Elicitor mediated enhancement of shoot biomass and lupeol production in Hemidesmus indicus (L.) R. Br. ex. Schult. and Tylophora indica (Burm. F.) Merrill using yeast extract and salicylic acid

Abstract Hemidesmus indicus (L.) R. Br. ex Schult. and Tylophora indica (Burm. F.) Merrill shoot cultures were treated with different concentrations of yeast extract (YE; 25–200 mg/L) and salicylic acid (SA; 50–200 µM), and their effect on lupeol production was assessed. The maximum dry weight (DW) biomass was recorded when H. indicus shoots were treated with SA (50 µM) and T. indica shoots with YE (200 mg/L). Highest lupeol yield (335.40 ± 0.04 µg/g DW) was obtained in H. indicus shoots after treatment with 50 µM of SA for 3 weeks. Whereas in T. indica, maximum lupeol content (584.26 ± 8.14 µg/g DW) was recorded by giving treatment with 25 μM of SA for 6 weeks. Graphical Abstract


Introduction
Hemidesmus indicus (L.) R. Br. ex Schult. and Tylophora indica (Burm. F.) Merrill are important medicinal climbers of Apocynaceae family, and contain pentacyclic triterpene lupeol (Figure 1). This compound is reported to have anticancer properties against different cancer types and it is non-toxic to normal cells, hence now considered as a potential chemopreventive agent. It also possesses antioxidant, antiasthmatic, antiarthritic, and venom neutralising activities (Saleem 2009). Lupeol can be extracted from plant samples using conventional techniques involving solvents along with intermittent shaking and heating, although nowadays other techniques such as ultrasound assisted extraction (UAE), high hydrostatic pressure extraction (HHPE), microwave assisted extraction (MAE), and enzyme assisted extraction (EAE) are used which are comparatively faster than conventional methods (Ramos-Hernandez et al. 2018). Similarly, quantification of metabolites can be done using various techniques, but high-performance thin layer chromatography (HPTLC) is one of the methods which simultaneously detect variation between different botanical samples (Toniolo et al. 2014).
Large amount of plant materials are indiscriminately harvested to extract secondary metabolites, but it hampers the populations of plants in wild (Sharifzadeh Naeini et al. 2021). This problem can be overcome by use of in vitro cultures which can produce metabolite throughout the year (Komaikul et al. 2019;El-Hawary et al. 2020). One of the advantage of these cultures is that the secondary metabolite production can be increased by application of biotic (Arman 2011) and abiotic (Santamaria et al. 2010) elicitors like yeast extract (YE) and salicylic acid (SA), however the response varies with species to species (Namdeo 2007). Exogenous application of elicitor proved advantageous in increasing the production of important metabolites through in vitro cultures (Zare et al. 2014;Rahamouz-Haghighi et al. 2022). There are many studies on regeneration of both these species (da Silva and Jha 2016; Kher et al. 2020), but reports on enhancing lupeol production through cultures is scanty (Misra and Mehrotra 2006). Thus, the aim of the present study was to evaluate the effect of YE and SA on shoot biomass, and production of lupeol in shoot cultures of H. indicus and T. indica, and it is being carried out for the first time.

Shoot biomass and lupeol quantity in control media
Liquid medium containing 6-benzyladenine (BA, 10 mM) with kinetin (Kn, 5 mM) was utilised for H. indicus, and the biomass [fresh weight (FW) and dry weight (DW)] of shoot reached to maximum [3.54 ± 0.42 g FW and 0.47 ± 0.05 g DW] at the end of three weeks ( Figure S1a, supplementary material). In T. indica, medium fortified with Kn (10 mM) with adenine sulphate (AdSO 4 , 15 mM) was taken up, in which maximum shoot biomass (0.70 ± 0.09 g FW and 0.13 ± 0.01 g DW) was obtained after six weeks ( Figure  S1b, supplementary material). HPTLC analysis confirmed the presence of lupeol in both the plants. After calculating area under curve (AUC) it was observed that the lupeol contents obtained in H. indicus and in T. indica were 260.58 ± 0.07 mg/g DW ( Figure S2a, supplementary material) and 457.08 ± 2.79 mg/g DW ( Figure S2b, supplementary material), respectively.

Influence of yeast extract on biomass and lupeol synthesis
YE is a rich source of vitamin B-complex, chitin, b-glucan, glycopeptides, and ergosterol, which induces plant growth and triggers defence responses (Cai et al. 2012). Treating H. indicus shoots with YE (25-200 mg/L) evoked better growth in comparison with control. The highest biomass in terms of FW (3.85 ± 0.10 g) and DW (0.48 ± 0.03 g) was achieved when shoots were treated with 50 mg/L of YE for three weeks ( Figure  S1a, supplementary material). Whereas in T. indica the maximum biomass (1.08 ± 0.05 g FW and 0.23 ± 0.01 g DW) was achieved after shoots treated with 200 mg/L of YE for six weeks ( Figure S1b, supplementary material). Evaluating lupeol content showed that 50 mg/L of YE treated H. indicus shoots (after three weeks) gave 1.05 fold increase in the quantity (275.56 ± 0.06 mg/g DW). Further increase in YE concentrations (100 and 200 mg/L) adversely affected the lupeol production ( Figure S2a, supplementary material). In T. indica 25 mg/L of YE elevated the lupeol content in four weeks, i.e. 420.33 ± 4.75 mg/g DW and it was 1.65 fold higher as compared to control. Increasing YE concentration from 50 to 200 mg/L as well as treatment time showed gradual decline in the lupeol content ( Figure S2b, supplementary material). The results suggested that lower concentration of YE proved to be beneficial for significantly elevating the amount of lupeol, whereas higher concentrations adversely affected its synthesis. This may be due to the utilisation of YE for different primary or secondary metabolites synthesis which might decreased the lupeol content, and similar observations are reported for Panax quinquefolium L. (Kochan et al. 2017). The beneficial effect of YE could be attributed to the presence of metal ions like Zn, Ca, and Co which act as abiotic stress, it also activates reactive oxygen species (ROS) and jasmonate pathways which ultimately triggers metabolite synthesis (S anchez-Sampedro et al. 2005).

Influence of salicylic acid on biomass and lupeol synthesis
SA is a defence signalling molecule, known to participate in the regulation of physiological processes as well as in inducing immune responses in plants by evoking systemic acquired resistance (SAR) and synthesising pathogenesis related (PR) proteins (Durrant and Dong 2004;Ferrari 2010). Results suggested that H. indicus shoots grew better in SA (25-200 mM) containing medium as compared to YE treatment, and maximum shoot biomass [DW (0.589 ± 0.02 g)] was recorded in presence of 50 mM of SA after three weeks ( Figure S3a, supplementary material). On the contrary, the production of biomass from T. indica shoots with 200 lM of SA [FW (0.53 ± 0.05 g) and DW (0.11 ± 0.01 g)] was minor as compared to YE treatment ( Figure S3b, supplementary  material). The elicitor effect varies from plant to plant, as according to present result it was inferred that the utilisation of SA was beneficial for shoot growth as well as lupeol production in H. indicus. Whereas SA did not work as nutrient supplement for the growth and only aided in enhancing the lupeol yield. This is in accordance with the study on Bacopa monnieri (L.) Pennell. where elicitor treatment showed inhibitor effect on shoot growth but enhanced metabolite production (Sharma et al. 2013). When HPTLC chromatograms were analysed it showed presence of lupeol in SA treated samples for both plants ( Figure S4a,b, supplementary material). In H. indicus, when shoots were treated with SA (50 mM) for three weeks, it gave highest lupeol content (335.40 ± 0.04 mg/g DW) which was 1.28 fold higher than control ( Figure S5a, supplementary material). Whereas in T. indica the maximum lupeol yield (584.26 ± 8.14 lg/g DW) was achieved after treating the shoots with SA (25 lM) for six weeks, which was also 1.28 fold higher than control ( Figure S5b, supplementary material). Recently, Darbahani et al. (2022) reported that SA increased the expression of several genes related to biosynthesis of sesquiterpene parthenolide in Tanacetum parthenium L. Present results also suggested that lower concentrations of SA were beneficial for maximum lupeol production in both the plants, which is in corroboration with previous results for daidzin production in Psoralea corylifolia L. (Zaheer et al. 2016). Similar observation is also noted in Bacopa monnieri (L.) Wettst. where lower concentration enhanced but higher concentration adversely affected the bacoside accumulation (Sharma et al. 2015).
The experimental findings suggested that both YE and SA significantly enhanced lupeol production in shoots, but elicitor type, its concentration, and duration of treatment affected the content, which is in accordance with previous report (Salehi et al. 2019). The increase in lupeol synthesis by YE and SA might be due to up-regulation of terpenoid biosynthetic pathway genes (Alex et al. 2000;Li et al. 2016). Present findings also confirmed that SA proved to be superior to YE, and it is in corroboration with report of Reis et al. (2019) in Trifolium pratense L. The reason for more production of lupeol with SA than YE is because SA is mainly known to trigger metabolite synthesis in plant by activating defence responses (Namdeo 2007) whereas YE is used as an additive (undefined supplement) in culture media (George and Sherrington 1984). Our previous study on in vivo shoots of H. indicus and T. indica suggested that the quantity of lupeol were 185 ± 0.0 lg/g and 24.43 ± 1.28 lg/g, respectively (Pathak et al. 2017;Patel 2021). Thus, from the present study it can be inferred that via shoot cultures, 1.81 fold elevation in lupeol content was achieved after treatment with 50 lM of SA for H. indicus, similarly in T. indica 23.91 fold lupeol yield was attained by 25 lM of SA treatment. This suggests that the in vitro technique will be an alternative way to increase the production of this important anticancer compound. It will also decrease threat on wild plants population and in turn help in conservation of natural resources (Komaikul et al. 2019;El-Hawary et al. 2020;Sharifzadeh Naeini et al. 2021).

Experimental
All experimental procedures are described in the supplementary material.

Conclusions
To the best of our knowledge, this is the first report on analysing the effects of YE and SA on shoot growth and lupeol content in both the plants. Both elicitors have enhanced the shoot biomass as well as lupeol production, but its type, concentration, and treatment time played a major role. Treatment of H. indicus shoots with 50 mM of SA for three weeks induced maximum biomass and enhanced lupeol content by 1.28 fold. On the contrary, YE (200 mg/L) was beneficial only for shoot biomass in T. indica, but SA (25 lM) favoured 1.28 fold increase in lupeol yield. Although SA has up-regulated lupeol production in shoot cultures of both plants, overall quantity was higher (1.74 fold) in T. indica shoots as compared to H. indicus shoots. It can also be concluded that shoot cultures of both these plants are promising alternative to wild plants for harnessing this commercially important anticancer compound in short duration.