The effects of Labisia pumila extracts on bone microarchitecture of ovariectomized-induced osteoporosis rats: A micro-CT analysis

Abstract

BACKGOUND: Labisia pumila (LP) is a popular herb used by women over the past few decades. This herb has shown potentials as an alternative agent for treatment and prevention of postmenopausal osteoporosis. It was observed in previous studies that supplementation to ovariectomized rats were associated with increased bone antioxidative enzymes and reduced lipid peroxidation activity. It had also improved bone formation markers in ovariectomized rats. This study aimed to evaluate the effects of giving different forms of LP extracts on the trabecular bone microarchitecture of ovariectomised rats.

METHODS: Forty-eight female Sprague-Dawley rats were randomly divided into sham-operated (Sham), ovariectomized control (OVX), ovariectomized and given estrogen at 64.5 μg/kg (ERT), ovariectomized and given LP aqueous extract (LP _aq ), LP methanol extract (LP _met ) and LP ethanol extract (LP _et ) at 100 mg/kg, respectively. Treatments were given daily via oral gavages for nine weeks. Following sacrifice, femora were dissected out for bone microarchitectural analysis using an in vitro micro-CT, which provided three dimensional informations on bone microarchitecture.

RESULTS: LP _aq was the most effective extract found to improve the bone microarchitectural paramaters which comprised ofBone volume fraction (BV/TV), Trabecular separation (Tb.Sp), Trabecular number (Tb.N), Connective density (Conn.dens), Structure model index (SMI) and Degree of anisotropy (DA).

CONCLUSION: LP _aq was effective in protecting the bone of postmenopausal osteoporosis rat model against microarchitectural deterioration.

Keywords

osteoporosis bone microarchitecture micro-CT imaging

1 Introduction

The use of traditional or complementary medicine is gaining popularity over recent years due to the adverse effects of conventional medicine. To date, in Malaysia, over 2000 species of lower plants with therapeutic values have been identified and used in various health care. Approximately, 17.1% Malaysians were reported to use herbs to treat health problems while another 29.6% consumed herbs for health maintenance [1]. Amongst the famous herbs that are widely used in Malaysia by the locals are Labisia pumila (Kacip Fatimah), Eurycoma longifolia Jack (Tongkat Ali), Orthosiphon stamineus (Misai Kucing), Quercus infectoria (Manjakani) and Piper sarmentosum (Daun Kaduk). Some of these herbs were used as traditional medicine for the treatment of fever, asthma, diabetes, post-partum problems, infertility, inflammatory disorders as well as bone disorders like osteoporosis [2, 3].

Osteoporosis is an important threat to the health and well-being of the population. Osteoporosis is defined as a systemic skeletal disease, characterized by low bone mass and microarchitectural deterioration with a consequent increase in bone fragility and susceptibility to fracture [4]. The prevalence of osteoporosis nowadays has reached to endemic proportions [5]. Statistics showed that up to one in two women and one in five men above the age of 50 may suffer from osteoporotic fractures during their lifetime. As projected by WHO, approximately 200 million people worldwide are known to suffer from osteoporosis and 9 million fractures occured due to osteoporosis, every year [6].

Osteoporosis generally affects women to a greater extent than men because women achieve lower peak bone mineral density (BMD) and they are more prone to deleterious effects of estrogen deficiency following menopause [7]. Estrogen plays an important role in maintaining bone remodelling, thus bone mass declines rapidly following menopause [8]. This explains the fact that postmenopausal osteoporosis contributes to the major prevalence of osteoporosis. The main treatment of postmenopausal osteoporosis is estrogen replacement therapy (ERT). Although ERT has been shown to be an effective bone-sparing agent, prolonged intake may increase risk of stroke, myocardial infarction, thromboembolism and breast cancer [9, 10]. Another form of treatment for postmenopausal osteoporosis are selective estrogen receptor modulators (SERMs) such as raloxifene and biphosphonates such as alendronate, risedronate and zoledronic acid [11, 12]. Despite being effective in preserving bone and inhibiting bone resorption, these treatments may also cause adverse effects such as thromboembolism and osteonecrosis of jaw [13, 14]. These unfavourable conditions have led to the wide search for alternative treatments.

Labisia pumila (LP) or also known as Kacip Fatimah is a famous herb used by women for many generations to treat menstrual irregularities, promote birth channel contraction as well as to promote sexual health functions [15, 16]. It has also been used to treat gonorrhoea, rheumatism and sickness in bone [17, 18]. In recent years, it was reported that LP produced similar effects to estrogen on bone biomarkers of postmenopausal induced-osteoporotic rats [19]. This may be associated with its phytoestrogenic properties, as reported by Husniza (2002) in which LP was able to inhibit estradiol binding to antibodies raised against estradiol [20]. Other than phytoestrogenic, LP also exerts antioxidative properties due to the presence of flavonoid, β-carotene, anthocyanins and phenolic compounds [21].A recent study reported that LP supplementation was able to increase the antioxidative enzymes (superoxide dismutase and glutathione peroxidase) and reduced the lipid peroxidation activity in the bones of ovariectomized rats [22].

The positive actions of LP on bone parameters have led to an insight that it has potential as an alternative to the conventional anti-osteoporotic agents. The gold standard measurement of bone mineral density (BMD) is dual X-ray absorptiometry (DXA) [23]. Osteoporosis definition derived from DXA assessments is often argued as it focuses too much on bone mass, rather than on bone microarchitecture and strength. Osteoporosis has long been considered solely a question of low bone mass based on densitometry [24]. However in 2001, WHO added the concept of ‘microarchitectural changes’ leading to decreased bone strength, which resulted in an increased risk of fractures [25]. Hence, it is now understood that bone microarchitecture assessment is vital in determining the risk of osteoporosis. To study bone quality as a whole, it is essential to develop a sensitive, accurate and non-invasive tool which can measure bone mass, density and microarchitecture simultaneously. Micro-computed tomography (micro-CT) has become the new state of art tools in bone research as it can detect early bone changes and provide a three-dimensional (3D) informations on bone microarchitectural changes [26].

Previous study has shown that crude extract of LP supplementation was able to improve bone parameters [27]. Present study was carried out to evaluate the effects of different forms of LP extracts supplementation on bone microarchitecture of postmenopausal osteoporosis rat model. To study bone quality as a whole, it is essential to develop a sensitive, accurate and non-invasive tool which can measure bone mass, density and microarchitecture simultaneously. Micro-computed tomography (micro-CT) has become the new state of art tools in bone research as it can detect early bone changes and provide a three-dimensional (3D) informations on bone microarchitectural changes [25]. Hence, in this study, we have used micro-CT to evaluate the microarchitecture of postmenopausal osteoporosis rat model. This study would determine which of the LP extract with the most bioactive compounds that may have contributed to the anti-osteoporotic properties, particularly in reversing the microarchitectural deteriorations induced by ovariectomy.

2 Materials and methods

The study was approved by Universiti Kebangsaan Malaysia Animal Ethics Committee. A number of 48 female Sprague-Dawley rats aged 3 to 5 months, weighing between 200–250 grams were obtained from the Universiti Kebangsaan Malaysia Laboratory Animal Research Unit. The rats were housed in plastic cages at the temperature of 29±3°C, under natural day/night cycle. They were fed with commercial food pellets (Gold Coin, Port Klang, Malaysia) and deionised water ad libitum. They were allowed to adapt to the new environment for a week prior to the study. They were then randomly divided into six groups of sham-operated (Sham), ovariectomized control (OVX), ovariectomized and given oestrogen (Premarin) at 64.5 μg/kg (ERT), ovariectomized and given Labisia pumila aqueous extract at 100 mg/kg (LP _aq ), ovariectomized and given Labisia pumila methanol extract at 100 mg/kg (LP _met ) and ovariectomized and given Labisia pumila ethanol extract at 100 mg/kg (LP _et ). All the treatments were given daily via oral gavages for nine weeks.

Raw powdered form of Labisia pumila var. alata (LP) extracts was supplied by Delima Jelita Herbs (Alor Setar, Kedah). It was obtained from the Labisia pumila var. alata whole plant and was grinded and dried prior to extraction. Three different extracts were prepared; aqueous, methanol and ethanol. The extraction procedures were done following the methods by Harborne (1973) [28]. To prepare the aqueous extract, samples of dried, grounded LP were extracted with a laboratory scale extractor known as Soxhlet apparatus (Buchi Labortechnik, Switzerland) in water at 100°C for 4 hours. The extraction ratio between the dried, grounded raw material and water was 1 : 10 by mass. Following extraction, the solid residue was removed by filtration and the liquid part was transferred into rotary evaporator for evaporation process. The residue was then immediately freeze dried using Buchi Spray Drier (Buchi Labortechnik, Switzerland) to produce LP powdered form. The inlet temperature of the freeze dryer was set at 180°C and the outlet was at 103°C. To prepare the methanolic and ethanolic extract of LP, samples of dried, grounded LP were extracted with Soxhlet apparatus in methanol or ethanol respectively at 100°C for 4 hours. The extraction ratio between the dried, grounded raw material and methanol or ethanol was 1 : 10 by mass. Following extraction, the solid part was removed by filtration and the liquid part was evaporated at 78°C. Each of the LP extract was dissolved in deionised water and given accordingly to their assigned groups via oral gavages at the dose of 100 mg/kg rat weight daily at 9 am for 9 weeks. Estrogen Premarin^® (Wyeth-Ayerst, Canada) tablet which contains 0.625 mg of conjugated oestrogen was crushed, dissolved in deionised water and given via oral gavage at the dose of 64.5 μg/kg rat weight daily at 9 am for 9 weeks.

Rats were euthanized upon completion of their treatments. Femora were dissected and cleaned from all soft tissues. They were then stored in 10% formalin solution prior to analysis. Then, microarchitectural bone parameters were assessed using in-vitro micro-CT (μCT80 scanner, Scanco Medical, Switzerland). Before scanning, femora were placed in a sample holder which can fit up to 8 femora per scanning and the measurement protocols were created to define parameters such as source of energy, intensity and filter. The source of energy and intensity selected were 70 kVp and 114 μA respectively. The filter used was 0.5 mm Al. High resolution of 10 μm was set in order to obtain the best images. The trabecular bone parameters were obtained from the distal end of the femur. Number of slices were set at about 200 slices and the region of interest (ROI) chosen was at the metaphyseal area located approximately 1.5 mm below the epiphyseal growth plate, extending 2.0 mm towards the proximal direction. The selected region is rich in blood supply and has high bone turnover activity. Trabecular bone was chosen because it has a greater surface-to-volume ratio and metabolically more active than the cortical bone [29]. This analysis involved the 3D morphometric evaluation comprising of both metric and non-metric bone structural parameters. The metric structural parameters were similar to histomorphometry analysis which included bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N). The non-metric parameters measured were connectivity density (Conn.dens), structural model index (SMI) and degree of anisotropy (DA).

3 Results

Figure 1 shows three dimensional (3D) images of bone microarchitecture of all groups, which were acquired using a Scanco Medical μCT 80 scanner equipped with a visualization software that perfoms a sophisticated 3D rendering of large data sets using high-quality ray-tracing algorithms. The bone of sham and LP _aq groups showed the densest microarchitecture with minimal trabecular separation, followed by ERT, LP _et and LP _met . The bone of OVX group showed the most porous microarchitecture with maximal trabecular separation. Trabecular separation and thickness can also be indicated by colour coding, ranges from blue to red (lowest to highest).

Generally, the Sham and all treated groups showed higher Bone volume fraction (BV/TV) values than the OVX group. However, only the Sham and LP _aq groups showed significant differences when compared to the OVX group (Fig. 2). Figure 3 also shows that there were no significant differences in TbTh for all groups, while Fig. 4 shows that the Tb.Sp of the Sham group and all the treated groups (ERT, LP _aq , LP _met and LP _et ) were significantly lower than the OVX group. Next, Fig. 5 shows that the Tb.N of the Sham, ERT and LP_aq groups were significantly higher than the OVX group.

After 9 weeks of treatment, the Conn.dens of the Sham, ERT, LP _aq and LP _met groups were significantly higher than the OVX group. As a result, Group LP _et showed significantly lower Conn.dens compared to the ERT and LP _aq groups (as shown in Fig. 6). Figure 7 shows that the structure model index (SMI) of the LP _aq group was significantly lower than the OVX group, while the SMI value of the LP _met group was significantly higher than the LP _aq group. Last, the OVX group showed a trend of higher degree of anisotropy (DA) value than all the other groups albeit not significant. There were no significant differences in DA values of all the groups (as shown in Fig. 8).

4 Discussion

Postmenopausal women are prone to osteoporosis, which is a skeletal disease characterized by low bone mass and microarchitectural deterioration with a consequent increase in bone fragility and susceptibility to fracture. According to the Study of Osteoporotic Fractures, 28% of hip fractures, 25% of vertebral fractures, and 13% of all fractures occurred in osteoporotic women [30]. These have elicited a devastating effect in terms of higher medical expenditures and mortality. Hip fractures in postmenopausal women accounted to approximately 25% increase in the mortality rate within the first year of incident [31]. This serious health issue has led to many discoveries on pharmacological interventions on postmenopausal osteoporosis.

In recent years, a herb known as Labisia pumila (LP) was found to possess estrogen-like effects on bone [32]. It has been used by the locals for many generations, to treat menstrual irregularities, as post-partum medication, rheumatism as well as sickness in bone. Previous studies have found that LP possesses phytoestrogenic effects which contribted to its therapeutic values on women’s health [33]. Nazrun et al. (2012) reported that LP supplementation at the dose of 17.5 mg/kg was able to increase bone formation marker and reduced bone resorption marker in ovariectomized rats [19].

In another study by Nadia et al. (2014), two doses of crude LP extract (20 mg/kg and 100 mg/kg) were given to ovariectomized rats for three, six and nine weeks treatment. It was found that the dose of 100 mg/kg for 9 weeks was the best regimen in reversing the OVX-induced bone changes [27]. Following these positive results, the present was performed to evaluate the effects of different forms of LP extracts on bone microarchitectural parameters. Previously it was reported that the bioactive compounds contributing to LP’s therapeutic values were mostly found in its aqueous, methanolic and ethnolic extracts [34, 35]. Hence, the present study was carried out to determine which of the three forms of LP extracts is the best in reversing and improving bone deteriorations induced by ovariectomy.

In the present study, micro-CT analysis was performed to evaluate the trabecular microarchitectural changes in bone of ovariectomized rats. The microCT (μCT 80 scanner, Scanco Medical) was equipped with a software which provided clear 3D images of trabecular thickness and trabecular separation, represented by coding of colors (Fig. 1). The coding ranges from blue >green >yellow >red where red represents high separation and thickness. Based on Fig. 1, the trabecular bone of the Sham group was found to be the most dense compared to other groups. Ovariectomized rats showed a massive loss in trabecular branches and high in trabecular separation. Rats treated with LP aqueous extract had comparable trabecular bone microarchitecture to the Sham group which was illustrated by a very dense, poorly separated and thick trabecular.

Micro-CT not only provides 3D images of bone microarchitecture, it also measures bone structural parameters [36]. In contrast to conventional histomorphometry which only provides information on structural parameters, micro-CT gives better insight on microarchitectural changes by providing both metric and non-metric bone parameters [37]. The metric parameters include bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N). Non-metric parameters include connectivity density (Conn.dens), degree of anisotropy (DA) and structure model index (SMI). This ability of micro-CT has overcome the limitations of the convensional 2D histomorphometry [38].

In the present study, the OVX group showed deteriorations in BV/TV, Tb.N and Tb.Sp values. The Sham and LP _aq groups showed significantly higher BV/TV compared to the OVX group. Tb.Th of all the groups showed no significant difference. The Sham and all the treated groups showed significantly lower Tb.Sp compared to the OVX group. This could also be observed in the 3D images as shown in Fig. 1. The Sham, ERT and LP _aq groups showed significantly higher Tb.N than the OVXgroup.

Conn.dens is defined as the maximum number of trabecular connections that can be broken before the structure breaks into parts [39]. Conn.dens does not only represent the connection of the trabecular branches, but it also represents the bone density. Hence, it is one of the important determinants of bone mechanical properties [40]. The Sham, ERT and LP _aq groups showed significantly higher Conn.dens compared to the OVX group. Ovariectomy has led to massive deteriorations in trabecular connectivity density, which may be contributed by the tremendous decline in estrogen. This is because estrogen deficiency has been shown to inhibit bone formation and simultaneously increase bone resorption activities [41]. This was also supported by previous studies which reported that ovariectomised rats had significantly lower bone structural parameters compared to sham-operated rats [42].

Structure model index (SMI) is another important parameter in assessing the evolution of osteoporosis. SMI quantifies the plate versus rod characteristics of trabecular bone by measuring the surface convexity based on dilation of the 3D voxel model [43, 44]. Trabecular structure changed radically from plate-like to rod-like during aging or remodeling. Plate-like trabeculae are thicker, denser and contribute to the overall quality of the bone structure. Meanwhile, rod-like trabeculae are bones that have become thinner due to loss of mineral density. SMI value of purely plate-like and rod-like bone is 0 and 3 respectively. In this study, it was shown that LP _aq had a significantly lower SMI value than OVX. This means that LP aqueous extract supplementation was able to improve trabecular branches which led to a more stabilized bone shape.

Degree of anisotropy (DA) is a measure of how highly oriented substructures are within a volume. It is calculated using mean intercept length (MIL) method which a line is sent through a 3D image volume containing binarised objects, dividing the length of line through the analysed volume [45]. Anisoptopic bone indicates that the bone structure depends on the load direction. Many studies have reported that DA is one of the important parameters to estimate fracture risk. The higher DA suggests greater anisotropy, which theoretically resulted from loss of directional trabeculae [46]. In this study, the OVX group showed a trend of higher DA value compared to other groups albeit not significantly so. This is because OVX bone is porous, hence it is prone to fracture when load is applied either longitudinally or over the bone surface. However, due to the non significant results, all groups might not be affected by loading directions. This may be due to the small size of rats’ bone.

Our findings were in accordance with previous histomorphometric studies which reported that LP supplementation was able to restore the structural bone parameters [47]. The present study provided better insights to microarchitectural bone changes, using the high resolution, non-destructive micro-CT technique. Micro-CT was able to analyse input from bone region as small as the trabeculum of a small rodent. Therefore, micro-CT could provide an alternative to the conventional 2D histomorphometric analysis of bone structural parameters. The bone histomorphometry analysis was not only destructive but also time consuming as it requires tedious serial staining of thin sections [48]. Hence, comparison between 2D histomorphometry and the high resolution 3D micro-CT showed that the advantages of micro-CT by far outweighs histomorphometry [49].

LP has long been known for its phytoestrogenic property [50, 51]. Bioactive compounds that are responsible for its phytoestrogenic property are polar in nature, hence, they are highly expressed in more polar solvent [52]. Among the three extracts of LP used in this study, aqueous extract may contain more bioactive compounds compared to other two less polar extracts. LP was reported to contain flavonoids, saponins, tanins, triterpenes and phenolic compounds [53, 54]. These compounds are able to increase the expression of osteoprotegrin (OPG), a potent anti-osteclastogenic factor which will then up-regulate osteoblasts differentiation [55]. The ability to up-regulate the osteoblast activity may be the contributing factor to the positive results seen in this current study.

Postmenopausal osteoporosis can be associated with oxidative stress following estrogen deficiency [56, 57]. Following menopause, reduction in estrogen will suppress the antioxidant defense and increase reactive oxygen species, resulting in oxidative stress. Oxidative stress up-regulates receptor activator of nuclear factor-κB ligand (RANKL), an important factor for bone resorption and down-regulates osteoprotegerin (OPG), which is important for bone formation [58, 59]. This would lead to imbalances in bone remodeling and eventually, bone loss. LP supplementation was reported to improve the bone oxidative status in ovariectomized rats [22]. It was known to possess anti-oxidative property due to the presence of flavonoids, beta-carotene, ascorbic acids and phenolic compounds [60]. The antioxidative property of LP indirectly increased bone formation and reduced bone resorption activities.

5 Conclusion

Based on the micro-CT analysis, aqueous extract of LP was shown to be the most effective extracts in reversing the damaging effects of ovariectomy. LP aqeuous extract may contain the most active ingredients to improve the bone microarchitectural parameters.

6 Disclosure

The authors declare to have no conflicts of interest whatsoever. The authors are responsible for the content and writing of this paper.

References

Elliot

Pharmacy needs tropical forests, manufacturing chemist, Nat Toxins 1 (1986), 185–196.

Kaur

, Hamid

, Ali

, Alam

M.S.

and Athar

, Antiinflammatory evaluation of alcoholic extract of galls of Quercus infectoria, J Ethnopharmacol 90(2-3) (2004), 285–292.

Whelan

A.M.

, Jurgens

T.M.

and Bowles

S.K.

, Natural health products in the prevention and treatment of osteoporosis: Systematic review of randomized controlled trials, Ann Pharmacother 40(5) (2006), 836–849.

NIH, Osteoporosis and Related Bone Disease, National Resource Centre, USA, 2011.

Ribeiro

, Blakeley

and Laryea

, Women knowledge and practices regarding treatment and prevention of osteoporosis, Health Care for Women Intl 21(4) (2000), 347–353.

WHO Scientific Group on the Assessment of Osteoporosis at Primary Health Care Level. Summary Meeting Report. Brussels, Belgium, 2004.

Ohta

, Sugimoto

, Masuda

, Komukai

, Suda

, Makita

, Takamatsu

, Horiquichi

and Nozawa

, Decreased bone mineral density associated with early menopause progresses for at least ten years: Cross-sectional comparisons between early and normal menopausal women, Bone (1996), 227–231.

Manolagas

S.C.

, O’Brien

C.A.

and Almeida

, The role of estrogen and androgen receptors in bone health and disease, Nat Rev Endocrinol 9(12) (2013), 699–712.

Santen

R.J.

, Pinkerton

, McCartney

and Petroni

G.R.

, Clinical review 121: Risk of breast cancer with progestins in combination with estrogen as hormone replacement therapy, J Clin Endocrinol Metab 86(1) (2001), 16–23.

10.

Lane

N.E.

The Osteoporosis Book: A Guide for Patients and Their Families, Oxford University Press, New York, 2001.

11.

Stevenson

, Jones

M.L.

, De Nigris

, Brewer

, Davis

and Oakley

, A systematic review and economic evaluation of alendronate, etidronate, risedronate, raloxifene and teriparatide for the prevention and treatment of postmenopausal osteoporosis, Health Technol Assess 9 (2005), 1–160.

12.

Marcea

, Jia

, Theresa

and George

, Biphosphonates for Osteoporosis- Where do we go from here? N Eng J Med 366 (2012), 2048–2051.

13.

Reiner

, Bertha

, Emmo

V.T.

and Christoph

, Biphosphonates in Medical Practice, Springer, New York, 2007.

14.

Khosla

, Burr

, Cauley

, Dempster

D.W.

, Ebeling

P.R.

, Felsenberg

, Gagel

R.F.

, Gilsanz

, Guise

, Koka

et al., Bisphosphonate-associated osteonecrosis of the jaw: Report of a Task Force of the American Society for Bone and Mineral Research, J Bone Miner Res 22(10) (2007), 1479–1491.

15.

Zakaria

and Mohd

M.A.

, Traditional Malay Medicinal Plants, 8th ed., Penerbit Fajar Bakti, Kuala Lumpur, Malaysia, 1994.

16.

Bodeker

Health and Beauty from the Rainforest: Malaysian Traditions of Ramuan, Editions Didier Millet Pty, Kuala Lumpur, Malaysia, 1999.

17.

Fasihuddin

, Rahman

A.H.

and Hasmah

, Medicinal plants used by bajau community in sabah, in: Chan

K.L.

(Eds.), Chan Trends in Traditional Medicine Research, The School ofharmaceutical Sciences, University of Science Malaysia, Penang, Malaysia, 1995, pp. 493–504.

18.

Jamal

J.A.

, Houghton

P.J.

and Milligan

S.R.

, Testing of labisia pumila for oestrogenic activity using a recombinant yeast screen, J Pharm Pharmacol 50(S9) (1998), 79.

19.

Nazrun

A.S.

, Lee

P.L.

, Norliza

, Norazlina

and Ima Nirwana

, The effects of Labisia pumila var. alata on bone markers and bone calcium in a rat model of post-menopausal osteoporosis, J Ethnopharmacol 133(2) (2011), 538–542.

20.

Husniza

Estrogenic and Androgenic Activities of Kacip Fatimah (Labisia pumila), Abstracts of Research Projects, Institute of Medical Research, Ministry of Health Malaysia, Kuala Lumpur, Malaysia, 2002.

21.

Norhaiza

, Maziah

and Hakiman

, Antioxidative properties of leaf extracts of a popular Malaysian herb, Labisia pumila, J Med Plant Res 3(4) (2009), 217–223.

22.

Nadia

M.E.

and Nazrun

A.S.

, Time and dose-dependent effects of Labisia pumila on bone oxidative status of postmenopausal osteoporosis rat model, Nutrients 6 (2014), 3288–3302.

23.

Winzenberg

and Jones

, Dual energy X-ray absorptiometry, Aust Fam Physician 40(1) (2011), 43–44.

24.

Kanis

J.A.

, Melton

L.J.

3rd , Christiansen

, Johnston

C.C.

and Khaltaev

, The diagnosis of osteoporosis, J Bone Miner Res 9(1994), 1137–1141.

25.

Anonymous, Osteoporosis prevention, diagnosis, and therapy, JAMA 285 (2001), 785–795.

26.

Odgaard

Three-dimensional methods for quantification of cancellous bone architecture, Bone 20(4) (1997), 315–328.

27.

Nadia

M.E.

, Fadhli

M.K.

, Ima Nirwana

and Nazrun

A.S.

, The fffects of Labisia pumila on postmenopausal osteoporotic rat model: Dose and time-dependent micro-CT analysis, J X-Ray Sci Technol 22(2014), 503–518.

28.

Harborne

J.B.

Photochemical Methods: A Guide to Modern Techniques of Plant Analysis. Chapman A. & Hall. London, 1973, pp. 279.

29.

Barbara

N.W.

, Imaging of arthritis and metabolic bone disease, in: Judith

E.A.

, Imaging evaluation of osteoorosis, Saunders Elsevier, Philadelphia, USA, 2009, p. 602.

30.

Wainwright

S.A.

, Marshall

L.M.

, Ensrud

K.E.

, Cauley

J.A.

, Black

D.M.

, Hiller

T.A.

, Hochberg

M.C.

, Voqt

M.T.

, Orwoll

E.S.

and for the Study of Osteoporotic Fractures Research Group, Hip fracture in women without osteoporosis, 90 (2005), 2787–2793.

31.

Anonymous, Management of osteoporosis in postmenopausal women: 2010 position statement of The North American Menopause Society, Menopause 27(1) (2010), 25–54.

32.

Melissa

P.S.W.

, Navaratnam

and Yin

C.Y.

, Phyoestrogenic property of Labisia pumila for use as an estrogen replacement therapy agent, Afr J Biotechnol 11(50) (2012), 11053–11056.

33.

Fazliana

Studies on Labisia pumila var. alata extract with phytoestrogenic effects: Impact on biological activities and gene expression; Karolinska Institutet: Stockholm, Sweden, 2010, pp. 1–4.

34.

Khairul

F.K.

, Haniza

M.N.

and Zahari

, Antioxidative properties of various extracts of Labisia pumila Kacip Fatimah, Proceeedings Of The Seminar On Medicinal And Aromatic Plants, Forest Research Institute, Malaysia, 2005, pp. 306–312.

35.

Chua

L.S.

, Abdul

, Lee

S.Y.

, Lee

C.T.

, Sarmidi

M.R.

and Abdul

, Flavonoids and phenolic acids from Labisia pumila (Kacip Fatimah), Food Chem 127(3) (2011), 1186–1192.

36.

Hsu

J.T.

, Wang

S.P.

, Huang

H.L.

, Chen

Y.J.

, Wu

and Tsai

M.T.

, The assessment of trabecular bone parameters and cortical bone strength: A comparison of micro-CT and dental cone-beam CT, J Biomech 46(15) (2013), 2611–2618.

37.

Nadia

M.E.

, Fadhli

M.K.

and Nazrun

A.S.

, Micro-CT assessments of potential anti – osteoporotic agents, Curr Drug Targets 12 (2013), 1542–1551.

38.

Muller

, Van

C.H.

and Van

D.B.

, Morphometric analysis of human bone biopsies: A quantitative structural comparison of histological sections and micro-computed tomography, Bone 23(1) (1998), 59–66.

39.

DeHoff

R.T.

, Aigeltinger

E.H.

and Craig

K.R.

, Experimental determination of the topological properties of three-dimensional microstructures, J Microsc 95 (1972), 69–91.

40.

Odgaard

and Gundersen

H.J.

, Quantification of connectivity in cancellous bone with a special emphasis on 3-D reconstruction, Bone 14 (1993), 173–182.

41.

Manolagas

S.C.

Birth and death of bone cells: Basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis, Endocr Rev 21 (2000), 115–137.

42.

Kabel

, Odgaard

, Rietbergen

B.V.

and Huiskes

, Connectivity and the elastic properties of cancellous bone, Bone 24(2) (1999), 115–120.

43.

Hildebrand

and Ruegsegger

, A new method for the model independent assessment of thickness in three dimensional images, J Microsc 185 (1997a), 67–75.

44.

Hildebrand

and Ruegsegger

, Quantification of bone microarchitecture with the structure model index, Comput Methods Biomech Biomed Engin 1 (1997b), 15–23.

45.

Van der Linden

J.C.

and Weihans

, Effects of microarchitecture on bone strength, Curr Osteoporos Rep 5(2) (2007), 56–61.

46.

Brunet-Imbault

, Lemineur

, Chappard

, Halba

and Benhamou

C.L.

, A new anisotropy index on trabecular bone radiographic images using the Fas Fourier transform, BMC Med Imaging 5(4) (2005), 1–11.

47.

Fathilah

S.N.

, Nazrun

A.S.

, Norazlina

, Norliza

and Ima

, Nirwana, Labisia pumila protects the bone of estrogen-deficient rat model. A histomorphometric study, J Ethnopharmacol 142 (2012), 294–299.

48.

Yeom

, Blanchard

, Kim

, Zunt

and Chu

T.M.

, Correlation between micro-computed tomography and histomorphometry for assessment of new bone formation in a calvarial experimental model, J Craniofac Surg 19(2) (2008), 446–452.

49.

Jiang

, Zhao

, Liao

E.Y.

, Dai

R.C.

, Wu

X.P.

and Genant

H.K.

, Application of μCT-assessment of 3D bone microstructure in preclinical and clinical studies, J Bone Miner Metab 23 (2005), 122–131.

50.

Nadia

M.E.

, Nazrun

A.S.

, Norazlina

, Isa

N.M.

, Norliza

and Ima

, Nirwana, The anti-inflammatory, phytoestrogenic and antioxidative role of labisia pumila in prevention of postmenopausal osteoporosis, Adv Pharmacol Sci (2012), Article ID 706905.

51.

Norhanisah

, Siavash

H.C.

, Chua

L.S.

and Mohammad

R.S.

, Labisia pumila: A review on its traditional, phytochemical and biological uses, World Appl Sci J 27(10) (2013), 1297–1306.

52.

Melissa

P.S.W.

, Navaratnam

and Yin

C.Y.

, Estrogenic assessment of Labisia pumila extracts using a human endothelial cell line, Int J Pharm Pharm Sci 5(2) (2013), 448–452.

53.

Al-Wahabi

, Nazaimoon

W.M.

, Farihah

H.S.

and Azian

A.L.

, Effect of ovariectomy, Labisia pumila var alata treatment and estrogen replacement therapy on the morphology of adipose tissue in ovariectomized Sprague Dawley rats, J Med 1(1) (2007), 1–7.

54.

Avula

, Wang

Y.H.

, Ali

, Smillie

T.J.

and Khan

I.A.

, Quantitative determination of triperpene saponins and alkenated-phenolics from Labisia pumila by LCUV/ELSD method and confirmation by LC-ESI-TOF, Planta Medica 76 (2010), 25.

55.

Sharan

, Siddiqui

J.A.

, Swarnkar

, Maurya

and Chattopadhyay

, Role of phytochemicals in the prevention of menopausal bone loss: Evidence from in vitro and in vivo, human interventional and pharmacokinetic studies, Curr Med Chem 16 (2009), 1138–1157.

56.

Almeida

, Han

, Martin-Millan

, Plotkin

L.I.

, Stewart

S.A.

, Roberson

P.K.

, Kousteni

, O’Brien

C.A.

, Bellido

, Parfitt

A.M.

, Weinstein

R.S.

, Jilka

R.L.

and Manolagas

S.C.

, Skeletal involution by age-associated oxidative stress and its acceleration by loss of sex steroids, J Biol Chem 282 (2007), 27285–27297.

57.

Sendur

O.F.

, Turan

, Tastaban

and Serter

, Antioxidant status in patients with osteoporosis: A controlled study, Joint Bone Spine 76(5) (2009), 514–518.

58.

Maziere

, Salle

, Gomila

and Maziere

J.C.

, Oxidized low density lipoprotein increases RANKL level in human vascular cells. Involvement of oxidative stress, Biochem Biophys Res Commun 440(2) (2013), 295–199.

59.

Swarna

, Dinesh

, Lavanya

B.S.

and Naidu

V.G.M.

, Interplay of RANKL in oxidative stress induced bone loss, Curr Trends Pharma Sci 1 (2014), 12–16.

60.

Lee

S.C.

, Norliza

A.L.

, Sze

Y.L.

, Chew

T.L.

, Mohamad

R.S.

and Ramlan

A.A.

, Flavonoids and 463 phenolic acids from Labisia pumila (Kacip Fatimah), Food Chem 127 (2011), 1186–1192.