ملخص الرسالت INTRODUCTION

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1 ملخص الرسالت رسالت ماجستير بعنوان : دراساث كيميائيت حيويت النتاد بعض المركباث الخانويت فى مختلف النباتاث بأستخذام تقنياث زراعت االنسجت BIOCHEMICAL STUDIES ON PRODUCTION OF SECONDARY METABOLITES FROM DIFFERENT PLANTS BY USING TISSUE CULTURE TECHNIQUES By Heba Mahmoud Mohamed Mahmoud Under supervision: Dr. Osama Konoswa Ahmed Professor of Biochemistry, Fac. Agric., Cairo University Dr. Eman Ahmed Hanafy Lecturer of Biochemistry, Fac. Agric., Cairo University INTRODUCTION Ginkgo biloba is known as living fossils as it is the only surviving member of ancient trees (Sadaf et al., 2016). Ginkgo biloba is a dioecious tree which has been used in traditional Chinese medicine for about 5000 years (Saquires, 1999). This species has a long history of use in medicine and as an ornamental plant in the landscape in different countries (Mantovani et al., 2013). Ginkgo possesses a high degree of resistance to fungal, viral, and bacterial diseases, as well as to ozone and sulfur dioxide pollution, making it an excellent choice for planting in urban areas (Sinclair et al., 1987). The term of medicinal plants include a various types of plants in herbalism and some of these plants have a medicinal activity. These medicinal plants consider as a rich resources of ingredients which can be used in drug development and synthesis. Besides that these plants play a significant role in 1

2 the development of human cultures around the whole world. Moreover, some plants consider as important source of nutrition and as a result of that these plants recommended for their therapeutic values. These plants include ginger, green tea, walnuts and some others plants. Other plants and their derivatives consider as important source for active ingredients which are used in aspirin and tooth paste (Hassan, 2012). Nature has provided a rich source of herbal medicines to cure various ailments of mankind. Plants are one of the most important sources of drugs, because every species in nature is of potential value to human beings. Medicinal plants are of great interest as pharmaceutical industries depend in part on plants for the production of secondary compounds (Molnar et al., 2011). A large number of medicinal plants are exploited from the natural flora for the commercial production of drugs. During the last few decades as a result of technology advancement and increasing demand of phytomedicines, medicinal plants used in various traditions were investigated chemically and pharmacologically for the active chemical constituents and to elaborate new stand recipes for more effective treatments for specific ailments. In view of growing world populations, increasing anthropogenic activities, rapidly eroding natural ecosystems the natural habitat for a great number of herbs and trees are decreasing and many of them are facing extinction (Kumar et al., 2009). Now plant tissue culture become an important part of biotechnology, which can be of immense help to overcome the problem of reduction of natural herbal wealth through rapid micropropagation methods. There are number of constraints for the propagation and conservation of many taxa through conventional methods like vegetative and seed propagation, which includes slow rate of multiplication, climatic factors and edaphic, dormancy, low percentage of seed set and germination. Sometimes, despite a high rate of germination, clonal uniformity cannot be maintained through seeds. Under these conditions, in vitro techniques have been successfully applied to solve various constraints related to conventional clonal propagation in a large number of medicinal plants (Sridhar and Aswath, 2014). 2

3 The main compounds synthesized by Ginkgo are diterpenelactones, for example ginkgolides A, B, C, J and M, a sesquiterpenebilobalide and the flavonol glycosides quercetin, kaempferol and isorhamnetin. These compounds are found in the bark, roots and in greater amount in the leaves of plants cultivated in the field (Van Beek, 2002) and moreover in in vitro regenerated plants and in cell cultures (Hao et al., 2009). Ginkgo s extracts are generally used in medicine because the therapeutic properties mainly linked to the regulation of cerebral blood flow and protection against free radicals (Cheng et al., 2009). Studies also suggest that extracts of ginkgo may reduce the progression of dementia (Weimann et al., 2010) and diabetes (Zhou et al., 2011). Various studies have shown a beneficial role of Ginkgo biloba in Alzheimer s disease (AD), although some have refuted its role in AD. In the absence of effective therapy in dementia particularly in AD, Ginkgo biloba seems to have a therapeutic value in dementia and it needs further clinical trial to recommend it as an effective drug in AD Dash (2015). The various compounds found in ginkgo improve blood circulation by opening up blood vessels, commonly in the brain. That is why they can be used for treating dementia and other vascular regulating problems in older age. Ginkgo preparations have been shown to help slow down Alzheimer s disease (Weinmann et al., 2010). Ginkgo leaves contain also antioxidant properties, which are principally connected to their polyphenolic constituents, particularly phenolic acids, flavonoids (Van Beek and Montoro, 2009). Ginkgo leaf extracts have been widely sold as phytomedicines in Europe and as dietary supplements worldwide. However, there remain some difficulties in supplying the demand for ginkgo extracts. Cultivation of the trees to harvest is lengthy, and the content of ginkgolides and bilobalide is highly variable. Additionally, leaf collection is labor intensive, but chemical synthesis of gingkolides, as an alternative source, is complex (Kim et al., 1996). Also, the content of ginkgolide B (GB), the most wanted component due to its 3

4 strongly antagonistic effects on platelet activating factors (PAFs) is very low in cultivated trees compared with other compounds such as bilobalide (B) and ginkgolide A (GA). Therefore, to provide a sufficient supply of these compounds, strategies to enhance production of the compounds by in vitro culture are needed. G.biloba has great ornamental and medicinal value. The ginkgo extract EGB761 contains two important active pharmaceutical components, flavonoids and terpene lactones (Van Beek and Montoro, 2009), which can promote blood circulation and cerebral metabolism. These compounds make EGB761 as an effective action for cerebrovascular diseases, such as coronary heart disease and high blood pressure (Lu et al., 2011). According to Ramirez-Estrada et al. (2016) in plant cell cultures, an elicitor is defined as a compound introduced in small concentrations to a living system to promote the biosynthesis of the target metabolite. Traditionally, elicitors have been divided into two types, abiotic or biotic, according to their chemical nature and exogenous or endogenous origin, and notably include methyl jasmonate, yeast extract, salicylic acid, vanadyl sulphate and chitosan. Wu et al. (2007) demonstrated that NO is a free radical with multiple physiological implications in plants. According to previous studies, NO is believed to play important roles in signal transduction and plant defense by enhancing the secondary metabolism, as NO production in plant tissues and cells usually occurs in response to abiotic stresses (Garcês et al., 2001). NO is the one of the key signaling molecules in elicitor-induced secondary metabolite biosynthesis in medicinal plant cells (Zhang et al., 2011). Nitric oxide (NO) is a free gaseous radical with a wide variety of physiological implications in animal and plant cells (Lamattina et al., 2003 and Tuteja et al., 2004). Moreover, Manivannan et al, (2016) showed that SNP (sodium nitroprusside the donor of nitric oxide) elicited the accumulation of total phenols, flavonoids, and acacetin, a flavonoid compound with multiple pharmaceutical values, as several studies have suggested its signaling role in the regulation of plant 4

5 growth, development, and defense responses (Flores et al., 2008 and Hong et al., 2008). Xue and He (2015) showed that cyanobacteria characterize a promising stage for the production of plant secondary metabolites. Their ability to express plant P450 proteins, which include essential functions in the biosynthesis of many plant secondary metabolites, makes cyanobacteria ideal for this function, and their photosynthetic capacity allows cyanobacteria to grow with simple nutrient inputs. On the other hand, Amin et al. (2009) reported that the increase in crop yields as a result of algal inoculation can not only be attributed to the nitrogen-fixing property of cyanobacteria, but may be largely due to the growth regulating substances endogenously produced by these algae, so the use of cyanobacteria as a cheaper substitute for the used phytohormones and vitamins to promote the growth and production of secondary metabolites in vitro. This study investigated the effect of various culture conditions on the growth of callus cell line from Ginkgo biloba leaves and the effect of elicitors by using elicitation via nitric oxide, micro algal elicitors (Spirulina Sp. and Anabaena Sp.) on phenols, flavonoids, ginkgo flavone glycosides and ginkolides production as well as growth in Ginkgo biloba suspension cells. MATERIALS AND METHODS MATERIALS 1. Plant materials The ginkgo (Ginkgo biloba L.) leaves were collected from International Garden, Nasr city, Cairo between the mid of April and the end of July (2013). 2. Chemicals All chemicals and reagents were obtained from Sigma Chemical Co. (London, Lab. Poole), England (Cairo branch) and distilled before use. 3. Algal cultures 5

6 Spirulina Sp. were obtained from Alga department, National Research Center and Anabeana Sp. from alga department, faculty of agriculture, Ain shams university. METHODS Tissue culture technique The present study was carried out in Tissue Culture Research Laboratory, Horticulture Research Institute, A.R.C., Giza, Egypt. 1. Culture media Murshige and Skoog (1962) basal nutrient media (MS) was used during this study. The medium was supplemented with 3% sucrose. The medium was solidified with 0.7 % agar and ph was adjusted to 5.8 before autoclaving. The prepared media were poured into heat sterilized 150 ml glass containers, each contained about 25 ml solidified medium. Liquid media were used without agar and ph was adjusted to 5.8 before autoclaving. Media were autoclaved at 121 º C for 25 min. Callus induction phytohormones stock solutions were prepared and stored at 4 C. 2. Leaves sterilization Leaves of Ginkgo biloba were surface sterilized by a brief immersion in 70 % ethanol for 10 sec. followed by 8 min. in 0.25 % active chlorine containing a few drops of Tween 80, and then rinsed four times with sterile distilled water under aseptic condition to release bleach (Joen et al., 1993). 1. Callus initiation a. Medium types used for callus induction Leaf explants (size ~ cm) were excised from the lamina. These were placed in small jars containing 25 ml MS basal medium. Two different supplementations of growth regulators; 6- benzyl adinine (BA) and α- naphthalene acetic acid (NAA) were used to study the effect of different types and concentrations of growth regulators on Ginkgo biloba callus induction. They were added to MS medium to obtain the best callus formation. Cultures 6

7 were kept in the light (50µmol m -2 s -1 ) with a 16/8 h light/dark photoperiod at 22 ± 1º C. Callus formation was monitored over a month period and subsequent growth (in terms of fresh weight) was followed until the stationary phase was reached. Once established, callus fresh weight was recorded the formed callus, callus was rebcultured at 3 weeks intervals on the same medium and under the same conditions described above. The phytohormones used in this experiment are shown in Table 1. Table 1. Different hormone combination of MS medium. NAA(mg/L) BA(mg/L) 1 A1 B1 C1 D1 2 A2 B2 C2 D2 3 A3 B3 C3 D3 4 A4 B4 C4 D4 The callus of Ginkgo biloba leaf explants was used to initiate the suspension culture as described by Cheng et al. (2014) using liquid medium B2 (NAA (2 mg / L) + BA (2 mg / L)) (without agar) in this experiment (Table 2). b. Initiation of suspension cultures Callus (0.5 g of callus fresh weight (FW) / 20 ml medium) was transferred into MS liquid medium supplemented with 3 % (w / v) sucrose and 2 mg / L NAA with 2 mg / L BA to induce proliferation. The cell suspension cultures were shaken (100 rpm) on an incubator in dark and maintained at 25º C (Hao et al., 2010). c. Measurement of cell suspension culture growth curve Ginkgo biloba cell suspension culture material weighing 0.5 g FW was transferred into a 100 ml conical flask containing 20 ml culture medium. Cell growth was monitored at every 3 rd day (up to 20 days) by determining the fresh 7

8 weight. Briefly the cell cultures were harvested on every 3 rd day to collect cells by filtration. The fresh weight of the cells was recorded with the help of a physical balance. All data are expressed as an average of three separated experiments, cell growth precisely was measured and performed the sedimented cell volume (SCV) measurement (Kang et al., 2006). 4. Chemical analysis a. Chemical characterization of Ginkgo biloba leaves and its callus 1. Determination of moisture The moisture content was determined according to A.O.A.C. (2000). 2. Determination of crude protein Total nitrogen (TN) was determined according to A.O.A.C. (2000) using microkjeldahl method. Crude protein was calculated by multiplying TN by a factor of Determination of ash Ash content of 2 g of samples was ignited in muffle furnace at 550 C to a constant weight and the percentage of ash was calculated A.O.A.C. (2000). 4. Determination of total Carbohydrates Total carbohydrate was spectrophotometrically estimated according to Dubois et al. (1956). b. Elicitation of secondary metabolites production Preparations and addition of elicitors Nitric oxide elicitor Sodium nitroprusside (SNP) was used as the donor of NO in this study. This compound was sterilized by filtering through 0.22 µm sterile filters (Millipore) and added directly to the fresh medium prior to inoculation at final concentration of 25, 50, 100 and 150 ppm (Xu et al., 2004). 8

9 Algal extract preparation Dry algal cultures (500mg) were suspended in 50 ml of distilled water and heated to 95º C for 60 min. After centrifugation at 8,000 g for 30 min., the supernatant was filtered through a 0.22 µm Millipore membrane filter then, the extracts obtained from each species was dissolved in known amount of sterilized distilled water and added after filtration at different concentrations of 25, 50, 100 and 150 ppm. Elicitors were introduced at the beginning of cell culture. Control culture treatment was elicitor-free (Rao et al., 2001). c. Determination of total phenolic compounds The phenolic compounds were determined using the Folin-Ciocalteu method, based on the reduction of phosphor-wolframate-phosphomolybdate complex by phenolics to a blue reaction product (Singleton and Rossi, 1965). The Folin-Ciocalteu reagent, diluted 10 times (2.5 ml) was mixed with 2 ml of sodium carbonate (75 g / L) and 50 μl of sample (supernatant) and homogenized for 10 sec. and heated for 30 min. at 45 C. The absorbance was measured at 765 nm after cooling at room temperature. The data were calculated by comparison between μmol gallic acid / L and the absorbance of each sample. The data were expressed as gallic acid equivalents (mg gallic acid / g dried extract). d. Determination of total flavonoids content The total flavonoids content of each plant extract was determined by method described by Zhishen et al. (1999). Based on this method, each sample (1.0 ml) was mixed with 4 ml of distilled water and subsequently with 0.30 ml of a NaNO 2 solution (10 %). After 5 min., 0.30 ml AlCl 3 solution (10 %) was added followed by 2.0 ml of NaOH solution (1 %) to the mixture. Immediately, the mixture was thoroughly mixed and absorbance was then determined at 510 nm versus the blank. The results were expressed as quercetin equivalents (mg quercetin / g dried extract). 9

10 e. Extraction and quantification of ginkgo flavone glycosides and ginkgolides The suspended cells were separated from the medium by filtration. The separated cells were washed with distilled water to remove any adhering medium to the cell surface to record the fresh weight; Fresh cell samples (0.1 g) were ground with a mortar and pestle to extract the phytochemicals by solvent extractions. The various ginkgolides were extracted into 10 ml n- hexane by sonication for 1 h. After centrifugation the upper n-hexane layer was withdrawn with a pipette. The bottom layer of cells was extracted with 10 ml ethyl acetate for in an ultra-sonicator (Jinwoo Engineering, Korea) for 2 h. The resulting cell extracts were centrifuged at 12,000 g for 10 min and the supernatant ethyl acetate layer was concentrated by rotary vacuum evaporation (EYELA, Japan). The combined ethyl acetate portions were concentrated using the rotary vacuum evaporator. The residue obtained was dissolved in 200 µl methanol (HPLC grade), filtered through a pre-filter (0.2 µmpore size, Supelco, Bellefonte, PA) and the quantification of ginkgo flavone glycosides and ginkgolides was carried out with HPLC Hewllet Packared (series1050) equipped with auto-sampling injector, solvent degasser, ultraviolet (UV) detector set at 280 nm and quaternary HP pump (series 1100). Isocratic mobile phase consisted of a mixture of methanol and H 2 O (50:50, v / v). After the injection of 20 µl of the sample solution, the column was operated with a flow rate (0.5 ml min 1 ) (Kang et al., 2006 a and b). Statistical analysis All experimental measurements were carried out in triplicate and are expressed as average of three analyses ± standard deviation using a SPSS (Chicago, IL) statistical software package (SPSS for Windows, ver. XII, 2004). 11

11 RESULT AND DISCUSSION Ginkgo biloba culture is the new route for large scale secondary metabolites production because of their fast and pleiotropic growth of Ginkgo biloba callus. The aim of this work is optimization of callus culture conditions from leaf explants of Ginkgo biloba and finding suitable protocol to increase the productivity of secondary metabolites of Ginkgo biloba suspension cells through elicitation via nitric oxide elicitor, micro algal elicitors (Spirulina Sp. and Anabaena Sp.). This work included the effect of different hormones combinations of MS on fresh weight (g) of Ginkgo biloba callus cells for 30 day. After that, the best callus of Ginkgo biloba leaf explants with best medium were used to initiate the suspension cell cultures using liquid medium B2 [NAA (2 mg / L) + BA (2 mg / L)] beside measurement of Ginkgo biloba cell suspension culture growth curve. Determination of chemical composition of Ginkgo biloba dry leaves and callus (g /100 g dw).the present study also determined secondary metabolites as total phenols, total flavonoids in Ginkgo biloba cell cultured under elicitors Anabaena Sp. and Spirulina Sp. elicitors at concentrations of 25, 50, 100, 150 ppm, nitric oxide elicitor at concentrations of 25, 50, 100, 150 ppm with determined their effect on G. biloba cells growth in suspension cultures. Finally, determination of ginkgo flavone glycosides and ginkgolides of Ginkgo biloba extract by using HPLC through elicitation of NO, Spirulina Sp. and Anabaena Sp.) at concentrations of 25, 150 ppm in suspension cultures. The results of the present study can be summarized as follows 1. MS medium with different plant growth regulators showed a significant effect on the quality and growth of callus. The optimal medium for inducing leaf-derived callus was medium B2 [NAA (2 mg / L) + BA (2 mg / L)] which was the best medium for callus growth and callus subculture. After initiation of suspension cultures and measurement of cell suspension culture growth curve these 11

12 results found that cell growth line entered their exponential growth phase, during this time, the cell mass increased and reached stationary phase around the day 13 of culture, the stability time for the cells in suspension culture was 2 days, then the cells entered their death phase (cell death gradually). 2. The results of chemical composition of G. biloba leaves and callus was evaluated, and indicated that carbohydrates were the most abundant macronutrients (62.1 g /100 g dw), but in G. biloba callus total carbohydrates were g / 100 g dw. Otherwise, lipids was the macronutrient present in lower amount (2.01 g /100 g dw), but in G. biloba callus total lipid was g / 100 g dw. The levels of total proteins were 23 and g / 100 g dw in G. biloba leaves and callus, respectively whereas the levels of ash were ( and 8.49) g / 100 g dw in G. biloba leaves and callus, respectively. 3. Results showed that the best growth of cultured G. biloba cells on day 13 which was 0.69 g fw with the treatment of Anabeana Sp. extract at concentration 150 ppm which showed a significant increase of 1.2 fold in biomass over their respective control (0.596 g fw) from the initial inoculum of 0. 5 g fw. Also the results reported that the highest content of phenols and flavonoids (899.3 and mg / 100 g fw, respectively) were obtained in culture cells treated with Anabeana Sp. extract at concentration 100 ppm which showed a significant increase 2.5 and 2 fold over their respective control ( and mg / 100 g fw on day 22, respectively). 4. The Spirulina Sp. extract at concentration of 150 ppm showed the best increasing in biomass of G. biloba cell cultures (0.7 g fw on day 13) which showed a significant increase of 1.9 fold over the control. While Spirulina Sp. extract at concentration of 100 ppm showed the maximum phenols and flavonoids content of Ginkgo biloba cultures ( and mg / 100 g fw, respectively) on day 22, which 12

13 showed a significant increase of 1.72 and 2.4 fold over the control, respectively. 5. The results showed that the using of SNP at concentration of 150ppm resulted the maximum phenols and flavonoids content of Ginkgo biloba cultures and mg / 100 g fw on day 22, which showed a significant increase of 2.8 and 2.2 fold increase over control, respectively with slightly inhibition of cells growth. The best growth was obtained by using 25 ppm SNP on day13 which was g fw which showed a significant increase than the control. 6. HPLC results strongly indicated that total contents of ginkgo flavone glycosides and ginkgolides in G. biloba cultured cells with elicitation of Anabeana Sp. at concentration of 150 ppm showed an increase of ginkgo flavone glycosides and ginkgolides contents ( and ppm, respectively) which was a significant increase of 1.9 and1.4 fold over the control ( and ppm, respectively). Similarly Spirulina Sp. extract at concentration of 150 ppm treated G. biloba cultured cells showed an increase of ginkgo flavons glycosides and ginkgolides ( and ppm, respectively) which was 1.9 and 2.3 fold over the control and ppm, respectively.whereas the accumulation of ginkgo flavons glycosides and ginkgolides increased of and ppm which reported 2.3 and 3.2 fold over the control ( and ppm, respectively) in cultures treated with SNP at concentration of 150 ppm. The result showed that the maximum production of ginkgo flavone glycosides and ginkgolides obtained with NO elicitation. 13

14 SUMMURY The present study was carried out in Tissue Culture Research Laboratory, Horticulture Research Institute, A.R.C., Giza, Egypt, during 2011 to Because of the importance of Ginkgo biloba and industrial needs of ginkgo secondary metabolites significantly directed this study towards elicitation using tissue culture for over production of ginkgo metabolites via using sodium nitroprusside (SNP) which utilized as the donor of nitric oxide (NO) and using micro algal elicitors (Anabaena Sp. and Spirulina Sp.) to investigate its effect as elicitor on phenols, flavonoids, ginkgo flavone glycosides and ginkgolides accumulation in G. biloba cell suspension cultures and the effect of these elicitors on cells growth. Moreover, study the effects of different hormone combinations on callus induction for production the best callus in Ginkgo biloba cell cultures. The results showed that the optimal medium for inducing leaf-derived callus and the reculture of callus was MS media with 2.0 mg/l naphthalene acetic acid (NAA) mg/l 6-benzylaminopurine (6-BA). It was found that using SNP at concentrations of 25 and 50 ppm showed the highest increase in biomass of cultured Ginkgo biloba on day 13 which was and 0.58 g FW, respectively from the initial inoculums of 0.5 g FW. Whereas the highest accumulation of phenols and flavonoids content with 150 ppm SNP on day 22 showed an increase of 2.8 and 2.2 fold relative to control. Similarly, Anabaena Sp. extract at concentration of 150 ppm which showed the highest accumulation of phenols and flavonoids content (2.5 and 2 fold on day 22 of the control, respectively). On the other hand, Anabaena sp. extract at concentration of 150 ppm also influenced the highest increase in biomass G. biloba cultured cells on day 13 day which was 0.69 g FW. Moreover, G. biloba cultures treated with Spirulina Sp. extract at concentrations of 150 ppm observed the maximum increase in biomass on day 13 over the control which was g FW. Also, the highest accumulation of phenols and flavonoids content increased to 1.7 and 2.4 fold, respectively during day 22 in cultures treated with 100 ppm of Spirulina Sp. extract. In addition, the highest contents of ginkgo flavone glycosides and ginkgolides compounds found in G. biloba cell suspension culture was 1.9 and 1.4 fold of the control by using 150 ppm Anabeana Sp. extract and the highest contents of the content of these compounds were 1.9 and 2.3 fold with 150ppm Spirulina Sp. extract, respectively. While, the highest content of ginkgo flavone glycosides and ginkgolides was increased to 2.3 and 3.2 fold relative to control with 150 ppm SNP elicitation. 14

15 CONCLUSION The optimal medium for inducing leaves-derived callus and the subculture of callus was MS with 2.0 mg / L of NAA and 2.0 mg / L of 6-BA. Maximum phenols and flavonoids content were obtained in cultures treated with 150 ppm SNP. The best treatment of micro alga-spirulina sp. was 100 ppm but with Anabaena sp. treatment of 150 ppm. These treatments were achieved the maximum production of phenols and flavonoids with best growth. In conclusion micro alga-spirulina sp. (100 ppm) and Anabaena sp. (150 ppm) extracts influenced elicitation of secondary metabolite in Ginkgo biloba cell suspension cultures and enhanced the growth whereas nitric oxide (100 and 150 ppm) elicited secondary metabolites production in Ginkgo biloba cultures with slightly inhibition in growth respectively. In addition the Spirulina Sp. and Anabeana Sp. extracts treatments produced increases of ginkgo flavons glycosides and ginkgolides contents in G. biloba cultured cells with 150 ppm whereas, the maximum production of flavonoids, phenols, ginkgo flavone glycosides and ginkgolides was obtained with NO elicitation while Anabeana Sp. and Spirulina Sp. showed the best growth in Ginkgo biloba cell suspension cultures with flavonoids, phenols, ginkgo flavone glycosides and ginkgolides elicitations. These results of the present study conclude that the maximum metabolites (flavonoids, phenols, ginkgo flavone production of secondary glycosides and ginkgolides) obtained with NO elicitation with slightly inhibition of cells growth. While, Anabeana Sp. and Spirulina Sp. showed the best. فترة دراست الموضوع: حالث سنواث تاريخ البحج : /12/ 15

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