Callus Production and Analysis of Isoflavonoids and Some Terpenes from Bituminaria bituminosa

Bituminaria bituminosa is traditionally used for feeding goats in the Canary Islands (Spain) (Davis 1970; Stirton 1981; Sternberg et al., 2006). It tolerates drought conditions and is known for its heavy metal-hytostabilization capacity in contaminated or degraded soils (Walker et al., 2014; Tava et al., 2007). Thus, the presence of toxic compounds in this plant can limit its use and must be considered. In Madeira Island, it is commonly used in folk medicine as a decoction with alcohol and iodine and applied externally for hair restoration. The infusion from fresh leaves is also used to treat fever and urinary infections (Llorent-Martínezet_al2015).

B.bituminosa have nearly 150 secondary metabolite. These bioactive compounds were flavonoids, coumarins,  furanocoumarins, chalcones, quinines, terpenoids. These species exhibit significant anti-oxidant, anti-bacterial, anti-fungal, anti-viral, anti-helmintic, anti-diabetic, diuretic, hepatoprotective, anti-cancer and anti-tumor activities (Bouque et al., 1998; Tava et al., 2007; Azzouzi et al., 2014; Llorent-Martínezet_al2015; Koul et al., 2019; Tram et al., 2021; Florvil et al., 2022). Angelicin and psoralen protect against infection and herbivory and phototoxic subtance in B.bituminosa  (Forestoet_al2021). The effects on angelicin and psoralen accumulation of the exposure of two populations of B. bituminosa to abiotic stress (cold, heat and drought) under field conditions were determined in the leaf and fruits (Walker et al., 2010; Walker et al., 2012).

The clastogenic activity of Mitomycin C (MMC) was significantly reduced by both erybraedin C and bitucarpin A. They were obtained from B.bituminosa and chemically characterised, they were used either a pre-incubation schedule or simultaneous treatment. These results suggest that the protective effects displayed by the two anti-clastogenic compounds against MMC could be due to the induction or inhibition of cellular reductive metabolic enzymes (Maurich et al., 2004; Maurich et al., 2006). Juan et al., (2003, 2004) has confirmed the self-pollinated breeding system of B. bituminosa. The pterocarpans bitucarpin A and B can be isolated from the aerial parts of B. bituminosa (Pistelli 2003). It has several potential uses such as; forage crop, phytostabilisation of heavy metal contaminated or degraded soils, synthesis of furanocoumarins (psoralen, angelicin, xanthotoxin and bergapten), compounds of broad pharmaceutical interest. Angelicin and psoralenused in sun-tan products and for photochemotherapy of vitiligo and psoriasis (Tava et al., 2007). The furanocoumarins psoralen and angelicin which act as repellents and feding deterrents and also are commercial interest due to the iruse in cosmetics and photochemotherapy. Terpens have similar properties, too (Atar and Çölgeçen 2013; Çölgeçenet_al2018).

Aucubin, catalpol and verbascoside a glycoside iridoid, is a class of monoterpenoids and the most common iridoid glycoside is aucubin. It has been revealed through different studies that aucubin could be a useful agent in the prevention of pancreatic cancer. In our literature search, aucubin, catalpol and verbascoside have never been studied before in B.bituminosa. The aim of the present study was to investigate and analysis the production of callus and secondary metabolites from B. bituminosa herba and in vitro culture.

Plant material

The seed is covered by the fruit (fruits beaked). Fruits of B. bituminosa were collected from Zonguldak Bulent Ecevit University, Science and Art Faculty, Department of Biology experiment garden in August 2020. Fruits were sterilizated with 10% sodium hypochlorite solution for 20 minutes. Then fruits were rinsed three times with distile water. Beaked fruits and cut-beaked fruits were germinated in glass jars (100 mm × 200 mm) on hormone-free MS medium (Murashige and Skoog 1962) (Fig 1). Samples were incubated at 22-24°C with a 16/8 hour photoperiod.  



In vitro callus culture

In vitro aseptically 15-day-old seedling were excised into hypocotyl (0.5-1 cm), cotyledon (0.5-1 cm), apical meristem (1-2 mm), epicotyl (0.5-1 cm) and young primary leaves (0.5-1 cm). The explants were cultured in Petri dishes (100×200×20 mm) (Fig 1). MS media containing 3% sucrose and 0.8% agar was used for in vitro germination. Different concentrations and combination of 2,4-dichlorophenoxyacetic acid (2,4-D), benzylaminopurine (BAP) and Kinetin were used for callus induction (Parameswari and Srimathi 2008); 2,4-D 0.75 mg/L + BAP 1.5 mg/L, 2,4-D 1 mg/L+ BAP 2.5 mg/L, 2,4-D 1.25 mg/L+ BAP 3.5 mg/L, 2,4-D 0.5 mg/L+Kinetin 2 mg/L, 2,4-D 0.75 mg/L+Kinetin 2.5 mg/L, 2,4-D 1.25 mg/L+Kinetin 3 mg/L. All media were adjusted pH 5.80 before autoclaving. Samples were incubated at 22-24°C in dark under a 16 h light /8 h dark photoperiod. Due to the darkening of calli after the third week, the shooted calli were subcultured onto the same media. Callus growth index (GI) was calculated by using the following formula:
 

  (Sánchez-Sampedro et al., 2005).

Callus obtaining from all B. bituminosa explants were used for analysis of isoflavanoids and terpens.
 
Chemicals and equipment

Chemicals and solvents were purchased from Sigma or Merck and were of the highest analytical grade. For isoflavonoid analysis, schimic acid, gallic acid, caffeic acid, p-coumaric acid, taxifolin, rosmarinic acid, kaempferol, genistein, quercetin, biochanin A, formononetin, daidzein standards (>98% purity) were purchased from Sigma or Merck. For terpen analysis catalpol (50839, Sigma), aucubin (5561, Sigma) and verbascoside (V4015, Sigma) were obtained as standard compounds. Chromatographic grade-double ultrapure water; gradient grade for liquid chromatography methanol (Merck-1.06007) were used.

Thermo Scientific Dionex Ultimate 3000 variable Wavelength Detector, WPS-3000SL Analytical system was used for all HPLC experiments. 
 
Phytochemical analysis of isoflavonoids
 
Phenolic extraction was done according to the modified procedure of Kiselev 2007. The freeze-dried and powdered 100 mg of callus culture sample were shaken twice in 25 ml of 80% MeOH at 180 rpm on a rotary shaker for 30 min and 24 hours. And all materials were filtered and 80% aqueous MeOH were evaporated under vacuum in a rotary evaporator at 45°C to dryness (Buchi Rotavapor). The dry residue was dissolved in 2 mL of pure methanol. The sample solution was membrane filtered (0.45 μm) and 20 μL aliquots were used for HPLC analysis.

The instrument used in the present study were Shimadzu 1200 series. Liquid chromatographic system equipped with UV-Vis detector and analysed by using Shimadzu CLASS-VP software. Chromatographic separation was performed on a C18 (5 μm 4.6 mm × 250 mm) column. Working standard solutions, 2,5-250 µg/ml, were prepared by diluting the stock standard solutions with methanol/water (30:70, v/v). A good linear correlation was obtained between the gradient retention time values. Retention times of standard in the examined plant extracts are presented in Table 1.



The analysis of gallic acid, caffeic acid, p-coumaric acid, taxifolin, rosmarinic acid, kaempferol, genistein, quercetin and biochanin A were resolved by using a mobile phase  (aseto nitrile) and B (ultrapure water+1% formic acid) at a flow rate of 0.9 ml/min for gradient elution program shown in Table 2. The detection UV wavelength was set at 254 nm. The analysis of were resolved by using a mobile phase [A (MeOH) and B (ultrapure water)] at a formononetin and daidzein flow rate of 0.5 ml/min for the gradient elution program shown in Table 3. The detection UV wavelength was set at 250 nm. The analysis of shikimic acid were resolved by using a mobile phase [A (MeOH) and B (ultrapure water)] at a flow rate of 0.5 ml/min for the gradient elution program shown in Table 4. The detection UV wavelength was set at 230 nm.






 
Phytochemical analysis of terpenes

B. bituminaria dried calli were weighed of one hundred milligrams and extracted with 10 mL of methanol (MeOH) on a shaker for 48 h (Alipieva et al., 2007). The extract was filtred and then evaporated with a rotary evaporator at 40°C (Crişan et al., 2010). The sample was dissolved by adding 10 ml of methanol. Finally, all samples were filtrated by 0.45 μm filter. HPLC analyses were performed by using a sample injection volume of 20 μL (Alipieva et al., 2007; Crişan et al., 2010). 

In the HPLC system (Thermo Scientific Dionex UltiMate 3000), a Thermo Scientific-Acclaim TM 120-C18, 3 μM, 4.6 ×150 mM  Dionex Bonded Silica Products column was used for catalpol, aucubin and verbascoside. The sample injection volume was 20 μL. For data acquisition, the flow rate was adjusted to 0.5 mL min^-1 for catalpol and aucubin and 0.8 mL min^-1 for verbascoside (Table 5-6).

A gradient elution of A (methanol) and B (ultrapure water) was used as in the following Table 5 and Table 6. On-line UV spectra were recorded with a diode array detector as 200 nm for catalpol and aucubin and 330 nm for verbascoside (Wang et al., 2010; Xie et al., 2012). Standard solutions: catalpol 200, 100, 50, 25, 15, 10 and 5 ppm; aucubin 10, 5, 2.5, 1 and 0.5 ppm; verbascoside 15, 10, 5 and 2.5 ppm concentrations were prepared with methanol. Primary testing showed that these compounds corresponded to peaks with retention times of 2.307 for catalpol, 3.420 for aucubin and 27.04 for verbascoside.




 
Data analysis

The data were analysed using a one-way analysis of variance (One-way ANOVA) and the means were compared using the Duncan’s multiple range test at 5% level of significance (p<0.05). All obtained data are average of the three measurements.

The seed is covered by the fruit (fruits beaked). About 70% of seeds are hard and do not imbibe. No germination was observed in beaked fruits and cut beaked fruit. A small scalpel cut that can reach the seed coat improved imbibition and a germination rate of 85% was obtained (Fig 1). In some studies B. bituminosa seeds were scarified with pure H₂SO₄ for remove external coats. In our studies seeds were cut by scalpel because B. bituminosa seeds can taken with hands. Germinations were conducted in hormone-free MS medium different from others (D'Angiolilloet_al2014).

In our study, 15-day-old in vitro seedlings are the most suitable explant sources. B. bituminosa aseptic seedlings used different ages as 25-day-old explant sources. However, the studies display variations in the ages of the seedlings. The apical meristem, epicotyls, young primary leaves, cotyledon, hypocotyl were used in experiments for B. bituminosa. An apical tip and hypocotyl explants have been used commonly in almost other the studies and our study (D'Angiolilloet_al2014). Young seedlings are prefered generally due to their totipotency properties.

The first callus formation started on the first leaf in 2 days. Callus growth was observed within 3-5 days on of the hypocotyl, cotyledon, apical meristem, epicotyl andyoung primary leaves. Callus is friable and yellow. The highest callus production was in 0.75 mg/L 2,4-D+2.5 mg/L kinetin medium (Table 7). 2,4-D and kinetin hormones were used at a ratio of 1:3. It was observed that the media using 2,4-D and BAP were not very successful in terms of callus production. In one study, unlike our data, the auxin ratio was 4 times the cytokinin ratio. They were able to produce callus slowly with NAA (mature leaves) and fast and well with 2,4-D (young leaves) (in young and mature leaves). They obtained 4-week growth curves similar to our study. However, the production of green and compact callus obtained after 3 weeks by exposure to light is different from our study (D'Angiolilloet_al2014). In vitro regeneration protocol was developed from hypocotyl-derived callus of Psoralea corylifolia L. green compact nodular calli were induced from 3 day-old hypocotyl explants on Phillips and Collins (L2) medium containing 25 g/L sucrose, 7 g/L agar and supplemented with 10 mM NAA and 2 mM TDZ. Higher shoot regeneration (89.5 %) was achieved in enriched L2 medium supplemented with 2 mM BA, 4 m M TDZ and 50 mg/L BVN (Baskarana and Jayabalan 2009). In our study, the calli produced by subculture in 3-4 weeks were yellow and friable. To assess the production of isoflavonoids, the best callus producing different concentration of 2,4-D+Kinetin media and 1.25 mg/L 2,4-D+3.5 mg/L BAP medium were selected for analysis.



HPLC analysis

In some studies, B. bituminosa extracts of in vitro shoots and in wild shoots contained the flavonoid daidzein, while plicatin B, erybraedin C and bitucarpin A were found. The furanocoumarins angelicin and psoralen were found in in vivo and in vitro plants, but in the callus were not detectable. The highest level of cytotoxicity in the extracts was found in in vitro shoot extracts in HeLa cell culture (D'Angiolilloet_al2014). The aeral part and in vitro products of B. bituminosa were analysed for different phenol and terpene group secondary metabolites. In general, it is seen that furanocoumarins, pterocarpans and additionally flavonoids have been detected in studies. The main furanocoumarins studied are angelicin and psoralen (Pistelli et al., 2003; D'Angiolilloet_al2014).

Considering the analyses of flavonoids that can be produced in vitro, it is seen that 0.75 mg/L 2,4-D+2.5 mg/L kinetin medium is more successful in the production ofses of flavonoids that can be produced in vitro, it is seen that 0.75 mg/L 2,4-D+2.5 mg/L kinetin medium is more successful in producing these phenols. The very high production of Schimic acid must be due to the fact that it is one of the first synthesised products in the phenyl propanoid pathway. Using kinetin as three times of 2,4-D produced positive results in in vitro isoflavonoid production. The use of auxin/cytokinin ratio of 1/3 provided efficient production from this plant in vitro. Extracts from callus cultures showed especially the presence of daidzein (2.88 mg/g FW), even though its glucoside, daidzein, was found only in young leaf (D'Angiolilloet_al2014). Our results detected daidzein in a similar quantity with previous studies on in vitro cultures in Bituminaria sp. Bouque et al., (1998) found more lower amount daidzein.

In terpene production, it is seen that 1.25 mg/L 2,4-D+3.5 mg/L BAP, which is the best callus producing medium containing BAP, is the most successful medium among all tested mediums (Table 8). In the literature review, no terpene measurement has been made in B. bituminosa before and no data has been found. Therefore, our study is the first in this regard. In some family, terpenoid analysis similar results have Çölgeçenet_al(2018) catalpol, aucubin and verbascoside content were analysed in Globularia trichosantha subsp. trichosantha callus.

The limited consideration of the peculiar ecological role and of the biological accumulation of flavonoids and terpenoids in herba surfaces is often overlooked in the development of extraction and analytical methods, thus leading to an underestimation of their presence in natural products, resulting in a difficult evaluation of their risk assessment. In conclusion, a flavonoid and terpenoid analysis method was developed for B. bituminosa. With this study, we done first time terpenoid analysis for B. bituminosa. In particular, the highest amount terpen in the callus is catalpol. This result revealed that B. bituminosa may be an essential plant for catalpol production.