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Anti-osteoporotic constituents from Indian medicinal plants.

ABSTRACT

The objective of this study was to determine the in vitro osteogenic activities of selected medicinal plants used traditionally in India. The compounds isolated from three plants viz. Allophylus serratus, Cissus quadrangularis and Vitex negundo were evaluated for their in vitro osteogenic activities. Primary cultures of osteoblasts were used to determine the effects of these components on osteoblast functions. Five (4, 6, 9, 12 and 14) of the fourteen compounds isolated led to increase in osteoblast differentiation and mineralization. These findings lend support to the use of Allophylus serratus, Cissus quadrangularis and Vitex negundo in traditional medicine.

ARTICLE INFO

Keywords:

Allophylus serratus

Cissus quadrangularis

Vitex negundo

Osteogenic

Osteoblast

Differentiation

[C]2010 Elsevier GmbH. All rights reserved.

Introduction

Osteoporosis, a silent epidemic, is characterized by decreased bone mineral density (BMD), increased risk of fractures and is associated with micro architectural deterioration of bone tissue that results in low bone mass. It is a major age-related health problem that threatens the mobility and increases mortality in elderly (Kung 2004). Osteoporosis is especially common in women after menopause as a result of reduced estrogen level (Lai et al. 2007). Although postmenopausal osteoporosis is epidemic, the choices of treatment are limited. For women at menopause, a widely used timely estrogen hormone replacement therapy (HRT) has been discontinued due to cardio-vascular side effects (Yusuf and Anand 2003). All currently available, approved therapies for osteoporosis are anti-resorptive. Anti-resorptive agents reduce fracture risk that is not more than 50% of the baseline risk. However, despite their great value, the anti-resorptive agents are not generally associated with increases in bone mass to any significant extent. Increase in bone mass requires discovery of agents that will enhance osteoblast functions - popularly ascribed as bone anabolic/osteogenic therapy by which bone formation is directly stimulated. The most popular anabolic therapy currently available includes PTH. PTH enhances the recruitment of pre-osteoblasts from marrow stromal cells, induces the maturation of lining osteoblasts and also reduces osteoblast apoptosis (Rosen and Bilezikian 2001). Recombinant human PTH (Teriparatide) has been approved in USA as monotherapy for the treatment of postmenopausal women with osteoporosis and men with low bone density and osteoporosis. Intermittent PTH has demonstrated increase in cancellous bone mass at several sites, but at the cost of cortical bone (Hodsman et al. 1999; Linkhart and Mohan 1989; Watson et al. 1995; Jilka et al. 1999; Baumann and Wronski 1995; Chang et al. 1995; Rosen and Donahue 1998). Teriparatide (rhPTH [1-34]) is effective for treating osteoporosis, but it requires daily injection and concerns about osteosarcoma has led to a recommendation of a maximum 2 years treatment course (Cappuzzo and Delafuente 2004). Very recently, strontium ranelate (Protelos) has been introduced as dual action agent that enhances osteoblast function as well as inhibits osteoclast function. Since, strontium acts by activating calcium-sensing receptor (a cell surface G-protein-coupled receptor) which acutely regulates PTH secretion, strontium could pose the risk of adversely modulating systemic calcium homeostasis (Brown 2003).

Natural products for the management of osteoporosis are largely phytoestrogens (Anderson and Garner 1998; Fitzpatrick 1999) which include isoflavones, lignins, flavonoids, and coumestans that share structural and functional similarities with naturally occurring or synthetic estrogens. Phytoestrogens exhibit estrogen-like effects at various reproductive and non-reproductive tissues (Anderson and Garner 1998; Fitzpatrick 1999). Traditional medicines have been re-evaluated by clinicians (Phillipson and Anderson 1989), because these medicines have fewer side effects and because they are more suitable for long-term use as compared to chemically synthesized medicines. Most of plant-derived medicines have been developed on the basis of traditional knowledge in health care and in many cases; there is a correlation between the indications of pure substances and those of respective crude extracts used in traditional medicine (Farnsworth et al. 1985). Allophylus serratus, Cissus quadrangularis and Vitex negundo were collected, due to their traditional uses, in a general biological screening program of the institute for the identification of osteogenic agent. We evaluated osteogenic activities of the pure compounds isolated from three species viz. A. serratus, C. quadrangularis and V. negundo.

Allophylus serratus (Roxb) Kurz, family Sapindaceae is astringent, better, sweet, anti-inflammatory, vulnerary, digestive, carminative and constipating. It is useful in bone fractures, dislocations, inflammations, ulcers, wounds, dyspepsia, anorexia, diarrhoea. The fruits are sweet, cooling and nourishing tonic (Dharmani et al. 2005). Traditional healers use this plant for promoting fracture healing but the effects on osteoporosis and total osteo-health and related disorders have not been scientifically explored. So far this plant has not been chemically investigated. Five compounds (1-5) (Fig. 1) quercetin (1) (Boligon et al. 2009), pinitol (2) (Dominguez et al. 1975), luteolin-7-O-[beta]-D-glucopyranoside (3) (Ulubelen et al. 1971), rutin (4) (Boligon et al. 2009) and apigenin-4'-O-[beta]-D-glucoside (5) (Ghanta et al. 2007) were isolated from Allophylus serratus. All these compounds are the first report from this plant.

[FIGURE 1 OMITTED]

Cissus quadrangularis is the most common species belonging to the family Vitaceae, commonly known as Hadjod or bonesetter in Hindi due to its bone fracture healing property (Prasad and Udupa 1964). But so far its chemical constituents (6-8) (Singh et al. 2007) have not been evaluated for this activity.

Vitex negundo (Linn.) family Verbenaceae is a large shrub grown throughout the India. It is one of the common plants used in traditional medicine and reported to have variety of biological activities. The preparation of leaves is used in catarrhal fever and applied to sinuses and scrofulous sores. Aqueous extract and oil of seeds possess anti-oxidant and anti-inflammatory property. Anti-genotoxic effects were reported from the leaves of V. negundo in combination with other constituents. Flavonoid-rich fraction of the seeds showed anti-fertility effect in adult dogs. Plant also exhibited CNS depressant activity and antihistamine release property. Flavonoids, iridoids, terpenes, and steroids are the major classes of compounds isolated from this plant. Six compounds (9-14) (Sathiamoorthy et al. 2007) were isolated from this plant.

The isolated compounds 1-14 were evaluated for their in vitro antiresorbing and osteogenic activities using chick fetal bone chase culture assay. Compounds 4, 6, 9, 12 and 14 showed significant osteogenic activity.

Materials and methods

Materials

The selected parts of the plant Allophylus serratus leaves, Cissus quadrangularis stem and Vitex negundo leaves were collected, and allowed to dry in shadow. Collection and identification of plants were made by botany division of Central Drug Research Institute Lucknow, Voucher specimens are preserved in the herbarium of the institute.

Optical rotations were measured on a Perkin-Elmer model 241 digital polarimeter. UV spectra were obtained on a Perkin-Elmer [lambda]-15 UV spectrophotometer. IR spectra were recorded on a Perkin-Elmer RX-1 spectrophotometer using KBr pellets. (1) H and (13) C NMR spectra were recorded on a Bruker DRX 300 MHz NMR spectrometer. ESMS on an Advantage Max LCQ Thermo-Finnigan mass spectrometer and FABMS were carried out on a JEOL SX 102/DA-6000 mass spectrometer. CC was performed using silica gel (230-400 mesh). TLC was carried out on precoated silica gel Plates 60 F254 or RP-18 F254 plates (Merck). Spots were visualized by UV light or by spraying with [H.sub.2][SO.sub.4]-MeOH or anisaldehyde-[H.sub.2][SO.sub.4] and vanillin-[H.sub.2][SO.sub.4] reagents. HPLC was performed using a Water 515 (Millford, USA) pump, photodiode array detector which was set at 220 nm for Allophylus serratus, 279 nm for Cissus quadrangu-laris and 254 nm for Vitex negundo, automatic 717 plus injector, and Millennium Empower 2 software. All the reagents used were HPLC grade, and samples filtered over regenerated cellulose membrane [0.45-mm poredia-meter (Schleicher & Schuell, Dassel, Germany)].

Preparation of extracts and isolation of compounds

Powdered leaves of Allophylus serratus (10 kg), Vitex negundo (5 kg) and Cissus quadrangularis (5 kg), were separately extracted with in a glass percolator with ethanol (251) and allowed to stand at room temperature overnight. The percolate was collected. This process of extraction was repeated four times. The combined extract was filtered, concentrated at 45[degrees]C under vacuum, afforded ethanol extracts of A serratus (900g), V. negundo (800 g), C. quadrangularis (500 g).

These ethanol extracts were suspended in hexane to separate oily impurities and then extracted with n-butanol saturated with water (500 ml 7x). The n-butanol fraction of each plant was subjected to silica gel CC.

A portion of the n-butanol-soluble fraction (200 g) of A. serratus was subjected to column chromatography over silica gel (230-400 mesh), eluted with a gradient solvent system composed of [CHCl.sub.3]-MeOH (95:05) to MeOH-[H.sub.2]O (95:05) and afforded six major fractions (F-1 to F-6). Column chromatography of F-2 over silica gel (60-120 mesh), using ethyl acetate saturated with water as mobile phase, yielded compound 1 (250 mg). Further purification of fraction F-3 over silica gel (230-400 mesh), using a gradient solvent system of [CHCl.sub.3]-MeOH (95-50%), afforded seven further fractions (F-7 to F-13) on the basis of their TLC profiles. Purification of fraction F-7 over silica gel (60-120 mesh) using [CHCl.sub.3]-MeOH (95:5) as eluting system afforded 2 (40 mg). Compound 4 (50mg) was obtained as an amorphous solid from fraction F-4 at room temperature. Fraction F-8, containing a mixture of two compounds, was purified over Sephadex LH-20, eluted with water-methanol (1:1) and yielded compound 3 (45 mg) and 5 (30 mg).

The isolated compounds (Fig. 1) from C. quadrangularis were identified as 6'-O-trans-cinnamoyl-catalpol (6), 6-0-[2, 3-dimethoxy]-t-cinnamoyl-catalpol (7) and 6-O-m-methoxy-benzoyl catalpol (8) (Singh et al. 2007).

The isolated compounds (Fig. 2) from V. negundo were identified as, agnuside (9), 5'-hydroxy-3',4',3,6,7-pentamethoxyflavone (10), 4',5,7-trihydroxy-3'-O-[beta]-D-glucuronic acid-6"-methyl ester (11), negundoside (12), iso-orientin (13) and luteolin (14) were characterized by comparing their spectroscopic data with those reported in the literature (Sathiamoorthy et al. 2007). The purity of all these compounds (1-14) was more than 98.0% tested by RP-HPLC.

[FIGURE 2 OMITTED]

HPLC-analysis

The ethanolic extract of all plants were also analyzed by HPLC. The chromatographic separation was carried out on a Thermo hypurity 5[micro] C8 column (4.6 mm x 150 mm i.d.; 5 [micro]m particle size) and eluted with the mixture of water: acetonitrile (80:20) in case of Allophylus serrates and in case of Cissus quadrangularis and Vitex negundo eluted with the mixture of water: acetonitrile (60:40). The isocratic program was set for 60min in all three cases. The flow rate was 0.5 ml/min and sample concentration was 5 mg/ml and injected 5 [micro]l for analysis. The chromatograms showed a good separation profile for the compounds. The peaks for compounds 1-14 depicted in chromatograms (Fig. 3) were identified by co-chromatography and comparison of retention time with isolated compounds.

[FIGURE 3 OMITTED]

Biological assay

Culture of calvarial osteoblasts

Rat calvarial osteoblasts were obtained following our previously published protocol of sequential digestion (Trivedi et al. 2008). Briefly, calvaria from ten to twelve 1 - to 2-day-old Sprague-Dawley rats were pooled. After surgical isolation from the skull and the removal of sutures and adherent mesenchymal tissues, calvaria were subjected to five sequential (10-15 min) digestions at 37 C in a solution containing 0.1% dispase and 0.1% collagenase P. Cells released from the second to fifth digestions were collected, centrifuged, resuspended, and plated in T-25 [cm.sup.2] flasks in [alpha]-MEM containing 10% FCS and 1% penicillin/streptomycin (complete growth medium).

Osteoblast differentiation

For Alkaline phosphatase (ALP) activity measurement, osteoblasts at ~80% confluence were trypsinized and 2 x [10.sup.3] cells/well were seeded in 96-well plates. Cells were treated with different compounds at varying concentrations for 48 h in [alpha]-MEM supplemented with 5% FCS, 10 mM [beta]-glycerophosphate, 50 [micro]g/ml ascorbic acid and 1% penicillin/streptomycin (osteoblast differentiation medium). At the end of incubation period, total ALP activity was measured using p-nitrophenylphosphate (PNPP) as substrate and quantitated colorimetrically at 405 nm (Ishizuya et al. 1997). Bone morphogenetic protein-2 (BMP-2) is a known stimulator of osteoblast differentiation. We used BMP-2 (100 ng/ml) as positive control to compare the differentiation promoting effects of compounds (Dijke 2006).

Mineralization of calvarial osteoblast cells

For mineralization studies, calvarial derived osteoblast cells were cultured in medium consisting of [alpha]-MEM, supplemented with 10% fetal bovine serum, 50 [micro]g/ml ascorbic acid, and 10 mM [beta]-glycerophosphate. Cells were cultured with and without compounds for 7 days at 37 C in a humidified atmosphere of 5% [CO.sub.2] and 95% air, and the medium was changed every 48 h. After 7 days, the attached cells were fixed in 4% formaldehyde for 20 min at room temperature and rinsed once in PBS. After fixation, the specimens were processed for staining with 40 mM Alizarin Red-S, which stains areas rich in nascent calcium. BMP2 was used as a positive control.

For quantification of staining, 800 [micro]l of 10% (v/v) acetic acid was added to each well, and plates were incubated at room temperature for 30 min with shaking. The monolayer, now loosely attached to the plate, was then scraped from the plate with a cell scraper and transferred with 10% (v/v) acetic acid to a 1.5-ml tube. After vortexing for 30s, the slurry was overlaid with 500 [micro]l mineral oil (Sigma-Aldrich), heated to exactly 85 C for 10 min, and transferred to ice for 5 min. The slurry was then centrifuged at 20,000 x g for 15 min and 500 [micro]l of the supernatant was removed to a new tube. Then 200 [micro]l of 10% (v/v) ammonium hydroxide was added to neutralize the acid. In some cases, the pH was measured at this point to ensure that it was between 4.1 and 4.5. OD (405 nm) of 150 [micro]l aliquots of the supernatant were measured in 96-well format using opaque-walled, transparent-bottomed plates (Maurya et al. 2009).

Results

Stimulatory activity of compounds on osteoblastic ALP activity

Alkaline phosphatase (ALP), which is one of the important markers of osteoblast differentiation, was measured spectropho-tometrically. Table 1 and Fig. 4 show that in the range of [10.sup.-12] to [10.sup.-8] M, five compounds (4, 6, 9, 12 and 14) increased ALP activity. BMP-2 was used as a positive control. Most importantly these compounds increased ALP activity at concentrations of ~ 1000-fold less than that of daidzein.

[FIGURE 4 OMITTED]
Table 1
Effects of compounds from Allophylus serratus, Cissus quadrangularis
and Vitex negundo on osteoblast.

     Compounds         Concentration  Osteoblastic ALP activity
                                         Absorbance (405 nm)

Compound 4 from        [10.sup.-8]M   0.784 [+ or -] 0.08 **
Allophylus serratus    [10.sup.-10]M  0.651 [+ or -] 0.07
                       [10.sup.12]M   0.925 [+ or -] 0.10 ***

BMP-2                  l00ng/ml       1.276 [+ or -] 0.09 ***
Control                               0.587 [+ or -] 0.07

Compound 6 from        [10.sup.-8]M   0.093 [+ or -] 0.007
Cissus quadrangularis  [10.sup.-10]M  0.123 [+ or -] 0.006 **
                       [10.sup.-12]M  0.125 [+ or -] 0.005 **

BMP-2                  l00ng/ml       0.148 [+ or -] 0.006 ***
Control                               0.068 [+ or -] 0.010

Compound 9 from        [10.sup.-8]M   0.855 [+ or -] 0.12 *
Vitex negundo          [10.sup.-10]M  0.876 [+ or -] 0.12 *
                       [10.sup.-12]M  1.158 [+ or -] 0.16 *

BMP-2                  l00ng/ml       1.276 [+ or -] 0.09 ***
Control                               0.587 [+ or -] 0.07

Compound 12 from       [10.sup.-8]M   0.104 [+ or -] 0.007 *
Vitex negundo          [10.sup.-10]M  0.105 [+ or -] 0.008 *
                       [10.sup.-12]M  0.123 [+ or -] 0.008 **

BMP-2                  l00ng/ml       0.148 [+ or -] 0.006 ***
Control                               0.068 [+ or -] 0.010

Compound 14 from       [10.sup.-8]M   1.061 [+ or -] 0.083 **
Vitex negundo          [10.sup.-10]M  0.968 [+ or -] 0.125 **

BMP-2                  [10.sup.-12]M  0.874 [+ or -] 0.082 **
Control                100ng/ml       1.276 [+ or -] 0.09 ***
                                      0.587 [+ or -] 0.07

* P < 0.05 compared with control.
** P < 0.01 compared with control.
*** P < 0.001 compared with control.


Stimulatory activity of compounds on formation of mineralization nodules

These compounds were further evaluated for the formation of mineralized nodules at effective concentrations by alizarin staining. Alizarin Red S is an anthraquinone derivative used to identify calcium. Calcium forms an alizarin red S-calcium complex in a chelation process and the end product is birefringent. Osteoblast cells were treated with or without compounds for seven days following which cells were stained with alizarin for formation of mineralized nodules. Subsequently, Alizarin stains was extracted and mean O.D. was determined at 405 nm (Fig. 5). All the five compounds (4, 6, 9, 12 and 14) enhanced ALP activity also stimulated the formation of mineralized nodules. BMP was used as positive control.

[FIGURE 5 OMITTED]

Discussion

Estrogen deficiency is a major risk factor for osteoporosis in postmenopausal women (Dempster and Lindsay 1993; Mundy 1993; Tolstoi and Levin 1992). Though hormone replacement therapy (HRT) lowers the risk for coronary heart disease and osteoporosis but it is associated with fears of increased risk of certain types of cancer like endometrial and breast cancers (Johannes et al. 1994). Phytoestrogens are compounds that have weak estrogenlike properties and exert their effect in a SERM-like manner (Brzezinski and Debi 1999). Phytoestrogens are non-steroidal plant compounds of diverse structures found in many fruits, vegetables, and grains. Substantial body of data generated from cultured bone cells and rat models of osteoporosis supports a significant bone conserving effect of phytoestrogens (Sharan et al. 2009). Observational studies have found a lower prevalence of hip fracture rates among Asian women (Chiechi et al. 1999) where diets are typically high in phytoestrogens. As a result of these studies, a great deal of interest has been generated in the United States and Europe about the health benefits of phytoestrogens.

In this study Allophylus serratus, Cissus quadrangularis and Vitex negundo were collected, owing to their traditional uses for the identification of osteogenic agent. Osteogenic activities of the pure compounds isolated from these three species were evaluated. In vitro, five of the fourteen compounds isolated from Allophylus serratus, Vitex negundo and Cissus quadrangularis increased ALP activity with varying potency. These were compound 4 from Allophylus serratus (maximally active at [10.sup.-12] M), compound 9, 12 and 14 from Vitex negundo (maximally active at [10.sup.-12] M) and compound 6 from Cissus quadrangularis (maximally active at [10.sup.-10]M). In addition, all these compounds increased osteoblast mineralization as assessed by alizarin extraction. Moreover the osteogenic effects of these compounds were observed at concentrations of ~1000-fold less than that reported for isoflavones like daidzein (Yamaguchi and Sugimoto 2000). Based on these data, we suggest that these pure compounds exhibit significant osteogenic activity and could be pursued further for use in menopausal osteoporosis.

Acknowledgements

The authors Manmeet Kumar, Preeti Rawat, Preeti Dixit and Devendra Mishra are thankful to the CSIR and Abnish K. Gau-tam and Rashmi Pandey are thankful to UGC-CSIR, New Delhi for Research Fellowship. This investigation received financial assistant from Department of Biotechnology, New Delhi, India.

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* Corresponding author. Tel: +91 522 2612411 18x4235; fax: +91 522 2623405/2623938/2629504.

E-mail address: mauryarakesh@rediffmail.com (R. Maurya).

(1) CDRI Comm. No. 7399.

0944-7113/$ - see front matter [C] 2010 Elsevier GmbH. All rights reserved.

Manmeet Kumar (a), Preeti Rawat (a), Preeti Dixit (a), Devendra Mishra (a), Abnish K. Gautam (b), Rashmi Pandey (b), Divya Singh (b), Naibedya Chattopadhyay (b), Rakesh Maurya (a), *

(a) Medicinal and Process Chemistry Division, Central Drug Research Institute, (1) CSIR, M. G. Road, Lucknow 226 001, Uttar Pradesh, India

(b) Endocrinology Division, Central Drug Research Institute, CSIR, M. C. Road, Lucknow 226 001, Uttar Pradesh, India

doi:10.1016/j.phymed.2010.03.014
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Author:Kumar, Manmeet; Rawat, Preeti; Dixit, Preeti; Mishra, Devendra; Gautam, Abnish K.; Pandey, Rashmi; S
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:Nov 1, 2010
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