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, orange to orangey red (Lim, 2012).

Major phytochemical constituents, biotechnology and biological activities of plants belonging to genus Diospyros are summarized as follows:

A- Phyto constituent studies.

B- Biotechnological studies.

C- Nutritional composition and biological activities.

A- Phytochemical studies:

The phytochemical researches on Diospyros species was started almost a decade ago. The results of these studies indicated that the genus Diospyros elaborates a great diversity of compounds ranging from hydrocarbons, steroids, terpenoids, napthaquinones to naphthalene

(Yoshihira et al., 1971).

Distribution of chemical constituents in various parts of Diospyros species were summarized in the following Table 1 (Mallavadhani et al.,1998).

Several phytoconstituents has been isolated and identified from the different parts of the Diospryos kaki L. belonging to the category of glycosides, flavonoids, tannins, sterols and triterpenoids (Singh and Joshi,

2011).

Table 1: Class of chemical compounds in different parts of the species of Diospyros kaki L. (Mallavadhani et al., 1998).

No. Class of compounds Plant parts 1 Carotenoids Fruit 2 Tannins Fruit, Leaf 3 Sugars Fruit, seed, root 4 Hydrocarbons Fruit, seed, root 5 Lipids Fruit, seed, bark 6 Aromatics Fruit, root, bark 7 Flavonoids / Coumarins Fruit, leaf, root, sapwood 8 Terpenoids Fruit, leaf, calyx, seed, root, bark, heartwood, ebony, 9 Steroids Leaf, root, bark, heartwood 10 Naphthoquinones Fruit, leaf, root, bark, heartwood

1- Phenolic compounds:

Matsuo and Itoo (1978) identified kaki- tannin in a Japanese persimmon and found that the tannin consisted of catechin, catechin-gallate, gallocatechin, and gallocatechin-gallate. Catechins are polyphenolic compounds which may offer potential benefits to human health. Catechins have been reported to exhibit antioxidant, anticarinogenic, antimutagenic, and cardioprotective effects. (Middleton and Kondaswami, 1992; Renaud and De Lorgeril, 1992; Hertlog et al., 1993; Kondo et al., 1999). Customers usually categorize persimmon fruits into two types-astringent and non-astringent. The former taste bitter because they contain soluble tannins

that occur in high concentrations at maturity: the latter are sweet and have low levels of soluble tannins when ripe (Yonemori et al., 2000; Chen et al., 2008; George and Redpath, 2008). Tannin compounds can be sub-divided into two groups that are hydrolysable tannins (low amounts in plants) and proanthocyanidins that often called condensed tannins (more widely distributed). (Yonemori et al., 2000; Park et al., 2004; Wolf and Lui, 2003;

Li et al., 2006; Kim et al., 2010).

Bei et al. (2009) extracted flavonoids from leaves of Diospyros kaki L. in which more than 77.4% flavonoids including 34.6% quercetin and it’s glycosides (hyperin and isoquercitrin) as well as 42.7% kaempferol and it’s glycosides (astargalin), characterized by HPLC and LC-MS.

Chen et al. (2009) described the isolation and structural elucidation of a novel C-glycosylflavone, 8-C-[α-L-rhamnopyranosyl-(1-4)]-α-D- glucopyranosyl apigenin together with the six known compounds 2-O- rhamnosyl vitexin ,kaempferol-3-O-α-L-rhamnopyranoside, myricetin-3-O-α- rhamnopyranoside, myricetin-3-O-β-D-gluco-pyranoside, blumeol C glucoside, and byzantionoside B from the BuOH extract of persimmon leaves.

Kawakami et al. (2010) identified polyphenols proanthocyanidin, consisting of catechin, epigallocatechin, epigallocatechin-3-O-gallate, epicatechin, and epicatechin-3-O-gallate in persimmon leaves.

2 – Sterols and triterpenes:

Fan and He (2006) developed HPLC method to simultaneously assess the three bioactive triterpene acids: barbinervic acid and it’s epimer, rotungenic acid, along with 24-hydroxy ursolic acid. The HPLC assay was performed on a reversed-phase C18 column with methanol and aqueous

H3PO4 as the mobile phase and using a monitoring wavelength at 210 nm.

Chen et al. (2012) isolated a new triterpene compound known as 18, 19-secoursane using 90% EtOH.

3 - Volatile oil:

Kameoka et al. (1989) studied several cultivars of Diospyros kaki: dried medicinal herb [A] and fresh leaves of Fuyugaki [B], Fujigaki [C] and

Jitsuseisibugaki (Senboro) [D]. The fresh leaves of the latter were collected at Nishikamo, Aichi in October 1986. The volatile oils were obtained by steam distilation with the following yields: [A] 0.05%, [B] 0.02%, [C] 0.02% and [D] 0.02%. The components of the volatile oils were investigated by IR, GC and GC-MS. The volatile oils were composed of alkanes, alcohols, carbonyl compounds, carboxylic acids, esters, phenols and heterocyclic compounds, in which the major common components were p-cresol, phenethyl alcohol, hexanoic acid and methyl-4-pentenoate. The eugenol and dihydroactinidiolide found predominantly in [A] oil were minor components of [C] oil. Eugenol content in [C] oil was less than 4% of [A] oil, and dihydroactinidiolide abundant in [A] oil was at a trace level in [C] oil.

Rong and Feng (1999) analyzed volatile compounds in persimmon leaves by means of programmed temperature capillary GC/MS. Eighty compounds were separated in which 75 volatile compounds were identified, occupied 98.13% of the total. The oxygen containing chemical compounds occupy 95.22% . There are 5 compounds, their content is more than 3%, occupied 76.37% of the total. They are (E)-2-hexenal; (Z)-2-hexenal; (E)-3- hexen-1-ol; 1,6-octadien-3-ol; 3,7-dimethyl-,2-hexadecen-1-ol; 3,7,11,15- tetramethyl-, their content are 42.22%,8.14%,3.63%,4.56%,17.82% respectively, they are the main volatile components of persimmon leaves.

Wang et al. (2012) found that persimmon fruit which is grown in various countries and is increasingly appreciated for its nutritional value, health benefits and rich flavour, surprisingly little research has been conducted to uncover the components responsible for its unique flavour. An aroma extract of persimmon fruit (Diospyros kaki L., var. Triumph) was obtained by hydrodistillation under vacuum followed by solid phase extraction. (GC-MS) analysis of the extract led to the positive identification of 50 compounds, among which aldehydes emerged as the most important class of volatile compounds. The six most intense aroma-impact compounds were methional, (E)-2-hexenal, phenylacetaldehyde, (E,Z)-2,6-nonadienal, hexanal and Furaneol.

4- Structure of major phytoconstituents isolated from Genus Diospyros:

Table 2: Compounds isolated from Genus Diospyros. Species/ No. Name Structure Reference Plant part Flavonoids Glycosides OH

OH O OH Diospyros kaki L. Myricetin 3-O-β-D- (Chou et al., 1 (Leaves) glucopyranoside 1984, 1985) OH D-glucopyranoside OH O H

OH O OH Diospyros kaki Kampferol 3-o-α-L- (Chou, 1984, 2 L. rhamnopyranoside 1985) (Leaves) H O--L-rhamnopyranoside OH O

Species/ No. Name Structure Reference Plant part H

OH O OH Diospyros kaki Kampferol 3-O-β-D- (Chou, 1984, 3 L. xylopyranoside 1985) (Leaves) H O--D-xylopyranoside OH O

Diospyros kaki Kampferol 3-O-α-L- (Chou, 1984, 4 L. arabinopyranoside 1985) (Leaves)

Species/ No. Name Structure Reference Plant part OH

OH O OH Diospyros kaki Quercetin 3-O-α-L- (Chou, 1984, 5 L. arabinopyranoside 1985) (Leaves) H L-arbinopyranoside OH O H

OH O (Chen et al., OH 1989b, Kampferol 3-O-(2”-O- Diospyros kaki L. Chou et al., 6 galloyol)-β-D- (Leaves) 1984, 1985, glucopyranoside H Takahisa et al., "-o-galloyl)--D-glucopyranoside 1987, 1988) OH O

Species/ No. Name Structure Reference Plant part OH (Chen et al., OH O Diospyros kaki L. 1989b; Quercetin 3-O-β-D- OH Diospyros Gafner and 7 glucopyranoside(isoquerctri .zombensi Rodrignez, 1988, n) H (Leaves) 1989) D-glucopyranoside OH O OH

OH O OH (Chen et al., Quercetin 3-O-(2”-O- Diospyros kaki L. 1989b; 8 galloyol)-β-D- (Leaves) Chou et al., 1984, glucopyranoside H 1985; Takahisa "-o-galloyl)D-glucopyranoside et al., 1987, 1988) OH O

Species/ No. Name Structure Reference Plant part

Kampferol 3-O-β-D- Diospyros kaki L. (Chen et al., 9 galactopyranoside(trifolin) (Leaves) 2002a)

Kaempferol 3-O-β-D- Diospyros kaki L. (Chen et al., 10 glucopyranoside (astragalin) (Leaves) 2002a)

Species/ No. Name Structure Reference Plant part

Diospyros kaki Isorhamnetin 3-O-β-D- (Chen et al., 11 L. glucopyranoside 2002a) (Leaves)

Diospyros kaki Quercetin 3-O-β-D- (Chen et al., 12 L. galactopyranoside (hyperin) 2002a) (Leaves)

Species/ No. Name Structure Reference Plant part

Quercetin 3-O-β-D- Diospyros kaki L. (Chen et al., 13 glucosyl-(6 1)-α-L- (Leaves) 2002a) rhamnoside (Rutin)

Diospyros Kaempferol 3-O-β-(2-O-α - cathayensis rhamnopyranosyl-3-O-β - (Furusawa et al., 14 Diospyros glucopyranosyl)-β- 2005) rhombifolia glucuronopyranoside. (Leaves)

Species/ No. Name Structure Reference Plant part

Kaempferol 3-O-(2-O-α- Diospyros rhamnopyranosyl-3-O-(6-O- cathayensis (Furusawa et al., 15 α-rhamnopyranosyl-β- Diospyros 2005) glucopyranosyl)-β- rhombifolia glucopyranoside (Leaves)

b- Aglycone

Diospyros kaki L. (Chen et al., 16 Myricetin (Leaves) 2009)

Species/ No. Name Structure Reference Plant part

Diospyros kaki L. (Xin-tang et al., 17 Annulatin (Leaves) 2014)

Diospyros kaki L. 18 Kampferol (Xie et al., 2015) (Leaves)

Species/ No. Name Structure Reference Plant part

Diospyros kaki L. 19 Quercetin (Xie et al., 2015) (Leaves)

Species/ No. Name Structure Reference Plant part Coumarins

OH O O Diospyros ebenaster (Dominguez et Diospyros al., 1979, Zhou, 20 Scopoletin gracilipes et al., 1983) Diospyros kaki L. (Leaves) OCH3

OCH3 O O (Zhou et al. Diospyros kaki L. 6-Hydroxy-7-methoxy- 1987) 21 (Leaves) coumarin

OH

Species/ No. Name Structure Reference Plant part Aromatic hydrocarbon

4', 5-dimethoxy-3-β-D- Diospyros kaki L. (Chen et al., glucopyranosyloxy-4- (Leaves) 22 2005a) hydroxy-biphenyl

(Kakispyrol)

Sterols and Triterpenes

Diospyros ebenum Diospyros evena (Dutta et al., Diospyros kaki 23 Stimasterol 1972; Lin et al., Diospyros mollis 1988, 1989) Diospyros Montana (Leaves)

OH

Species/ No. Name Structure Reference Plant part

H Diospyros kaki L. (Matsura et al., Diospyros 1977; 24 β- Sitosterol glucoside CH3 morrisiana Chen et al., (Leaves) 1989a)

O-Glu.

Diospyros kaki L. (Matsura et al., Diospyros castanea 1977; Singh and 25 Oleanolic acid Diospyros Prakash, 1988) malanonilau

(Leaves)

Species/ No. Name Structure Reference Plant part

Diospyros (Andiamasy and ebenaster Foursate 1978; Diospyros indica Dominguez et 26 Betulin Diospyros kaki L. al.,1979) Diospyros peregrina

Diospyros ebenaster H Diospyros ebenum (Dominguez et Diospyros indica al., 1979; Lin et 27 β-Sitosterol CH3 Diospyros kaki al., 1988, 1989) Diospyros kirkii Diospyros kaki L. (Leaves) OH

Species/ No. Name Structure Reference Plant part

H Diospyros kaki L. (Lin et al., 1988, 28 Campesterol H (Leaves) 1989)

OH

Diospyros greeniway (Lin et al. 1988, 29 Lupeol Diospyros kaki L. 1989;Khan and Diospyros Rwekika ,1992) montana

Species/ No. Name Structure Reference Plant part

Diospyros (Lin et al., 1988, greeniway 1989, ;Khan 30 Betulinic acid Diospyros kaki L. and Rwekika, Diospyros 1992) montana

Diospyros kaki L. 31 Barbinervic acid (Ahmad, 1994) (Leaves)

Species/ No. Name Structure Reference Plant part

32 Pomolic acid Diospyros kaki L. (Ahmad, 1994)

(Chen et al., Diospros kaki L. 33 α- Amyrin 2002b) (Leaves)

Species/ No. Name Structure Reference Plant part

(Chen et al., Diospros kaki L. 34 Ovaol 2002b) (Leaves)

(Chen et al., 19α, 24-dihydroxy ursoloic Diospros kaki L. 35 2002b) acid (Leaves)

Species/ No. Name Structure Reference Plant part

(Chen et al., Diospyros kaki L. 36 Kakisaponin 2007) (Leaves)

Diospyros kaki L. (Fan and He, 37 24-hydroxy ursolic acid (Leaves) 2006).

Species/ No. Name Structure Reference Plant part

Diospyros kaki L. (Fan and He, 38 Rotungenic acid (Leaves) 2006)

18, 19-Seco-3β-Hydroxy- Diospyros kaki L. (Chen et al., 39 urs-12-en-18-one (Leaves) 2012)

Species/ No. Name Structure Reference Plant part

Diospyros kaki L. (Xin-tang et al., 40 β- Amyrin (Leaves) 2014)

Diospyros kaki L. (Xin-tang et al., 41 Betulinaldehyde (Leaves) 2014)

Species/ No. Name Structure Reference Plant part

(Xin-tang et al., Diospyros kaki L. 42 Rosamutin 2014) (Leaves)

Tannins

Diospyros kaki L. (Nakatsubo et Diospyros lotus al., 2002; Zhang Diospyros 43 Catechin et al., virginiana 2011) (Fruits and leaves)

Species/ No. Name Structure Reference Plant part

Diospyros kaki L. (Nakatsubo et Diospyros lotus al., 2002; Zhang 44 Epicatechin Diospyros et al., virginiana 2011) (Fruits and leaves)

Diospyros kaki L. (Nakatsubo et Diospyros lotus al., 2002; Zhang Catechin-3-gallate 45 Diospyros et al., (Epicatechin-3-O-galate) virginiana 2011) (Fruits and leaves)

Species/ No. Name Structure Reference Plant part

Diospyros kaki L. (Nakatsubo et Diospyros lotus al., 2002; Zhang Gallocatechin 46 Diospyros et al., (Epigallocatechin) virginiana 2011) (Fruit and leaves)

Diospyros kaki L. (Nakatsubo et Gallocatechin-3-gallate Diospyros lotus al., 2002; Zhang 47 (Epigallocatechin-3-O- Diospyros et al., gallate) virginiana 2011)

(Fruit and leaves)

V-Naphthalene OH O

OCH3 (Yoshihira et al., 48 3-methoxy-7-methljuglone Diospyros kaki L. 1971)

CH3 O

Species/ No. Name Structure Reference Plant part

(Yoshihira Diospyros ferrea et al., 1971; 49 8-Hydroxyisodiospyrin Diospyros kaki L. Tezuka et al., 1973)

Species/ No. Name Structure Reference Plant part

(Yoshihira et al. 1971; Diospyros kaki L. 50 Maritinone Richomme et al., D.samoensis 1991)

OH O

Diospyros kaki L. (Tezuka et al., and Diospyros kaki 51 Shinanolone 1972; Lin et var.sylvestris al.1988, 1989)

CH3 OH

Species/ No. Name Structure Reference Plant part OH O O OH D.kaki and D.kaki (Lillie et al., sylvestris 1976; Lin et al., D.mollis 1988, 1989) D.montana

52 Mamegakinone

CH3 CH3

O O

O Diospyros canaliculata (Zakaria et al., CH3 Diospyros ebenum 1984; Lin et al., Diospyros kaki L. 53 Plumbagin 1988, 1989) Diospyros siamang

(Blackened

heartwood and O OH leaves)

Species/ No. Name Structure Reference Plant part OH O Diospyros OH O (Zhong et al., cinnabarina 1984; Diospyros fragrans 54 Diospyrin Lin et al., 1988, CH3 Diospyros kaki L. 1989) Diospyros O CH3 virginiana

O

(Zakaria, et al., Diospyros kaki L. 1984; Lin et al., 55 Neodiospyrin Diospyros ismailii 1988, 1989)

Species/ No. Name Structure Reference Plant part O

CH3 Diospyros ismailii Diospyros kaki L. (Carter et al., 56 7-Methyljuglone Diospyros 1978; Lin et al., virginiana 1988, 1989,) (Wood and bark)

OH O

D.dendo (Lin et al., 1988, D.kaki L. and 1989; Zhong and 57 Isodiospyrin D.kakisylvestris Feng, 1987, D.montana 1988).

Species/ No. Name Structure Reference Plant part

(Matsushita et 4-Hydroxy-5,6- dimethoxy Diospyros kaki L. 58 al., 2010). -2-naphthaldehyd (Root)

(Matsushita et 5,6,8-trimethoxy-3-methyl- Diospyros kaki L. 59 al., 2010). 1-naphthol (Root)

Species/ No. Name Structure Reference Plant part

Diospyros kaki L. (Matsushita et 4,8-dihydroxy-5-methoxy- 60 (Root and al., 2010). 2-naphthaldehyde heartwood)

(Matsushita et 4-hydroxy-5,8-dimethoxy- Diospyros kaki L. 61 al., 2010). 2-naphthaldehyde (Root)

Species/ No. Name Structure Reference Plant part

(Matsushita et 4-hydroxy-5-methoxy-2- Diospyros kaki L. 62 al., 2010). naphthaldehyde (Root)

4-hydroxy-5,8- dimethoxy- Diospyros kaki L. (Matsushita et 63 2-(2-oxopropyl)- (Blackened al., 2011) naphthalene heartwood)

Species/ No. Name Structure Reference Plant part

Diospyros kaki L. (Matsushita et 2-glycidyl-4-hydroxy-5,8- 64 (Blackened al., 2011) dimethoxy naphthalene heartwood)

Volatile oil

Eugenol (Kameoka et al., 65 Diospyros kaki L. 1989)

Species/ No. Name Structure Reference Plant part

OH

Diospyros kaki (Kameoka et al., 66 P-cresol L. 1989)

CH3

CH3 CH=CH (Horvat et al., 67 (E)-2-hexenal Diospyros kaki L. 1991) (CH2)2 CHO

CH3 CH3 CH3 (Horvat et al., 68 bornyl acetate H Diospyros kaki L. 1991)

OCOCH3

B- Biotechnological studies:

I) Plant biotechnology: History of plant tissue culture:

The science of plant tissue culture took its roots from the discovery of cell followed by propounding of cell theory. In 1838, Schleiden and Schwann proposed that cell is the basic structural unit of all living organisms. They visualized that cell is capable of autonomy and therefore it should be possible for each cell if given an environment to regenerate into whole plant. Based on this premise, in 1902, A German physiologist, Gottlieb Haberlandt (Figure 2) for the first time attempted to culture isolated single palisade cells from leaves in knop’s salt solution enriched with sucrose. The cells remained alive for up to one month, increased in size, accumulated starch but failed to divide. Though he was unsuccessful but laid down the foundation of tissue culture technology for which he is regarded as the father of plant tissue culture (Hussain et al., 2012).

Figure 2: Dr. Gottlieb Haberlandt )1885 – 1932).

What is tissue culture?

Plant tissue culture, or the aseptic culture of cells, tissues, organs, and their components under defined physical and chemical conditions in vitro, is an important tool in both basic and applied studies as well as in commercial application. It owes its origin to the ideas of the German scientist,

Haberlandt, at the beginning of the 20th century

(http://www.ncbi.nlm.nih.gov/pubmed/17914178).

Plant tissue culture is the process by which parts of plant can be grown in vitro condition in a sterile culture medium (Figure 3). The nutrient media used in plant tissue culture contains macro, micro salts, vitamins, carbohydrates and plant growth regulators. Plant tissue culture plays an important role in the area of plant conservation, crop improvement, mass propagation and secondary metabolite production. This technique also helps to propagate economically important plants, medicinal aromatic plants, orchids and other ornamental plants in large scale through in vitro method

(Balakrishnan et al., 2009).

Figure 3 .Scheme of plant tissue culture.

The advantages of in vitro propagation of medicinal plants are listed below:

Diseases, drought and resistance are major aim in plant breeding programs of many crop species. In vitro selection and gene transfer studies have been successfully used to produce many number of improved crop species. The propagation of valuable economic plants through tissue culture is based on the principle of totipotency. Single cell cultures, plant cell without cell wall (protoplast), tissues of leaves, or roots can be used to generate plants on culture media given the required nutrients and growth regulators. Plant cell and tissue culture has contributes significantly to crop improvement and has great potential for the future (Hasler et al., 2003). These advantages are

(Prakash and Van, 2007):

1. Higher rate of multiplication.

2. Environment can be controlled or altered to meet specific needs of the plant.

3. Plant available all over year round (independent of regional or seasonal

variation).

4. Identification and production of clones with desired characteristics.

5. Production of secondary metabolites.

6. New and improved genetically engineered plant can be produced.

7. Conservation of threatened plant species.

8. Preservation of genetic material by cryopreservation

Plant cell culture as a source of secondary metabolites:

Plant cell cultures are an attractive alternative source to whole plant for the production of high-value secondary metabolite. Plant cells are biosynthetically totipotent, which means that each cell in culture retains complete genetic information and hence is able to produce the range of chemicals found in the parent plant as shown in Figure 4.

The advantages of this technology over the conventional agricultural production are the possibility to produce novel compounds that are not normally found in parent plant.

Strategies to increase secondary metabolite production in plant cell cultures:

1. Obtaining efficient cell lines for growth.

2. Screening of high-growth cell line to produce metabolites of interest

a. Mutation of cells

b. Amenability to media alterations for higher yields.

3. Immobilization of cells to enhance yields of extracellular metabolites and to facilitate biotransformation.

4. Use of elicitors to enhance productivity in a short period of time.

5. Permeation of metabolites to facilitate downstream processing.

6. Adsorption of the metabolites to partition the products from the medium and to overcome feedback inhibition.

7. Scale-up of cell cultures in suitable bioreactors (Ramachandra and Ravishankar, 2002).

Figure 4: Use of plant tissue culture in micropropagation of plants used in pharmaceutical products.

Major stages of plant micropropagation as illustrated in Figure 5.

Stage I: Selection of suitable explants, their sterilization and transfer to nutrient media for establishment, i.e. initiation of a sterile culture of the explant.

Stage II: Proliferation or multiplication of shoots from explant on medium.

Stage III: Transfer of shoots to a rooting medium followed later by planting into soil (Chawla, 2000).

Figure 5: Major stages of plant micropropagation.

Tissue culture media:

Culture media contains vital nutrients and elements for in vitro growth of plant tissues (Murashige and Skoog, 1962; Gamborg et al., 1976; Fowler et al., 1993). Choosing the right media composition is important for successful tissue culturing (Gamborg et al., 1976; Huang and Murashige 1977; Bhojwani and Razdan 1996). Medium contains a carbon source (sucrose), macro and micro nutrients, vitamins, hormones and other organic substances (Gamborg et al., 1976; Bhojwani and Razdan 1996). A wide range of media are available for plant tissue culture, but MS

(Murashige and Skoog, 1962) medium is commonly used (Murashige and Skoog 1962; Gamborg et al., 1976; Bhojwani and Razdan, 1996). Other media used are LS (Linsmaier and Skoog, 1965), SH (Schenk and Hilderbrandt, 1972), WPM (Lioyd and McCown, 1980), and the NN (Nitsch and Nitsch, 1969). Agar is not essential media component but is used as gelling agent (Rayns et al., 1993; Bhojwani and Razdan , 1996). It prevents death of cultured cells due to submerging and lack of oxygen in liquid medium (Bhojwani and Razdan, 1996). The pH of culture media is normally between (5.0-6.0), and is also very important as it affects uptake of ions (Bhojwani and Razdan, 1996).

The effects of media composition were demonstrated when tissue culturing Vitis thunbergii using WPM, MS and NN medium. WPM medium was found to enhance shoot proliferation. Whereas, explants cultured on MS medium showed increased plant growth, leaf formation and root induction. NN medium showed explants contained higher amounts of chlorophyll but showed lower growth (Lu, 2005). A comparison of the chemical composition of the frequently used plant tissue culture media appears in Table 3 which was given in the appendix of the proceedings of the technical meeting of the IAEA and WPM (Ali et al., 2009).

Table 3: Composition of the most frequently used media (mg/l). Medium components MS G5 W LM VW Km M NN WPM (mg/l) Macronutrients

Ca3(PO4)2 200.0

NH4NO3 1650.0 400.0 720.0 400.00

KNO3 1900.0 2500.0 80.0 525.0 180.0 180.0 950.0

CaCL2.2H2O 440.0 150.0 96.0 166.0 96.00

MgSO4.7 H2O 370.0 250.0 720.0 370.0 250.0 250.0 250.0 185.0 370.00

KH2PO4 170.0 170.0 250.0 150.0 150.0 68.0 170.00

NH4 )SO4)2 134.0 500.0 100.0 100.0

NaH2PO4.H2O 150.0 16.5

CaNO3.4H2O 300.0 556.0 200.0 200.0 556.00

Na2SO4 200.0 KCl 65.0

K2SO4 990.0 990.00 Micronutrients KI 0.83 0.75 0.75 80.0 0.03

H3BO3 6.20 3.0 1.5 6.2 6.2 0.6 10.0 6.20

MnSO4. 4H2O 22.30 7.0 0.075 25.0 22.30

MnSO4. H2O 10.0 29.43

ZnSO4.7 H2O 8.6 2.0 2.6 8.6 0.05 10.0 8.60

Na2MoO4.2HO 0.25 0.25 0.25 0.25 0.05 0.25 0.25

CuSO4.5 H2O 0.025 0.025 0.25 0.025 0.025 0.25

CoCl2.6 H2O 0.025 0.025 0.025

Co(NO3)2.6 H2O 0.05

Na2EDTA 37.3 37.3 37.3 74.6 37.3 37.3 37.30

Medium components MS G5 W LM VW Km M NN WPM (mg/l)

FeSO4.7 H2O 27.8 27.8 27.8 25.0 27.8 27.8 27.80

MnCl2 3.9 0.4 Vitamins and other supplements Inositol 100.0 100.0 100.0 100.0 100.00 Glycine 2.0 2.0 3.0 2.0 2.00 Thiamine HCl 0.1 10.0 0.1 1.0 0.3 0.3 0.5 1.00 Pyridoxine HCl 0.5 0.1 0.5 0.3 0.3 0.5 0.50 Nicotinic acid 0.5 0.5 0.5 1.25 5.0 0.50 Ca-Panthotenate 1.0 Cysteine HCl 1.0 Riboflavin 0.3 0.05 Biotin 0.05 0.05 Folic acid 0.3 0.5

Where, MS= Murashige and Skoog, G5= Gamborg, W= White, LM= Lloyd and McCown, VW = Vacin and Went, Km = Kudson modified, M= Mitra et al., NN= Nitsch and Nitsch media, WPM = woody plant media.

Plant growth hormones:

Growth hormones regulate various physiological and morphological processes in plants and are also known (PGRs) or phytohormones (Bhojwani and Razdan 1996; Srivastava 2002) they were collected and summarized in Table 4. Plant growth regulators are synthesized by plants, therefore many plant species can grow successfully without external medium supplements (Bhavisha and Jasrai, 2003; Baksha et al., 2005). Hormones can also be added into cultures to improve plant growth and to enhance metabolite synthesis as shown in Figure 6 (Bhojwani and Razdan 1996; Rayns et al., 1993).

Table 4: Plant growth hormones and their functions. Growth Categories Functions References hormones Auxins Cell division, IAA (Rayns et al. 1993, elongation and root IBA Bhojwani and differentiation. NAA Razdan 1996 and NOA Rout et al. 2000). Cytokinins Cell division, shoot BAP (Rayns et.al. 1993, induction, IPA or 2ip Bhojwani and development and Kn Razdan, 1996 and proliferation ZT Rout et al. 2000)

Gibberellins Growth, elongation GA3 (Fowler et al. 1993 and flowering and Bhojwani and Razdan, 1996) Florigen Flowering inducing Paradigm (Shalit et al., 2009) hormone

Figure 6: Four hormones and their functions Sterilization of explant: Chemical sterilization:

It is eradication of microorganisms with the aid of chemical. Plant material can be surface sterilized by some of the commonly used chemical sterilants and their effectiveness is shown in a comparative form in the Table 5 (Chawla, 2000).

Table 5: A comparison of the effectiveness and properties of common surface sterilants for explant. Sterilizing Concentration Ease of Treatment Remarks agent used removal time (min) Sodium 1-1.4% +++ 5-30 Very hypochlorite effective Calcium 9-10% +++ 5-30 Very hypochlorite effective Hydrogen 10-12% +++++ 5-15 Effective peroxide Bromine 1-2% +++ 2-10 Very water effective Silver nitrate 1% + 5-30 Effective Mercuric 0.01-1% + 2-10 Satisfactory chloride Antibiotics 4-50mg/l ++ 30-60 Effective

(+) weak effect, (++) moderate effect, (+++) very effective.

Sterilization of media:

Is routinely achieved by autoclaving at the temperature ranging from 115°C – 135°C (Torres, 1989).

Phenolic exudation:

A blackening or browning of tissues excised from many woody species of the tropics can be observed. This process is called phenolic oxidation inactivates the growth of the tissues in culture media (Pree and Compton, 1991 and Loomis and Battaile, 1996).

Browning of tissue is caused by the oxidation of tannin and polyphenols and the formation of quinones which are highly reactive and toxic to the plant tissues (Monaco et al., 1977).

Quinine substance which are produced as a result of oxidation of phenolic compounds will gradually enter the tissues cultured on the medium and further represses the activities of other enzymes and as a result, poison other contents of the medium (Tao et al., 2007).

Factors affecting production of phenolic compounds:

1-As production of phenolic compounds indirectly stimulated by several factors like age of the plant, size of plant, biotic and abiotic stresses, probably young seedlings do not synthesize higher quantities of phenolics when grown in a growth chamber (Chandra et al., 2005).

2-Light appears to induce flavonol synthesis in the chloroplasts and cytoplasm.

3-Some nutrients, especially carbohydrate supplies influence the phenolic composition (Biswas et al., 1999).

Browning was controlled by:

1-Culturing different types of explants on tissue culture media supplemented with an adsorbent (activated charcoal) and antioxidants (ascorbic acid,

cysteine and silver nitrate) (Ahmad et al., 2013).

2-Glutathione (Matkowski, 2000).

3-Phenol absorbing polymers like PVP (Matkowshi, 2008).

4-Liquid media can be used to reduce phenolic oxidation (MacRae and Van,

1990).

5-In addition frequent subculturing (Francisco et al., 2004)

As browning of media prevents further progress in biotechnology of woody plants especially, therefore it is necessary to explore a suitable protocol for successful micropropagation (Sauls and Campbell, 1994).

In vitro propagation of Diospyros kaki L.

Fukui et al. (1990) determined the best season for shoot tip culture of Japanese persimmon, shoot tips were cultured monthly from February 1987 to January 1988 in 1/2N-MS basal medium, Murashige and Skoog's medium containing half strength nitrogen, with zeatin. No shoot growth was observed in the medium without zeatin. In any month of the year, the shoot growth was enhanced by zeatin. The activity of shoot tips in shoot elongation increased from late April to late June, and shoot length after six weeks of culture was

maximum on 22 June. Thereafter, activity decreased until late October and then remained constant until early April. These changes in shoot growth in vitro coincided with the changes of depth of internal dormancy. Leaf number was high in zeatin only on 30 May and 22 June. Shoot tip growth in June had, therefore, better shoot elongation, leaf differentiation, callus formation and mortality rate, so the appropriate season for shoot tip culture of Japanese persimmon was considered to be June.

Tao and Sugiura (1992) found that genus persimmon (Diospyros L.) was interesting for biotechnological research so they focused mainly on quality improvement and preservation of the cultivars popular among growers. Because of the small number of cultivars utilized, there has been a loss of persimmon genetic variability. Further, much attention has been paid to persimmon in vitro propagation.

On the other hand, Bellini and Giordani (1997) continued the establishment, regeneration, elongation, and rooting of shoots of different species. Cultivars of different species such as D. lotus and D. virginiana , as well as many persimmon cultivars, were very difficult to in vitro propagation. The high amount of polyphenols in D. kaki , D. lotus, and D. virginiana tissues, including axillary buds, often causes browning and oxidation of media and cut tissues, strongly hindering in vitro establishment.

Whereas Gondoa et al. (1999) assumed that calli of Diospyros kaki Thunb were induced on half-strength Murashige-Skoog solid medium supplemented with 1.0 mg/L IAA and 0.1 mg/L BA in the dark and successfully subcultured on the same medium. A new phenolic metabolite, 7-

methyl-1, 4, 5-trihydroxy-naphthalene 4-O-(6'-O-b-xylopyranosyl)-b- glucopyranoside, was isolated from MeOH extract of the calli cultures and its chemical structure was elucidated by NMR spectroscopic analysis.

On the other hand, Salomao et al. (1999) cultivated buds of Diospyros kaki in half-strength of MS medium with addition of eight combinations of growth regulators as follows: 10 µM of ZT (medium 1); 10 µM of ZT+0.05

µM of IAA (medium 2); 10µM of ZT+ 0.10 µM of IAA (medium 3); 10 µM of ZT 2 µM of IAA (medium 4); 10 µM of ZT+ 0.04 µM of IAA (medium 5); 10 µM of ZT+ 0.01 µM of NAA (medium 6); 0.01 µM of Kinetin (medium7); and half-Strength MS (medium 8). Fifty percent of the explants obtained in each passage were subcultured in the same medium. The other fifty percent were transferred to MS medium with 0.20 µM of IBA for rooting. Explants introduced onto MS medium or MS medium with added Kinetin did not develop shoots and formed only callus. The best explants for development were found on medium with ZT or ZT + IAA. Explants cultured on MS medium containing 0.02 µM of IBA and kept in the dark for rooting developed roots or callus but did not develop shoots.

In the same respect Jun-lian et al. (2004) compared the effects of basal mediums, hormones and their concentrations on the shoot regeneration from leaves. Modified Murashige and Skoog (MS) [MS(1/2N)] was the most optimum for the regeneration and 1/2MS was better than MS. Shoot percentage in the medium containing 4.0 mg/l ZT was much higher than other two concentrations, of which 2.0 mg/l ZT was much better than 1.0

mg/l ZT and shoot percentage in the 1.0 mg/l ZT was only 4%. There were no any positive effect when supplementing IAA in the medium, and shoot percentage and average shoots per explants were dramatically decreased in the 2.0 mg/l IAA. Data in the orthogonal trials indicated that ZT was the most effective factor in the shoot regenerating of Uenishwase persimmon and basic medium was important too, but IAA had no any beneficial effects at all.

Also, Xiao-Xin et al. (2004) studied the effects of basic media, plant growth regulators and their concentration, and parts of leaf on callus formation and adventitious bud regeneration in persimmon of Mopanshi (Diospyros kaki Thunb). The results showed that modified MS medium supplied with 4.0 mg/L ZT (or 1.0 mg/L TDZ) and 0.1 mg/L IAA was the optimal protocol for adventitious bud regeneration. The effects of ZT and

TDZ on the generation from leaf disks were different. Applied ZT could benefit to the generation directly. But the leaf disks with TDZ treatments need to be transferred into the medium supplied with lower concentration of cytokinin for further culture to allow the adventitious bud growth. Dark incubation for two to three weeks could increase the percentage of adventitious bud regeneration. Leaf disks from near the petioles were the easiest to generate adventitious buds, followed by that from the middle of leaves and the leaf tips.

However, Xi-zhu et al. (2005) studied the effect of concentrations and combinations of plant growth regulators on the proliferation and rooting of shoot cuttings of Diospyros kaki (Persimmon). The results showed that the combination of ZT 1.0 mg/L+ BA 0.2 mg/L+IAA 0.02 mg/L in the modified

MS (1/2N) culture medium was benefitial to bud propagation and the combination of ZT 1.0 mg/L+BA 0.1mg/L+IAA 0.05 mg/L in the modified MS (1/2N) culture medium was benefitial to shoot growth when the shoot cuttings were shorter than 1.0 cm. The combination of NAA 0.5 mg/L +IBA 0.25 mg/L in the 1/2 MS culture medium was best for rooting when the shoot cuttings were longer than 1.0 cm, the rooting rate was higher and there were more roots at every shoot cuttings than other combinations. The result also showed that the effect of inducing rooting in the presence of NAA was better than that of IBA and IAA.

Moerover, Xi-zhu et al. (2005) mentioned that combination of ZT 1.0mg/l + BA 0.2 mg/L+ 0.02 IAA mg/L in the modified MS (1/2N) culture medium was beneficial to bud propagation and the combination of ZT 1.0 mg/L+ BA 0.1mg/L+IAA 0.05 mg/L in the modified MS (1/2N) culture medium was beneficial to shoot growth. The combination of NAA 0.5 mg/L + IBA 0.25 mg/L in the 1/2 MS culture medium was best for rooting.

However, Kai and Chun-feng (2007) investigated the effect of different media on "Fuyu" using micropropagation, shoot regeneration and rooting. The result indicated that: MS medium supplemented with ZT 2.0 mg/L and IAA 0.1 mg/L was the best medium for micropropagation. The best regeneration was achieved in MS(1/2N)+TDZ 0.4 mg/L +ZT 1.0 mg/L.After 30 days culture,the callus were subdivided and transferred to MS(1/2N)medium supplemented with ZT 2.0 mg/L and IAA 0.1 mg/L.

While, Wang el al. (2010) used dormant buds, sprout buds and shoot tips as explants of Boaibayuehuang Diospyros kaki , effect of sampling time,

disinfection time, disinfectants and plant growth regulators on culture of their organizations were investigated. The results showed that highest germination ratio of dormant buds, sprout buds and shoot tip reached 70%, 50%, 30%, respectively, that of dormant bud (dormant period explants) is the highest, and its proliferation ability is stronger; best sampling period is around December 6, using dormant buds, sprout buds as explants could establish the beginning of culture, while the beginning of shoot tip culture is not easy to be established. Using 0.1% Hg2Cl2 to disinfect 5 min will be best to explant at its earlier growing period, its pollution rate, the browning rate is relatively lower, while its survival rate is the highest; it would be better to use 0.1%

Hg2Cl2 to disinfect explants for 10 min, although its pollution rate is higher, but its browning rate is the lowest, the survival rate is the highest;

Comparison of disinfectants (NaClO and Hg2Cl2) indicated the optimal level of disinfectant to explant in the dormant period is 0.1% Hg2Cl2, the disinfection time is 15min,the survival rate is higher, reaching 70.2%. The most suitable culture medium for Boaibayuehuang Diospyros kaki is 1/2 MS+1 mg/L ZT +0.1 mg/L IAA +4.0 mg/L 6-BA.

Where's, Yokoyama et al. (2011) reported that the adventitious buds of the Japanes persimmon grew poorly on the medium with TDZ, but resumed growth and developed into shoots after transfer to the medium containing 2% (w/v) sucrose and 10 mM zeatin.

Further, Giordani et al. (2013) suggested for bud development and shoot establishment, place the explants on modified MS basal medium,

containing 20 g/L sucrose and 5 mg/L BA or 2.2 mg/L of zeatin (depending on the cultivars) and solidified with 2 % (w/v) of gellan gum. As for shoot multiplication, use the same basal medium with 2.2 mg/L BA or 4.4 mg/L zeatin. For elongation replace BA or zeatin with 5 mg/L 2iP. The medium for rooting is half-strength MS hormone-free.

II) Genetic Profiling of Diospyros kaki L:

DNA is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms. The main role of DNA molecules is the long term storage of information where DNA is often compared to a set of blue prints or a recipe, or a code, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or involved in regulating the use of this genetic information

(Nathan, 2009).

Authentication of botanicals which are medicinally valuable is an important issue globally because of unavailability/ underutilization of appropriate tools for standardization. Since DNA is more stable and does not vary seasonally and with age of the plant. DNA based fingerprinting techniques have greater role in the authentication of botanicals which are medicinally important. DNA markers are reliable for information on genetic polymorphism as the genetic composition is unique for each species irrespective of plant part used and is not affected by age, physiological condition as well as environmental factors (Nikam et al., 2012). Therefore the DNA fingerprint of Diospyros kaki was done as a contribution to the macro- and micro- morphological identification and characterization of the plant.

Methodology of DNA fingerprinting:

The basic methodology of DNA profiling in plants involve first the isolation of DNA from plant cells, quantification and quality assessment of isolation. The important steps involved in DNA fingerprinting are:

1) PCR based. e.g.- RAPD, ISSR and SSR

2) Non PCR based. e.g. – RFLP.

1- DNA Isolation:

DNA from plant tissue was isolated by removal of cell wall and nuclear membrane around the DNA and the separation of DNA from other cell components such as cell debris, proteins, lipids or RNA without affecting the integrity of the DNA. The DNA was isolated from tissues of plants, generally fresh leaves were preferred.

2- DNA Quantification and Quality assessment:

DNA quantification and quality assessment was done by using UV- VIS spectrophotometry. Normally quality check is performed through the

A260/A280 ratio that is 1.8 values shows the highest purity, if more than 1.8 shows the presence of RNA contamination and less than that indicates protein contamination.

3- Polymerase Chain Reaction (PCR): The DNA amplification by thermal cycling called Polymerase Chain Reaction is in vitro method that can be used to amplify a specific DNA

segment from small amounts of DNA template or duplex into millions of copies.

Steps involved in PCR were:

• Heat Denaturation. • Annealing. • Primer Extension.

Then Application of different genotyping methods like RAPD, AFLP, RFLP and ISSR were carried out according to described method by Kumar et al., (2014).

Guo and Luo (2006) reported that nine polymorphic simple sequence repeat primers were develop in Japanese persimmon using ISSR suppression polymerase chain reaction. These primers were tested on 30 individuals from Japan and China.

Yyldy et al. (2007) stated that genetic relationships among Diospyros kaki L. growing around Hatay province in Turkey were assessed by randomly amplified polymorphic DNA (RAPD) assay. Ten decamer primers were selected from 50 primers. The study suggests that the morphological differences among cultivars of persimmon might be the result of genetic differences rather than the ecological or growing conditions. The study forms a basic contribution to the characterization of Diospyros kaki L. population in Turkey.

C- Chemical evaluation and Biological activities:

Plant-derived foods for example fruits, vegetables, grains, oils, nuts and many more have become an essential part of the human diet for their beneficial health promoting effects (Tayade et al., 2013). Plants (fruits, vegetables, medicinal herbs, etc.) contain a wide variety of free radical scavenging molecules, such as phenolic compounds, nitrogen containing compounds, vitamins, minerals and some other endogenous metabolites, which have antioxidant activity. Epidemiological studies have shown that many of these antioxidant compounds possess anti-inflammatory, antiatherosclerotic, antitumor, antimutagenic, anticarcinogenic, antibacterial, or antiviral activities (Pe’rez-mature et al., 2009).

Nutrients are divided into macro-nutrients such as proteins, fats and carbohydrates. Micronutrients such as vitamins and minerals (Ogbonnaya and Chinedum, 2013). Persimmon is a source of some active compounds that have both nutritional and nutraceutical values. Persimmons have a high level of dietary fiber, minerals, trace elements and some antioxidants (β- carotene (pro-vitamin A), β- cryptoxanthina, lutein, zeaxanthin, and lycopene). Persimmons are rich in vitamin C and polyphenols, all of which are powerful antioxidants that protect against free radicals, prevent risk of cardiovascular disease, diabetes and cancer (Nazirl et al., 2013).

Persimmon is also a good source of some active compounds such as ascorbic acid and condensed tannins and these are related to various physiological functions including a protective role against oxidative stress-

related diseases, antimutagenic and anticarcinogenic capacities. (Suzuki et al., 2005). Persimmon leaves contain flavonoids, amino acids, protein and vitamins, which possess a wide range of pharmacological activity, has anti- bacterial, anti-inflammatory, blood pressure-lowering, lipid-lowering and anti-cancer effect. It is also to promote the body metabolism and the role of cough and phlegm. It can be used to prepare drugs for the prevention and treatment of atherosclerosis, hypertension, and coronary heart disease

(Zhang et al., 1983).

I) Chemical evaluation:

1- Minerals:

Mineral elements are generally classified either as microelements or macroelements. The macroelements are those present in relatively higher amounts (750 ppm) in animal tissues. They include Ca, K, Na, Mg, S., P and Cl. The microelements, also called trace elements, are present at less than 50 ppm. Trace elements essential for human, they are Fe, Zn, Mn, Se, F and I2. Minerals cannot be synthesized in the body so they must be obtained from diet. Minerals are constituents of skeletal tissues, cofactors for some enzymes, carrier proteins, protein hormones and electrolytes in body fluids and cells (Mertz, 1980). A summary of major and trace minerals are listed in the following Tables 6, 7 and Figure 7 (Boyle, 2014).

Table 6: Major minerals, food sources, biological roles, and deficiency and toxicity symptoms. Mineral Sources Roles Deficiency Symptoms Toxicity Symptoms Milk and milk products, Principal mineral of bones Stunted growth in Excess calcium is small fish (with bones), and teeth; involved in children; bone loss usually certain green vegetables, muscle contraction and (osteoporosis) in adults. excreted except in legumes, fortified juices. relaxation, nerve function, hormonal imbalance Calcium blood clotting and blood states. pressure.

Meat, poultry, fish, dairy Part of every cell; involved Muscle weakness and May cause calcium Phosphorus products, soft drinks, in acid–base balance and bone pain (rarely seen). excretion. processed foods. energy transfer. Nuts, legumes, whole Involved in bone Weakness, confusion, Excess intakes (from grains, dark green mineralization, protein depressed pancreatic overuse of laxatives) vegetables, seafoods, synthesis, enzyme action, hormone secretion, has caused low blood Magnesium chocolate, cocoa. normal muscular growth failure, pressure, lack of

contraction, hallucinations, coordination, coma, nerve transmission. muscle spasms. and death.

Salt, soy sauce; Helps maintain normal Muscle cramps, mental High blood pressure. Sodium processed foods such as fluid and acid–base apathy, loss of appetite.

cured, canned, pickled, balance; nerve impulse

and many boxed foods. transmission.

Mineral Sources Roles Deficiency Symptoms Toxicity Symptoms

Salt, soy sauce; Part of hydrochloric acid Growth failure in Normally harmless processed foods. found in the stomach, children, muscle cramps, (the gas chlorine is a Chloride necessary for proper mental apathy, loss of poison but

digestion, fluid balance. appetite. evaporates from water); vomiting. All whole foods: meats, Facilitates many reactions, Muscle weakness, Causes muscular milk, fruits, vegetables, including protein synthesis, paralysis, confusion; can weakness; Potassium grains, legumes. fluid balance, nerve cause death; triggers vomiting; if

transmission, and accompanies given

contraction of muscles. dehydration. into a vein, can stop the heart. All protein-containing Component of certain None known; protein Would occur only if foods. amino acids; part of biotin, deficiency would occur sulfur Sulfur thiamin, and insulin. first. amino acids were eaten in excess; this (in animals) depresses growth.

Table 7: Trace Minerals, food sources and their actions. Mineral Sources Roles Deficiency Symptoms Toxicity Symptoms Iodine Iodized salt, seafood, Part of thyroxine, which Goiter, cretinism. Depressed thyroid bread. regulates metabolism. activity.

Iron Red meats, fish, poultry, Hemoglobin formation; Anemia: weakness, pallor, Iron overload: shellfish, eggs, legumes, part of myoglobin; energy headaches, reduced infections, liver dried fruits, fortified utilization. immunity, inability to injury, acidosis, cereals. concentrate, cold shock. intolerance. Zinc Protein-containing foods: Part of insulin and many Growth failure in Fever, nausea, meats, fish, shellfish, enzymes; involved in children, delayed vomiting, diarrhea, poultry, grains, making genetic material development of sexual kidney failure. vegetables. and organs, loss of taste, poor proteins, immunity, wound healing. vitamin A transport, taste, wound healing, making sperm, fetal development. Copper Meats, seafood, nuts, Aid in hemoglobin Anemia, bone changes Nausea, vomiting, drinking water formation; part of several (rare in human beings) diarrhea

Mineral Sources Roles Deficiency Symptoms Toxicity Symptoms enzymes Fluoride Drinking water (if Formation of bones and Susceptibility to tooth Fluorosis fluoride containing or teeth; helps make teeth decay (discoloration of fluoridated), tea, seafood. resistant to decay. teeth); nausea, vomiting, diarrhea

Selenium Seafood, meats, grains, Helps protect body Fragile red blood cells, Nausea, abdominal vegetables (depending on compounds from cataracts, growth failure, pain; soil oxidation; works with heart damage. nail and hair changes; conditions). vitamin E. liver and nerve damage.

Chromium Meats, unrefined foods, Associated with insulin Abnormal glucose Occupational vegetable oils. needed for release of metabolism. exposures damage skin and energy from glucose. kidneys.

Molybdenum Legumes, cereals, organ Facilitates, with enzymes, Unknown. Enzyme inhibition. meats. many cell processes.

Mineral Sources Roles Deficiency Symptoms Toxicity Symptoms

Manganese Widely distributed in Facilitates, with enzymes, In animals: poor growth, Poisoning, nervous foods. many cell processes. nervous system disorders, system abnormal reproduction Disorders.

Figure 7: Recommended dietary minerals.

Gorinstein et al. (2001) mentioned that the contents of Na, K, Mg, Ca, Mn, and Fe were higher in whole persimmons than in whole apples.

USDA (2010) reported that Nutrient values of raw Japanese persimmon per 100 g edible portion as Ca 8 mg, Fe 0.15 mg, Mg 9 mg, P 17 mg, K 161 mg, Na mg, Zn 0,11 mg, Cu 0.113 mg, Mn 0.355 mg, Se 0.6 mg.

Butta et al. (2015) mentioned that the nutritional value of persimmons (mg/100 g) were as shown in Table 8.

Table 8: The nutritional value of persimmons (mg/100g).

Nutrient Persimmons, Persimmons, Persimmons, Japanese, dried Japanese, raw Chines , raw Calcium 25 8 27 Iron 0.74 0.15 2.50 Phosphorus 81 17 26 Potassium 802 161 310 Sodium 2 1 1

2- Fatty acids:

An and Guo (2000) listed the flavor substances of persimmon leaves of fatty acids as myristic acid, palmitic acid, stearic acid, 10-octadecenoic acid, cerotic acid, linoleic acid, linolenic acid,.etc.

USDA (2010) classifed fatty acids in persimmon fruits as follows, total saturated fatty acids of fruits 0.020 g, 14:0 (myristic acid) 0.001 g, 16:0 (palmitic acid) 0.0016 g, 18:0 (stearic acid) 0.003 g; total monounsaturated fatty acids 0.037 mg, 18:1 (oleic acid) 0.037 g, total polyunsaturated fatty acids 0.043 g, 18:2 (linoleic acid) 0.039 g, 18:3 (linolenic acid) 0.004 g, phytosterols 4 mg.

Puwastien et al. (2011) mentioned that fatty acids are one of the components of lipid that is present in a class called triglycerides. Fatty acids are either saturated or unsaturated. Boyle (2014) classified types of fats, Dietary sources and their effects on blood lipids as mentioned in the following Table 9.

Table 9: Dietary sources of different types of fat and their effects on blood lipid profiles.

Type of Fat Dietary Sources Effects on Blood Lipids

All animal meats, beef tallow, Increases total cholesterol butter, cheese, chocolate, coconut, Increases LDL-cholesterol. cocoa butter, coconut oil, cream, hydrogenated oils, lard, palm oil,

stick margarine, shortening, whole Saturated Fat Saturated

milk, and ice cream.

Almonds, corn oil, cottonseed oil, If used to replace saturated fat filberts, fish, liquid/soft margarine, in the diet, polyunsaturated fat mayonnaise, pecans, safflower oil, may: sesame oil, soybean oil, sunflower Decrease total cholesterol oil, walnuts Decrease LDL-cholesterol

Polyunsaturated Fat Polyunsaturated Increasing HDL-cholesterol.

Almonds, avocados, canola oil, If used to replace cashews, olive oil, olives, saturated fat in the diet, peanut butter, peanut oil, peanuts, monounsaturated fat may: poultry. Decrease total cholesterol Decrease LDL-cholesterol

without decreasing HDL- Monounsaturated Fat Monounsaturated cholesterol. Canola and soybean oils, flaxseed, If used to replace oily coldwater fish (salmon, saturated fat in, the diet, mackerel, tuna), shellfish, omega-3 fat may:

3 Fat 3 soyfoods, walnuts, wheat germ. - Decrease total cholesterol

Decrease LDL-cholesterol Omega Increase HDL-cholesterol Decrease triglycerides.

3- Vitamins and carotenoids:

a) Vitamins: Food is the main source of vitamins for human and animals. The human diet often does not contain the appropriate amount of vitamins needed for the normal development of body functions. Absence of these vitamins causes serious physiological problems (Lebiedzinska et al., 2007).

Vitamins can be broadly classified in two major groups (Tayade et al., 2013). Water- soluble vitamin includes: B group vitamins [thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), Biotin (B7), folic acid (B9), cyanocobalamin (B12) and ascorbic acid (C).

Thiamine (B1): Thiamine is involved in many body functions, including nervous system and muscle function, the flow of electrolytes in and out of nerve and muscle cells, digestion, and carbohydrate metabolism. Very little thiamine is stored in the body and depletion can occur within 14 days. Severe thiamine deficiency may lead to serious complications involving the nervous system, brain, muscles, heart, and stomach and intestines. http://www.mayoclinic.org/drugs- supplements/thiamine/background/hrb-20060129 Riboflavin (B2): Is involved in the regulatory functions of some hormones that are connected with carbohydrate metabolism.

Niacin (Vitamin B3): Is essential for the normal functioning of the skin, intestinal tract and the nervous system (Vasundev, 2006).

Ascorbic acid (vit.C): Ascorbic acid has been widely used in the pharmaceutical applications, chemicals, cosmetics and food industries due to bioactivity and antioxidant properties. Natural ascorbic acid is vital for the body performance and also increases the organism resistance against microorganism and participate in the antibody formation. Lack of ascorbic acid impairs the normal formation of intracellular substances throughout the body including collagen, bone matrix and tooth dentine also, deficiency include fatigue and disability against blood vessels, teeth, bones (Hussain et al., 2010).

Fat soluble vitamin includes: Retinol (A), Ergocalciferol (D2), Cholecalciferol (D3),Tocopherol (E), Phylloquinone (K1) and menaquinone (K2).

Vitamin A: Vitamin A (a fat-soluble vitamin) occurs in many forms; as retinol

(alcohol), retinal (aldehyde), retinyl acetate or palmitate (esters) and vitamin

A carotenoids (β-carotene, α-carotene) (Wirakartakusumah and Hariyadi, 1998). In plants, it occurs in the precursor, or provitamin form as carotenoids, which animals convert into vitamin A after ingestion in the diet (Bloem and Dranton-Hill, 1999). Vitamin A has been identified as essential for vision, growth, cell differentiation, immune system and reproduction (Guthrie,

1989).

Vitamin E: Vitamin E is a powerful antioxidant that protects cells against oxidative effects by neutralizing molecules very unstable, known as free radicals. Acting as an antioxidant, it protects the lungs against damage caused by air pollution, protects the whole body against free radicals and prevents tumors (Alert, 2002). Tocopherol protects the red blood cell from hemolysis, boosts the immune response, and reduces the risk of myocardial infarction by reducing the oxidation of LDL as well as acting as an anti-mutagen. It also functions synergistically with other antioxidants like vitamin A and C and selenium. (Wangner et al., 2004). List of water soluble vitamins and fat soluble vitamins is shown in Tables 10 and 11 (Boyle, 2014).

Table 10: The different food sources, biological roles and the deficiency symptoms of some water soluble vitamins. Vitamin Food Sources Biological Roles Deficiency Symptoms Meat, pork, liver, fish, poultry, Helps enzymes release energy Beriberi: edema, heart- whole-grain and, from carbohydrate; supports irregularity, mental confusion, Thiamin (B1) enriched breads, cereals normal appetite and nervous musle weaknes.

and grain products, nuts, legumes. system function.

Milk, leafy green vegetables, Helps enzymes release energy Eye problems, skin disorders yogurt, cottage cheese, liver, from carbohy drate, fat, and around nose and mouth, Riboflavin meat, whole-grain enriched breads, protein, promotes healthy skin hypersensitivity to light. (B2) and normal vision. cereals and grain products.

Meat, eggs, poultry, fish, milk, Helps enzymes release energy Pellagra: flaky skin rash on parts whole-grain, enriched breads, from energy nutrients, exposed to sun, loss of appetite, cereals, grain products, nuts, promotes health of skin, nerves dizziness, weakness, irritability, Niacin (B3) legumes, peanuts. and digestive system. fatigue, mental confusion, indigestion, delirium.

Pyridoxine Meat, poultry, fish, green leafy Protein and fat metab olism; Nervous disorders, skin rash,

Vitamin Food Sources Biological Roles Deficiency Symptoms (B6) vegetables. formation of muscle weakness, anemia, convulsions, kidney stones. antibodies and blood cells helps convert tryptophan to niacin.

Green leafy vegetables, liver, Red blood cell formation, Anemia, heartburn, legumes, seeds, citrus fruits, protein metabolism, new cell Folate diarrhea, poor growth, depression, melons, enriched breads, grain division. (folacin, folic increased risk products. acid) of heart disease, stroke, and certain cancers. Citrus fruits, cabbage, tomatoes, Synthesis of collagen Scurvy: anemia, depression, potatoes, dark green vegetables, (helps heal wounds, maintains frequent infections, bleeding peppers, lettuce, strawberries, bone and teeth, strengthens gums, loosened teeth, muscle Ascorbic acid mangoes, papayas. degeneration, rough skin, bone ( C) blood vessel walls) antioxidant, strengthens fragility, poor wound resistance to infection, helps healing. body absorb iron.

Table 11: The different food sources, biological roles and the deficiency symptoms of some fat soluble vitamins.

Vitamin Food Sources Biological Roles Deficiency Symptoms

Vitamin A Retinol: fortified milk , margarine, cream, Visison: growth and repaire Night blindness, rough skin, cheese, butter, eggs, liver of blood tissues; maintenance susceptibility to infection, Beta-carotene: spinach and other dark leafy of mucous membranes; bone impaired bone growth, greens, broccoli, deep orange fruits (apricots, and tooth formation; abnormal tooth and jaw peaches, cantaloupe), and vegetables (squash, immunity; hormone alignment, eye problems carrots, sweet potatoes, pumpkin). synthesis; antioxidant. leading to blindness, impaired growth. Vitamin E Vegetable oils, green leafy vegetables, wheat Protects blood cells, Muscle wasting, weakness, red germ, whole grain products, liver, egg yolk, antioxidant, stabilization of blood cell breakage, anemia, salad dressings, mayonnaise, margarines, all membranes. hemorrhaging. nuts, seeds. Vitamin K Bacterial synthesis in digestive tract, liver, Synthesis of blood clotting Hemorrhaging, decreased green leafy and cabbage type, vegetables, protein that regulates blood calcium in bones. soyabeans, milk, vegetables oil. calcium.

Water- soluble vitamins in Diospyros kaki L: USDA (2010) reported water soluble vitamins in Diospyros kaki L. fruits were thiamin 0.030 mg, riboflavin 0.020 mg, niacin 0.100 mg, vitamin B-6 0.100 mg, and total folate 8 mg. Bin et al. (2000) mentioned that vitamin C was another main compound in persimmon leaves. Its content was reported to be 13.9 mg/g among the determined 410 kinds of leaves.

Fat soluble vitamins Diospyros kaki L.: USDA (2010) reported fat soluble vitamins in Diospyros kaki L. fruits were, total vitamin A 1627 IU, vitamin E ( a- tocopherol) 0.73 mg, vitamin K (phylloquinone) 2.6 mg .

b) Carotenioids:

Carotenoids are antioxidant compounds found in plants that can enhance the human health immune response by playing preventive roles against degenerative diseases such as: cancer, cardiovascular diseases, vision related abnormalities, Parkinsonism, infertility, etc. (Eleazu and Eleazu,

2012). Carotenoids are responsible for many of the red, orange, and yellow colors of plant leaves, fruits, and flowers. (Pfander, 1992). Examples of carotenoids are Lycopene, β- carotene, α-carotene, β-cryptoxantin, Zeaxenthin, lutein, antheraxanthin, violaxanthin, canthaxanthin and capsanthin which are listed in the following Table 12 (Oliver and Palou,

2000).

Some 600 different carotenoids are known to occur naturally, and new carotenoids continue to be identified. Carotenoids are classified according to the structure as follows: The hydrocarbon carotenoids which are known as carotenes example β-carotene and the oxygenated carotenoids which are derivatives of these hydrocarbons known as xanthophylls, examples of these compounds are a zeaxanthin and lutein (hydroxy), spirilloxanthin (methoxy), echinenone (oxo), and antheraxanthin (epoxy) (Eldahshan and Singab, 2013). USDA (2010) mentioned the different types of carontenoids in Diospyros kaki L. fruit as follows ; β -carotene 253 mg, β -cryptoxanthin 1447 mg, lycopene 159 m g, lutein + zeaxanthin 834 mg.

Table 12: Chemical structures of some carotenoids.

OH

OH Lycopene Zeaxanthin OH

O

OH Antheraxanthin B-Carotene

OH

O

O

OH Violaxanthin Alpha-Carotene OH O

O B-Cryptoxanthin Canthaxanthin

OH OH

O

OH Lutein OH Capsanthin

II) Biological activities:

During the last few decades, there has been an increasingly intensified search for biologically active compounds of natural origin. Over this period, a worldwide interest in drugs derived from plants has been developed to produce a daily increasing of public demand for these drugs possess multiple biological effects as anticancer, antiviral, antihyperglycemic or immuno- modulating agent (Dahanukar et al., 2000). Persimmon is an abundant source of amino acids, carotenoids, flavonoids, sugars, tannins, terpenoids, and vitamin A (Thuong et al., 2008). The components of Persimmon have wide range of pharmacological activities, anti-bacterial, anti-inflammatory, blood pressure-lowering, lipid lowering and anti-cancer effect. It can be used to prepare drugs for the prevention and treatment of atherosclerosis, hypertension, coronary heart disease drug and cough. Moreover the remedy has been reported to possess significant efficacy and few side-effects in the treatment of stroke patients such as cerebral atherosclerosis (Yin et al., 2010).

Toxicity studies:

Toxicity cases were not observed in various uses of persimmon leaves during past hundred years. Modern toxicity studies on animals did not indicate toxicity of the leaves (Wu et al., 2012). In Chen’s study sub-chronic toxicity test was performed on 100 rats with oral administration of different concentrations of ethanolic extract of persimmon leaves for 90 days at a dose

of 6.4 g/kg, no signs of maternal toxicity, embryo toxicity and teratogensis were observed (Chen et al., 2005b).

Anitoxidant activity:

Free radicals are naturally produced in the body through normal metabolism of carbohydrates, amino acids and fats. Other factors known to increase free radicals in our body include chronic diseases, smoking, environmental poisons, alcohol and ionizing radiation. Antioxidants are chemical compounds that can bind to free oxygen radicals preventing these radicals from damaging healthy cells (Halliwell, 2011) and compounds that when added to food products, especially to lipids and lipid-containing foods, can increase the shelf life by retarding the process of lipid peroxidation, which is one of major reasons for deterioration of food products during processing and storage. Synthetic antioxidants such as BHT, have restricted to be carcinogenic. Therefore, the importance of the search for and exploitation of natural antioxidants, especially of plant origin, has greatly increased in recent years (Singh et al., 2002).

Also α-carotene, β-carotene, lutein, and zeaxanthin, are from familiar plant sources (Parejo et al., 2002).

Antioxidant-based drug formulations are used for the prevention and treatment of complex diseases like atherosclerosis, stroke, diabetes, Alzhemers’s disease and cancer (Khalaf et al., 2008).

Shi et al. (1999) reported that Diospyros kaki L. leaves exerted potential antioxidant activity in vitro. Ethanolic extracts from persimmon

leaves (600 mg/kg) was effective in retarding lard deterioration, which showed a little higher antioxidative activity than hesperidin (100mg/kg) and tea polyphenols (100mg/kg).

Chen et al. (2002a) isolated five flavonoid compounds from the leaves of Diospyros kaki (kaempferol 3-O- β -D-galactopyranoside, kaempferol 3-O- β -D-glucopyranoside, isorhamnetin 3-O- β - D- glucopyranoside, quercetin 3-O- β – D-galactopyranoside and quercetin 3-O- β -D- glucopyranosyl-(6 → 1)- α -αL-rhamnopyranoside) were found to suppress stimulus-induced superoxide generation and tyrosyl phosphorylation and may have pharmaceutical application.

Chen et al. (2002b) isolated five triterpenoid compounds, from persimmon leaves: α -amyrin, uvaol, ursolic acid, 19 α -hydroxy ursolic acid and 19 α ,24-dihydroxy ursolic acid were found to be significantly suppress superoxide generation induced by N-formyl-methionylleucyl-phenylalanine in human neutrophils in a concentration-dependent manner.

Sakanaka et al. (2005) reported the scavenging activity against superoxide anion radicals of comparing different persimmon leaves extract.

The effect of methanol extract of persimmon leaves was stronger than that of the water extract. DPPH radical-scavenging activity of water and methanol extracts was strong and 0.1% water extract showed an inhibition rate of more than 90%. The hydroxyl radical-scavenging activity of the 0.1% extracts was nearly equal to that of 10 mM ascorbic acid.

Suzuki et al. (2005) reported that the epigallocatechin contents in three astringent persimmons: Hiratanenashi, Tone-wase, Ishibashi-wase were higher than those of two non-astringent persimmons Maekawa-jiro and Matsumoto-wase-fuyu. The epigallocatechin content in the astringent Hiratanenashi was the highest among the five Japanese persimmons. The epigallocatechin content in the non-astringent Maekawa-jiro was the lowest among the five Japanese persimmons. Therefore, astringent persimmons may be better sources of the natural antioxidant, epigallocatechin than non- astringent persimmons, and the astringent Hiratanenashi may be the best source of epigallocatechin among the five Japanese persimmons.

Singh and Joshi (2011) reported that Diospyros kaki L. exhibited strong radical scavenging activity which can be attributed to the presence of catechin, epicatechin, epigallocatechin, chlorogenic acid, caffeic acid and gallic acid.

Anticancer activity:

Achiwa et al. (1997) demonstrated that persimmon extract and related polyphenol compounds such as catechin , epicatechin, epicatechingallate, epigallocatechin , and epigallocatechingallate (EGCG) inhibited the growth of human lymphoid leukemia.

Chen et al. (2005a) isolated a new biphenyl derivative Kakispyrol with three known compounds vitexin, 2’- O-rhamnosyl vitexin and isorhamnetin-3-O- β - D-glucopyranoside from Diospyros kaki L. leaves exhibited cytotoxic effects against several cancer cell lines.

Chen et al. (2007) reported that kakisaponin and kakispyrone together with 11 known compounds, from Diospyros kaki L. leaves exhibited cytotoxic effects against several cancer cell lines.

Kawakami et al. (2010) found that water-soluble polyphenols reached a maximum (2.40% w/w) in June, and then gradually decreased. The major components were unique proanthocyanidin oligomers consisting of four heterogeneous extension units, including epigallocatechin- 3-O-gallate. Persimmon leaf tea also contained similar proanthocyanidins with similar compositional units. Oral administration of starch with polyphenol concentrate of persimmon leaf tea resulted in a significant and dose- dependent decrease in the blood glucose level in Wistar rats.

Khanal et al. (2010) found that Persimmon extract and its constituents like 24-hydroxyursolic acid, a triterpenoid was found to have potent antitumour activity against human cancer cells.

Matsushita et al. (2011) isolated from the blackened heartwood 4,8- Dihydroxy -5-methoxy-2-naphthaldehyde which exhibited stronger cytotoxic activity against cancer cells.

Antidiabetic Activity:

Deng et al. (2011) studied ethanol fraction, ethylacetate fraction, n- butanol fraction and water extract fraction precipitated by alcohol from persimmon leaves significantly decreased blood glucose in STZ-induced

diabetic mice, with the decreasing inslulin resistance index and significantly increased insulin sensitivity index.

Wang et al. (2011) isolated from leaves of Diospyros kaki L. vomifoliol 9-O-a-arabinofuranosyl (1-6)-β-D-glucopyranoside with peripheral glucose utilization effect.

Antihypercholesterolemic/ Hypolipidaemic Activity:

Rho et al. (2003) isolated a new type of cholesterol acyl transferase inhibitor from a methanol extract of Diospyros kaki L. On the basis of spectral and structural evidence, the compound was identified as pheophorbide A-methyl ester.

Matsumoto et al. (2006) reported that diets supplemented with dried and powdered young and mature fruits of two cultivars of persimmon, Fuyu- kaki and Hachiya-kaki significantly decreased the rise in plasma lipids, including total cholesterol, triglyceride, and LDL cholesterol.

Xie et al. (2015) Persimmon leaves extract and leaf tea showed anti- atheroscloresis effect by decreasing the triglyceride and total cholesterol and

LDL-C level, whereas increasing the ratio of HDL level in serum of high-fat diet fed rats.

Cardioprotective and Antithrombotic Activity:

Ouyang et al. (2003) indicated that flavone extracted from persimmon leaves could inhibit the apoptosis of in vitro cultured neonatal rat

cardiac myocytes induced by hypoxia-reoxygenation and advanced glycation end products.

Sa et al. (2005) reported that an anticoagulant fraction was purified from persimmon leaves. It retarded thrombin time, activated partial thromboplastin time, and prothrombin time using human plasma.

Xie et al. (2015) mentioned that Persimmon leaves were used to manufacture tea for drink in China and Japan, which has been popular with local people for many years. It is because that persimmon leaves and it is preparations possess a great many effective activities on cardiovascular system. Several reports have proved that persimmon leaves increased the coronary artery blood flow in the hearts of rabbits and frogs in vitro and the coronary blood circulation in anesthetic dogs. Solutions made from persimmon leaves markedly reduced the incidence of arrhythmia.

Neuroprotective Activity:

Beia et al. (2009) reported that quercetin and kaempfetol were discovered to be the main aglycones of FLDK-P70 in LC–MS analysis. The glucosides of quercetin and kaempfetol, such as hyperin and astragalin were also found in FLDK-P70. These flavonoids containing phenolic hydroxyl which shows very potent antioxidant effects and can scavenge ROS, inhibit the formation of ROS, and alleviate oxidative damage in neuronal cells.

Antiaging/Antiwrinkle activity:

An et al. (2005) reported that polyphenols isolated from the persimmon leaf can be used as natural materials or additives for human skin owing to their beneficial biological functions, including the anti-wrinkle effect and the inhibition of skin problems.

Immunomodulating Activity:

Duan et al. (2010b) isolated the major pectic polysaccharide from Diospyros kaki L. leaves which is responsible for immunomodulatory activity.

Antifunal, Antimicrobial and Antiviral Activity:

Yasumasa et al. (1999) isolated and identified of antimicrobial compound kaempferol against Streptococcus mutans from the leaves of

Diospyros kaki.

Ji et al. (2003) detected the Minimum inhibition concentration and the minimum bactericidal concentrations of persimmon leaves were determined as 0.313 and 0.625 g/mL against tested food spoilage and food- borne pathogens (Escherichia coli, Staphylococcus aureus, Bacillus subtilis,

Bacillus cereus, Proteus vulgaris) respectively, and the antimicrobial rate were over 90%.

Kawase et al. (2003) reported that Diospyros kaki L. peels extracts possess significant anti-human immunodeficiency virus activity.

Singh et al. (2012) isolated Plumbagin and isodiospyrin from root bark of Diospyros kaki L. which exhibited remarkably good antifungal activity against all the fungi.

GENERAL SUMMARY Diospyros kaki Thunb is named the food of the Gods (from Greek, Dios meaning God and Spyros meaning food). It is 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

The most important commercial varieties cultivated in Egypt are Fuyu, Hashiya, Costata, Triumph and Hannah Fuyu.

Diospyros kaki 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.

The present study includes the investigation of phytochemical constituents, biotechnological studies, chemical evaluation and biological activities.

Part 1: Genetic profiling of Diospyros kaki L.

Genetic profiling of Diospyros kaki L.

Five random primers (AO1, A13, BO5, DO1 and DO9) out of 20 were screened in RAPD analysis and five random primers (HO8, HO9, H11, H13 and H15) out of 10 were screened in ISSR analysis for their ability to produce sufficient amplification products.

It can be concluded that primer AO1 has highly distinguish effects in RAPD analysis while primer H15 has highly distinguish effects in ISSR analysis to evaluate Disopyros kaki L. cv. Costata DNA using PCR.

Part 2: Phytochemical study of Diospyros kaki L.

Chapter I: Phytochemical screening of Diospyros kaki L.

Preliminary phytochemical screening revealed that the dried powdered leaves and fruits of Diospyros kaki L. contain traces of volatile constituents, carbohydrates and/or glycosides, tannins, flavonoids, coumarins, anthraquinones, sterols and/or triterpenes; while saponins were absent.

Chapter II: Investigation of the volatile constituents of Diospyros kaki leaves L.

The volatile constituents isolated from leaves of Diospyros kaki L. under investigation by hydro-steam distillation comprises 6 identified components constituting 83.12% of the total oil composition. Butylated hydroxytoluene is the major constituent, constituted 61.31% followed by Bis(2-propylpentyl) phthalate which constituted 14.32% of the total oil composition.

Chapter III: Investigation of lipid content of Diospyros kaki L. fruits and leaves:

The unsaponified matter and fatty acid methyl esters were subjected to GC/MS analysis.

GC/MS analysis of the unsaponifiable matter of Diospyros kaki fruits , revealed that the presence of 13 identified compounds, representing 85.61% of the total unsponifiable matter.

GC/MS analysis of the unsaponifiable matter of Diospyros kaki leaves, revealed the presence of 10 identified compounds, representing 87.16 % of the total unsponifiable matter.

GC/MS analysis of the fatty acids methyl esters of Diospyros kaki fruits, revealed the presence of 13 components, representing 84.79 % of the total fatty acids methyl ester where the major saturated fatty acid was methyl palmitate which constituted 17.4% while the major unsaturated fatty acid was methyl palmitoleate which constituted 9.68 %.

GC/MS analysis of the fatty acids methyl esters of Diospyros kaki leaves, revealed the presence of 10 components, representing 91.07 % of the total fatty acids methyl ester where the major saturated fatty acid was methyl palmitate which constituted 40.74 % while the major unsaturated fatty acid was methyl 9-octadecenoate which constituted 17.61%.

Chapter IV: Investigation of phenolic constituents of Diospyros kaki L.:

Air dried powdered leaves were defatted with petroleum ether then the defatted marc was extracted by percolation in 80% methanol at room temperature. The methanolic extract obtained was evaporated under reduced pressure to yield a dark greenish brown residue and the residue was suspended in distilled water left overnight in refrigerator, filtered, and extracted with successive portions of chloroform then ethyl acetate and finally with n-butanol.

 Investigation of chloroform fraction:

The chloroform fraction was dissolved in the least amount of methanol, subjected onto the top of chromatographic glass column (2x80 cm) packed with sephadex LH-20. Elution of the column was carried with 100% methanol. From the spectroscopic data, scopoletin and kaempferol were isolated and identified.

 Investigation of ethyl acetate fraction:

The residue of the ethyl acetate fraction was dissolved in the least amount of methanol, mixed with silica gel and allowing the solvent to evaporate. The powder was introduced onto the top of chromatograhic glass column (4x 120 cm) packed with silica gel (400 g). Gradient elution was carried out using first, 100% methylene chloride, then increasing polarity by stepwise addition of methanol. Further fractionation, purification of the ethyl acetate fraction and from the spectroscopic data the following compounds were identified, luteolin, and rutin .

 Investigation of the main phenolic constituents in n-butanol fraction:

The n-butanol fraction was dissolved in the least amount of methanol, introduced onto the top of chromatographic glass column (4x120 cm) packed with polyamide 30-60 mesh. Gradient elution of the column was carried out using distilled water and decreasing the polarity by 10% stepwise addition of methanol. Further fractionation, purification of the n-butanol fraction and from the spectroscopic data was identified apigenin7-O-glucoside.

Part 3: Biotechnological study of Diospyros kaki L.

In vitro culture on Diospyros kaki L.:

I- Development stage:  Sterilization of plant organs:

Using Hg2Cl2 at 0.2% gave the best results of survival explants for internode explants compared with leaf explants which showed that using

0.1% of Hg2Cl2 gave the best reslults of leaf survival.

 Media used:

It could be noticed that culturing of internode explants on WPM supplemented with 2 mg/l ZT+ 5 mg/l 2ip recorded the best results of explant development compared using different medium.

II- Callus induction:

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 (%) as well as calli fresh and dry weights compared used other nutrient media and supplementations.

III- Regeneration stage:

Supplementation of ½ MS + 1 mg/l Zeatin + 2 mg/l IAA + 4 mg/l BA + 0.5 g/l PVP recorded the best results of percentage of regenerated shootlet compared used other nutrient media and supplementations.

IV- Quantitative determination of the total flavonoid and other phenolic contents of leaves extract, callus internodes and regenerated shootlets (80% MeOH) of Diospyros kaki L. using HPLC analysis:

The highest amount recorded using HPLC analysis of flavonoids in leaves extract was kaempferol (80.9 μg/g DW) while calli derived internode explants was luteo.6-arbinose 8-glucose (65.1 μg/g). The lowest amount recorded in leaves extract was apig.7-O-neohespiroside ( 6.86 μg/g ) while in callus internodes, was kampferol and rhamnetin (0.7 μg/g) that, luteolin was detected in leaves extract (80%) and not detected in callus internodes, while

quercetin-3-O-glucoside was not detected in leaves extract and detected in callus internodes.

The highest amount of scopoletin (57.08 and 26.42 μg/g DW) was recorded using HPLC analysis for phenolics in leaves extract and regenerated shootlets respectively.

The lowest amount of a compound recorded in leaves extract was pyrogallol (8.8 μg/g DW) while callus internodes was reversetrol and coumarin (0.5 μg/g DW). Part 4: Chemical studies and Biological activities

Chapter 1: Chemical evaluation of fruits and leaves of Diospyros kaki L.

I) Determination of moisture, ash and minerals in fruits and leaves of Diosopyros kaki L.:

The moisture content was higher in fruits and ash % was higher in leaves.

Concerning macro-elements:

 Potassium and calcium were the major elements in fruits.  Sodium, magnesium and phosphorous were major in leaves.

Concerning the micro- elements:

 Iron and zinc were major in leaves. II) Determination of tannins, catechin and antioxidant activity of fruit and leaves of Diospyros kaki L.: Leaves showed the higher level of tannins where fruit sample showed the higher amount of catechin and antioxidant activity of leaves recorded the higher activity than the fruit.

III) Amino acids content of fruits and leaves of Diospyros kaki L.

Essential amino acids content of the tested samples was higher in leaves. Fruits showed the lower level of essential amino acids. For non- essential amino acids the fruits showed the higher level while leaves were the lower level.

IV) Carotenoids content of Fruits and leaves of Diospyros kaki L.

This study concluded that Diospyros kaki fruits are a good source for β-carotene and lycopene. V) Fat and water soluble vitamins content of fruits and leaves of Diospyros kaki L.

The results for the levels of fat soluble vitamins in the tested parts indicated that Diospyros kaki L. leaves are good source for Vitamin A and E.

The obtained result encourages the usage of the fruits as good source for water soluble vitamins such as vitamin B1 and C. For the level of B2 leaves indicated the highest value.

Chapter II: Biological activities of fruits and leaves of Diospyros kaki L.

The biological evaluation for the kaki fruit and leaves at the level of 10 % indicated that this plant can provide a good nutritional value and it is save with regard to the kidney and liver functions, good source that help in enhancing the antioxidant defense against free radical that proved by values of the activity of the antioxidant enzymes. No bad effect was found in lipids profile and good effect was found in the ratio of HDL and LDL Cholesterol. Also this plant can help in decreasing the blood sugar as found in this biological evaluation and can help in enhancing the level of the blood hemoglobin according to the raise in the hemoglobin values that was observed in the rats that fed on the plant.

General Conclusion and Recommendations

General conclusion:

Preliminary phytochemical screening revealed that the air dried powdered leaves and fruits of Diospyros kaki L. contain, traces of volatile constituents, carbohydrates and/or glycosides, tannins, flavonoids, coumarins, sterols, triterpenes and anthraquinones; while saponins were absent.

Could be concluded that primer H15 has highly distinguish effects to evaluate Diospyros kaki L. DNA using ISSR PCR.

Eugenol was identified from GC/MS analysis with other compounds from Diospyros kaki L. leaves.

GC/MS analysis of the unsapoifiable matter, Table 18 and 19 of Diospyros kaki L. fruits revealed the presence of 13 identified compounds, representing 85.61% of the total unsponifiable matter.

Oxygenated compounds constituted the major percent of the unsaponifiable matter; they reached 84.38% of the unsaponifiable matter, in which aromatic compounds were the major (57.52%) and aliphatic compounds were the minor (2.1%). Phthalic acid bis(2-ethylhexyl) phthalate (42.9%) was predominant as aromatic oxygenated compound.

Nonoxygenated compounds constituted the minor percent of the unsaponifiable matter; they reached 1.23%.

GC/MS analysis of the unsapoifiable matter of Diospyros kaki L. leaves revealed that the presence of 10 identified compounds, representing 87.16 % of the total composition of the unsponifiable matter.

The oxygenated compounds constituted the major percent of the unsaponifiable matter; they reached 70.67 % of the unsaponifiable matter, while the non-oxygenated compounds were minor (16.49%), in which terpene compounds were the major (56.81%) and aliphatic compounds were the minor (7.85%).

α –amyrin (42.39) constituted the major terpene compound in the oxygenated fraction of unsap. of Diospyros kaki L. which coincided with reported data by Xie et al. (2015).

 GC/MS analysis of the fatty acids methyl esters of Diospyros kaki L. fruits revealed the presence of 13 fatty acids, representing 84.79 % of the total fatty acids methyl ester.

 The percent of saturated and unsaturated fatty acids methyl esters were

57.35 % and 27.44 % respectively.

 The major saturated fatty acid methyl esters was ethyl stearate which constitutes 26.87% while the major unsaturated fatty acid methyl esters was methyl palmitoleate which constituted 9.68 %

Myristic acid, palmitic acid and stearic acid of saturated fatty acids of Diospyros kaki L. are coincided with those reported by USDA (2010)

whereas linolenic acid, linoleic acid and palmitoleic acid of unsaturated fatty acids of Diospyros kaki L. are coincided with Singh et al. (2011).

. GC/MS analysis of the fatty acids methyl esters of Diospyros kaki L. leaves revealed the presence of 10 fatty acids, representing 91.07 % of the total fatty acids methyl ester.

. The percent of saturated and unsaturated fatty acids methyl ester

were 54.89 % and 36.18% respectively.

. The major saturated fatty acid methyl ester was methyl palmitate which constituted 40.74 % while the major unsaturated fatty acid methyl ester was methyl 9-octadecenoate which constituted 17.61%.

Myristic acid, palmitic acid, stearic acid, 10-octadecenoic acid, cerotic acid and linolenic acid were identified by An and Guo (2000) from Diospyros kaki L. Methylated compounds of myristic acid, palmititc acid, stearic acid and linolenic acid were identified using GC/MS analysis in the present study. scopoletin, kaempferol and rutin were, isolated and identified.

It is the first record to isolate apigenin 7-O-glucoside and luteolin from Diospyros kaki L. cultivated in Egypt. Protocols for in vitro propagation of Diospyros kaki L.

have been proposed by many authors but at present no reliable method has been defined for time selection for culture of Diospyros kaki L, development stage, callus stage and regeneration stage for Diospyros kaki L. cultivated in Egypt.

Cultivated lateral buds on April sterilized with 70% ethanol for 5 seconds and 0.2% of Hg2Cl2 gives the lowest contamination and the highest survival rate where this result has been modified from using 0.1% of Hg2Cl2 for 15 min. suggested by Wang et al. (2010).

In contrast with Giordani et al. (2013) who used MS basal medium for internodes development and elongation and agreement of using BA or zeatin with 5 mg/l 2ip where WPM medium in combination with growth regulators as 2 mg/l ZT + 5 mg/l 2ip in dark then transferred to light gives the highest percentage of shoots development from the explant.

Calli of Diospyros kaki L. were highly induced on MS medium supplemented with 10 mg/l ZT + 10 mg/l IAA +500 mg/l PVP + 0.1 mg/l Thiamine HCL. This result was in agreement with Tao and sugiura (1992) who used ½ MS supplemented with 2.19 mg/l and 0.22 mg/l IAA with modification of MS and hormones added to obtain callus.

The best regeneration was achieved and developed in ½ MS + 1mg/l ZT + 2 mg/l IAA + 4 mg/l BA + 500 mg/l PVP and this result has been agreed with Wang et al. (2010) with addition of 2mg/IAA instead of 0.1 mg/l

IAA, 4 mg/l BA instead of 6 mg/l BA and adding 500mg/l PVP.

The highest amount recorded using HPLC analysis of flavonoids in leaves extract was kaempferol (80.9 μg/g DW) while calli derived internode explants was luteo.6-arbinose 8-glucose (65.1 μg/g). The lowest amount recorded in leaves extract was apig.7-O-neohespiroside ( 6.86 μg/g ) while in callus internodes, was kampferol and rhamnetin (0.7 μg/g) that, luteolin was

detected in leaves extract (80%) and not detected in callus internodes, while quercetin-3-O-glucoside was not detected in leaves extract and detected in callus internodes.

The highest amount of scopoletin (57.08 and 26.42 μg/g DW) was recorded using HPLC analysis for phenolics in leaves extract and regenerated shootlets respectively.

The lowest amount of a compound recorded in leaves extract was pyrogallol (8.8 μg/g DW) while callus internodes was reversetrol and coumarin (0.5 μg/g DW). The moisture content was higher in fruits and ash % was higher in leaves.

Concerning macro-elements:

 Potassium and calcium were the major elements in fruits.  Sodium, magnesium and phosphorous were major in leaves.

Concerning the micro- elements:

 Iron and zinc were major in leaves. Leaves showed the higher level of tannins where fruit sample showed the higher amount of catechin and antioxidant activity of leaves recorded the higher activity than the fruit.

Essential amino acids content of the tested samples was higher in leaves. Fruits showed the lower level of essential amino acids. For non- essential amino acids the fruits showed the higher level while leaves were the lower level.

This study concluded that Diospyros kaki fruits are a good source for β-carotene and lycopene.

The results for the levels of fat soluble vitamins in the tested parts indicated that Diospyros kaki L. leaves are good source for Vitamin A and E.

The obtained result encourages the usage of the fruits as good source for water soluble vitamins such as vitamin B1 and C. For the level of B2 leaves indicated the highest value.

The biological evaluation for the kaki fruit and leaves at the level of 10 % indicated that this plant can provide a good nutritional value and it is save with regard to the kidney and liver functions, good source that help in enhancing the antioxidant defense against free radical that proved by values of the activity of the antioxidant enzymes. No bad effect was found in lipids profile and good effect was found in the ratio of HDL and LDL Cholesterol. Also this plant can help in decreasing the blood sugar as found in this biological evaluation and can help in enhancing the level of the blood hemoglobin according to the raise in the hemoglobin values that was observed in the rats that fed on the plant.

RECOMMENDATIONS

1- This study recommended that we should widely encourage the cultivation of this plant to be available for Egyptian consumers since this evaluation proved the health benefits of Diospyros kaki L. cv. Costata.

2- Using Diospyros kaki L. leaves as a good source for herbal medicine as used in China (Tang and Eisenbrand, 1992) to reduce blood glucose level.

3- Chemical and biological evaluations of Diospyros kaki L. cv. Costata (fruits and leaves) indicated the good health effect of using this plant since it is rich in some effective nutrients and antioxidants that may help in the prevention of some nutritional diseases.

4- Application of plant biotechnology on large scale; using bioreactor technique, to improve and increase the rate of accumulation of secondary metabolites in Diospyros kaki L. cv. Costata.

5- Formulation of Disopyros kaki L. cv. Costata fruits and leaves active extracts as natural products in pharmaceutical industry as natural antioxidant.