Research Journal of Applied Biotechnology (RJAB) BIOSYNTHETICAL CAPACITY OF KAEMPFEROL FROM IN VITRO PRODUCED ARGEL (SOLENOSTEMMA 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 plants 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 plant 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 Special volume for the first International Conference of Genetic Engineering and Biotechnology, Sharm el Shiekh, Egypt. 26-29 April, 2016. 134 Research Journal of Applied Biotechnology (RJAB) (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 Special volume for the first International Conference of Genetic Engineering and Biotechnology, Sharm el Shiekh, Egypt. 26-29 April, 2016. 135 Research Journal of Applied Biotechnology (RJAB) 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