Rapid mass propagation of endangered valuable medicinal plant Salacia chinensis L. and GC-MS/LC-MS analysis of active compounds produced in callus and leaf extracts

A protocol for indirect organogenesis of Salacia chinensis was established. Normally S. chinensis is propagated through seeds. However, due to the difficulty to obtain uniform plants in a short time period by seed germination, micro-propagation is a possible alternative method. For the micro-propagation, media with different concentration combinations of cytokinins and auxins were used to induce callus formation in three explants types: leaf segment, nodal segment and seeds. The rate of recurrence of callus formation from leaf on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) 0.5mg and TDZ 1mg was 100% and the maximum percentage of shoot let development from nodal segment on MS basal medium supplemented with BAP 6-(benzylamino purine) 3.5+ indole3-butyric acid (IBA) 1 mg/l was 78.3% when compared to other plant growth regulators (PGR) combinations used. The highest shoot regeneration response (85%) and the determined shoots (12.33 ± 0.33%) per callus were attained from leaf explants on MS medium containing 1-napthaleneacetic acid (NAA) 1mg/l + BAP 0.5 mg/l. The seeds showed highest percentage of shoot formation on MS medium supplemented with BAP 2mg + indole 3-acetic acid (IAA) 2mg/l and BAP 2mg + isopentenyl adenine (2iP) 2mg/l. Highest root formation (70±1.3%) was found in shoot regenerated using leaf segment on MS medium supplemented with IBA 0.5mg. The gas chromatography mass spectrometry (GC-MS) analysis of methanolic extract of callus showed more compounds at higher percentage. HPLC-MS analysis of methanolic extract of callus showed higher concentration of Mangifera than in leaf extracts are reported for the first time.

Salacia chinensis L. is a medicinally important plant used as herbal medicine in Ayurveda (Ghanam et al., 2016) and different parts of this plant have been extensively used to treat variety of ailments (Sikarwar et al., 2012). Biologically active compounds such as mangiferin, salacinol, kotanalol, phenolic compounds, glycosides and triterpenes have been isolated from the plant (Matsuda et al., 2005). For the treatment of diabetes, the compound Salacinol along with other compounds, present in S. chinensis is increasingly being used (Patwardhan et A protocol for indirect organogenesis of Salacia chinensis was established. Normally S. chinensis is propagated through seeds. However, due to the difficulty to obtain uniform plants in a short time period by seed germination, micro-propagation is a possible alternative method. For the micro-propagation, media with different concentration combinations of cytokinins and auxins were used to induce callus formation in three explants types: leaf segment, nodal segment and seeds. The rate of recurrence of callus formation from leaf on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) 0.5mg and TDZ 1mg was 100% and the maximum percentage of shoot let development from nodal segment on MS basal medium supplemented with BAP 6-(benzylamino purine) 3.5+ indole3-butyric acid (IBA) 1 mg/l was 78.3% when compared to other plant growth regulators (PGR) combinations used. The highest shoot regeneration response (85%) and the determined shoots (12.33 ± 0.33%) per callus were attained from leaf explants on MS medium containing 1-napthaleneacetic acid (NAA) 1mg/l + BAP 0.5 mg/l. The seeds showed highest percentage of shoot formation on MS medium supplemented with BAP 2mg + indole 3-acetic acid (IAA) 2mg/l and BAP 2mg + isopentenyl adenine (2iP) 2mg/l. Highest root formation (70±1.3%) was found in shoot regenerated using leaf segment on MS medium supplemented with IBA 0.5mg. The gas chromatography mass spectrometry (GC-MS) analysis of methanolic extract of callus showed more compounds at higher percentage. HPLC-MS analysis of methanolic extract of callus showed higher concentration of Mangifera than in leaf extracts are reported for the first time.

Research Article
Open Access Sri Lankan Journal of Biology 6(2) June 2021June al., 2014. Extracts of S. chinensis have preventive effect on various metabolic disorders (Waltenberger et al., 2016).
Due to the lack of proper cultivation practices, devastation of plant habitats, excessive and unselective collection to fulfil global demands, especially for its anti-diabetic property, this medicinal plant is severely threatened and categorized as endangered species (Bhagya et al., 2011, Maheswari et al., 2016, Majid et al., 2016, florakarnataka.ces.iisc.ac.in., www.iucnredlist.org 2015. There is a need for its conservation and largescale production. Very little efforts have been reported on propagation (Dhanasri et al., 2015;Majid et al., 2016). In this study, we report a reproducible and reliable micro-propagation protocol developed for its effective propagation.

Explants source and surface sterilization
Young and healthy leaves and nodal segments were collected from four-year old S. chinensis plants maintained in Botanical garden of College of Forestry, Sirsi and seeds were collected from Amruta herbals, Bargal Karwar. The undamaged collected explants were washed thrice under running tap water and were pre-washed with concentrated dish washer gel (Sodium LAS, Disodium EDTA, SLES, Concentrated Lime Juice, CI 19140, CI 42051, Water) (4-5drops/100ml double distilled water (ddH2O) for 5minutes followed by rinsing five times in ddH2O. Subsequently, the explants were submerged in 70% (v/v) ethanol for 2 minutes. Nodal segments were surface sterilized with 0.1% of mercuric chloride (HgCl2) while leaf explants were sterilized with 1.0% (w/v) of Sodium hypochlorite (NaOCl) plus Tween 20 (2-3 drops/100ml ddH2O) and seeds were sterilized in 2% NaOCl solution for 15minutes. The solutions were discarded and surface sterilized explants were washed thoroughly with ddH2O for four times under aseptic conditions and placed on a sterilized petridish covered with autoclaved filter paper to remove excess moisture.

Medium composition and culture conditions
Basal medium used for inoculation was Murashige and Skoog's medium (MS) (Murashige and Skoog 1962) supplemented with 3% (w/v) sucrose and 0.8% agar. The pH of the medium was adjusted to 5.7 before autoclaving at 121°C at 15 psi pressure for 20 min for medium sterilization. The explants were cut into suitable sized pieces (1.0 cm for leaf and 1.5cm for nodal segment). Each excised explant was incubated in pre-sterilized test tubes, petridishes or suitable containers containing 20ml medium supplemented with various combinations of auxins and cytokinins.

Preparation of stock solutions of plant growth hormones (PGRs)
The desired PGRs stock solutions (1.0mg/l) were prepared by weighing 13mg of desired PGR dissolving in 13ml of ddH2O with the help of respective solvents (Table 1). The stock solutions were stored in micro centrifuge tubes at suitable temperature (at 4°C). IAA was stored in amber coloured bottle in order to prevent photodecomposition.
Seed explants were inoculated on MS medium supplemented with different combinations of PGR's (Table 1). Treatments were arranged in a completely randomized design 20 seeds per treatment one seed/tube and the experiment was repeated thrice.

Indirect organogenesis:
Callus induction, shoot differentiation, shoot proliferation and rooting of regenerated shoots from leaf explant: For callus induction, the excised healthy mature leaves were inoculated on MS medium supplemented with different growth regulators viz., 0.5 or 2.0mg/l Indole-3-butyric acid (IBA) or αnaphthalene acetic acid (NAA) or 2,4dichlorophenoxyacetic acid (2,4-D) or 6benzylaminopurine (BAP) or thidiazuron (TDZ) or 6-(γ-γ, dimethylallyamino purine) (2iP). Leaf explants were cultured on the media in a growth chamber at 25°C under 16 h light/8 h dark photoperiod provided by white fluorescent tubes. Effect of various auxin/cytokinins on callus induction was evaluated based on the appearance and consistence of callus formation in each treatment. After 4-weeks of callus induction, only well-developed regenerating calli (2-3 mm diameter pieces) were transferred to half strength MS media supplemented with 0.5-1.0mg/l NAA in combination with 0.5-1.0mg/l 2iP or 0.5-1.0mg/l TDZ or 0.5-1.0mg/l BAP. The regenerating callus cultures were maintained in growth chambers on the same medium with controlled conditions for 8 weeks for shoot induction. There were four replicate culture flasks with 5 explants each per treatment and the experiment was repeated thrice. The mean percentage of shooting response, mean number of shoot buds formed, mean shootlet length and mean number of leaves per callus were recorded after 90 days of subculture. Two-three cm long shoots were isolated from shoot clusters from elongation medium and transferred individually to half strength MS medium supplemented with IBA, NAA or IAA (0.5-2mg/l) and incubated for 4 weeks for root development. After root initiation micro shoots were transferred to half strength MS medium supplemented with IAA (2.0mg/l) for normal growth of roots.

Preparation of leaf extract for GC-MS and HPLC-MS analysis:
The fine powdered granules from shade dried leaves of S. chinensis were subjected to extraction. In 400-500ml methanol about 30g of dried powdered granules were soaked and extracted at temperature between 60-65 °C by continuous hot percolation method using Soxhlet apparatus. The extraction was continued for 72 hours. The extract was then filtered and concentrated using rotary evaporator to the volume of 25ml. The dark yellowish brown concentrate was obtained (Jayaraj et al., 2016). This concentrate was used for phytochemical analysis using Gas chromatography mass spectrometry (GC-MS). The dark yellowish brown concentration was further dried to obtain powder which was used for High performance liquid chromatography mass spectrometry (HPLC-MS) technique analysis of mangiferin concentration in root and stem samples (Dhanasri et al., 2015). The experiments were repeated three times.

Preparation of leaf extract for GC-MS and HPLC-MS analysis:
Callus was dried at 40˚C for 24hrs, ground into powder using mortar and pestle, then subjected to extraction. About 30g of callus powder was extracted with 400-500ml methanol using Soxhlet apparatus for 72 hours at 60°C. The solution was then evaporated to dryness as mentioned in 2.6, and then the extract was stored at 4°C for future use.

Mangiferin concentration analysis of leaf and callus developed from leaf explant:
In this study we have analysed the phytoconstituents using GC-MS MS (Shimadzu model number QP2010S) equipped with Rxi-5Sil MS fused silica capillary column (30 m X 0.25 mm ID, film thickness 0.25µm). As carrier gas pure helium gas was used at constant flow rate of ±1 mL/min. Injector temperature was set at 260 °C. The oven temperature was programmed initially at 80°C and then programmed to increase at 280°C at a rate of 10°C ending with a 20min. The spectrums of the components were compared with database of spectrum of known components stored in GC MS libraries NIST 11 & WILEY 8. Measurement of peak areas and data processing were carried out by GC MS solutions software (Sivakumar & Dhivya 2015).
The concentration of mangiferin was determined using HPLC-MS (Shimadzu LC-10 AT vp) equipped with luna 5u C18 analytical column (250 x 4.6 mm) and SPD-M10A vp photo diode array detector (PDA). Mobile phase used for mangiferin quantification was A: Acetonitrile (15%) and B: 0.1% Ortho Phosphoric acid (85%). The 20µl (1mg sample dissolved in 1ml methanol) of sample was injected for flow of 1ml/min with total 12min run time at wavelength of 257nm. Calibration was carried out using standard Mangiferin (Jayaraj et al., 2016) in callus part derived from leaf explant for comparative analysis of mangiferin between plants collected naturally and callus culture of S. chinensis.

Statistical analysis:
All the experiments described above were conducted in a completely randomized design and repeated thrice. All the tissue culture experiments such as callus induction, shoot regeneration, shoot number and shoot length under different plant growth regulator concentrations were computed and analysis of variance (ANOVA) was used for data analysing using the IBM SPSS statistical package (Version 20.0. Armonk, NY, USA:IBM Corp.) The significant differences between means were scored using Duncan's Multiple Range Test (P = 0.05).

Leaf explants:
Leaf, nodal and seed explants cultured on MS medium without PGRs failed to produce shoots even after 4 weeks of inoculation. Whereas swelling and expansion was observed in MS medium supplemented with PGR's and callus induction was started at the cut ends after 6-7 days of incubation of leaf segment explants of S. chinensis. Two types of calli were formed at the cut ends of leaf explants.   Soft, green nodular organogenic callus sub-cultured on MS medium with different combinations of BAP, TDZ and IBA in combination with NAA (0.5-1mg/l) produced embryogenic callus and shoots were induced after few days. Maximum shoot regeneration was observed when callus cultured on MS medium supplemented with BAP (1.0mg/l) along with NAA (0.5mg/l). Responses of different concentration of PGRs on shoot bud regeneration from callus cultures, mean of shooting response, mean number of shoot buds observed and mean height of shootlet of S. chinensis were listed in Table  3. Elongated shoots were then transferred to half strength MS medium containing different concentration of PGR's i.e., IBA, IAA or NAA (0.5-1mg/l) (Table 4).

Nodal explants:
Nodal explants cultured for regeneration, on full strength MS medium supplemented with different combinations of PGRs i.e. high concentration of GA3 (2-3mg/l), TDZ + Kn (1-3+0.5mg/l), Kn + IBA (2.5-3+0.5-1mg/l), Kn + IAA (2-3+0.5-1mg/l) showed significant changes in the first week of inoculation i.e., small protuberance. Plate 2 shows different stages of micro propagation of S. chinensis L nodal explants. The percentage of explants showing the same response at the axils is more or less similar in all the PGRs combinations used. The sprouts in the axils increased in size to form shootlets in 2 weeks. The maximum percentage of shootlets development was observed on the phytohormones combinations BAP 3.5mg/l with combination of IBA 1mg/l (78.3%) and BAP 3mg/l with combination of IAA 1mg/l (43%) when compared to other PGRs combinations used. Full strength medium supplemented with GA3 (0.5-1.5mg/l) induced shoots but the shoots were dwarf.
Full strength medium supplemented with different concentration of BAP in combination with IAA or IBA induced shoot formation but BAP 3.5 mg + IBA 1 mg gave better results in regeneration of number of shoots 15±0.6 and enhancement of shoot elongation (table 5, Plate 2b). The shootlets increased in length during the 3 rd week and the maximum shoot length of 2.83 ± 0.44 was observed in BAP 3.5mg/l + IBA 1mg/l.  Table 5. Opening of small leaves from the shootlets was observed during 4 th week (Plate 2b). In few explants, the explant started yellowing from the bottom and the yellowing reached to top, causing death of that explant in 8-9 weeks. Shoots longer than 3cm, with leaves opened were transferred to half MS rooting media, supplemented with different concentrations of PGRs i.e. IBA (0.5-1mg/l) or NAA (0.5-1mg/l) or IAA (0.5-1mg/l). Maximum number of rooting observed in half MS media supplemented with IAA 0.5mg/l. small roots were initiated in explants that were grown on BAP (3.5mg/l) + IBA (1mg/l) (Plate 2c) where the explants from all other PGRs combinations failed to root and they showed yellowing of leaf and browning of tissue.

Seed explant:
Seed explants cultured on full strength MS medium supplemented with BAP (1-3mg/l) with different combinations of PGRs i.e. IAA (1-3mg/l) or 2iP (2mg/l) or NAA (1-3mg/l). Even at the lowest concentration BAP alone induced adventitious shoot bud from seed explants. However, The seeds showed highest percentage of shoot on MS medium supplemented with BAP 2mg + IAA 2mg/l and BAP 2mg + 2iP 2mg/l whereas the former medium induced shoot regeneration after the callusing of the However, the differentiation of shoots was preceded by callusing of the explants. At lower concentration, IAA (68%) and NAA (1mg/l) (64%) direct shoot buds regeneration was observed with an occurrence comparable to BAP (3mg/l) + 2iP (2mg/l). Nevertheless, the intensity of shoot bud formation was high with the latter treatment (Table 6). Shoots were then transferred to MS medium containing only BAP 1mg/l for well and healthy development. On this medium shoots were well developed and attained height of 2.63 ± 0.29 after 20 days (Plate 3b). For the initiation of roots shoots were individually transferred to MS medium supplemented with NAA 0.5mg/l. In this treatment 90% of shoots produced roots directly at the bottom within 7 days, with an average of five roots per explants (Plate 3c).    After 5 weeks of incubation in light, the rooted shoots were carefully removed from culture vessels and washed in running tap water to free agar. Healthy rooted shoots were transferred to plastic cups containing sand: soil: vermiculite in the proportion of (1:1:1) and covered with polythene covers to maintain humidity under aseptic conditions inside the culture room. After 2 weeks the bag was carefully removed and transferred to earthen pots with garden soil (plate 1f, 2d and 3d).

Figure 1: Representative of GC-MS chromatograms of leaf (a) and callus extract (b) of S. chinensis
HPLC analysis has shown the presence of mangiferin in both leaf and callus extracts of S. chinensis (Figure 2). The study showed that S. chinensis callus extract (26mg/ml) contain higher quantity of mangiferin as compared to leaf extract (4mg/ml) (Figure 3).

Discussion
The present study comprehensively investigated the effects of various kinds and concentrations of PGRs on 3 different explants of S. chinensis micropropagation. The ability to regenerate complete plants from mature leaf derived callus, nodal segment and seeds of S. chinensis has been found.
Leaf explants and nodal explants are the most suitable plant source for regeneration. BAP among the different cytokinins that have the higher potential for both shoot and root induction and IAA for root initiation among the auxins (Elangomathavan et al., 2017). An increase in the auxin concentration with the higher concentration of cytokinins resulted in the production of callus at cut ends of leaf segments, proliferation of shoot and root for the leaf derived callus, nodal segment and seed explants which may be due to the cell proliferation stimulated by the accumulation of auxins in the presence of cytokinins (Marks and Simpson 1994;Dhanshri et al., 2015). In the present study, cytokinins promoted intensive shoot multiplication. Cytokinins are reported to overcome apical dominance, induce high number of shoot buds and also release lateral buds from dormancy (Rout et al., 2000;Rout, 2005).
The MS medium supplemented with BAP 3.5 mg/l + IBA 1 mg/l and NAA 0.5mg/l +TDZ 0.5mg/l resulted highest number of shoot elongation and root formation. The negative effect on shoot regeneration was observed when the MS medium supplemented with BAP 4 mg/l and continued exposure of explants to high concentration of BAP during shoot induction inhibited the growth of shoots. The results of the present study are in accordance with Deepak et al., (2015) with the use of BAP for shoot multiplication. The presence of auxins at lower concentration in medium facilitated better root formation. MS medium supplemented with 15 mg/l IAA was found superior to IBA and NAA in inducing roots.
In our previous study we have analysed the phytoconstituents and mangiferin concentration in different species of Salacia (Kamat et al. 2019). We have noticed that the leaf, stem and root extract of S. chinensis showed the higher concentration of mangiferin compared to other species. Although many compounds were reported from different species of Salacia, it is an eminent fact that mangiferin is important for its antidiabetic property and is a well explored molecule in in vitro and preclinical studies. Mangiferin projected to be the most active principle from Salacia spp.
The phenolic compounds are known to be synthesized by plants in response to microbial infection. It is therefore possible that they can act as effective antimicrobial substances against a wide array of microorganisms. However, the antimicrobial activity of plant extracts depends not only on phenolic compounds but also by the presence of different secondary metabolite (Gordana et al., 2007) like hydroxyl groups on the active constituents, because these substances are capable of binding to bacterial adhesions and interacting with the supply of receptors on the surface. The phenolic compound observed in this study is phenol, 2,4-bis (1,1-dimethylethyl) in natural as well as callus extract.

Conclusions
In the present study, the successful protocol for callus induction and shoot regeneration from leaf derived callus of S. chinensis has been established. About 64% seeds exhibited regeneration. Leaf explants and nodal segments are the most suitable plant materials for regeneration. The protocol presented in this study may provide a high efficiency regeneration system for successful regeneration of adventitious shoots for ex situ preservation of S. chinensis as well as genetic improvement studies for pharmaceutical uses and future research investigations. This optimized protocol helps in providing a promising method for the large scale propagation of this species. Like mangiferin, other compounds identified in leaf and callus extract have their own biological importance and further study of this plant's phytochemicals by in silico and in vitro methods can prove its medicinal importance in future and can be effective and efficient drug source.