SCREENING FOR VIETNAMESE PLANT EXTRACTS WITH POTENTIAL BENEFIT FOR ANTI-OSTEOCLASTOGENESIS

Bone's homeostasis is only achieved when there is a balance between bone formation and bone resorption. A metabolic disorder of bone-resorbing osteoclasts can lead to osteoporosis. Long-term use of anti-osteoporosis drugs can lead to undesirable side effects so traditional herbs can be a potential source of alternative medicine. In the present study, forty-one Vietnamese plants (seventy methanol extracts) were screened for osteoclastogenesis inhibitory activities on RAW264.7 mouse macrophage cells. For the first time, 29 extracts from 24 species: Thunbergia grandiflora (Roxb. ex Rottl.) Roxb., Saurauia nepaulensis DC, Choerospondias axillaris (Roxb.) Burtt., Xylopia vielana Pierre, Ilex godajam Colebr. ex Hook.f., Ilex kaushue S.Y. Hu, Pothos repens (Lour.) Druce, Carallia brachiate (Lour.) Merr., Oroxylum indicum (L.) Kurz, Bombax ceiba L., Stixis fasciculata (King) Gagn., Viburnum lutescens Blume, Garcinia multiflora Champ. ex Benth., Garcinia oblongifolia Champ. ex Benth., Amischlotype mollissima (Blume) Hassk., Trichosanthes rubriflos Thorel ex Cayla, Dichapetalum longepetalum (Turcz) Engl., Cleistanthus tonkinensis Jabl., Homonoia riparia Lour., Macaranga henryi (Pax & K.Hoffm.) Rehder, Croton tiglium L., Chaetocarpus castanocarpus (Roxb.) Thwaites, Phyllanthus emblica L., Hydnocarpus hainanensis (Merr.) Sleum, Hydnocarpus anthelminhicus Pierre, Illigera rhodantha Hance, Cratoxylum cochinchinense (Lour.) Blume, Machilus thunbergii Sieb. & Zucc., Saraca dives Pierre, Piper longum L., Nageia fleuryi (Hickel) de Laub., Gouania javanica Miq., Psychotria rubra (Lour.) Poir., Mussaenda pubescens W. T. Aiton, Mycetia balansae Drake, Clausena excavata Burm.f., Zanthoxylum avicennae (Lam.) DC., Turpinia montana (Blume) Kurz, Helicteres hirsuta Lour., Symplocos cambodiana (Pierre) Hallier f., Eurya cerasifolia (D.Don) Kobuski, Pellionia repens (Lour.) Merr. showed potential as effective inhibitors of osteoclastogenesis.


INTRODUCTION
Bone is a connective tissue that mechanically supports movement and also acts as a metabolic organ with large reserves of calcium and phosphate. Bone remodeling is an important physiological process in which old bones are degraded and replaced, performed by a combination action of 4 types of bone cells: bone lining cells, osteocytes, osteoclasts, and osteoblasts. The bone surface is made up of bone lining cells and the osteocytes are the main mechanical cells that play a role in controlling the initial stage of bone remodeling. Osteoclasts are large multinucleated cells that dissolve the bone whereas osteoblasts are the bone-forming cells [1]. The balance of bone formation and resorption processes play a key role in bone development and homeostasis. The imbalance between bone forming cells and bone resorbing cells may lead to abnormal bone conditions: too much bone formation (osteopetrosis) or too little bone (osteoporosis) [2]. Osteoporosis is one of the major bone health problems today, and as a result of estrogen deficiency after menopause, it occurs most commonly in women. Approximately 50 % of women suffer from osteoporotic fractures throughout their lifetime [3]. Osteoclastogenesis is mainly regulated by two essential cytokines: Macrophage colonystimulation factor (M-CSF) and Receptor activator of NF-κB (RANK) ligand (RANKL promotes the proliferation and survival of osteoclast precursors while RANKL participates in the osteoclastogenesis by interacting with RANK on the precursor's surface and driving the osteoclast precursor into osteoclast [4]. Drugs that target the inhibition of the formation or activity of osteoclasts are valuable for osteoporosis treatment [5]. However, the long-term use of medications such as bisphosphonates and estrogen therapy results in several adverse effects [6,7]. Because of patients' concerns, it is essential to search for new anti-osteoporotic agents. In developing countries, medicinal plants may bring important sources of health care. Viet Nam has high biodiversity as a source of medicine with more than 12,000 species. In this study, forty-one Vietnamese plants (seventy methanol extracts) were collected from the Me Linh station for Biodiversity and screened for the inhibitory activities of osteoclastogenesis in RAW264.7 murine macrophages.

Plant materials
A total of 41 plants were randomly collected in Me Linh Station for Biodiversity, Vinh Phuc, Viet Nam in Spring 2019. The plant samples were identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology. A voucher of specimens was deposited at the Department of Life Sciences, University of Science and Technology of Hanoi.

Preparation of extracts
Different parts of the collected plants including branches (B), leaves (L), roots (R), stems (S), fruits (Fr), and flowers (F) were classified. After drying by oven and cutting into pieces, the samples were extracted with the ratio sample:MeOH 1 : 1 (w/v) for 20 minutes, followed by15 minutes in an ultrasonicator. This process was repeated 3 times, the collected solvent was dried by the vacuum evaporator and the corresponding residues were concentrated. The crude extracts were stored at -10 °C until assayed.

Assay for in vitro differentiation of osteoclasts
RAW264.7 cells were seeded at a density of 5 × 10 4 cells/ml onto 96-well plates. For osteoclast differentiation, the cells were stimulated with receptor activator of NF-κB ligand (RANKL, 100 ng/ml; R&D Systems, Minneapolis, MN, USA) in the presence or absence of the extracts at the concentration of 25 µg/ml for 4 days. The extracts and culture media were replaced every two days for four days.

Tartrate-resistant acid phosphatase (TRAP) staining assay
Histochemical staining of the cells was performed with an acid phosphatase leukocyte kit (Sigma-Aldrich; EMD Millipore, Billerica, MA, USA). The assayed cells were fixed with 3.7 % formalin for 15 min, permeabilized with 0.1 % Triton X-100. TRAP staining was performed following the instructions of the manufacturer. TRAP-positive multinucleated cells with more than three nuclei were defined as osteoclasts. The cells were photographed under microscopy model CKX-53 (Olympus, Tokyo, Japan).
The percentage of cell viability was calculated using the formula: %Cell viability = AS/AC × 100, %Inhibition = 100 % -%Cell viability, where: AS is the absorbance of sample, AC is the absorbance of negative control.

Statistical analysis
Data are expressed as mean ± standard error (SE). Statistical significance was assessed by Student's t test. In all analyses, P values of less than 0.05 were considered statistically significant.

RESULTS AND DISCUSSION
In the present study, 41 plants ( Fig. 1) divided into 70 extracts were screened (Table 1) for the inhibition of osteoclastogenesis in RAW264.7 murine macrophages. The cell line has proven to be an important primary osteoclast precursor for in vitro studies of osteoclast formation. By interacting with RANK on the RAW264.7 cell's surface, RANKL promotes the differentiation of osteoclast precursors into osteoclasts [8]. At the concentration of 25 µg/ml, the negative effects on osteoclastogenesis inhibition were recorded in 23 samples, 17 methanol extracts revealed the cytotoxicity effects (data not shown), and 29 extracts inhibited the osteoclast differentiation of RAW264.7 cells without significant impact on the cell survival (Table 2).   Among 29 positive samples, 23 samples exhibited very strong inhibitory effects with more than 75 % osteoclastogenesis inhibition, and 6 samples displayed the moderate effect with the suppression of osteoclastogenesis by 50 -75 % compared to the control (Fig. 2).
According to our results, both leaf and stem extracts of Phyllanthus emblica L. (S39 and S40) have osteoclastogenesis inhibitory effects in RAW264.7 cells (66.5 % and 97.5 % at 25 µg/ml, respectively). The previous study showed geraniin and quercetin were two of the chief components of the fruits of this plant [9]. Geraniin has been shown effective in preventing osteoporosis whereas quercetin is a potent inhibitor of osteoclastogenesis. The ability of quercetin in increasing the new bone formation was also recorded when this compound was presented in a collagen matrix [10 -12]. Belonging to the Euphorbiaceae family with high inhibitory effects like P. emblica L., but based on our knowledge, there is no information about the chemical components or the effect on the bone disease on C. castanocarpus (Roxb.) Thwaites (S38).
Piper longum L., known as long pepper, has not only been used as an important spice but also as an important ingredient in traditional medicine. In this study, the methanol extracts from Piper longum L. leaves and stems (S51 and S52) showed anti-osteoclastogenesis potential with a very strong effect of more than 95% osteoclastogensis inhibition (Fig. 1). Piperinean alkaloid presented in this plant was shown to inhibit the osteoclast formation in RAW264.7 macrophages and human CD14+ monocytes by suppressing the p38/NFATc1/c-Fos signaling axis [13].
Psychotria (Rubiaceae) is a genus potentially used in pharmacology due to the discovery of many active compounds. The phytochemical components of Psychotria rubra were well investigated but no study about anti-osteoclastogenic activity of these metabolites was recorded [14]. Our results indicated that P. rubra stems and leaves (S57 and S59) may be a potential source for the discovery of anti-osteoclastogenetic agents. Belong to the same family Rubiaceae as P. rubra, however, the information about the chemical contents Mussaenda pubescens W. T. Aiton (S59 and S60) still has been undiscovered. Previous study showed that lupeol -one of the chief components of Pellionia repens (Lour.) Merr (S70) [15], Cratoxylum cochinchinense (Lour.) Blume (S47) [16], and Bombax ceiba L. (S16) [17] exhibits strong inhibitory effects on osteoclastogenesis [18]. Machilus thunbergii Sieb. & Zucc. (S48) has been used in folk medicine for a long period. Machilin A, one of the bioactive components of this plant, has been proven to be capable of stimulating osteoblast differentiation in MC3T3-E1 cells [19,20]. In our study, the extracts of these species significantly inhibited the differentiation to osteoclasts in RAW264.7 cells with the inhibition percentage ranging from 98.5 % and 83.1 % at the tested concentration (Fig. 2).
Oroxylum indicum with the main components from seeds including chrysin, baicalein, and quercetin [21] could be the best explanation of its anti osteoclastogenesis properties. The three compounds have been shown as having promising preventive effects for bone diseases related to excessive bone resorption and bone formation [12,22,23] Our experimental data showed that O. indicum fruit extract (S14) has a strong osteoclastogenesis inhibitory effect with 91 % inhibition. Thunbergia grandiflora (S2) is also a promising candidate for anti-osteoporosis drug development. Major constituents of this plant are caffeic acid which showed powerful inhibitory effects on osteoclastogenesis on the rat [24], and other prominent elements are quercetin which enhances bone cell proliferation [25].
Clausena excavata Burm.f. (S63) is widely used in traditional medicine for the cure of many diseases. The morphological, phytochemical, and pharmacological aspects of this plant were profoundly studied. However, the information about the anti-osteoporosis properties of this plant is still limited [26]. Several compounds were isolated from Garcinia oblongifolia Champ. ex Benth.(S25), Ilex kaushue S.Y. Hu (S9), and Xylopia vielana Pierre (S7), however no evidence on bone effects of these chemicals were recorded [27 -29]. Our data about the positive effects of these samples may be a benefit for further studies on the discovery of the antiosteoclastogenetic components from the plants. , Viburnum lutescens Blume (S19 and S20), and Stixis fasciculate (King) Gagn. (S17). This is both an opportunity and a challenge for us in further research on these plants.

CONCLUSIONS
In our present study, 70 plant extracts were screened for the osteoclastogenesis inhibitory effects in RAW264.7 murine macrophages. Twenty-nine extracts from 24 plants have potential as effective agents against osteoclastogenesis. This is the first but important assessment step, providing a preliminary overview of the anti-osteoclastogenesis ability of these plants. In a future study, potential plant species will be studied further, especially species whose chemical constituents have not yet been studied. The phytochemical components of these plants should be analyzed and the active components of these plants should be evaluated.