Effect of Ascorbic, Folic acids and Hibiscus Extract on Geranium (Pelargonium graveolens)

The present investigation was carried out in Sabahia Horticulture Research Station, Alexandria, Egypt during 2015/2016 2016/2017 seasons to study the effect of foliar application with ascorbic acid, folic acid as well as hibiscus extract on quality of Geranium (Pelargonium graveolens L.). Foliar application of ascorbic acid and folic acid as well as extract of hibiscus were sprayed twice in each cut (first spry before cut 60 days and second spray before cut 30 days). Treatments included: control "water only", ascorbic acid at (100 and 200 ppm), folic acid at (50 and 100ppm) and hibiscus extract at (5% and 10%). The essential oil (%) of Geranium was determined by hydro-distillation as well as the main constituents of the volatile oil were determined by (GC). The Results showed that, all treatments (tested) caused a great increase in vegetative growth, the essential oil (%), oil yield (ml/plant and L/Fed.) and active compounds; Citronerlol, Citronelly formate, Isomenthone, B-caryophyllene and Geranyl formate, while it decreased α-pinene and Geraniol. In addition, ascorbic acid at 200ppm, folic acid at 100ppm and hibiscus extract at (5, 10%) are sharp efficacy on increasing the vegetative growth and volatile oil yield than other treatments. Furthermore, it could be illustrated that natural drying caused pronounced changes (acceptable) in the volatile oil% and main constituents.


Introduction
Pelargonium graveolens L. (Geranium, family; Geraniaceae). Major areas of the production (about 5795 fed.) are primarily in mainly Bani Sweif (4057 fed.) followed by Giza (1500 fed.) and Minya (110 fed.) Governorates. Two harvests (cuts) are taken per year; a spring cut from April to June and a summer cut from September to October. Generally, the production averaged by 105581 ton in 2016 year [1]. The volatile oil of Geranium is widely used in the perfumery and cosmetic industries [2][3][4]. It is an indispensable aromatherapy oil since geranium oil and its major constituents citronellol, geraniol, and linalool [5]. Geranium volatile oil has also become important skin care oil because it is good in opening skin pores and cleaning oily complexions [6,7]. This oil has also been found to have use in reducing pain due to postherpetic neuralgia, treating dysentery, hemorrhoids, inflammation, heavy menstrual flows, and cancer [6]. The French community is currently treating diabetes as well as diarrhea, gallbladder problems, gastric ulcers, jaundice, liver problems, sterility, and urinary stones with this oil [6,8,9]. Moreover furthermore, in Chinese homeopathy, it is thought to open up the liver chakra and promote the expulsion of toxins, helping to achieve a balanced body [10]. Also Geranium essential oil exhibited efficacy on keep the treated peach fruits during cold storage and market life periods [11].
Many biochemical effects of vitamins, one that has factor more recent attention is the improvement of yield and quality of many plants. And especially, enhancing growth and productivity will be of more importance to increase the yield [12][13][14][15]. Vitamin Ascorbic acid (C) has a regulatory role in promoting productivity in many plants such as pepper [16]. Balbaa and Talaat (2007) who concluded that with ascorbic acid, phenylalanine and ornithine treatments significantly increased plant height (cm) and number of branches/plant of Rosmary plants [17]. Thymus treated with phenylalanine, Hibiscus Extract on Geranium (Pelargonium graveolens) tryptophan or ascorbic acid at different concentrations caused a significantly increment of plant height (cm) and number of branches/plant compared with untreated plants [18]. However, Ascorbic acid (vitamin C) is one Important from vitamins, it is play important roles in plant growth, cell division, cell wall ex-pansion, and other developmental processes [19][20][21]. And also is synthesized in the higher plants and affects growth of plant and development, it is Dglucose metabolism product which affects some nutritional cycles activity in higher plants and play an important role in the electron transport system [22]. On the other hand, folic acid (vit. B9) has become the most prominent of B complex vitamins despite its essential biochemical function in amino acids metabolism and nucleic acids synthesis [23]. Folic acid (vit. B9) was first isolated from Spinacia oleracea L. (1941) and characterized as pteroylmonoglutamic acid, a complex water soluble vitamin B [24,25]. Foliar treatment of strawberry plants with both concentrations of folic acid, significantly improved agronomic properties of the treated plants including yield, primary and secondary weight of fruits and number of their achenes as compared to the control treatment [26].
Rosella "Karkadeh in Arabic", (Hibiscus sabdariffa L., family; Malvaceae), and is mainly cultivated in tropical and subtropical regions of Asia and Africa. The water extract from the calyx is consumed as hot and cold drink. It is also used in the production of Karkadeh jam and as a food colouring agent [27]. Major areas of the production in Egypt (about 12232 fed.) are primarily in mainly Luxor (6058fed.) followed by Aswan (5861 fed.). Generally, the production averaged by 12809 ton in 2016 year [1]. The in vivo pharmacological activities as antipyretic, antioxidant as well as anticholesterol products were screened in rats and rabbits [28]. It was also proved effective in regulating human blood pressure [29,30].
The main target of the present study was to improve the growth and productivity of Germanium by some vitamins and Hibiscus extract and evaluate the effect of natural drying on the content and chemical composition of the essential oil.

Plant Material
The research was carried out at the Sabahiya, Horticulture Research Station in, Alexandria, Egypt during two successive seasons of 2015/2016 and 2016/2017, to study the effect of ascorbic, folic acid and hibiscus extract on quality of Geranium (oil%, active compounds and vegetative growth). Seedlings were planted at the end (April/2015) in pots. Foliar application of ascorbic acid and folic acid as well as extract of hibiscus were sprayed twice in each cut (first spry before cut 60 days and second spray before cut 30 days) and taken two cut in each season.

Preparation of the Hibiscus Extract
Collect the fresh hibiscus (Roselle) calyx and natural drying at room temperature until constant weight. The dry calyx was powder using automatic blender and taken 50g and 100g of dried calyx was placed in 1000 mL water under stirring and then left for 12 hours then filtration and used to spray plants.

Harvest
The plants were harvested first cut (October) and second cut (May) in both seasons.

The Essential Oil Percentage (%)
A hundred (g) fresh (after 24 h. from cut) and dry Geranium herb (after 2 weeks from natural drying at room temperature 20 ± 2°C until constant weight) were subjected to three-hours of hydro-distillation using a Clevenger-type apparatus as described by [34].

Chemical Composition (Active Compounds) of Essential Oil
The GC analysis of volatile oil samples was carried out using gas chromatography instrument stands at the Medicinal and Aromatic Plants Res. Dept., Hort. Res. Inst. With the following specifications. DsChrom 6200Gas Chromatograph equipped with a flame ionization detector, Column: BPX-5, 5% Phenyl (equiv.) polysillphenylene-siloxane 30m x 0.25mm ID x 0.25 µm film., Sample size: 1 µl, Temperature program ramp increase with a rate of 10°C /min from 70°C to 200°C, Detector temperature (FID): 280°C, Carrier gas: nitrogen, Flow rate: N2 30ml/min; H2 30ml/min; air 300ml/min. Main compounds of the volatile oils were identified by matching their retention times with those of the authentic samples injected under the same conditions. The relative percentage of each compound was calculated from the area of the peak corresponding to each compound.

Experimental Design and Statistical Analysis
Data of the present study were subjected to the analysis of variance test (ANOVA) as Randomized Complete Block Design. Where the first factor was for seven treatments mentioned before, the second factor was for cuts. The least significant differences (LSD at the 5%) of probability were calculated by a computer program (Costat, version 6.303, 2005) according to the study [35].

Plant Height (cm) and Number of Branches (N/plant)
Data of studying the effect of ascorbic acid, folic acid and hibiscus extract on plant height (cm) and number of branches/ plant of Geranium during 2015/2016 -2016/2017 seasons are reported in (Table 1) and ( Figure 1) in two seasons of study, results showed that all treatments caused a significantly increase in plant height (cm) and number of branches/ plant compared with control. In addition, ascorbic acid at 200 ppm, folic acid 100ppm and hibiscus extract at 5% and 10% in both seasons were sharp efficacy on increasing plant height (cm) and number-branches/ plant than other treatments and control.
Regarding the effect of cuts date, statistical analysis show that, plant height (cm) and number of branches/ plant were gradually increase and significantly with the second cut compared with first cut in both seasons. The interactions among treatments and cuts dates revealed that significantly decreased plant height (cm) and number of branches/ plant in first cut. Ascorbic acid, folic acid and hibiscus extract treatments were more effective on increasing plant height (cm) and number of branches/ plant than first cut in both seasons.
Plant height (cm) Hibiscus Extract on Geranium (Pelargonium graveolens) Many biochemical effects of vitamins, one that has factor more recent attention is the improvement of yield and quality of many plants. And especially, enhancing growth and productivity will be of a more importance to increase the yield [12][13][14][15]. The results obtained showed that foliar application of Geranium plants with ascorbic acid, folic acid, hibiscus extract, significantly increase plant high (cm) and number of branches/ plant the experimental period. Similar results were obtained by Balbaa and Talaat (2007) who found that ascorbic acid treatment significantly increased plant height (cm) and number of branches/plant of Rosmary plants [17]. And also Ghazal (2015) reported that plant (Thymus vulgaris L.) treated with ascorbic acid at different concentrations caused a great increased of plant height (cm) and number of branches/ plant at (100 and 150ppm) compared with control [18]. However, Ascorbic acid (vitamin C) is one Important from vitamins, it is play important roles in plant growth, cell division, cell wall expansion, and other developmental processes [19][20][21]. On the other hand, folic acid (vitamin B9) has become the most prominent of B complex vitamins despite its essential biochemical function in amino acids metabolism and nucleic acids synthesis [23]. The present results are in a general harmony with those of Emam et al., (2011) who found that foliar application of flax plants with either folic acid or ascorbic acid significantly stimulated growth and development throughout the experimental [36]. Chemical composition of Roselle: ash 8-12%, protein 5-10%, carbohydrate 60-62%, vitamin C 92.0 mg/100g, organic acids (hibiscic acid 25.5%, citric acid 14.15%, vitamin C 92.7 mg/100g, oxalic acid 0.6%), mineral contents (Ca 0.56%, mg 0.24%, fe 0.08%, na 0.23%, k 0.95%, p 0.19%) and total anthocyanin 0.9 to 1.6% [32]. The effect of hibiscus extract treatment on plant height (cm) and number of branches/plant may be due to its effect on enzyme activity as a (cofactor) and metabolism, as it contains; ascorbic acid, mineral elements and anthocyanin.

Fresh and Dry Weights (g/plant)
Data presented in (Table 2) indicated that when foliar application with ascorbic acid, folic acid and hibiscus extract, they significantly increased of fresh and dry weighs (g/plant) when compared with the control treatment in both seasons. On the other hand ascorbic acid at 200ppm, folic acid 100ppm and hibiscus extract at 5% and 10% in two seasons were more effective on increasing fresh and dry weights (g/plant) than other treatments. Data showed that, fresh and dry weight (g/plant) was sharp increased with the second cut compared with first cut in two seasons. Looking to the interaction effect between the tested treatments and cuts, it is obvious that all treatments had the lowest fresh and dry herb weights (g/plant.) in 1 st Cut in both seasons.
The results obtained showed that foliar application of Geranium plants with ascorbic acid, folic acid as well as hibiscus extract, significantly increase fresh and dry weight the experimental period. These results are in agreement with those obtained by Ghazal (2015) who showed that fresh weight of (Thymus vulgaris L.) herb, significantly responded to ascorbic acid foliar application at different concentrations compared with untreated plant [18]. Similar results were obtained by Balbaa and Talaat (2007) who observed that ascorbic acid treatments significantly increased fresh and dry herb weights of rosemary plants [17]. Foliar treatment of strawberry plants with both concentrations of folic acid, significantly improved agronomic properties of the treated plants including yield, primary and secondary weight of fruits and number of their achenes as compared to the control treatment [26]. The effect of hibiscus extract treatment on fresh and dry weight (g/plant.) may be due to its effect on enzyme activity (cofactors) and metabolism, as it contains; ascorbic acid, mineral elements and anthocyanin.

Essential Oil% and Essential Oil Yield (ml/plant) of Fresh Herb
Results of the present investigation, presented in (Table 3) and (Figure 2) showed that effect of spray application of ascorbic acid, folic acid and hibiscus extract, on essential oil% and essential oil yield (ml/plant) of fresh Geranium herb in 2015-2016 and 2016-2017 seasons. Data showed that, oil percentage and oil yield of all treatments significantly increase compared with control in both seasons. Moreover, ascorbic acid at 200 ppm, and hibiscus extract at 5% and 10% in two seasons were more effective on increasing essential oil% and essential oil yield (ml/plant) of Geranium.
Regarding the effect of first and second cuts date, results recorded in second cut of the two seasons gave significantly higher essential oil% and essential oil yield (ml/plant) than first cut.
Evaluating the interaction effect between treatments and tested cuts, data show that the interactions registered the lowest values of oil percentage and oil yield with first cut in both seasons.  [40] on geranium reported that foliar application of ascorbic acid caused significantly increased in the essential oil% and oil yield. Ascorbic acid treatment significantly increased oil percentage during the 1st season where ascorbic acid at 150 mg / l gave the maximum mean values of volatile oil% in the 1st and 2nd cuts on Thymus vulgaris L plant [18]. The effect of hibiscus extract treatment on essential oil% and essential oil yield (ml/plant) may be due to its positively affected on enzyme activity and metabolism, as it contains; ascorbic acid, mineral elements and anthocyanin. Table 4 indicated that effect of ascorbic acid, folic acid as well as hibiscus extract on essential oil% and essential oil yield (ml/plant) from dry herb of Geranium during 2015/2016 -2016/2017 seasons. Different treatments increased the% and yield of essential oil (ml/plant) of dry herb in two seasons than treatment control. For essential oil% and essential oil yield (ml/plant), the higher value from spray ascorbic acid at 100, 200ppm, and hibiscus extract at 5% and 10% in first seasons. And also ascorbic acid at 100, 200ppm, folic acid at 100ppm and hibiscus extract at 5% and 10% gave increased% and yield of volatile oil (ml/plant) of dry herb in second season.

Essential Oil% and Essential Oil Yield (ml/plant) of Dry Herb
Regarding the effect of first and second cuts date, Data show that, essential oil% and essential oil yield (ml/plant) of dry herb in first season were significantly increased with the second cut compared with first cut. In Second season not significantly increase between first and second cut. The interaction effect between the treatments and cuts date, it is obvious that all treatments had the lowest essential oil% and essential oil yield (ml/plant) at first cut in two seasons.

Fresh and Dry Herbs Yield (ton/fed.)
Data in (Table 5); generally revealed that when spray application of ascorbic acid, folic acid and hibiscus extract, on fresh and dry herbs yield (ton/fed.) of Geranium in 2015-2016 and 2016-2017 seasons. Data showed that all treatments increased fresh and dry herbs yield (ton/fed.) than control. And also in the first season ascorbic acid at 200ppm, folic acid 100 ppm and hibiscus extract 5, 10% achieved significantly the highest yield [fresh and dry (ton/fed.)] expressed as weight. Besides, the highest yield in second season with ascorbic acid at 100, 200ppm, folic acid 100 ppm and hibiscus extract 5, 10%.
Regarding the effect of first and second cuts date, results recorded in second cut of the first season gave significantly higher fresh and dry herbs yield (ton/fed.) than first cut. On the other side, in the 2 nd season no significant differences were observed between first and second cut.
The interaction effect between the treatments x cuts date, it is obvious that all treatments had the lowest fresh and dry yields (ton /fed.) at 1 st Cut in two seasons. The results obtained showed that foliar application of Geranium plants with ascorbic acid, folic acid, hibiscus extract; a great increase fresh and dry weight (ton/fed.) the experimental period. These results are in agreement with those obtained by Ghazal (2015) showed that fresh weight herb, significantly responded to ascorbic acid at different concentrations compared with untreated plants (Thymus vulgaris L.) [18]. Similar results were obtained by Balbaa and Talaat (2007) who showed that ascorbic acid treatment significantly fresh and dry herb weights of rosemary plants [17]. Foliar treatment of strawberry plants with both concentrations of folic acid significantly improved agronomic properties of the treated plants including yield, primary and secondary weight of fruits and number of their achene's as compared to the control treatment and also found that folic acid enhances fruit quality and yield of strawberry [26]. The effect of hibiscus extract treatment on fresh and dry weight (g/plant.) may be due to its positively affected on enzyme activity and metabolism, as it contains; ascorbic acid, mineral elements and anthocyanin. Table 6 indicated that effect of ascorbic acid, folic acid as well as hibiscus extract on essential oil yield (L/fed.) from fresh and dry herbs of Geranium during 2015/2016-2016/2017 seasons. Different treatments increased the essential oil yield (L/fed.) in first and second seasons than control treatment. For essential oil yield (L/fed.) from fresh herb, the higher value from spray ascorbic acid at 200 ppm and hibiscus extract at 5% and 10% in two seasons. And also ascorbic acid at 200ppm, folic acid at 100 ppm and hibiscus extract at 5% and 10% gave increased essential oil yield (L/fed.) from dry herb in in both seasons. Regarding the effect of first and second cuts date, results recorded in second cut of the first season gave significantly higher essential oil yield (L/fed.) from fresh and dry herbs than first cut. On the other hand in the second season no significant differences were observed between first and second cut of essential oil yield (L/fed.) from dry herb. But essential oil yield (L/fed.) from fresh herb showed that significant differences between first and second cut in two seasons.

Essential Oil Yield (L/fed.) from Fresh and Dry Herbs
Evaluating the interaction effect between treatments x tested cuts, data show that the interactions registered the lowest values of essential oil yield (L/fed.) from fresh and dry herbs with 1 st Cut in both seasons. The results obtained showed that foliar application of Geranium plants with ascorbic acid, folic acid, hibiscus extract; a great increase volatile oil yield (L/fed.) from fresh and dry herbs. These results are in agreement with those obtained by Taraf et al., reported that foliar application of ascorbic acid caused pronounced increased in the yield of essential oil. Ascorbic acid treatment significantly increased oil percentage during the 1st season where ascorbic acid at 150 mg / l gave the maximum mean values of volatile oil% in the 1st and 2nd cuts on Thyme plant [18]. Pronounced increases in the growth characters of Ammi visnaga L. plants were obtained as a result of ascorbic acid treatments [41]. This synergistic effect of ascorbic acid might be due to its involvement in the main metabolic processes, especially with energy coenzymes [42]. Foliar of strawberry plants with both concentrations of folic acid, significantly improved agronomic characteristics of the treated plants including yield, primary and secondary weight of fruits and number of their achenes as compared to the control treatment [26]. The effect of hibiscus extract treatment on yield of essential oil may be due to its positively affected on enzyme activity and metabolism, as it contains; ascorbic acid, mineral elements and anthocyanin.

Effect of Ascorbic Acid, Folic Acid, Hibiscus Extract and Natural Drying (NA) on Oil Yield Production (L/fed.)
From data in (Table 7) and ( Figure 3) show that NA (natural drying) increased oil loss from dry herb (L/fed.) compared to fresh herb during 2015/2016-2016/2017 seasons. On the other hand the oil loss by NA 5.00-6.00 (L/fed.) and weight loss of herb to 70% from fresh herb Table  5 reducing transport and distillation costs percentage 70%. The Geranium product should compare oil lost (5-6) L/fed. to reducing costs (distillation and transportation). The herb may be left to wilt in the field for 72 hours (3 days) to allow for better vaporization of moisture and greater packing of biomass in the distillation vessel [43]. Extending the drying time over 3-4 days caused reduction in oil yield, which may be attributed to volatile oil loses caused by wind, temperature and plant part loses [44]. Oil yield (L/fed.)

Effect of Natural Drying on Active Compounds
Data were presented in (Table 8) and (Figure 4): effect of NA (natural drying) on active compounds of Geranium during 2016/2017 season. The percentages of components showed that, the Citronellol represented the main compound, followed by Geraniol, Eugenol, Geranyl formate, Citronelly formate, Iso-menthone, B-caryophyllene, Linalool and, α-pinene ordered from high to low. Moreover observed that natural drying caused pronounced increasing in the main constituents (Citronellol, Eugenol, Geranyl formate, Citronelly formate and Iso-menthone) on the other beside caused pronounced decreasing of Geraniol, B-caryophyllene, Linalool and, α-pinene.
Drying herbal yield of geranium up to 3 to 4 days does not affect the physicochemeical traits of the volatile oil, rather increases slightly the yield and quality, reduces the staking volume and thus the transportation and distillation costs; consequently encourage geranium plantations to extend in new areas far from distillation factories [44]. Active compounds%

Effect of Ascorbic Acid, Folic Acid and Hibiscus Extract on Active Compounds
Data concerning the effect of ascorbic acid, folic acid and hibiscus extract on active compounds of Geranium essential oil during 2 nd Cut, 2016/2017 season are reported in (Table  9). Results showed that active compounds were 9 compounds representing about 88.11% -97.09% as a result of different treatments during 2016-2017 season. Citronerlol was identified as the major compound in the different treatments ranging from 31.55% to 40.99%. Geraniol the second main component, ranged 19.29% -27.11% in the essential oil followed with Eugenol which was identified as the third compounds in the volatile oil and its relative percentage accounted for 8.30% -10.18%. And results indicated that all treatments caused increase main constituents; Citronerlol, Citronelly formate, Iso-menthone, B-caryophyllene and Geranyl formate. On the other beside all treatments caused a reduction in Geraniol and α-pinene. The effect of ascorbic acid on volatile oil constituents may be due to its effect on enzyme activity and metabolism of essential oil production [18]. Folic acid (vitamin B9) has become the most prominent of Bcomplex vitamins despite its essential biochemical function in amino acids metabolism and nucleic acids synthesis [23]. The effect of hibiscus extract treatment on essential oil and its constituents may be due to its positively affected on enzyme activity and metabolism of essential oil production, as it contains; Ascorbic acid, mineral elements and anthocyanin.

Conclusion
In the present study, it could be concluded that using ascorbic acid at 200 ppm, folic acid at 100 ppm and extracthibiscus 5 and 10% were better treatments in increasing vegetative growth, higher volatile oil content and active compounds than other treatments. Effect of natural drying; it increased oil loss from dry herb (L/fed.) Compared to fresh herb. On the other hand the oil loss by natural drying/ fed. 5.00-6.00 (L/fed.) and weight loss of herb to 70% from fresh herb reducing transport and distillation costs by 70%. The Geranium product should compare the lost oil (5-6, L/fed.) to reducing costs (distillation and transportation).