Lidah buaya (Aloe vera L.)

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

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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