Menurutnya, organisasi merupakan salah satu komponen penting dalam membangun karakter personal dari masing – masing mahasiswa. Beliau menceritakan, bagaimana pengalamannya dalam menjalani hari – hari sebagai mahasiswa, salah satunya ketika beliau menjabat sebagai Ketua Senat Biologi (sekarang Badan Eksekutif Mahasiswa) yang bergerak dan mewadahi kegiatan kemahasiswaan. “Saya ga pinter-pinter amat dalam bidang Biologi, jadi ya saya rasa saya harus mengembangkan diri dan yang penting lulus cepat”, sahut Pak Ario.

Inilah yang menarik dari yang beliau sampaikan, kalau persaingan ke depan di dunia, bukan hanya pada akademik, tapi juga persaingan waktu. Saat ini, persaingan yang terjadi selain membutuhkan kualitas sumberdaya manusia, juga harus tepat dalam mengatur waktu yang kita punya, kuncinya pada manajemen waktu. Dalam sesi ini, selain berbagi cerita tentang pengalamannya dimasa kuliah, beliau juga menyampaikan pengalamannya dalam dunia kerja. Mulai dari terlambat mengikuti test lowongan pekerjaan, hingga mempertahankan idealisme ditempat kerjanya, disekolahkan di luar negeri oleh perusahaan, hingga sukses membangun cabang perusahaan menjadi pengalaman yang sangat menarik ketika beliau bercerita.

“Wether you ready or not, uncertainty will come…” sambung Pak Ario. Ketidaktentuan itu menjadi kesempatan kita untuk dapat melakukan perubahan yang sesuai dengan prospek serta rencana yang kita atur sebelumnya. Goal setting kita sedari sekarang sudahlah harus diatur, seperti apa kita akan menghadapi masa depan. “Apa yang membentuk kita dapat kita prediksi dari apa yang kita baca, dan dengan siapa kita bergaul..” sambungnya. Dalam mencapai kesuksesan pun, kita perlu adanya keberanian, berani untuk berinovasi, berani untuk mengambil resiko dan berani untuk keluar dari comfort zone kita. Inilah yang dimaksud dengan berani merubah mindset kita yang masih dalam kondisi kekinian. Prinsip ini berlaku juga dalam mengatur kehidupan sosial masyrakat kita, “Everyone is a customer. If you don’t serve it, the others will…” kata Pak Ario. Sudah sepantasnya kita memperlakukan semua orang dengan pelayanan terbaik yang kita miliki.

Berbekal pengalaman ini, saat ini beliau sudah menjadi freelance trainer yang bergerak dalam training dan seminar motivasi, lebih khusus pada beberapa bidang yakni competitive marketing, marketing communication, marketing management, international marketing, leadership, change management dan menjadi President dalam sebuah perusahaan terbatas yang bergerak dibidang bisnis. Selain itu, berbekal pengetahuan selama menimba ilmu di Fakultas Biologi ini, Pak Ario juga menjadi Dosen Pengajar di Post Graduate Studies MMR UGM, yang bergerak dalam pengkajian manajemen rumah sakit.

Ada empat hal menarik yang beliau sampaikan, sebagai pesan penutup bagi kita selaku mahasiswa “Kerja-kerja kita perlulah disesuaikan dengan 4 “AS” berikut : kerja dengan ker-as, kerja dengan cerd-as, kerja dengan tunt-as, dan kerja dengan ikhl-as….“. Inilah yang membekali kita menjadi pribadi – pribadi yang dapat merubah diri kita sendiri, berproses menuju hal yang lebih baik lagi.

Pada acara ini juga diawali dengan Pemberian Bantuan Pendidikan bagi 12 Mahasiswa aktif Fakultas Biologi UGM, serta Launching Buku Ekpedisi Biodiversitas Tropika dari Kelompok Studi yang ada di Fakultas Biologi UGM. Launching buku ini dibersamai oleh Wakil Dekan Bidang Riset dan Alumni Dr. Endang Semiarti, M.S., M.Sc.

Harapannya dengan disampaikannya bantuan, sharing motivasi, serta adanya launching buku Ekspedisi Biodiversitas Tropika ini, dapat memberikan inspirasi dan menjadi bara api perubahan bagi kita sebagai civitas akademika  Fakultas Biologi UGM, untuk senantiasa mengasah kepekaan, menajamkan riset, dan menggunakan potensi kita sebagai institusi dengan lebih baik lagi.(raas)

Jaya Almamater!

Gambar 1. Foto bersama dengan narasumber (Drs. Ario Setra Setiadi, MM, Ph.D. CPM, DipM ACIM

Edukasi mangrove dan biota laut ini dilakukan oleh mahasiswa Biologi yang tergabung dalam Kelompok Studi Kelautan (KSK) yang bekerjasama dengan SDN Pasir Mendit dengan mengajari anak-anak kelas 3 hingga kelas 5 tentang bagaimana pentingnya laut bagi kehidupan manusia. Murid-murid kelas 3 hingga kelas 5 ini sangat antusias dalam menyambut acara yang diberikan oleh kakak-kakak KSK ini, bahkan anak-anak kelas 1 dan kelas 6 pun turut berpartisipasi menonton dan belajar bersama. Menurut salah satu anak sd kelas 3 SD yang pendiam “aku senang di ajarin sama mbak dan masnya, seneng bisa belajar laut” seru Bima sambil tersenyum polos. Tidak hanya belajar di dalam ruang kelas, kami juga membuat stand di lapangan sekolah yang terdiri dari lima kelas keilmuan KSK yaitu Algae, Crustacea, Echinodermata, Mollusca, dan Pisces. Stand ini diharapkan dapat memberikan wawasan lebih tentang biota laut dengan suasana berbeda karena KSK menghadirkan langsung biota asli dari Laut yang di koleksi untuk pembelajaran.

Gambar 1. Edukasi diruang kelas bercerita tentang keunikan laut di dunia

Gambar 2. Saat anak-anak sedang diluar menyebar berkelompok masuk ke tiap stand kelas kelimuan

Gambar 3. Foto KSK bersama anak-anak SDN Pasir Mendit

Rangkaian selanjutnya adalah magrove replant yang dilaksanakan pada tanggal 21 April 2013 di Pasir Mendit, Kulon Progo, Yogyakarta. Kegiatan ini bekerja sama dengan beberapa lembaga dan kelompok pecina lingkungan. Acara yang terbuka untuk umum ini dihadiri oleh sekitar 150 peserta yang terdiri dari berbagai elemen. Tidak hanya civitas biologi UGM saja, akan tetapi umum bagi siapa saja baik dari dalam civitas UGM maupun luar UGM. Peserta yang berpartisipasi antara lain INSTIPER, KANOPI, UIN, UNY, WALHI dan mahasiswa UGM dari berbagai fakultas. Tidak hanya dari wilayah Yogyakarta, acara ini dihadiri pula peserta dari UNS. Cuaca yang sangat terik tidak memadamkan semangat para peserta untuk menanam mangrove. Saat acara berlangsung peserta melewati jalur-jalur menuju tempat penanaman yang telah disiapkan oleh panitia. Pada tiap jalur tersebut terdapat stand atau pos yang telah terisi oleh panitia yang bertugas di pos tersebut. Tiap-tiap pos tersebut memberikan materi yang berbeda-beda serta games yang seru dan membutuhkan kekompakan. Hal ini  dimaksudkan agar peserta memiliki rasa kesatuan untuk membangn dan menjaga bumi ini bersama. Setelah sampai pada tempat penanaman peserta bisa dapat langsung terjun menanam bibit 1000 bibit mangrove. Dengan 1000 bibit mangrove yang ditanam semoga dapat memberikan goresan perbaikan bumi walau kecil. Karena yang tepenting dalam sebuah penanaman adalah “Bukan berapa banyak yang ditanam tapi berapa banyak yang tumbuh” (Tjut, 2011).

Gambar 4. Peserta saat menanam mangrove

Diumumkan kepada mahasiswa yang mengambil mata kuliah seminar bahwasanya akan diadakan pertemuan pada:

Hari Jumat, tanggal 19 April 2013 jam 08.00 WIB di Ruang I.

Atas perhatiannya diucapkan terimakasih.. (a.n. Pengelola Seminar Dr. Maryani, M.Sc.)

 

1. Tumbuh dan berkembangnya kemampuan masyarakat Desa Kemadang dalam mengembangkan potensi bidang ekonomi, dan sosial budaya, khususnya bidang pertanian tanaman melon dan hortikultura, kelautan, usaha mikro, kecil dan menengah (UMKM),  dan kepariwisataan secara berkelanjutan;
2. Terwujudnya peningkatan kesejahteraaan masyarakat Desa Kemadang pada khususnya dan masyarakat Gunungkidul pada umumnya.
3. Terlaksananya upaya pelestarian lingkungan di lahan pantai Porok, Desa Kemadang;
4. Terlaksananya upaya pengembangan komoditas unggulan Desa Kemadang;
5. Terwujudnya peningkatan kesejahteraaan masyarakat Desa Kemadang pada khususnya dan masyarakat Gunungkidul pada umumnya.

Di sela-sela pertemuan ini Pak Syaref juga mempromosikan Kebun Buah Nglanggeran Gunung Kidul. Supriyadi juga menambahkan,”Rencananya embung di area Kebun Buah akan dikembangkan menjadi daerah wisata yang menarik, dan Fakultas Biologi UGM diharapkan dapat menjadi mitra kerjasama dalam mewujudkan gagasan tersebut”.  Dalam perjalanan pulang menuju kampus UGM, rombongan Fakultas Biologi diajak berkunjung sebentar di area Kebun Buah Nglanggeran Gunungkidul buah yang dilatarbelakangi dengan pemandangan yang indah, seperti dapat dilihat pada foto berikut :

Foto 1. Penandatangan Naskah Kerjasama Fakultas Biologi UGM dengan BAPPEDA Gunungkidul DIY dan Distanhor Gunungkidul DIY, Senin, 8 April 2013, Gunungkidul Yogyakarta

Foto 2. Embung Nglanggeran dengan latar belakang gugus gunung purba yang berlokasi di Kebun Buah Nglanggeran

Foto 3. Kawasan Kebun Buah Nglanggeran

Selamat kepada dosen muda Fakultas Biologi UGM Lisna Hidayati, M.Biotech. sebagai salah satu pemenang dalam kompetisi kewirausahaan yang diselenggarakn oleh Kementrian Koperasi dan UMKM Republik Indonesia. http://www.spiritgkn.com/

Daftar pengumuman pemenang bisa dilihat di tautan/link di bawah ini dalam bentuk file pdf.

Daftar Pengumuman Pemenang

Gambar 1. Penyerahan kenang-kenangan dari Fakultas Biologi UGM oleh Dr. Endang Semiarti kepada Direktur IBRC, Prof.Dr.drh. IGNK Mahardika.

Indonesia memiliki keterbatasan dalam hal jumlah penelitian dan publikasi keanekaragaman hayati di negaranya sendiri. “Berdirinya IBRC diharapkan dapat memberikan rekomendasi bagi pemerintah Indonesia dalam mengelola sumberdaya alam Indonesia. IBRC terbuka bagi para peneliti dari luar lembaga IBRC termasuk dosen dan mahasiswa untuk mengadakan penelitian di  IBRC“, demikian diungkapkan Prof. Dr. drh. I Gusti Ngurah Kade Mahardika, Guru Besar Fakultas Kedokteran Hewan, Universitas Udayana yang juga salah satu inisiator berdirinya IBRC dalam acara Stadium General yang berjudul Penyegaran Biologi Molekuler dan Pengantar Bioinformatika di Ruang Sidang Bawah Fakultas Biologi UGM, Senin, 1 April 2013. Stadium General ini diikuti secara antusias oleh civitas academica Fakultas Biologi UGM, dosen dan mahasiswa serta civitas academica dari Fakultas lain di lingkungan UGM. Stadium general ini sekaligus membuka rangkaian acara kursus bioinformatika mengenai filogenetik yang dilaksanakan selama 5 hari mulai Senin, 1 April 2013 – Jumat, 5 April 2013 dan dipandu oleh para instruktur peneliti dari IBRC, diikuti dengan diskusi MoU kerjasama antara IBRC dan Fakultas Biologi UGM. (SA)

Abstract

Genus Dendrobium is widely known as traditional medicine that has potency as antihyperglicemia, antiinflammation, and anticancer. This genus has a lot of antioxidant compounds that potencial as anticancer. Dendrobium crumenatum Swartz. (pigeon orchid), one of tropical orchids is not widely explore yet. This orchid is guessed having anticancer potency. Thus this research was aimed to study the effect of ethanolic extract of Dendrobium crumenatum on growth inhibition and apoptosis induction on HeLa cells, as well as antiangiogenesis activity on chorio allantoic membrane (CAM) of chicken. Cell growth inhibition was done using MTT assay. Apoptotic assay was done using double staining method (ethidium bromide-acridine orange). Antiangiogenesis test was done on chorio allantoic membrane of chicken with bFGF as inductor of angiogenesis. The results showed that the IC₅₀ of three part of D. crumenatum i.e leaves, pseudobulb, and whole plant on HeLa cell growth inhibition were 153.828; 166.284; and 192.298 µg/mL respectively. The apoptosis induction and antiangiogenesis activity were done using leaves ethanolic extract of D. crumenatum because of the smallest IC₅₀ value. The percentage of apoptotic cells on the treatment showed pattern based on concentration. Nevertheless at the highest concentration of leaves ethanolic extract of D.crumenatum the mean of apoptotic cells just 27.69 ± 10.22%. In this research angiogenesis was induced by bFGF as a positive control. Antiangiogenesis activity of leave ethanolic extract of D. crumenatum showed that inhibition value was 64.77% at the concentration of 76.914 µg/mL. Based on this research it could be concluded that ethanolic extract of D. crumenatum has potency as anticancer agent.

 

Keywords: Type your keywords not more than 5 keywords 

 

INTRODUCTION

Several kind of orchids are believed can curing tuberculosis, gastric and duodenal ulcer, impotence, cough, malaria, fever, nervous diseases, headaches, tumor, antiinflammation, anticancer, and increase fertility [1, 2, 3, 4]. One of the orchid’s genus that most used as medicine is Dendrobium. Dendrobium has a lot of compounds such as polysaccharide, bibenzyls, phenanthrenes, fluorenones, simple aromatic acids [5], 1,4,5-tihydroxy-7-metoxy-9H fluoren-9-one, dendroflorin, denchrysan [6], denthyrsin, denthyrsinol, denthyrsinone, denthyrsinin [7], and moscatilin [8]. Those compounds have antioxidant activities, potential as antitumor and anticancer. Dendrobium has anticancer activities both in vivo and in vitro. Wang et al. [9] reported that different polysaccharides fraction of Dendrobium nobile could inhibit cell growth of Sarcoma 180 cells and HL-60 leukemia cells. Chen et al. [6] stated that compounds from Dendrobium chrysotoxum have potency to inhibit the growth of human leukemia K562, HL-60 cells, human lung adenocarcinoma A 549, human hepatoma BEL-7402 and human stomach cancer SGC-7901. Dendrobium thyrsiflorum contain several compounds which showing significant cytotoxicity toward HeLa, K562, and MCF-7 cells [7]. Moscatilin, derivative from bibenzyl, one of bioactive compounds of Dendrobium loddigesii could suppress tumor angiogenesis [8].

Cancer growth involved angiogenesis or neovaskularisasi. It is a process of blood vessel forming from the former blood vessels in the tissues which enables cancer cells to gain nutrien and oxygen supply from host cells [10,11]. Angiogenesis is a complex process which involves the balance between proangiogenic and antiangiogenic factors [12]. Fibroblast growth factor (FGF) can stimulate angiogenesis and cells proliferation in vivo [13]. Basic fibroblast growth factor (bFGF) is a potential inductor for endothelial cells proliferation and migration, as well as blood capillar tube formation in vitro. This growth factor also plays a role as a promoter on blood vessel repair in wounded tissue and in angiogenesis process which is induced by tumor in vivo [14]. Basic fibroblast growth factor is active during angiogenesis procces of tumor formation. This growth factor has important role on the increasing of VEGF receptor density and production on the blood capilar endothel cells [13,14].

D. crumenatum Swartz. commonly known as pigeon orchid or sparrow orchid is one kind of special orchids in the tropical country. In an empirical manner, this orchid is popular as traditional medicine material. However its potency is not completely study yet. This orchid is guessed having potency as anticancer. So far, there is no research to give evidence that D. crumenatum can be use as anticancer. Thus, this research was aimed to study the effect of ethanolic extract of Dendrobium crumenatum on growth inhibition and apoptosis induction on HeLa cells, as well as antiangiogenesis activity on chorio allantoic membrane (CAM) of chicken.

MATERIALS AND METHOD

1. Plant material and preparation of plant extract

D. crumenatum was collected from Yogyakarta. The orchid which is used in this research have been flowered and collected on April, 2011 (rainy season). The D.crumenatum was devided into adult leaves, adult pseudobulb, and whole plant. Sample extraction were done by maceration methods [15], using 96% ethanol as solvent. The orchid was rinsed using tap water and air dried. After that the sample was cut into pieces and dried in the oven 50°C. Dried sample was powdered become simplicia using blender. Ten gram simplicia put into the Erlenmeyer and added 250 mL 96% ethanol. The sample was kept for 24 h and stir occasionally. After 24 h, supernatan were filtrated and obtained filtrate I. This procces was repeated twice using residue of the extract and obtained filtrate II and III. Those three filtrates were mixed in the porcelain dish and air dried using fan. Ethanolic extract of D. crumenatum were stored in the sterile flacon at the 4°C refrigerator.

2. Cell growth inhibition and apoptotic assay of ethanolic extract of D. crumenatum on HeLa cells

a. Cell growth inhibition assay

HeLa cells were seeded into 96 well microplate (2 x 10⁴ cells/100 mL/well). Each treatment was made triplicate. Groups of this research were:

1. Medium control group (HeLa cells suspension were incubated in the RPMI 1640 complete media with 10% FBS). It was used as negative control.
2. Solvent control (HeLa cells suspension + media + DMSO). It was used to make sure that the result of this research was caused by Dendrobium crumenatum ethanolic extract not because of the solvent. Within this research we used 0.1; 0.2; 0.4, and 0.8% DMSO.
3. Positive control groups (HeLa cells suspension + media + Doxorubicine): the concentrations of doxorubicine which were given to the each cells suspension: 3.125; 6.25; 12,5; and 25 mg/mL.
4. Treatment groups: the concentration of Dendrobium crumenatum ethanolic extract (leaves, pseudobulb, and whole plant) which was given to the each cells suspension was: 12,5; 25; 50; 100; 200; 400; and 800 mg/mL. Each well was added by 100 mL of Dendrobium crumenatum ethanolic extract.

HeLa cells
were incubated at 37 ºC in a humidified atmosphere with 5% CO₂. Cells were permitted to adhere for 24 h. Then into each of wells was given the test solution according to its groups and cells were incubated at 37 ºC, CO₂ in a humidified atmosphere with 5% CO₂. Cells were permitted to adhere for 24 h. Cell growth inhibiton assay were done by MTT assay as described by [16, 17]. At the end of incubation period, each wells was added with MTT (5 mg/mL) 10 mL. Then cells suspension were incubated at 37 ºC, CO₂ in a humidified atmosphere with 5% CO₂. Cells were permitted to adhere for 4 h. SDS 10% in 0,1 N HCl, 100 mL/well was added as stop reaction. Optical Density (OD) at 550 nm was determined by ELISA reader. The inhibitory rate of cell proliferation was calculated by the following formula:

Cell growth inhibition (%) = OD medium control – OD treatment x 100%

OD medium control

The result of IC₅₀ of cell growth inhibition was used as the base for apoptotic assay and antiangiogenesis test.

b. Apoptotic assay

Apoptotic assay was done using solution mixture of ethidium bromide-acridine orange. For apoptotic assay HeLa cells were incubated into 24 well microplate on coverslip. Each treatment was made triplícate. HeLa cells were incubated at 37 ºC in a humidified atmosphere with 5% CO₂. All groups were incubated for 24 h. After the period of incubation solution in the well was poured, coverslip was take and put on the glass object. Five to ten microliter of etidium bromide-acridine orange were dropped on coverslip. Slides were observed under light microscope with 10×10-10×40 magnification. Intact cell nucleus with bright green color was a viable cell. Cell nucleus showing chromatin condensation with orange color was an apoptotic cell [18]. The amount of apoptotic cells are count every 100 cells (represent as %). The apoptotic assay were done for leaves ethanolic extract of Dendrobium crumenatum Swartz. based on the IC₅₀ value.

3. Antiangiogenesis activity on the chorio allatoic membrane

Antiangiogenic activity was done using 11^st day embryonated eggs of chicken which were obtained from Ngaglik, Yogyakarta. bFGF was used as inductor of angiogenesis. Extract and bFGF were implanted on the chorio allantoic membrane (CAM) of chicken embrio. In this research there were 8 treatment groups as follow (each group was made triplicate):

a)  Group A: CAM with paper disc implantation which contain 30 µL buffer solution Tris-HCl 10 mM pH 7,5;

as paper disc control.

b)  Group B: CAM with paper disc implantation which contain bFGF 30 ng, as control group of bFGF or

positive control group.

c)  Group C: CAM with paper disc implantation which contain bFGF 30 ng + 30 µl DMSO 0.8%; as bFGF +

solvent control group.

d)  Group  D,  E,  F,  G  and H  were the treatment groups. The concentration of this test based on the IC₅₀ value

of leaves extract. CAM with paper disc implantation which contain bFGF 30 ng + leaves ethanolic extract of

D. crumenatum with 5 series of concentration there were ½ IC₅₀ (76.914  µg/mL), ¾ IC₅₀ (115.371  µg/mL),

IC₅₀ (153.828 µg/mL), 1¼ IC₅₀ (192.285 µg/mL), and 1½ IC₅₀ (230.742 µg/mL).

Firstly the 10^th day embryonated eggs were incubated in the incubator with the temperature of 39ºC and relative humidity 60% for eggs conditioning. At the 11^st day these eggs were implanted as above research design. The eggs were incubated again for 72 h at the 39ºC and relative humidity 60%. After 72 h (at the 14^th day), the eggs were removed to the refrigerator for one day to inactive the embryo metabolism and to freeze the blood capillars. After one day in the refrigerator, blood vessels which were attached on the chorio allatoic membrane detached carefully from the egg shell and sticked on the filter paper and then fixed in 4% formaldehyde. The new formed blood vessels were examined using stereo microscope. Evaluation of antiangiogenic activity of leaves ethanolic extract of D. crumenatum were done descriptively by counting the number of new formed blood vessels on paper disc or around the paper disc with radial patern (CAM 2 cm²). Angiogenesis inhibition percentage was counted based on this formula:

Angiogenesis formation (%) = average number of the blood vessels on the treatment groups  x 100%

average number of blood vessels on bFGF control group

Angiogenesis inhibition (%) = 100% – angiogenesis formation (%)

[11, 12, 19, 20].

4. Data analysis

Growth inhibiton and apoptosis percentage mean value were compared for significance using one way analysis of variance (ANOVA) with SPSS 13 or 19 for Windows followed by Tuckey’s HSD test (p<0.05). Determination of IC₅₀ on cell growth inhibiton was analysed using probit analysis. Quantification of antiangiogenesis activity on CAM were analyzed statistically using non-parametric analysis Kruskal Wallis and followed by Duncan Multiple Range Test (DMRT) (p<0.05). Antiangiogenesis data were analyzed using SPSS 16.0 for Windows.

RESULTS AND DISCUSSION

Cell growth inhibition of ethanolic extract of D. crumenatum Swartz. on HeLa cells

The results showed that leaves, pseudobulb, and whole plant ethanolic extract of D. crumenatum could inhibit the growth of HeLa cells with pattern depend on the concentration (increasing of concentration will increase the growth inhibition). Leaves, pseudobulb, and whole plant ethanolic extract showed growth inhibition more than 60% at the concentration of 200, 400, and 800 µg/mL (Figure 1.). Nevertheless those three concentrations did not showed significant differences (p>0.05).

Within this research doxorubicine was used as positive control. Doxorubicine has been used widely as anticancer medicine. The result showed that doxorubicine at the concentration of 3.125-25 µg/mL could inhibit the growth of HeLa cells around 60-70% (Figure 1.). Based on the stastitical analysis, there were no significant differences between those concentrations. In this research DMSO was used as solvent control at the concentration of 0.1 up to 0.8%. There were no significant differences between all the concentrations of DMSO that were used in the research (Figure 1.). The growth inhibition of DMSO was low enough so it could be said that DMSO did not affect the cell growth. Thus it could be said that the growth inhibition of all the treatments were cause by doxorubicine or ethanolic extract of D. crumenatum.

 

Figure 1. Cell growth inhibition of ethanolic extract of D. crumenatum on HeLa

cells. Note: the line which was followed
by different letter showed

significant differences (p<0.05)

 

Based on the probit analysis, IC₅₀ values of leaves, pseudobulb, and whole plant ethanolic extract of D. crumenatum Swartz. were 153.828; 166.18; and 192.289 µg/mL respectively.

Apoptotic induction on HeLa cells

In this research apoptotic assay was evaluated using leaves ethanolic extract of Dendrobium crumenatum based on the probit analysis result. The results showed that the percentage of apoptotic cells followed the pattern depend on the concentration from the concentration of 12.5-100 µg/mL (the higher of the concentration, the higher of the apoptotic cells). Nevertheless, at the concentration above of 100 µg/mL did not show those pattern (Figure 2.). At the highest concentration, the apoptotic cells were 27.69 ± 10.22%. Percentage of apoptotic cells on the treatment with doxorubicine also showed the pattern depend on concentration. Percentage of apoptotic cells on the solvent control (DMSO) at the concentration of 0.1; 0.2; 0.4; and 0.8% less than 1% (Figure 2.). This result confirmed that DMSO did not affect the HeLa cells. Apoptosis assay with double staining method using ethidium bromide-acridine orange showed green color for a viable cell. Cell nucleus showed chromatin condensation with orange color was an apoptotic cell (Figure 3.).

 

Figure 2. Percentage of apoptotic HeLa cells in all control and treatment groups

Note: the bar which was followed by different letter showed significant

differences (p<0.05)

 

Figure 3. Control media group (A). Leaves ethanolic extract 100 µg/mL (B).

Doxorubicine (C). Early apoptotic (arrow head). Late apoptotic

(arrow). (Magnification 10 x 10)

Antiangiogenesis activity on chorio allatoic membrane

Antiangiogenesis activity test showed that leaves ethanolic extract of D. crumenatum with 5 series could inhibit angiogenesis which was induced by bFGF (Table 1 and Figure 4.). At the concentration of 76.914; 115.371; 153.828; and 192.285 µg/mL there were no significant differences (p>0.05) for the angiogenesis inhibition. The highest inhibition response was achieved at the concentration of 76.914 µg/mL with the inhibition percentage of 64.77%. Whereas at the highest concentration of the extract (230.742 µg/mL), the angiogenesis inhibition response only 34.09%.

Table 1. Angiogenesis response on chorioallantoic membrane after treatment with

ethanolic extract of leaves of D. crumenatum

 

Treatment	Concentration	Average number of new blood vessels	Percentage of growth (%)	Percentage of inhibition (%)
Paper disc control	–	9.33	1	1
bFGF control	30 ng	29.33	100	0a
bFGF control + DMSO	30 ng 0.8%	28.33	96.59	3.41a
bFGF 30 ng + ethanolic extract of leaves of D. crumenatum	76.914 µg/mL 115.371 µg/mL 153.828 µg/mL 192.285 µg/mL 230.742 µg/mL	10.33 11.67 12 17 19.33	35.23 39.77 40.91 57.95 65.91	64.77cd 60.23cd 59.09cd 42.05bc 34.09b

Note: the number in the same coloum which was followed by different letter in the

same column has significant differences (p<0,05).

Figure 4. Microscopical feature of angiogenesis respons on CAM of chicken. (A). paper disc

control, (B). bFGF control, (C). bFGF control + DMSO, (D). bFGF + ethanolic

extract of leaves of D. crumenatum 76.914 µg/mL. (è) blood vessel of CAM, (®)

new blood vessel, and (i) implant

DISCUSSION

Based on the research it was known that ethanolic extract of D. crumenatum could inhibit the growth of HeLa cells in the pattern depends on the concentration (Figure 1.) Leaves ethanolic extract of D. crumenatum has the highest growth inhibition potency followed by pseudobulb and whole plant ethanolic extract. IC₅₀ values of leaves, pseudobulb, and whole plant ethanolic extract of D. crumenatum were 153.828; 166.18; and 192.289 µg/mL respectively. Kamuhabwa et al. [21] reported that extract with IC₅₀ ≤ 100 µg/mL could be said having potency as antiproliferation. Whereas Manosroi et al. [22] categorized essential oils with IC₅₀ < 0.125 mg/mL having high potency to be developed as anticancer agent and essential oils with IC₅₀ 0.125-5 mg/mL having moderate potency as anticancer agent. Based on those two categories, ethanolic extract of D. crumenatum could be said having moderate potency to develop as anticancer agent. The three parts of D. crumenatum had little bit differences on the growth inhibition activities. It might be because there were differences in the compositions and concentrations of bioactive compounds in the three parts of D. crumenatum. Some researches reported
that the bioactive compounds of genus Dendrobium varies depend on its species. Flavonoids, reducing sugars, terpenes, and bibenzyl were found in some species of Dendrobium [23, 24]. The composition of those compounds was influenced the bioactivity of the species.

Wang et al. [9] reported that polysaccharides fraction at the concentration of 25, 50, 100, and 200 µg/mL have high growth inhibition on HL-60 cells. This inhibition was showed the pattern depends on the concentration. The differences at the inhibition activity were guessed because there were differences in the monosaccharides which compose the polysaccharides. Chen et al. [6] showed that fluorenones, dendroflorin, and dendhrysan which were isolated from D. chrysotoxum could inhibit the growth of cancer cells selectively depend on the type of its cancer. Phenolics from orchid Bulbophyllum odoratissimum also showed the same pattern [25].  The potency of each compound of orchid’s relative wide in range from IC₅₀ value < 0.5 µg/mL up to > 100 µg/mL  [5, 6, 7]. Bioactivity of stem chloroform fraction of Dendrobium lasianthera J.J.  Sm was higher than bioactivity of stem chloroform extract of Dendrobium lasianthera J.J. Sm on breast cancer cells (T47D cells) [26]. That research showed that the increasing of bioactivity of compounds depends on its purity. So the isolation of compounds of D. crumenatum was guessed will have higher growth inhibition activity especially on HeLa cells.

In this research was studied about the possibility of growth inhibition through apoptosis induction. Apoptosis is the programmed cell death which happens naturally in the body of organisms. This mechanism is important to regulate the homeostasis, development, and elimination of “unhealthy cells” such as in cancer [27]. In apoptosis there are cells shrinkage, fragmentation of nucleus, and formation of apoptotic bodies [28].

Several anticancer medicines are purposed to induce apoptosis. It is hoped to eliminate adverse effect. The results of this research showed that leaves ethanolic extract of D. crumenatum Swartz. did not much induced apoptosis. Folmer et al. [29] reviewed that programmed cells death could happen through 3 mechanisms: apoptosis, autophagy, and oncosis/necrosis, depend on the cellular signaling pathway. Autophagy is similar with apoptosis but mainly caspase-independent process. There is organelles degradation at the early phase of autophagy but the cytoskeleton still intact until the late phase of cells death. Autophagy usually happened because of fasting or genotoxic stress. Necrosis happened because of severe cytotoxicity which marked with cell swelling and bursting of cell membrane. Growth inhibition of ethanolic extract of D. crumenatum Swartz. on HeLa cells might be through the others mechanism such as the lysis of cells membranes or autophagy. In this research the test compound was used as crude extract which contain a lot of compounds. Those compounds could be proapoptotic or antiapoptotic factors so those compounds will work antagonistically on HeLa cells. It caused cell death through apoptosis was low. Whereas doxorubicine is a cancer medicine which work through apoptosis induction so the cells which undergo apoptosis is higher. It is important for further study to investigate the mechanism of compounds in Dendrobium to inhibit cancer cells growth.

Based on the antiangiogenesis activity of leaves ethanolic extract of D. crumenatum was known that this extract was potential as anticancer agent. The extract showed angiogenesis inhibition was 64.77% at the concentration of 76.914 µg/mL. Angiogenesis is a complex mechanism which involves the balance of proangiogenic and antiangiogenic factors. bFGF as a growth factor will interact with endothelial cell through tyrosine kinase and heparin sulphate proteoglycans receptors on the cell surface. Inhibition toward one of proangiogenic factors such as bFGF will disturb angiogenesis process which direct to angiogenesis inhibition.

In this research, the leaves ethanolic extract of D. crumenatum was known could inhibit angiogenesis but the inhibition mechanism was not known yet. The research which was done by Tsai et al. [8] showed that moscatilin (bibenzyl derivative) from D. loddigesii showed could inhibit angiogenesis in vivo and in vitro. Angiogenesis inhibition of moscatilin was known through blocking of angiogenic factor signaling pathway which involves ERK1/2, Akt, and eNOS. At the angiogenesis inhibition procces, the endothelial cells proliferation, migration, and tube formation which was induced by bFGF-VEGF was blocked. Tsai et al. [30] also showed that orchid Ephemerantha lonchophylla contain denbinobin which potensial to inhibit endothelial cell proliferasi, migrasi, dan capillary-like tube formation via suppressesion of IGF-1-induced IGF-1 R phosphorylation. It is important to study whether angiogenesis inhibition mechanism of leaves ethanolic extract of D. crumenatum also though the same procces.

Based on the growth inhibition activity, apoptosis induction, and antiangiogenesis activity, it could be said that ethanolic extract of D. crumenatum especially the leaves extract potencial to develop as anticancer agent.

CONCLUSION

Based on the results it could be concluded that ethanolic extract of Dendrobium crumenatum has potency to inhibit the growth of HeLa cells. The growth inhibition was not through apoptosis induction. Leaves ethanolic extract of D. crumenatum on chorioallantoic membrane could inhibit angiogenesis. Ethanolic extract of D. crumenatum especially the leaves extract potency to develop as anticancer agent.

ACKNOWLEDGMENT

This work was supported by Research Grant from Indonesia-Managing Higher Education for Relevance and Efficiency (I-MHERE) Project, Letter of Agreement No. UGM/BI/1304/I/05/04, April, 2011. Our gratitude thanks are also given to Mrs. Istini for technical assistance.

REFERENCES

[1]   R.M.P. Gutiérrez, 2010. Review Paper. Orchids: A review of uses in traditional medicine, its

phytochemistry and pharmacology. Journal of Medicinal Plants Research. 4 (8) 592-638.

[2]   N. Khouri, M. Nawasreh, S., M. Al-Hussain and A.S. Alkofahi, 2006. Effect of orchids (Orchis anatolica)

on reproductive function and fertility in adult male mice. Reproductive Medicine and Biology Journal. 5

269-276.

[3]   K. Jin-Ming, G. Ngoh-Khang, C. Lian-Sai and C. Tet-Fatt, 2003. Recent advances in traditional plant drugs

and orchids. Acta. Pharmacol. Sin. 24 (1) 7-21.

[4]   J. Arditti, Fundamental of Orchid Biology, John Wiley & Sons, Inc. USA, 1992.

[5]   Y. Chen, Y. Liu, J. Jiang, Y. Zhang and B. Yin, 2008. Dendronone, a new phenanthrenequinone from

Dendrobium cariniferum. Food Chemistry. 111 11–12.

[6]   Y. Chen, Y. Li, C. Qing, Y. Zhang, L. Wang and Y. Liu, 2008. 1,4,5-Trihydroxy-7-methoxy- 9H-fluoren-9-

one, a new cytotoxic compound from Dendrobium chrysotoxum. Food Chemistry. 108 973–976.

[7]   G. Zhang, L. Zhong, S.W.A. Bligh, Y. Guo, C. Zhang, M. Zhang, Z. Wang and L. Xu, 2005. Bi-bicyclic

and bi-tricyclic compounds from Dendrobium thyrsiflorum. Phytochemistry. 66 1113–1120.

[8]   A. Tsai, S. Pan, C. Liao, J. Guh, S. Wanga, H. Sun, Y. Liu, C. Chen, C. Shen, Y. Chang and C. Teng, 2010.

Moscatilin, a bibenzyl derivative from the
India orchid Dendrobrium loddigesii, suppresses tumor

angiogenesis and growth in vitro and in vivo. Cancer Letters. 292 163–170.

[9]   J. Wang, J. Luo, X. Zha and B. Feng, 2010. Comparison of antitumor activities of different polysaccharide

fractions from the stems of Dendrobium nobile Lindl. Carbohydrate Polymers. 79 114–118.

[10] R. Gomez-Flores, H. Hernández-Martínez, P. Tamez-Guerra, R. Tamez-Guerra, R. Quintanilla-Licea, E.

Monreal-Cuevas and C. Rodríguez-Padilla, 2010. Antitumor and immunomodulating potential of

Coriandrum sativum, Piper nigrum and Cinnamomum zeylanicum. Journal of  Natural Products. 3 54-63.

[11] R.I. Jenie, E. Meiyanto, dan R. Murwanti, 2006. Efek antiangiogenik ekstrak etanolik daun sambung nyawa

(Gynura procumbens (Lour.) Merr.) pada membran korio alantois (CAM) embrio ayam. Majalah Farmasi

Indonesia. 17 (1) 50-55.

[12] N. Salamah, Sugiyanto, M.S. Hartati, F. Hayati, dan P. Jumariyatno, 2009. Isolasi dan identifikasi

eurycomanone akar pasak bumi (Eurycoma longifolia, Jack) serta uji antiangiogenik. Majalah Farmasi

Indonesia. 20 (3) 118-126.

[13] F. Nussenbaum and  I.M.  Herman, 2010. Tumor angiogenesis: insights and innovations. Journal of

Oncology. 11-34.

[14] M. Klagsbrun and M.A. Moses, 1999. Molecular angiogenesis. Chemistry and Biology. 6 (8) 217-224.

[15] D.P. Saxena, S.K. Shukla, K. Kumar, R. Saxena, S. Saxena, S. Shukla, V. Gupta, R. Stephen, H. Kumar

and L. Kumar, 2011. Efficacy studies of in vitro screening  of  antiplasmodial  activity by crude extracts of

Diospyros melanoxylon. Research Journal of Medicinal Plant. 5 (3) 312- 320.

[16] Sismindari, 2003. Cytotoxic effect of methanol extract isolated from Erythrina fusca lour leaves on cancer

cell-lines. Berkala Ilmu Kedokteran. 35 (2) 75-80.

[17] Y.V. Yuan, M.F. Carrington and N.A. Walsh, 2005. Extracts from dulse (Palmaria palmata) are effective

antioxidants and inhibitors of cell proliferation in vitro. Food and Chemical Toxicology. 43 1073-1081.

[18] M. Sharma, S. K. Agrawal, P. R. Sharma, M. K. Khosla and A. K. Saxena, 2009. Cytotoxic and apoptotic

activity of essential oil from Ocimum viride towards COLO 205 cells. Food Chem. Toxicol.

doi:10.1016/j.fct.2009.10.021

[19] P. Indrayudha, 2010. Aktivitas Antiangiogenesis dan Antivirus Fraksi Protein MJ-C Ribosome Inactivating

Protein Asam Mirabilis jalapa L. Master Thesis. Sekolah Pascasarjana, Universitas Gadjah Mada,

Yogyakarta, Indonesia.

[20] N.  Klauber-DeMore, 2008. Characterization of Gene Expression in Human Breast Tumor Endothelium,

Annual Summary, U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland.

[21] A. Kamuhabwa, C. Nshimo and P. de Witte, 2000. Cytotoxicity of some medicinal plant extracts used in

Tanzanian tradisional medicine. J. Ethnopharmacol. 70 143-149.

[22] J. Manosroi, P., Dhumtanom and A. Manosroi, 2006. Anti-proliferative activity of essential oil extracted

from Thai medicinal plants on KB and P388 cell lines. Cancer Letters. 235 114-120.

[23] L. Yang, Z.Wang and L. Xu, 2006. Phenols and a triterpene from Dendrobium aurantiacum var.

denneanum (Orchidaceae). Biochemical Systematics and Ecology. 34 658-660.

[24] M. Maridass, M.I. Zahir Hussain and G.  Raju, 2008. Phytochemical Survey of Orchids in the Tirunelveli

Hills of South India. Ethnobotanical Leaflets. 12 705-712.

[25] Y. Chen, J. Xu, H. Yu, C. Qing, Y. Zhang, L. Wang, Y. Liu and J. Wang, 2008. Cytotoxic phenolics from

Bulbophyllum odoratissimum. Food Chemistry. 107 169–173.

[26] S. Handayani, 2012. Identifikasi  Golongan  Senyawa  Potensial  Penghambat Pertumbuhan Sel Kanker

Payudara (T47D) Organ Vegetatif Dendrobium lasianthera J.J. Sm. Master Tesis. Sekolah Pascasarjana,

Universitas Gadjah Mada, Yogyakarta, Indonesia.

[27] G.P. Young, F. Chai and P. Zalewski, 1999. Polysaccharide fermentation, butyrate and apoptosis in the

colonic epithelium. Asia Pasific J Clin Nutr. 8(Suppl) 527-531.

[28] V. Kumar, A.K. Abbas, N. Fausto and R.N. Mitchell, Robbins Basic Pathology. 8^th Saunders Elsevier Inc.

Philadelphia, USA, 2007.

[29] F. Folmer, M. Jaspars, M. Dicato and M. Diederich, 2009. Marine cytotoxins: callers for the various dances

of death. Gastroenterology and Hepatology From Bed to Bench. 2 S34-S50.

[30] A-C. Tsai, S-L. Pan, C-Y. Lai, C-Y. Wang, C-C. Chen, C-C. Shen and C-M. Teng, 2011. The inhibition of

angiogenesis and tumor growth by denbinobin is associated with the blocking of insulin-like growth factor-

1 receptor signaling. Journal of Nutritional Biochemistry. 22 625– 633.

 

Arista melaksanakan penelitian yang merupakan bagian dari skripsinya berjudul “Analisis  Molekular Mekanisme Terhadap  Spesifikasi  Sel Adaksial Pada Daun Tanaman Arabidopsis thaliana” di bawah bimbingan Dr. Endang Semiarti dan Prof. Chiyoko Machida. Penelitian yang didanai oleh JASSO ini dapat terselenggara oleh karena Fakultas Biologi UGM memiliki ikatan kerjasama (MoU) dengan Chubu University untuk pengembangan penelitian. Arista mengatakan bahwa syarat mengajukan beasiswa untuk seleksi secara umum dapat dilihat di website JASSO, dan seleksi dilaksanakan di tingkat fakultas dengan mengalahkan satu orang mahasiswa lainnya.

Selama mengikuti penelitian di Jepang, Arista mengatakan bahwa sebenarnya keunggulan di Jepang tidak jauh berbeda dengan di Fakultas Biologi. Hanya saja kedisiplinan memang sangat  tinggi, Kesadaran dari masing-masing pribadi untuk mengikuti segala peraturan yang ada itu sangat penting, sehingga semuanya berjalan dengan baik.

Menutup perbincangan, Arista menyatakan bahwa manfaat yang paling dirasakan adalah dapat belajar hal-hal baru yang berhubungan dengan penelitian yang sedang berkembang  saat ini. Kemudian, bisa belajar bahasa jepang dengan intensif sehingga membantu sekali dalam berkomunikasi. Selain itu, juga belajar tentang kebudayaan jepang seperti “sado (Upacara minum teh jepang) dan “Kadou”/Ikebana (Seni merangkai bunga), pergi ke tempat-tempat bersejarah di Jepang, dan lainnya.

Diberitahukan kepada mahasiswa S1, S2, dan S3 Fakultas Biologi, bahwa Fakultas Biologi bekerjasama dengan Indonesian Biodiversity Research Center (IBRC) akan menyelenggarakan Stadium General tentang “Penyegaran Biologi Molekular dan Pengantar Bioinformatika” oleh Prof. Dr. Drh. IGNK Mahardika dari IBRC, pada :
Hari : Senin, 1 April 2013
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Tempat : Ruang Sidang Bawah, Fakultas biologi UGM

Stadium General ini diselenggarakan secara free/ tidak dipungut biaya

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