Research Journal of Applied Biotechnology (RJAB)

BIOSYNTHETICAL CAPACITY OF KAEMPFEROL FROM IN VITRO PRODUCED ARGEL ( ARGEL (DEL.) HAYNE) CALLUS

1Mohamed R. Abd Alhady, 1Ghada A. Hegazi, 1Reda E. Abo El-Fadl , 2#Samar Y. Desoukey

1Tissue Culture Unit, Genetic Resources Department, Ecology and Dry Land Agriculture Division, Desert Research Center, 11753 El-Matariya, 1 Mathaf El-Matariya St., Cairo, Egypt 2Pharmacognosy Department, Faculty of Pharmacy, Minia University, 61519 Minia, Egypt

ABSTRACT Solenostemma argel (Del.) Hayne (family Asclepiadaceae) is an Egyptian natural perennial shrub. The study attempted to establish callus from S. argel and to investigate its biosynthetic potentiality to produce the flavonoid kaempferol, which has a wide range of pharmacological activities. Seeds and aerial parts were collected from naturally grown at Saint Catherine, Sinai. Callus from different explants of in vitro germinated seedlings (stem, leaf and root) was successfully initiated on Murashige and Skoog (MS) medium supplemented with each of 2,4-dichlorophenoxyacetic acid (2,4-D) and β- naphthalene acetic acid (NAA), separately, at concentrations of 1.0, 2.0 and 3.0 mg/L, in addition to 0.5 mg/L kinetin (KIN). However, the fresh weights of leaf-derived callus were the highest. Casein hydrolysate (CH) and yeast extract (YE), as elicitors, at concentrations of 0.5, 1.0 and 2.0 g/L, were examined for elevating the in vitro production of kaempferol in callus cultures. The total methanol extracts of the aerial parts of wild plants, four-week-old in vitro germinated seedlings’ explants and their derived callus were analyzed qualitatively and quantitatively for kaempferol, using HPLC. A significant difference in the content of kaempferol was observed. Callus produced from tested explants showed a capacity for production and accumulation of kaempferol. The highest concentration of kaempferol was in callus obtained from seedlings’ stem explants on MS medium containing 1.0 mg/L 2,4-D and 0.5 mg/L KIN, in addition to 2.0 mg/L YE. In conclusion, S. argel callus cultures showed a capacity for synthesizing kaempferol and could be an alternative renewable natural source for its large-scale production. Keywords: Asclepiadaceae, in vitro, flavonoid, elicitor, casein hydrolysate, yeast extract

INTRODUCTION bronchitis, for gastrointestinal cramps, Medicinal plants are the most exclusive stomach ache, colic, cold and urinary tract natural source of life saving drugs for the infections (Hegazi et al., 1994). In addition, majority of the world’s population. Bioactive this is used in herbal mixtures for the compounds currently extracted from plants treatment of viral hepatitis B and C, as an are used as pharmaceuticals, food additives, immunostimulant and for pigments, dyes, insecticides, cosmetics, hypercholesterolemia (Shawkat, 1997). Hegazi perfumes and fine chemicals, but et al. (2006) investigated the hepatotoxic and unfortunately many are directed to extinction nephrotoxic effect of S. argel. The plant also and should be conserved. showed significant anti-cancer and anti- Solenostemma argel (Del.) Hayne (family oxidant activities (Nassr-Allah et al., 2009 and Asclepiadaceae) is an Egyptian species, known Hanafi and Mansour, 2011) as “Argal” or “Harghel” (El-Hadidi and Fayed, Phytochemical investigations on S. argel have 1994), commonly growing in the Eastern led to the isolation of compounds belonging Desert and along the Nile in South Egypt to several classes of secondary metabolites, (Täckholm, 1974). It is a wild perennial shrub, such as flavonoid glycosides (El-Askary, 2003 with white flowers in auxiliary umbles, fruits and Heneidak et al., 2006), monoterpenes ovate green-purple. S. argel leaves are used in (Kamel et al., 2000 and El-Askary, 2003), Egyptian folk medicine as purgative, acylated phenolic glycosides (Kamel, 2003), antipyretic, expectorant and as a remedy for pregnane and secopregnane glycosides

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(Hamed, 2001; Hassan et al., 2001; El-Askary, al., 2012). Numerous preclinical studies have 2003; Plaza et al., 2003, 2004, 2005a & b and shown that kaempferol and some of its Perrone et al., 2006]. The pharmacological glycosides have a wide range of studies on S. argel evidenced spasmolytic and pharmacological activities, including anaesthetic (El-Tahir et al., 2005), fungitoxic antioxidant, anti-inflammatory, antimicrobial, (Abd El-Hady and Ouf, 1993 and Abd El-Hady anticancer, cardioprotective, neuroprotective, et al., 1994a & b) and antimicrobial activity antidiabetic, anti-osteoporotic, against selected gram-positive and gram- estrogenic/antiestrogenic, anxiolytic, analgesic negative bacteria (Hegazi et al., 1994 and and antiallergic activities (Calderon-Montaño Tharib et al., 1986). Topical anti-inflammatory et al., 2011). Kaempferol consumption is activity of extracts of S. argel leaves were also correlated with a reduced lung cancer reported (Innocenti et al., 2005). Pregnane incidence (Irwin, 2008). It may be also a and secopregnane glycosides isolated from potent prophylactic against NOX-mediated this species have shown interesting neurodegeneration (Jang et al., 2011). antiproliferative activity (Plaza et al., 2005a & In general, plants produce very small b). Fifteen new 14,15-secopregnane quantities of secondary metabolites and their glycosides, namely argelosides A-J, and nine production may only occur under certain new 15-ketopregnane glycosides, namely circumstances. However, in vitro culture stemmosides C-K, have been isolated from the techniques could be the alternatives for the pericarps, hairy seeds and leaves (Perrone et mass production of these metabolites, which al., 2008). is not season dependent. The principle Kaempferol, kaempferol monoglycoside, and advantage of this technology is that it its derivatives, isolated along with other provides a continuous, reliable source of pure flavonoids from a preparation of the S. argel, and natural plant pharmaceuticals on a large were found to have a potent spasmolytic scale, where plant cell cultures give activity (Khalid et al., 1992). Kaempferol metabolites that are easily extracted derivatives inhibited oedema by 30-40%, (Abraham et al., 2011). these values were quite comparable to the In plant tissue culture, accumulation of activity of indomethacin (59% oedema secondary metabolites could be enhanced by reduction). This confirms and justifies the the treatment of various kinds of elicitors, topical anti-inflammatory activity of this which can be biotic and abiotic. Elicitors could species in Egyptian folk medicine (Innocenti et be applied to unorganized plant cell and callus al., 2005). Also, Shafek et al. (2012) cultures for the production of specific investigated the antibacterial and antioxidant compounds. They control the metabolism of activities of two new kaempferol glycosides receptive plant cells. Previous studies showed isolated from S. argel stem extract. that the accumulation of different secondary Kaempferol is a natural flavonoid that has metabolites can be efficiently induced by using been isolated from many plant sources. It is a elicitor (Ekiert and Gomolka, 2000 and Ekiert, yellow crystalline solid with a melting point 2001). Casein hydrolysate (CH) and yeast 267-278 °C. It is a strong antioxidant extract (YE) are well known as complex compound and helps to prevent oxidative mixtures of organic compounds that have been damage of pure cells, lipids and DNA. It used in plant tissue culture due to their ability prevents arteriosclerosis by inhibiting the to induce and promote secondary metabolism oxidation of low density lipoprotein and the (Marsik et al., 2014). formation of platelet in the blood; Callus cultures have been carried out in concomitantly it also inhibits the formation of several plants for the production of flavonoids cancer cell, thus meaning a reduced risk of (Bharati and Bansal, 2014). However, few developing several disorders such as studies were found on the in vitro production cardiovascular diseases and cancer (Konam et of active compounds from S. argel. Amariei et

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al. (1991) tested the biosynthetical capacity of plant growth regulators (Sigma Cell Culture, the active principles of in vitro regenerated S. min. 90%, St. Louis, USA). Naphthalene acetic argel callus and shoots and found that both acid (NAA) or dichlorophenoxy acetic acid the callus and shoots showed a capacity for (2,4-D), at concentrations of 1.0, 2.0 and 3.0 synthesizing some medicinal compounds. mg/L, as auxins, and kinetin (KIN), at a Also, El-Tigani and Ahmed (2009) showed that concentration of 0.5 mg/L, as a cytokinin, in vitro cultured tissues of S. argel are capable were used for callus initiation, in addition to to produce and accumulate alkaloids, MS medium without growth regulators. Callus cardinolides and flavonoids. The present study tissues were transferred to freshly prepared aimed to produce kaempferol through in medium for proliferation. Each treatment vitro callus cultures of S. argel, and to contained at least 10 replicates and was determine its yield in the callus tissue. CH and repeated twice. Percentage of callus initiation, YE as elicitors were examined for the fresh weight of callus (g) and callus color and enhancement of kaempferol production in texture were recorded after eight weeks of callus cultures of S. argel. culture. MATERIALS AND METHODS Elicitation Plant Material and Culture Conditions Two elicitors; CH (OXOID, OXOID Limited, S. argel seeds were collected from mature Hampshire, England) and YE (Sigma Aldrich) at plants grown naturally in Saint Catherine, concentrations of 0.5, 1.0 and 2.0 g/L, were Sinai. Seeds were washed under running tap added to the most favorable callus initiation water for 30 min. Surface sterilization of seeds medium for each type of explants, in order to was carried out in a transfer hood by soaking improve the yield of kaempferol in the callus them in a 50% (v/v) Clorox bleach solution cultures. The control medium was made (2.5% sodium hypochlorite) for 30 min, without elicitors. Growth of the callus tissue (g providing gentle agitation, followed by three fresh weight/jar) and kaempferol content sequential rinses for one min in sterilized (ng/g fresh weight) were measured. Callus distilled water. All seeds were placed directly tissues were oven dried at 35°C for 48 h for on the surface of full-strength Murashige and the quantification of kaempferol. Skoog (MS) (Murashige and Skoog, 1962) Extraction and Determination of Kaempferol medium (Duchefa, Haarlem, the Netherlands) Kaempferol was extracted from dry powdered supplemented with 100 mg/L myo-inositol samples, of callus, tissue of S. argel seedling's and 30 g/L sucrose. The pH of the medium explants (stem, leaf and root) and the aerial was adjusted to 5.7-5.8 before being solidified parts of wild plant. Extraction was carried out with 3.0 g/L phytagel (Duchefa, Haarlem, the with methanol (HPLC grade), till exhaustion. Netherlands). Media were dispensed in large The filtered extract was dried under vacuum, jars capped with autoclavable polypropylene and a known weight of each of the dried lids, then autoclaved at a pressure of 1.06 extracts was diluted with methanol (1:1 w/v), kg/cm2 and 121°C for 15 min. The cultures into a round bottom flask, sonicated for five were incubated at approximately 25±2°C with min. Four ml of conc. HCl (HPLC grade) was a 16-h photoperiod under cool white added and refluxed for 2.5 h, cooled to room florescent tubes (F140t9d/38, Toshiba). Four- temp. A known quantity was transferred to a week-old in vitro germinated seedlings of S. volumetric flask and completed to a known argel were used as sources of explants for volume with HPLC H2O, then filtered over a initiation of callus cultures. 0.45 µm syringe. Callus Initiation and Proliferation A standard solution of kaempferol (3,5,7- Leaves, stems and roots were excised from trihydroxy-2-(4-hydroxyphenyl)-4H-1- the seedlings of S. argel and cut into small benzopyran-4-one; Sigma Aldrich) was pieces (0.5-1 cm), then cultured aseptically on prepared by dissolving kaempferol in solid MS medium supplemented with various acetonitrile (2.0 mg in 10 ml), sonicated for

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five min and filtered over a 0.45 µm syringe. both seedlings’ stem and leaf explants, while Standard solutions were prepared in the range seedlings’ root explants responded better in of 50-2000 ng/ml by serial dilution method. the presence of NAA. The results show also Presence and content of kaempferol was that callus on the most favourable medium for determined in the different methanolic each explant had a yellowish green color and a extracts by RP HPLC column (phenomenex, friable nodular desirable appearance. ODS-3,5 micron; 4.6x250) using; mobile phase In general, all explants had the capability to 50% acetonitrile : water, 50 µl injection initiate callus, and this callus successfully volume, wave length of 365 nm, 1.5 ml/min proliferated on the same medium, which is flow rate, 40ºC column temp, running time promising for the production of high yield of 4.5 min and PDA photodiode array detector. tissue and consequently secondary

The retention time (Rt= 3.4 min) and online UV metabolites. The capability to cultivate plant spectra of kaempferol was compared to those callus cells and produce homogeneity of an in of the different samples. vitro cell population, with large availability of Statistical Analysis material, and high rate of cell growth provide Experiments were subjected to the completely a valuable platform for the production of high- randomized design. Variance analysis of data value secondary metabolites and other was carried out using ANOVA for statistical substances of commercial interest analysis. The differences among means for all (Moscatiello et al., 2013). treatments were tested for significance at 5% Effect of Elicitors on Callus Growth and level, by using Duncan´s multiple range test Kaempferol Content (Duncan, 1955), as described by Snedecor and Two elicitors; CH and YE were added, Cochran (1990). Means followed by the same separately, to the culture medium and their letter are not significantly different. influence on the growth of callus cultures of S. argel and kampferol content was studied RESULTS AND DISCUSSION (Table 2). Generally, CH negatively affect Callus Initiation callus biomass and had non-significant effect Hunderd percent of explants of S. argel on kaempferol accumulation, compared to the produced callus on media containing all the control, except the concentration of 0.5 mg/L, growth regulators’ combinations examined in it increased the kaempferol production in the present study. As presented in Table 1, callus derived from seedlings’ root to 276.050 comparing the effect of different treatments ng/g dry weight, but this amount decreased on each explant type, the maximum callus with the increase of CH concentration. fresh weight from seedlings' stem explants Treatment with YE also resulted in a lower was obtained on MS medium containing 1.0 callus biomass, compared to the control, for mg/L 2,4-D and 0.5 mg/L KIN (Figure 1a), both callus derived from seedlings’ stem and while, seedlings' leaf explants initiated callus root, but it gave higher biomass of callus of the highest biomass on MS medium derived from seedlings’ leaf over the control containing 3.0 mg/L 2,4-D and 0.5 mg/L KIN at concentrations of 0.5 and 1.0 mg/L. The (Figure 1b). However, seedlings' root explants results in Table 2 show that there were produced the maximum biomass of callus on significant differences in the content of MS medium containing 2.0 mg/L NAA and 0.5 kaempferol between the induced callus and mg/L KIN (Figure 1c). Comparing between the wild and seedlings’ explants. The highest explants, it was found that the ability of content of kaempferol was in callus derived seedlings' leaf explants to produce and from seedlings’ stem on medium containing proliferate callus was higher than that of the 2.0 mg/L YE (624.4356 ng/g dry weight) with a other tested explants (Figure 1d), followed by significant difference with all the other the seedlings' root tissue. Moreover, 2,4-D treatments, it enhanced kaempferol content was found to stimulate callus formation from to about 13-fold over the control. Followed by

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Table 1. Callus initiation from seedlings’ explants of S. argel on MS medium containing 0.5 mg/L KIN in addition to different concentrations of 2,4-D or NAA (5 explants/jar) after 8 weeks of culture with 100% callus initiation on all tested growth regulators’ combinations.

Seedlings’ stem derived callus Seedlings’ leaf derived callus Seedlings’ root derived callus 2,4-D NAA KIN Fresh weight of Colour of Shape Fresh weight of Colour of Shape Fresh weight of Colour of Shape (mg/L) (mg/L) (mg/L) callus (g/jar) callus callus (g/jar) callus callus (g/jar) callus 0.0 0.0 0.0 ------1.0 0.0 0.5 1.633a Yellowish Friable 1.623b Yellowish Friable 0.847d Whitish Compact white nodular green nodular creamy nodular 2.0 0.0 0.5 0.993b Yellowish Friable 1.443b Yellowish Friable 2.797c Whitish Compact creamy nodular green nodular creamy nodular 3.0 0.0 0.5 1.527ab Creamy Friable 9.03a Yellowish Friable 2.300c Whitish Compact nodular green nodular creamy nodular 0.0 1.0 0.5 1.433ab Whitish Compact 2.723b Green Compact 3.743b Yellowish Compact green nodular nodular green nodular ad a 0.0 2.0 0.5 1.230 Whitish Compact 1.577b Whitish Compact 4.920 Yellowish Friable green nodular creamy nodular green nodular 0.0 3.0 0.5 1.3633ab Green Compact 2.643b Whitish Compact 0.847d Creamy Friable nodular green nodular nodular

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its content in the callus derived from the same complication found in in vitro secondary explants treated with 0.5 g/L YE (307.211 ng/ metabolites production (Maharik et al., 2009). g dry weight). However, the content of Difference in contents of kaempferol between kaempferol was the highest in the wild plants callus cultures and wild and seedlings’ explants (11885.125 ng/g dry weight) than in the could be explained by the influence of seedlings’ explants and in the callus. In general, overlapping growth regulators depending on the highest contents of kaempferol were in the type and concentration of the used auxin callus treated with YE. On the other hand, CH and cytokinin. They could increase or decrease had the lowest effect on kaempferol the amount of accumulated compound, due to accumulation, except for callus derived from the fact that growth regulators increase the seedlings’ root at the concentration of 0.5 mg/L. absorption of minerals and accumulation of It is noticed that there is a reverse correlation carbohydrates and amino acids (Besher et al., between cell growth and kaempferol 2014). production. This represents a general

Table 2. Fresh weight and kaempferol concentration in callus of S. argel after 8 weeks of culture on MS medium supplemented with the best callus induction medium for each explant type in addition to different concentrations of CH and YE as elicitors (5 explants/jar). Explant type CH YE Fresh weight Kaempferol (ng/g (g/L) (g/L) (g/jar) dry wt.) Callus derived from 0.0 0.0 3.593b 48.322ef seedlings’ stem1 0.5 0.0 0.758k 11.561f 1.0 0.0 1.055ij 55.758ef 2.0 0.0 1.010ijk 5.830f 0.0 0.5 1.190hi 307.211b 0.0 1.0 1.705ef 55.416ef 0.0 2.0 1.623efg 624.435a Callus derived from 0.0 0.0 1.880e 45.426ef seedlings’ leaf2 0.5 0.0 1.220hi 47.814ef 1.0 0.0 1.520fg 20.775f 2.0 0.0 1.215hi 47.896ef 0.0 0.5 2.150d 15.065f 0.0 1.0 2.750c 62.354ef 0.0 2.0 1.820e 102.946de Callus derived from 0.0 0.0 4.055a 120.820d seedlings’ root3 0.5 0.0 0.875jk 276.050b 1.0 0.0 2.210d 7.143f 2.0 0.0 1.390gh 57.700ef 0.0 0.5 0.812jk Not detected 0.0 1.0 2.585c 169.060c 0.0 2.0 2.715c 130.245d Seedlings’ stem 8790.520 Seedlings’ leaf 4002.226 Seedlings’ root 2061.622 Wild plant 11885.125

1Callus derived from seedlings’ stem cultured on MS medium+1.0 mg/L 2,4-D+0.5 mg/L kinetin supplemented with elicitors. 2Callus derived from seedlings’ leaf cultured on MS medium+3.0 mg/L 2,4-D+0.5 mg/L kinetin supplemented with elicitors. 3Callus derived from seedlings’ root cultured on MS medium+2.0 mg/L NAA+0.5 mg/L kinetin supplemented with elicitors.

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Although, CH did not enhance kaempferol pathways. Elicitiation is one of the important production in the present study, it is well strategies to get better productivity of known that CH is a source of calcium, several bioactive secondary products and to lower mico-nutrients, vitamins and a mixture of up production costs. The secondary metabolites to 18 amino acids. It was are released due to defense responses, which investigated that CH contains some unknown are triggered and activated by elicitors (Bharati growth promoting factors (Khaleda and Al- and Bansal, 2014). Forkan, 2006). Thus, the response of callus to In conclusion, medicinal plants are the main a particular elicitor may vary between source of secondary metabolites, which are different plants and cell lines, therefore, it industrially important as they constitute becomes crucial to determine the suitable pharmaceutically important drugs. As a result, type and concentration of elicitors for product there is a huge demand for these plants, which optimization. are overexploited from their natural habitat. Currently, YE is commonly employed as a biotic Therefore, plant tissue culture is being elicitor for the induction and enhancement of potentially used as an alternative strategy for secondary metabolites production. Reports production of secondary metabolites. suggest that YE is used as a supplement in Flavonoids are important secondary order to promote plant growth, due to its high metabolites having immense medicinal amino acid content (George et al., 2008). properties ranging from anti-oxidant, anti- However, YE did not affect the fresh biomass of cancerous, anti-inflammatory etc. To enhance callus, the addition of YE into the culture the rapid and controlled, continuous medium increased kaempferol. This indicates production of this class of compounds, in vitro that YE did not perform as nutrient supplement culture strategies application would ensure for the growth of callus. This might be the large scale and stable production throughout result of stress response of the callus to the the year, under controlled environmental accumulation of secondary metabolites due to conditions. The present study is a stepping the YE that acted as elicitor (Abraham et al., stone in in vitro production of the flavonoid 2011). kaempferol from callus cultures of S. argel. Generally, elicitors could be used to enhance Callus showed a potentiality for accumulation plant secondary-metabolite synthesis and could of kaempferol, especially from seedlings’ stem play an important role in biosynthetic explants.

Figure 1. Callus of S. argel. (a) Callus derived from seedlings’ stem cultured on MS medium+1.0 mg/L 2,4-D+0.5 mg/L kinetin. (b) Callus derived from seedlings’ leaf on MS medium+3.0 mg/L 2,4-D+0.5 mg/L kinetin. (c) Callus derived from seedlings’ root on MS medium+2.0 mg/L NAA+0.5 mg/L kinetin. (d) Proliferated callus of S. argel.

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