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. 2025 Apr 7;109(1):83. doi: 10.1007/s00253-025-13453-x

Engineering Bacopa monnieri for improved bacoside content and its neurological evaluation

Gajendra Singh Jeena 1,#, Sunil Kumar 1,#, Sachi Bharti 5,6, Neeti Singh 1,5, Ashutosh Joshi 1, Vaibhavi Lahane 4,5, Roshni Meghani 3, Akhilesh Kumar Yadav 4,5, Shubha Shukla 5,6, Vineeta Tripathi 2,5, Rakesh Kumar Shukla 1,5,✉
PMCID: PMC11976368  PMID: 40195155

Abstract

Abstract

Bacosides are triterpenoidal saponins with numerous pharmacological benefits. One of the significant drawbacks is the low availability of these bacosides. The bacoside pathway is not well elucidated, and there is no prior report of a metabolic engineering approach in this plant. In this study, we have over-expressed the active isoform of Bacopa monnieri squalene synthase (BmSQS1-OE) and silenced the B. monnieri G10H (BmG10H-1-KD), the competitive metabolic pathway, to divert the flux towards triterpene biosynthesis. Absolute quantification of bacosides in these BmSQS1(OE)-BmG10H1(KD) lines has identified improved content of bacoside A3, bacopaside II, and bacoside A. Moreover, the engineered plant extract was also found to have better efficacy on locomotor activity, neuromuscular coordination, and social interaction in a 6-hydroxydopamine (6-OHDA)-induced rat model of Parkinson’s disease (PD). Immunohistochemistry of the brain tissues indicates that an extract of enhanced bacoside contents reduces 6-OHDA-induced dopaminergic depletion, implying a potential utility in neurological disorders.

Key points

• The engineered Bacopa monnieri extract has improved amounts of various bacoside.

• The engineered Bacopa extract has shown enhanced effectiveness in a 6-hydroxydopamine (6-OHDA)-induced rat model of Parkinson’s disease (PD).

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-025-13453-x.

Keywords: Bacopa monnieri, Terpenoidal saponin, Bacosides, Overexpression, RNAi, Triterpene

Introduction

Bacopa monnieri (L.) Wettst, or ‘Brahmi’, is a well-recognised medicinal plant containing pharmaceutically essential metabolites such as triterpene bacosides. The B. monnieri shoot region is the primary site of biosynthesis and accumulation of bacoside (Naik et al. 2012). The traditional Indian medicinal system widely uses B. monnieri as a memory-enhancing and stress-relieving medication (Sivaramakrishna et al. 2005; Saini et al. 2019; Sekhar et al. 2019). Bacosides are diversified from the two isomers, jujubogenin and pseudojujubogenin (Bhandari et al. 2020). These two isomers are formed after cyclisation of 2,3, oxidosqualene into dammarane type triterpene using dammarenediol synthase, followed by the catalytic action of cytochrome P450s, leading to the formation of jujubogenin and pseudojujubogenin. This jujubogenin and pseudojujubogenin are further enzymatically catalysed by UGTs, forming different bacosides (Kumar et al. 2024; Bhandari et al. 2020). B. monnieri contains different bacosides, a triterpenoidal saponin, which contains the addition of different sugar molecules in the isomer jujubogenin and pseudojujubogenin (Fig. 1).

Fig. 1.

Fig. 1

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The pathway engineering approach to enhance the production of bacosides in Bacopa monnieri plants. Here, we have knocked down the BmG10H-1 using RNAi to block the monoterpenoid biosynthesis and overexpressed the BmSQS-1, diverting the flux towards triterpenoid biosynthesis

Squalene synthase (SQS) is a vital rate regulatory and branch point enzyme in the isoprenoid biosynthetic pathway. It is a potential regulator controlling the carbon sequestration towards triterpenoidal and steroidal saponins (Huang et al. 2007; Upadhyay et al. 2018). G10H is a member of the CYP76B superfamily that catalyses the hydroxylation of geraniol to form 10-hydroxy geraniol, which is a key committed and rate regulatory step in the biosynthesis of monoterpenes (Collu et al. 2001). Ectopic expression of various biosynthetic pathway genes, such as terpene synthases or cyclases, produces pharmaceutically important terpenoids in different plant species. Increasing the total metabolic flux of the triterpene precursors has been widely used to enhance triterpene production in plants (Kempinski et al. 2019). Although B. monnieri is well known for its production of pharmaceutically important triterpenes bacosides, the main disadvantage of this plant is the low availability and post-harvest loss of total bacoside content. The bacoside pathway is not well elucidated, and no prior report of any metabolic engineering approach utilised to enhance bacoside content in this plant is available. A substantial quantity of biomass is harnessed in pharmaceutical preparations due to the plant’s production of comparatively modest concentrations of bacosides. The overall yield of bacosides in B. monnieri is subject to considerable variation influenced by many factors, encompassing plant age, environmental conditions, geographical location, and the specifics of extraction methodologies. Nevertheless, on a broad scale, the average bacoside content within B. monnieri extract is typically confined to 3–6%, representing a notably low proportion (Kunjumon et al. 2023; Deepak et al. 2005). Given these aforementioned constraints, our approach involved the strategic application of metabolic engineering to surmount these limitations and augment the overall bacoside content. We overexpressed the methyl jasmonate (MeJA) and wound-responsive BmSQS-1 and silenced the BmG10H-1 transcript based on their substrate affinity so that the overall metabolic flux diverts towards the enhancement of bacosides (Fig. 1). This further supports our strategy that enhancement in bacoside is due to the increased flux towards triterpene. Moreover, we also studied the effect of enhanced bacoside content on the neuroprotective role in 6-hydroxydopamine (6-OHDA) induced experimental Parkinsonism in rats.

Materials and methods

Plant material, cloning, and enzyme assay of recombinant BmSQS proteins from yeast BY4741 proteins

Fresh cuttings of B. monnieri (var: CIM-JAGRITI) were obtained from our experimental farm and grown under controlled conditions in a glasshouse for two months for experimentation. From our previously published B. monnieri transcriptome data, we were able to isolate the BmSQS and BmG10H transcripts (SRA Study accession number SRP065301). Gene-specific primers were used to amplify the BmSQS transcripts from the cDNA of leaves and roots (Table S2). For protein induction and purification of BmSQS from yeast, positive clones were transformed with standard lithium acetate (LiAc) protocol into yeast Saccharomyces cerevisiae (strain BY4741) (Gietz and Schiest 2007). The recombinant BmSQS-1 and BmSQS-2 proteins in yeast transformants were induced using raffinose and 2% (w/v) galactose incubated at 30 °C. The recombinant protein was purified with nickel affinity agarose beads (Clontech) column as described in Jeena et al. 2021b. The purified protein was resolved on a 10% (w/v) SDS-PAGE gel (Fig. S1). The enzyme activity of BmSQS-1 and BmSQS-2 was determined as described in an earlier study (Su et al. 2017). Formation of squalene by BmSQS-1 and BmSQS-2 in vitro was detected by using GC–MS (Perkin Elmer, Clarus-680GC, SQC-MS, Germany) equipped with a GC-column (Elite 5MS, dimensions 30 m × 0.25 mm, 0.25-µm film thickness) with helium carrier gas at 1.0 ml/min and were executed from m/z ratio of 50 to 700. All incubations were performed in triplicate. The calibration plot of standard squalene (Sigma) at different concentrations was plotted (Fig. S11) to quantify the amount of squalene.

Agrobacterium-mediated genetic transformation of B. monnieri for generation of BmSQS-1 (OE) and BmG10H-1 (KD) lines

B. monnieri plants were transformed using Agrobacterium tumefaciens (GV3101 strain), harbouring the cloned BmSQS-1 in a pBI-121 binary vector. The transformation protocol was followed as described by Jeena et al. 2021a (Fig. S2a). In addition, Kinetin (2.5 mg/l) supplemented MS media promoted root development and shoot elongation (Fig. S2b). For the preparation of the silencing construct in the pGSA1131 vector, the target site was selected from the unique region of the BmG10H-1 transcript. The target site was amplified using a forward primer containing SpeI and AscI restriction sites and a reverse primer containing BamHI and SwaI restriction sites (Table S2) (Mishra et al. 2016). The target site was cloned immediately after the CaMV35S promoter in both sense and antisense orientation, separated by a GUS spacer (Fig. S3). DNA sequencing finally confirmed the successful generation of BmG10H-1 RNAi constructs (Jeena et al. 2021b). We have four positive co-transformants with overexpression of BmSQS-1 and silencing of BmG10H-1. These four lines are hardened and maintained in the greenhouse for further experiments.

Quantification of squalene, 10-hydroxy geraniol, and bacosides in B. monnieri

To estimate squalene in plant samples, 1 g of fresh shoot tissue was crushed in liquid nitrogen and suspended in 1 ml of the solvent containing hexane: ethyl acetate (85: 15) ratio. For extraction of squalene, the mixture was incubated overnight at 28 °C under continuous shaking. The extract was centrifuged at high speed for 10 min, and the supernatant was passed with filter paper to avoid unwanted particles in the crude extract. The filtered crude extract was passed under a nitrogen gas stream to evaporate the hexane: ethyl-acetate solvent. The concentrated extract was finally suspended in the hexane, and GC–MS was performed as described above. For the detection of 10-hydroxy geraniol content in the B. monnieri plant samples (1 g fresh tissue crushed in 1 ml of methanol), HPLC system Waters 2695 equipped with a Hypersil ODS column (250 × 4.6 mm, the particle size of 5 μm, Thermo Scientific) was used along with a photodiode array detector. Extracted fractions were filtered through a nylon membrane of pore size 0.45 mm under reduced pressure. The sample was separated isocratically with methanol: water (60:40) at a 1.0 ml/min flow rate. The analysis was performed at a UV absorbance of 210 nm. The 10-hydroxy geraniol (Sigma) was used as a reference standard (1 mg/ml).

Quantification of bacoside A, bacoside A3, and bacopaside II in B. monnieri using HPLC

Shoot parts of BmSQS1 (OE) and BmG10H1 (KD) (BmSQS1(OE)-BmG10H1(KD)) lines and vector control plant were collected from the glass house. One gram of fresh sample was crushed in liquid nitrogen using an ice-cold mortar pestle to make a fine powder. We used the extraction solvent of methanol/water (80/20v/v; HPLC-grade, Merck, Germany). Afterwards, the extraction was carried out according to Taamalli et al. 2015 with minor modifications. The finely crushed powder was dissolved in 10 ml extraction solvent and kept on the rotatory shaker for 6 h. Subsequently, after 10 min of centrifugation at 6000 RPM, the supernatant was transferred to a fresh tube. This process was repeated three times, and all supernatants were collected and further evaporated using a rotatory evaporator at 35 °C and low pressure. The remnant was then reconstituted in 2 ml LC–MS grade methanol and stored at − 20 °C. To prepare the samples for HPLC analysis, we centrifuged the sample extracts at 12,000 RPM for 15 min to remove particles. Then, we transferred 200 µl of supernatant in 800 µl HPLC grade methanol (100 mg/ml final conc.) and were filtered via a 0.2-mm nylon syringe filter prior to HPLC analysis. HPLC was performed using waters 2695 equipped with a LiChroCART Purospher-W STAR RP-18 (250 × 4.6 mm, 5 mm particle size) column containing a guard column (Purospher STAR RP 18e 4.0 × 4.0 mm, 5 µm). The analysis was done at a UV absorbance of 205 nm. Bacoside A, bacoside A3 and bacopaside II were used as reference standards (Sigma), and the calibration plot at different concentrations was plotted (Fig. S13). The mobile phase containing the gradient of acetonitrile (A) and 0.05% (v/v) orthophosphoric acid in water (B) was utilised in HPLC. A total of 2 µl of filtered methanolic extract was syringe injected into the HPLC system. Isocratic methanol: water (60:40) separation at a 1.0 ml/min flow rate was used to purify the sample.

Liquid chromatography-mass spectrometry (LC–MS/MS analysis) to identify and relatively quantify bacosides

To prepare the samples for LC–MS/MS, 200 µl of the above-extracted sample was supernatant in 800 µl LC–MS grade methanol (100 mg/ml final conc.) and was filtered via a 0.2-mm nylon syringe filter prior to LC–MS/MS analysis. A liquid chromatographic system hyphenated with an AB SCIEX TripleTOF®5600 mass spectrometer (AB Sciex, USA) was used for the LC–MS analysis. Gradient elution of mobile phase A (water with 0.1% formic acid, v/v) and B (acetonitrile with 0.1% formic acid, v/v) was used on C18 column (100 × 2.1 mm 1.7 µm, Phenomenex, USA) to achieve the chromatographic separation at a temperature of 40 °C. The separation gradient was set as solvent B 10–80% from min 0–15, which was brought back to the initial composition of 10% B at min 15.5 and continued till min 18. Source parameters for the mass analysis were optimised as 40, 40, and 25 psi for nebuliser, drying, and curtain gases, respectively. Data was recorded in the positive ionisation mode (ESI +) at source temperature 600 °C and IS voltage 5000 V with declustering potential (DP) and collision energy (CE) values set at 100 V and 5 V, respectively. Software Analyst 1.8, MultiQuant 2.0, and MarkerView 3.1.1 were used to analyse the data.

Neurological assessment of BmSQS1(OE)-BmG10H1(KD) lines in comparison to vector control plants

Animals

Adult male Sprague Dawley (SD) rats weighing 200–250 g were obtained from the National Laboratory Animal Centre (NLAC) of CSIR-Central Drug Research Institute, Lucknow, India. Rats were housed in a pathogen-free facility with free access to food and water at a constant temperature (23 °C ± 1) and a 12/12-h light/dark cycle (8:00 am to 8:00 pm) environment. All animal protocols and studies were approved by our Institutional Animals Ethical Committee (IAEC) in accordance with CPCSEA (Committee for the Purpose of Control and Supervision of Experiment on Animals) criteria that comply with international norms of INSA (Indian National Science Academy).

6-Hydroxydopamine (6-OHDA) lesioning

6-OHDA lesioning was carried out in rats, as described in our previous publication (Singh et al. 2017a, b). Animals were anaesthetised with a combination of ketamine (80 mg/kg) and xylazine (10 mg/kg) before being mounted on a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). A 30-gauge needle-fitted Hamilton syringe (Hamilton Company, Switzerland) was used to unilaterally inject 6-OHDA (16 μg; Sigma Aldrich, St. Louis, USA) dissolved in 0.02% ascorbic acid into the right medial forebrain bundle (MFB), using coordinates related to Bregma, anterior–posterior (AP) = + 4.4, medial–lateral (ML) = + 1.2, dorsal–ventral (DV) = − 7.8 mm, at a flow rate of 0.3 μl/min. After injecting 6-OHDA, the needle was maintained in place for an additional 5 min to prevent reflux.

Behavioural tests

Open-field activity test, rotarod test, and social interaction test

Rats were evaluated in the OptoM-3 open-field arena of Optovarimeax (Columbus Instruments, USA) to measure locomotor activity. To evaluate horizontal and rearing activity, each animal was placed into a locomotor box (42.5 cm × 42.5 cm). Rats were acclimated for 10 min before the experiment, and locomotor activity was monitored for 30 min, with the distance travelled being recorded. The open-field arena was periodically swabbed with 10% alcohol before the addition of the following animal to the locomotor boxes in order to prevent odour interference from the preceding animal (Mishra et al. 2019). Rotarod apparatus (Rotamex-5, Columbus Instruments, USA), set at 20 rpm, was used to assess the neuromuscular coordination for 5 min. The animals underwent 3 days of training to stay on the rotating rod before receiving 6-OHDA injections to ensure they could stay upright for 300 s. The final test was carried out on 14 and 21 days, post-6-OHDA injection, and latency to fall was observed.

As one of the non-motor symptoms of Parkinson Disease (PD), a social interaction test was used to examine sociability in rats (Crawley 2004). The test was carried out on days 7, 14, and 21 after injection of 6-OHDA in a rectangular box made of transparent plexiglass with three compartments (Length -112 cm, Width -56 cm, and Height- 40 cm) and a dividing wall with small retractable portals allowing free access to all three compartments. The experiment was divided into two phases. The rat was placed in the central chamber and given five minutes to explore all three chambers during the habituation phase. During the sociability phase, a stranger rat similar to the test rat was placed in a chamber with an inverted wire cup on the socially isolated chamber. The experimental rat was free to move around the arena and interact with conspecific (unfamiliar) rats for ten minutes. The time spent by experimental rats engaged in close proximity in terms of active interaction was measured by a blind observer. Before introducing the next animal, the arena was swabbed with 10% alcohol.

Immunohistochemistry (IHC)

As per our lab’s previously published methodology, IHC was carried out (Singh et al. 2018; Singh et al. 2017a, 2017b). Rats were euthanised by using an overdose of pentobarbital sodium (100 mg/kg, ip) and perfused transcardially with 0.9% normal saline, followed by 4% ice-cold PFA (paraformaldehyde) in phosphate buffer saline (PBS). In brief, the brain was cryoprotected in sucrose solution at three different concentrations (10%, 20%, and 30%) for three consecutive days. Using a cryostat (Thermo Scientific, USA), serial coronal Sects. (30 μ thick) were cut, and sections encompassing the striatum and substantia nigra were taken for immunohistochemistry examination. Next, sections were incubated in tris buffer saline in Tween-20 (TBST) for 30 min at room temperature and kept with 5% bovine serum albumin (BSA) for 2 h at room temperature. After washing, sections were incubated overnight at 4 °C in rabbit anti-tyrosine Hydroxylase (TH) antibody (1:400, Millipore, USA) as the primary antibody, followed by incubation with Alexa Fluor-594 conjugated (1:400, Invitrogen, USA) secondary antibody for 2 h at room temperature. The slices were mounted on slides with Fluoroshield diamidino phenylindole (DAPI) (Sigma Aldrich, USA), which stain DNA Images were acquired using 20 × objective employing a Leica inverted fluorescence microscope containing a digital CCD camera (Leica, Watzlar, Germany) and analysed using ImageJ software (NIH (Singh et al. 2018).

Drug Administration

Animals were assigned to subsequent groups at random. Group I—Control group received 0.02% ascorbic acid (2 µl) in the mid-forebrain bundle (MFB) stereotaxically. Group II received 6-OHDA (16 µg in 2 µl) in the MFB. Group III—6-OHDA lesioned rats received vector control extract (250 mg/kg, p.o.), 7 days post-6-OHDA administration for 21 days. The dose was selected based on the dose-dependent effect of vector control on the behavioural paradigm (supplementary data). Group IV—6-OHDA lesioned rats received BmSQS1(OE) + BmG10H1(KD)-L1 extract (50 mg/kg, p.o.), 7 days post-6-OHDA administration for 21 days. Group V—6-OHDA + [BmSQS1(OE) + BmG10H1(KD)]-L1 (100 mg/kg, p.o.) extract in 6-OHDA lesioned rats, 7 days after 6-OHDA administration for 21 days. Group VI—6-OHDA lesioned rats received L-DOPA treatment (25 mg/kg, p.o.) 30 min after benserazide administration (15 mg/kg. i.p) and 7 days post-6-OHDA administration for 21 days. All drug administration started from the seventh day of 6-OHDA lesioning.

Statistical analysis

There were two biological and experimental replicates of each experiment in the study. The study presents data as mean ± SD. To determine statistical significance, one-way ANOVA was run in GraphPad Prism (version 9.5) software; results with a p-value ≤ 0.05 are considered significant and are indicated by an asterisk above the relevant bar graph. With a p-value ≤ 0.01, a double asterisk denotes an increased significance level. For animal studies, results with a p-value ≤ 0.05 are considered significant and are indicated by different symbols according to the comparison between the groups above the relevant bar graph.

Results

Overexpression of BmSQS-1(OE) and RNAi-mediated gene silencing of BmG10H-1(KD) redirects metabolic flux to triterpene biosynthesis

Based on our earlier published transcriptome data, we have selected two transcripts of BmG10H (BmG10H-1 and BmG10H-2) that might be involved in monoterpene indole alkaloid biosynthesis (Jeena et al. 2017). In vitro enzyme assays demonstrated that BmG10H-1 has a higher Vmax than BmG10H-2 (Jeena et al. 2021b). The silencing construct was then transformed in Agrobacterium tumefaciens (GV3101) and followed by confirmation through colony PCR. The BmG10H-1 silencing lines were generated in the background of BmSQS1 overexpression lines of B. monnieri using tissue culture transformation protocol. BmSQS1 and BmG10H1 protein expression and enzyme analysis were performed to optimise the substrate concentration, optimum pH, and temperature. The substrate saturation curve looks allosteric, which shows that BmSQS-1 is enzymatically more active than BmSQS-2, having a higher affinity with its substrate FPP at the optimum concentration (Table S1). Multiple shoot regeneration and elongation were observed in the selection media supplemented with BAP and Kinetin (Fig. S2a, b). Overexpression and silencing lines of BmSQS-1 and BmG10H-1 were confirmed by qRT-PCR using gene-specific primers (Table S2). The increased transcript level of BmSQS-1 and reduced transcript of BmG10H-1 were observed in the overexpression and silencing lines of B. monnieri compared to the vector control (VC), which confirms the successful generation of co-transformants (Fig. 2a). To determine whether the silencing of rate regulatory enzyme BmG10H-1 diverts the metabolic flux towards triterpene biosynthesis, we quantified total squalene and 10-hydroxygeraniol content in different transgenic lines. In the GC–MS, peaks generated from the extract were compared with a significant peak corresponding to standard squalene for the same retention time (11.8 min). A significantly higher peak intensity of squalene was found in the co-transformed transgenic line compared to the VC. However, there is a slight increase in the peak intensity of squalene in the overexpression line of BmSQS-1 compared to the VC (Fig. S4a). Further, the MS spectra analysis confirmed the compound peak squalene was the same as that of the standard (m/z ratio of squalene is 69) (Fig. S5). The plant’s total squalene content was quantified using the relative peak area obtained from the GC chromatogram. It was found that the squalene content was significantly increased (4 folds) in the co-transformed transgenic lines. In contrast, in the BmSQS-1 overexpression lines, there is only a twofold increase compared to the VC (Fig. 2b). Moreover, we also determined the 10-hydroxy geraniol content in the co-transformed transgenic lines using HPLC. The corresponding peaks generated from the methanolic plant extract were compared with the standard 10-hydroxy geraniol at the same retention time (4.59 min). HPLC data showed reduced peak intensity (up to 50%) in the co-transformed transgenic lines compared to the VC (Fig. S4b). Further, we quantified the 10-hydroxy geraniol content in the co-transformed transgenic lines and found that it is significantly reduced (2 folds) compared to the VC (Fig. 2c).

Fig. 2.

Fig. 2

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Expression and metabolite analysis of the B. monnieri co-transformants. a qRT-PCR was used to examine the expression of BmSQS-1 and BmG10H-1 transcripts in different co-transformant lines. The experiments were carried out in two independent biological and experimental triplicates, and the results were normalised relative to actin and ubiquitin as endogenous controls. The error bars indicate mean ± SD. ANOVA, **, p < 0.01. b Total squalene content (μg/g of fresh weight) was determined in the VC and transgenic lines. c Total 10-hydroxy geraniol content (μg/g of fresh weight) was determined in the VC and co-transformed transgenic lines. The experiments were performed in two independent biological and experimental triplicates. Error bars indicate mean ± SD. ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Quantification of bacoside confirms their enhancement in BmSQS1(OE)-BmG10H1(KD) lines

These co-transformants were analysed by high-performance liquid chromatography (HPLC) to determine the content of bacoside A, bacoside A3 and bacopaside II. For the same retention time, the peaks generated by the plant extract were compared to the corresponding peaks of the bacoside A, bacoside A3, and bacopaside II analytical standards (Fig. S13). The total peak area was calculated and compared to quantify the bacoside A, bacoside A3, and bacopaside II content in co-transformed transgenic plants compared to the VC (Fig. 3; Table 1). Quantification of bacosides in BmSQS1(OE)-BmG10H1(KD) line1 and line 2 revel 332.79 ± 28.41 µg/g fresh weight and 356.93 ± 25.34 µg/g fresh weight of bacoside A3 and 562.87 ± 35.79 µg/g fresh weight and 584.24 ± 35.40 µg/g fresh weight of bacopaside II, respectively which was found to be significantly high compared to vector control plant (bacoside A3, 118.45 ± 14.21; and bacopaside II, 226.13 ± 17.46) (Table 1). We also found that bacoside A was significantly increased in the co-transformed transgenic lines (up to 2.6 to 2.8 folds) compared to the VC (Fig. 3 g; Table 1). These results showed that overexpression of shoot-specific BmSQS-1 and silencing of BmG10H-1 resulted in enhanced production of bacoside A, bacoside A3, and bacopaside II content in B. monnieri.

Fig. 3.

Fig. 3

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Absolute quantification of bacoside content in BmSQS1(OE)-BmG10H1(KD) lines. Quantification of bacoside A, bacoside A3, and bacopaside II content (µg/g fresh weight) in two different co-transformed transgenic lines of B. monnieri was compared with the vector control. HPLC chromatograms of a crude methanolic extract of BmSQS1(OE)-BmG10H1(KD) line 1, b crude methanolic extract of BmSQS1(OE)-BmG10H1(KD) line 2, c crude methanolic extract of vector control line, d standard bacoside A3, e) standard bacopaside II, and f bacoside A. Quantification of g bacoside A, h bacoside A3, and i bacopaside-II content in two different co-transformed transgenic lines of B. monnieri. The experiments were performed in three independent biological and experimental replicates. Error bars indicate mean ± SD. ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Table 1.

Bacoside quantification in BmSQS1(OE)-BmG10H1(KD) line1 and line2 along with vector control plants. All the experiments are proceeded in biological triplicate (n = 3) and experimental replicates

Quantification of bacosides
Bacoside Vector Control (in µg/g FW) BmSQS1(OE)-BmG10H1-(KD)-L1 (in µg/g FW) BmSQS1(OE)-BmG10H1-(KD)-L2 (in µg/g FW)
Bacoside A 631.38 ± 60.75 1621.07 ± 190.66 1766.76 ± 102.60
Bacoside A3 118.45 ± 14.21 332.79 ± 28.41 356.93 ± 25.34
Bacopaside II 226.13 ± 17.46 562.87 ± 35.79 584.24 ± 35.40
Relative quantification of bacosides in co-transformed lines compared to vector control
Bacoside BmSQS1(OE)-BmG10H1-(KD)-L1 (in Fold change) BmSQS1(OE)-BmG10H1-(KD)-L2 (in fold change)
Bacopaside N1/N2 2.67 2.98
Bacopaside N1/N2 2.34 2.69
Bacopaside I 2.30 2.47
Bacopasaponin B/G 1.94 2.06
Bacopasaponin B/G 2.37 2.24
Bacopaside X/Bacopasaponin C 1.80 2.36
Bacopaside X/Bacopasaponin C 1.64 1.90
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Identification and relative quantification of different bacosides using LC–MS/MS suggest increased content in BmSQS1(OE)-BmG10H1(KD) lines

We have further performed the identification and relative estimation of bacosides using LC–MS/MS analysis in these BmSQS1(OE)-BmG10H1(KD) lines in comparison with the vector control plants. The analysis of the MS/MS fragmentation patterns led to the final identification of the metabolites. After comparing the TIC chromatogram of BmSQS1(OE)-BmG10H1(KD) lines with vector control, apart from bacoside A3 and bacopaside II, seven differential peaks were found at retention time (RT) 8.0 min, 8.2 min, 8.24 min, 8.3 min, 8.5 min, 9.0 min, and 11.4 min, which were enhanced in BmSQS1(OE)-BmG10H1(KD) lines compared to vector control plants (Fig. S6). After analysing the MS/MS fragmentation patterns, the metabolites were identified as bacopaside N1 and N2 (RT 8.0 and 11.4 min, m/z 473.3856, 635.4498, 797.5091), bacopaside I (RT 8.2 min; m/z 437.3651, 455.3776, 979.5405, 997.5537), bacopasaponin B and G (RT 8.25 and 8.5 min; m/z 473.3529, 605.3911, 737.4320), and bacopaside X and bacopasaponin C (RT 8.3 and 9.0 min; m/z 473.3529, 605.3918, 767.4416, 899.4834) (Fig. S7 and S14). The absolute identity of bacosides like bacopaside N1/N2, bacopasaponin B/G, and bacopasaponin C/bacopaside X, being the structural isomers having similar molecular masses and mass fragmentation patterns, was left ambiguous. The relative amounts of these bacosides were found to be enhanced in BmSQS1(OE)-BmG10H1(KD) lines compared to vector control (Fig. 4). The enhanced content of identified bacosides was further confirmed by MarkerView software using a T-test (Fig. S8). GC–MS analysis of hexane extract of the co-transformed line was performed to check whether the diversion of flux leads to alteration in other metabolite contents. Five differential peaks were observed in vector control as compared to co-transformed BmSQS1(OE)-BmG10H1(KD) transgenic lines. However, those peaks remain unidentified (Fig. S9 a, b, c). These results showed that overexpression of BmSQS-1 and silencing BmG10H-1 are able to divert the flux towards triterpene biosynthesis and enhance the relative bacoside content in B. monnieri.

Fig. 4.

Fig. 4

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LC–MS/MS data analysis of a methanolic extract of BmSQS1(OE)-BmG10H1(KD) co-transformants. Relative quantification of a bacopaside N1/N2, b) bacopaside N1/N2, c bacopaside I, d bacopasaponin B/G, e bacopasaponin B/G, f bacopaside X/bacopasaponin C, and g bacopaside X/bacopasaponin C. All the relative quantification was performed in two independent biological and experimental replicates. Error bars indicate mean ± SD. ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001

Enhanced bacoside content in BmSQS1(OE)-BmG10H1(KD) transgenic lines improves locomotor activity, neuromuscular coordination, and sociability in rats

Locomotor activity

Some reports have claimed that a single 6-OHDA injection into MFB triggers behavioural impairment in rats. We, therefore, performed a battery of behavioural tests to compare the effects of both BmSQS1(OE)-BmG10H1(KD)-L1 and vector control extract on 6-OHDA-induced behavioural deficits in rats. First, we conducted an open-field activity test to measure locomotor activity using Opto-Varimex over 30 min on days 7, 14, and 21. When compared to the control (gum acacia-treated rats), 6-OHDA treatment significantly decreased the total distance travelled on the 14th (S12B.b p < 0.05) and 21st (Fig. 5a p < 0.01) days. In contrast, rats treated with BmSQS1(OE)-BmG10H1(KD)-L1 extract (at two different dosages of 50 mg/kg and 100 mg/kg, p.o.) and vector control extract (at 250 mg/kg, p.o.) which was chosen based on behavioural experiments carried out using various doses (Fig. S12A) after 6-OHDA lesioning; we found that extract of co-transformed B. monnieri lines enhanced total distance travelled compared to vector control B. monnieri extract in 6-OHDA lesioned rats. Our findings indicate that BmSQS1(OE)-BmG10H1(KD)-L1 extract is more potent and effective than vector control extract at the dose of 50 mg/kg on both days 14 (Fig. S12 B.b, p < 0.001) and day 21 (Fig. 5a p < 0.01). Similarly, rats treated with L-DOPA showed significantly higher distance travelled than rats treated with 6-OHDA alone (Fig. 5a, p < 0.05).

Fig. 5.

Fig. 5

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Treatment with enhanced bacoside content improved locomotor activity, neuromuscular coordination, and sociability behaviour after 6-OHDA lesioning in rats. a The bar graph shows a fold change in the distance travelled in open field activity on the 21st day. b The bar graph shows the fold change of latency to fall on the 21st day during the assessment of neuromuscular coordination. c The bar graph shows the fold change in interaction time during the sociability test on the 21st day. Data are expressed as mean ± SEM of n = 9–10 rats/group. Data were analysed by one-way ANOVA followed by Fisher’s LSD test (*p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01, ###p < 0.001, &p < 0.05, &&p < 0.01, &&&p < 0.001, $p < 0.05, $$p < 0.01, $$$p < 0.00, @p < 0.05, @@p < 0.01, @@@p < 0.0011) * = control vs. 6-OHDA, # = 6-OHDA vs. 6-OHDA + BmSQS1(OE)-BmG10H1(KD)-L1 (50 mg/kg) & = 6-OHDA vs. 6-OHDA + BmSQS1(OE)-BmG10H1(KD)-L1 (100 mg/kg), $ = 6-OHDA vs. 6-OHDA + Vector control (250 mg/kg), @ = 6-OHDA vs. 6-OHDA + L-DOPA (25 mg/kg). d The bar graph shows the number of TH+ cells in the striatum. e The bar graph shows the number of TH+ cells in the SNpc region. Data are expressed in ± SEM of n = 4 rats/group and analysed by one-way ANOVA followed by Tukey test

Neuromuscular coordination

In order to assess possible motor alterations induced by 6-OHDA and the protective role of extracts that could modulate the motor performance in rats, we recorded latency to fall in the rotarod apparatus at three-time intervals on days 7, 14, and 21 after the B. monnieri extract administration. 6-OHDA administration significantly reduced latency to fall when compared to control on the 7th (Fig. S12C.a, p < 0.001), 14th (Fig. S12C.b, p < 0.001), and 21st day (Fig. 5b, p < 0.001), as compared to control rats. Further, to compare the efficacy and potency between BmSQS1(OE)-BmG10H1(KD)-L1 M-BME and vector control, rats were treated with BmSQS1(OE)-BmG10H1(KD)-L1 (at two different doses of 50 mg/kg and 100 mg/kg, p.o) and vector control (at 250 mg/kg; p.o. Fig. S12A) after 6-OHDA lesioning, and we found all the B. monnieri extract doses were able to significantly enhance latency to fall on days 14 and 21 as compared to 6-OHDA lesioned rats. However, our results show BmSQS1(OE) + BmG10H1(KD)-L1 extract at a dose of 50 mg/kg to have higher potency and efficacy as compared to vector control extract (Fig. 5b p < 0.01). Likewise, L-DOPA treatment significantly enhanced latency to fall after 6-OHDA lesioning as compared to 6-OHDA-lesioned rats (Fig. S12C.b and Fig. 5b, p < 0.001 on day 14 and 21, respectively).

Social interaction

Next, we assessed the effect of B. monnieri extract on sociability behaviour in 6-OHDA lesioned rats; interaction time was recorded on three time points on days 7, 14, and 21. 6-OHDA administration significantly reduced interaction time as compared to control on the 7th (Fig. S12D.a, p < 0.001), 14th (Fig. S12D.b, p < 0.05), and 21st day (Fig. 5c, p < 0.001) as compared to control (gum acacia treated) rats. Further rats treated with BmSQS1(OE)-BmG10H1(KD)-L1 and vector control, after 6-OHDA lesioning, showed enhanced interaction time on day 21 (Fig. 5c, p < 0.05) as compared to 6-OHDA lesioned rats, suggesting that BmSQS1(OE)-BmG10H1(KD)-L1 extract has higher potency and efficacy as compared to vector control. Similar results were observed with L-DOPA treatment that significantly enhanced interaction time after 6-OHDA lesioning as compared to 6-OHDA-administered rats (Fig. 5c, p < 0.001) on day 21.

BmSQS1(OE)-BmG10H1(KD) extract enhances dopaminergic neuronal population

Tyrosine hydroxylase (TH) immunostaining was performed to examine the effect of Bacopa extract treatment on the dopaminergic neuronal population in the striatum (Fig. 5d and S10.a) and substantia niagra pars compacta (SNpc) (Fig. 5e and S10 b). We observed significantly decreased TH intensity and TH+ cells in striatum and SNpc brain regions in rats with 6-OHDA respectively (Fig. 5d, e, p < 0.0001), whilst treatment with vector control (250 mg/kg, p < 0.0001) and BmSQS1(OE)-BmG10H1(KD)-L1 (50 mg/kg, p < 0.0001) after 6-OHDA neurotoxicity in rats, enhanced TH intensity and TH+ cells in both brain regions suggesting that Bacopa extract attenuated 6-OHDA-induced dopaminergic depletion. In a comparative view, BmSQS1(OE)-BmG10H1(KD)-L1 extract-treated rats had a higher TH + dopaminergic neuronal population and intensity in comparison to vector control extract. Similarly, positive control L-Dopa treatment also significantly showed a neuroprotective effect in terms of ameliorating dopaminergic loss in Parkinsonian rats.

Discussion

B. monnieri is well-regarded in the cure of numerous neurological deficits due to the presence of pharmaceutically important bacosides (Farooqui et al. 2018). The major limitation associated with this important medicinal plant is the low availability and post-harvest loss of bacoside content. To enhance the pharmaceutically important metabolite, mainly the bacoside content in B. monnieri, we target the important rate-limiting enzyme of the triterpenoidal pathway. We engineered the bacoside biosynthesis by overexpressing MeJA and wound-responsive BmSQS-1 transcript and silencing the BmG10H-1 having higher substrate affinity so that the overall metabolic flux gets diverted towards the biosynthesis of triterpenoidal saponins. B. monnieri extract defend the brain cells against oxidative damage and age-related disorders (Witter et al. 2018; Saini et al. 2019). Therefore, we studied the outcome of enhanced bacoside content against Parkinson’s disease using rat models.

Based on the higher substrate affinity with FPP, we utilised BmSQS-1 protein for further study. The SQS protein has been proven to have a wide range of Vmax values, indicating that SQS has different substrate binding affinity in different plant species (Huang et al. 2015). Different plant species were studied for a positive correlation between SQS overexpression and the amount of triterpenes produced. For example, overexpression of SQS from Panax ginseng resulted in the enhanced production of triterpene ginsenoside in the adventitious roots, and overexpression of SQS from Withania somnifera increases the production of withanolides (Lee et al. 2019; Grover et al. 2013). The co-transformation of squalene epoxidase and terpene cyclase genes from Cucurbita pepo increases the biosynthesis of various triterpenes in yeast and plants (Dong et al. 2018). In a recent study, three OSCs were isolated and characterised from Tripterygium wilfordii, involved in celastrol biosynthesis, and enhanced the production of friedelin in engineered yeast (Zhou et al. 2019). Coexpression of HMG-CoA reductase and biotin carboxyl carrier protein in Nicotiana benthamiana enhanced the production of both sesquiterpenes and triterpenes (Lee et al. 2019). The overexpression of FPPS and silencing of the CAS gene enhanced the production of triterpenoidal saponins in the Panaxnoto ginseng (Yang et al. 2017). Similarly, our study utilised the novel metabolic engineering approach by overexpressing BmSQS-1 and silencing the BmG10H-1 transcript to divert the total metabolic flux towards bacoside biosynthesis. We found a total fourfold increase in the squalene content and a twofold decrease in the 10-hydroxy geraniol content in the co-transformed transgenic lines, whereas there is only a twofold increase in the squalene content in only BmSQS-1 overexpression lines indicating that silencing the BmG10H-1 plays an important role in diverting the metabolic flux towards squalene biosynthesis which further enhances the bacoside content in B. monnieri.

We further explored the effects of B. monnieri extract (BME) with enhanced bacoside content compared to the vector control in the 6-OHDA-induced rat model of Parkinson’s disease. Previous reports have shown the neuroprotective role of Bacopa extract in different neurodegenerative disorders (Singh et al. 2021; Islam et al. 2020). Similarly, our results show improved locomotor activity, neuromuscular coordination, and social behaviour in the genetically modified Bacopa extract, BmSQS1(OE)-BmG10H1(KD)-L1 treated rats, reflecting that it may be useful as a therapeutic intervention. Dopaminergic neurons are essential for motor function and are often damaged in Parkinson’s disease; hence, to further explore the effect of the modified extract on the dopaminergic neuronal population, we performed immunohistochemistry in brain tissue sections to identify the neuronal population expressing the rate-limiting enzyme of dopamine biosynthesis pathway, tyrosine hydroxylase (TH). The increased bacoside leaf extract treatment in rats significantly lessened the dopaminergic depletion caused by 6-OHDA. These findings are supported by earlier studies that indicate the neuroprotective effects of bacoside in both in vivo and in vitro models (Sekhar et al. 2019). Bacosides contain anti-inflammatory, anti-apoptotic, and antioxidant properties that may help maintain neurons’ health (Fatima et al. 2022). The enhanced BmSQS1(OE)-BmG10H1(KD)-L1 extract potency and therapeutic efficacy increased due to increased bacoside content in engineered plants. The findings of this study offer a compelling case for future research to identify the precise molecular pathways behind its neuroprotective benefits and assess its long-term safety and effectiveness in animal models.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 4331 KB) (4.2MB, pdf)

Acknowledgements

The plants used in this study were obtained from the national gene bank of the Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow. GSJ, SK, AJ, and NS acknowledge CSIR-UGC for fellowship. CSIR-CIMAP publication communication number for this manuscript is CIMAP/PUB/2023/04.

Author contribution

GSJ has performed the cloning and characterisation of Squalene synthase, enzyme assay, plant transformation, and analysis for squalene and geraniol. SK has performed the sub-cloning of the BmSQS-2 transcript in pYES-2. SK has cloned G10H-2 in pYES2.0 NTB and purified the recombinant protein G10H-2, HPLC analysis of extract. SK, RM, VL, and AKY have analysed the LC–MS/MS data. SK has made LC–MS/MS figures for the bacoside analysis. NS, AJ, maintained the plants and prepared the extract for analysis. SK, NS, and AJ have isolated the extract in a replicate and sent it for animal experiments. SB has performed and analysed in vivo experiments in SD rats. SS has planned and supervised in vivo experiments in rats. VT was involved in the planning of experiments. SK, NS, and AJ have modified the draft manuscript and its figures. RKS has supervised the experiments and edited the manuscript. All authors finally read and approved the manuscript.

Funding

This study was funded by CSIR-FBR MLP005. RKS would like to thank the CSIR and the SERB for funding support to his lab.

Data availability

BmG10H-1- MT438687, BmG10H-2- MT438686, BmSQS-1- MT446023, BmSQS-2- MT438685, BmSQS-1 promoter- MT438690, β-Actin- MW389548, E3-Ubiquitin ligase- MW389549, SRA Study accession number SRP065301.

Declarations

Ethical approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed.

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Gajendra Singh Jeena and Sunil Kumar contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (PDF 4331 KB) (4.2MB, pdf)

Data Availability Statement

BmG10H-1- MT438687, BmG10H-2- MT438686, BmSQS-1- MT446023, BmSQS-2- MT438685, BmSQS-1 promoter- MT438690, β-Actin- MW389548, E3-Ubiquitin ligase- MW389549, SRA Study accession number SRP065301.

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