Cytotoxicity Effect of Zingiber officinale, Curcuma aeruginosa and Curcuma xanthorhiza Extracts on Adipose-Derived Stem Cells (ADSCs) Rilianawati 1, Mutia Hardhiyuna 1, Angelia Yulita 1, Ago Halim 2 1 Center of Pharmaceutical and Medical Technology Agency for the Assessment and Application of Technology 2 JMB Clinic, Jakarta Corresponding Author Email: mutia.hardhiyuna@bppt.go.id Abstract In the past few decades, attention and research in the field of stem cell are progressing very rapidly. Hospitals in Indonesia have been using stem cells as an alternative to cure some illnesses like diabetes, heart disease, fractures and joints, dental implants, and asthma. The purpose of this research was to characterized and to produced stem cells from human subcutaneous adipose tissue (ADSC), which can be used for assessing cytotoxicity effects potency of Zingiber officinale (red ginger), Curcuma aeruginosa (temu ireng) and Curcuma xanthorhiza (temulawak) on ADSC proliferation. The cytotoxic effect was done using MTT assay. The results showed Z. officinale var. and C. xanthorhiza Roxb. extracts have no cytotoxic effect to ADSC at 25 ppm, 50 ppm and 100 ppm concentrations, however the proliferation inhibition of Z. officinale extracts gradually rises to 0.05 % at 200 ppm and slightly fall at 600 ppm, after that it sharply raises unto 74.84% at 1000 ppm. The proliferation inhibition of C. xanthorhiza extracts at 200 ppm is about 28% and significantly increase to 90 % at 1000 ppm which is the highest percentage on the figure. The C. aeruginosa extracts has no inhibition effect to ADSC from 25 ppm until 800 ppm concentration of extract, however it modestly goes up at 1000 ppm reaching 9% of inhibition. Further studies are required to investigate the metabolites in these extracts that are highly correlated with its toxic effects. Key word: adipose derived stem cells, adherent stem cells, cytotoxicity, plant extract Introduction Research activities towards the development of traditional medicines like Jamu as standardized extracts and phytopharmaca have been initiated and attracted public interest for Volume 8, No.1, Agustusn 2015 19
finding herbal medicinal plant species to cure major diseases such as cancer. Zingiber officinale (red ginger), Curcuma xanthorhiza (temulawak) are Indonesian medicinal plants commonly used as traditional folk medicine in Indonesia which has been widely known having potential anticancer activity (Batugal, 2004; Kirana, 2003), while Curcuma aeruginosa (temuireng) has less anticancer activity (Kirana, 2003). Mesenchymal stem cells (MSC) are a prospective object for the use in cell therapy and intensely studied by many research groups. MSC are characterized primarily by expression of surface markers and differentiation potential. MSC express a series of specific markers (CD44, CD90, CD105, CD13, etc.) and should differentiate into cells of mesodermal origin such as adipocytes, osteoblasts and chondroicytes. Due to prospective clinical application, MSC from different sources are actively studied. For example, MSC isolated from the bone marrow and fat tissue (Musina, 2006). Due to the easy accessible anatomical location and the abundant existence of subcutaneous adipose tissue, ADSC hold the advantage of a simple and above all less invasive harvesting technique. Some studies have been conducted to explore the potential of toxicity of Zingiber officinale (red ginger), Curcuma aeruginosa (temuireng) and Curcuma xanthorhiza (temulawak). These plants are well-known Indonesian medicinal herb that was used extensively as anticancer agents. However, the influence of these three extracts on the cytotoxic effect of stem cell have not been widely explored. Therefore, on this study to investigated the cytotoxic effects of Curcuma xanthorhiza Roxb., Zingiber officinale var. and Curcuma aeruginosa on human Adipose-Derived Mesenchymal Stem Cells (ADSCs) derived from lipoaspirate using MTT assay. 1. Methodology 1.1. Plant Extraction The crude extracts of Z. officinale, C. aeruginosa, and C. xanthorrhiza were obtained from the collection of the Laboratory of Pharmaceutical and Medical Technology, Agency of the Assessment and Application of Technology, Indonesia. The extracts were fractionated using ethanol and dissolved with DMSO prior to use for cells treatment. 1.2. Isolation of Adipose-Derived Stem Cells (ADSCs) The human ADSCs were isolated from fat tissue which obtained from JMB Clinic in Jakarta, Indonesia. The contaminating layers were removed, the remaining adipose tissue was washed using an autoclaved PBS. The washed adipose was transferred into a sterile tube, then enzyme digestion was used. The mixture was incubated in 5% CO 2 and 37 o C incubator for an Volume 8, No.1, Agustusn 2015 20
hour. The layers formed at the end of incubation time: an upper adipose layer and an infranatant of enzyme. The upper layer was aspirated off and the remaining was transferred into a new sterile tube. The filtered infranatant was then centrifuged at 1200 rpm for 10 minutes. Supernatant was discarded and the pellet was collected. Pellet was resuspended in lysis buffer and was centrifuged at 1200 rpm for 10 minutes. Stromal vascular fraction (SVF) pellet was resuspended in 1 ml of culture media. Stem cells in SVF were seeded in 75 cm 2 flasks containing 10 ml of culture media. The cells were incubated in 5% CO 2 incubator at 37 o C and allowed to grow confluently. 1.3. Cell Counting Using Haemocytometer Stem cells were trypsinized then plated to 96 well-plates after confluently. This process was initiated by removing the culture media from the flask and 2 ml of PBS was added. The flask was gently shaken then the liquid was discarded. Stem cells were trypsinized with Trypsin- EDTA. Subsequently, Stem cells were incubated in a 5% CO 2 incubator at 37 o C for 5 minutes. The culture media served as a stop solution to cease the trypsin activity. Following the addition of stop solution, stem cells were transferred into a new tube and centrifuged at 1200 rpm for 10 minutes at 20 o C. The resulting supernatant was discarded and the pellet was resuspended in culture media. A 10 µl volume was taken from the resuspended pellet and mixed with 10 µl of trypan blue. The mixture was loaded to a hemocytometer then counted under a microscope. The desired concentration of cells was 10 5 cells per 100 µl, for MTT assay. 1.4. Plating and Culture of ADSCs The ADSCs were plated on 96 well-plates respectfully for MTT assay. Stem cells were plated on 96 well-plates for 1 x 10 5 cells/well. 96 well-plates were incubated in a 5% CO 2 incubator at 37 o C for 24 hours to allow the stem cells to adherent. 1.5. Treatment of ADSCs with Plant Extracts Stem cells were treated with different concentration of Z. officinale, C. aeruginosa, and C. xanthorrhiza extracts (25 ppm, 50 ppm, 100 ppm, 200 ppm, 400 ppm, 600 ppm, 800 ppm, and 1000 ppm). Each concentration was prepared in triplicate wells for MTT assay. Culture media Volume 8, No.1, Agustusn 2015 21
without stem cells and stem cells in culture media without addition of extracts were used as controls. 1.6. Cell Cytotoxicity Assay The cells containing plant extracts were incubated for 24 hours. Hence, MTT reagent was added directly to the cells followed by incubation of the cells. The insoluble colored formazan was dissolved in SDS and incubated for a night. Cells were processed on ELISA reader for absorption at 570 nm. 2. Result 2.1. Isolation of Adipose-Derived Stem Cells (ADSCs) Adipose tissue was considered as a rich source of stem cells, especially with the increased incidence of obesity in modern populations (Kuhbier, 2010). In comparison, 1 g of adipose tissue has greater number of MSCs than 1 g of bone marrow (Kitagawa, 2006). Isolation ADSC involves centrifugation, digestion, and filtration, resulting in an adherent cell population containing mesenchymal stem cells. Figure 1 shows the Adipose-Derived Stem Cells (ADSC) which isolated using enzyme digestion and incubated for 4 days. ADSC was observed under inverted microscope after treated by Z. officinale var., C. xanthorhiza Roxb. and C. aeruginosa extracts and MTT solution (Fig.1). Addition of 1000 ppm concentration of extracts sharply reduced number of ADSC, especially in the addition of Curcuma xanthorhiza Robx. (temulawak) extract which inhibit 90% proliferation of ADSC. This cells were mostly detached, grew poorly, lost normal spindle shape and acquired round and collapsed appearance. Volume 8, No.1, Agustusn 2015 22
control 1: culture media without ADSC control 2: ADSC without addition of extracts. Red Ginger 25 ppm Temulawak 25 ppm Temuireng 25 ppm Red Ginger 1000 ppm Temulawak 1000 ppm Temuireng 1000 ppm Figure 1. ADSC treated by plant extracts 2.2. Cytotoxixity Effects of Zingiber officinale, Curcuma aeruginosa and Curcuma xanthorhiza on ADSC proliferation. Stem cell survival was evaluated by MTT test. Cytotoxicity effect of three different herbal medicine extracts were evaluated (Fig.2). Z. officinale var. and C. xanthorhiza Roxb. extracts mostly inhibited ADSC proliferation, in contrast with Curcuma aeruginosa which almost has no inhibition effect to ADSC proliferation. Figure 2. shows that Z. officinale var. and C. xanthorhiza Roxb. extracts have no cytotoxic effect to ADSC at 25 ppm, 50 ppm and 100 ppm concentrations, however the proliferation inhibition of Z. officinale extracts gradually rises to 0.05 % at 200 ppm and slightly fall at 600 ppm, after Volume 8, No.1, Agustusn 2015 23
that it sharply raises unto 74.84% at 1000 ppm. The proliferation inhibition of C. xanthorhiza extracts at 200 ppm is about 28% and significantly increase to 90 % at 1000 ppm which is the highest percentage on the figure. The C. aeruginosa extracts has no inhibition effect to ADSC from 25 ppm until 800 ppm concentration of extract, however it modestly goes up at 1000 ppm reaching 9% of inhibition. Volume 8, No.1, Agustusn 2015 24
Figure 2. Comparison of ADSC proliferation inhibition by C. xanthorhiza (temulawak), Z. officinale (jahe merah) and C. aeruginosa (temuireng) extracts Overall, the higher concentration of extracts showing the greater proliferation inhibition of ADSC. C. xanthorhiza extracts was the most cytotoxic followed by Z. officinale. Lower concentration of C. xanthorhiza and Z. officinale extracts have no effect for proliferation of ADSC (<100 ppm), meanwhile high concentration of both extracts (>100 ppm) have inhibition effect on proliferation of ADSC. According to reference (Batugal, 2004), these extracts were potential as an anticancer, according to this study these extracts were cytotoxic for adiposederived stem cell. According to reference (Kirana, 2003), C. aeruginosa was less potential for anticancer, it is in line with this study that it has potential to stimulate ADSC proliferation. 3. Conclusion This study showed that C. xanthorhiza Roxb., and Z. officinale var. extracts have potency as cytotoxic agent by inhibiting the proliferation of ADSC at greater than 100 ppm concentration of extracts. However, C. aeruginosa has potency effect to stimulate the proliferation of ADSC at low and high concentration and can be used in ingredient of jamu. Further studies are required to investigate the metabolites in these extracts that are highly correlated with its toxic effects. 4. References Fraser JK, Wulur I, Alfonso Z, Hedrick MH. 2006. Fat Tissue: An Underappreciated Source of Stem Cells for Biotechnology. Trends Biotechnol. 24: 150 154. Kuhbier JW, Weyand B, Radtke C, and Vogt PM. 2010. Isolation, Characterization, Differentiation, and Application of Adipose-Derived Stem Cells. Adv Biochem Engin/Biotechnol. 123:55-105 Kirana, Chandra. 2003. Potential Anticancer Activity in Rhizomes of Ginger Species (Zingiberaceae family). Thesis. University of Adelaide, Dept of Medicine & Dept. of Horticulture, Viticulture and Oenology. Australia. Kitagawa Y, Korobi M, Toriyama K, Kamei Y, Torii S. 2006. History of Discovery of Human Adipose-Deived Stem Cells and Their Clinical Applications. Jpn J Plast Reconstr Surg. 49:1097 1104. PA Batugal, J Kanniah, Lee SY and JT Oliver, editors. 2004. Medicinal Plant Research in Asia, volume I: The Framework and Project Work. International Plant Genetic Resources Institute. Volume 8, No.1, Agustusn 2015 25
R. A. Musina, E. S. Bekchanova, A. V. Belyavskii*, and G. T. Sukhikh. 2006. Differentiation Potential of Mesenchymal Stem Cells of Different Origin. Cell Technologies in Biology and Medicine, 2(1):147-151. Safety Data Sheets (SDS), Certificates of Analysis. www.lifetechnologies.com/support. 2013. Life Technologies Corporation. Volume 8, No.1, Agustusn 2015 26