Research Article

Identification of Secondary Metabolites from Callus Orthosiphon aristatus (Blume) Miq by Thin Layer Chromatography

Fahrauk Faramayuda1,2*, Totik Sri Mariani3, Elfahmi1,4 and Sukrasno1

1School of Pharmacy, Institut Teknologi Bandung (ITB), Bandung, West Java, Indonesia, 40132; 2Faculty of Pharmacy, Universitas Jenderal Achmad Yani (UNJANI), Cimahi, West Java, Indonesia, 40532; 3School of Life Sciences and Technology, Institut Teknologi Bandung (ITB), Bandung, West Java, Indonesia, 40132; 4Biosciences and Biotechnology Research Center, Institut Teknologi Bandung (ITB), Bandung, West Java, Indonesia, 40132.

Received | February 04, 2021; Accepted | April 03, 2021; Published | August 03, 2021

*Correspondence | Fahrauk Faramayuda, School of Pharmacy, Bandung Institute of Technology (ITB), Bandung, West Java, Indonesia; Email: ramayuda.f@gmail.com

Citation | Faramayuda, F., T.S. Mariani, Elfahmi and Sukrasno. 2021. Identification of secondary metabolites from callus Orthosiphon aristatus (Blume) Miq by thin layer chromatography. Sarhad Journal of Agriculture, 37(3): 1081-1088.

DOI | https://dx.doi.org/10.17582/journal.sja/2021/37.3.1081.1088

Keywords | In-vitro culture, White-purple, Purple variety O. aristatus, Growth media, Phytochemistry of callus

Introduction

The potential of the O. aristatus plant as a medicinal plant has been widely studied, including as diuretics (Arafat et al., 2008), treatment of gastric disorders (Yuniarto et al., 2017), antidiabetic (Mohamed et al., 2011), antihypertensive (Matsubara et al., 1999), hepatoprotective (Yam et al., 2007; Maheswari et al., 2008), antimicrobial (Ho et al., 2010; Hossain et al., 2008), antioxidants (Alshawsh et al., 2012; Akowuah et al., 2004), anti-epilepsy (Kar et al., 2018), memory enhancer (George et al., 2015), treatment of cardiovascular disorders (Abraika et al., 2012), rheumatoid treatment and osteoarthritis (Adawiyah et al., 2018), anticancer (Pauzi et al., 2018; Halim et al., 2017), antiviral (Ripim et al., 2018; Abdelwahed et al., 2020; Li et al., 2020; Sarkar and Das, 2020; Wondmkun and Mohammed, 2020; Lin et al., 2019; Hsieh et al., 2020; Faramayuda et al., 2021b) and immunomodulatory (Harun et al., 2015; Woottisin et al., 2011; Friedman, 2015; Kim et al., 2008; Takano et al., 2004; Sanbongi et al., 2004; Youn et al., 2003). The pharmacological activity of O. aristatus is inseparable from its main compounds, namely rosmarinic acid, sinensetin, and eupatorin (Guo et al., 2019). Not only these three compounds, but O. aristatus also contains rhamnazin (Malterud et al., 1989), danshensu (Nuengchamnong et al., 2011) and Orthosiphol D (Takeda et al., 1993; Awale et al., 2002).

According to Febjislami et al. (2019), three varieties of O. aristatus grow in Indonesia, namely purple, white and white purple. The levels of sinensetin are more significant in purple varieties’ than in white varieties (Lee, 2004; Anggraeni, 1992). Recently, the white-purple and purple O. aristatus decreased, especially in Indonesia (Febjislami et al., 2019). Therefore it is necessary to produce active compounds without directly processing them from the original plant and efforts to micropropagate O. aristatus using plant tissue culture technique. Several studies on tissue culture of O. aristatus have previously been reported, including induction of the callus from O. aristatus, which can grow on MS medium with 0.4 ppm 2,4-D;1 ppm NAA + 1 ppm 2,4-D; 2 ppm 2, 4-D; 1.0 ppm Kinetin + IAA 1.0 ppm (Faramayuda et al., 2020; Wai and Lai, 2004; Bordbar et al., 2015; Sheena and Jothi, 2015; Ali et al., 2017; Faramayuda et al., 2021a). However, these studies did not report identification of active compounds in the callus of the O. aristatus, which were derived using Gamborg, Schenk, and Hildebrandt (SH) and N6 media. The findings of this research are expected to provide new knowledge on the secondary metabolites content of callus derived from two varieties of O. aristatus.

Materials and Methods

Chemicals and reagents

Rosmarinic acid, sinensetin, eupatorin, silica gel pre-coated plate 60 F254 were purchased from Sigma. Acetone, ethanol, ethyl acetate, toluene, formic acid were purchased from Loba Chemie (Mumbai, India).

Collection of plants

White-purple and purple O. aristatus leaves and stems (wild type) were obtained from the Manoko experimental garden in West Bandung, Indonesia. The plants were taxonomically identified with letter number 6115/I1.CO2.2/PL/2019. Callus of two varieties of O. aristatus was obtained from the Laboratory of Plant Tissue Culture, Center for Research and Innovation, ITB.

Callus and plant (wild type) extraction

The callus was removed from the medium and dried in a 70°C oven (Memmert, Germany). The dry callus was then crushed. Callus powder was extracted separately by maceration using n-hexane, acetone, ethyl acetate, and ethanol as solvent. Crude extract from of 1 g of O. aristatus (wild type) was extracted with acetone, ethyl acetate, and ethanol (the amount of each solvent was 15 mL).

Identification of secondary metabolites

On silica gel GF 254 plates, thick extracts from two varieties of O. aristatus were applied. The mobile phase used was ethyl acetate-toluene-formic acid and water (3:3:1:0.2) (Craciun et al., 2014). The plate was then inserted for the development. TLC profile analysis was performed under UV light 366 nm.

Data analysis

For each compound, the Rf value was calculated by dividing the distance traveled by the component with the distance traveled by the eluent (mobile phase).

Results and Discussion

Morphological identification and characterization

The results of the identification of two plant varieties of O. aristatus explained that two plant samples were identified as white purple and purple varieties of O. aristatus. The most striking difference among the two varieties is the flowers morphology (Keng and Siong, 2006; Almatar et al., 2013). The purple variety has a purple crown color, while the white-purple variety is white-purple. These results were in line with the studies reported (Febjislami et al., 2019; Batubara et al., 2020; Faramayuda et al., 2020).

Analysis of secondary metabolite content in callus of two varieties of O. aristatus

The callus of two varieties of O. aristatus came from MS, Gamborg, SH, and N6 media with growth regulators 2.4 D 0.4 ppm. Callus derived from purple varieties of O. aristatus on all media had a soft texture and greenish-white. The callus was white-brown using white-purple variety, while callus induced on the N6 and SH media were white-green (Figures 1, 2).

Regeneration of O. aristatus has been carried out using nodal segment, where some shoots (6.1 shoots per explant) were observed from explants nodal stems on MS medium with added plant growth regulators BAP 6.7 μM and four weeks culture period (Lee and Chan, 2004).

 

 

When leaf explants were cultured in MS supplemented with 2,4-D 1.0 ppm and NAA 1.0 ppm as growth regulators, callus development was observed. Callus cells in the MS liquid medium supplemented with 2,4-D 1.0 ppm, proved to be the best conditions for O. aristatus cell culture suspension (Lee and Chan, 2004). Research conducted by Faramayuda et al. (2020) reported that callus of two varieties of O. aristatus grown on MS + 2.4 D 0.4 ppm resulted in optimal growth. This research is a continuation of Faramayuda’s research reported in 2020, where callus induction of white purple, and purple varieties of O. aristatus was carried out on media other than MS and secondary metabolites compounds were identified. This research contributes to understanding of the phytochemical content of the callus derived from two varieties of O. aristatus using in Gamborg, SH, and N6 media.

The concentration for each standard compound and the test sample are 100 mg/L and 1500 mg/L, respectively. The volume of bottling on TLC for standard and sample is 10 µL. In the ethanol, ethyl acetate, and callus acetone extracts of purple varieties derived from MS, Gamborg, N6, and SH media, the presence of sinensetin compounds was detected at Rf 0.5 (Figure 3). Sinensetin fluorescence was brighter in callus growing on N6 media (NU1; NU2; NU3) (Figure 3). Rosmarinic acid was detected in MU2, NU1, NU3, SU1, SU2, and SU3. Eupatorin compounds were detected in ethanol and ethyl acetate extracts of white-purple variety (wild type) O. aristatus under the observation of a 254 nm UV lamp. Eupatorin was not detected in all callus extracts (Figure 4).

 

Observation of TLC profiles wild-type leaf and O. aristatus extract and callus of purple variety showed that sinensetin was detected in ethanol, ethyl acetate, and acetone extracts of O. aristatus leaves and stems with bright blue fluorescence. Rosmarinic acid in purple variety callus looks clear and brighter than wild type (Figure 5). There were some spots in the callus of purple variety but not in wild types (Figure 5). Sinensetin was detected in all extracts on leaves and stems of O. aristatus, but the bands for stems were fainter than the leaves. Sinensetin in white-purple variety callus from SH medium appeared more dim than the wild type, but for rosmarinic acid, the fluorescence appeared brighter than the wild type (Figure 6).

 

Several hypotheses clarify how secondary metabolites in callus can be detected. Callus is totipotent, which means that each cell has the same complete genetic information and is capable of producing the same secondary metabolites (Efferth, 2019). Plant tissue culture can affect plant cells biosynthetic pathways to produce secondary metabolites derivatives (Sarfaraj et al., 2012). Several studies have reported that there are active compounds in callus, including resveratrol (Nandagopal et al., 2017), shikonin (Hao et al., 2014), hypericin (Walker et al., 2002), and justicidin b (Ionkova et al., 2013).

In research conducted by Faramayuda et al. (2020), the content of sinensetin and rosmarinic acid was observed in the acetone extract of callus from purple and white-purple varieties of O. aristatus using MS medium supplemented with 2, 4-D 0.4 ppm. From the comparison of TLC profiles between wild type and callus from two varieties of O. aristatus, there are several compound spots in the callus that can be further identified and characterised.

 

 

Conclusions and Recommendations

The callus derived from two varieties of O. aristatus contain rosmarinic acid and sinensetin. In the callus originating from N6 and SH media, rosmarinic acid spots were visible with blue fluorescence.

Acknowledgments

The Ministry of Science and Technology / National Agency for Research and Innovation sponsored this research through the “Doctoral Dissertation Research” contract number 2 / E1 / KP.PTNBH / 2020.

Novelty Statement

This study provides new information regarding the secondary metabolite content of callus from two varieties of O. aristatus grown on Gamborg, N6, and SH media. So far, research on callus induction from O. aristatus was limited to MS media.

Author’s Contribution

Fahrauk Faramayuda performed the experiment and wrote the manuscript with assistance and supervision from Prof. Sukrasno, Dr. Elfahmi, and Dr. Totik Sri Mariani.

Conflict of interest

The authors have declared no conflict of interest.

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