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Clinacanthus nutans (Acanthaceae), generally known as ‘snake grass’, has diverse uses in customary system of herbal medicine. The species is endowed with various bioactive compounds exhibiting extensive pharmacological properties. The present investigation focused on elicitor-intervened in vitro shoot biomass cultivation and scale-up production of the anti-cancerous compound ‘lupeol’, one of the foremost constituents in this species. For the augmented production of lupeol, the shoot cultures were elicited with various concentrations of yeast extract (YE), chitosan and methyl jasmonate (MeJA). Maximum shoot biomass yield and production of lupeol was detected in MS medium supplemented with 1.0 mgl -1 BA and 400 mgl -1 YE. The petroleum ether extracts of selected samples upon TLC analysis proved R f values corresponding to lupeol. HPTLC analysis revealed that the sample treated with YE displayed relatively higher amount (975.50 ng) of lupeol than the in vivo plant (713.69 ng). Hence the in vitro shoot culture system with elicitor (YE) treatment propose an appropriate method for the elevated synthesis of lupeol which can be scaled up via bio-reactor technology in doing so profiting the pharmaceutical appliances.

Enhanced production of Enhanced production of Enhanced production of Enhanced production of l l l lupeol through elicitation in upeol through elicitation in upeol through elicitation in upeol through elicitation in in vitro in vitro in vitro in vitro shoot shoot shoot shoot cultures cultures cultures cultures of of of of snake grass snake grass snake grass snake grass ( ( ( (Clinacanthus nutans Clinacanthus nutans Clinacanthus nutans Clinacanthus nutans) ) ) ) Introduction

Introduction Introduction Introduction
Clinacanthus nutans (Burm. f.) Lindau belonging to family Acanthaceae commonly known as "Sabah snake grass" is indigenous to the tropical provinces of Southeast Asia, particularly Thailand, southern China and some temperate region (Haida and Hakiman, 2019). The taxa have immense pharmaceutical properties and has been utilized traditionally as anti-venom, anti-inflammatory, analgesic, anti-diabetic, anti-rheumatic, antiviral and antioxidant. Fresh leaves have been suggested as an effective remedy in treating venomous snake and insect bites, burns, allergic reactions and skin rashes. Leaves are chewed for internal consumption and crushed leaves are applied on the area of snake bite (Thongharb and Tejasen, 1977;Tejasen and Thongthaap, 1978). The dried leaves are used to treat fever, diarrhoea and dysuria (Sriwanthana et al., 1996;Shim et al., 2013); while the infusion or decoction of dried leaves and stem is recommended for hepatitis infection (Teshima et al., 1998). The leaf extracts also employed for the cure of varicella-zoster virus (VZV) gashes (Tuntiwachwuttikul et al., 2004). Besides, this species has gained considerable attention amongst the Malaysian populace for the treatment cancer (P'ng et al., 2013). In Malaysia, the fresh leaves are boiled in water and consumed as herbal tea; several manufacturers are there selling the herbal tea product of snake grass tea. A greater part of the bioactive compounds reported from C. nutans includes triterpenes, C-glycosyl flavones and sulfur containing glucosides. Occurrence of six known C-glycosyl flavones namely, schaftoside, vitexin, isovitexin, orientin, isoorientin and isomollupentin-7-O-β-D-glucoside have also been described in the dried leaves and stem segments of this species (Teshima et al., 1997). Preceding studies disclosed the presence of lupeol and β-sitosterol in the stem (Wanikiat et al., 2008), betulin, lupeol and β-sitosterol in dried up rhizomes (Dampawan et al., 1977).
Apart from few phytochemical studies, the literature related to in vitro multiplication of this species is scanty and the plant is often vegetatively propagated by stem cuttings (Chen et al., 2015). However, the demand for quality raw materials to the pharmaceutical industry as well as herbal tea manufacturers is very high and conventional multiplication could not meet the widening gap between demand for quality raw material and supply. Consequently, development of a rapid propagation scheme through biotechnological intervention is an attractive proposition to bridge the demand-supply gap. Furthermore, the popularity of C. nutans has encouraged several investigators to execute in vitro culture technique, a more rapid propagation means than stem cuttings in order to increase the consistent supply of true to type clones (Chen et al., 2015) whereas merely callus induction from explants was reported in some (Ng, 2013;Gunasekaran, 2014), while Bong et al. (2021) developed callus and cell suspension cultures in this species. Also, Haida et al. (2020) reported in vitro responses of plant growth factors on growth, yield, phenolics content and antioxidant activities of C. nutans. Conversely, perusal of literature reveals that there are no attempts carried on high frequency elicitor mediated in vitro biomass cultivation for the enhanced production of bioactive compounds followed by bioassay of in vitro biomass, which forms the basis of the present research; wherein the established in vitro shoot cultures of C. nutans were subjected to treatment with different concentrations of elicitors viz., yeast extract, chitosan and methyl jasmonate in order to examine the production of lupeol using chromatographic techniques.

Plant material for in vitro studies
Healthy shoots collected from the natural habitat of Thiruvananthapuram district were planted and reared in green house of the Botany Department of University College, Thiruvananthapuram, Kerala, India. The plant species were identified and the voucher specimens were deposited in the herbarium. Explants were taken from the green house grown plants to develop the in vitro system in the present study.
In vitro shoot culture establishment Top cuttings of the shoots with 4-5 nodes collected from mature plant were washed in running water and sliced to appropriate size. Subsequently, the explants were cleansed in 0.5% (v/v) labolene for 10 min and then in running tap water for 30-45 minutes along with 4-5 times rinses in distilled water, before taken to the laminar air flow hood. During surface sterilization inside the airflow chamber, the explants were treated with 10% (v/v) sodium hypochlorite solution for 20 min. Following rinsing 3-4 times in sterile distilled water, the explants were trimmed to appropriate sizes (0.8-1.0 cm) and inoculated vertically into Murashige and Skoog 3 (MS) medium (Murashige and Skoog, 1962) fortified with 0.5 mgl -1 BA. The shoots/shoot buds induced from nodal cultures were excised out for fostering auxiliary biomass harvesting in medium complemented with 1.0 mgl -1 BA. Shoot biomass cultures maintained in MS basal medium were used in elicitation experiments.

Experimental design, data collection and sstatistical analysis
The whole experiment was performed in randomized block design. Each treatment consisted of ten replicates and was repeated thrice. The cultures were consistently examined and the remarks on number of shoots, shoot length (cm) and number of nodes per shoot were recorded after 4 weeks. Results were presented as mean value ± standard error. Data were subjected to ANOVA and the means were compared by Duncan's multiple range test (p ≤ 0.05) using the computer software SPSS (PC + version 10; SPSS Inc., Chicago, USA 1999).

Chemical analysis for the elucidation of lupeol
Extract preparation and Thin Layer Chromatography (TLC) Four samples (control and treated samples) i.e., in vivo plant (absolute control), in vitro plant (control), in vitro plant treated with yeast extract and chitosan were dried up and powdered using mortar and pestle and afterward extracted with petroleum ether. These extracts were used for loading on TLC plates. Authentic standard of lupeol purchased from Sigma Aldrich was used for confirmation. The solvent system used was petroleum ether: methanol (9:1) and the spots developed were visualized under ultra-violet chamber (Kemi) (365 nm).
HPTLC (High Performance Thin Layer Chromatography) HPTLC method was used for the quantification of lupeol in dried powdered samples of C. nutans plantlets (both in vivo and in vitro), that was used for TLC. The samples were analyzed using HPTLC system where they were spotted in the form of bands of width 7 mm and 10 mm apart with a Camag microlitre syringe on precoated silica gel aluminium Plate 60F254 having 10 cm × 10 cm dimensions (E. Merck, Darmstadt, Germany) using a Camag Linomat IV sample applicator (Muttenz, Switzerland). The mobile phase consisted of toluene, ethyl acetate, glacial acetic acid in the volume ratio of (6:1.5:0.1). Linear ascending development was carried out in 10 × 10 cm twin trough glass chamber (Camag, Muttenz, Switzerland) saturated with the mobile phase. The optimized chamber saturation time for mobile phase was 20 min at room temperature. The length of chromatogram run was 80 mm. Subsequent to the development; applied with methanolic sulphuric acid and plate was dried at 110 °C for 5 minutes. Densitometric scanning was performed on Camag TLC scanner III in the reflectance absorbance mode at 580 nm and was operated by CATS software (Camag). The amount of lupeol present in the sample was determined in nano gram units as per the system generated values using the standard curve.

Res Res Res Results ults ults ults and Discussion and Discussion and Discussion and Discussion
The axenic shoots of C. nutans derived from established in vitro shoot cultures while treated with different elicitors exhibited varied responses with regard to the concentrations and type of elicitors being tested.

Effect of elicitors in shoot multiplication
An elicitor can stimulate any kind of plant defense, upholding secondary metabolism to protect the cell and the whole plant and once it is introduced in minute concentrations to a living cell system, it initiates or boost up the biosynthesis of specific compounds. Therefore, elicitation is the induced or enhanced biosynthesis of metabolites due to addition of trace amount of elicitors (Radman et al., 2003). Growth, regeneration and moreover the production of bioactive molecules of in vitro cultured plants can be enhanced by elicitation. Biotic and abiotic elicitors equally stimulate product accretion in whole plants, plant organs and in plant cell cultures as a consequence of their defensive, protective or offensive reactions. The quantity of these substances required for elevated product accumulation varies with species and genotype as well. The addition of elicitors for the enhanced production of secondary metabolites is well documented in many medicinal plants (Namdeo, 2007;Naik and Al-Khayri, 2016;Shakya, 2017). Protocol optimization by way of accomplishment of elicitors for the bioproduction of secondary metabolites is highly promising in medicinal herbs for their sustainable utilization. However, no such attempts have been made yet in C. nutans concerning this aspect and in this study the effect of three elicitors viz., chitosan, YE and methyl jasmonate were examined to check their role in the synthesis of bioactive metabolites.

Effect of Chitosan on shoot morphogenesis
The explant exhibited varied response with regard to the treatment with different concentrations of chitosan. The in vitro shoot explants displayed a linear decrease in the mean number of shoots and mean number of axillary shoots with the increase in concentration of chitosan (Figure 1a). Maximum 4.4±0.16 shoots only were produced per explant in MS medium supplemented with 1.0 mgl -1 BA+ 50 mgl -1 chitosan within 6 weeks against the control, which evoked the production of 5-6 shoots. This was analogous to the findings in Lilium longiflorum, wherein the number of shoots decreased with the increase of chitosan concentration and the combinations of chitosan and BA were less effective in promoting shoot multiplication when compared to chitosan or BA alone (Kanchanapoom, 2012). A mean number of 3.3±0.79 axillary shoots obtained at 50 mgl -1 concentration of chitosan was dropped down to 1.3±0.15 when the concentration was increased to 200 mgl -1 (Table 1).

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The length of regenerated shoots increased as the chitosan milieu increased (Figure 1a); the mean shoot length of 5.26±0.04 cm was recorded at 200 mgl -1 chitosan, while it was 3.26±0.03 cm at 50 mgl -1 concentration. Maximum number of 2.5±0.16 nodes per shoots was observed at 200 mgl -1 chitosan. Thus, higher concentrations of chitosan in the growth medium supported shoot growth in C. nutans as a consequence of which the shoots remained elongated with proliferation in the mean number of nodes per shoot. In strawberry also, the highest number of shoots, diameter of shoots, number of leaves and dry weight of plant mass were obtained in 1.0 or 2.0 mgl -1 BA without chitosan; but the highest length of shoot was observed in combination of 1.0 or 2.0 mgl -1 BA along with 30 mgl -1 chitosan (Jalili Marandi et al., 2011). Basal rooting (4-5 cm length) was noticed in 50 mgl -1 chitosan (Table 1) and these roots were having root hairs with powdery appearance. In 100 and 150 mgl -1 chitosan, the shoots were devoid of basal roots and they exhibited merely aerial lateral roots. However, in 200 mgl -1 chitosan, basal roots (1-2) were present and the aerial lateral roots were seen around the sub-aerial parts (Figure 1a).

Effect of yeast extract (YE) on shoot morphogenesis
Yeast extract, a source of amino acids and vitamins in particular inositol and thiamine is used as an additive in plant tissue culture media (George, 2007). In C. nutans, the treatment with YE exhibited good range of influence on the explants than control. The in vitro shoot explants differed in the mean number of shoots in different concentrations of YE (100, 200, 300, 400 mgl -1 ) (Figure 1b). Maximum 8.5±0.22 shoots were produced in MS medium incremented with 1.0 mgl -1 BA (SM) + 400 mgl -1 YE which was 7.0±0.21 at 100 mgl -1 YE (Table 2). Though statistically insignificant, the mean number of shoots obtained with 300 mgl -1 YE was found to be 7.2±0.20, while at 200 mgl -1 YE 7.7±0.15 shoots were proliferated. The mean shoot length enhanced linearly with increase in YE concentrations (Figure 1b). Maximum 6.38±0.03 cm long shoots were obtained at 400 mgl -1 whilst the shoot length was 5.54±0.03 cm at 100 mgl -1 YE ( Table 2). The mean number of nodes per shoot remained constant at 100 and 200 mgl -1 , i.e., 2.7±0.15. Axillary shoots were produced only at 200 and 300 mgl -1 YE and it was missing in 100 and 400 mgl -1 concentrations. Presence of basal roots was invariably observed in all the concentrations of YE tested in the present study. Among the four concentrations of YE examined, 400 mgl -1 was exceedingly favourable which increased the shoot proliferation, shoot length and number of nodes per shoot in C. nutans. In agreement with this, the promotive effect of YE on shoot multiplication was reported earlier in Lavandula latifolia (Gras and Calvo, 1996), Eryngium foetidum, Stevia rebaudiana, etc. (Sridhar and Aswath, 2014).

Effect of methyl jasmonate (MeJA) on shoot morphogenesis
The treatment with MeJA at different concentrations (50, 100, 150 and 200 µM) induced strong stress on the explants so that all of them turned glassy and died within 2-3 days after inoculation. The explants exhibited abscission and become pale coloured (data not shown). The reason for this may be the lethal effect occurred due to the high concentration of MeJA selected for the experimentation. So other minute concentrations need to be experimented to check the efficiency of the same.

Comparative analysis of elicitor-mediated shoot multiplication in C. nutans
Among the three types of elicitors analyzed, the explants responded to only two of them i.e., YE and chitosan. The values obtained for different parameters at various chitosan concentrations as well as YE showed significant differences from that of the control. The mean number of shoots rose with increase in concentration of YE, but decreased upon chitosan treatments (Figures 2a and 2b). The mean shoot length increased 7 concurrently as the amount of chitosan and YE increased. The results of this study demonstrated that YE is complementing the vigorous growth of C. nutans compared to chitosan.     (Table 3). This is the first and foremost report regarding the presence of lupeol in the in vitro shoot cultures of C. nutans. The partitioning of lupeol using the same solvent system viz., petroleum ether: methanol (9:1) was earlier reported in Wrightia tinctoria (Jain and Bari, 2010) thereby substantiating our study. Fig  Fig Fig  Figure ure   High performance thin layer chromatography (HPTLC) HPTLC, a sophisticated and automated separation technique of TLC is a valuable quality measurement tool for the assessment of botanical materials proficiently, thereby offering high degree of selectivity, sensitivity and rapidity combined with single-step sample preparation. It is a consistent method for the quantification of nanogram level of sample components. In the present study, HPTLC chromatogram of samples in petroleum ether extract revealed the presence of lupeol (Figures 4a-d) and the band having Rf value 0.62 corresponds to lupeol as per the HPTLC scan report.

Quantification of lupeol
The amount of lupeol in the selected samples was determined as per the standard procedures ( Figure 5). In the in vivo plants of C. nutans (absolute control), lupeol was detected with an Rf value of 0.62. It also showed other three unknown compounds with Rf values of 0.18, 0.24 and 0.48 and the amount of lupeol found in this sample was 713.69 ng ( Figure 5). While, in in vitro plants (control, without elicitation) lupeol was detected at Rf value 0.62. This sample showed five compounds including lupeol and the other four compounds seen were having Rf values 0.18, 0.43, 0.48 and 0.77; the amount of lupeol in the same was quantified as 1067.0 ng. In elicited samples, in vitro shoots of C. nutans treated with YE also showed lupeol at Rf value 0.62; with 975.50 ng lupeol in it. In addition to this, five other unknown compounds were detected at Rf values at 0.18, 0.40, 0.48, 0.77 and 0.78. Lupeol in in vitro shoots of C. nutans treated with chitosan was 761.26 ng ( Figure 5 All the samples analysed here contained lupeol and showed characteristic Rf value of 0.62. Most of the unknown compounds present in different samples showed same Rf values such as 0.18, 0.24, 0.47, 0.48 and 0.77. Among the treated samples, elicitation with YE resulted in comparatively higher amount (975.50 ng) of lupeol. However, the values corresponding to lupeol content was moderately higher in the control (1067 ng) than YE elicitation (975.50 ng). This may be because in the control i.e., the shoot cultures raised in BAsupplemented medium, where the plant growth regulator, cytokinin (BA) itself may be acting as the elicitor thereby eliciting the production of lupeol. Similarly, in Hypericum perforatum, modified MS medium, with 50% reduction in ammonium nitrate and potassium nitrate with supplements of BA (0.44 μM) and indole 3butyric acid (0.049 μM), have resulted in the increased production of hypericins (Liu et al., 2007). In the present study, the in vitro shoots (both control and treated samples) exhibited higher production of lupeol than the absolute control i.e., the in vivo plant (713.69 ng). On the other hand, YE-treated plants exhibited higher shoot multiplication rate than control. Hence the in vitro shoot culture system with elicitor treatment (especially YE treatment) offers an enhanced shoot biomass production as well as elevated synthesis of lupeol, compared to the in vivo plants (absolute control).