TR2023014297A2 - A PRODUCT TO HELP SLIMMING CONTAINING HERBAL EXTRACELLULAR VESICULES - Google Patents
A PRODUCT TO HELP SLIMMING CONTAINING HERBAL EXTRACELLULAR VESICULESInfo
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- TR2023014297A2 TR2023014297A2 TR2023/014297 TR2023014297A2 TR 2023014297 A2 TR2023014297 A2 TR 2023014297A2 TR 2023/014297 TR2023/014297 TR 2023/014297 TR 2023014297 A2 TR2023014297 A2 TR 2023014297A2
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- extracellular vesicles
- product
- pineapple
- obesity
- fat
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Abstract
Bu buluş, obezite, aşırı kilo ve yağ foliküllerinin tedavisi için potansiyel sunan bitkisel eksozomlar içeren bir ürünle ilgilidir. Bu buluş, insan vücudunda toksisiteye yol açmayan, yağ foliküllerini azaltarak hücrenin eski formuna dönüşmesini teşvik eden, sağlıklı hücrelere zarar vermeyen, cerrahi müdahale gerektirmeyen ve bu nedenle enfeksiyon riski taşımayan, maliyeti düşük bir ürünün geliştirilmesini amaçlamaktadır. Bu bağlamda, bitkisel kaynak olarak ananas tercih edilebilir. Bu buluş, sağlık sorunlarına etkili bir çözüm sunma potansiyeline sahiptir ve özellikle kilo yönetim ve yağ metabolizmasıyla ilgili sorunları olan bireyler için umut verici bir yaklaşım sunmaktadır.This invention relates to a product containing herbal exosomes that offers potential for the treatment of obesity, overweight and fat follicles. This invention aims to develop a low-cost product that does not cause toxicity in the human body, encourages the cell to revert to its old form by reducing fat follicles, does not damage healthy cells, does not require surgical intervention and therefore does not carry the risk of infection. In this context, pineapple can be preferred as a herbal source. This invention has the potential to provide an effective solution to health problems and offers a particularly promising approach for individuals with problems with weight management and fat metabolism.
Description
TARIFNAME BITKISEL EKSTRASELÜLER VEZIKÜL IÇEREN ZAYIFLAMAYA YARDIMCI BIR Teknik Alan Bu patent basvurusu, insan yag kaynakli kök hücrelerin biyolojik canliligina toksik bir etki olusturmadan, olusan yag foliküllerinin morfoloj ik ve hücresel düzeyde küçülmesini destekleyen bir ürünün kesfine odaklanmaktadir. Bu ürün, bitkisel kaynakli ekstraselüler vezikül kompleksi içermekte olup, vücut agirliginin düzenlenmesi ve obeziteyle iliskili sorunlarin yönetilmesi amaciyla tasarlanmistir. Bu bulus, obezite ve vücut kompozisyonu iyilestirmesi konusunda bilimsel bir ilerlemeyi temsil etmektedir. Önceki Teknik (Teknigin Bilinen Durumu) Dünya Saglik Örgütü (DSÖ), obeziteyi, çevresel ve genetik etmenlerin karmasikligina dayali olarak kronik, ilerleyici ve bulasici olmayan bir hastalik olarak resmen tanimistir. Obezite, dünya genelinde hizla artan ve bireylerin ve toplumlarin sagligini ciddi sekilde tehdit eden bir küresel salgindir. Bu salgin, mücadelesi ömür boyu süren ve siki bir kontrol gerektiren bir saglik sorununu temsil etmektedir. DSÖ verilerine göre, dünya çapinda 500 milyon yetiskin obezite ile mücadele etmektedir ve dünya nüfusunun %65'i, obezitenin yaygin oldugu bölgelerde yasamaktadir. Bu bölgelerde obeziteye bagli ölüm oranlari endise verici bir düzeydedir. Ayrica son yapilan arastirmalar, dünya genelinde 250 milyon çocugun obezite sorunu ile karsi karsiya oldugunu göstermektedir. Obezitenin prevalansinin son 20-25 yilda hizla arttigi kaydedilmektedir. Obezite, kilo artisi ile birlikte uyku apnesi ve diger solunum problemlerini tetikleyebilirken, uyku kalitesini ve süresini bozarak istahi artirabilir. Ayni zamanda hipertansiyon, diyabet, kalp hastaliklari, osteoartrit ve depresyon gibi diger ciddi saglik sorunlarinin gelisme riskini artirir. Bu baglamda, ananasin içerdigi yüksek miktarda C vitamini ve fenolik bilesikler, obezite kaynakli komplikasyonlarin önlenmesine yönelik antioksidan tabanli tedavilerde önemli bir potansiyele sahiptir. Ananas suyu ile yapilan arastirmalar, yüksek kalorili beslenmeye bagli obeziteyi azaltarak Vücuttaki yag miktarini ve karacigerdeki yag birikimini azalttigini kanitlamistir. "Ananas suyunun, lipid metabolizmasini düzenlemedeki potansiyel etkileri, lipoj enezin baskilanmasi ve lipit metabolizmasi genlerinin yag asidi oksidasyonunu artirma mekanizmalariyla Vücutta gözlemlenmistir. Bu bulgular, ananas suyunun belirgin bir zararli etkiye sahip olmadigini ve obeziteyle mücadelede potansiyel bir ajan olarak kabul edilebilecegini göstermektedir (Samir ve ark., 2018). Öte yandan, ekstraselüler veziküller hücreler arasi iletisimde ve uzak mesafelerde iletisimde göreV alarak, içlerinde hücreye özgü DNA, RNA ve proteinler gibi biyokimyasal bilesenleri tasimaktadir. Ekstraselüler veziküllerin hastaliklarin tanisinda kullanilabilecek önemli biyobelirteçleri içermesi, hastaliklarin erken teshisine olanak saglayarak ekstraselüler veziküllere olan ilgiyi artirmistir (Inanir ve ark., 2020). Bu tür çalismalar, temel arastirma alaninda önemli bilgiler sunmakla birlikte, klinik uygulamalara dönüsmeden önce daha fazla deneysel ve klinik çalismanin yapilmasini gerektirir. Ayrica, herhangi bir besinin veya bilesenin saglik üzerindeki etkilerini degerlendirirken, uzman bir saglik profesyonelinin rehberligi ve kapsamli bir inceleme önemlidir. Laboratuvar ortaminda gerçeklestirilen preklinik çalismalarin sonuçlari, ananas suyu ve ananastan elde edilen ekstraselüler nanopartiküllerin yag folikülleri üzerindeki etkilerini karsilastirmis ve ananastan elde edilen partiküllerin yag folikülleri üzerinde daha etkili oldugu gözlemlenmistir. Bizim bulusumuzun en yenilikçü ve diger rünlerden en önemli farki bitkinin özütü yerine 0 bitkiden özel yöntemlerle elde edilen ekstraselüler vesiküllerin kullanilarak bulusun etkinliginin arttirilmasidir. Bu sonuçlar, yag foliküllerinin küçülmesi yoluyla zayiflamayi destekleyebilecek bir ajan olarak kullanilabileceklerini öngörmektedir. Bu bulgular dogrultusunda, ekonomik açidan erisilebilir bir çözüm olarak tasarlanabilecek bitkisel bir ürün gelistirme hedeflenmektedir. Bu arastirma, obeziteye karsi yenilikçi bir yaklasim sunma potansiyeline sahiptir. Bitkiler tarafindan salinan ekstraselüler veziküllerin anti-inflamatuar ve anti-kanser potansiyeli, bir dizi çalisma ile ortaya konmustur. Sarimsak, üzüm, greyfurt, zencefil ve havuç gibi bitkilerin bu tür veziküllerin sagladigi faydali etkilere sahip oldugu bilimsel olarak kanitlanmistir. Ancak, ananas bitkisinin ekstraselüler veziküllerinin, zayiflamaya katki saglama veya obezitenin temel süreçlerine olan etkileri hala arastirmanin öncelikli konularindan birini olusturmaktadir. Bu bulgu, bitkisel kaynakli ekstraselüler veziküller içeren bir zayiflama teknolojisi stratejisinin gelistirilmesi olarak sunulmaktadir. Ayrica, ekstraselüler veziküllerin hücresiz ürünler olmalari, hücresel terapi yöntemlerinin beraberinde getirebilecegi potansiyel zorluklarin üstesinden gelme açisindan son derece önemlidir. Ekstraselüler veziküller, kullanimi kolay ve hizla uygulanabilir terapötik araçlardir. Ayrica, toksik kriyoprezervatif maddelerin ve ultra-dondurucularin kullanilmasina gerek kalmadan düsük sicakliklarda depolanabilirler ve hastalarin güvenli bir sekilde erisimine sunulabilirler. Bu bilimsel bilgiler isiginda, bu teknolojinin alternatif tipta Ananas Comosus'tan elde edilen bitkisel ekstraselüler komplekslerin kullanildigi, zayiflamayi destekleyen bir ürün olarak potansiyel uygulamalarini degerlendirmeyi amaçlamaktayiz. Bu, saglik alaninda önemli bir ilerleme ve yeni bir terapötik yaklasimin kapisini aralamaktadir. Ilk olarak, ekstraselüler veziküller ananas bitkisinden izole edilmis ve bu partiküllerin gerçekten ekstraselüler veziküller oldugunu dogrulamak için karakterizasyon deneyleri gerçeklestirilmistir. Bu karakterizasyon deneylerinden biri, ekstraselüler veziküllerrin boyutlarini ve sekillerini elektron mikroskopi kullanarak göstermeyi içermistir. Ayrica, ekstraselüler veziküllerin yüzeyinde bulunan özel belirteçlerin tespiti için akis sitometrisi yöntemi de kullanilmistir. Elde edilen ekstraselüler veziküllerin farkli konsantrasyonlarinin, in Vitro ortamda yag kök hücreleri üzerinde sitotoksik bir etkiye sahip olmadigi, hücre canliliginin MTS, apoptoz ve aneksin V boyama testleriyle gösterildigi tespit edilmistir. Ayni zamanda, yag kirmizisi boyamasi kullanilarak hücre içinde olusan yag foliküllerinin ekstraselüler veziküller tarafindan azaltildigi gözlemlenmistir. Daha sonra, ekstraselüler veziküllerle tedaVi edilen hücrelerden RNA izolasyonu yapilarak Polimer Zincir Reaksiyonu (RT-PCR) yöntemi kullanilmis ve PPARgamma, adiponektin, LPL ve aP2 genlerinin ekspresyon düzeyleri incelenmistir. PPARgamma, bu çalismalarda farelerde ve maymunlarda aktivasyonunun artmasiyla F-GF21'in arttirildigini ve bu artan F-GF21'in kan sekerini, kolesterolü ve Vücut agirligini azalttigini göstermistir. F-GF21'in artan enerji tüketimi, PPARgamma aktivasyonu ile iliskilendirilmis ve bu nedenle yaglarin tüketiminde ve Vücutta atilmasinda önemli bir rol oynar. Ayrica literatürde, LPL enziminin yag hücreleri tarafindan yag depolarini olusturmak için salgilandigi ve kilo alimi sirasinda LPL aktiVitesinin arttigi belirtilmektedir. Obez kisilerde adiponektin düzeylerinin normal kilolu kisilere göre daha düsük oldugu ve adiponektin düzeyinin düsmesinin olumlu bir faktör oldugu bulunmustur. Sonuç olarak, bu çalisma sonuçlarina dayanarak, ananas ekstraselüler veziküllerini içeren ve zayiflamayi destekleyen bir biyoteknoloji ürününün gelistirildigi sonucuna varilmistir Bulusun Kisa Açiklamasi Bu bulusun temel hedefi, obezite tedavisi ve kilo kaybi yönetimi için kullanilabilen bitkisel kökenli ekstraselüler veziküllerden türetilmis etkili bir ürünün gelistirilmesidir. Bu ürün, yüksek maliyetlerle iliskilendirilmeyen ve vücuda sentetik ilaçlarin neden oldugu kimyasal kirlenmeye maruz kalmadan dogal bir yaklasim sunacaktir. Ayrica, bu bulusun bir diger önemli hedefi, ürünün üretim sürecini pratik ve erisilebilir hale getirmektir. Bu amaçla, bitkisel kaynaklarla kolayca yetistirilebilen bitkilerin kullanilacagi bir üretim süreci gelistirilmistir. Bu bitkiler, hizli büyüme ve yüksek verimlilik özelliklerine sahip olduklari için büyük ölçekte ekstraselüler veziküller üretimi için idealdir. Bu yöntem, ekstraselüler veziküllerin toplanmasi ve saIlastirilmasi süreçlerini optimize ederek, son ürünün maliyetini düsürmeyi ve daha genis bir kitleye ulasilabilir hale getirmeyi amaçlamaktadir. Bulusun potansiyel etkileri oldukça büyüktür, çünkü obezite ve kilo yönetimi, modern toplumun saglik sorunlarindan biridir. Bu dogal ve ekonomik olarak sürdürülebilir ürün, obezite ile mücadelede yeni bir perspektif sunabilir ve ayni zamanda çevresel sürdürülebilirlik ilkesini benimseyerek kimyasal kirliligi azaltma potansiyeline sahiptir. Bu nedenle, bulus, saglik ve çevresel açidan olumlu bir etki yaratma potansiyeline sahiptir. Bulusun Faydalari Bu ürün, obezite sorununa karsi etkili bir çözüm sunmanin ötesinde, yag kök hücrelerinin canliligina zarar vermeden ve toksik etkilere yol açmadan obezite yönetimine yeni bir yaklasim getirir. Ilerleyen asamalarda, bu ürünün sagladigi avantajlar daha ayrintili olarak incelenmelidir. Bu ürünün benzersiz özelliklerinden biri, yag kök hücrelerine zarar vermeden kilo kaybina katkida bulunabilmesidir. Bu, obeziteyle mücadelede önemli bir adim olarak kabul edilmelidir çünkü yag kök hücreleri, vücutta yag depolama ve metabolizma süreçlerinde kritik bir rol oynar. Geleneksel yaklasimlar genellikle bu hücrelere zarar verirken, bu ürün bu olumsuz etkileri ortadan kaldirarak daha saglikli ve sürdürülebilir bir kilo kaybi saglar. Ayni zamanda, bu ürünün yag foliküllerini küçültme yetenegi obeziteyle dogrudan iliskilendirilmistir. Yag foliküllerinin boyutunun azaltilmasi, kilo kaybinin saglanmasinda etkili bir yol olarak kabul edilmektedir. Bu, ürünün obezite sorununu kökten çözme potansiyeline isaret eder. Ek olarak, bitkisel ekstraselüler vezikül kompleksi, obezite ile iliskili saglik sorunlarinin azaltilmasina yardimci olur. Bu, metabolik sendrom, tip 2 diyabet, kardiyovasküler hastaliklar ve diger obezite ile iliskilendirilen saglik risklerine karsi koruma saglama potansiyeline isaret eder. Sonuç olarak, bu ürün, obeziteye karsi sadece etkili bir çözüm sunmakla kalmaz, ayni zamanda saglik açisindan olumlu etkiler sunar ve bu da obezite ile mücadelede önemli bir ilerleme olarak kabul edilebilir. Bu ürün, gelecekte obezite epidemisinin kontrol edilmesine ve saglik sorunlarinin azaltilmasina katkida bulunabilir Bulusun Ayrintili Açiklamasi Bu inovasyon, kilo kaybini desteklemeyi hedeIleyen bir ürünün gelistirilmesine odaklanmaktadlr. Bu ürün, bitkisel ekstraselüler veziküllerin içerisinde yer almaktadir ve özellikle ananas bitkisinden türetilen ekstraselüler vezikülleri bünyesinde barindirmaktadir. Bu baglamda, bilimsel ve etkileyici bir ifadeyle asagidaki açiklama sunulmaktadir: Bu bulus, obezite gibi yaygln estetik ve saglik sorunlarina karsi yeni bir çözüm sunma amacini tasimaktadir. Ürün, bitkisel kökenli ekstraselüler veziküllerin (EVs) kullanimi ile öne çikmaktadir. Özellikle ananas bitkisinden elde edilen ekstraselüler veziküller, bu EV'lerin kaynagini olusturmaktadir ve saglik ve estetik alanindaki sorunlarin ele alinmasinda önemli bir potansiyele sahiptir. Ekstraselüler veziküller, hücreler arasi iletisim ve sinyal iletiminde kritik bir rol oynayan biyolojik yapilar olarak bilinir. Bu ürün, özellikle ananas bitkisinden türetilen ekstraselüler vezikülleri içermesi nedeniyle, dogal bir kaynaktan gelen bu önemli bilesenleri kullanarak obeziteye ve selülite karsi etkili bir yaklasim sunar. Ayrica, bu ürünün bitkisel ekstraselüler vezikülleri kullanarak sentetik kimyasallarin zararli etkilerinden kaçinmasi, saglik ve çevre açisindan daha sürdürülebilir bir yaklasim sunmaktadir. Bu, kullanicilar için daha güvenli ve dogal bir çözüm sunmanin yani sira, çevresel sürdürülebilirlige de katki saglama potansiyeline isaret etmektedir. Sonuç olarak, bu bulus, obezite ve selülit gibi yaygln sorunlara karsi dogal, etkili ve bilimsel temellere dayali bir çözüm sunma potansiyeline sahip olan önemli bir inovasyonu temsil etmektedir. Ananas bitkisinden elde edilen ekstraselüler veziküller, bu ürünün gücünü ve potansiyelini belirlemektedir ve gelecekte bu tür sorunlari ele almak isteyen bireyler için umut vaat etmektedir. Bulusun amacina ulasmak için gelistirilmis "BITKISEL EKSTRASELÜLER VEZIKÜL IÇEREN ZAYIFLAMAYA YARDIMCI BIR ÜRÜN" ile ilgili sekiller ekte gösterilmis olup; Sekil 1A: Ananas bitkisinden elde edilen bitki ekstraselüler veziküllerin morfolojisini izlemek için taramali elektron mikroskop görüntüsü. Sekil 1B: Ananas bitkisinden elde edilen bitki ekstraselüler veziküllerin CD63, CD9, CD81 ve HSP70 yüzey 10 belirteçleri kullanilarak ananas ekstraselüler veziküllerinin akis sitometrisi Sekil 2: HADSC insan adipoz kaynakli kök hücrelerinin belirli bir konsantrasyonda (100ug/mL) uygulanan ananas ekstraselüler vezikülleri ile muamele edildikten 24, 48 ve 72 saat sonrasindaki hücre canliligi analizidir. Sekil 3: HADSC insan adipoz kaynakli kök hücrelerinin, adipojenik indüksiyon altinda in vitro olarak 14 gün kültür ortaminda farklilastirildi ve farklilasma sonrasi 11 gün ekstraselüler veziküller tedavisi uygulandi. In vitro ortamda yag foliküllerinin azalmasini degerlendirmek için kontrol grubu ve 100ug/ml ananas ekstraselüler vezikülleri ile muamele edilmis grubunun adipojenik farklilasmanin tamamlandigi anda ve 11 günlük ananas ekstraselüler veziküller tedavisinden sonraki Iloresan mikroskop görünümüdür. Sekil 4-A: 100 ug/ml ananas EV,lerin 11 günlük tedavisinde her iki günde bir toplanan 20 medyalardan HADSC insan adipoz kaynakli kök hücrelerinin LPL ELISA analizi. Sekil 4-B: 100 ug/ml ananas EV,lerinin 11 günlük tedavisinde her iki günde bir toplanan medyalardan HADSC insan adipoz kaynakli kök hücrelerinin PPAR ELISA analizi. Sekil 5: HADSC insan adipoz kaynakli kök hücreleri, StemPro Adipogenesis Differentiation Kit kullanilarak in vitro kosullarda 14 gün süreyle kültürlendi ve adipojenik farklilasmanin ardindan hücreler, 100 ug/ml ananas EV konsantrasyonu ile 11 gün muamele edildi. Insan adipoz kaynakli kök hücrelerinin(HADSc) PPAR-gamma, adiponektin, LPL ve aP2 ekspresyon seviyelerinin kantitatif RT-PCR analizi gösterilmistir. Bu sonuçlar dogrultusunda bitkisel ekstraselüler veziküllerin insan adipoz kaynakli kök hücrelerinin (hADSc) öldürmedigi, bu hücrelerde tamamlanan adipoj enik farklilasmanin ardindan yag foliküllerini azalttigi görülmüstür. Tedavinin Gelistirilmesi Hücre Toksisitesinin Belirlenmesi Bu çalismada, bulusun potansiyel toksik etkilerini degerlendirmek amaciyla literatürde yaygin olarak kabul edilen MTS (3-(4,5-dimetiltiyazol-2-il)-5-(3-karboksimetoksi fenil)-2-(4-sülfonat fenil)-2H-tetrazol-3-iyum) yöntemi kullanilmistir (Yalvaç ve ark, 2009). Çalisma için, belirtilen konsantrasyonlarda kimyasal maddeler besiyeri içinde hazirlanmis ve ardindan 96 kuyucuklu kültür kaplarina ekimi yapilan Insan adipoz kaynakli kök hücreleri (HADSC) üzerine uygulanmistir (her bir kuyucukta 5000 hücre bulunmaktadir). Bu kimyasal maddelerin hücreler üzerindeki toksik etkisi, 3 gün boyunca hücre canliliginin ölçülmesi yoluyla belirlenmistir. Hücre canliligi analizi, MTS yöntemine dayanmaktadir. MTS, mitokondriyal enzim aktivitesini ölçmek için kullanilan bir kolorimetrik bir yöntemdir. Önceden kültür kaplarina ekilen hücrelere farkli dozlarda ananas EV,leri uygulanarak, belirli bir zaman araliginda uygulanan maddenin hücre canliligi üzerindeki etkisi degerlendirilmistir. MTS ajansi hücrelerle karistirildiginda, canli hücre sayisinin artmasiyla birlikte daha koyu bir renk olusturur. Bu renk degisimi ELISA okuyucusu kullanilarak absorbans ölçümü ile degerlendirilmistir. Bulus kapsaminda gelistirilen ürün, saglikli hücreler üzerinde farkli konsantrasyonlarda edilmistir. Bu analizler, bulusun olasi toksik etkilerini bilimsel ve detayli bir sekilde degerlendirmek amaciyla gerçeklestirilmistir. Deneysel Çalisma Bitkisel Kaynakli Ekstraselüler Veziküllerin Hazirlanmasi Bu çalismanin odak noktasi bitkisel ekstraselüler veziküller içeren ürünün bitki kaynaginin seçilmesi ve ekstraselüler vezikül izolasyon sürecinin ayrintilaridir. Bu nedenle, asagida daha bilimsel ve etkileyici bir ifadeyle sunulmustur: Bulusun konusu olan bitkisel ekstraselüler vezikül içerikli ürünün gelistirilmesi için farkli bitki kaynaklari arastirilmis ve deneylerde tercihen ananas kullanilmistir. Ekstraselüler veziküllerin izolasyonu için, ananaslarin suyu sikilmis ve bu su, öncelikle 10 dakika boyunca 1000xg, edilerek ananas suyu kaynakli ekstraselüler veziküller çift fazli polimer sistemleri kullanilarak izole edilmistir. Izole edilen ekstraselüler veziküller daha sonra %0,9'luk izotonik serum içinde çözülmüstür. Elde edilen ekstraselüler veziküller, taramali elektron mikroskobu kullanilarak görsel olarak incelenmistir (Sekil 1A). Daha sonra, bu ekstraselüler veziküller CD63, CD9, CD81 ve HSP70 markörleri kullanilarak islenmis ve akis sitometrisi ile karakterizasyonlari gerçeklestirilmistir. Bu yöntemler, bitkisel ekstraselüler veziküllerin etkili bir sekilde izole edilmesi ve karakterize edilmesi için bilimsel olarak saglam bir temel olusturur. Bu çalisma, bitki kaynakli ekstraselüler veziküller potansiyel uygulamalari üzerine daha fazla bilgi edinmek ve bu alandaki ilerlemelere katki saglamak için önemli bir adimdir. (Sekil 1B) Kantitatif RT-PCR analizi Insan adipoz kaynakli kök hücrelerinde adipoj enik farklilasma orani, RNA'larin izolasyonu ve gen ifadelerindeki farkliliklar ile arastirililmistir.. Bu hADSC'ler 6 kuyucuklu plate,e her bir kuyucukta 100.000 hücre olacak sekilde ekilmistir. Ilk olarak, her bir kuyucuk, adipojenik farklilasmasi için StemPro Adipogenesis Differentiation Kit kullanilarak in Vitro kosullarda 14 gün süreyle kültürlenmistir. Adipoj enik farklilasmadan sonra, 6 kuyucuklu platede ki iki kuyu, 11 gün boyunca 100 ug/ml konsantrasyonunda ananas EV,si ile tedaVi uygulandi ve her iki günde bir hücreler, yeni ortam-ananas EV çözeltileri ile tazelendi. Plate,in diger iki kuyucugu, 11 gün boyunca sadece DMEM düsük glikoz ortami (%10 PES ve %5 PSA) ile muamele edildi. Total RNA izolasyonu için Macherey-Nagel firmasina ait NucleoSpin RNA Izolasyon Kiti, RNA izolasyonundan sonra cDNA eldesi için BIORAD firmasina ait iScript cDNA Sentez Kiti kullanildi. Adiponektin, LPL, PPAR-gama ve aP2 genlerinin ekspresyonu, RealTime-PCR kullanilarak ölçüldü. Yag kirmizi boyamasi Bu çalismada, adipojenik farklilasmayi indüklemek için hADSC'ler StemPro Adipogenesis Differentiation Kit kullanilarak in vitro kosullarda 14 gün süreyle kültürlendi ve adipoj enik farklilasmanin tamamlanmasindan sonra hücreler, 11 gün boyunca DMEM (10%FBS, 5%PSA) ve 100 ug/ml ananas EV,si ile tedavi uygulandi. Yag hücrelerindeki yaglar, sekil 3'te Oil-Red boyama ile incelenmistir. Sekil 3'te görüldügü gibi, kontrol hücreleri sadece DMEM ile inkübe edilmis ve adipo hücrelerinde normal yag olusumu ve morfoloj isi görülmüstür. Sekil 3, e bakildiginda ananas EV,lerin 100 ug/ml konsantrasyonu ile yag hücrelerindeki yag folikülü olusumu azalmistir. Deneysel Sonuçlar Bu çalisma, gelistirilen izolasyon yöntemiyle tercih edilen ananas kaynakli ekstraselüler veziküller elde edilmesini ve bu EV,lerin karakterizasyonunu içermektedir. Asagida, bu süreçlerin daha bilimsel ve etkileyici bir dil kullanilarak ifadesi sunulmustur: Bulusun konusu olan bitkisel ekstraselüler veziküller içerikli ürün, bitki kaynagi olarak tercih edilen ananas kullanilarak izole edilmistir. Ekstraselüler veziküllerin elde edilmesi, ananas olarak 60 dakika boyunca 15000xg hizlarinda santrifüj edilmesi yoluyla çift fazli polimer sistemleri kullanilarak gerçeklestirilmistir. Izole edilen ekstraselüler veziküller daha sonra Elde edilen ekstraselüler veziküller, taramali elektron mikroskobu kullanilarak görsel olarak incelenmistir (Sekil 1). Bu mikroskopik görüntüler, elde edilen yapilarin literatürde tanimlanan ekstraselüler veziküller elektron mikroskop görüntüleriyle benzerlik gösterdigini göstermektedir. Ayrica, akis sitometrisi kullanilarak yapilan karakterizasyon sonuçlari, ekstraselüler veziküllerin en az %79.91 oraninda yüzey markörlerini ifade ettigini ortaya koymustur. Bu sonuçlar, MISEV2020 Standartlarina dayanarak elde edilen molekülün ekstraselüler veziküller oldugunun bir kanitidir (Sekil 1). Saglikli hücreler, bitki ekstraselüler veziküller içeren serum ile maruz birakildiginda, MTS sonuçlariyla kanitlanan hücre proliferasyonunda artis gösterir. Artan absorbans miktari, canliligin arttigini gösterir. Bu analizler, ananas EV,lerinin saglikli hücrelerde bölünme hizini artirdigini kanitlar (Sekil 2). Adipojenik farklilasma sonrasinda ekstraselüler veziküllerle tedavi edilen hücreler, Real-Time PCR ve Oil-Red boyama yöntemi kullanilarak karakterize edilmistir. Oil-Red boyama, hidrofobik lipitlerin tanimlanmasinda kullanilan bir yöntemdir. Sekil 3, ananas EValeri ile tedaVi edilen hADSC'lerde olusan yaglarin Oil-Red boyama ile gözlendigini göstermektedir. Sekil 5'te ise ananas EV,lerinin PPAR, aP2, adiponektin ve LPL genlerinin ekspresyon seViyelerini göstermektedir. Bu gen ekspresyonlari, literatüre dayali olarak, PPAR, aP2 ve adiponektin seViyelerinin arttigini ve LPL ekspresyonunun seViyesinin azaldigini göstermelidir. Bu gen ekspresyonlarinin protein seViyeleri, ELISA metodu kullanilarak LPL (Sekil 4A) ve PPAR (Sekil 4B) proteinleri ile de dogrulanmistir. Elde edilen deneysel sonuçlar, bulus konusu ürünün Vücuttaki yag hücrelerinin azalmasina neden oldugunu göstermektedir. Yag kirmizisi boyamasi ve PCR sonuçlarina dayali deney modeli, ananas ekstraselüler vezikülleri kaynakli ürünün yag foliküllerini belirgin bir sekilde azalttigini kanitlamistir. Bu çalisma, bitkisel ekstraselüler veziküllerin hücresel düzeydeki etkilerini bilimsel ve deneysel olarak dogrulayan önemli bir adimi temsil etmektedir. Elde edilen sonuçlar, gelecekte saglikli bir sekilde kilo yönetimi ve yag metabolizmasi ile ilgilenen bireylere umut vaat etmektedir. Bulusun Uygulanmasi Söz konusu bulus, insanoglu adipoz kaynakli kök hücrelerinde uygulama potansiyeline sahiptir. Bulusun bu ürünü, siVi halde serum formunda basta olmak üzere kati formlarda da üretilebilir, örnegin; serum, surup, tablet, ilaç, jel, krem gibi farkli formülasyonlarda sunulabilir. Ayrica, nano tasiyici molekül olarak kullanilarak, hücre içine etken baska bir maddenin (örnegin ilaç veya kimyasal madde) tasinmasina olanak tanir. Bu tasima yöntemi de yukarida belirtildigi gibi çesitli formlar ve formülasyonlarla kullanilabilir. Bu açiklama, bulusun çesitli uygulama ve tasima yöntemlerini bilimsel ve etkileyici bir dille ifade eder. Bu, bulusun çok yönlü ve özgün kullanim potansiyelini vurgulamaktadir ve gelecekte farkli saglik ve biyomedikal alanlarda kullanilabilirligini göstermektedir. TR DESCRIPTION A SLIMMING AID CONTAINING HERBAL EXTRACELLULAR VESICULAR Technical Field This patent application focuses on the discovery of a product that supports the shrinkage of formed fat follicles at the morphological and cellular level, without creating a toxic effect on the biological viability of human fat-derived stem cells. This product contains a plant-derived extracellular vesicle complex and is designed to regulate body weight and manage obesity-related problems. This discovery represents a scientific advance in obesity and body composition improvement. Prior Art (State of the Art) The World Health Organization (WHO) has officially recognized obesity as a chronic, progressive and non-communicable disease based on the complexity of environmental and genetic factors. Obesity is a global epidemic that is rapidly increasing worldwide and seriously threatens the health of individuals and societies. This epidemic represents a health problem that lasts a lifetime and requires strict control. According to WHO data, 500 million adults worldwide struggle with obesity and 65% of the world's population lives in regions where obesity is common. Obesity-related death rates are at an alarming level in these regions. In addition, recent research shows that 250 million children around the world face the problem of obesity. It is noted that the prevalence of obesity has increased rapidly in the last 20-25 years. While obesity can trigger sleep apnea and other breathing problems with weight gain, it can increase appetite by impairing sleep quality and duration. It also increases the risk of developing other serious health problems such as hypertension, diabetes, heart diseases, osteoarthritis and depression. In this context, the high amounts of vitamin C and phenolic compounds contained in pineapple have an important potential in antioxidant-based treatments to prevent obesity-related complications. Research conducted with pineapple juice has proven that it reduces the amount of fat in the body and fat accumulation in the liver by reducing obesity due to high-calorie nutrition. "The potential effects of pineapple juice in regulating lipid metabolism have been observed in the body through the mechanisms of suppressing lipogenesis and increasing fatty acid oxidation of lipid metabolism genes. These findings indicate that pineapple juice does not have a significant harmful effect and can be considered a potential agent in the fight against obesity (Samir et al. On the other hand, extracellular vesicles take part in intercellular communication and communication over long distances, carrying biochemical components such as cell-specific DNA, RNA and proteins. Extracellular vesicles contain important biomarkers that can be used in the diagnosis of diseases, allowing early diagnosis of diseases. It has increased interest (Inanir et al., 2020). Although such studies provide important information in the field of basic research, they require further experimental and clinical studies before being translated into clinical applications. Health professional guidance and a thorough examination are important. The results of preclinical studies conducted in a laboratory environment compared the effects of pineapple juice and extracellular nanoparticles obtained from pineapple on fat follicles, and it was observed that the particles obtained from pineapple were more effective on fat follicles. The most innovative and most important difference of our invention from other products is that the effectiveness of the invention is increased by using extracellular vesicles obtained from the plant with special methods instead of the extract of the plant. These results suggest that they can be used as an agent that can promote weight loss through the shrinkage of fat follicles. In line with these findings, it is aimed to develop a herbal product that can be designed as an economically accessible solution. This research has the potential to offer an innovative approach to obesity. The anti-inflammatory and anti-cancer potential of extracellular vesicles released by plants has been demonstrated by a number of studies. It has been scientifically proven that plants such as garlic, grapes, grapefruit, ginger and carrots have the beneficial effects provided by such vesicles. However, the effects of the extracellular vesicles of the pineapple plant on contributing to weight loss or the basic processes of obesity still constitute one of the priority topics of research. This finding is presented as the development of a slimming technology strategy involving plant-derived extracellular vesicles. Additionally, the fact that extracellular vesicles are cell-free products is extremely important in overcoming the potential difficulties that cellular therapy methods may bring. Extracellular vesicles are therapeutic tools that are easy to use and rapidly administered. Additionally, they can be stored at low temperatures and safely accessible to patients without the need for the use of toxic cryopreservatives and ultra-freezers. In the light of this scientific information, we aim to evaluate the potential applications of this technology as an alternative type of product that supports weight loss, using herbal extracellular complexes obtained from Pineapple Comosus. This opens the door to a significant advance in healthcare and a new therapeutic approach. First, extracellular vesicles were isolated from the pineapple plant and characterization experiments were performed to confirm that these particles were indeed extracellular vesicles. One of these characterization experiments involved demonstrating the sizes and shapes of extracellular vesicles using electron microscopy. Additionally, flow cytometry method was used to detect specific markers found on the surface of extracellular vesicles. It was determined that different concentrations of the obtained extracellular vesicles did not have a cytotoxic effect on fat stem cells in vitro, and cell viability was demonstrated by MTS, apoptosis and annexin V staining tests. At the same time, using oil red staining, it was observed that the fat follicles formed inside the cell were reduced by extracellular vesicles. Then, the Polymer Chain Reaction (RT-PCR) method was used to isolate RNA from cells treated with extracellular vesicles, and the expression levels of PPARgamma, adiponectin, LPL and aP2 genes were examined. In these studies, PPARgamma showed that F-GF21 was increased by increasing its activation in mice and monkeys, and this increased F-GF21 reduced blood sugar, cholesterol and body weight. Increased energy consumption of F-GF21 has been associated with PPARgamma activation and therefore plays an important role in the consumption and excretion of fats in the Body. Additionally, it is stated in the literature that the LPL enzyme is secreted by fat cells to create fat depots and that LPL activity increases during weight gain. It has been found that adiponectin levels in obese people are lower than in normal weight people and that the decrease in adiponectin level is a positive factor. As a result, based on the results of this study, it was concluded that a biotechnology product containing pineapple extracellular vesicles and supporting weight loss has been developed. Brief Description of the Invention The main goal of this invention is to develop an effective product derived from plant-derived extracellular vesicles that can be used for obesity treatment and weight loss management. This product will offer a natural approach that is not associated with high costs and without exposing the body to chemical contamination caused by synthetic drugs. In addition, another important goal of this invention is to make the production process of the product practical and accessible. For this purpose, a production process has been developed in which plants that can be easily grown with plant resources will be used. These plants are ideal for the production of extracellular vesicles on a large scale, as they have the characteristics of rapid growth and high efficiency. By optimizing the collection and purification processes of extracellular vesicles, this method aims to reduce the cost of the final product and make it accessible to a wider audience. The potential implications of the invention are huge, as obesity and weight management are among the health problems of modern society. This naturally and economically sustainable product can offer a new perspective in the fight against obesity and also has the potential to reduce chemical pollution by adopting the principle of environmental sustainability. Therefore, the invention has the potential to create a positive impact in terms of health and the environment. Benefits of the Invention: Beyond providing an effective solution to the obesity problem, this product brings a new approach to obesity management without damaging the viability of fat stem cells and without causing toxic effects. In the following stages, the advantages provided by this product should be examined in more detail. One of the unique features of this product is that it can contribute to weight loss without damaging fat stem cells. This should be considered an important step in the fight against obesity because fat stem cells play a critical role in fat storage and metabolism processes in the body. While traditional approaches often damage these cells, this product eliminates these negative effects and provides a healthier and sustainable weight loss. At the same time, this product's ability to shrink fat follicles has been directly linked to obesity. Reducing the size of fat follicles is considered an effective way to achieve weight loss. This indicates the product's potential to radically solve the problem of obesity. Additionally, the herbal extracellular vesicle complex helps reduce obesity-related health problems. This indicates its potential to protect against metabolic syndrome, type 2 diabetes, cardiovascular disease, and other health risks associated with obesity. As a result, this product not only offers an effective solution against obesity, but also offers positive health effects, which can be considered a significant advance in the fight against obesity. This product may contribute to controlling the obesity epidemic and reducing health problems in the future. Detailed Description of the Invention This innovation focuses on the development of a product that aims to support weight loss. This product is contained in vegetal extracellular vesicles and especially contains extracellular vesicles derived from the pineapple plant. In this context, the following explanation is presented in a scientific and impressive way: This invention aims to offer a new solution to common aesthetic and health problems such as obesity. The product stands out with the use of extracellular vesicles (EVs) of plant origin. In particular, extracellular vesicles obtained from the pineapple plant constitute the source of these EVs and have an important potential in addressing problems in the field of health and aesthetics. Extracellular vesicles are known as biological structures that play a critical role in intercellular communication and signal transduction. This product offers an effective approach against obesity and cellulite by using these important ingredients from a natural source, especially since it contains extracellular vesicles derived from the pineapple plant. Additionally, this product avoids the harmful effects of synthetic chemicals by using plant extracellular vesicles, offering a more sustainable approach in terms of health and the environment. This points to the potential to provide a safer and more natural solution for users, as well as contributing to environmental sustainability. In conclusion, this invention represents a significant innovation that has the potential to provide a natural, effective and scientifically based solution to common problems such as obesity and cellulite. Extracellular vesicles derived from the pineapple plant determine the potency and potential of this product and hold promise for individuals looking to address such issues in the future. The figures related to "A SLIMMING AID PRODUCT CONTAINING HERBAL EXTRACELLULAR VESICULE", which was developed to achieve the purpose of the invention, are shown in the appendix; Figure 1A: Scanning electron microscope image to monitor the morphology of plant extracellular vesicles obtained from pineapple plant. Figure 1B: Flow cytometry of pineapple extracellular vesicles obtained from pineapple plant using CD63, CD9, CD81 and HSP70 surface 10 markers. Figure 2: HADSC after treatment of human adipose derived stem cells with pineapple extracellular vesicles applied at a certain concentration (100ug/mL). It is cell viability analysis after 24, 48 and 72 hours. Figure 3: HADSC human adipose-derived stem cells were differentiated in culture medium for 14 days in vitro under adipogenic induction, and extracellular vesicles treatment was applied for 11 days after differentiation. Illuminance microscope view of the control group and the group treated with 100ug/ml pineapple extracellular vesicles at the time of completion of adipogenic differentiation and after 11 days of pineapple extracellular vesicles treatment to evaluate the reduction of fat follicles in vitro. Figure 4-A: LPL ELISA analysis of HADSC human adipose-derived stem cells from 20 media collected every second day over an 11-day treatment of 100 µg/ml pineapple EVs. Figure 4-B: PPAR ELISA analysis of HADSC human adipose-derived stem cells from media collected every second day during an 11-day treatment of 100 µg/ml pineapple EVs. Figure 5: HADSC human adipose-derived stem cells were cultured for 14 days in vitro using the StemPro Adipogenesis Differentiation Kit, and after adipogenic differentiation, the cells were treated with 100 µg/ml pineapple EV concentration for 11 days. Quantitative RT-PCR analysis of PPAR-gamma, adiponectin, LPL and aP2 expression levels of human adipose-derived stem cells (HADSc) is shown. In line with these results, it was observed that vegetal extracellular vesicles did not kill human adipose-derived stem cells (hADSc), but reduced fat follicles after adipogenic differentiation was completed in these cells. Development of Treatment Determination of Cell Toxicity In this study, in order to evaluate the potential toxic effects of the invention, MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfonate), which is widely accepted in the literature, was used. phenyl)-2H-tetrazol-3-ium) method was used (Yalvaç et al., 2009). For the study, chemical substances at the specified concentrations were prepared in the medium and then applied to Human adipose-derived stem cells (HADSC) seeded in 96-well culture dishes (each well contains 5000 cells). The toxic effect of these chemicals on cells was determined by measuring cell viability for 3 days. Cell viability analysis is based on the MTS method. MTS is a colorimetric method used to measure mitochondrial enzyme activity. By applying different doses of pineapple EVs to the cells previously planted in culture dishes, the effect of the applied substance on cell viability within a certain time interval was evaluated. When the MTS agency is mixed with cells, it creates a darker color as the number of living cells increases. This color change was evaluated by absorbance measurement using an ELISA reader. The product developed within the scope of the invention was applied to healthy cells at different concentrations. These analyzes were carried out in order to evaluate the possible toxic effects of the invention in a scientific and detailed way. Experimental Study Preparation of Plant-derived Extracellular Vesicles The focus of this study is the selection of the plant source of the product containing plant extracellular vesicles and the details of the extracellular vesicle isolation process. For this reason, it is presented below in a more scientific and impressive way: To develop the herbal extracellular vesicle-containing product that is the subject of the invention, different plant sources were researched and preferably pineapple was used in the experiments. For the isolation of extracellular vesicles, the juice of pineapples was squeezed and this juice was first heated at 1000xg for 10 minutes and extracellular vesicles originating from pineapple juice were isolated using dual-phase polymer systems. The isolated extracellular vesicles were then dissolved in 0.9% isotonic serum. The resulting extracellular vesicles were examined visually using a scanning electron microscope (Figure 1A). Then, these extracellular vesicles were processed using CD63, CD9, CD81 and HSP70 markers and characterized by flow cytometry. These methods provide a scientifically sound basis for effectively isolating and characterizing vegetal extracellular vesicles. This study is an important step to gain more information on the potential applications of plant-derived extracellular vesicles and to contribute to advances in this field. (Figure 1B) Quantitative RT-PCR analysis The adipogenic differentiation rate in human adipose-derived stem cells was investigated by the isolation of RNAs and differences in gene expressions. These hADSCs were seeded in a 6-well plate with 100,000 cells in each well. First, each well was cultured for 14 days in vitro using the StemPro Adipogenesis Differentiation Kit for adipogenic differentiation. After adipogenic differentiation, two wells in the 6-well plate were treated with pineapple EV at a concentration of 100 µg/ml for 11 days, and cells were refreshed with new medium-pineapple EV solutions every second day. The other two wells of the plate were treated with DMEM low glucose medium (10% PES and 5% PSA) alone for 11 days. NucleoSpin RNA Isolation Kit from Macherey-Nagel was used for total RNA isolation, and iScript cDNA Synthesis Kit from BIORAD was used to obtain cDNA after RNA isolation. Expression of adiponectin, LPL, PPAR-gamma and aP2 genes was measured using RealTime-PCR. Oil red staining In this study, to induce adipogenic differentiation, hADSCs were cultured for 14 days in in vitro conditions using the StemPro Adipogenesis Differentiation Kit, and after completion of adipogenic differentiation, the cells were cultured in DMEM (10%FBS, 5%PSA) and 100 µg/kg for 11 days. Treatment was applied with ml pineapple EV. The fats in the fat cells were examined with Oil-Red staining in Figure 3. As seen in Figure 3, control cells were incubated with DMEM alone and normal fat formation and morphology were observed in adipo cells. Looking at Figure 3, fat follicle formation in fat cells decreased with 100 ug/ml concentration of pineapple EVs. Experimental Results This study involves obtaining preferred pineapple-derived extracellular vesicles with the developed isolation method and characterization of these EVs. Below, the expression of these processes using a more scientific and impressive language is presented: The product containing herbal extracellular vesicles, which is the subject of the invention, was isolated using pineapple, which is preferred as the plant source. Obtaining extracellular vesicles was achieved using dual-phase polymer systems by centrifuging pineapples at speeds of 15000xg for 60 min. The isolated extracellular vesicles were then examined visually using a scanning electron microscope (Figure 1). These microscopic images show that the structures obtained are similar to the electron microscope images of extracellular vesicles described in the literature. Furthermore, characterization results using flow cytometry revealed that at least 79.91% of extracellular vesicles expressed surface markers. These results are evidence that the molecule obtained based on MISEV2020 Standards is extracellular vesicles (Figure 1). Healthy cells, when exposed to serum containing plant extracellular vesicles, show increased cell proliferation as evidenced by MTS results. Increasing absorbance amount indicates increased viability. These analyzes prove that pineapple EVs increase the rate of division in healthy cells (Figure 2). Cells treated with extracellular vesicles after adipogenic differentiation were characterized using Real-Time PCR and Oil-Red staining. Oil-Red staining is a method used to identify hydrophobic lipids. Figure 3 shows that the fats formed in hADSCs treated with pineapple EValer were observed by Oil-Red staining. Figure 5 shows the expression levels of PPAR, aP2, adiponectin and LPL genes in pineapple EVs. These gene expressions, based on the literature, should indicate increased levels of PPAR, aP2, and adiponectin and decreased levels of LPL expression. The protein levels of these gene expressions were also confirmed with LPL (Figure 4A) and PPAR (Figure 4B) proteins using the ELISA method. The experimental results obtained show that the product of the invention causes the fat cells in the body to decrease. The experimental model based on oil red staining and PCR results proved that the product derived from pineapple extracellular vesicles significantly reduced fat follicles. This study represents an important step in scientifically and experimentally confirming the effects of herbal extracellular vesicles at the cellular level. The results obtained promise hope for individuals interested in healthy weight management and fat metabolism in the future. Application of the Invention The invention in question has the potential to be applied in human adipose-derived stem cells. This product of the invention can be produced in liquid form, primarily in serum form, and also in solid forms, for example; It can be offered in different formulations such as serum, syrup, tablet, medicine, gel and cream. Additionally, it can be used as a nanocarrier molecule, allowing another active substance (for example, a drug or chemical) to be transported into the cell. This delivery method can also be used with various forms and formulations as stated above. This description expresses the various application and transportation methods of the invention in a scientific and impressive language. This highlights the versatile and unique use potential of the invention and demonstrates its usability in different healthcare and biomedical fields in the future.TR
Publications (1)
| Publication Number | Publication Date |
|---|---|
| TR2023014297A2 true TR2023014297A2 (en) | 2024-02-21 |
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