified and the main components included (Z,Z)9,12-octadecadienoic acid (33.47%), n-hexadecanoic acid (15.02%), (E)-9-octadecenoic acid (9.03%), αcadinol (6.51%), 4-hexyl-2,5-dihydro-2,5-dioxo-3-furanacetic acid (4.93%) and (E)-8-octadecenoic acid methyl ester (2.69%).


INTRODUCTION
Osteoporosis, a disease characterized by low bone mass and microarchictectural deterioration of bone tissues, is due to the persistent excess of osteoclastic bone resorption over osteoblastic bone formation (Rodan and Martin, 2000;Kong et al., 2000;Teitelbaum et al., 2000).Therefore, both stimulators of bone formation and specific suppressors of bone resorption are significant for the treatment of osteoporosis.
Ironically, data indicated that their long-term use is accompanied by potentially malignant effects (Davison and Davis, 2003;John 2010).In addition, their costs are too high to benefit a large population in the developing or even developed countries for the prevention and treatment of osteoporosis (Kaufman and Goemaere, 2008).For more than a millennium, Chinese herbal medicine has been extensively used, apparently safely and effectively, in Asian countries, especially in China, Japan and Korea, to alleviate various symptoms of diseases (Zhang et al., 2009;Wu et al., 2009;Chena et al., 2009).So, it will undoubtedly be a cost-effective alternative to commercial pharmaceutical products.
The fruits of Ligustrum lucidum Ait.(Oleaceae) (FLL, Chinese name, Nvzhenzi), are well known as tonic for kidney and liver in the traditional Chinese medicine.Previous papers reported that the aqueous and ethanolic extracts of FLL could improve bone properties by enhancing the mineralization process on osteoblast cells or maintaining the calcium balance, and accelerate the osteoblast differentiation of mesenchymal stem cells (MSCs) (Zhang et al., 2006(Zhang et al., , 2008;;Li et al., 2008).Many researches showed that volatile components obtained from many plants are responsible for their pharmacological activities just as non-volatile components in herbs (Lograda et al., 2010;Ho et al., 2010;Da et al., 2010).Moreover, the quality and quantity of volatile components, related with pharmacological activities, are highly influenced by genetic and environmental factors (Cardile et al., 2010).FLL contain rich volatile components, from which we could always smell the strong fragrance.However, as far as our literature survey could ascertain, there is no report on any pharmacological investigation on the volatile components from FLL.Therefore, in this study, we investigated the effects of volatile components of FLL on the proliferation and alkaline phosphatase (ALP) activity (the expression of ALP is closely associated with osteoblastic differentiation) of rat calvarial osteoblasts and identified the composition of the volatile components of FLL.

MATERIALS AND METHODS
The fruits of L. lucidum Ait.(Oleaceae) (FLL, Chinese name, Nvzhenzi) (20080601) were purchased from Fujian Tianren Pharmaceutical Company and identified by Professor Cheng-zi Yang of the Department of Pharmacy, Fujian University of Traditional Chinese Medicine.The voucher specimens of these fruits were deposited at the Herbarium of the Department of Pharmacognosy, Fujian University of Traditional Chinese Medicine, Fuzhou, P. R. China.

Extraction of volatile components
Dry fruits (200 g) were crushed (40 mesh), then soaked in 2000 ml water for about 12 h before they were subjected to hydrodistillation in a Clevenger type apparatus.The contents were distilled for 3 h to obtain the volatile oil in a 0.31% (w/w) yield (on a dry mass) of yellowish colour and with a pleasant smell.The oils were dried over anhydrous sodium sulphate and stored at 4°C in the dark until they were tested and analyzed.

Preparation of test samples
Volatile components were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mg/ml, and diluted in culture medium of the working solution before use.To avoid DMSO toxicity, the concentration of the solvent was less than 1% (v/v).For effects of steroids on growth or differentiation, culture media was charcoal stripped and without phenol red.Wu et al. 8663 Cell cultures Sprague-Dawley rats, which were 2 to 3 days old, were purchased from the Experimental Animal Center of the Fujian Medical University, Fuzhou, P. R. China.Rat calvarial osteoblasts were prepared from the calvarias of newborn rats following the sequential enzymatic digestion method (Idris et al., 2008).Briefly, skull (frontal and parietal bones) were dissected; then, the endosteum and periosteum were stripped off, and the bone was cut into approximately 1 to 2 mm 2 pieces and digested sequentially using trypsin (0.25%, w/v) for 30 min and collagenase II (1.0 mg/mL) containing 0.05% trypsin (w/v) for 2 h.The cells were collected and cultured in phenol red free DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, for 24 h in a humidified atmosphere of 5% CO2 in air at 37°C, then the media was changed.

Assay for osteoblast proliferation and ALP activity
The rat calvarial osteoblasts (2 × 10 4 cells/well) were subcultured into 96-well culture plates, and incubated 24 h before the addition of test samples or control (DMSO, final concentration was 1% v/v.), then cultured for another 48 h.Prior to the end of the culture, MTT (20 µl, 5 mg/ml) was added to each well and incubated for 4 h, after which the medium was discarded, and 150 µl of DMSO was added to each well.The cells were incubated for 20 min.The UV absorbance was measured at 490 nm at a microplate spectrophotometer (Bio-rad Model 680, USA) with a reference at 630 nm and used as an indicator of osteoblast proliferation.Proliferation (%) was calculated as 100 × (OD of volatile components -treated / OD of control), where OD is the average absorbance of six experiments with 8 replicates.Primary osteoblasts were seeded at 2 × 10 4 cells/well in 96-well culture plates, and treated with test samples or control for 9 days (media was changed every three days).The ALP activity was measured according to the literature (Owen, 1990).Total protein was assayed by the method of Bradford (1976).The ALP activity was expressed as micromoles of 4-nitrophenol liberated per milligram protein.

GC-MS analysis
GC-MS analysis was performed on an Agilent 6890N Network GC System, fitted with a HP-5MS capillary column (30 m × 0.25 mm i.d.× 0.25 µm film thickness; maximum temperature, 350°C), coupled to an Agilent 5975 inert XL Mass Selective Detector.Ultrahigh purity helium (99.999%) was used as carrier gas at a constant flow of 1.0 ml/min.The injection, transfer line and ion source temperatures were 250, 250 and 200°C, respectively.The ionizing energy was 70 eV.Electron multiplier (EM) voltage was obtained from autotune.All data were obtained by collecting the full-scan mass spectra within the scan range of 35 to 500 amu.The splitless injection was employed for the analysis.The diluted sample (10 mg/ml, in redistilled diethyl ether) volume injected with an Agilent 7683B series injector was 1 µl.The oven temperature program was 90 -2.5°C/min -130 -1.2°C/min -170 -2°C/min -230°C -2°C/min -250°C (5 min).

Identification and quantification of volatile components
Volatile components were first identified by comparing the spectra obtained with a mass spectrum library (NIST 05.L).Corroboration of the identification was then sought by matching the mass spectra of compounds with those present in the literatures and the retention indexes of the compounds reported on equivalent column (Cardile et al., 2010;Lv, 2005;Zhang et al., 1993;Li and Li, 1990).Components relative percentages were calculated from the TIC of the automated integrator.

Statistical analysis
Data were expressed as the mean ± standard deviation.Statistical significances were analyzed by using the Student's t-test.A value of p<0.01 was considered significant.Linear regression analysis was performed by the correlation coefficient.

RESULTS AND DISCUSSION
Volatile components with different concentrations (1 to 100 µg/ml) dose-dependently stimulated the proliferation of rat calvarial osteoblasts (p<0.01)(Figure 1).The maximal effect was observed when cells were incubated with volatile components (100 µg/ml).To ascertain whether FLL are capable of affecting osteoblastic cell differentiation, we examined the changes of ALP activity.As shown in Figure 2, volatile components significantly (p<0.01)increased ALP activity in osteoblasts over the 9 days, and their maximal effects were observed when cells were incubated with volatile components (1 µg/ml).Therefore, volatile components from FLL could stimulate osteoblastic differentiation at least in part by enhancing the synthesis of ALP.Such findings accredited the FLL as a potential candidate that might be useful in bone engineering and in treating bone defects including osteoporosis.
Essential fatty acids (EFAs) can be divided into two families viz.omega-6 or n-6 and omega-3 or n-3 families.Linoleic acid (LA) is the parent molecule of the n-6 series of EFAs metabolites while α-linolenic acid (ALA) is the  parent molecule of the n-3 series.Both of them are converted by alternating desaturation and elongation reactions to their respective active metabolites (Schlemmer et al., 1999).There are some evidences that EFAs, as well as their metabolites play critical roles in regulating bone metabolism and may have potential in the treatment or prevention of osteoporosis.They may enhance the effectiveness of calcium, alter cell-to-cell signaling processes, and impact transcription factors in vivo (Poulsen et al., 2007(Poulsen et al., , 2008;;Das et al., 2000).Furthermore, EFAs might modulate other mechanisms also involved in the regulation of bone parameters.A recent study that examined dietary intake of the two families of EFAs reported that postmenopausal women with a high dietary ratio of n-6:n-3 fatty acids had the lowest bone mass density (BMD) (Poulsen and Kruger, 2006), which indicated that high n-6 EFAs intake rather than high total EFAs intake may be detrimental to bone mass.Hence, in light of this study, the effects of volatile components from FLL on the proliferation and ALP activity of rat calvarial osteoblasts might be not only related to the presence of (Z,Z)-9,12-octadecadienoic acid (LA) (33.47%), but the synergistic effects of the diversity of major and minor constituents present in the volatile components.

Figure 2 .
Figure 2. Effect of volatile components of FLL on the ALP activity of rat calvarial osteoblasts (n = 8, x ±SD; *p<0.01,compared with control)

Table 1 .
Volatile components of the fruits of L. lucidum Ait.RI: Retention indexes relative to n-alkanes C8-C40 on HP-5MS column; a b Peak area (%)