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Assessment of bacoside production, total phenol content and antioxidant potential of elicited and non-elicited shoot cultures of Bacopa monnieri (L.)

  • Nupur Jauhari
  • Rakesh Bharadwaj
  • Neelam Sharma
  • Navneeta BharadvajaEmail author
Original Article
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Abstract

Thirteen accessions of Bacopa monnieri (L.) Wettst. were assessed for in vitro propagation, total bacosides, total phenol content and antioxidant potential. Nodal explants were cultured on Murashige and Skoog (MS) medium with 0.4 mg/L benzyl aminopurine (BA) to identify best accession based on growth parameters. The selected accession (IC 554588) was elicited with 1 mg/L of jasmonic acid, salicylic acid or malt extract to assess its efficacy in enhancing secondary metabolites production in in vitro cultures. Elicitor-treated plants showed increased production of bacosides (2.7–3.9 fold), total phenol content (5–18 fold) and higher antioxidant potential (7.9 fold) in 4 weeks. According to statistical analysis, antioxidant potential is highly correlated to total phenol as R-sq (adj) is 90.1%. Data was subjected to correlation, Principal Component Analysis (PCA) and cluster analysis to understand the relationship between different variables and identify major contributors of variability. Strong correlation between total bacoside, total phenol, and antioxidant activity indicate that elevated antioxidant potential was the result of overall increase in bacoside and phenol content, which can be enhanced by the application of elicitors. The study suggested that accessions IC 554588, IC 344312 and IC 554585 are elite and may be utilized for various pharmacopeias after further in vivo research and clinical trials.

Keywords

Antioxidant Bacoside Bacopa monnieri Elicitor Phenols Principal component analysis 

Introduction

Secondary metabolites from herbal plants are important source of medicinal compounds. Pharmacological effects of herbal plants has been identified from earliest annals of human habitancy. Therapeutic properties of plants are attributed to secondary metabolites including antioxidant properties (Chen et al. 2009). Antioxidants support human defense system to fight age-related degenerative diseases and other oxidative damages (Atoui et al. 2005). Synthetically produced antioxidants are commonly being used presently. The major drawback of synthetic antioxidants is their adverse side effects (Chen et al. 1992). Although the living creatures have the capacity to balance the fatalistic effect of free radicals, it is necessary to provide some supplements through diet to maintain the radical concentration to lesser extent. Considering the above facts, consumption of herbal drugs rich in antioxidants is an alternate way to strengthen the antioxidant potential of body. Secondary metabolites of plants offer a safe alternative for antioxidants (Walton and Brown 1999).

Bacopa monnieri (L) Wettst. (Family Scrophulariaceae), commonly known as ‘Brahmi’, ‘Neerbrahmi’ and ‘the thinking person’s herb’, has been reported to possess antioxidant properties (Kapoor 1990). Whole plant is widely used in Ayurvedic formulations since ancient times to enhance memory functions or cognitive functions (Kumar et al. 2016). Brahmi is also utilized as hepatoprotective, cell stabilizer (Sairam et al. 2002), antidepressant, sedative and cardiotonic (Roodernrys et al. 2002). Shahid et al. (2017) suggested the use of B. monnieri in neuropathic pain associated with allodynia and hyperalgesia. These pharmacological effects are attributed to the presence of biologically active secondary metabolites such as brahmins, stigmasterol, beta-sitosterol, triterpenoid saponins, bacosides and bacosaponins (Basu et al. 1967). Researchers have reported the antioxidant property of bacosides (Kapoor 1990; Meena et al. 2012; Alamgir et al. 2014) including free radical scavenging potential (Anbarasi et al. 2005). Bacoside A3 and bacosaponine C acts as inhibitor of superoxide release from the polymorphonuclear cells (Pawar et al. 2001). Madhu et al. (2019) reported the effectiveness of bacoside A against progression of experimental autoimmune encephalomyelitis by reducing neutrophil infiltration and demyelination. The requirement of medically useful secondary metabolites bacosides has increased significantly during last two decades. Demand of this key active herbal principle is increasing continuously as researches have reported its potential to cure various neuro-degenerative diseases and improve cognitive functions. Estimated annual trade of brahmi is 2000–5000 MT with the price range of Rs. 30–35 per kg (NMPB 2019). Unorganized collection and over-exploitation of plant from its natural habitat are responsible for threat to its existence (Verma et al. 2012). Therefore, there is an urgent need to manage important medicinal plants for present and future generations. To facilitate sustainable use, germplasm collection and use of different accessions for plant tissue culture technique is essential. Plant tissue culture technique has tremendous potential for sustainable use of overexploited medicinal plants like B. monnieri. Plant tissue culture based propagation methods play an important role in the production of secondary metabolites (Ahmad et al. 2013). Higher bacoside A content was reported in in vitro cultures of B. monnieri as compared to field grown plants (Sharma et al. 2012). The yield of secondary metabolites is strongly affected by environmental conditions (Gorelick et al. 2014). According to literature, elicitors increase the yield of secondary metabolites (Hussain et al. 2012; Karuppusamy, 2009). Although, several abiotic chemical and plant hormones such as jasmonic acid (JA) (Farmer et al. 2003; Sharma et al. 2015), salicylic Acid (SA) (Ali et al. 2006; Sharma et al. 2015), malt extract (ME) (Kundu et al. 2016) and CuSO4 (Sharma et al. 2015) have been used as elicitors to enhance the secondary metabolite production, their effects are species specific. JA and SA are hormonal elictors involved in the stress response and affect the production of secondary metabolites like terpenoids, flavonoids and alkaloids (Farmer et al. 2003; Shabani et al. 2009). ME can be added as natural mixture in special cases to support culture growth (Mangathayaru 2013).

High therapeutic value of the plant and less availability for future use, justify the sustainable use of B. monnieri. Therefore, investigating different accessions of B. monnieri in order to select accessions with higher bacoside content and strategy to further enhance bacosides for commercial exploitation is desirable.The present study aims to find elite accession with maximum number of shoots, shoot length, maximum bacosides production and highest antioxidant potential. In addition, elicitation of the selected accession with JA, SA or ME for the assessment of their effect on total bacosides, total phenol content and antioxidant potential compared to non-elicited plants has been evaluated in order to achieve high amount of therapeutic principle.

Materials and methods

Plant material

Thirteen accessions of B. monnieri procured from ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR) and maintained in tissue culture laboratory, at Delhi Technological University, Delhi, India were used in the present study. All the accessions belong to different states of India (Table 1).
Table 1

List of B. monnieri accessions used for the study

Number representation

Accession no.

State of collection

Alphabetical representation

Latitude and longitudee

Temperature (°C)

Annual rainfall (mm)

1

IC 439118

Jharkhand

A

23.610°N

85.279°E

26

1397

2

IC 426442

Punjab

B

31.147

75.341

24

200

3

IC 426447

Madhya Pradesh

C

22.974

78.656

32

1017

4

IC 468878

Kerala

D

10.850

76.271

32

3055

5

IC 373640

Kerala

E

10.850

76.271

32

3055

6

IC 344312

Karnataka

F

15.317

75.713

27

1248

7

IC 531621

Jharkhand

G

23.612

85.279

24

1397

8

IC 375976

Jammu & Kashmir

H

34.083

74.797

21

1238

9

IC 353203

New Delhi

I

28.704

77.102

28

800

10

IC 554588

Madhya Pradesh

J

22.974

78.656

32

1017

11

IC 554586

Madhya Pradesh

K

22.974

78.656

32

1017

12

IC 554587

Madhya Pradesh

L

22.974

78.656

32

1017

13

IC 554585

Amarkantak

M

22.682

81.753

24

800

In vitro shoot induction and elicitation

In vitro cultures were maintained on Murashige and Skoog (MS) (Murashige and Skoog 1962) medium supplemented with 0.5 mg/l benzyl aminopurine (BA) (Sharma et al. 2007). For elicitation study, stock solutions were prepared in 90% ethanol for JA and sterile Mili-Q water (Mili-Q, USA) for SA and ME (Sigma-Aldrich USA). Filter sterilized 1.0 mg/l of JA, SA or ME were added to the autoclaved medium. Stem segments 1.0-2.0 cm (2-3 nodes) of selected accession from in vitro propagated plants were transferred to Erlenmeyer flasks (100 mL) containing MS + 0.5 mg/l BA (20 mL) with or without elicitor (control). The cultures were maintained at 25 ± 2 °C under 16-h photoperiod. After 4 weeks the elicitor-treated and non-elicited cultures were evaluated for growth parameters (number of shoots/explant and shoot length), moisture content, antioxidant potential and biochemical parameters for the presence and quantity of major secondary metabolites (total bacosides and total phenol content). All treatments were performed in triplicate and each treatment consisted of 10 samples.

Biochemical analyses

For estimation of total bacosides, total phenol content and antioxidant potential, elicited and non-elicited in vitro cultured B. monnieri accessions were analyzed. Cultures from the following treatments were used for sample preparation: (i) non-elicited 4-week-old cultures of 13 accessions grown on MS + 0.5 BA and (ii) 4-week-old cultures of selected accession grown on MS + 0.5 BA supplemented with elicitors and control (without elicitor).

Sample preparation

Approximately 5 g of green shoots were harvested, shade-dried and crushed to fine powder. The powdered sample was extracted with HPLC grade methanol (Merck Chemicals) using Soxhlet apparatus (5 cycles) at room temperature. The extracted solvent was dried on rotary evaporator and volume was made up to 2 mL. The sample was filtered through 0.22-micron filter and used for estimation of total bacosides, total phenol content and antioxidant potential by 1,1-diphenyl-2-picrylhydrazyl (DPPH) and cupric ion reducing antioxidant capacity (CUPRAC) assays.

Quantification of total bacosides

Total bacosides were quantified through high performance liquid chromatogrpahy (HPLC) using the method described by Murthy et al. (2006) and Mishra et al. (2013). HPLC experiments were performed on Agilent HPLC system equipped with 1260 Infinity Binary Pump (G1312B). ZORBAX ODS C18 column (5 μm 4.6 × 250 mm), 1260 infinity diode array detector (G4212B). The mobile phase consisted of sodium acetate buffer and acetonitrile 0.1 M (65:35 v/v), pH 3.2 was adjusted using acetic acid. The flow rate was 0.5 mL/min for isocratic elution of bacosides with 30 min run time. Detection was done at 205 nm and sample injection volume was kept at 2.5 µL. Bacoside mixture (1 mg) (Sigma-Aldrich) comprising of bacoside A, bacoside A3, bacoside II, bacoside X and bacosaponin C was dissolved in 2 mL methanol and used as a standard solution to quantify total bacosides.

Linearity and precision

Standard curve was drawn by injecting different concentrations of bacoside. Linear standard curve with R2 > 0.9 was obtained. Good precision based on three replications was observed. Standard deviation within the reasonable level was noticed.

Limit of detection and limit of quantification

Limit of detection and limit of quantification were calculated 0.0306 µg/5 mL and 0.0918 µg/5 mL respectively.

Determination of total phenol content (TPC)

TPC was estimated by Folin-Ciocalteu (FC) reagent using gallic acid as standard solution (Singleton et al. 1999) with slight modifications. 100 µL of the extract was added to 900 µL of distilled water and 200 µL of 1 N FC reagent and 2.0 mL of sodium carbonate (7% w/v). Contents were mixed and allowed to stand for 30 min at room temperature (25 ± 1 °C) in dark. Absorbance was measured at 750 nm using UV–Vis spectrophotometer (LAMBDA 25 UV/Vis Systems, PerkinElmer, Inc. MA 02451, USA). Total phenol content was expressed as µg/g gallic acid equivalent (GAE).

Determination of antioxidant potential

Antioxidant potential of samples was assessed using DPPH and CUPRAC assay.

DPPH assay was done according to Brand-Williams et al. (1995) with slight modification. DPPH assay was performed by pipetting out 800 µL (100 mM) of Tris HCl buffer (pH 7.4) to which 200 µL of standard solution (gallic acid) of varying concentration was added. Different volumes of sample extract were dried under nitrogen and redissolved in 200 µL of methanol, which was added to 0.8 mL of DPPH reagent. The reaction mixture was vortexed and left for 30 min in dark. Absorbance was recorded at 517 nm using UV–vis spectrophotometer (LAMBDA 25 UV/Vis Systems, PerkinElmer, Inc. MA 02451, USA) with methanol as the blank.
$$50\% {\text{Inhibition}}\, = \,\left\{ {A \, \left( {\text{control}} \right){-}A \, \left( {\text{sample}} \right)/A \, \left( {\text{control}} \right)} \right\}\, \times \,100$$
where, A (control) is absorbance of blank control (containing all reagents except the extract) and A (sample) is absorbance of the test sample.

For CUPRAC assay method of Apak et al. (2004) was used. In reaction mixture 1 mL CuCl2 solution (0.01 M), 1 mL neocuproine alcoholic solution (7.5 mM), 1 mL ammonium acetate buffer (1.0 M) of pH 7.0, 100 µL extract and 1 mL water were added (total volume, 4.1 mL). Absorbance against reagent blank was measured at 450 nm after 30 min.

Statistical analysis

Data for in vitro propagation and elicitation was analyzed using ANOVA with SPSS version 21.0 statistical software package. Results were expressed in terms of means and standard error, while means were compared with Duncan’s multiple range test (DMRT, 9) at P < 0.05 level. Analysis of variance was carried out using PROC GLM to determine significant differences in parameters studied among B. monnieri accessions. Multilinear regression (MLR) has been used for statistical analysis to check the correlation among total bacoside, total phenol and antioxidant potential.

Simple linear correlation analysis was performed to indicate the measure of correlation and strength of relationship between variables using Pearson’s correlation. Sample similarities were calculated on the basis of pair-wise Euclidean distance and unweighted pair-group method with arithmetic averaging (UPGMA) algorithm was used for establishing cluster to search natural groupings among accessions for different parameters. Principal component analysis (PCA) using correlation matrix was carried out to evaluate relative contribution of moisture, CUPRAC, DPPH, total bacosides and total phenols to total variability in B. monnieri accessions.

Result and discussion

In vitro shoot induction and elicitation

Shoot induction and proliferation were assessed for thirteen accessions of B. monnieri on MS medium supplemented with 0.5 mg/L BA using nodal explants. Various researchers have reported the growth of B. monnieri on different media (Sharma et al. 2007; Jauhari et al. 2016). In the present study, maximum shoot length was observed in IC554588 (2.8 cm) followed by IC375976 (2.2 cm) whereas maximum number of shoots/explant were recorded in IC344314 (8.7 shoots/explants) followed by IC554588 (8.3 shoots/explants) (Table 2). Overall, maximum shoot growth was observed in IC554588 which was selected for elicitation experiments. No alteration in the morphology of the plant and no callus formation were observed in elicitor-supplemented medium. Leonard et al. (2018) reported that inoculum size, sucrose concentration and concentration of KH2PO4 are some potent factors for maximum biomass production. Slight reduction in number of shoots/explants and shoot length was observed in elicitor-treated plants (Figs. 1 and 2). This may be due to increase in the level of enzyme β-amyrin synthase, responsible for increasing structural components, up to 2–3 weeks of elicitation with methyl jasmonate. Subsequent reduction of the enzyme after 4 weeks results in reduced number of shoots/explants and shoots length (Mangas et al. 2009). Similar elicitation effect has been reported in earlier studies for bacoside A and triterpenoid saponins (Namdeo 2007; Sharma et al. 2015). Explants grown on media with ME often displayed necrosis of meristametic area. Thus, the growth of ME-treated plants was inhibited and exhibited less number of shoots/explants and shoot length compared to SA and JA. Plants elicited with SA, exhibited higher number of shoots/explant compared to JA and ME. Shoot length of SA-treated plants was comparable to control while slight decrease was observed with JA and ME. Maximum number of shoot regeneration has been reported in Centella asiatica elicited with ME as compared to JA and SA (Kundu et al. 2016). Thus, the response to elicitors may be species specific. Moisture content was more or less constant across all the accessions and only non-significant differences were observed in non-elicited plants (Table 2).
Table 2

Total bacoside amount, total phenol content and antioxidant potential (DPPH and CUPRAC) studied in 13 accessions of B. monnieri (culture period—4 weeks)

Accession No.

Moisture (%)

CUPRAC (µg/g)

DPPH (µg/g)

Bacosides (µg/g DW)

Phenol (µg/g)

No of shoots/explant

Shoot length (cm)

IC 439118

86.0 ± 0.5e

60.7 ± 0.4j

0.978 ± 0.6g

324.1 ± 0.6h

17.7 ± 0.6b

7.0 ± 0.8cde

2.1 ± 0.6ab

IC 426442

89.0 ± 0.4b

67.8 ± 0.6i

0.856 ± 0.4g

250.9 ± 0.5k

9.81 ± 0.7e

8.1 ± 0.7abc

2.1 ± 0.6ab

IC 426447

87.5 ± 0.5d

69.9 ± 0.2h

0.681 ± 0.4g

434.0 ± 0.7c

7.58 ± 0.4f

5.6 ± 0.8fg

1.2 ± 0.7bc

IC 468878

88.4 ± 0.7cb

35.1 ± 0.4l

2.32 ± 0.6f

433.4 ± 0.4c

1.49 ± 0.5i

4.5 ± 0.6g

1.1 ± 0.5bc

IC 373640

88.4 ± 0.4cb

51.7 ± 0.9k

3.82 ± 0.5e

407.2 ± 0.5f

5.57 ± 0.5g

6.4 ± 0.6def

1.3 ± 0.6bc

IC 344312

90.1 ± 0.6a

193.0 ± 0.6b

6.49 ± 0.6b

455.1 ± 0.6b

18.3 ± 0.6b

8.7 ± 0.6a

2.1 ± 0.4ab

IC 531621

87.6 ± 0.3dc

88.9 ± 0.4f

3.79 ± 0.6e

328.5 ± 0.7g

5.77 ± 0.7g

5.4 ± 0.7fg

2.1 ± 0.5ab

IC 375976

87.4 ± 0.4d

126.1 ± 0.5d

5.26 ± 0.4c

313.5 ± 0.9j

3.31 ± 0.7h

5.3 ± 0.5fg

2.2 ± 0.7ab

IC353203

89.1 ± 0.5b

108.2 ± 0.4e

4.37 ± 0.9e

208.9 ± 0.ll

15.3 ± 0.4c

7.2 ± 0.6bcd

1.6 ± 0.4bc

IC 554588

88.0 ± 0.6dc

173.4 ± 0.7c

7.15 ± 0.5a

515.4 ± 0.6a

11.9 ± 0.5d

8.3 ± 0.7ab

2.8 ± 0.5a

IC 554586

82.7 ± 0.5g

126.4 ± 0.5d

3.62 ± 0.5e

432.2 ± 0.6d

8.98 ± 0.8e

2.2 ± 0.5h

0.8 ± 0.3c

IC 554587

84.7 ± 0.3f

78.1 ± 0.6g

2.07 ± 0.6f

412.9 ± 0.5e

16.2 ± 0.5c

4.9 ± 0.6g

1.2 ± 0.5bc

IC 554585

85.5 ± 0.6e

231.0 ± 0.5a

4.25 ± 0.4d

318.7 ± 0.4i

22.3 ± 0.6a

5.8 ± 0.5efg

1.5 ± 0.4bc

Fig. 1

Effect of elicitors JA, SA and ME (1.0 mg/L) on number of shoots/explant of B. monnieri (IC 554588) after 4 weeks of culture. Data represents mean ± standard error of three replicates; each experiment was repeated thrice (P < 0.05)

Fig. 2

Effect of elicitors JA, SA and ME (1.0 mg/L) on shoot length (cm) of B. monnieri (IC 554588) after 4 weeks of culture. Data represents mean ± standard error of three replicates; each experiment was repeated thrice (P < 0.05)

Quantification of total bacosides

Bacoside content in in vitro propagated shoots of elicited and non-elicited plants using HPLC is depicted in Table 2. Wide variation in bacoside amount (216.93–515.47 µg/g dry weight (DW) basis) was observed in 13 accessions based on DMRT. Although accession IC554588 with maximum number of shoots/explants and highest shoot length produced maximum amount of total bacosides, there was no correlation of growth parameters on yield of bacosides. Accessions belonging to Madhya Pradesh (IC 554588) and Karnataka (IC 344312) produced highest amount of bacosides followed by Kerala (IC 648878), Madhya Pradesh (IC 426447, IC 554586) and Jharkhand (IC 531621, IC 439118). The results of present study are in accordance with earlier reports (Mishra et al. 2013; Mundkinajeddu et al. 2005; Pal et al. 1998). In the present study, IC554588 produced maximum amount of total bacosides (515.4 µg/g DW), which was remarkably influenced by elicitors. Bacoside standard is a mixture of five types of bacosides (bacoside A, bacoside A3, bacoside II, bacopaside X and bacosaponin C) shown at Retention Time 1.7, 2.3, 20.3, 22.8, 27.3 min in chromatogram (Fig. 3a). After analyzing the peaks of 13 accessions, few peaks were below Limit of Quantification (LOQ) but above Limit of Detection (LOD). Out of five peaks of accession IC 426447, four can be quantified while one was only detected. Likewise, in accessions IC 375976 and IC 353203, two out of three peaks and three out of five peaks were quantified respectively.
Fig. 3

Chromatogram of bacosides obtained through HPLC a standard, b control, c elicitor treated (JA) for the accession IC554588

Elicitors (JA, SA and ME) analyzed in the present investigation, exhibited different effects on bacoside production. Five peaks were observed in bacoside standard as given in Fig. 3a. Two types of bacosides are present in control at Retention Time 1.7, 2.3 min (Fig. 3b). After elicitation of JA, increased amount of bacoside was recorded at Retention Time 1.7, 2.3, 22.8, 27.3 min (Fig. 3c). Despite slight reduction in growth parameters, increased bacoside content was recorded in all elicited samples of accession IC554588 compared to control (Fig. 4a). Maximum amount of total bacosides was obtained in plant elicited with JA (3.9 fold) followed by SA (3.2 fold) and ME (2.7 fold) after 4 weeks of elicitation. Sharma et al. (2015) reported 3.08 and 1.32 fold increase in bacoside content after elicitation with JA and SA respectively in B. monnieri after 9 days of elicitation. A 2–3 fold enhancement in bacoside A content was achieved with MeJ (50 µM) and SA (50 µM) respectively, after 3 weeks of elicitation by Largia et al. (2015). 2374 unique transcripts (UTs) were noticed to be signaling up-/down regulated with 24 h elicitation of MeJA in Polygonum minus leaves. These UTs contains many genes related to plant secondary metabolites (Rahnamaie-Tajadod et al. 2017). Saeed et al. (2017) reported enhanced levels of phytoecdysteroids with 14 days of methyl jasmonate treatment in Ajuga bracteosa. Lee et al. (2004) reported that treatment with methyl jasmonate over express the gene PgSS1, which is responsible for upregulation of triterpene saponins. This may be the possible reason for enhanced production of bacoside in JA treated roots. The results of present study are in conformity of earlier findings and exhibit significant elicitation on bacoside content. Sharma et al. (2013) analyzed shoots (in vitro) for bacoside A and reported 4.4 mg/g dry weight of bacoside A and 1.8 fold increase with 50 µM MJ treatment for 1 week. In the present study, high amount of bacoside i.e. 515.4 µg/g DW was obtained in accession IC554588 whereas different accessions showed varied bacoside content (Table 2). The yield of bacoside depends on various factors such as extraction method, media composition on which in vitro plants are grown, age of plants used for the extraction of bacosides and environmental conditions under which the experiment has been performed. In previous reports researchers have analyzed yield of bacoside A only while in our study total bacoside content (bacoside A, bacoside A3, bacoside II, bacoside X and bacosaponin C) was analyzed. Methods for analyzing the amount of bacoside play important role in its yield; many researchers have used high performance thin-layer chromatography (HPTLC) while HPLC was used in present investigation. A 1.8 fold increase in the amount of bacoside A was obtained with treatment of 50 µM MJ for 1 week whereas higher amount of bacoside (3.9 fold) with less amount of elicitor (1 mg/L) but for comparatively long duration (4 weeks) was noticed in the present investigation. Sharma et al. (2015) observed increase in bacoside content after 1 and 4 weeks of elicitor treatment with copper sulfate, JA and SA, whereas, increase in bacoside content was analyzed after 4 weeks of elicitor treatment with ME in place of copper sulphate in the present study. Therefore, variation in experimental conditions in different experiments play major role in the yield of bacosides. Thus, it can be summarized that difference in yield of bacoside and increase in amount of bacoside after elicitation depends on method of metabolite extraction and experimental conditions, which are responsible for variation in bacoside content among accessions.
Fig. 4

Effect of elicitors JA, SA and ME (1.0 mg/L) on total bacoside content, total phenol content, CUPRAC and DPPH of B. monnieri (IC 554588) after 4 weeks of culture. Data represents mean ± standard error of three replicates; each experiment was repeated thrice (P < 0.05)

Determination of total phenol content (TPC)

Bacopa accessions were investigated for TPC, responsible for antioxidant property (Gottlieb and Borin 2000). Phenol plays an important role in free radical scavenging potential of plants. The present study reported 1.5–22.4 µg/g GAE TPC in thirteen accessions of B. monnieri (Table 2). Maximum phenol content was noticed in accession belonging to Amarkantak (IC 554585) and Karnataka (IC 344312). Jharkhand (IC 439118) Madhya Pradesh (IC 554587), New Delhi (IC 353203) and other states showed comparatively less amount of phenol content. Elicitors were applied on selected accession IC554588 having 11.9 µg/g GAE phenol content, which was further increased to 219.9, 203.2 and 61.2 µg/g GAE in plant cultures elicited with JA, SA and ME respectively (Fig. 4b). Results of present investigation confirmed earlier reports of phenol concentration in ethanolic and aqueous extract of B. monnieri, which was 3.18 µg/mL and 3.71 µg/mL GAE respectively (Mukherjee et al. 2011). Saeed et al. (2017) reported enhanced TPC with the use of methyl jasmonate and phenyl acetic acid in root suspension of A. bracteosa. Mendoza et al. (2018) reported that elicitation with 3 μM MeJA after 96 h of treatment produced highest amount of phenol compounds 49.25% higher than control in Thevetia peruviana. The present study clearly indicates that elicitation increases accumulation of total phenol content in in vitro cultures of B. monnieri.

Antioxidant potential

In the present study, presence of significant amount of bacosides and phenol in the samples of B. monnieri suggested that these might be the contributors to antioxidant activity, which can be enhanced with the use of elicitors. Evaluation of antioxidant potential of the plants using different methods (DPPH, CUPRAC, 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS), ferric reducing antioxidant power (FRAP)) show variation in antioxidant activity (Thaipong et al. 2006). Different methods vary in terms of principle and environmental conditions. It is difficult to precisely measure the antioxidant activity of a system having various components using single test. Therefore, different mechanisms are needed to confirm antioxidant potential of the plants. In the present study, antioxidant potential was studied using DPPH and CUPRAC method. Maximum antioxidant activity was 7.15 µg/g GAE (IC 554588) for DPPH and 231 µg/g GAE (IC 554588) for CUPRAC, among all the thirteen accessions of B. monnieri. Antioxidant potential (CUPRAC and DPPH) of all the samples of B. monnieri is given in Table 2. Considering both the methods (CUPRAC and DPPH), Madhya Pradesh (IC 554588), Karnataka (IC 344312) and Amarkantak (IC 554585) accessions exhibited maximum antioxidant potential compared to other states among the thirteen accessions of B. monnieri. Jain et al. (2017) studied free radical scavenging activity with CUPRAC assay resulting 0.226 TE mM at 1000 µg/mL. Few researchers have evaluated effect of bacosides on antioxidant potential. Hazra et al. (2017) reported stimulated reactive oxidation species (ROS) production and plastid terminal oxidase (PTOX) accumulation by increasing their mRNA stability with the treatment of methyl jasmonate in Podophyllum hexandrum. Tripathi et al. (1996) compared the alcoholic extract of B. monnieri with Tris, ethylenediaminetetraacetic acid (EDTA) and vitamin E and found that 100 µg of antioxidant potential is equivalent to 247 µg of EDTA and 58 µg of vitamin. Russo et al. (2003) reported antioxidant potential using DPPH method and reduced hydrogen peroxide-induced cytotoxicity of methanolic extract of whole plant. Administration of 10 mg/kg aqueous bacoside A remarkably enhanced levels of glutathione, Vitamin C, Vitamin A and Vitamin E in brain of rats exposed to cigarette smoke. Bacoside A has also been found to increase superoxide dismutase, catalase, glutathione peroxide and glutathione reductase (Anbarasi et al. 2006). Dey et al. (2019) noticed higher levels of total bacoside content, total phenol content and antioxidant potential in in vitro grown plants treated with polyamines as compared to mother plant. Free radicals produced during the normal metabolic reaction of aerobic cells are extremely reactive and at unsteady state that have tendency to react with food lipids, nucleic acid, sugars and sterols (Lee et al. 2004), which lead to various physiological modifications. Chemical elicitation treatment significantly enhanced free radical scavenging potential of extract.

Ahmad et al. (2013) reported that enhanced phenol content leads to increased antioxidant capacity. Saxena et al. (2016) reported that phenol content (using Folin-Ciocalteu reagent) present in methanolic and ethnolic extract of plants Eclipta and Plumbago is responsible for antioxidant potential. It was observed that an increase or decrease in total bacosides, phenol content and antioxidant potential was not influenced by growth parameters. All the tested elicitors, when used separately, stimulated yield of total bacosides, total phenols and antioxidant potential, with a significant difference (Fig. 4). Total bacosides and phenol content were much higher with JA-elicited plants while elicitation with SA showed highest increase in antioxidant potential. Antioxidant activity for CUPRAC assay showed highest 231 µg/g GAE (IC 554588), which was enhanced to 1119.8, 1762 and 563 µg/g GAE by the application of elicitors JA, SA and ME respectively (Fig. 4c). Antioxidant activity of accession IC554588 through DPPH assay was noticed 7.15 µg/g GAE, which was increased to 37.6 µg/g, 56.5 µg/g and 14.6 µg/g GAE with the elicitation of JA, SA and ME respectively (Fig. 4d). The present study clearly indicates that elicitation increases accumulation of total bacosides, TPC and antioxidant potential of in vitro cultures of B. monnieri.

According to statistical analysis (MLR), antioxidant potential is highly correlated to total bacoside and total phenols as R-sq(adj) is 65.8% and 90.1% respectively. It is also evident that phenol is more correlated to antioxidant potential than bacoside (statistical study). As per regression equation, the coefficient of phenol is higher than that of bacoside, which employs that antioxidant potential, is more sensitive to phenol as compared to bacoside. The sign of coefficients indicate that both the factors are directly correlated to antioxidant potential. The R-adj value for the regression equation is 89%, which shows very strong correlation of both the factors with antioxidant potential.

The present study is a comprehensive study of 13 accessions for bacoside, phenol and antioxidant potential, which were collected from different geographical locations. Elite accessions were analyzed on the basis of various parameters such as maximum number of shoots, highest shoot length, total yield of bacosides, TPC and antioxidant potential. Apart from this, JA, SA and ME were used for elicitation which have not been tested for the studied accessions by varying experimental conditions. Elicitation with JA, SA and ME has been performed on all the above tested parameters and noticeable increase was found in amount of bacoside, phenol content and antioxidant potential, which is undoubtedly enhancing their therapeutic potential.

Correlation, variation, principal component and cluster analysis

Statistical analysis revealed negative correlation of moisture content with bacoside content, antioxidant potential and TPC (Table 3), which is beneficial as high moisture content can lower the pharmacological effect of plant. Antioxidant by CUPRAC method exhibited strong correlation with DPPH, bacoside and phenol content. Phenols and bacosides are highly correlated with strong positive correlation. Among all components phenol is the major contributor to variation. Elicited samples exhibited increase in bacoside content as well as phenol component.
Table 3

Linear correlation between moisture content, antioxidant potential using CUPRAC and DPPH method, bacosides and phenol content of 13 B. monnieri accessions

Moisture

CUPRAC

DPPH

Bacosides

Phenol

 

Moisture

     

 Pearson correlation

1

− 0.815**

− 0.820**

− 0.574**

− 0.832**

 Sig. (2-tailed)

 

0.000

0.000

0.000

0.000

 N

48

48

48

48

48

CUPRAC

     

 Pearson correlation

− 0.815**

1

0.994**

0.811**

0.951**

 Sig. (2-tailed)

0.000

 

0.000

0.000

0.000

 N

48

48

48

48

48

DPPH

     

 Pearson correlation

− 0.820**

0.994**

1

0.798**

0.954**

 Sig. (2-tailed)

0.000

0.000

 

0.000

0.000

 N

48

48

48

48

48

Bacosides

     

 Pearson correlation

− 0.574**

0.811**

0.798**

1

0.871**

 Sig. (2-tailed)

0.000

0.000

0.000

 

0.000

 N

48

48

48

48

48

Phenol

     

 Pearson correlation

− 0.832**

0.951**

0.954**

0.871**

1

 Sig. (2-tailed)

0.000

0.000

0.000

0.000

 

 N

48

48

48

48

48

**Correlation is highly significant at an error of p < 0.001 level

PCA was carried out to find total variance among all samples. In the present study, five parameters have been taken into account for the same that are moisture content, total bacosides, TPC and antioxidant potential with DPPH and CUPRAC methods. PCA revealed that moisture, CUPRAC, and DPPH together governed 83% of total variability among all five components (Table 4). Therefore, these three components are considered as Principal Components (PC). Component matrix is shown in Table 5 where PC1 represents moisture while PC2 and PC3 represent CUPRAC and DPPH respectively. In PC1, CUPRAC (0.644) and DPPH (0.689) have high positive loading and are considered as major contributors to variability. In PC2, CUPRAC is major contributor followed by phenol and bacoside. Maximum contribution of bacoside was noticed in PC3. Antioxidant potential as assessed by CUPRAC and DPPH methods are major traits responsible for variability amongst all the tested accessions (Table 5). Three dimensional plot for PCA is shown in Fig. 5. Antioxidant potential was major contributor in cluster grouping, and its influence is distinctly reflected in the primary grouping of 13 B. monnieri accessions.
Table 4

B. monnieri: eigenvalues for all the five components in 13 accessions

Component

Initial eigen values

Total

% of variance

Cumulative %

Moisture

2.816

40.232

40.232

CUPRAC

1.725

24.643

64.875

DPPH

1.261

18.016

82.891

Bacosides

0.628

8.971

91.862

Phenols

0.432

6.172

98.034

Extraction method: principal component analysis

Table 5

Component matrix of five components in 13 accessions

 

Component

PC1

PC2

PC3

Moisture

0.584

− 0.679

0.229

CUPRAC

0.644

0.692

− 0.095

DPPH

0.689

0.419

0.431

Bacosides

− 0.015

0.431

0.725

Phenol

0.448

0.453

− 0.683

Extraction method: principal component analysis

PC1 principal component 1, PC2 principal component 2, PC3 principal component 3

Fig. 5

Three dimensional plot for PCA

The dendrogram generated, based on judged parameters revealed five distinct clusters containing five accessions in Cluster I, two accessions in each Cluster II and Cluster III, three in Cluster IV and one accession in Cluster V at the linkage distance of 10 (Fig. 6). In general, accessions present in Cluster I, Cluster IV and Cluster V have moderate amount of bacoside content. Accessions of Cluster II showed the highest amount of bacoside while accessions of Cluster III had lowest amount. Accessions of Cluster II (IC 344312 and IC 554588), besides having highest bacoside content also had high TPC and antioxidant potential. IC554585 of Cluster V showed high antioxidant potential (by CUPRAC method) and phenol content with moderate amount of bacoside. Hence, amongst the 13 studied accessions, IC 554588, IC 344312 and IC 554585 are best suited for exploitation for pharmaceutical purposes.
Fig. 6

Clustering of 13 accessions of B. monnieri based on components moisture content, antioxidant potential using CUPRAC and DPPH methods, bacosides and phenol content, number of shoots/explants and shoot length. (Refer Table 1 for the corresponding accessions of the shown numbers in the Fig. 4

The present study has been conducted with in vitro cultures of B. monnieri to enhance the bacoside production, TPC and antioxidant potential, which contributes to its therapeutic value. However, it is an easy growing plant but wild collection from its natural habitat by various pharmaceutical companies has resulted in overexploitation. Use of in vitro cultures and elicitors to enhance pharmaceutically important compounds in culture conditions can protect the natural habitat of this medicinally important plant contributing to its sustainability in the environment.

Conclusion

The study revealed wide variability among thirteen accessions of B. monnieri, collected from different states of India. Variability was defined with respect to total bacoside content, TPC and antioxidant potential. Highly significant and strong correlation was found between total bacosides, total phenols and antioxidant activity, which were the major contributors in cluster grouping. IC 554588, IC 344312 and IC 554585 have been identified elite accessions with higher bacoside production, TPC and antioxidant potential as compared to others. Use of elicitors enhanced the total bacosides and TPC, which are responsible for higher antioxidant potential. Hence, elicitor-treated and superior accessions can be cultivated by farmers to increase the raw material for drug manufacturing.

Notes

Acknowledgements

We are grateful to Vice Chancellor of Delhi Technological University, Delhi, India for constant support and encouragement in conducting this study. We are also thankful to Dr. Ram Singh, Assistant Professor (Applied Chemistry) and his advisee Dr. Atya and Dr. Gitika (Applied Chemistry), Delhi Technological University for their help in performing HPLC.

Authors contribution statement

Experimental strategy belongs to NB, NS and NJ. NJ and RB performed experiment. RB did statistical analysis.

Funding

This research did not receive any grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

42398_2019_71_MOESM1_ESM.pdf (116 kb)
Supplementary material 1 (PDF 115 kb)

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Copyright information

© Society for Environmental Sustainability 2019

Authors and Affiliations

  1. 1.Department of BiotechnologyDelhi Technological UniversityDelhiIndia
  2. 2.ICAR-National Bureau of Plant Genetic Resources, Pusa CampusNew DelhiIndia

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