EVALUATION OF THE CHEMICAL CONSTITUENTS OF WILD AND CULTIVATED MARIGOLD PLANTS

Heba Ibrahim Abd El-Moaty 1 and Hanan Ali El-Sayed 2 . 1. Assistant professor of Phytochemistry,Medicinal and Aromatic Plants Department, Desert Research CenterElMataria, Cairo, Egypt. 2. Researcher of cultivation of Medicinal and Aromatic Plants Medicinal and Aromatic Plants Department, Desert Research CenterEl-Mataria, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

For evaluation of different sowing dates and foliar fertilization effects on cultivated marigold, a factorial experiment with split plot design and three replications was carried out. Marigold (Calendula officinalis) was cultivated at Baloza Research Station, Desert Research Center, North Sinai Governorate, Egypt,during cropping seasons (2014/2015 and 2015/2016). Three sowing dates (15 September, 1 October and 15 October) and three foliar fertilization (Control, Ever Full Grow and Biomagic product) and their interaction were studied. Results showed that, sowing dates and foliar fertilization have significant effects on plant height, flower dry weight, primary metabolite constituents in marigold flowers such as (carbohydrates, nitrogen, protein and lipids) and flavonoids as secondary metabolites when comparing with the estimated component of wild C. officinalis. However, the best interaction treatment was the sowing date at 1 October and biomagic, which gave a highly significant effect on evaluated traits compared to other ones. So, this treatment was chosen to investigate the separated primary and flavonoid compounds compared with wild plants. The obtained data declared that the highest concentrations of the separated free and combined sugars was inulin. Meanwhile the lysine was the highest percentages of the separated protein amino acids. On the other hand, the highest percentage of fatty acids was palmitic acid. Investigation of flavonoids using HPLC analysis revealed that the plant contained 22 flavonoids compounds in the best interaction treatment and wild C. officinalis. It was noticed that, the obtained major compounds for the chosen cultivated plants were Apigenin-6-arbinose -8-glactose (169.790mg/100g), Hespiridin (102.330mg/100g) and Luteolin-6-arbinose-8-glucose (85.565mg/100g). While the obtained major flavonoids compounds for the wild C. officinalis were Hespirtin(117.340 mg/100g), Luteolin-6-arbinose-8-glucose (21.587 mg/100g) and Apignin-6-glucose -8rhamnose (11.778 mg/100g).
Sowing date play an important role in plant growing and effect on active substance in medicinal plants, significantly (Ghani, 2011).Applying various sowing dates results in facing to different temperature, solar radiations and day length by plant growing processes, so that impact on plants growth and yield (Dadashi and Khajepour, 2004). Seeds of Marigold (Calendula Officinalis L.) plants were kindly provided from SEKEM Company of Medicinal and Aromatic Plants. The seeds were sown in the nursery bed. Meanwhile, seedlings were transplanted in the experimental area after 45 days from swing dates for the two seasons in sandy soil. The mechanical and chemical properties of the used soil are shown in Table (A) according to Chapman and Pratt (1971).  1609 Investigation of primary metabolites in flowers:- Determination of total carbohydrates percentageand content according to (Chaplin and Kennedy, 1994).  Identification of free sugars and combined sugars for cultivated plant under the best interaction treatment in this study and wild plant by HPLC according to (Zielinski et al., 2014).  Determination of total nitrogen percentageand content by using Kjeldahl method according to James (1995).  Determination of total protein percentageand content according to James (1995).  Investigation of total amino acids for cultivated plants under the best interaction treatment in this study and wild plantsaccording to Csomos and Simon-Sarkadi (2002), using Amino Acid Analyzer.  Determination of total lipids percentageand content according to Faraget al. (1986).  Determination of saponifiable matter (fatty acids) for cultivated plants under the best interaction treatment in this study and wild plants.They were determined using GLC (Faraget al., 1986).

Materials and methods:-
Investigation of flavonoids as secondary metabolitesin flowers:-Estimation of Flavonoid percentageand content according to Karawya and Aboutable (1982).
Qualitative and quantitative of flavonoids for cultivated plants under the best interaction treatment in this study and wild plantsby HPLC The ethanol extracts of Calendula Officinalis flowers were analyzed usingHPLC. The employed HPLC system consisted of HP 1090M Series ІІ high performance liquid chromatography equipped with an HP 1090M Series ІІ diode array and an eight-channel electrochemical coulometric array detector ((EC); Esa Inc., USA). The EC was operated using 100-800 mV potentials (100mV intervals). The detector array was housed in a temperature-regulated compartment at 35ºC.
Flavonoid separation was done by ODS-3 (4.0 × 150 nm, 3µm) column with a C-18 guard column, with temperature set at 35ºC. The flow rate of the mobile phase was 0.7mL/min, and the injection volumes were 10µL of the standards and sample extracts. All flavonoids were quantified using the external standard method. Quantification was based on peak area (DAD) or beak hight (EC). (Mattilaet al., 2000).

Design and Statistical Analysis:-
The experimental design was factorial experiment between sowing date and complete fertilizer or biomagic product treatments in Split blot design (Main plots were consisted of sowing date and sub plots included the complete fertilizer or biomagic product) with three replicates. Data of the present study were statistically analyzed and the differences between the means of the treatments were considered significant when they were more than least significant differences (L.S.D.) at the 5% or 1% levels according to Steel and Torrie(1980).  Imholte and Carte (1987) reported that delay of sowing caused a decline in plant height.

Results and discussion:-
Moreover, spraying marigold plants with complete fertilizer (Ever Full Grow) or biomagic product gave a highly significant increase in plant height compared to control. Moreover, the tallest plants were obtained from biomagic treatment, then decreased gradually with Ever Full Grow followed by control treatments. These results are in agreement with those found by Khalid and Shedeed(2015) on Nigella sativa L. plants by using foliar nutrition, (Hafez, 2013) on Jerusalem Artichoke by using biomagic and Hashem (2007) on thyme plants by using complete fertilizer and biomagic. These results may be due to the Biomagic contents of proteins, amino acids, vitamins and hormones, as well as some micro nutrients (Hafez, 2013).

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Meanwhile, the interaction between the second sowing date (1 October ) and biomagic treatment recorded the tallest plants and gave a highly significant increase in this regard compared to the other ones. Furthermore, the interaction treatment between second sowing date and control recorded the shortest plants in comparison with other interaction treatments in the first season, but in the second season the interaction between the third date (15 October) and control showed the shortest plants compared with other treatments. most studies about sowing date indicate that delaying in sowing date leads to decrease qualitative and quantitative yield. In an investigation on Calendulaofficinalis L. medicinal plant and Menthapiperitaindicated that sowing date effect on dry weight of Calendula officinalis L. and Menthapiperita wassignificant and delaying in sowing decreased growth parameters and chemical constituents (TahmasbpourandMohamadin, 2006). Also, the overall response for planting marigold in September was better due to the availability of favorable temperature and day length (duration of light) before the onset of flower bud initiate and flowering (Singh, 2015) on TageteserectaLinn plant .

Flowers dry weight / plant:-
Data recorded in Table (1) reveal that, the treatment of biomagic was the best one and gave a highly significant increase in flowers dry weight per plant compared to Ever Full Grow and control treatments. These results may be due to the Biomagic contents of proteins, amino acids, vitamins and hormones, as well as some micro nutrients (Hafez, 2013). The increase of flowers dry weight per plant might be due to the application of complete fertilizer which consist of macro and microelements such as nitrogen for its importance to consist the amino acids to form the protein which participate in cell enlargement and cell division. While, phosphorus have an important role in producing energy for the physiological processes as synthesis proteins by formation the coenzyme adenine triphosphate (ATP). Furthermore, potassium plays a direct or indirect role in plant metabolism, as explained by (Devlin, 1979).
In addition, such promoting effect on all parameters might be due to that Zn had an important role in indole acetic acid synthesis in plant tissues as it is an activator of enzymetryptophane synthesis (the precursor of auxin), (Krishnamoorthy, 1981). Also, manganese had a regulatory role in biosynthesis of proteins of photosystem II, as reported by (Khmara, 1984). At the same time, Fe might play an important role in the prophyrin structure of chlorophyll. In green plants, there is often a good correlation between the level of supply and the chlorophyll content, (Dekocket al., 1960). Generally, the used fertilizer might play an active role (direct or indirect) in cell division and / or cell enlargement (elongation) in stem tissue leading to more growth parameters (flowers dry weight).
Moreover, the interaction between biomagic and second sowing time (1 October ) treatment recorded the highest flowers dry weight per plant and gave a highly significant increase in this respect compared to other interaction treatments. Under each sowing date flowers dry weight per plant was increased gradually by using complete fertilizer ( Ever Full Grow) flowed by biomagic. These results were similar in both seasons.

Investigation of primary metabolites:-
Total carbohydrates percentage and content:-Table (1), indicate that the best sowing date was (15 September) which gave the best value of total carbohydrates percentage and content compared to other planting times and wild plant. Moreover, total carbohydrates content decrease gradually by delaying the sowing from 1 October and followed by 15 October. Also, the first sowing date (15 September) showed a highly significant increase in total carbohydrates content in comparison with second and third dates. These results are in similar with those stated by (Al-Doghachiet al., 2016) onWithaniasomniferaL. plant.
On the other hand, total carbohydrates percentage and content increased by using complete fertilizer (Ever Full Grow) or biomagic product compared to control. Meanwhile, spraying marigold plants with Ever Full Grow gave the highest value in total carbohydrates percentage during the first season, but in the second season biomagic treatment recorded the best value in total carbohydrates percentage and content in comparison with other treatments. The treatment of biomagic gave a highly significant increase in total carbohydrates content compared to Ever Full Grow and control treatments. These results are in harmony with those reported by (Hafez, 2013) on Jerusalem Artichoke and (El-Hifny and El-Sayed, 2011) on sweet pepper plant.
Furthermore, the highest increase in total carbohydrates percentage was obtained from the interaction treatment between the first sowing date and biomagic during the first season but, in the second season it observed by interaction treatment between the third sowing date and biomagic compared to other interaction treatments. Moreover, the best interaction treatment was that between the second sowing date (1 October) and biomagic which recorded a highly significant increase in carbohydrates content per plant flowers compared to other interaction treatments.

Investigation of free sugars:-
The data illustrated in Table ( 2 ) show that, the separation of the free sugars contents achieved using High Pressure Liquid Chromatography (HPLC), where twelve of free sugars were detected at cultivated marigold plant, which produced from the best interaction treatment in this study (1 October and biomagic) , while eleven of free sugars were detected at wild plants . It was noticed that, the highest concentrations of the separated free sugars at cultivated and wild plants was inulin (9.71% and 13.82 %, respectively). Table ( 2 ) reveal that, the separation of the hydrolyzed combined sugars were achieved using HPLC, where eleven of combined sugars were detected at cultivated marigold plant which produced from the best interaction treatment in this study (1 October and biomagic), while ten of combined sugars were detected at wild plants. The highest percentage of the separated sugars at cultivated and wild plants was inulin (2.95% and 1.29 %, respectively).  Table ( From the same Table (3) it could be noticed that total nitrogen percentage and content increased gradually by using complete fertilizer (Ever Full Grow) followed by biomagic treatment. Moreover, spraying marigold plants with Ever Full Grow or biomagic recorded an increase in total nitrogen percentage and content per plant flowers compared to control. Furthermore, the best treatment was that spraying marigold plants with biomagic which gave a highly significant increase in total nitrogen content per plant flowers compared to other treatments. These results were found in both seasons. These results are in agreement with those stated by (Khalid and Shedeed, 2015) on Nigella sativa L. plants by using foliar nutrition and (Hashem, 2007) on thyme plants by using complete fertilizer and biomagic.

Investigation of combined sugars:-Data recorded in
Furthermore, the highest values of total nitrogen and protein percentage were observed by interaction treatment between the third planting date (15 October) and biomgic in the two seasons compared to the other interaction treatments and the wild plant. Moreover, the best interaction treatment was that between first sowing date and Ever Full Grow which recorded highly significant increase in total protein content per plant flowers during the fist season but, in the second season was that between the second sowing date and biomagic in this respect compared to other interaction treatments. Also, under each of sowing date total nitrogen and protein content were increased gradually by Ever Full Grow followed by biomagic.

Investigation of total amino acids (protein-amino acids):-
From data presented in Table ( 4), results show that the investigation of hydrolyzed protein-amino acids, achieved using amino acid analyzer, where fifteen amino acids of different types were detected at cultivated marigold plant which produced from the best interaction treatment in this study (1 October and biomagic), while fourteen amino acids of different types were detected at wild plant . The highest percentage of the separated amino acids was that of lysine at biomagic and wild plants (15.86 % and 25.00 %, respectively). Total lipids percentage and content:-Data recorded in Table (5) revealed that the total lipids percentage and content per plant flowers decreased gradually by delaying sowing date 1 October followed by 15 October. Moreover, the best sowing date in this regard was that 15 September compared to other sowing dates. However, the first sowing date recorded a highly significant increase in total lipids content in comparison with other ones. These results coincided with those found by (El-Saadyet al.

2013) on Lallemantiaibericaplant.
Meanwhile, total lipids percentage and content of marigold flowers increased gradually by using Ever Full Grow followed by biomagic treatments. Since, spraying marigold plants with biomagic gave a highly significant increase in total lipids content compared to Ever Full Grow and control. Also, the treated plants with Ever Full Grow or biomagic led to an increase in this regard compared to control. These results are in agreement with those recorded by (Khalid and Shedeed, 2015) on Nigella sativa L. plants by using foliar nutrition and (El-Sherbenyet al., 2007) on RutagraveolensL. plant by using foliar fertilizers.
Furthermore, the best interaction treatment was that between the first sowing date (15 September) and complete fertilizer (Ever Full Grow) which recorded the highest value of total lipids percentage compared to other interaction treatments but the wild plant recorded the highest value in this respect in comparison with all interaction treatments during the two seasons. Moreover, the highest content of total lipids was obtained from the interaction treatment between the second sowing date (1 October) and biomagic which gave a highly significant increase in this regard compared to other ones. Moreover, there were no differences between complete fertilizer (Ever Full Grow), biomagic, control treatments and wild plants in total flavonoids percentage. Since, spraying marigold plants by biomagic was the best treatment which gave a highly significant increase in total flavonoids content per plant flowers in comparison with Ever Full Grow or control treatments. Spraying marigold plants by Ever Full Grow or biomagic led to an increase in this respect compared to control. These results were similar in both seasons.
As regard to total flavonoids content per plant flowers, it could be noticed from Table (5) that, the best interaction treatment was that between the second sowing date (1 October) and biomagic product. Meanwhile, there were no differences between all interaction treatments and wild plant in total flavonoids percentage. On the other hand, the interaction treatment between the second sowing date and biomagic gave a highly significant increase in this regard compared to other ones.
Qualitative and quantitative of flavonoids by HPLC:-Data at Table (7) indicate that, investigation of flavonoids by HPLC revealed the presence of 22 compounds of the cultivated marigold plant, which produced from the best interaction treatment in this study (1 October and biomagic) and wild Calendula Officinalis, where the major compounds at cultivated plant were Apigenin-6-arbinose -8glactose (169.790mg/100g), Hespiridin (102.330mg/100g) and Luteolin-6-arbinose-8-glucose (85.565mg/100g). While the major compounds at wild plants were Hespirtin (117.340 mg/100g), Luteolin-6-arbinose-8-glucose (21.587 mg/100g) and Apignin-6-glucose -8-rhamnose (11.778 mg/100g). The significant decrease in all parameters with delaying in sowing date could be attributed to shorter period of vegetative growth of plants. Also, the used fertilizer might play an active role (direct or indirect) in cell division and / or cell enlargement (elongation) within stem tissue leading to more growth parameters and chemical constituents (carbohydrates, nitrogen, protein, lipids and flavonoids).

Conclusion:-
It could be concluded that, the sowing date of 1 October and spraying plants by biomagic product is suitable for producing the highest flower yield /plant and chemical constituents content of cultivated marigold at North Sinai conditions. These results demonstrated that we can use the cultivated marigold instead of wild plant to keep out the wild plant from extinction and our autecology.