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Phytochemical and Biotechnological Studies on Diospyros kaki (Family Ebenaceae) Cultivated in Egypt Thesis Submitted By Iman Abdel Aziz El Seody Abdel Gaffar El Sheikh Research Assistant, Phytochemistry Department Pharmaceutical Industries Research Division National Research Centre For the Degree of Master in Pharmaceutical Sciences "Pharmacognosy" Under the Supervision of Prof. Dr. Seham S. El-Hawary Prof. Dr. Soad Hanna Tadros Pharmacognosy Department Pharmacognosy Department Faculty of Pharmacy Faculty of Pharmacy Cairo University Cairo University Prof. Dr. Medhat M. Seif El-Nasr Phytochemistry Department Pharmaceutical Industries Research Division National Research Centre Pharmacognosy Department Faculty of Pharmacy Cairo University, Egypt 2016 Abstract Diospyros kaki L. or Costata cultivar is the main persimmon variety, progressively consumed in the Egyptian market and exportation, it is grown in clay soil under flood irrigation system. The volatile constituents isolated from leaves, comprised 6 identified components, constituting 83.12% of the total oil. GC/MS analysis of the unsaponifiable matter of fruits revealed the presence of 13 compounds, representing 85.61% of the total identified compounds, while that of leaves revealed the presence of 10 compounds, representing 87.16% of the total identified compounds. GC/MS analysis of the fatty acids methyl esters of fruits showed the presence of 13 components, representing 84.79% of the total identified compounds, while that leaves indicated the presence of 10 components, representing 91.07% of the total identified compounds. In addition, scopoletin, kaempferol, luteolin, rutin and apigenin 7-O-glucoside were isolated and identified by TLC, UV and 1H-NMR spectroscopic analysis. Biotechnological study showed that supplementation of MS medium with 10 mg/l ZT + 10 mg/l IAA + 500 mg/l PVP + 0.1 mg/l Thiamine HCL recorded the best results of percentage of calli induction, and supplementation of 1/2 MS + 1 mg/l Zeatin + 2 mg/l IAA + 4 mg/l BA + 0.5 g/l PVP gave the best results of percentage of regenerated shootlets. The quantitative determination of the flavonoid and phenolic constituents of leaves and calli extracts (80% MeOH, each) using HPLC analysis, showed kaempferol (80.9 µg/g Dw) as the highest amount of flavonoid in leaves, and luteolin-6-arbinose 8-glucose (65.1 µg/g) in calli derived internode explants, while scopoletin was the highest phenolic compound recorded (57.08 and 25.3 µg/g) in both, respectively. The biological evaluation of kaki fruits and leaves at the level of 10% indicated that the plant could provide a good nutritional value, save with regard to kidney and liver functions. Also, it could help in decreasing blood sugar, enhancing blood hemoglobin level with good effect in the ratio of HDL and LDL cholesterol. Key words: Diospyros kaki L., phenolic compounds, biotechnology, biological evaluation. INTRODUCTION Africa is the world’s second largest continent after Asia, both in terms of area and population. It has a unique diversity of geographic and climatic factors and exceptionally rich, varied flora with an estimated 68,000 plant species, of which about 35,000 are known to be endemic (Vasisht and Kumar, 2004). Unlike humans and animals, plants are not mobile which makes them very susceptible to attack from pests and predators. To overcome this problem; during metabolism plants produce enormous number of compounds as part of defense mechanism (Bennett and Wallsgrove, 1994 ; Oksman- Caldentey and Inzé 2004). These compounds are not essential for primary functions like growth, photosynthesis and reproduction and are called secondary metabolites (Oksman-Caldentey and Inzé, 2004; Ramachandra and Ravishankar, 2002). Plants which possess therapeutic properties or exert beneficial pharmacological effects on the human body are generally designated as medicinal plants, they naturally synthesize and accumulate some secondary metabolites, like alkaloids, sterols, terpenes, flavonoids, saponins, glycosides, tannins, resins, lactones, quinines, volatile oils etc. as shown in Figure 1 (Ramachandra and Ravishankar, 2002). 1)Anthroquinones 1) Carotenes 2) Benzoquinones 1) Cardiac glycosides 2) Monoterpenes 3) Naphthoquinon 3) Sesquiterpenes 2) Pregnenolone derivative 4)Diterpenes 5) Triterpenes Quinones Steroids Terpenoids Medicinal Plants Phenylpropanoids Alkaloids 1)Anthocyanins 1) Acridines 6) Isoquinolines 6)Liganins 2)Coumarins 2)Quinolizidines 7)Indoles 7)Phenolenons 3)Flavonoids 3)Betalianes 8)Purines 8)Proanthocyanidins 4)Isoflavonids 4)Furonoquinones 9)Pyridin 5)Hydroxycinnamoyl 9)Stilbenes 5)Harringtonines 10)Tropane alkaloids derivatives 10)Tannins Figure 1: Adapted schematic showing the classification of plant derived compounds. Medicinal plants have been used for the treatment of illness and diseases, since the dawn of the time. Egyptian papyrus hieroglyphics and Ancient Chinese scriptures describe medicinal plant uses for treatment , While others developed traditional medical systems (e.g. Ayurvedic and Traditional Chinese Medicine) in which herbal therapies were used (Motaleb et al., 2011) also the production of herbal drugs is a growing industry in Egypt (Fabricant and Farnsworth, 2001). Egypt has a long history of use of medicinal and aromatic plants and drugs. The earliest written record of herbal medicines practice in Egypt was found in the medical books “Ebers Papyrus”, dating back to the sixteenth century B.C. These books contain 877 prescriptions and recipes based on many medicinal and aromatic plants (WHO, 1992). Family Ebenaceae including four genera Diospyros, Euclea, Lissocarpa, and Royena. It consists of woody shrubs and trees distributed in the tropical, sub-tropical and temperate areas and is known worldwide for it’s biological activities. The largest genus of Ebenaceae is Diospyros with approximately 300 species; occur in Asia and Pacific area (De Vera and Santiago, 2014). Diospyros is economically the most important genus of Ebenaceae (Matsushita et al., 2010). Most Diospyros species are important tropical forest resources, yet have not been used efficiently (Utsunomiya et al., 1998). It is native to China, India, Japan and Myanmar. It is exotic to Afghanistan, Algeria, Australia, Brazil, Egypt, France, Indonesia, Israel, Italy, Korea, Palestine, Philippines, Russian Federation, Union of Soviet Socialist Republic (Former), United States of America and Vietnam (Singh and Joshi, 2011). Oriental persimmon, Japanese persimmon or Kaki (Diospyros kaki L. or Diospyros kaki Thunb., according to different authors) is named the food of the Gods (from Greek, Dios meaning God and Spyros meaning food) (Sugiura, 1997). The species seems to have originated in China and it was introduced to Japan in the 7th century and to Korea in the 14th century. In Europe, it was introduced in the 17th century and later. In the 18th century, it was already known world-wide (Yin et al., 2010). In Egypt the cultivated area increased specially in last few years, since it reached 1826 feddans and the total annual production reached nearly 10118 tons of fruits (Fathi et al., 2011). The most important commercial varieties cultivated in Egypt are Fuyu, Hashiya, Costata, Triumph and Hannah Fuyu (Ministry of Agriculture and Land Reclamation agricultural researches Center, 2003). Diospyros kaki L. or Costata cultivar is the main persimmon variety progressively consumed in the Egyptian market and exportation and is grown in clay soil under flood irrigation system, in a private orchard at Aga district, Dakahlia Governorate (Fathi et al., 2011). Diospyros kaki L. is well known in Chinese herbal medicine and used for the treatment of hypertension, cancer, diabetes and atherosclerosis. Tannins, phenols and flavonoids are known to be amongst the active constituents of this plant (Tang and Eisenbrand, 1992). The fruit is a rich source of dietary fiber, minerals, vitamin C, and phenolics (Li et al., 2011) and also condensed tannins (Nakatsubo et al., 2002). In recent years, the leaves of Diospyros kaki L. have been favored as a tea for healthcare in Southeast Asia (Duan et al., 2004). The objective of this work was thus together towards assessing the efficacy of the locally cultivated Diospyros kaki L. as a source of potential medicinal in order to further increase their propagation. Aim of work 1- Reviewing literature of the Diospyros kaki L. 2- Isolation and identification of some active constituents. 3- Studying the effect of some plant growth regulators on in vitro establishment of calli and regeneration cultures from different explants. 4- Determination of some active constituents in different obtained cultures using HPLC. 5- Chemical evaluation of fruits and leaves of Diospyros kaki L. 6- Biological activities of fruits and leaves of Diospyros kaki L. REVIEW OF LITERATURE Family Ebenaceae including four genera Diospyros, Euclea, Lissocarpa, and Royena (De Vera and Santiago, 2014). It is distributed in tropical and warm regions with few species in temperate regions (Mabberley, 1997), in addition to some cultivated species of Diospyros, cultivated as ornamentals or for their edible fruit (Diospyros kaki) (Boulos, 2009). Diospyros kaki L. has other synonyms as Oriental persimmon, Diospyros amara, Diospyros chinensis, Diospyros costata, Diospyros sinensis and others (Meyer and Walker, 1965; Li et al., 1996 ). It is deciduous, branched tree growing to 27 m high with densely pubescent to glabrous young branchlets, with reddish brown lenticels. Leaves are alternate and fruits yellow,