lidah buaya (aloe vera l.)
TRANSCRIPT
Lidah buaya (Aloe vera L.)
• Tanaman asli Afrika (Ethiopia)
• Tanaman keabadian
• Tanaman kerajaan
• Hadiah dari Tuhan
• Tanaman kecantikan: Cleopatra, Ratu Nefertiti
• Holtikultura
• Liliaceae
• Tahan hidup di daerah kering, dimusim kemarau
Produk-produk aloe vera
• Kecantikan/kosmetik
• Makanan
• Minuman
• Kesehatan
✓ Gel/krim
✓ Tablet
✓ Suppositoria
✓ Nanopartikel
Senyawa bioaktif dalamaloe vera
Seny
awa
bio
akti
fd
alam
alo
e ve
ra
Senyawa bioaktif bisa juga dicari secara bioinformatik
• Dr. Duke’s Phytochemical and Ethnobotanical Databases
https://phytochem.nal.usda.gov/phytochem/search/list
• KNApSAcK Core System http://www.knapsackfamily.com/knapsack_core/top.php
Dr. Duke’s Phytochemical and Ethnobotanical Databaseshttps://phytochem.nal.usda.gov/phytochem/search/list
KNApSAcK Core System http://www.knapsackfamily.com/knapsack_core/top.php
Schematic
outlining the
biological
properties of Aloe
vera (Kumar et al.
2019).
Immunomodulation studies of Acemannan
• Increase white blood cell, macrophage and T cell numbers
• Increase the activity on dendritc cells by inducing maturation of these cells
• Upregulate function and generation of cytotoxic T-lymphocytes
• Enhance wound healing by activation of macrophages
• Acemannan upregulates the phagocytosis and candidicidal activity
• This activity is attributed to the recognition of terminal mannose by macrophages as a foreign substance due to it being common on the polysaccharides and oligosaccharides produced by microorganisms
• Acemannan allows the production of cytokines, interleukin-6, interferon gamma and tumor necrosis factor, the release of nitric oxide, which in turn is related to receptors for mannose monosaccharide, and candicidal activity by phagocytes
• Acemannan possesses antiviral activity by modifying glycosylation of both virally infected cells and glycoprotein coats of viruses, thus inhibiting virus replication and infectivity
Riset: AKTIVITAS REACTIVE OXYGEN SPECIES MAKROFAG AKIBAT STIMULASI GEL LIDAH BUAYA PADA INFEKSI Salmonella typhimurium
KontrolAloe vera 0,0 ml/ekor/hari (9hari)
P1Aloe vera 0,5 ml/ekor/hari(9hari)
P2Aloe vera 0,5 ml/ekor/hari(9hari)
P3Aloe vera 0,5 ml/ekor/hari(9hari)
hari ke-6, mencit diinfeksi bakteri patogen Salmonella typhimurium (virulen phage type 510) intraperitoneal 105 CFU.
hari ke-10 mencit didislokasi dan dibedah, diambil makrofagdari peritoneum untuk dianalisis produksi ROS-nya
24 ekor mencit BABL/c betina umur 8-10 minggu berat 20-30 g
LD50 S. typhimurium → 106
Daun A. vera dicucibersih dan dibuangbagian latex leaf lining. Bagian daging(gel) daun diblender(tanpa penambahanair), menjadi gel.
Isolasi Makrofag-Peritoneal Exudate Cell (PEC)
• Mencit dimatikan dan diletakkan dalam posisi terlentang
• kulit bagian perut dibuka dan selubung peritoniumnya dibersihkan denganalkohol 70%.
• 100 cc larutan RPMI dingin diinjeksikan ke dalam rongga peritonium. Peritonium dipijat sambil ditepuk-tepuk pelan untuk mendapatkanmakrofag.
• cairan disedot kembali sampai habis dan dimasukkan dalam tabung falcon 15 cc.
• Cairan disentrifus 1200 rpm pada suhu 4 0C selama 10 menit. Bila cairanterkontaminasi darah, sel dicuci dengan Phosphate Buffered Saline (PBS) sampai bersih.
• Supernatan dibuang
• Pelet ditambahkan 3 ml medium Roswell Park Memorial Institute (RPMI) 1640, Foetal Bovine Serum (FBS) 10%, ditambah penicilin dan streptomycin
• Sel-sel dihitung dengan hemositometer
• Resuspensi dengan medium komplit RPMI → kepadatan 2,5 x 106
sel/ml
Isolasi Makrofag-Peritoneal Exudate Cell (PEC)
Kultur sel makrofag
• Sel makrofag dikultur pada mikroplate 24 well yang bawahnya datardan dasarnya diberi kaca benda (coverslip)
• Setiap sumuran 200 µl (kepadatan 5 x 105 sel/ml)
• Diinkubasi dalam inkubator CO2 pada suhu 37 0C selama 30 menit
• ditambah medium RPMI komplit 1 ml tiap sumuran dan diinkubasi lagiselama 2 jam
• Sel dicuci RPMI 2 kali dan ditambahkan medium komplit 1 ml tiapsumuran dan inkubasi dilanjutkan sampai 24 jam
Pemeriksaan produksi Reactive Oxygen Species (ROS) denganNitroblue Tetrazolium (NBT) Reduction Assay
• Prinsipnya, makrofag distimulasi dengan PMA (Phorbol 12-Miristat 13-Acetate) sehingga mensekresi anion superoxide (O2 - ) yang akanmereduksi NBT menjadi presipitat formazan (tidak larut) yang berwarnabiru (counterstain neutral red)
• Presipitat biru dihitung presentasenya pada tiap sel makrofag
• Derajat presipitat didasarkan pada persentase terbentuknya presipitat:
Derajat 1: presipitat < 25%,
Derajat 2: presipitat 25-50%,
Derajat 3: presipitat 50-75%,
Derajat 4: presipitat >75%
Hasil
ROS detection method
Acemanan:β-(1, 4)-acetylated polymannose
• Found in the inner leaf gel of the aloe plant
• Produced by a specialized cells called leucoplasts
• Polisakarida utama pada Aloe vera
• Composition of acemannan is 31 β-(1, 4)-linked mannoses, 1 β-(1, 4)-linked glucose, and 1 α-(1, 6)-linked galactose
• Activity: immune-stimulating, wound-healing, antimicrobial and antiviral activity, anticancer activity, hypoglycemic activity
Schematic representation of extraction, separation purification and structure characterization of acemannan from Aloe vera (Liu et al 2019)
Acemannan→macrophage activity
• Peningkatan aktivitas makrofag melalui ikatan ligan (residu manosa dari acemannan) dengan receptor manosa pada makrofag
• Mannose receptor (MR) (Cluster of Differentiation 206, CD206) is a C-type lectin primarily present on the surface of macrophages, dendritic cells, and specific lymphatic or endothelial cells
• The mannose receptor is a type I transmembrane protein, with an extracellular N-terminus and an intracellular C-terminus
Mannose Receptor family
Binding properties of the Mannose Receptor (CD206) Schematic representation of the domain structure and binding properties of the MR.
• The MR can mediate endocytosis and phagocytosis, as well as activation of macrophages and antigen presentation
• resulted in increased NO and IL-6 production
• the mannose receptor is a professional receptor for phagocytosis.
• The engagement and phagocytosis of microorganisms by the macrophage mannose receptor actively stimulates the release of secretory products including IL-1, TNFα and reactive oxygen intermediates, further enhancing the clearance of antigen
• next
PolarisasiMakrophag
Classically M1 activated → ROS meningkat
ROS : Reactive Oxygen
Species
• O2 − (superoxide anion)
• hypochlorous acid (HOCl)
• OH − (Hydroxyl ion)
• Nitric oxide radicals (NO ⋅ )
• peroxynitrite (ONOO − )
• Myeloperoxidase (MPO)
• inducible nitric oxide synthase 2
(iNOS)
• natural resistance-associated
macrophage protein 1
(NRAMP-1)
• type IIA secreted phospholipase
A2 (sPLA2-IIA)
Simpulan
• Aloe vera beraktivitas meningkatkan produksi ROS makrofag
• Peningkatan aktivitas makrofag melalui ikatan ligan (residu manosa dari acemannan) dengan receptor manosa pada makrofag, menstimulasi sekresi IL1, TNF alpha, mengaktivasi makrofaq secara klasik (M1) sehingga produksi ROS menin