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Türkiye kökenli çörek otu tohumlarından timokinon eldesi, saflaştırılması ve enkapsülasyonunun incelenmesi

Extraction, isolation and encapsulation of thymoquinone from Turkey based black cumin seeds

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  1. Tez No: 603447
  2. Yazar: EZGİ ECE SANCAKDAR
  3. Danışmanlar: PROF. DR. MELEK TÜTER
  4. Tez Türü: Yüksek Lisans
  5. Konular: Kimya Mühendisliği, Chemical Engineering
  6. Anahtar Kelimeler: Belirtilmemiş.
  7. Yıl: 2019
  8. Dil: Türkçe
  9. Üniversite: İstanbul Teknik Üniversitesi
  10. Enstitü: Fen Bilimleri Enstitüsü
  11. Ana Bilim Dalı: Kimya Mühendisliği Ana Bilim Dalı
  12. Bilim Dalı: Kimya Mühendisliği Bilim Dalı
  13. Sayfa Sayısı: 75

Özet

Çörek otu (Nigella Sativa), tohumlarından çoğalan, yıllık otsu bir bitkidir. Özellikle Akdeniz ve Ortadoğu bölgelerinde asırlardır şifalı olduğu bilinmekte ve insanlar tarafından çeşitli hastalıkların tedavisinde kullanılmaktadır. Yapılan araştırmalarda, çörek otu yağında bulunan biyoaktif maddelerin; antioksidan, antiviral, antibakteriyel, antidiyabetik, antialerjik ve antitümör etkikeri olduğu kanıtlanmıştır. Bu aktif maddeler arasında en önemli biyoaktiviteyi gösteren timokinondur (TK). Çörek otu uçucu yağı içerisinde % 20-60 oranında bulunan TK'nin, özellikle son yıllarda yapılan çalışmalarda çeşitli kanser hücrelerine karşı oldukça etkili olduğu görülmüş ve kanser tedavisinde kullanılabilecek doğal bir alternatif olarak öne çıkmıştır. Bu çalışmada, öncelikle Denizli yöresinde yetişen çörek otu tohumlarından uçucu yağlar, doğrudan öğütülmüş tohumlardan distilasyonla ve çözücü ekstraksiyonu (hekzan, metanol) yöntemi ile elde edilen sabit yağın distilasyonu ile elde edilmiştir. Öğütülmüş çörek otu tohumlarının su ile distilasyonu çalışmalarında, tohum/su oranı 1:5, 1:6 ve 1:8 olarak değiştirilmiş, en yüksek uçucu yağ miktarı 1:8 tohum/su oranında % 0.041olarak bulunmuştur. Öğütülmüş tohumlar 24 saat suda bekletildikten sonra 1:8 tohum su oranında distilasyona tabi tutulmuş ve uçucu yağ verimi % 0.076 olarak bulunmuştur. Çörek otu tohumlarından hekzan (soxhlet) ve metanol (soğukta) kullanılarak elde edilen sabit yağın 1:8 yağ/su oranında gerçekleştirilen distilasyonu sonucunda en yüksek uçucu yağ verimi, hekzan ile elde edilen sabit yağdan % 0.432 olarak bulunmuştur. Balıkesir yöresinden temin edilen çörek otu tohumu sabit yağından (hekzan ile soxhlet ekstraksiyonu) elde edilen uçucu yağ verimi ise % 0.067 olarak bulunmuştur. Çörek otu uçucu yağından TK'nin saflaştırılması için kolon kromatografisi uygulanmıştır. Yürütücü sistem olarak hekzan/etil asetat (15:1 v/v) kullanılmıştır. Kolon kromatografisi sonucunda elde edilen TK'nin saflığı UV-Vis Spektrofotometresinde 254 nm dalga boyunda saf ticari TK ile karşılaştırılarak % 90.03 olarak belirlenmiştir. Denizli yöresi tohumlarının uçucu yağında % 48.81 oranında TK bulunduğu görülmüştür. Elde edilen TK'nin farklı yöntemler (karbon nanotüp, poliüretan film, PEG3000) kullanılarak enkapsülasyon işlemleri gerçekleştirilmiş ve en iyi sonuç PEG3000 ile yürütülen çalışmalar sonucunda elde edilmiştir. Enkapsülasyon verimi % 84.3 olarak bulunmuştur. pH 2.3 ve 6.4 olmak üzere iki farklı ortamda yapılan ilaç salım çalışmalarında ise, TK'nin 72 saatlik süre içerisinde kontrollü ve pH'dan bağımsız olarak salındığı belirlenmiştir.

Özet (Çeviri)

Black cumin (Nigella Sativa) is an annual, herbaceous plant that grows from its seeds. It is known to be curative for centuries, especially in the Mediterranean and Middle East regions and has been used by people for the treatment of various diseases. The bioactive substances found in black seed oil; has been proven to have antioxidant, antiviral, antibacterial, antidiabetic, antiallergic and antitumor effects. Among these active substances, the most important one is thymoquinone (TQ). TQ, which is found in 20-60% of black seed essential oil, has been proven to be highly effective against various cancer cells especially in recent studies and has emerged as a natural alternative for cancer treatment. TQ is the most abundant bioactive molecule in Nigella Sativa essential oil. It has an isopropyl group attached to the quinone ring at the 2nd position and a methyl group attached at the 5th position. It is a yellow, crystalline substance at room temperature. TQ was first isolated from black seed essential oil by El-Dakhakhny in 1963. In his study, El-Dakhakhny obtained volatile oil by vapor distillation from fixed oil of black seed extracted with petroleum ether, and then he succeeded in crystallizing the TQ molecule by column chromatography. Determining the structure of the molecule using ultraviolet and infrared spectroscopy, El-Dakhakhny stated that this molecule may be responsible for the pharmacological effects of black seed. This study led to an intensive research on TQ molecule. Despite the promising pharmacological properties, in particular the anticancer effects, of TQ, there are some obstacles to the clinical use of it as a drug. The hydrophobic nature of the molecule results in low water solubility and leads to excretion from the body without the desired effects. Furthermore, it can easily decompose when exposed to high temperature, high pH or light. These properties lead to low bioavailability of TQ molecule. Various drug delivery systems are used to convert bioactive substances with low bioavailability such as TQ into drugs and helps to be used in treatments. The most important advantages of drug delivery systems are that, they increase the solubility and bioavailability of hydrophobic active substances, minimize toxic effects that may occur against healthy cells, increase the stability of active substances and prevent degradation, as well as capable of controlled and targeted drug release. Many researches were done on the encapsulation of TQ using these drug carriers in recent years, but none of them have been clinically used yet. In this study, experiments were conducted in order to obtain essential oil of Turkey based black cumin seeds with high efficiency and TQ was isolated from the essential oils. Then, encapsulation studies were done with the isolated TQ, using three different drug carriers. Initially essential oil of black cumin seeds grown in Denizli region was obtained by direct distillation from ground seeds and distillation of fixed oils obtained by solvent extraction (hexane, methanol) methods. In the direct distillation studies, Clevenger apparatus and simple distillation systems were used and different seed/water ratios (1:5, 1:6, 1:8) were studied in order to obtain the highest essential oil yield. The highest yield was obtained with 1:8 seed/water ratio in simple distillation system as 0.041 %. Then, the ground seeds were soaked in water for 24 hours before distillation and the obtained essential oil yield was 0.076 %. In the solvent extraction studies, soxhlet extraction (with hexane) and cold extraction (with methanol) methods were used. The obtained fixed oils from these extraction techniques were both distilled by simple distillation, with 1:8 oil/water ratio, as it was the method with the highest yield in direct distillation studies. The obtained fixed oil yields from soxhlet and cold extraction techniques were 39.0 % and 23.5 %, and the essential oil yields were 0.432 % and 0.251 %, respectively. It is known that, the origin of the black cumin seeds effects the amount and the chemical composition of the essential oils. Thus, two different black cumin seeds obtained from local suppliers with different origins, Denizli and Balıkesir, were examined. Both seeds were subjected to soxhlet extraction followed by simple distillation. Essential oil yields of Denizli and Balıkesir seeds were 0.432 % and 0.067 %, respectively. TQ was isolated from the obtained essential oils by column chromatography. Hexane: ethyl acetate (15:1 v/v) solvent system was used as the mobile phase. The obtained yellow crystals from the column was analyzed by UV-Vis spectroscopy at 254 nm, according to the prepared calibration graph with standard TQ, in order to determine its purity. Purity of the obtained TQ crystals were found to be 90.03 %. Characterization of the obtained TQ was done by Fourier Transform Infrared Spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC). Finally, encapsulation and drug release studies were done with the obtained TQ from black cumin seeds. Carbon nanotubes (CNT), polyurethane (PU) films and polyethylene glycol (PEG3000) were used as drug carriers. Encapsulation efficiency obtained from the carbon nanotubes was 5.24 %. No further drug release test was conducted because the efficiency was low. Encapsulation of TQ into PU films were performed in two different solvent media. Drug loading studies and swelling and degradation tests of PU films were carried out simultaneously, in order to properly explain the drug loading behavior. In the first experiments with ethanol, it was found that PU films swell in ethanol by 53.4 % and after 20 hours weight loss of the PU films was 10.8 %. In drug loading experiments performed in ethanol medium, the drug concentration was continuously increasing and decreasing up to about 5 hours. It showed that TQ molecules enter into PU discs, but were released again into ethanol solution in a short time. The second drug loading medium was ethanol-water (1:1), which gave swelling ratio of 47.8 % and a weight loss of 1.5 % in 20 hours. In contrast to the results in ethanol medium, in the experiments using ethanol-water (1: 1) solution, there was a controlled increase in the amount of TQ loaded on PU discs. The loading efficiency obtained after 72 hours of operation was 22.5%. It was concluded that, the reason of the higher drug loading ratio in ethanol-water medium than the ethanol medium despite the low swelling ratio of PU films, was the lower solubility of TQ in ethanol-water medium. Since TQ has a very high solubility in ethanol, the diffusion of TQ from this medium into PU films was low. Third drug carrier used for the encapsulation studies was PEG. PEG3000 was used in the experiments and the obtained encapsulation efficiency was 84.3 %. This was the highest efficiency obtained in this study. Obtained PEG-TQ particles were subjected to drug release tests in two different pHs. pH 2.3 buffer solution modeling the stomach medium and pH 6.4 buffer solution modeling the wound liquid (exudate) medium was used. The results of the drug release tests showed that there was a controlled release of TQ in 72 hours, in both mediums. In acidic medium, percentage release of TQ was 62.4 % and 61 % in basic medium. It was discovered that pH has no significant effect on the release of TQ from PEG-TQ. Overall, TQ was successfully extracted and isolated from black cumin seeds with high yield and purity, proving that Turkey based black cumin seeds are suitable for TQ extraction. Encapsulation experiments with PU showed that drug loading medium signicantly effects the encapsulation efficiency. It is concluded that, synthesised PEG-TQ particles are potential candidates to be used in both basic and acidic media with the high encapsulation efficiency and drug relase rates.

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