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Research

greens+ bone builder : Rao LG study (progress report)

By Genuine Health

greens+™, bone builder™ and greens+bone builder™ all have stimulatory effects on bone formation in human osteoblast cells

(submitted by Rao LG September 30, 2009)

ABSTRACT

INTRODUCTION

Osteoporosis is a skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and consequent increase in fracture risk (1-3). The high prevalence of this disease, and the resulting morbidity  associated with fracture incidence, costs the Canadian health care system 1.3 billion dollars per year (2). It has been estimated that among the current Canadian population over the age of 55, 1 in 2 women and 1 in 4 men have osteoporosis (3). Given that many nutrients have been identified as being beneficial to bone health (4), there is strong scientific support for the potential benefits of incorporating therapeutic nutritional interventions with contemporary pharmaceutical treatments. A dynamic equilibrium between bone formation and bone loss is essential in maintaining healthy, strong bones. Diet is now recognized as an important life-style factor in the management of bone health. A number of epidemiological studies have shown an association between fruit and vegetable intake and bone mineral density (BMD) (5-7). This may be attributed to the nutritive and nonnutritive plant compounds that help support the formation of new bone or decrease the removal of old bone. Nutritional supplements, such as greens+™, bone builder™ and greens+bone builder™, can serve to assist in achieving adequate intakes of  nutrients which otherwise may be lacking in the diet.

Oxidative stress has been implicated in the progression of osteoporosis. Excess of reactive oxygen species, which are produced normally in the body through metabolic processes results in oxidative stress (8). Studies have shown that there is an inverse correlation between oxidative stress biomarkers and bone mineral density (BMD) (9) and in patients with fractures, oxidative stress parameters are higher than those found in healthy controls (10). Antioxidants capable of counteracting this effect, by quenching reactive oxygen species, have been demonstrated to be important in decreasing the risk of osteoporosis (11). An accumulation of reactive oxygen species can be counteracted by antioxidants which are obtained primarily through foods such as fruits and vegetables, as well as plant-based beverages and nutritional supplements. Our laboratory has been studying two types of antioxidants that have been shown to have beneficial effects on bone health – the lipid-soluble lycopene and the water-soluble polyphenols. Recently, we have shown that a high serum lycopene is associated with decreased protein oxidation and bone resorption in postmenopausal women (12), suggesting a potential benefit of lycopene in reducing the risk of osteoporosis. At the cellular level, we showed that lycopene stimulates the growth and differentiation of osteoblasts (13,14) and inhibit the formation and  resorption activity of osteoclasts (15). Lycopene is one of the components of bone builder™ . The polyphenols have also now been shown to have beneficial effects on bone health (16). The nutritional supplement greens+™ is a blend of several herbal and botanical products containing a substantial amount of polyphenols which act as antioxidants and therefore should be able to counteract oxidative stress. Our laboratory has shown that the polyphenolic extracts from greens+™ have stimulatory effect on mineralized bone nodule formation in human osteoblast cells in a dose and time dependent manner (17). We have further shown that this stimulatory effect is accompanied by decreases in the reactive oxygen species H2O2 (18), thus proving that greens+™ is able to counteract oxidative stress in human osteoblastic cells and may therefore be a good candidate as a nutritional supplement to prevent the risk of osteoporosis.

Two additional nutritional supplements have since been formulated which may prove to be good for bone health. These are the bone builder TM and the greens+bone builder ™ ; the latter is the original greens+ TM product that has been supplemented with the bone builder formula containing several compounds including vitamins, minerals, and antioxidants. These various components have been separately shown to have some beneficial effect on bone. Calcium is the main bone forming mineral. Numerous studies have shown that intake of calcium is positively associated with increased bone mineral density (BMD) across the majority of population worldwide (19-21). However, studies have shown that calcium alone is not sufficient, but requires other nutritional components, in increasing bone formation in vivo (22). Thus, the components of bone builder™ other than the three absorbable calciums (Citrate-malate, Formate and Bisglycinate) including the vitamins vitamin D, vitamin C and B vitamins; the antioxidant lycopene; magnesium, selenium, the trace minerals, such as zinc, copper and manganese; the essential amino acid, L-lysine, boron and silicon have all been shown separately to be important for the bone formation pathway, mechanisms of which is not fully understood (Reviewed in 22). However, their deficiencies in animal models result in bone defects. Overall, it is clear from current literature that other nutrients, in addition to the standard recommendation of calcium and vitamin D, may be required for bone health. The hypothesis for our study is that a combination of the antioxidant polyphenols in greens+™ and the components of bone builder will, together, have a more positive effect on bone formation compared to greens+™ or bone builder™ alone. The objective of our study was to compare the separate effects of the polyphenol extract of greens+ ™ and the water-soluble component of bone builder TM with that of the combination of the two extracts (greens+bone builder) on bone formation by human osteoblasts in vitro.

MATERIALS and METHODS

The nutritional supplements greens+ ™, bone builder ™ and greens+ bone builder ™ were supplied by Genuine Health Inc., Toronto, Canada. All other chemicals were purchased from Sigma.

Sample preparation

Preparation of polyphenol extract of greens+™ – greens+ ™ was subjected to polyphenol-acetone extraction, modified from Sun et al. (23). Briefly, 1 g of greens+ was stirred for one hour with chilled 80% acetone using a magnetic stirrer, then filtered through number 2 Whatman paper in a Buchner funnel under vacuum. The acetone-H2O was evaporated to dryness and the filtrate recovered with 50ml distilled H2O (greens+extract). A parallel extraction was performed without greens+ to be used as a control. Aliquots were stored at -20ºC. The total phenolic content of the extract was analyzed using the Folin-Ciocalteu method (24).

Preparation of water-soluble extract of bone builder ™ – The water-soluble components of the bone builderTM supplement were dissolved in double distilled water. One g of bone builder per 100ml solution was prepared to yield stock solutions of 10mg/ml by mixing on a magnetic stirrer for 10min, centrifuged for 10min at 2500rpm, then filtered through 0.2 uM syringe filters under sterile conditions. The sterile extract was aliquoted into 5ml tubes to be frozen and stored at -40°C until needed for experiments. (bone builder or bb extract).

Cell culture – The human osteoblastic cells, SaOS-2, was obtained from the American Type Culture Collection (ATCC, Rockville, MD). Te CD34+ cells were cloned in our laboratory by limiting dilution from a commercial preparation of hematopoetic cells. The cells were maintained in a CO2 incubator at 37 ºC with a 5% CO2 atmosphere and passaged weekly in a 75cm2 flask in Ham’s F12 medium supplemented with 28mM HEPES buffer, pH 7.35, 1.1mM CaCl2, 2 mM glutamine, 1% antibiotic-antimycotic solution, and 10% fetal calf serum (medium) as previously described (25). When cells were to be used for experiments, the cells were plated in 12-well dishes in a medium supplemented with 10nM dexamethasone and 50ul/ml ascorbic acid. The medium was also supplemented with 10mM b-glycerophosphate at day 8 and the addition was repeated at every medium change until the end of culture.

Treatment with greens+ extract (g+), water-soluble bone builder solution (bb)  and combination of greens+ extract and bone builder solution (greens+bone builder or g+bb) – SaOS-2 cells or CD34+ cells were plated at a density of 1×104 cells/well in each well of a 12-well plates. Varying doses of the g+, bb or g+bb were added at day 8 and every medium change thereafter. The cells were incubated at 37o C and the culture stopped at days 13 to 20 for assays.

Determination of cell number – A modification of methylene blue assay by Genty et al. (26) was used. At the end of the incubation period, cells were washed with PBS and fixed overnight with 4% paraformaldehyde. Cells rinsed with PBS and stained with 1% methylene blue dye, prepared in 0.01M borate buffer, pH 7.5. The excess stain was rinsed every hour with the same buffer until solution remained clear. The blue colour of the nuclei was eluted with ethanol-0.01M HCl (1:1). The sample was diluted in ethanol- 0.01M HCl (1:1) and the optical density of the colour was measured in a Multiscan spectrophotometer (Flow Laboratories, McLean, VA). Results were calculated from a  standard curve of known cell number vs optical density reading and corrected for the dilution factor.

Alkaline phophatase (ALP) activity assay – Cells were rinsed twice with 50mM Tris(hydroxymethyl)aminomethane (Tris), pH 7.4, harvested by scraping, and sonicated in 0.5ml of Tris containing 0.05% Triton X-100. ALP activity was determined according to the method of Lowry (27) using appropriate dilutions of cell sonicates as previously described (28). The release of p-nitrophenol from 10nM p-nitrophenyl phosphate in a buffer containing 1.0mM MgCl2.6H2O in 2-amino-2-methyl-1-propanol, pH 10.3, was carried out at 35ºC. The reaction was stopped after 5 minutes with 0.5 N NaOH and the absorbance was meatured at 405nm using a Multiscan plate reader (Flow Laboratories, McLean, VA). The protein content of the cell sonicates was determined using a commercial protein dye reagent (Biorad). The ALP activity was expressed as nanomoles per min/mg protein

Mineralized bone nodule formation assay – The cells were washed with PBS and fixed overnight with 4% paraformaldehyde. They were stained in situ with 5% silver nitrate and exposed to UV light for 1 hour. The cells were then rinsed with distilled H2O and exposed to 5% sodium thiosulphate for 5-10 minutes to remove background, according to the von kossa technique previously described (25). Cells were then rinsed and covered with 50% glycerol. The mineralized nodule areas and numbers were quantified using an image analyzer (Fluorchem 8900, Alpha Innotech Corporation, California).

Statistical analysis – Growth analysis was carried out in two experiments, each with 3 replicates. The results were expressed as mean+-SD or SEM, according to the experiments. All graphs were plotted using Prism ver 5 (Graphpad Sotware, San Diego, CA). For statistical analysis, one way analysis of variance (ANOVA) were used to determine the  statistical significance of treatments and time course. p<0.05 was considered statistically  significant. T test or Post hoc tests included Tukey’s were carried out.

RESULTS

Publications
Papers published and abstract presentations to Local/International Meeting/ Symposium related to the project are as follows:

  1. Rao LG, B Balachandran, Rao AV 2009 Polyphenol extract of greens+TM nutritional supplement stimulsatrd bone formation in cultures of human osteoblast-like SaOS-2 cells. Journal of Dietary Supplements 5(3); 264-282;2008.
  2. Balachandran B, Rao V, Murray T, Rao LG 2004 Polyphenols in the extract of greens+TM herbal preparation have effects on cell proliferation and differentiation
    of human osteoblast cell line SaOS-2. Presented at the 26th Annual Meeting of the American Society for Bone and Mineral Research, Oct 1-5, 2004, Seattle,
    Washington. (Journal of Bone and Mineral Research 2004 19 (suppl): S403.
  3. Rao LG, Balachandran B, Rao AV. The stimulatory effect of the polyphenols in
    the extract of greens+TM herbal preparation on the mineralized bone nodule
    formation (MBNF) of SaOS-2 cells is mediated via its inhibitory effect on the
    intracellular reactive oxygen species (iROS). Presented at the 27th Annual
    Meeting of the American Society of Bone and Mineral research, Nashville,
    Tennesse, September 23-27, 2005.
  4. Rao LG, Beca J. 2007 greens+ bone builderTM nutritional supplement stimulates
    bone formation in an in vitro human SaOS-2 cell cultures. Presented to the
    Canadian Herb, Spices, & Natural health Products Coalition: Tradition to
    Technology, Saskatoon, Saskatchewan, May 10-13, 2007.
  5. Rao LG greens+ bone builderTM nutritional supplement stimulates bone
    formation in an in vitro human SaOS-2 cell cultures” presented to Gelda
    Scientific and Sunstar Inc, Mississauga, Ontario, February 14, 2007.
  6. Rao LG “Natural food components/nutritional supplements and bone health”,
    presented at the Canadian College of Naturopathic Medicine, Grand rounds,
    November 19, 2007.
  7. Rao LG “Natural food components, nutritional supplements, and lifestyle in the
    prevention of osteoporosis” presented to the Osteoporosis Support & Information
    Group, Scarborough Village Recreation Centre, Kingston Rd, Scarborough,
    March 31, 2008.
  8. Rao AV, Snyder DM, Mackinnon ES, Rao LG 2008 Trace elements present in
    nutritional supplements stimulate bone formation in human osteoblasts SaOS-2
    cells in vitro, oral presentation to the 5th International Meeting of the Advances
    in Antioxidants (trace elements, vitamins and polyphenols):molecular
    mechanisms, nutritional and clinical aspects to be held Oct 11 -15, 2008 at
    Monastir-Sousse (Tunisia).

SUMMARY OF PREVIOUSLY PUBLISHED STUDIES ON GREENS+™

The results of these studies have been published (17), and the manuscript is appended. The important findings of these studies are that cell proliferation was stimulated at early time points (days 2 to 4) and inhibited at later time point (after day 7) of treatment with the polyphenolic extract of greens+ TM. The inhibition of cell number was followed with the initial stimulation of ALP by lower concentration of greens + extract (days 9 to 11). At a later time point of treatment (day 13), greens+ TM inhibited ALP and stimulated mineralized bone nodule formation (day 13 to 17) in a time- and dose-related fashion. The results were consistent with the effect of greens+ TM on the faster maturation of  osteoprogenitors toward progression to a bone forming stage compared to cells that did not receive any greens+ treatment.

STUDIES ON BONE BUILDER™

2 types of osteoblast-like cells were used for this study. First, we used SaOS-2 cells and later the CD34+ cells in order to confirm our results on another cell line. To study the effect of bone builder on the cells, we used the water-soluble component of bone builder (bb) as described under method. This part of the study concentrated on the effects of bb on bone formation. Bone formation by osteoblasts in culture can be visualized with vonKossa staining of the mineralized bone nodules. An example is shown in the photomicrograph of vonKossastained cells that were treated with increasing concentrations of bb from 0 to 1.0 mg/ml (Figure 1). It can be observed that the intensity of staining, representative of bone formed by osteoblasts, increased with increasing concentration, starting from 0.5 mg/ml to 1.0 mg/ml. The area of the stained nodules can be quantified using an image analyzer. In the subsequent figures, effects of treatments are presented as the area of bone mineralized nodules quantified by image analysis. Figure 2 shows the time and dose-dependent effects of varying concentrations of bb on mineralized bone nodule area in SaOS-2 cells. At day 17, treatment concentrations of 0.5 – 1.0 mg/ml were significantly different in the area of mineralized nodules, compared to controls. By day 20, a greater range of treatment concentrations were significantly different from controls (0.3 – 1.0 mg/ml). The treatment effect appears to peak at 0.8 mg/ml. However, since there are no significant differences between the majority of treatments ranging from 0.7 – 1.0 mg/ml, it is not possible to be sure of this trend. There is however, a significant dose-dependent effect, both after 17 and 20 days of treatment (One-way ANOVA, p<0.0001), with up to 500-fold increase at day 17 and 1000-fold increase at day 20 in nodule area compared to  control. The significant timecourse effect (P<0.05), had similar drastic changes with 3 fold  increases in nodule area between day 17 and day 20. The stimulatory effect of bb on mineralized bone formation in osteoblast-like CD34+ cells was examined and results presented in Figure 3. Similar to the effect on SaOS-2 cells, bb had a dose- and time-dependent effects on mineralized bone formation in CD34+ cells. Results revealed that although calcium was as effective as bb at all concentrations at days 13 and 15 of  treatment, it became obvious that treatment with bb at a concentration of 1.0 mg/ ml was  more effective than calcium treatment after 17 days. Thus, calcium stimulated the area of mineralized bone nodule by 250-fold while bb at 1.0 mg/ml stimulation was over  1000-fold. This could indicate that the other components of bone builder are necessary for  the stimulation of bone formation.

STUDIES ON GREENS+BONE BUILDER – COMPARISON WITH GREENS+™
OR BONE BUILDER ALONE

To study the effect of greens+ and bone builder in osteoblast-like SaOS-2 or Cd34 cells, we used the acetone-extracted polyphenols of greens+ (g+) and the water-soluble extract of bone builder (bb), respectively. To study the effect of greens+bone builder (g+bb), the extracts of g+ and bb were added together and simultaneously to the cells. The effects of the three treatments on the maturation of the cells towards bone-forming stage, we studied their effects on proliferation, alkaline phosphatase activity (ALP) and mineralized bone nodule formation. As shown in Figure 4, treatment of SaOS-2 cells with g+, bb and g+bb all had resulted in increased cell numbers after day 13. After day 15, no further increase was observed, with g+ or bb treatment and an inhibition was observed only in bb treatment. Figure 5 demonstrates the effects of the various treatments on ALP of SaOS-2 cells. On day 13, bb and g+bb had significantly decreased the activity while on day 17, all treatments resulted in significantly lower ALP compared to control. The statistical significance in p-values are given in the text. The effect of g+bb was compared with g+ alone by treating the cells with varying doses of g+ alone and varying doses of g+ in the  presence of 0.5 mg/ml bb. A dosedependent stimulatory effects of varying concentrations  of g+ (One-way ANOVA, P<0.001) and varying concentrations of g+ 0.5 mg/ml bb (One-way ANOVA, p<0.0001) on mineralized bone nodule formation in SaOS-2 cells are illustrated in Figure 6. At any given concentration of g+ used, the effect was higher in the presence of 0.5 mg/ml bb. The effect of g+bb was next compared with bb alone by treating the cells with varying doses of bb and varying doses of bb in the presence of 1.2 mg/ml g+. Figure 7 shows that varying concentrations of bb without (One-way ANOVA, P<0.0005) or with 1.2 mg/ml g+ (One-way ANOVA, p<0.001) have stimulatory effects on mineralized bone nodule formation in SaOS-2, but the presence of 1.2 mg/ml greens+ with bb at concentrations of 0.5 and 0.8 mg/ml was more effective. At concentrations of 1.0 mg/ml bb, the stimulatory  effect was not significantly different in the presence or absence of 1.2 mg/ml g+.
The effects of g+bb compared to g+ alone or bb alone were next studied in the osteoblast-like Cd34+ cells. Figure 8 illustrates that addition of 0.5 mg/ml bb to varying doses of g+ increased the effect 10-fold at 0.8 mg/ml g+ (p<0.005), 15-fold at 1.2 mg/ml g+ (p<0.005) and 22-fold at 2.0 mg/ml g+ (p<0.0.001). A dose-dependent effects of g+bb (0.5 mg/ml bb) was shown to be significant by One-way ANOVA (p<0005). Figure 9 illustrates that addition of 1.2 mg/ml g+ to varying doses of bb increased the effect 5-fold at 0.5 mg/ml bb (p<0.05), 15-fold at 0.7 mg/ml bb (p<0.05) and 20-fold at 1.0 mg/ml bb (p<0.005). A dose-dependent effects of g+bb (0.5 mg/ml bb) was shown to be significant by One-way ANOVA (p<001). This particular batch of CD34+ cells had very low level of mineralization, having only 250 pixels compared to the value of over 1500 pixels in previous study (Figure 3). Inspite of this, however, the synergistic effects between g+ and bb could still be observed.

DISCUSSION

The most important finding in this study was that although greens+ and bone builder  separately had significant effects in stimulating the mineralized bone nodule formation in both SaOS-2 cells and CD34+ cells, the effects were significantly higher with combination of the two. We have shown in previous study that greens+TM (17) had stimulatory role on mineralized bone nodule formation in SaOS-2 cells. However, this is the first time we are reporting that a combination of the two was more effective than either product. The results were obtained using two different cell lines, the SaOS-2 cells and the CD34+ cells, showing that the effects are reproducible. The three treatments had stimulatory effects on cell proliferation earlier in the culture. Cells were stimulated to grow more quickly, such that at earlier points the cell number was higher for treated cells compared to controls. Near to the end of the culture periods, the control cells reached statistically similar numbers as the treated cells, suggesting that the control cells might have still continued proliferating while the treated cells have now stopped growing and beginning to differentiate. These results are consistent with results on alkaline phosphatase and mineralized bone nodule formation. It suggest that the treated cells have proliferated earlier, matured more quickly, and were in a position to slow growth in favour of differentiating and mineralizing at an earlier time point than controls. It was important to observe marked decreases in ALP during later time points of treatment with greens+, bone builder and greens+bone builder. There is evidence in the literature that SaOS-2 cells phenotypically behave like osteoblast cells by undergoing different stages of proliferation followed by differentiation in the presence of dexamethasone. The differentiation stages are marked by increases in osteoblastic markers including ALP. Following differentiation, the ALP decreases and mineralized bone formation takes place. Thus, our study demonstrated that at later time points of 17 days in culture there was a significant reduction in ALP for all three treatments compared to control. The finding that greens+, bone builder and greens+bone builder had inhibitory effect on ALP, a marker of bone formation, is consistent with their effects on osteoblasts maturation and differentiation towards bone-forming stage. Thus, the decrease in ALP signals the ability of osteoblasts to form mineralized bone nodules. This suggests that the treated cells underwent differentiation earlier in culture compared to control cells, and that at the time of assay the cells had completed their differentiation stages and began forming bone. To study the effects of greens+bone builder, we had to resort to combining the polyphenolic extract of greens+ by acetone extraction and the water extract of bone builder since the various herbal components in greens + and the components of bone builder are not amenable to a single method of extraction. Indeed, only the water-soluble component of bone builder has been studied so far, and the effect of the lipid-soluble components such as vitamin D and lycopene need to be carried out in future studies. Therefore, the stimulatory effect of bone builder reported here would have been much greater were lycopene and vitamin D were also added. The effect of the water-soluble component of bone builder was higher compared to calcium alone. This could indicate that the other components of bone builder are necessary for the stimulation of bone formation.

The reason for our finding that greens+bone builder had a greater capacity to stimulate bone formation than the greens+TM or the bone builderTM alone could be because the antioxidant polyphenol in greens+TM and the various components of bone builder, individually shown to have beneficial effect on bone health, are all necessary for the osteoblasts to form bone. The mechanisms of such synergy remains unclear, but our findings could have important implication in the management of osteoporosis and may suggest that greens+bone builder can be considered as alternative supplement in the prevention of osteoporosis in both men and postmenopausal women.

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Concentration of bb, mg/ml

Figure 1. Photomicrograph of VonKossa-stained cells were taken under the microscope  after 17-days of treatment with increasing concentrations of water-soluble preparation of  bone builder, and the areas of the darkly stained mineralized nodules were quantified  using FluorChem 8900 Image Analysis System (Alpha Innotech Corporation, California).Concentration of bone builder chart

Figure 2. Time and Dose-dependent Effects of bone builder on mineralized bone nodule area in SaOS-2 cells. indicates significant difference from vehicle: p<0.0001; p<0.0005; p<0.005, #, p<0.01 and ##, p< 0.05. There is a significant dose-dependent effect both at day 17 and day 20, according to One-way ANOVA; p<0.0001.

concentration of bone builder chart

Figure 3. The effect of bone builder solution on bone formation in CD34+ cells, treated from day 8 in culture for 13,15 and 17 days. One-way Anova revealed a dose-dependent effect of bone builder at all time points (p<0.0001). The effect of 1.0 mg/ml bone builder is greater than that of calcium alone after 17 days of treatment, *p<0.005.

Cell proliferation chart

Figure 4. Cell proliferation assay of SaOS-2 cells treated continuously beginning day 8 with  medium ©, greens+ extract (g+), water soluble component of bone builder (bb) and a  combination of g+ and bb (g+ plus bb). Cell numbers were determined using the Methylene Blue assay as described in the text. The cultures were stopped and assayed on days 10, 13 and 15. Results are expressed as mean ± SEM of three replicates each from two separate experiments (n=6). * p<0.05 compared to control.

Alkaline phosphatase activity

Figure 5. Alkaline phosphatase assay of SaOS-2 cells treated continuously beginning day
8 with medium control ©, greens+ extract (g+), water soluble component of bone builder
(bb) and a combination of g+ and bb (g+ plus bb). The cultures were stopped and assayed
on days 13 and 17. Results are expressed as mean ± SEM of three replicates each from
two separate experiments (n=5 or 6). Significant decreases in ALP activity was observed
as follows: Day 13: c vs g, p = 0.54 (marginal significance); ***p<0.01 for c vs bb and c
vs g+ plus bb and Day 17: # p< 0.05, for c vs g+,; ** p< 0.005, c vs bb, and *p<0.0005, for c vs g+ plus bb.

Mineralized bone nodule area

Figure 6. Dose Dependent Effect of greens + (g+) with and without 0.5 mg/ml of bone   builder (bb) on the area of mineralized bone nodules in osteoblasts SaOS-2 Cells.   Significant differences were found compared to respective controls as follows:  *p<0.0005,  **p<0.005; ***p<0.05; #p<0.0001; ## p<0.001; ###p<0.01; Significance differences were  found when treatment with g+ plus 0.5 mg/ml bb was compared to treatment with g+  alone as follows: a p< 0.0001; b p< 0.005; c p< 0.01; d p< 0.05. One-way ANOVA showed a  dose-dependent effect of greens+ alone at p<0.001 and a dose-dependent effect of  greens+plus 0.5 mg/ml bb at p<0.0001.

Area of mineralized bone nodules chart

Figure 7. Dose-dependent effects of bone builder (bb) with and without 1.2 mg greens+  (g+) on the area of mineralized bone nodules in osteoblast SaOS-2 cells. Significant differences were found compared to respective controls as follows: **p<0.005; ***p<0.05; ###p<0.01; Significance differences were found when treatment with bb plus 1.2 g+ was  compared to treatment with bb alone as follows: b p< 0.005; c p< 0.01; d p< 0.05. Dose-dependent stimulatory effects of varying concentrations of bb (One-way ANOVA, P<0.0005) and varying concentrations of bb plus 1.2 mg/ml g+(One-way ANOVA, p<0.001) were observed.

Area of mineralized bone nodules chart

Figure 8. Dose Dependent Effect of greens + (g+) with and without 0.5 mg/ml of bone builder (bb) on the area of mineralized bone nodules in osteoblast Cd34+ Cells. Significant differences were found compared to respective controls as follows: Significant difference compared to respective control: *p<0.0005, **p<0.005. Significance differences were found when treatment with g+ plus 0.5 mg/ml bb was compared to treatment with g+ alone as
follows:  bp< 0.005; a p< 0.001. One-way ANOVA showed a dose-dependent effect of  greens+plus 0.5 mg/ml bb at p<0.0005.

Area of mineralized bone nodules chart

Figure 9. Dose-dependent effects of bone builder (bb) with and without 1.2 mg greens+  (g+) on the area of mineralized bone nodules in osteoblast CD34+ cells. Significant differences were found compared to respective controls as follows: **p<0.005 and  *p<0.0005; Significance differences were found when treatment with bb plus 1.2 g+ was compared to treatment with bb alone as follows: b p< 0.005; and cp< 0.05. Dose-dependent stimulatory effects of varying concentrations of bb plus 1.2 mg/ml g+ (One-way ANOVA, p<0.001) was observed.

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