Dammarane triterpenoid glycosides in Bacopa monnieri: A review on chemical diversity and bioactivity

Phytochemistry 172 (2020) 112276

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Review

Dammarane triterpenoid glycosides in Bacopa monnieri: A review on chemical diversity and bioactivity

T

Pamita Bhandari∗, Nitisha Sendri, Shinde Bhagatsing Devidas Natural Product Chemistry & Process Development, CSIR-Institute of Himalayan Bioresource Technology, Palampur, India

ARTICLE INFO

ABSTRACT

Keywords: Bacopa monnieri Plantaginaceae Dammarane triterpenoids Structure elucidation Biological activity

Bacopa monnieri (L.) is a reputed medicinal herb in traditional system of medicine of India, where it is used as nervine tonic to sharpen intellect and memory. This review discusses chemical characterization of dammarane triterpenoid glycosides which are well accepted for improvement in memory and for potential pharmacological activities. In addition, this review provides information on the chemical composition of specialized metabolites of B. monnieri and in the formulations by different analytical techniques. This comprehensive review covers literature up to 2019 with an emphasis on structural characterization of dammarane triterpenoid glycosides by spectroscopic techniques, chemical composition by analytical methods and pharmacological activities.

1. Introduction Bacopa monnieri (L.) Wettst, commonly known as “Brahmi”, is a well-respected medicinal herb of Ayurveda comprising of 100 species. It is found throughout the Indian subcontinent in wet and muddy places (Kapoor, 1990). B. monnieri is a high value medicinal plant recognized as brain tonic and is capable to improve memory and intellect (Garai et al., 1996a; Christopher et al., 2017). Different research groups had explored B. monnieri for treatment of dementia (Saini et al., 2010), amnesia (Kishore and Singh, 2005), memory disfunction (Dwivedi et al., 2013), Parkinson's disease (Jadiya et al., 2011; Srivastav et al., 2017), Alzheimer's disease (Uabundit et al., 2010), epileptic seizures (Giramkar et al., 2013) and schizophrenia (Piyabhan et al., 2018). Besides neuroprotection, B. monnieri has been reported to possess sedative (Kothandapani et al., 2005), antimicrobial (Chaudhuri et al., 2004), anti-inflammatory (Channa et al., 2006), anticonvulsant (Mathew et al., 2011), anti-ageing (Rastogi et al., 2012), broncho-vasodilatory (Channa and Ahsana, 2012), anticancer (Mallick et al., 2015), antidepressant (Rauf et al., 2014), anti-emetic (Ullah et al., 2017) and antiulcer (Manoj et al., 2013) activities. Although this wellknown ayurvedic medicinal plant contains a vast range of specialized metabolites i.e. dammarane triterpenoid glycosides, sterols (Ahmed and Rahman, 2000), sterol glycoside (Bhandari et al., 2006a,b) phenylethanoid glycosides (Chakravarty et al., 2002; Ohta et al., in 2016) and cucurbitacins (Bhandari et al., 2007). However, the potential effects of B. monnieri may be attributed due to the presence of closely related structures i.e. dammarane triterpenoid glycosides (bacosides). Hence,

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the present review is focused on extraction methods, chemical characterization of dammarane triterpenoid glycosides by 1H & 13C NMR, qualitative and quantitative analysis of specialized metabolites (dammarane triterpenoid glycosides) by HPLC, LC-MS and LC-NMR and pharmacological activities of B. monnieri. The dammarane triterpenoid glycosides (bacosides) are the specialized metabolites typically comprised of aglycone [jujubogenin (1) or psuedojujubogenin (2) (Fig. 1)] and sugar moiety (glycan). The type of aglycone (1) or (2) is distinguished by the linkage of isobutylene unit i.e. in jujubogenin aglycone, the isobutylene unit is linked to the 23 position of carbon skeleton, and in pseudojujubogenin, the isobutylene unit is attached to 22 position of carbon skeleton. The sugar moieties present in B. monnieri includes β-D-glucopyranosyl, α-L-arabinofuranosyl, α-L-arabinopyranosyl and 6-O-sulphonyl-β-D-glucopyranosyl. These dammarane triterpenoid glycosides are further classified as mono- and bi-desmosides on the basis of linkage of sugar units to the available sites of aglycone i.e. at C-3 and C-20. Although majority of dammarane triterpenoid saponins in B. monnieri are monodesmosides (attachment of sugar moieties only to C-3 of the aglycone). However, till date, a few number of bidesmosidic saponins (attachment of sugar moieties to C-3 & C-20) have been repoted from this plant. The upcoming subsections of this review will discuss the extraction, structure characterization, analytical methods and pharmacological activities of dammarane triterpenoid glycosides.

Corresponding author. E-mail addresses: [email protected], [email protected] (P. Bhandari).

https://doi.org/10.1016/j.phytochem.2020.112276 Received 10 October 2019; Received in revised form 11 January 2020; Accepted 13 January 2020 0031-9422/ © 2020 Elsevier Ltd. All rights reserved.

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B. monnieri paves the way of conventional methods such as soxhlet and maceration (Bhandari et al., 2006a,b, 2007; 2009a; Ahmed and Rahman, 2000; Garai et al., 1996a; Chakravarty et al., 2002, 2003; Pawar and Bhutani, 2006). Due to polar nature of dammarane triterpenoid glycosides, the selection of extractive solvent is also limited. As a result, solvents employed for the extraction of dammarane triterpenoid glycosides are combinations of either methanol/ethanol or methanol-water. In most of the cases, the plant material is defatted with n-hexane prior to extraction with selective solvent (Bhandari et al., 2009a, 2006; 2007; Mahato et al., 2000; Chakravarty et al., 2001, 2002) and partitioned with chloroform/ethylacetate and n-butanol to obtain the extracts of different polarities respectively. The dammarane triterpenoid glycosides were obtained by purification of n-butanol fractions using a combinations of normal phase, reverse phase column chromatography and preparative thin layer chromatography on silica gel G followed by crystallization. Some of the dammarane triterpenoid glycosides were isolated from macroporous resin chromatography followed by Sephadex LH-20 and reverse phase silica gel (ODS) chromatography (Zhou et al., 2009). One of the research group had isolated dammarane triterpenoid glycosides from the ethylacetate extract chromatographed over Diaion HP-20 (Bhandari et al., 2009a). Also, three, 20-deoxy dammarane triterpenoid glycosides were isolated from methanol extract (Pawar et al., 2007; Dang et al., 2018).

Fig. 1. Structures of jujubogenin (1) and pseudojujubogenin (2) aglycones.

2. Dammarane triterpenoid glycosides Dammarane triterpenoid glycosides illustrated from B. monnieri are tetracyclic compounds with β–orientated methyl group at C-8. The most commonally occurring sugar moieties in dammarane triterpenoid saponins are β-D-glucopyranosyl, α-L-arabinofuranosyl and α-L-arabinopyranosyl. Despite the presence of these sugars, three dammarane triterpenoid glycosides are reported to have 6-O-sulphonyl-β-Dglucopyranosyl sugar moiety (Chakravarty et al., 2001; Zhou et al., 2007). Number of reputed medicinal plants i.e. Zizypus species, Panax species, Bacopa monnieri plants of rhamnaceae, araliaceae and plantaginaceae family, reported to exhibit dammarane triterpenoid glycosides (Brandao et al., 1992; Garai et al., 1996a, 1996b; Ganapaty et al., 2006; Rambabu et al., 2011). Due to thrilling reputation of B. monnieri in ayurvedic system of medicine, systematic chemical studies of the plant have been conducted by different research groups. The foremost chemical exploration of this plant has been carried out by Bose and Bose in 1931 and reported the isolation of an alkaloid “Brahmine”. Afterwards, two alkaloids, namely, nicotine and herpestine (Chopra and Nayar, 1956), hersaponin and mannitol (Sastri et al., 1959) have also been reported from this plant. Subsequently, in 1965, a chemical constituent assisgned as 3-(α-L-arabinopyranosyl)-O-β-D-glucopyranoside-10,20dihydroxy-16-keto-dammar-24-ene commonly known as bacoside A was isolated and considered as main active constituent for memory enhancing activities (Chatterji et al., 1965). Previously, it was documented that bacoside A co-exist with bacoside B and differs only in optical rotation (Rastogi and Mehrotra, 1990; Chatterji et al., 1963: Basu, 1967) which on acidic hydrolysis reported to yield a mixture of artefacts, bacogenin A1 (3), A2 (4), A3 (5), (Kulshreshtha and Rastogi, 1973a; 1974; Chandel et al., 1977), bacogenin A4 (6) as ebelin lactone pseudojujubogenin (Kulshreshtha and Rastogi, 1973b). Later on, the structures of bacoside A and bacoside B were established as mixture of four triglycosidic saponins and four diglycosidic saponins (Deepak et al., 2005) respectively. The chemical composition of bacoside A was characterized as bacoside A3 (8), bacopaside II (19), bacopaside X (27) and bacopasaponin C (11) (Deepak et al., 2005) while the bacoside B was characterized as mixture of bacopaside N1 (30), bacopaside N2 (31), bacopaside IV (21) and bacopaside V (22) respectively. In spite of bacoside A and bacoside B, the other triterpenoid glycosides characterized were bacoside A1 (7), bacoside A3 (8), bacopasaponin A (9), bacopasaponin B (10), bacopasaponin C (11), bacopasaponin D (12), bacopasaponin E (13), bacopasaponin F (14), bacopasaponin G (15), bacopasaponin H (16), bacopasaponin I (17), bacopaside I (18), bacopaside II (19), bacopaside III (20), bacopaside IV (21), bacopaside V (22), bacopaside VI (23), bacopaside VII (24), bacopaside VIII (25), bacopaside IX (26), bacopaside X (27), bacopaside XI (28) and bacopaside XII (29), bacopaside N1 (30), bacopaside N2 (31), bacoside A4 (32) and bacoside A5 (33). Furthermore, two acylated dammarane triterpenoid glycosides namely, bacomosaponins A (34) and bacomosaponins B (35), had been isolated and elucidated with spectroscopic techniques. The isolated dammarane triterpenoid glycosides from the B. monnieri are described in Fig. 2 and Table 1.

3.1. Chromatographic analysis of B. monnieri Despite the isolation and characterization of specialized metabolites, dammarane triterpenoid glycosides were analysed in natural matrixes of the plant and different herbal formulations by using different analytical techniques (Renukappa et al., 1999; Ganzera et al., 2004; Shrikumar et al., 2004; Deepak et al., 2005; Murthy et al., 2006; Agrawal et al., 2006; Bhandari et al., 2006a,b, 2009a; Zehl et al., 2007; Srivastava et al., 2012; Ahmed et al., 2015). The various techniques used for analysis of specialized products in B. monnieri include TLC, HPLC (high performance liquid chromatography), HPTLC (high performance thin layer chromatography), SFC (super critical fluid chromatography) and LC-MS/MS (liquid chromatography mass spectrometry). The spectrophotometeric methods had also been developed for the quantification of bacosides in which bacosides were hydrolysed and aglycone was measured at 278 nm (Pal and Sarin, 1992). A HPLC-PDA method was described for the quantification of six bacosides and enlisted that the total saponin contents varied in the range of 1.06–13.03%. The findings showed that accumulation of dammarane triterpenoid glycosides was more in the leaves as compared to the stem part of the herb (Ganzera et al., 2004). For the determination of bacoside-A3 and bacopaside –I content in B. monnieri extract and monoherbal formulations, a HPTLC and SFC methods were developed by different research groups (Shrikumar et al., 2004; Agrawal et al., 2006). As discussed previously that the structures of bacoside A & B were conflicting and most of the pharmacological and clinical studies of the B. monnieri extract were considered due to bacoside A and bacoside B (Chatterji et al., 1963; Chatterji et al., 1965; Kawai and Shibata, 1978). To resolve this ambiguity, authors attempted to establish the chemical structures of bacoside A and accomplished that it was a mixture of four saponins (Deepak et al., 2005). Later on, the composition of bacoside B was also eastablished as a mixture of four diglycosidic saponins (Sivaramakrishna et al., 2005b). An analytical HPLC–DAD method had been developed for quantification of twelve saponins in plant extract and herbal formulations in which saponins were resolved on Luna C18 column (Phenomenex) with isocratic elution of 0.05 M sodium sulphate buffer and acetonitrile (68.3:31.5, v/v) with flow rate of 1.0 ml/min (Murthy et al., 2006). The results revealed that bacopaside II, bacopaside I, bacopaside X and bacopasaponin C dominate the bacopasaponin E and F. Bhandari et al., 2009 reported a high performance liquid chromatographic method using silica-based monolithic column coupled with evaporative light scattering detector (HPLC-ELSD) for

3. Extraction, isolation and analytical analysis An insight into extractions of dammarane triterpenoid glycosides in 2

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Fig. 2. Structures of isolated compounds from B. monnieri.

quantification of bacosides (bacoside A, bacopaside I, bacoside A3, bacopaside II, bacopaside X, bacopasaponin C) and apigenin in nine accessions of B. monnieri. The method was developed on five different reversed phase analytical columns and found that optimum separation of analytes was achieved on chromolith column. The same authors standardized a method for quantification of bacoside A in minimum amount of plant material i.e. up to 2 mg by HPLC (Bhandari et al., 2006a,b). Afterwards number of HPTLC methods were developed for quantification of bacosides in B. monnieri (Ahmed et al., 2015; Nuengchamnong et al., 2016). Furthermore, for rapid screening of specialized metabolites in B. monnieri, the techniques like HPLC coupled with mass spectrometry (HPLC-MS), and nuclear magnetic resonance spectroscopy (LC-NMR) had also been used. For the qualitative analysis of dammarane triterpenoid glycosides, a descriptive reversed phase high performance liquid chromatography coupled with nuclear magnetic resonance and mass spectroscopy (LC-NMR-MS) was used on LiChrosphere RP-18 HPLC column using acetonitrile-water as mobile phase with a flow rate of 0.8 ml/min (Renukappa et al., 1999). In the analysis, four major peaks were observed, of which three peaks were identified as bacoside A3, 3-β-[O-β-D-glucopyranosyl (1 → 3)-O-[α-L-arabinofuranosyl-(1 → 2)]-O-β-D-arabinopyranosyl) oxy] jujubogenin and bacopasaponin C. In another study, different MS techniques like ESI-ion trap (IT), APMALDI-IT, MALDI-IT/reflectron time-of-flight (RTOF) MS, all utilizing

low-energy collision-induced dissociation (CID) and high-energy CID analysis were used to understand the glycosidic linkages and distinction of aglycone (Zehl et al., 2007). The fragmentation behaviour observed from these techniques indicated that all the applied techniques produced the sequence and branching of the glycan and the molecular mass of the aglycone. Although low-energy collision induced dissociation (CID) provides the information about the interglycosidic linkages however, this study was not able to differentiate the type of aglycone (jujubogenin or pseudojujubojenin). In another study, Srivastava et al. analysed the B. monnieri for bacopaside-I and bacoside A content and suggested that the maximum yield of bacosides can be achieved at temperature up to 60 °C. To study the effect of geographical variations, a HPTLC method was used to study the content of bacoside A and concluded that highest content of bacoside A was observed in leaves collected from Jammu region while lowest content was examined in sample collected from Kerela region. In Addition to this, a liquid chromatography coupled with electrospray ionization quadrupole timeof-flight mass spectrometery (LC-ESI-QTOF-MS) was conducted to characterize dammarane triterpenoid glycosides in B. monnieri (Nuengchamnong et al., 2016). In this study authors reported that the aglycone (jujubogenin and pseudojujubogenin) of B. monnieri can be distinguished on the fragmentation pattren.

3

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Fig. 2. (continued)

4. Structure elucidation

recent past for the structure elucidation of dammarane triterpenoid glycosides in B. monnieri. The most frequently high resolution-electrospray ionization-time of flight-mass spectrometry (HRESI-TOF-MS) and Fast Atom Bombardment (FAB) in positive ion mode has been used for the determination of molecular weight of the triterpenoid glycosides. To distinguish the type of aglycone and glycosidic linkages, different mass spectroscopic techniques were atempted. These studies revealed that low energy-CID, PSD (post source decay) and high energy-CID yielded the fragment ions which evidently give the sequence and branching of the glycan. However, low energy CID of the precursor ion yielded exclusive fragment ions created from the cleavage of one or two glycosidic bonds. The diagnostic fragment ions obtained from this study provide the structural information of the ring systems and confirm the linkages of glycan part to aglycone. In one of the report, B. monnieri is analysed by electrospray ionization quadrupole time of flight mass spectrometry (ESI-QTOF-MS) technique in positive and negative ion modes where jujubogenin & pseudojujubogenin glycosides illustrated the molecular ion peaks at m/z 455 [aglycone + H–H2O]+ and m/z of 473 [aglycone + H]+ respectively (Nuengchamnong et al., 2016). In this study, ESI-QTOF-MS is used to crack the complexity of the glycan present in the compounds. For example, the loss of different monosaccharide units from the dammarane triterpenoid glycoside generate ion peaks to the corresponding molecular weight i.e. loss of a hexose moiety provides a fragment ion at [M-162]+ units. Similarly, subtraction of 132 and 146 mass units depicts the loss of pentose [M-132]+ and deoxyrhamnose [M-146]+ moieties.

The structure elucidation of dammarane triterpenoid glycosides involves the determination of both the units i.e. aglycone and glycan (sugars). The identification of dammarane triterpenoid glycoside involve methods such as Co-TLC with reference standard and comparison of spectroscopic data with reported values. The structure elucidation of complex dammarane triperpenoid glycosides becomes extremely difficult as the increase of sugar moieties and its branching points. Hence, the usage of chemical means as well as standard spectral techniques for complete and clear elucidated structures are required. 4.1. Spectroscopic techniques Being complex compounds, dammarane triterpenoid glycosides can be recognized on the basis of Co-TLC, MS, IR, 1H and 13C NMR spectral data. An attentive comparison of spectroscopic data of dammarane triterpenoid glycosides with literature may lead to the identification of compounds. However, where detailed structural analysis of compound is required, 1D and 2D NMR techniques are essential. 4.1.1. Infrared spectroscopy IR spectrum of dammarane triterpenoid glycosides exhibited a strong and broad absorption band at 3320-3600 cm−1 corresponding to hydroxyl groups. The absorption band near 1635 cm−1 indicates the presence of olefinic bond and the characteristic absorption band at 1220 and 809 cm−1 indicates the presence of sulphate group.

4.1.3. Nuclear magnetic resonance studies of dammarane triterpenoid glycosides The dammarane triterpenoid saponins of B. monnieri are tetracyclic

4.1.2. Mass spectrometric studies Different mass spectrometry experiments had been involved in the 4

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Fig. 2. (continued)

triterpenoids and displayed seven methyl resonances in 1H and 13C NMR spectrum. Among seven methyls, the tertiary methyls (CH3-18 & 19) are in β-orientation while CH3-21 is always in α-orientation thus directing the S configuration to C-20. The type of aglycone i.e. jujubogenin or pseudojujubogenin is differentiated by the chemical shifts of H-22, H-23, C-17, C-20, C-21, C-24 and C-25. In case of jujubogenin aglycone, methylene protons (H-22) resonates in the range ≈ δ 1.63 & 1.73 while the methine proton of H-23 resonates at ≈ δ 5.18. The 13C NMR spectrum of jujubogenin aglycone exhibits resonances for C-17, C20, C-21, C-23, C-24 and C-25 at δ 54.0, 68.5, 30.0, 68.0, 127.0 and 134.0 respectively (Garai et al., 1996a; Mahato et al., 2000; Chakravarty et al., 2003). The jujubogenin carbon skeleton is further reaffirmed by HMBC correlations of H-22 (methylene protons) with C17, C-20, C-21 & C-23 carbons and correlations of H-23 (methine proton) with C-24 and C-25. In pseudojujubogenin aglycone, methine proton (H-22) showed downfield shift of ≈ δ 2.57 whereas H-23 (methylene protons) resonate at ≈ δ 3.86 & 4.70. Also, a significant upfield chemical shift value of approximately 3–4 ppm in C-24 and about 2–3 ppm upfield shift in C-25 are observed (Mahato et al., 2000; Garai et al., 1996b; Chakravarty et al., 2001). In terms of 1H-NMR spectroscopy, the dammarane triterpenoid glycosides are characterized with olefinic H-24 (≈δ 5.84) protons as a doublet or broad doublet and seven methyls as singlets (<2.0 ppm). In case of 13C-NMR spectrum, for olefinic carbons i.e. C-24 and C-25, displayed characteristic downfield shift values at δ 127.0 and δ 134.0 (jujubogenin) and approximately ≈2–3 ppm upfield (pseudojujubogenin) respectively. The carbon resonance at δ 110.3 showed the presence of a ketal group at C-16 and oxygenated carbon (C-20) at δ 71.8. Glycosylation at particular position (C-3 & C-20) of dammarane triterpenoid glycoside results in the downfield shift in 1H and 13C NMR signals of the respective proton and

carbon. For example, glycosilation at C-3 and C-20 of dammarane triterpenoid glycosides result in approximately 8–9 ppm downfield shift at C-3 and ≈ 4–5 ppm downfield shift at C-20 in 13C-NMR spectra (Sivaramakrishna et al., 2005a; Garai et al., 1996b; Bhandari et al., 2009a). The configurations at C-20 and C-22 in the aglycone pseudojujubogenin were determined to be 20 (S) and 22 (R) by ROESY. 4.1.4. Sugar moieties The complete assignment of sugar moieties existing in dammarane triterpenoid glycosides involves the identification of sugar unit, interglycosidic linkage and linkage of sugars to the aglycone. The linkage of the sugar moieties to C-3 and C-20, interglycosidic linkages and the sugar units form (pyranose form of the glucose, pyranose & furanose form of arabinose) were determined by 13C NMR chemical shift values (Tori et al., 1977; Kasai et al., 1979). For example, glycosylation on 3OH results in downfield shifts of approximately δ 3.16–3.31 and δ 75.1 to H-3 and C-3 respectively. Further, the linkage of sugar moieties to the aglycone, interglycosidic linkages and 1H & 13C chemical shifts of the specific sugar units were assigned from a combination of 1H–1H COSY, ROSEY, HMQC and HMBC experiments (Chakravarty et al., 2001; Garai et al., 1996a). The glycosidic chemical shifts observed for protons & cabons in the dammarane triterpenoid glycosides were in the range of δ 3.93–6.18 and ≈δ 98.7–110.1 respectively. The anomeric proton signals of the β-D- glucopyranosyl appeared in the range of approximately δ 4.51–5.05 with coupling constant 8.0 Hz and the anomeric proton of α-L-arabinopyranosyl was present in the range of around δ 4.76–4.85 with coupling constant ≈ 5.8–7.0 Hz. The α-L- arabinofuranosyl moiety had its protons ≈ δ 5.41–6.27 as broad singlet or doublet with coupling constant, J < 3 (Garai et al., 1996a, 1996b; Bhandari et al., 2009a; Hou et al., 2002; Chakravarty et al., 2003; 5

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Fig. 2. (continued)

Sivaramakrishna et al., 2005a). Besides the presence of above discussed sugar units, two compounds namely bacopaside I (18) and bacopaside XI (28) showed sulphonyl glucose. The vigilant study of the 1H and 13C chemical shifts of these compounds revealed deshielding of methylene protons (H2-6) and C-6 by more than 0.5 ppm and ≈4.5 ppm respectively. This was further supported by up field shift of ≈2.3 ppm of C-5 of glucopyranose due to γ-effect (Bhandari et al., 2009a; Chakravarty et al., 2001). In case of trisaccharide, the dammarane triterpenoid glycosides in B. monnieri possess two types of interglycosidic linkages i. e 1 → 3 (inner sugar), and 1 → 2 (inner sugar unit) i.e. one sugar unit is linked to C-2′ of inner sugar unit and another terminal sugar is attached to C-3'. The cabons involved in the glycosidic linkages showed substantial downfield shift and are used to determine the linkages between the sugar moieties in dammarane triterpenoid glycosides. For example, bacopasaponin C (a trisaccharide pseudojujubogenin consisting of arabinofuranose and glucose as terminal sugars and arabinopyranose as inner sugar unit) exhibited C-3 linkage to aglycone (pseudojujubogenin) which was characterized by means of HMBC experiments between arabinopyranose H-1' (δH 4.76) and pseudojujubogenin C-3 (δC 88.6). The signals of C-2′ and C-3′ of arabinopyranose was significantly shifted to a downfield value of δC 76.7 ppm (≈2 ppm) and δC 83.1 ppm (≈5–6 ppm) respectively. The terminal sugar moieties can be indicated by the absence of any glycosylation shift.

remarkable pharmacological activities like cognition, memory-enhancing, anxiolytic, antidepressant, anticonvulsive and antioxidant activities etc. Cognition-facilitating, antidementic, and acetylcholinesterase inhibiting effects were observed in animal models and indicated that the herb holds potential phytochemicals which could be used to relieve debilitating ailments such as Alzheimer's disease (Wang et al., 1993; Weinges and Künstler, 1977; Li et al., 1998; Aoyama et al., 1997; Kokubun and Harborne, 1995). In addition, the cognitive enhancing activity was supported by numerous clinical studies, which displayed memory and learning augmenting effects upon chronically administered to adults and children. B. monnieri was correspondingly examined to possess antidepressant and anticonvulsive (antiepileptic) activities. In addition to memory enhancing activity, it has also been described to show anti-inflammatory, antiulcerogenic, anti-Helicobacter pylori, anthelmintic, adaptogenic, anticancer, cardiac-antidepressant, bronchovasodilatatory, antipyretic, sedative and mast cell stabilizing activities (Jain et al., 1994; Russo et al., 2003a, 2003b). 5.1. Neuropharmacological activity Ample studies had been carried on alcoholic extracts and isolated compounds of B. monnieri by several researchers for their neuropharmacological effects. The conducted studies demonstrated that cognitive-enhancing effect was due to the presence of two active compounds, viz., bacosides A and B (Singh and Dhawan, 1982; Malhotra and Das, 1959; Rai et al., 2003). Besides, facilitating learning and memory enhancing activities these active principles repressed the amnesic effects of scopolamine, immobilization stress and electroshock (Dhawan and Singh, 1996). The mechanism of action has demonstrated that the dephosphorylation membrane induced by bacosides with

5. Biological activities The use of B. monnieri goes back from 3000 years or more and is described for its memory improving capacity in the Vedic scripts “Athar-Ved Samhita” (3:1) of 800 BCE and in Ayurveda. This herb attracted the attention of enthusiastic phytochemical researchers for its 6

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Fig. 2. (continued)

growth in protein and RNA turn over in particular brain areas (Srivastava and Yadav, 2016). It has been documented that bacosides enriched fraction retreated the breaking down of acetylcholine, decrease in cholinesterase activity and reduction in muscarinic acetylcholine receptor (mAChRs) binding in the frontal cortex and hippocampus induced by neurotoxin and colchicines (Bhattacharya et al., 1999). In adult wistar rats, experimental studies concluded that B. monnerri and bacoside A act synergistically with therapeutic application by reversing the alteration in GABA receptor binding in cerebral cortex neurons and also helpful for managing mood disorder, memory troubles during epilepsy (Mathew et al., 2012). Histopathological findings provided significant effect of neuroprotection by bacopaside I in cerebral ischemic injury in rats induced by middle cerebral artery occlusion (MCAO) (Liu et al., 2013). The neuroprotective activity of bacopaside I was blocked by PKC/PI3K inhibitors and the restoration of phospho-Akt (p-Akt) level, an anti-apoptotic factor. Thus, concluding that bacopaside I has a contribution towards neuroprotection in ischemia-induced cognitive deficits, via PKC and PI3K/Akt pathways (Le et al., 2015). A study demonstrated that bacopaside I has therapeutic effect on cognition and neuropathology in APP/PS1 transgenic mice via immune

stimulation of β-amyloid clearance. The authors reported the mechanism of Alzheimer's disease (AD) mouse model for the very first time, BS-I frees Amyloid-β via the induction of an appropriate degree of innate immune-mediated stimulation and phagocytosis (Li et al., 2016). Oral administration of ethanolic extract of B. monnieri in adult wistar rat model for 60 days showed encouraging results on long-term synaptic potentiation enhancement and strengthening of hippocampal synapses, which plays a significant role in learning and memory formation (Promsuban et al., 2017). Konar et al. (2015), evaluated the first molecular evidence by pre- and post-administration of CDRI-08 ameliorated amnesic effect of scopolamine via enhancement of both neuronal and glial plasticity markers and own-regulated acetyl cholinesterase activity. B. monnieri administration was also found to normalize neurotranmitter (dopamine, acetylcholine, glutamate, norepinephrine) levels and to contribute in improvement of scopolamine effect by modulating AChE, BDNF, CREB expression against scopolamine induced amnesia in hippocampus of rat brain (Pandareesh et al., 2016). Number of executed studies indicated that B. monnieri could be a frontline in prevention and restoration of cognitive deficit in schizophrenia (Piyabhan et al., 2016). A mice model induced by 1-methyl-47

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Table 1 Dammarane-type triterpenoid saponins reported from B. monnieri. Saponins

Structural name

Reference

Bacogenin A1 (3)

3,18-dihydroxy-20 → 25-epoxy-22 (or 23)-methyl-24-nor-dammar-22-en-16-one.

Bacogenin A2 (4)

Epimeric genin of Bacogenin A1

Bacogenin A3 (5) Bacogenin A4 (6)

– Ebelin lactone pseudojujubogenin

Bacoside A1 (7)

3-O-[α-L-arabinofuranosyl-(1 → 3)-O-α-L-arabinopyranosyl] jujubogenin

Bacoside A3 (8)

3-O-[β-D-glucopyranosyl-(1 → 3)-O-{α-L-arabinofuranosy-(1 → 2)}-O-β-D-glucopyranosyl)] jujubogenin

Bacopasaponin (9) Bacopasaponin (10) Bacopasaponin (11) Bacopasaponin (12) Bacopasaponin (13) Bacopasaponin (14) Bacopasaponin (15) Bacopasaponin (16)

A

3-O-α-L-arabinopyranosyl-20-O-α-L-arabinopyranosyl jujubogenin

Kulshreshtha and Rastogi (1973a) Kulshreshtha and Rastogi (1974) Chandel et al. (1977) Kulshreshtha and Rastogi (1973b) (Jain and Kulshreshtha, 1993) Rastogi and Mehrotra (1990) Garai et al. (1996a)

B

3-O-[α-L-arabinofuranosyl-(1 → 2)-O-α-L-arabinopyranosyl] pseudojujubogenin

Garai et al. (1996a)

C

3-O-[β-D-glucopyranosyl (1–3){α-L-arabinofuranosyl-(1 → 2)}-α-L-arabinopyranosyl] pseudojujubogenin

Garai et al. (1996a)

D

3-O-[α-L-arabinofuranosyl-(1 → 2)-β-D-glucopyranosyl] pseudojujubogenin

Garai et al. (1996b)

E

Mahato et al. (2000)

F

3-O-[β-D-glucopyranosyl-(1 → 3){α-L-arabinofuranosyl-(1 → 2)}- O -α -L arabinopyranosyl]-20-O-(α-L-arabinopyranosyl) jujubogenin 3-O-[β-d-glucopyranosyl (1 → 3){α-L- arabinofuranosyl (1 → 2)}β-d-glucopyranosyl]-20-O-α-L-arabinopyranosyl) jujubogenin

Mahato et al. (2000)

G

3-O-[α-L-arabinofuranosyl-(1 → 2)]- O - α-L-arabinopyranosyl jujubogenin

Hou et al. (2002)

H

3-O-α-L-arabinopyranosylpseudojujubojenin

(Mandal and Mukhopadhyay, 2004) Dang et al. (2018)

Bacopasaponin I (17) Bacopaside I (18)

3-O-[β-D-glucopyranosyl-(1 → 3)-O-{α-L-arabinofuranosyl-(1 → 2)}-O-β-D-glucopyranosyl]-20-deoxypsedojujubogenin 3-O-α-L-arabinofuranosyl-(1 → 2)-[6-O-sulphonyl-β-D-glucopyranosyl-(1 → 3)]-O-α-L-arabinopyranosyl] pseudojujubogenin

Bacopaside II (19)

3-O-α-L-arabinofuranosyl-(1 → 2)-[-O-β-D-glucopyranosyl-(1 → 3)]-O-β-D-glucopyranosyl pseudojujubogenin

Bacopaside III (20) Bacopaside IV (21) Bacopaside V (22)

3-O-α-L-arabinofuranosyl-(1 → 2)-O-β-D-glucopyranosyl jujubogenin

3-O-[6-O-sulfonyl-β-D-glucopyranosyl-(1 → 3)]-O-α-L-arabinopyranosyl pseudojujubogenin

Chakravarty et al. (2001) Chakravarty et al. (2001) Chakravarty et al. (2003) Chakravarty et al. (2003) Chakravarty et al. (2003) Zhou et al. (2007)

3-O-{β-D-glucopyranosyl-(1 → 3)-[α-L-arabinofuranosyl-(1 → 2)]-O-α-L-arabinopyranosyl} jujubogenin

Zhou et al. (2007)

3-O-{β-D-glucopyranosyl-(1 → 3)[α-L-arabinofuranosyl-(1 → 2)]-β-D-glucopyranosyl}-20-O-α-L-arabinopyranosyl jujubogenin

Zhou et al. (2007)

3-O-{β-D-glucopyranosyl-(1 → 4)[α-L-arabinofuranosyl-(1 → 2)]-O-β-D-glucopyranosyl}-20-O-α-L- arabinopyranosyl jujubogenin

Zhou et al. (2009)

3-O-{β-D-glucopyranosyl-(1 → 3)[α-L-arabinofuranosyl-(1 → 2)]-α-L-arabinopyranosyl} jujubogenin 3-O-[α-L-arabinofuranosyl (1 → 3)]-6-O-sulfonyl-β-D-glucopyranosyl pseudojujubogenin

Deepak et al. (2005) Bhandari et al. (2009a) Bhandari et al. (2009a) Sivaramakrishna et al. (2005b) Sivaramakrishna et al. (2005b) Pawar and Bhutani (2006) Pawar and Bhutani (2006) Ohta et al. (2016)

3-O-β-D-glucopyranosyl-(1 → 3)-O-α-L-arabinopyranosyl jujubogenin 3-O-β-D-glucopyranosyl-(1 → 3)-O-α-L-arabinopyranosyl pseudojujubogenin

Bacopaside VI (23) Bacopaside VII (24) Bacopaside VIII (25) Bacopaside IX (26) Bacopaside X (27) Bacopaside XI (28) Bacopaside XII (29) Bacopaside N1 (30) Bacopaside N2 (31) Bacoside A4 (32)

3-O-α-L-arabinopyranosyl jujubogenin

Bacoside A5 (33)

20-O-α-L-arabinopyranosyl jujubogenin.

Bacomosaponin A (34) Bacomosaponin B (35)

3-O-{β-D-glucopyranosyl (1 → 3)[α-L-arabinofuranosyl (1 → 2)]-β-D-glucopyranosyl}-20-O-(3,4-diacetyl-α-l-arabinopyranosyl) jujubogenin 3-O-{β-D-glucopyranosyl (1 → 3)[α-L-arabinofuranosyl (1 → 2)]-α-L-arabinopyranosyl}-20-O-(3,4-diacetyl-α-l-arabinopyranosyl) jujubogenin

3-O-{β-D-glucopyranosyl (1 → 3)[α-L-arabinofuranosyl (1 → 2)]- O -β-D-glucopyranosyl}-20-O-α-L-arabinopyranosyl pseudojujubogenin 3-O-[β-D-glucopyranosyl-(1 → 3)- O -β-D- glucopyranosyl] jujubogenin 3-O-[β-D-glucopyranosyl-(1 → 3)-O-β-D-glucopyranosyl] pseudojujubogenin

phenyl-1,2,3,6-tetrahydropyridine (MPTP) on Parkinsons disease found that an ethanolic extract of B. monnieri helps to improve motor behaviour, reduces oxidative stress, inhibits apoptotic pathways of dopaminergic neurons (Singh et al., 2017). Bacoside A also showed neuroprotective response against 6-hydroxy dopamine induced parkinsonism (Shobana and Sumathi., 2013) and reduced the change affected by

Ohta et al. (2016)

H2O2-induced oxidative stressed neuronal (N2a) cells, which may be due to suppression of ROS and N2a cell apoptosis (Bhardwaj et al., 2018). Bacopa extract and bacoside A significantly deterred the activity of caspase 1 and 3 and MMP3 in a cell free assay system that target reduction in neuroinflammation, and have the potential for treating a wide range of CNS disorders including Alzheimer's disease, depression, 8

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and schizophrenia (Nemetchek et al., 2017). An in silico study illustrated that bacopasaponin G and bacopaside N2 showed the favourable docking binding energy value compared to Donepezil on Caspase-3 and Tau protein kinase I receptors. These two compounds exhibit inhibitory effects on the studied receptors to prevent Alzheimer's disease (Roy et al., 2019). B. monnieri aqueous extract was found to activate caspase3 and downregulate Bcl-2 gene expression in EAT (Ehrlich ascites tumor) cell lines in vivo (Kalyani et al., 2013). Furthermore, B. monnieri extract supposedly decreased the amyloid levels in PSAPP mouse supporting the attainability of bacoside A in crossing the blood-brain barrier (Holcomb et al., 2006). Bacoside A showed the potential of neuroprotective action against Amyloid-β instigated neuronal toxicity in SH-SY5Y cells. The rescue for neurodegenerative disorder was because of being the after effect of its antioxidant nature (Limpeanchob et al., 2008). Malishev et al. (2017) elucidated the significant effects of bacoside A on Aβ42 induced cell toxicity, the peptide which leads to neuronal toxicity resulting in Alzheimer's disease. In contrast to extracellular Amyloid-β aggregates, intracellular aggregates of Tau are also considered as a cause of AD. B. monnieri extract (100 μg/ml) usage in nerve growth factor-deprived PC12 cells abate the phosphorylated-Tau levels and weakens Tau-interceded toxicity (Ternchoocheep et al., 2012). In contrast to extracellular Amyloid-β totals, intracellular aggregates of Tau are moreover considered as a reason for AD. B. monnieri extract or bacoside A supplementation significantly downregulated the cAMP content and ameliorated the dopaminergic imbalance which prompted a decreased activity in the cerebral cortex of hypoglycaemic rats (Thomas et al., 2013). A study described the high efficacy of bacoside A-loaded PLGA Poly (lactic-co-glycolic acid) nanoparticles in conveying bacoside A to the brain, crossing the blood brain barrier (Jose et al., 2014). B. monnieri loaded-Pt nanoparticles treatment reversed the MPTP-induced neurotoxicity in Parkinsonism fish model via suppression of mitochondrial complex I (Nellore et al., 2013). Sekhar et al. (2019) documented that the major constituents of this herb i.e. bacoside A and bacopaside I displayed neuroprotective effects by diminishing oxidative stress, persuading expression of antioxidant enzymes and regulating surface expression of different neuroreceptors.

expression and cytochrome P450 action in rat brain (Chowdhuri et al., 2002). Bacopaside I (Zu et al., 2017) and B. monnieri extract (80–120 mg/kg, dose) (Banerjee et al., 2014) displayed noticeable antidepressant effect in mice/rat deficits model induced by chronic unpredictable mild stress (CUMS) by activating brain derived neurotrophic factor (BDNF) signaling pathway and messenger RNA expressions in the hippocampus and frontal cortex. Singh et al. (2014) assessed the impact of constituents, bacoside A and bacopaside I and found that these compounds inhibited the human recombinant MAO enzymes. Monoamine oxidase (MAO) inhibitors are anti-depressant drugs which avert oxidative deamination of monoamine type neuro synapses (Sekhar et al., 2019). B. monnieri extract restored the regular level of brain derived neurotrophic factor (BDNF), Akt (protein kinase B), cyclic-AMP response element binding (CREB) on chronic unpredictable stress (CUS) induced depressive rat model (Hazra et al., 2017). B. monnieri preventive treatment significantly ameliorated oxidative homeostasis by restoring paraquat-induced oxidative stress markers such as reactive oxygen species (ROS), malondialdehyde (MDA), and hydro-peroxides (HP) levels. Oral supplementation of its extract for 4 weeks also renders the brain resistant to mitochondrial dysfunctions, and neurotoxicity of prepubertal mice (Hosamani et al., 2016). In another study bacopaside I was evaluated for the anti-depressant activity for the first time in mice model. A daily dose of bacopaside I (15 and 5 mg/kg) for seven progressive days, significantly reversed reserpine-induced depressive-like actions via the central noradrenergic neurotransmitter system although the detailed mechanism had not been elucidated (Liu et al., 2013a, 2013b). 5.4. Anticonvulsant activity Reported studies indicated that the water extract of B. monnieri controls epilepsy. Its extract revealed a sedative effect (Shanmugasundaram et al., 1991) and showed prolonged pentobarbitone hypnotic action, offer shield against electroshock seizures and chemoconvulsions (Shanker and Singh, 2000). An ayurvedic formulation containing major phytochemical bacoside A showed significant anticonvulsant, antischizophreniac and memory enhancing activity on pentylenetetrazol (ptz)-induced seizures in laboratory animals (Mishra et al., 2018).

5.2. Anxiolytic activity The anxiolytic properties of B. monnieri extracts assumes greater significance in the fact unlike benzodiazepine anxiolytic's amnesic action. The extracts at the level of 5, 10 and 20 mg/kg when compared with lorazepam, a standard benzodiazepine (0.5 mg/kg administered orally) revealed that B. monnieri exhibit better anxiolytic effects compared to lorazepam (Bhattacharya and Ghosal, 1998). The ethanolic extract of B. monnieri at the doses 200 mg/kg and 300 mg/kg exhibited significant protection against anxiety like behaviour which was increased during alcohol abstinence by regulating GABA signaling pathways in Rats (Lal and Sharma, 2019). The alterations in catalase enzyme activity, glutathione level and the content of thiobarbituric acid reactive substances (lipid peroxidation marker), reverted by an ayurvedic drug B. monnieri against oxidative stress methyl mercury toxicity induced in mitochondrial-enriched fractions of rat brain (Ayyathan et al., 2015).

5.5. Antioxidant activity Different oxidative stresses lead into imbalance of redox reactions resulting into loss in cellular functions. In such conditions antioxidants provide initial defence system to scavenge free radicals. Studies showed that the cognition promoting properties of B. monnieri are partially ascribed to the antioxidant effects in key areas of rat brain i.e. hippocampus, frontal cortex and striatum (Bhattacharya et al., 2000). It was reported to protect the liver (Singh et al., 2015) and brain from morphine induced inhibition antioxidant enzymes system (Sumathy et al., 2002) where its standardized methanolic extract contain bacoside-A [mixture of bacoside-A3 (37.5 μg/mg), bacopaside-II (4.62 μg/mg) and bacopasaponin-C (1.91 μg/mg)] has beneficial effect on opioid induced nephrotoxicity and hepatotoxicity (Shahid et al., 2016). This beneficial herbal remedy has strong antioxidant potential which may be efficient from toxicological influence of morphine and street heroin. This herb reported to protect DNA damage and cytotoxicity in human fibroblast by highly reactive free radical scavenging activity (Russo et al., 2003b). Bacosides provide neuroprotection against cigarrete smoke induced apoptosis and aluminium induced oxidative stress (Anbarasi et al., 2005a, Anbarasi et al., 2005, 2006; Vani and Devi, 2005; Jyoti et al., 2007). The bacosides rich extract has potent neuromodulatory, antioxidant and adaptogenic propensity. Bacoside rich extract prophylaxis improved histopathalogical changes, lipid peroxidation, ROS levels, acetylcholine esterase activity and brain neurotransmitter levels in rats via down regulating inducible nitric oxide synthase (iNOS) and

5.3. Antidepressant activity The standardized bacoside A extract of B. monnieri was examined for significant anti-depressant activity in rodent models and the effect was comparable to standard antidepressant drug, imipramine. Daily oral dose (20 and 40 mg/kg, once in a 5 day) of B. monnieri extract was found to have potential antidepressant activity in forced swim and learned helplessness test (Sairam et al., 2002). Pawar et al. (2001) mentioned that dammarane triterpenoids glycosides especially bacoside A3 exhibit superoxide inhibitory effect in polymorphonuclear (PMN) cells. It has potential to ease stress by Modulating SOD, Hsp70 9

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hemeoxygenase-1 (HO-1) expression, when exposed to crackers smoke (Pandareesh and Anand, 2014). The reported antioxidant effects of B. monnieri, considered as a therapy in neurodegenerative pathologies and age associated cognition decline (Mathew et al., 2010). B.monnieri loaded silver nanoparticles significantly reduced lipid peroxidation levels with a significant increment of SOD, CAT and GPx, results the end of oxidative stress and prevent impairment of tissue in Al exposed mice (Mahitha et al., 2015). The potent B. monnieri extract attenuates significantly depressive like behaviour and enhanced antioxidant activity of enzymes on chronic unpredictable stress induced rat model (Kumar and Mondal, 2016). Oral administration (50 mg/kg, daily) of B. monnieri extract for 15 days, reduces inflammatory and oxidative stress markers. Its supplementation improved cognition, suppress BACE1 activity and reversed colchicine-induced dementia in animals (Saini et al., 2019; Vani and Devi, 2005). One of the study reported that metal treated bacopa plant had no noticeable sign of toxicity due to the presence of sufficient antioxidants which prevent biological damage intervened by Reactive Oxygen Species. Sinha and Saxena (2006) concluded that the protective effect of Fe in stressed B. monnieri leaves estimated mainly because of incitement of ascorbate peroxidase (APX), non-enzymatic antioxidants and retention of bacoside A & bacopaside I content (Gupta et al., 2014).

induced ulcers via significant reduction in LPO in gastric mucosa (Sairam et al., 2001a). 5.9. Miscellaneous studies Apart from memory enhancing or mental functioning, the use of B. monnieri is described in other physiological conditions since ancient time. As a result, researchers have evaluated the other activities such as anti-inflammatory, cardiotonic, anticancer, bronchodilatory and vasodilatory effects of B. monnieri. B. monnieri effectively act as anti-inflammatory agent by inhibiting the prostaglandin synthesis (Jain et al., 1994). B. monnieri or its extracts stabilize the lysosomal membrane and did not cause gastritis at inflammation preventive doses. It showed protective effects against DNA damage in astrocytes and human fibroblasts. In vitro studies of B. monnieri determined that an anti-cancerous effect revealed via inhibition of DNA replication in cancer cell lines (Russo et al., 2003a, 2003b; Elangovan et al., 1995). In vitro, oral administration of standardized B. monnieri extract in the dose of 1 mg/ml exhibited anti-Helicobacter pylori activity (Goel et al., 2003). B. monnieri showed vasodilatory effects on calcium chloride-induced contraction in both ileum and jejunum of animal. The methanolic fractions of B. monnieri suppressed carbachol induced bronchoconstriction, bradycardia and hypotension in rat model (Channa et al., 2003). Kamkaew et al. (2019) studied the vasorelaxation elicited by bacoside A, bacopaside I and flavonoids (particularly luteolin and apigenin). However, bacoside A and bacopaside I are about 20th times more the contents of the flavonoids in B. monnieri extract. Thus, the dammarane triterpenoid glycosides would be relied upon to make a larger contribution to the vasorelaxation on Rat Mesenteric Arteries via endothelial nitric oxide synthase pathway. An In vitro study showed that methanolic extracts of B. monnieri exhibited mast cell stabilizing activity equivalent to disodium cromoglycate, a commonly used allergy treatment (Samiulla et al., 2001). Bacosine, a triterpene produced a noteworthy reduction in the blood glucose level in alloxan-induced diabetic rats and might have antihyperglycemic effect due to protection against oxidative damage as well as an increase in peripheral glucose consumption (Ghosh et al., 2011). B. monnieri extract (CDRI-08) and bacosine, enhanced recovery towards molecular basis of the memory impairments and anti-diabetic role in Streptozotocin-Induced Diabetes Mellitus Type 2 Mice (Pandey et al., 2015) and reduction of AGEs (advanced glycation end product) formation (Kishore et al., 2017). Bacoside A posess the chemopreventive potential in rodents against N-nitrosodiethylamine- instigated hepatocarcinogenesis by extinguishing lipid peroxidation and improving antioxidant status via free radical scavenging mode of action (Janani et al., 2010). Peng et al. (2010) demonstrated that the dammarane triterpene saponins, bacopaside I and bacopaside VII, had potential antitumor effect and showed cytotoxicity in methyl thiazole tetrazolium assay in vitro. Pei et al. (2016) reported that bacopaside I and bacopaside II was cytotoxic to HT-29 cancer cell lines and bacopaside II selectively blocked the AQP1 water channel. Further findings suggest that in vitro, Aquaporin1 (AQP1) modulator bacopaside II has antitumor potential by inhibiting growth of colon cancer cell via inducing cell cycle arrest and apoptosis (Smith et al., 2018). Whereas, the combination of bacopaside I and bacopaside II performed synergistically and reduced the viability, migration and proliferation of the breast cancer cell lines (MDA-MB-231, T47D, MCF7 and BT-474) (Palethorpe et al., 2019). Furthermore, the standardized extract of this herb additionally reduces allodynia and hyperalgesia in the CCI (chronic constriction injury) model of neuropathic throb in rats (Shahid et al., 2017). The findings demonstrated B. monnieri was equipotent to gabapentin (the positive control) and presents antinociceptive properties.

5.6. Hepatoprotective activity D-GalN induced liver damages reflect turbulences of liver cells metabolisms, leads to the characteristic alterations in the serum enzyme activities. Excess serum enzymes are signals of cellular leakage and loss of functional integrity of the liver cell membrane (Ryan et al., 2008). Oral administration of bacoside A (10 mg/kg, daily) for 21 days and then after the final administration of bacoside A, D-GalN (300 mg/kg) was injected on 21st day. It was observed that bacoside A prevent the elevated levels of enzyme activities viz., serum alanine transaminase (ALT), alkaline phosphatase (ALP), aspartate transaminase (AST), γglutamyl transferase (γ -GT), lactate dehydrogenase (LDH), 50 nucleotidase (5′ ND) against intoxicated rats. In addition, bacoside A knowingly behind the normalization of the decreased levels of vitamin-C, and vitamin-E against D-GalN both in liver and in plasma which suggests that bacoside A possesses hepatoprotective effect in contrary to D-GalN intoxicated in rats (Sumathi and Nongbri, 2008). 5.7. Anti-bacterial activity The antibacterial activity of B. monnieri (methanol, ethanol, chloroform and petroleum ether) was investigated against Bacillus amyloliquefaciens, Streptococcus pyogens (MTCC 1923), Vulgarica, Bacillus megaterium (MTCC 3353), Aspergillus niger (MTCC 281), Bacillus pumilus, Salmonella typhi, Bacillus subtilis and Micrococcus luteus. The conducted study revealed that methanol extract was considered to exhibit highest antibacterial activity (Joshi et al., 2013). 5.8. Gastrointestinal effects The In vitro studies of B. monnieri have demonstrated direct spasmolytic activity on intestinal smooth muscle predominantly through the suppression of calcium influx, applicable to both electrical impulsemediated and receptor-mediated calcium ion channels in the cell membrane. In vivo and In vitro, studies proposed that B. monnieri may have a shielding and healing effect on gastric ulcers (Sairam et al., 2001b; Goel and Sairam, 2002). Bacoside A enriched fraction was assessed for its prophylactic and curative effects in rat models of gastric ulcers (Sumathy et al., 2002). Bacoside A enriched fraction at a dose of 20 mg/kg for 10 days significantly cured acetic acid-induced penetrating ulcers as well as strengthened the mucosal blockade and reduced mucosal exfoliation. The standardized extract's antioxidant properties relieved stress-

5.10. Toxicological studies B. monnieri, a nootropic herb from centuries recorded to be safe for 10

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therapeutic use in Ayurveda medicines. The LD50 of aqueous and alcoholic crude extracts of B. monnieri were 1 g and 15 g/kg respectively, by intraperitoneal route in rats (Martis et al., 1992). The aqueous crude extract did not show any toxicity when given orally at a dose of 5 g/kg. The recommended daily dose of B. monnieri is 100–150 ml decoction of leaves twice (Poonam and Singh, 2009) for the treatment of epilepsy and in general traditional recommended daily dose is 5–10 g of extract powder, 30 ml of syrup and 8–16 ml of infusion (“Bacopa monniera,” 2004). Safety evaluation of B. monnieri composition employed on 23 healthy volunteers with 350 mg daily, did not shown any untoward indications in any of the treated volunteers at pre and post-treatment periods (Pravina et al., 2007). A single dose of this herb at 5000 mg/kg to female rats did not indicate any severe undesirable effects neither produce any toxicity (Sireeratawong et al., 2016). With improved memory retention and acquisition in healthy older persons, this herb has side effects related to digestive symptoms such as nausea, abdominal cramps, and diarrhoea (Morgan and Stevens, 2010; Singh and Dhawan, 1997).

activities. B. monnieri contains two types of saponins i.e. jujubogenin and pseudojujubogenin. Dammarane triterpenoid saponin of B. monnieri have typical glycosylation pattern with β-D-glucopyranosyl or α-Larabinofuranosyl sugar moiety directly attached to C-3 of the aglycone in the most of the cases. However, in few of the cases sulphated glucopyranosyl sugar moiety is present. This up-to-date review presented methods of extraction, isolation and chemical profiling of chemical constituents of B. monnieri. The extraction procedures used in B. monnieri are classical methods viz. soxhlet extraction and maceration. However, the advanced extraction techniques such as ultra-sonication, microwave extraction, accelerated solvent extraction and super critical fluid extraction techniques could intensify the overall effectiveness of the extraction procedures. In addition, analytical modern methods such as LC-MS/MS and LC-NMR-MS and other hyphenated techniques execute better analysis and structure elucidation of chemical constituents in B. monnieri. Recently, chemical constituents of B. monnieri were characterized by LC-ESI-OTOF-MS and concluded that thirty-two new saponins were tentatively characterized which provides a basis for further isolation of these compounds for their valuable activity (Nuengchamnong et al., 2016). The structure elucidation part describes the spectroscopic techniques such as 1D, 2D-NMR and high resolution mass spectroscopy. Various biological and pharmacological activities of B. monnieri and its products have been summarized. In lit of many reports, B. monnieri revealed that the wide varieties of neuropharmacological actions of this plant opens up enthralling prospects for future research and may offers innovative perceptions in the treatment of diseases. Clinical trials and future research is required to ensure the outcomes of this plant species. Future research should also have a focus on synergistic effects of different compounds of this herb rather than considering only bacoside A & B, structure activity relationships (SAR) for strategic drug development/formulation leads and translational research studies for clinical applications (Fishburn, 2013). Molecular modelling studies proves to be supportive in the preliminary evaluation and creation of SAR (Ali et al., 2012). This herb has a vast untapped potential in terms of undescribed constituents that may have promising pharmacological activities and may provide prospect for future studies.

5.11. Clinical studies Several clinical studies have been carried out to ascertain the efficiency of B. monnieri in state of absentmindness, low memory retention and learning. Clinically, a number of experiments have been conducted to study the acute and chronic effects on cognitive functions. A study conducted to examine the chronic effects of an extract of B. monnieri (Stough et al., 2001) via double-blind placebo-controlled plan and a series of well-validated neuropsychological tests, on cognitive function in healthy human subjects. To study the significant effect of B. monnieri on retention of new information, 76 adults aged between 40 and 65 years were volunteered. Follow-up tests showed that the retention rate of newly acquired information were significant, but the learning rate and anxiety level was unaffected (Roodenrys et al., 2002). The authors provide support for the previous published studies that chronic dosages of herb treatment for 90 days on healthy volunteers increases accuracy and memory consolidation in more complex cognitive clarity (Stough et al., 2008). A randomized double blind placebo controlled protocol for the first 16 weeks over male children (6–14 years age) with standardized B. monnieri extract (CDRI 08) supplementation, significantly indicated that it may be beneficial for the symptoms of hyperactivity or Attention Deficit Hyperactivity Disorder (ADHD) and improves cognitive performances (Kean et al., 2015). Another study of the alike propose tested with a beverage powder, combination of plant extract and multiple micronutrients in 300 children over 4 weeks, significantly improved spatial memory test on day 60 but did not shown any relevant results on short term memory neither on secondary outcomes (sustained attention, periodic memory) relative to “control” beverage (Mitra-Ganguli et al., 2017). Results from a randomized double-blind placebo controlled trial involving 60 medical students with 6 weeks administration of B. monnerri, showed statistically significant improvement relating to cognitive functions from conducted various neuropsychological tests (logical memory test and digit span memory task) but there was no change in attention and sensory motor performance of brain (Kumar et al., 2016). A plant based literature review which have evidence of anxiolytic activity through human clinical trial found that B. monnieri have anti-anxiety effects on secondary outcomes for people with cognitive decline (Sarris et al., 2013).

Declaration of competing interest Authors declare no conflict of interest. Acknowledgement Authors are grateful to the Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur, INDIA, for support. This work was supported by project MLP-0203.IHBT communication number is 4447. References Agrawal, H., Kaul, N., Paradkar, A.R., Mahadik, K.R., 2006. Separation of bacoside A3 and bacopaside II, major triterpenoid saponins in bacopa monnieri, by HPTLC and SFC. Application of SFC in implementation of uniform design for herbal drug standardization, with thermodynamic study. Acta Chromatogr. 17, 125–150. Ahmed, B., Rahman, A., 2000. Bacosterol, a new 13, 14-seco-steroid and bacosine, a new triterpene from Bacopa monniera. Indian J. Chem. 39B, 620–625. Ahmed, A., Ahmad, S., Ur-Rahman, M., 2015. Quantitative analysis of bacoside A from Bacopa, collected from different geographic regions of India, by high performance thin layer chromatography-densitometry. J Plan. Chrom 28 (4), 287–293. Ali, R., Mirza, Z., Ashraf, G.M.D., Kamal, M.A., Ansari, S.A., Damanhouri, G.A., Abuzenadah, A.M., Chaudhary, A.G., Sheikh, I.A., 2012. New anticancer agents: recent developments in tumor therapy. Anticancer Res. 32, 2999–3005. Anbarasi, K., Vani, G., Balakrishna, K., Devi, C.S.S., 2005. Creatine kinase isoenzyme patterns upon chronic exposure to cigarette smoke: protective effect of Bacoside. A. Vasc. Pharmacol. 42, 57–61. Anbarasi, K., Vani, G., Balakrishna, K., Devi, C.S.S., 2006. Effect of bacoside A on brain antioxidant status in cigarette smoke exposed rats. Life Sci. 78, 1378–1384. Anbarasi, K., Vani, G., Balakrishna, K., Devi, C.S.S., 2005a. Effect of bacoside A on membrane-bound ATPases in the brain of rats exposed to cigarette smoke. J. Biochem. Mol. Toxicol. 19, 59–65.

6. Conclusion and future prospects Over the years, researchers had looked at the reputed herb of ayurveda for its bioactive constituents and pharmacological activities with special emphasis on neuroprotection. The present review gave the summary of chemical constituents, especially, dammarane triterpenoid glycosides, extraction, isolation, structure elucidation strategies, chemoprofiling and biological 11

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Nitisha Sendri is a current Research fellow under the guidance of Dr. Pamita Bhandari in the department of Natural Product Chemistry & Process Development Division at the CSIR-Institute of Himalayan Bioresource and Technology, Himachal Pradesh, India. She did her postgraduate in Chemistry from Hemvati Nandan Bahuguna Garhwal University (Central University), Uttarakhand, India. Her present focus is on exploration of Western Himalayan flora for natural colors/dyes and bioactive molecules.

Pamita Bhandari has completed her doctorate degree in Natural Product Chemistry at CSIR- Institute of Himalayan Bioresource Technology, Palampur. During her Ph.D she joined National Institute of Pharmaceutical Education and Research (NIPER, Mohali, India) as Scientist in the Department of Natural Products. Currently, she is working as Senior Scientist at CSIR-IHBT Palampur, India and her research is focused towards Natural Product Chemistry in terms of exploring unique chemical diversity of Himalayan flora; specifically, the isolation and structure elucidation of the undescribed scaffolds of biological relevance, methodologies development for quality assurance and chemical fingerprinting of medicinal plants.

Shinde Bhagatsing Devidas did his M.S. (Pharm.) from the Department of Natural Products, National Institute of Pharmceutical Education and Research (NIPER Mohali (2014–2016). Currently he is working as Research fellow in Natural Product Chemistry and Process Development Division under the guidance of Dr. Pamita Bhandari, Senior Scientist at CSIR-Institute of Himalayan Bioresource and Technology, Himachal Pradesh, India. Presently, he is working on isolation and characterization of bioactive molecules from plants of Western Himalaya.

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