{"id":60106,"date":"2024-09-30T10:58:30","date_gmt":"2024-09-30T10:58:30","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=60106"},"modified":"2024-10-09T18:26:21","modified_gmt":"2024-10-09T18:26:21","slug":"the-effect-of-water-fraction-derived-from-green-clover-marsilea-crenata-c-presl-leaves-on-differentiation-and-maturation-of-human-osteoblast-cell","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/the-effect-of-water-fraction-derived-from-green-clover-marsilea-crenata-c-presl-leaves-on-differentiation-and-maturation-of-human-osteoblast-cell\/","title":{"rendered":"The Effect of Water Fraction Derived from Green Clover (Marsilea crenata C. Presl.) Leaves on Differentiation and Maturation of Human Osteoblast Cell"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

It is common for women over the age of 40 to suffer\nreduced bone mass as a result of estrogen shortage, which in turn increases the\nchance of developing osteoporosis. Data from the Central Bureau of Statistics\nof Indonesia 2020 indicate that the number of older individuals in Indonesia\nincreased by 9.92% to around 26 million. The proportion of elderly women in\nIndonesia is higher than that of elderly males (10.43% vs 9.42 in Indonesia),\n19.7% of the elderly population, which accounts for 3.6 million people, suffer\nfrom osteoporosis. In 2005, there were 18 million elderly individuals in\nIndonesia, and this number is projected to increase to 33 million by 2020, with\na life expectancy of 70 years. According to predictions, 30% of women who are\n50 years old or older will have osteoporosis, while 37-54% are expected to have\nosteopenia, and 54% are at risk of experiencing issues related to fractures1<\/sup>.\nIt is projected that by the year 2050, Asia, including Indonesia, would account\nfor almost 50% of global osteoporotic fractures2<\/sup>. According to\nGallagher and Sai (2010)3<\/sup>, the incidence of osteoporosis is twice as\ncommonly seen in females as it is in males.<\/p>\n\n\n\n

Hormones including estrogen, testosterone, and\nparathyroid hormone (PTH) have a significant role in the creation and\npreservation of bones. During menopause in women and in older men, a drop in\nhormones causes a gradual decline in bone mass4<\/sup>. This phenomenon is\nobserved in both types of individuals. It is common for women to experience\nmenopause after the age of 40, which is defined by the cessation of\nmenstruation and a decline in ovarian activity5<\/sup>. Menopause often\noccurs around the age of 40, and when weman become older, estrogen levels drop,\nwhich raises the likelihood that we may develop osteoporosis5,6<\/sup>.<\/p>\n\n\n\n

Bones go through a process of ongoing production and\nrepair while the remodeling process is taking place. During this process,\nestrogen plays a significant role by controlling the equilibrium between\nbone-forming cells (osteoblasts) and bone-destroying cells (osteoclasts)7<\/sup>.\nAdditionally, estrogen has a function in the formation of bone. Activation of\nthe transcription factor osterix\n(Osx) is necessary for osteoblast differentiation and maturation8<\/sup>.\nBone formation is dependent on osteoblast differentiation and maturation9<\/sup>.\nImportant proteins such as osteocalcin\n(Ocn), osteopontin (OPN), collagen type I (COL-I), and bone sialoprotein\n(BSP) are also altered by Osx10<\/sup>. <\/p>\n\n\n\n

There\nare phytoestrogens present in Marsilea\ncrenata<\/em> C. Presl. as well11<\/sup>. According\nto Putra and Laswati (2011)12<\/sup>, the amounts of phytoestrogens that\nare present in these leaves are relatively high13<\/sup>. It has been\ndemonstrated that the ethyl acetate fraction of M. crenata<\/em> have antiosteoporosis activity, which in turn has been shown\nto enhance bone density in female osteoporotic mice14<\/sup>. This plant, belonging to the\nMarsileaceae group, is found in Laos, Kalimantan, Java, the Lesser Sunda\nIslands, New South Wales, Malaya, New Guinea, the Philippines, South Australia,\nQueensland, Thailand, the Northern Territory, Victoria, Vietnam, and Western\nAustralia15,16<\/sup>.<\/p>\n\n\n\n

So, the\npurpose of this research is to determine the water fraction of M. crenata<\/em> leaves could increase the\nexpression of Osx and Ocn, which would indicate an increase in bone production.\nhFOB 1.19 cells were given water fractions in varying dosages, with genistein\nserving as a positive control. <\/p>\n\n\n\n

Material and methods<\/strong><\/p>\n\n\n\n

Materials<\/strong><\/p>\n\n\n\n

The leaves of M. crenata<\/em> were collected from Surabaya, East Java, Indonesia. The hFOB 1.19 cell line (CRL-11372) was purchased from ATCC, Virginia, USA. Genistein, paraformaldehyde (PFA), dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), and bovine serum albumin (BSA) were purchased from Sigma Aldrich, Missouri, USA. The anti-mice osteocalcin and anti-rabbit osterix antibodies were obtained from Abcam, Cambridge, UK. Penicillin-streptomycin antibiotics, and fetal bovine serum (FBS), G418, dulbecco’s modified eagle’s medium (DMEM), were supplied by Aretha, Bandung, Indonesia. Tween-80, paraformaldehyde, fluorescein isothiocyanate (FITC), rhodamine, and an anti-mouse and anti-rabbit secondary antibody were purchased from Universitas Brawijaya, Malang, Indonesia.<\/p>\n\n\n\n

Method<\/strong><\/p>\n\n\n\n

Extraction, Fractionation, and Preparation of Water Fraction <\/strong><\/p>\n\n\n\n

Using ultrasonic-assisted extraction, 1.5 kg of M. crenata<\/em> leaves was extracted with 96% ethanol solvent to get 60 g of extract17<\/sup>. Liquid-liquid extraction were used in the fractionation process. The extract was suspended in 700 ml of water and fractionated at a 1:1 ratio using n-hexane. After that, the resulting aqueous phase was separated and further fractionated using n-butanol and ethyl acetate. A Heidolph Hei-VAP ML\/G3 rotary evaporator was then used to evaporate the water phase.<\/p>\n\n\n\n

The samples were obtained by mixing 50 mg of water fraction with 0.5% Tween-80 in 0.5% DMSO (w\/v), and prepared at doses of 62.5, 125, and 250 \u00b5g\/L.<\/p>\n\n\n\n

Cell Culture<\/strong><\/p>\n\n\n\n

Complete medium formulated from DMEM, penicillin and streptomycin 1%, G418, and FBS 10%, were used to cultivate hFOB 1.19 cells in a culture flask. The cells were then incubated for six days at 37\u00b0C in an incubator with 5% CO2. Every 24 hours, the progress of the cells was monitored, and the medium was changed out until it had 80\u201390% content. Following that, the cells were moved to a microplate with 24 wells.<\/p>\n\n\n\n

Osx and Ocn Measurement<\/strong><\/p>\n\n\n\n

The hFOB 1.19 cells, which had reached confluence, were given a water fraction at doses of 62.5, 125, and 250 \u00b5g\/L, as well as 2.5 \u00b5g\/ml genistein as a positive control. Following the cultivation of cells to 80% confluence in a 24-well microplate, the administration of 10 ng\/ml TNF-\u03b1 was performed. The cells were subjected to a 48-hour treatment with samples and genistein. After a subsequent washing with PBS, the cells were subjected to fixation using a 4% PFA solution, Triton X-100, BSA, and Osx and Ocn primary antibodies for immunochemistry. The cells were then incubated at 4 \u00b0C overnight. The cells then received the secondary antibodies rhodamine and FITC, respectively. The samples were then subjected to analysis utilizing CLSM (Fluoview Olympus FV1000) at wavelengths of 488 nm and 543 nm. The immunofluorescence of the markers was analyzed using Olympus Fluoview Ver.4.2a software to determine the expression levels of Osx and Ocn18<\/sup>. <\/p>\n\n\n\n

Data analysis<\/strong><\/p>\n\n\n\n

Following the acquisition of data through CLSM, the subsequent procedure involves quantification to derive numerical data. This quantification will be performed through one-way ANOVA and followed with post-hoc LSD analysis utilizing Statistical Product and Service Solutions (SPSS) statistical software, with a significance threshold set at p<0.05.<\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

A water fraction of 70 g was produced from the extraction process\nusing 1.6 kg of M. crenata<\/em> leaves powder using 96% ethanol solvent.\nAfter that, extract 96% ethanol from the leaves of M. crenata<\/em> separated\nusing liquid-liquid extraction and produced 27 g water fraction. <\/p>\n\n\n\n

Figure 1 and Figure 2 show that after each dose was given, the amount of Osx immunofluorescence in hFOB 1.19 cells increased in all groups. The positive control group exhibited the highest intensity, followed by the treatment groups based on the dose level, while the negative control group displayed the lowest intensity. The 250 \u00b5g\/ml dose group had the best results. They significantly increased Osx expression compared to the negative control (p = 0.000), but not significantly compared to the genistein 2.5 \u00b5g\/ml dose group (p = 0.125). At the dose of 62.5 \u00b5g\/ml, Osx expression was higher compared to the negative control (p = 0.000) and genistein 2.5 \u00b5g\/ml (p = 0.000). It was also higher at the dose of 125 \u00b5g\/ml compared to the negative control (p = 0.000) and genistein 2.5 \u00b5g\/ml (p = 0.000).       <\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 1: Osx expression using FITC staining in a 488 nm wavelength.
(A) Negative control, (B) dose 62.5 \u00b5g\/ml, (C) dose 125 \u00b5g\/ml, (D) dose
250 \u00b5g\/ml, (E) genistein 2.5 \u00b5g\/ml.<\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 2: Expression of Osx in hFOB 1.19 cells after administration of M. crenata <\/em>leaves water fraction. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

A similar pattern was seen in Ocn observations, as depicted in Figure 3 and Figure 4, which show Ocn immunofluorescence in hFOB 1.19 cells in all groups. The positive control group showed the highest intensity, followed by the treatment group based on the dose level, and the negative control group showed the lowest intensity. In terms of increasing Ocn expression, the 250 \u00b5g\/ml dose group did the best. It did this significantly more than the negative control (p = 0.000) and also significantly more than genistein 2.5 \u00b5g\/ml (p = 0.000). The increase in Ocn expression at a dose of 62.5 \u00b5g\/ml was also significantly different from the negative control (p = 0.000) and genistein 2.5 \u00b5g\/ml (p = 0.000), as well as at a dose of 125 \u00b5g\/ml, which was significant compared to the negative control (p = 0.000) and genistein 2.5 \u00b5g\/ml (p = 0.000).<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 3: Ocn expression using Rhodamine staining in a 543 nm wavelength. (A) Negative control, (B) dose 62.5 \u00b5g\/ml, (C) dose 125 \u00b5g\/ml, (D) dose 250 \u00b5g\/ml, (E) genistein 2.5 \u00b5g\/ml. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\nFigure 4: Ocn expression in hFOB 1.19 cells after administration of M. crenata <\/em>leaves water fraction. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The results showed that the water fraction from the leaves of M. crenata<\/em> at a dose of 62.5; 125; and\n250 \u00b5g\/ml significantly and linearly increased the expression of Osx and Ocn in\nhFOB 1.19 cells compared with the negative control. Osx is a key transcription\nfactor that plays an important role in the process of osteoblast cell\ndifferentiation, activating the SP7 promoter gene during the maturation of\nosteoblasts into mature osteoblasts and osteocytes. Inactivation of Osx can\ninhibit the expression of osteocalcin, bone sialoprotein, and osteopontin,\nthereby reducing the activity of forming new bone cells19,20<\/sup>. The\nfunction of Osx is to regulate the production of bone formation proteins,\nincluding Ocn, which is the main non-collagen protein produced by mature\nosteoblast cells.<\/p>\n\n\n\n

The Ocn protein plays a crucial role in the bone mineralization\nprocess and maintaining calcium ion homeostasis. There are two forms of Ocn,\nthe carboxylated form which has a high affinity for calcium and hydroxyapatite\ncrystals, so they are in the bone matrix during the mineralization process with\nthe help of \u03b3-carboxyglutamic acid (Gla) which helps absorption into\nhydroxyapatite, which is a crucial step in bone mineralization. Uncarboxylated\nosteocalcin has a low affinity for bone matrix and is likely to enter the\nbloodstream to reach other organs20,21<\/sup>. The formation of the\nosteocalcin matrix plays an important role in increasing bone mineralization20,21,22<\/sup>.<\/p>\n\n\n\n

The administration of a water fraction derived from M. crenata<\/em>\nleaves resulted in an upregulation of Osx and Ocn expression. This observation\nserves as evidence supporting the involvement of this fraction in enhancing the\nprocess of bone growth. The observed phenomenon might perhaps be attributed to\nthe regulatory mechanism of phytoestrogens present in the aqueous portion of M.\ncrenata<\/em> leaves, which has resemblance to the mode of action of estrogen.\nPhytoestrogens are botanical substances exhibiting a structural resemblance to\n17\u03b2-estradiol, hence enabling them to serve as substitutes for estrogen.\nPhytoestrogen substances encompass a variety of chemicals, including flavonoids\nlike kaempferol and quercetin, isoflavonoids such as genistein and daidzein, as\nwell as triterpenoids and steroids23,24,25<\/sup>. Aditama et al., 2021\nalso conducted similar research. The ethyl acetate fraction of M. crenata<\/em> leaves can increase osterix with\nan optimal dose of 250 ppm26<\/sup>, and 96% ethanol extract of M. crenata<\/em> leaves can increase\nosteocalcin expression with an optimal dose of 125 ppm27<\/sup>.<\/p>\n\n\n\n

Conclusion<\/strong><\/strong><\/p>\n\n\n\n

The administration of\nwater fractions derived from M. crenata<\/em> leaves has the ability to\nenhance the expression of Osx and Ocn in hFOB 1.19 cells, the best dose that\ncan increase Osx and Ocn is 250 \u00b5g\/ml. This suggests that it has the potential\nto serve as a natural source for promoting differentiation and maturation of\nosteoblast through the upregulation of the Osx and Ocn genes.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

None<\/p>\n\n\n\n

Conflict of Interest <\/strong><\/p>\n\n\n\n

The authors declare there is no conflict of interest.<\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

No funding agency in the governmental, commercial, private, or not-for-profit sectors provided a particular grant for this research.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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     CrossRef<\/a><\/li><\/ol>\n","protected":false},"excerpt":{"rendered":"

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