|Year : 2021 | Volume
| Issue : 2 | Page : 175-179
Rhaponticin contained Rheum officinale root extract improved Postmenopause symptom of Ovariectomized Rat
Anton Bahtiar1, Herlina Tri Setyowati1, Retno Rela Mahanani1, Azizah Wati1, Ade Arsianti2, Fadilah Fadilah2
1 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Universitas Indonesia Kampus UI Depok 16424, Jakarta, Indonesia
2 Department of Medicinal Chemistry, Faculty of Medicine, Universitas Indonesia Kampus UI Jl Salemba Raya, Jakarta, Indonesia
|Date of Submission||15-Dec-2020|
|Date of Decision||16-Feb-2021|
|Date of Acceptance||17-Mar-2021|
|Date of Web Publication||27-Apr-2021|
Dr. Anton Bahtiar
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Universitas Indonesia Kampus UI Depok 16424, Depok, West Java
Source of Support: None, Conflict of Interest: None
Postmenopausal women have decreased levels of the hormone estrogen. Reduced estrogen levels will often involve many symptoms that reduced quality of life. This research aims to analyze the effects of Rheum officinale root extract on postmenopausal model rats. To this end, thirty rats underwent ovariectomy (OVX) surgery and six rats were operated without having their ovaries removed. The OVX was confirmed by body weight–uterus weight ratio and a vaginal swab. Six groups of the rats were performed: SHAM group and negative control groups are given vehicle; the positive control was assigned tamoxifen; and the extract has been given three doses 7, 35, and 175 mg/200 g BW, respectively, for 30 days. The calcium content of bone ash was measured using atomic absorption spectrophotometer. Blood pressure was evaluated using CODA®, and the metabolites in the blood were assessed using gas chromatography–mass spectrometry (MS) and high-performance liquid chromatography. As a result, using ultra-performance liquid chromatography (UPLC)-MS, we found that the extract's major component was rhaponticin and its metabolites. The bone calcium levels increased with increasing doses of the extract. In the OVX group, the bone calcium content was decreased significantly 51.56% ± 8.9% g compared with the SHAM group 62.97% ±5.6% g, and the administration of Rheum extract could restore the calcium content of the bone to become 69.27% ± 3.8% g. From the above data, we concluded that Rheum root extracts contain astrigin, rhaponticin, rhapontigenin, and desoxyrhaponticin. Rheum root extract could improve calcium content and lipid profiles of OVX rats by stimulation osteoblastogenesis. Rheum root extracts could control the blood pressure of OVX rats by reducing lipid profiles.
Keywords: Calcium, hypertension, Kalembak, lipid profiles, osteoporosis, postmenopausal, rhaponticin, Rheum officinale
|How to cite this article:|
Bahtiar A, Setyowati HT, Mahanani RR, Wati A, Arsianti A, Fadilah F. Rhaponticin contained Rheum officinale root extract improved Postmenopause symptom of Ovariectomized Rat. J Adv Pharm Technol Res 2021;12:175-9
|How to cite this URL:|
Bahtiar A, Setyowati HT, Mahanani RR, Wati A, Arsianti A, Fadilah F. Rhaponticin contained Rheum officinale root extract improved Postmenopause symptom of Ovariectomized Rat. J Adv Pharm Technol Res [serial online] 2021 [cited 2021 Jun 25];12:175-9. Available from: https://www.japtr.org/text.asp?2021/12/2/175/314680
| Introduction|| |
Postmenopausal women have decreased levels of the hormone estrogen. Reduced estrogen levels will increase bone remodeling and lead to imbalanced activity between osteoclasts and osteoblasts, as osteoblasts activity can not compensate for osteoclast activity, thereby decreasing bone mass and made osteoporotic. Hormone replacement therapy (HRT, a combination of estrogen and progestin) has been used for many years as the gold standard to deal with the symptoms of menopause. However, long-term use may increase breast cancer risk, endometrial cancer, and thrombosis., There are several alternatives to HRT; one of them is selective estrogen receptor modulators (SERMs). SERMs are compounds that do not have a steroid structure such as estrogen but have a tertiary structure that can bind to ERα and ERβ.,,
In 1993, dry extract of rhubarb (Rheum rhaponticum L.; Dahuang) was first used to treat symptoms of menopause. Rheum root dry extract mainly contains rhaponticin which has a stilbene backbone. Tamoxifen and other SERMS derivate compounds have stilbene's backbone., Rheum species in Indonesia are rhubarb, Rheum officinale. The present research wants to evaluate rhubarb extract's function on the animal model's bone, cardiovascular, and lipid profiles with the background described above.
| Materials and Methods|| |
Chemicals and reagents
All chemical reagents were purchased from Sigma-Aldrich. PT offers tamoxifen, Kalbe Farma, Indonesia. The Ketalar injection was purchased from PT, Pfizer, Indonesia. Carboxymethyl cellulose was purchased from Brataco, Indonesia.
Rhubarb root (R. officinale Baill.) obtained from Tawangmangu, Solo, Central Java. The plant was determined by the Center for Research and Development of Medicinal Plants and Traditional Medicine (certificate of determination No. 125/Dec/2013).
Preparation of Rheum roots extracts
The dried powder of Rheum roots was extracted according to the method of Bahtiar et al.
Chromatographic and mass conditions
Chromatographic analysis and mass spectrometry detection were carried out according to the method of Zhou et al. using Waters Acquity UPLC system (Waters Corp., Milford, MA, USA) at the Biocenter, Gyeonggido Business and Science Accelerator (GBSA), Suwon, Korea.
This research had certified by the Ethical Committee of Faculty of Medicine, University of Indonesia (UIFM No. 164b/H2.F1/Ethics). Thirty rats underwent ovariectomy (OVX) surgery, and six rats were operated without having their ovaries removed. The OVX was confirmed by uterus weight– body weight ratio and a vaginal swab. Six groups of the rats were performed: SHAM group and negative control groups are given vehicle; the positive control was assigned tamoxifen; and the extract has been given three doses 7, 35, and 175 mg/200 g BW, respectively, for 30 days.
The calcium content of bone
The calcium content of the bone was analyzed using femurs as calcium source according to the methods of Bahtiar et al., by atomic absorption spectrophotometry Shimadzu AA-700.
Determination of the lipid profile of serum
Lipid profiles had determined according to kits' procedure by an enzymatic colorimetric method (DiaSys, Germany).
Measurement of blood pressure
A noninvasive blood pressure gauge CODA® (Kent Scientific Corporation, USA) was used to measure blood pressure. The systolic and diastolic pressures were analyzed four times. They were as follows: before OVX, 21 days after OVX, and after 28 days of extract treatment.
Analysis of amino acids
Serum samples were measured and evaluated using gas chromatography (GC)-MSD 5975C, Agilent Technologies (USA)
| Results|| |
Identification of rhaponticin in Rheum extract
[Figure 1] shows the identification of the components of the extract using high resolution of MS. There are four significant peaks detected in the extract. Four components of Rheum extract were identified as astringin, rhapontin, rhapontigenin, and desoxyrhaponticin. Rhaponticin has a retention time of 10.25 min.
|Figure 1: Identification of extract component using ultra-performance liquid chromatography–mass spectrometry. Four components of Rheum extract identified as astringin, rhapontin, rhapontigenin, and desoxyrhaponticin|
Click here to view
Effects of Rheum extract on the calcium content of bone
[Table 1] shows that OVX increased rats' body weight but decreased the uterine index and calcium content of the OVX rats. The administration of tamoxifen reduced OVX rats' body weight and increased the uterine index and calcium content of OVX rats' bone. The administration of Rheum extract showed a reduced body weight gain of OVX rats but no effect on the uterine index. At a high dose, Rheum extract could increase the calcium content of the bone.
|Table 1: Bodyweight gain, uterine index, and calcium content of OVX Rats|
Click here to view
Effects of Rheum extract on the lipid profile of ovariectomy rats
[Table 2] shows that OVX increased cholesterol, triglycerides, and low-density lipoprotein (LDL) but decreased high-density lipoprotein (HDL) in animal models. The tamoxifen administration could reduce high cholesterol, triglycerides, and LDL of OVX rats with increased HDL. The administration of Rheum extracts was similar to the tamoxifen effect.
Effects of Rheum extract on blood pressure of ovariectomy rats
[Table 3] shows that OVX increased systole and diastole of OVX rats but can be reduced by tamoxifen administration. The administration of Rheum extracts reduced systole and diastole.
Effects of Rheum extract on 4-methylproline and l-proline
[Table 4] shows that OVX increased 4-methylproline and l-proline in model rats. Tamoxifen and Rheum extracts could reduce 4-methylproline and l-proline in OVX rats.
| Discussion|| |
Four components of Rheum extract were identified as astringin, rhapontin, rhapontigenin, and desoxyrhaponticin with retention time 8.70, 10.25, 12.49, and 13.49 minutes, respectively, as shown in [Figure 1]. Rhaponticin shows the highest peak compared with other components. This indicated that the extract's effect was dictated by the high components of the extracts, as shown in another experiment that found a positive correlation between the content and the effects.,,
[Table 1] is shown that OVX increased body weight compared with SHAM rats. These results are similar to Iwasa et al.; they found that OVX-treated rats showed higher serum leptin levels. The administration of tamoxifen and Rheum extracts does not affect the body weight of OVX rats. Some studies found that the estrogen effects are mediated through ERα and Erβ receptors, and lack of ERα in hypothalamic proopiomelanocortin neurons leads to overeating. There is a balanced ratio of these two receptors before menopause in women, but after menopause, this ratio is disrupted and is associated with increased ERβ signaling. Thus, it has been concluded that ERα signaling is involved in regulating food intake.,,
Tamoxifen has both antagonistic and agonistic effects on the ER. It acts as a complete antagonist in the mammary glands, a partial agonist in the uterus, and a complete agonist in the bone and cholesterol metabolism. Tamoxifen reduces fat mass through boosting ROS. R. officinale root extracts have no effects on body weight since R. officinale contained rhaponticin, a stilbene structure similar to tamoxifen. Therefore, R. officinale extract has a similar result with tamoxifen.
Moreover, OVX made uterine atrophy; decreasing in estrogen lead the uterus to become small and fibrotic. Tamoxifen could prevent uterine atrophy but not for R. officinale root extract-treated rats. This result indicated that the R. officinale root extracts showed a different mechanism of action from tamoxifen on uterine cell proliferation.
[Table 2] shows that the calcium content of the OVX rats decreases, and the administration of tamoxifen and R. officinale root extract could recover the calcium content. Tamoxifen prevented OVX by increasing urinary hydroxyproline or Ca and conserved bone. The mechanism of Rheum extract in bone calcium has not been elucidated yet, but rhaponticin that contained in Rheum extract has a similar molecular structure with tamoxifen; they have a stilbenoid group.,
OVX increased blood pressure both systole and diastole; as shown in [Table 3], plasma levels of NO metabolites, nitrites, and nitrates were reduced by the OVX. The administration of tamoxifen and Rheum extract could recover blood pressure to normal value.
The GC-MS result shows in [Table 4]; methyl-proline and L-proline were increased during OVX and recovered after administration of tamoxifen and R. officinale root extract. This result was similar to that of other researchers;, these amino acids increased, indicating that bone collagen type 1 synthesis increases the density of bone.
In our previous study, Rheum extract could stimulate osteoblastogenesis by increasing RUNX2, BMP2, and alkaline phosphatase., This current in vivo results confirmed the previous work: the bone density of osteoporotic rats could increase by the administration of R. officinale root extract.
We suspected that substances in the extracts that similar to tamoxifen could stimulate osteoblastogenesis and increase bone density. Tamoxifen contains a stilbene group which believes that it has selectivity when the acts on ERs.
We then evaluated the Rheum extract composition using ultra-performance liquid chromatography (UPLC)-MS [Figure 1] and found that the Rheum extract consists of astrigin, rhaponticin, rhapontigenin, and desoxyrhaponticin. All components have stilbene groups.
This result indicated that rhaponticin and derivatives in this extract could recover the bone density of OVX rats.
| Conclusions|| |
Rheum root extracts contain astrigin, rhaponticin, rhapontigenin, and desoxyrhaponticin. Rheum root extract could improve calcium content and lipid profiles of OVX rats by stimulation osteoblastogenesis. Rheum root extracts could control blood pressure of OVX rats by reducing lipid profiles.
We thank Prof. Jaehong Han and Mihyang Kim, Ph.D., from Chung-Ang University as host in Korea. We thank Jong-Suk Lee, Ph.D., from Biocenter, Gyeonggido Business and Science Accelerator, who evaluated the content of the extract by UPLC.
Financial support and sponsorship
This research was funded by International Collaboration Based Research Grant from Universitas Indonesia to AB.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Emam MN, Abo El Gheit RE. New treatment paradigm of combined raloxifene and conjugated estrogen for postmenopausal symptoms in VCD-induced menopausal rats. Alexandria J Med 2017;53:227-36.
Ushiroyama T, Ikeda A, Sakai M, Higashiyama T, Ueki M. Prevention of postmenopausal bone loss with exchange for short-term HRT for 1α-hydroxycholecalciferol. Maturitas 2003;45:119-27.
Saito N, Kawase K, Yamashita N, Tang Y, Wang Y, Wang J, et al
. Identification of 10-dehydrooxyglycyuralin E as a selective human estrogen receptor alpha partial agonist. Bioorg Chem 2019;88:102977.
Sandberg K. HRT and SERMs: The good, the bad … and the lovely? Trends Endocrinol Metab 2002;13:317-8.
Müller ST, Pählig S, Merabet A, Abdelsamie AS, van Koppen CJ, Marchais-Oberwinkler S, et al
. Effects of 17β-HSD2 inhibition in bones on osteoporosis based on an animal rat model. Steroid Biochem Mol Biol 2019;192:105405.
Keiler AM, Papke A, Kretzschmar G, Zierau O, Vollmer G. Long-term effects of the rhapontic rhubarb extract ERr 731® on estrogen-regulated targets in the uterus and on the bone in ovariectomized rats. J Steroid Biochem Mol Biol 2012;128:62-8.
Rupprich N, Hildebrand H, Kindl H. Substrate specificity in vivo
and in vitro
in the formation of stilbenes. Biosynthesis of rhaponticin. Arch Biochem Biophys 1980;200:72-8.
Makar S, Saha T, Swetha R, Gutti G, Kumar A, Singh SK. Rational approaches of drug design for the development of selective estrogen receptor modulators (SERMs), implicated in breast cancer. Bioorg Chem 2020;94:103380.
Amari G, Armani E, Ghirardi S, Delcanale M, Civelli M, Caruso PL, et al
. Synthesis, pharmacological evaluation, and structure-activity relationships of benzopyran derivatives with potent SERM activity. Bioorg Med Chem 2004;12:3763-82.
Bahtiar A, Gusmira A, Tjandrawinata R. Functional analysis of 70% ethanolic extract of Akar kelembak (Rheum officinale
Baill.) on 3t3 L1 preadipocyte cell lines in osteogenic medium. Int J Pharm Pharm Sci 2014;6:86-89.
Zhou R, Luo F, Lei H, Zhang K, Liu J, He H, et al
. Liujunzi Tang, a famous traditional Chinese medicine, ameliorates cigarette smoke-induced mouse model of COPD. J Ethnopharmacol 2016;193:643-51.
Bahtiar A, Nurazizah M, Roselina T, Tambunan AP, Arsianti A. Ethanolic extracts of babandotan leaves (Ageratum conyzoides
L.) prevents inflammation and proteoglycan degradation by inhibiting TNF-α and MMP-9 on osteoarthritis rats induced by monosodium iodoacetate. Asian Pac J Trop Med 2017;10:270-7.
Vamanu E, Nita S. Antioxidant capacity and the correlation with major phenolic compounds, anthocyanin, and tocopherol content in various extracts from the wild edible Boletus edulis
mushroom. Biomed Res Int 2013;2013:313905.
Zhang RX, Li MX, Jia ZP. Rehmannia glutinosa
: Review of botany, chemistry and pharmacology. J Ethnopharmacol 2008;117:199-214.
Kolodziejczyk-Czepas J, Liudvytska O. Rheum rhaponticum
and Rheum rhabarbarum
: A review of phytochemistry, biological activities and therapeutic potential. Phytochem Rev 2020; 21:1-19.
Iwasa T, Matsuzaki T, Kinouchi R, Gereltsetseg G, Murakami M, Nakazawa H, et al
. Effect of immune stress on body weight regulation is altered by ovariectomy in female rats. Reprod Immunol 2011;91:41-7.
Brown CM, Mulcahey TA, Filipek NC, Wise PM. Production of proinflammatory cytokines and chemokines during neuroinflammation: Novel roles for estrogen receptors alpha and beta. Endocrinology 2010;151:4916-25.
Xu Y, Nedungadi TP, Zhu L, Sobhani N, Irani BG, Davis KE, et al
. Distinct hypothalamic neurons mediate estrogenic effects on energy homeostasis and reproduction. Cell Metab 2011;14:453-65.
Tomicek NJ, Lancaster TS, Korzick DH. Increased estrogen receptor β in adipose tissue is associated with increased intracellular and reduced circulating adiponectin protein levels in aged female rats. Gend Med 2011;8:325-33.
Liu L, Zou P, Zheng L, Linarelli LE, Amarell S, Passaro A, et al
. Tamoxifen reduces fat mass by boosting reactive oxygen species. Cell Death Dis 2015;6:e1586.
Luo ZH, Liu ZW, Mao Y, Shu R, Fu LC, Yang RY, et al
. Cajanolactone A, a stilbenoid from cajanus cajan, prevents ovariectomy-induced obesity and liver steatosis in mice fed a regular diet. Phytomedicine 2020;78:153290.
Hu X, Wang J, Yin QZ, Lu H, Yie SM. You Gui Wan can reverse atrophic effect of ovariectomy on rat vaginal fold and blood vessels in the lamina propria. Biol Pharm Bull 2011;34:1808-14.
Sourla A, Luo S, Labrie C, Bélanger A, Labrie F. morphological changes induced by 6-month treatment of intact and ovariectomized mice with tamoxifen and the pure antiestrogen EM-800. Endocrinology 1997;138:5605-17.
Goulding A, Gold E. In the ovariectomized rat, tamoxifen conserves bone similarly in parathyroid-intact and parathyroidectomized animals. Bone 1994;15:497-503.
Mannal P, McDonald D, McFadden D. Pterostilbene and tamoxifen show an additive effect against breast cancer in vitro
. Am J Surg 2010;200:577-80.
Hernández I, Delgado JL, Díaz J, Quesada T, Teruel MJ, Llanos MC, et al
. 17β-Estradiol prevents oxidative stress and decreases blood pressure in ovariectomized rats. Am J Physiol Integr Comp Physiol 2000;279:R1599-605.
Borgo MV, Lopes AB, Gouvêa SA, Romero WG, Moyses MR, Bissoli NS, et al
. Effect of tamoxifen on the coronary vascular reactivity of spontaneously hypertensive female rats. Braz J Med Biol Res 2011;44:786-92.
Lubec G, Labudova O, Seebach D, Beck A, Hoeger H, Hermon M, et al
. Alpha-methyl-proline restores normal levels of bone collagen Type I synthesis in ovariectomized rats. Life Sci 1995;57:2245-52.
Nam SY, Yoou MS, Kim HM, Jeong HJ. Efficacy of proline in the treatment of menopause. Exp Biol Med (Maywood) 2016;241:611-9.
[Table 1], [Table 2], [Table 3], [Table 4]