Sains Malaysiana 51(6)(2022): 1833-1845

http://doi.org/10.17576/jsm-2022-5106-19

The Effects of the Fractions of Piper sarmentosum Leaves on Inhibition of Adipogenesis of 3T3 L1 Preadipocytes

(Kesan daripada Fraksi Daun Piper sarmentosum pada Perencatan Adipogenesis pada Sel 3T3L1 Pra-adiposit)

FAIRUS AHMAD¹, IMA NIRWANA SOELAIMAN², KHAIRANA HUSAIN³, FARIHAH HJ SUHAIMI¹, CHUA KIEN HUI⁴ & NORZANA ABD GHAFAR^1,*

¹Department of Anatomy, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaakob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

²Department of Pharmacology, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaakob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

³Drug and Herbal Research Centre, Faculty of Pharmacy, Jalan Raja Muda Abdul Aziz, 53000 Kuala Lumpur, Federal Territory, Malaysia

⁴Department of Physiology, Universiti Kebangsaan Malaysia Medical Centre,

Jalan Yaakob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

Diserahkan: 29 Jun 2021/Diterima: 2 November 2021

ABSTRACT

Piper sarmentosum Roxb. was reported to have anti-obesity, hypoglycaemic and anti-oxidant properties. The aim of this study was to identify the fractions of P. sarmentosum leaf extract in inhibiting adipogenesis of 3T3L1 preadipocytes. The crude extract of the P. sarmentosum leaves was fractionated to produce hexane, dichloromethane, methanol, and aqueous fractions. Various dilutions of the fractions; hexane (0.1 - 1 µg/mL), dichloromethane (9.76 - 97.6 µg/mL), methanol (3.6 - 36 mg/mL), and aqueous (1 - 10 mg/mL), were treated onto the 3T3L1 preadipocytes from 3^rd to 15^th day of culture. The crude extract (1 - 10 mg/mL) and glycyrrhizic acid (GCA) (0.24 - 2.4 mg/mL) were used as positive controls. The viability of the adipocytes was measured by MTT assay at the 15^th day of culture. The content of each fraction was quantified with reference standards of naringin, naringenin, pellitorine, sarmentosine and β-sitosterol by using HPLC. The results showed that 49.1% of the crude extract contained aqueous fraction, 0.12% in hexane fraction, 9.7% in dichloromethane fraction and 36% in methanol fraction. The aqueous fraction and crude extract at the dose of 7 mg/mL and GCA at the dose of 1.92 mg/mL showed potent inhibitory effects on the adipogenesis. However, none of the reference standards were identified from the fractions using HPLC analysis. In conclusion, the aqueous fraction was the main fraction in the crude extract of the P. sarmentosum and contributed a significant role in inhibiting adipogenesis of the 3T3L1 preadipocytes.

Keywords: Adipogenesis; aqueous fraction; P. sarmentosum; 3T3L1 preadipocytes

Abstrak

Piper sarmentosum Roxb. adalah tumbuhan yang mempunyai ciri-ciri anti-obesiti, hipoglisemia dan antioksidan. Tujuan kajian ini adalah untuk mengenal pasti fraksi ekstrak daun P. sarmentosum dalam menghalang adipogenesis pada sel 3T3L1 pra-adiposit. Melalui proses fraksinasi, ekstrak daun P. sarmentosum ini dapat menghasilkan fraksi heksana, diklorometana, metanol dan akueus. Pencairan fraksi heksana (0.1 - 1 μg/mL), diklorometana (9.76 - 97.6 μg/mL), metanol (3.6 - 36 mg/mL) dan akueus (1 - 10 mg/mL) digunakan untuk merawat sel 3T3 L1 pra-adiposit dalam kultur. Pencairan ekstrak (1 - 10 mg/mL) dan asid gliserizik (GCA) (0.24 - 2.4 mg/mL) digunakan sebagai kawalan positif. Kebolehhidupan sel adiposit pada hari ke-15 kultur diukur dengan menggunakan kaedah asai MTT. Kandungan setiap fraksi dianalisis dengan menggunakan kaedah HPLC untuk mengesan rujukan sebatian piawai naringin, naringenin, pellitorine, sarmentosine dan β-sitosterol. Keputusan menunjukkan 49.1% ekstrak mengandungi fraksi akues, 0.12% fraksi heksana, 9.7% fraksi diklorometana dan 36% fraksi metanol. Fraksi akues dan ekstrak pada dos 7 mg/mL dan GCA pada dos 1.92 mg/mL menunjukkan kesan perencatan yang kuat terhadap adipogenesis. Walau bagaimanapun, tidak terdapat rujukan sebatian piawai pada fraksi ekstrak melalui analisis HPLC. Kesimpulannya, fraksi akues adalah fraksi utama ekstrak P. sarmentosum dan ia memainkan peranan yang penting dalam merencat adipogenesis sel 3T3L1 pra-adiposit.

Kata kunci: Adipogenesis; fraksi akues; P. sarmentosum; sel 3T3L1 pra-adiposit

RUJUKAN

Agu, K.C. & Paulinus, N.O. 2017. Proximate composition, phytochemical analysis, and in vitro antioxidant potentials of extracts of Annona muricata (Soursop). Food Science & Nutrition 5(5): 1029-1036.

Alberti, L., Girola, A., Gilardini, L., Conti, A., Cattaldo, S., Micheletto, G. & Invitti, C. 2007. Type 2 diabetes and metabolic syndrome are associated with increased expression of 11 [beta]-hydroxysteroid dehydrogenase 1 in obese subjects. International Journal of Obesity 31(12): 1826-1831.

Arner, E., Westermark, P.O., Spalding, K.L., Britton, T., Rydén, M., Frisén, J., Bernard, S. & Arner, P. 2010. Adipocyte turnover: Relevance to human adipose tissue morphology. Diabetes 59(1): 105-109.

Aslam, M.S., Ahmad, M.S., Ahmad, M.A. & Akhlaq, M. 2017. An updated review on phytochemical and pharmacological properties of Piper sarmentosum. Current Trends in Biotechnology and Pharmacy 11(4): 345-356.

Atiax, E., Ahmad, F., Sirat, H.M. & Arbain, D. 2010. Antibacterial activity and cytotoxicity screening of sumatran kaduk (Piper sarmentosum Roxb). Iranian Journal of Pharmacology & Therapeutics 10(1): 1-5.

Azlina, A.A., Farihah, H.S., Qodriyah, H.M.S. & Nur Azlina, M.F. 2009. Effects of Piper sarmentosum water extract on 11-β hydroxysteroid dehydrogenase type 1 bioactivity in ovariectomy-induced obese rats. International Journal of Pharmacology 5(6): 362-369.

Azmi, M.F., Aminuddin, A., Jamal, J.A., Hamid, A.A. & Ugusman, A. 2021. Quantified Piper sarmentosum Roxb. leaves aqueous leaf extract and its antihypertensive effect in dexamethasone-induced hypertensive rats. Sains Malaysiana 50(1): 171-179.

Barchan, A., Bakkali, M., Arakrak, A., Pagán, R. & Laglaoui, A. 2014. The effects of solvents polarity on the phenolic contents and antioxidant activity of three Mentha species extracts. International Journal Current Microbiology and Applied Sciences 3(11): 399-412.

Baskar, R., Rajeswari, V. & Kumar, T.S. 2007. In vitro antioxidant studies in leaves of Annona species. Indian Journal of Experimental Biology 45(5): 480-485.

Berthiaume, M., Laplante, M., Festuccia, W., Gélinas, Y., Poulin, S., Lalonde, J., Joanisse, D.R., Thieringer, R. & Deshaies, Y. 2007. Depot-specific modulation of rat intraabdominal adipose tissue lipid metabolism by pharmacological inhibition of 11beta-hydroxysteroid dehydrogenase type 1. Endocrinology 148(5): 2391-2397.

Blüher, M. 2019. Obesity: Global epidemiology and pathogenesis. Nature Reviews Endocrinology 15(5): 288-298.

Brasaemle, D.L., Dolios, G., Shapiro, L. & Wang, R. 2004. Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes. The Journal of Biological Chemistry 279(45): 46835-46842.

Bujalska, I.J., Hewitt, K.N., Hauton, D., Lavery, G.G., Tomlinson, J.W., Walker, E.A. & Stewart, P.M. 2008. Lack of hexose-6-phosphate dehydrogenase impairs lipid mobilization from mouse adipose tissue. Endocrinology 149(5): 2584-2591.

Bujalska, I.J., Walker, E.A., Hewison, M. & Stewart, P.M. 2002. A switch in dehydrogenase to reductase activity of 11β-hydroxysteroid dehydrogenase type 1 upon differentiation of human omental adipose stromal cells. The Journal of Clinical Endocrinology & Metabolism 87(3): 1205-1210.

Caron, M., Auclair, M., Sterlingot, H., Kornprobst, M. & Capeau, J. 2003. Some HIV protease inhibitors alter lamin A/C maturation and stability, SREBP-1 nuclear localization and adipocyte differentiation. AIDS 17(17): 2437-2444.

Chandramouli, C., Ting, Y.S., Lyn, L.Y., Ha, T.S. & Kadir, K.A. 2011. Glycyrrhizic acid improves lipid and glucose metabolism in high-sucrose-fed rats. Journal of Endocrinology and Metabolism 1(3): 125-141.

Chapagain, A., Caton, P.W., Kieswich, J., Andrikopoulos, P., Nayuni, N., Long, J.H., Harwood, S.M., Webster, S.P., Raftery, M.J., Thiemermann, C. & Walker, B.R. 2014. Elevated hepatic 11β-hydroxysteroid dehydrogenase type 1 induces insulin resistance in uremia. In Proceedings of the National Academy of Sciences. pp. 3817-3822.

Davis, L.A. & Zur Nieden, N.I. 2008. Mesodermal fate decisions of a stem cell: The Wnt switch. Cellular and Molecular Life Sciences 65(17): 2658-2674.

Eu, C.H.A., Lim, W.Y.A., Ton, S.H. & Abdul Kadir, K. 2010. Glycyrrhizic acid improved lipoprotein lipase expression, insulin sensitivity, serum lipid and lipid deposition in high-fat diet-induced obese rats. Lipids in Health and Disease 9(1): 1-9.

Fairus, A., Ima, S.N., Elvy, M.S., Tan, M.H., Santhana, R. & Farihah, H.S. 2013. Piper sarmentosum is comparable to glycyrrhizic acid in reducing visceral fat deposition in adrenalectomised rats given dexamethasone. La Clinica Terapeutica 164(1): 5-10.

Fan, J.Y., Carpentier, J.L., Van Obberghen, E., Grunfeld, C., Gorden, P. & Orci, L. 1983. Morphological changes of the 3T3-L1 fibroblast plasma membrane upon differentiation to the adipocyte form. Journal of Cell Science 61(1): 219-230.

Fischbach, C., Spruß, T., Weiser, B., Neubauer, M., Becker, C., Hacker, M., Göpferich, A. & Blunk, T. 2004. Generation of mature fat pads in vitro and in vivo utilizing 3-D long-term culture of 3T3-L1 preadipocytes. Experimental Cell Research 300(1): 54-64.

Gavamukulya, Y., Abou-Elella, F., Wamunyokoli, F. & AEl-Shemy, H. 2014. Phytochemical screening, anti-oxidant activity and in vitro anticancer potential of ethanolic and water leaves extracts of Annona muricata (Graviola). Asian Pacific Journal of Tropical Medicine 7(1): S355-S363.

Green, H. & Meuth, M. 1974. An established pre-adipose cell line and its differentiation in culture. Cell 3(2): 127-133.

Hairi, H.A., Jamal, J.A., Aladdin, N.A., Husain, K., Mohd Sofi, N.S., Mohamed, N., Mohamed, I.N. & Shuid, A.N. 2018. Demethylbelamcandaquinone B (Dmcq B) is the active compound of Marantodes pumilum var. alata (Blume) Kuntze with osteoanabolic activities. Molecules 23(7): 1686.

Hamm, J.K., Park, B.H. & Farmer, S.R. 2001. A role for C/EBPβ in regulating peroxisome proliferator-activated receptor γ activity during adipogenesis in 3T3-L1 preadipocytes. Journal of Biological Chemistry 276(21): 18464-18471.

Han, J., Hamilton, J.A., Kirkland, J.L., Corkey, B.E. & Guo, W. 2003. Medium-chain oil reduces fat mass and down-regulates expression of adipogenic genes in rats. Obesity Research 11(6): 734-744.

Han, J., Farmer, S.R., Kirkland, J.L., Corkey, B.E., Yoon, R., Pirtskhalava, T., Ido, Y. & Guo, W. 2002. Octanoate attenuates adipogenesis in 3T3-L1 preadipocytes. The Journal of Nutrition 132(5): 904-910.

Harmon, A.W. & Harp, J.B. 2001. Differential effects of flavonoids on 3T3-L1 adipogenesis and lipolysis. American Journal of Physiology-cell Physiology 280(4): C807-C813.

He, Y.F., Liu, F.Y. & Zhang, W.X. 2015. Tangeritin inhibits adipogenesis by down-regulating C/EBPα, C/EBPβ, and PPARγ expression in 3T3-L1 fat cells. Genetics and Molecular Research 14(4): 13642-13648.

Hill, J.O., Wyatt, H.R. & Peters, J.C. 2012. Energy balance and obesity. Circulation 126(1): 126-132.

Hua, K., Deng, J. & Harp, J.B. 2004. Interleukin-4 inhibits platelet-derived growth factor-induced preadipocyte induced preadipocyte proliferation. Cytokine 25(2): 61-67.

Hussain, K., Ismail, Z., Sadikun, A. & Ibrahim, P. 2010. Standardization and in vivo antioxidant activity of ethanol extracts of fruit and leaf of Piper sarmentosum. Planta Medica 76(5): 418-425.

Hussain, K., Ismail, Z., Sadikun, A. & Ibrahim, P. 2009a. Antioxidant, anti-TB activities, phenolic and amide contents of standardised extracts of Piper sarmentosum Roxb. Natural Product Research 23(3): 238-249.

Hussain, K., Ismail, Z., Sadikun, A. & Ibrahim, P. 2009b. Proximate and qualitative analysis of different parts of Piper sarmentosum, and quantification of total amides in various extracts. Pharmacognosy Research 1(3): 113-119.

Ibanez, E., Kubátová, A., Señoráns, F.J., Cavero, S., Reglero, G. & Hawthorne, S.B. 2003. Subcritical water extraction of antioxidant compounds from rosemary plants. Journal of Agricultural and Food Chemistry 51(2): 375-382.

Institute of Public Health of Malaysia. 2019. National Health and Morbidity Survey (NHMS).

Institute of Public Health of Malaysia. 2006. National Health and Morbidity Survey (NHMS).

Joshi, N.P., Vaidya, V.V., Pawar, S.S. & Gadgil, J.N. 2013. Development and validation of HPLC method for simultaneous determination of bio-active markers corosolic acid, asiatic acid and β-sitosterol from leaves of Lagerstroemia speciosa Linn. and from marketed formulation. International Journal of Pharmacy and Pharmaceutical Sciences 5(4): 223-226.

Kumar, V., Yadav, S.C. & Yadav, S.K. 2010. Syzygium cumini leaf and seed extract mediated biosynthesis of silver nanoparticles and their characterization. Journal of Chemical Technology & Biotechnology 85(10): 1301-1309.

Lee, K.H., Padzil, A.M., Syahida, A., Abdullah, N., Zuhainis, S.W., Maziah, M., Sulaiman, M.R., Israf, D.A., Shaari, K. & Lajis, N.H. 2011. Evaluation of anti-inflammatory, antioxidant and anti-nociceptive activities of six Malaysian medicinal plants. Journal of Medicinal Plants Research 5(23): 5555-5563.

Lim, W.Y.A., Chia, Y.Y., Liong, S.Y., Ton, S.H., Kadir, K.A. & Husain, S.N.A.S. 2009. Lipoprotein lipase expression, serum lipid and tissue lipid deposition in orally-administered glycyrrhizic acid-treated rats. Lipids in Health and Disease 8(1): 31.

Malnick, S.D. & Knobler, H. 2006. The medical complications of obesity. Journal of the Association of Physicians 99(9): 565-579.

Mohd Zainudin, M., Zakaria, Z., Nordin, M.M., Anita, N. & Othman, F. 2013. Does oral ingestion of Piper sarmentosum cause toxicity in experimental animals? Evidence-Based Complementary and Alternative Medicine 2013: 705950.

Ng, M., Fleming, T., Robinson, M., Thomson, B., Graetz, N., Margono, C., Mullany, E.C., Biryukov, S., Abbafati, C., Abera, S.F. & Abraham, J.P. 2014. Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: A systematic analysis for the global burden of disease study 2013. The Lancet 384(9945): 766-781.

Nune, S.K., Chanda, N., Shukla, R., Katti, K., Kulkarni, R.R., Thilakavathy, S., Mekapothula, S., Kannan, R. & Katti, K.V. 2009. Green nanotechnology from tea: Phytochemicals in tea as building blocks for production of biocompatible gold nanoparticles. Journal of Materials Chemistry 19(19): 2912-2920.

Ribeiro, I.A. & Ribeiro, M.H. 2008. Naringin and naringenin determination and control in grapefruit juice by a validated HPLC method. Food Control 19(4): 432-438.

Rukachaisirikul, T., Siriwattanakit, P., Sukcharoenphol, K., Wongvein, C., Ruttanaweang, P., Wongwattanavuch, P. & Suksamrarn, A. 2004. Chemical constituents and bioactivity of Piper sarmentosum. Journal of Ethnopharmacology 93(2-3): 173-176.

Russell, T.R. & Ho, R.J. 1976. Conversion of 3T3 fibroblasts into adipose cells: Triggering of differentiation by prostaglandin F2alpha and 1-methyl-3-isobutyl xanthine. In Proceedings of the National Academy of Sciences of the United States of America. pp. 4516-4520.

Shobha, C.R., Prashant, V., Akila, P., Chandini, R., Suma, M.N. & Basavanagowdappa, H. 2017. Fifty percent ethanolic extract of Momordica charantia inhibits adipogenesis and promotes adipolysis in 3T3-L1 pre-adipocyte cells. Reports of Biochemistry & Molecular Biology 6(1): 22.

Spalding, K.L., Arner, E., Westermark, P.O., Bernard, S., Buchholz, B.A., Bergmann, O., Blomqvist, L., Hoffstedt, J., Näslund, E., Britton, T. & Concha, H. 2008. Dynamics of fat cell turnover in humans. Nature 453(7196): 783-787.

Student, A.K., Hsu, R.Y. & Lane, M.D. 1980. Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes. Journal of Biological Chemistry 255(10): 4745-4750.

Subramaniam, V., Adenan, M.I., Ahmad, A.R. & Sahdan, R. 2003. Natural antioxidants: Piper sarmentosum (Kadok) and Morinda elliptica (Mengkudu). Malaysian Journal of Nutrition 9(1): 41-51.

Sultana, B., Anwar, F. & Ashraf, M. 2009. Effect of extraction solvent/technique on the antioxidant activity of selected medicinal plant extracts. Molecules 14(6): 2167-2180.

Sun, X., Chen, W., Dai, W., Xin, H., Rahmand, K., Wang, Y., Zhang, J., Zhang, S., Xu, L. & Han, T. 2020. Piper sarmentosum Roxb.: A review on its botany, traditional uses, phytochemistry, and pharmacological activities. Journal of Ethnopharmacology 263: 112897.

Suzawa, M., Takada, I., Yanagisawa, J., Ohtake, F., Ogawa, S., Yamauchi, T., Kadowaki, T., Takeuchi, Y., Shibuya, H., Gotoh, Y. & Matsumoto, K. 2003. Cytokines suppress adipogenesis and PPAR-gamma function through the TAK1/TAB1/NIK cascade. Nature Cell Biology 5(3): 224-230.

Tang, Q.Q., Otto, T.C. & Lane, M.D. 2003. Mitotic clonal expansion: A synchronous process required for adipogenesis. In Proceedings of the National Academy of Sciences. pp. 44-49.

Thieringer, R., Balkovec, J.M., Chen, H.Y., Frazier, E., Le Grand C.B., Li, Z., Metzger, J.M., Mundt, S.S., Nunes, C.N., Strowski, M., Olson, S.H., Robertson, N., Strack, A.M., Schaeffer, J.M., Wright, S.D., Zhang, B.B. & Hermanowski-Votska, A. 2004. A novel non-steroidal inhibitor of 11beta-hydroxysteroid dehydrogenase type 1 improves features of metabolic syndrome in murine disease models. 23^rd Joint Meeting of the British Endocrine Societies with the European Federation of Endocrine Societies. Endocrine Abstract 7: P47.

Ugusman, A., Zakaria, Z., Hui, C.K., Nordin, N.A.M.M. & Mahdy, Z.A. 2012. Flavonoids of Piper sarmentosum and its cytoprotective effects against oxidative stress. EXCLI Journal 11: 705.

Wang, S.J.Y., Birtles, S., De Schoolmeester, J., Swales, J., Moody, G., Hislop, D., O’Dowd, J., Smith, D.M., Turnbull, A.V. & Arch, J.R.S. 2006. Inhibition of 11β-hydroxysteroid dehydrogenase type 1 reduces food intake and weight gain but maintains energy expenditure in diet-induced obese mice. Diabetologia 49(6): 1333-1337.

Wang, Z.G., Mi, J., Wang, X.R., Huo, Y.Y., Peng, Y.J., Zhang, H.M., Gao, Y. & Zhang, H.L. 2018. A new cinnamic acid glycoside from roots of Heracleum dissectum. Natural Product Research 32(2): 133-140.

Wongsa, P., Chaiwarit, J. & Zamaludien, A. 2012. In vitro screening of phenolic compounds, potential inhibition against α-amylase and α-glucosidase of culinary herbs in Thailand. Food Chemistry 131(3): 964-971.

WHO. 2020. Obesity and overweight. World Health Organization (WHO). https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight#:~:text=In%202016%2 C%2039%25%20of%20adults,tripled%20between%201975%20and%202016.

Yoon, S.O., Kim, M.M., Park, S.J., Kim, D., Chung, J. & Chung, A.S. 2002. Selenite suppresses hydrogen peroxide-induced cell apoptosis through inhibition of ASK1/JNK and activation of PI3-K/Akt pathways. The FASEB Journal 16(1): 111-113.

Yusof, A.M., Abd Ghafar, N., Kamarudin, T.A., Hui, C.K. & Yusof, Y.A.M. 2016. Gelam honey potentiates ex vivo corneal keratocytes proliferation with desirable phenotype expression. BMC Complementary and Alternative Medicine 16: 76.

Zebisch, K., Voigt, V., Wabitsch, M. & Brandsch, M. 2012. Protocol for effective differentiation of 3T3-L1 cells to adipocytes. Analytical Biochemistry 425(1): 88-90.

^*Pengarang untuk surat-menyurat; email: norzana@ukm.edu.my