Advances in dammarane-type triterpenoid saponins from Bacopa monnieri: Structure, bioactivity, biotechnology and neuroprotection

Chapter 15

Advances in dammarane-type triterpenoid saponins from Bacopa monnieri: Structure, bioactivity, biotechnology and neuroprotection Samapika Nandya, Abhijit Deya,* and Anuradha Mukherjeebb a

Department of Life Sciences, Presidency University, Kolkata, India MMHS, South 24 Pgs, West Bengal, India * Corresponding author: e-mail: [email protected]; [email protected] b

Chapter Outline Introduction Phytochemistry Biotechnology Pharmacological properties Adaptogenic/anxiolytic activity Anti-Alzheimer’s activity Anti-amnesic activity Anti-cancer activity Anti-cholinesterase activity Anti-dementia activity Anti-depressant activity Anti-diabetic activity Anti-epileptic activity Anti-fertility activity Anti-Helicobacter pylori activity Anti-hypercholesterolemic/ renoprotective activity Anti-inflammatory activity Anti-nociceptive activity Anti-muscarinic activity

490 491 491 506 506 506 507 507 508 508 508 509 509 509 510 510 510 511 511

Anti-oxidant activity Anti-Parkinson’s activity Antipsychotic activity Anti-rheumatic activity Anti-schizophrenic activity Anti-thyroid activity Cardioprotection Cognition enhancement Emetogenic activity Hepatoprotection Mast cell stabilizing activity Memory enhancement Neuropharmacological properties Neuroprotection Nootropic effect TLE antagonism Wound healing activity Activity of the active compounds Anti-amyloid activity Anti-anxiolytic activity Anti-autism activity

Studies in Natural Products Chemistry, Vol. 63. https://doi.org/10.1016/B978-0-12-817901-7.00015-0 Copyright © 2019 Elsevier B.V. All rights reserved.

512 512 512 513 513 513 513 514 514 515 516 516 516 517 519 519 519 520 520 520 520

489

490 Studies in Natural Products Chemistry Anti-cancer activity Anti-depressant activity Anti-dopaminergic/serotonergic effect Anti-hyperglycemic activity Anti-inflammatory activity Anti-nematode properties Anti-oxidant activity Cognition enhancement Cytogenetic effect Hepatoprotection

521 521 521 522 522 522 522 522 523 523

Neuropharmacological properties Nootropic effect Oxido-nitrosative stress related activity LDH-stabilizing activity Wound healing activity Concluding remarks Abbreviations References

524 524 524 524 525 525 525 529

Introduction Herbal medicine is frequently used by 80% of the world population and is gaining in popularity in Europe and North America [1]. In one survey during 2008, the National Institutes of Health (NIH) observed 4 in 10 adults reported using complementary and alternative medicine (CAM) in the last 1 year, of which 17.7% was herbal medicine [2] Active phyto-constituents of Bacopa monnieri (L.) Wettst. (Scrophulariaceae) (synonyms: Bacopa monniera and Herpestis monniera) were identified as the dammarane-type triterpenoid saponins such as bacosides A and B. Later, bacoside A was determined as a mixture of four saponins viz. bacoside A3, bacopaside II, bacopasaponin C and the jujobogenin, an isomer of bacopasaponin C whereas the identity of bacoside B is still dubious. Bacosides and/or bacopasides were reported against morphine dependence, memory deficit in epilepsy, hepatocarcinogenesis, hepatotoxicity, β-amyloid cytotoxicity, inflammation and oxidative stress. B. monnieri exhibits neuroprotective properties, nootropic and memory enhancing properties. Besides, it also improves concentration, comprehension, recall and alertness and considered as a natural antioxidant implicated in neuroprotection. Neuroprotective role of the plant was manifested against diseases such as epilepsy, Parkinson’s disease (PD), Alzheimer’s disease (AD) associated dementia, attention-deficit hyperactivity disorder (ADHD) in children and anxiety. Many of these neuroprotective properties of B. monnieri were attributed to the active phytochemicals bacosides and/or bacopasides. Unfortunately little is known about the underlying mechanism of neuroprotection manifested by these dammarane-type triterpenoid saponins and very little systematic structure-activity relationship studies have been carried out. Therefore, information on structure-bioactivity relationships of these saponins, and development of their semi-synthetic derivatives are of immense importance in facilitating rational designing of novel drugs based on leads from bacopasaponins. This review is focused on the origin, analysis of the pharmacological and medicinal properties and structural significance of bioactivities exerted by the bacopasaponins. Microbial regulation of bacoside A biosynthetic pathway and taking advantage of microbes for enhancing bacoside content are also

Advances in dammarane-type triterpenoid saponins Chapter

15 491

discussed. Moreover, to unfold future research openings, lacunae in the present understanding are also highlighted. Together, this review may further direct to the synthetic optimization of bacopasaponins as lead compounds for the designing of efficacious and clinically acceptable analogs. The present review incorporates the literature involving the structure, bioactivity, biotechnology and neuroprotection associated with the dammarane-type triterpenoid saponins from B. monnieri. Tables 15.1 and 15.2 represent the phytochemicals and pharmacological properties of active extracts and bioactive compounds from the plant. Fig. 15.1 represents the chemical structures of the bioactive alkaloids compiled from the well-known database Pubchem (www. Pubchem.com). Nomenclature of the plant has been authenticated from the Missouri Botanical Garden’s electronic database (www.tropicos.org).

Phytochemistry The major nootropic chemicals are dammarane types of triterpenoid saponins known as bacosides, with jujubogenin or pseudo-jujubogenin moieties as aglycone units [99]. Bacosides are known to be composed of a family of 12 known analogs. 39 Novel saponins such as bacopasides I–XII have been reported recently [5,8]. Besides, alkaloids such as brahmine, nicotine, and herpestine have also been reported. The plant is also known to contain D-mannitol, apigenin, hersaponin, monnierasides I–III, cucurbitacins and plantainoside B [14,100–106]. The most studied chemical bacoside A, is actually a mixture of bacoside A3, bacopacide II, bacopasaponin C, and a jujubogenin isomer of bacosaponin C.48. In another experiment, the bacoside profile was reported to be the following: bacopaside I (5.37%), bacoside A3 (5.59%), bacopaside II (6.9%), bacopasaponin C isomer (7.08%), and bacopasaponin C (4.18%) [91].

Biotechnology The plant was attributed a second rank in a priority list of the important Indian medicinal flora assessed considering medicinal properties, commercial uses and possible material for research and development studies [107]. However, indiscriminate harvesting of B. monnieri from natural populations has caused a fast downfall in the wild populations, and that is why the plant is included as a threatened species by the International Union for Conservation of Nature and National Resources (IUCN) [108,109]. The tentative requirement of B. monnieri for the year 1990 was estimated about 12,700 t of dry biomass with an estimated cost of Rs. 1.5 billion [110]. Because of its widely accepted pharmacological, commercial and ornamental properties, both wild and cultivated plants are in constant requirement [111] and that is why large scale production of this plant by high frequency in vitro multiplication of shoots is in high demand [112,113]. A number of tissue culture or plant biotechnological protocols have been reported for in vitro production of this important medicinal plant [114,115].

TABLE 15.1 Saponins in Bacopa monnieri. Derivative

Name

Compound

Reference

Jujubogenin derivatives

Bacoside A1

3-O-[α-L-arabinofuranosyl(1 ! 3)]-α-L-arabinopyranoside

[3]

Bacoside A3

3-O-α-L-arabinofuranosyl-(1 ! 2)-[β-D-glucopyranosyl-(1 ! 3)]-β-Dglucopyranoside

[4]

Bacopasaponin A

3,20-di-O-α-L-arabinopyranoside

[5]

Bacopasaponin E

3-O-α-L-arabinofuranosyl-(1 ! 2)-[β-D-glucopyranosyl-(1 ! 3)]-α-Larabinopyranoside, 20O-α-L-arabinopyranoside

[6]

Bacopasaponin F

3-O-α-L-arabinofuranosyl-(1 ! 2)-[β-D-glucopyranosyl-(1 ! 3)]-β-Dglucopyranoside,20-Oα-L-arabinopyranoside

[6]

Bacopasaponin G

3-O-[α-L-arabinofuranosyl-(1 ! 2)]-α-L-arabinopyranoside

[7]

Bacopaside III

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

[8]

Bacopaside IV

3-O-β-D-glucopyranosyl-(1 ! 3)-α-L-arabinopyranosyl

[8]

Bacopaside IX

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

[9]

Pseudojujubogenin derivatives

Bacopasaponin B

3-O-[α-L-arabinofuranosyl-(1 ! 2)]-α-L-arabinopyranoside

[5,10]

Bacopasaponin C

3-O-α-L-arabinofuranosyl-(1 ! 2)-[β-D-glucopyranosyl-(1 ! 3)]-α-Larabinopyranoside

[5,10]

Bacopasaponin D

3-O-[α-L-arabinofuranosyl-(1 ! 2)]-β-D-glucopyranoside

[5,10]

Bacoside A2

3-O-α-L-arabinopyranosyl-(1 ! 5)-[α-L-arabinofuranosyl-(1 ! 6)]-αD-glucofuranoside

[11]

Bacopaside III

3-O-[6-O-sulfonyl-β-D-glucopyranosyl-(1 ! 3)]-α-L-arabinopyranoside

[7]

Bacopaside I, Bacopaside V

3-O-α-L-arabinofuranosyl-(1 ! 2)-[6-O-sulfonyl-β-D-glucopyranosyl-(1 ! 3)]-αLarabinopyranoside, 3-O-β-D-glucopyranosyl-(1 ! 3)-α-L-arabinofuranosyl

[8,12]

Bacopaside II

3-O-α-L-arabinofuranosyl-(1 ! 2)-[β-D-glucopyranosyl-(1 ! 3)]-β-Dglucopyranoside

[8,12]

Bacopasaponin H

3-O-[α-L-arabinopyranosyl]

[13]

Bacopaside XI

3-O-[β-D-arabinofuranosyl (1 ! 3)]-6-O-sulfonyl-β-D-glucopyranosyl

[14]

Bacopaside XII

3-O-{β-D-glucopyranosyl(1 ! 3)[β-D-arabinofuranosyl(1 ! 2)]-β-Dglucopyranosyl}-20-Oβ-D-arabinopyranosyl

[14]

TABLE 15.2 Pharmacological properties of extracts and bioactive compounds.

Bioactivity

Crude powder/extract/ fraction/formulation

Animal model and/or cell line

Adaptogenic

BME

SD rat

Anti-AD

BME

Anti-amnesic

Inductive chemical an/or mechanism; standard drug(s) (if any)

Mode of action

References

Acute and chronic stress

#Ulcer index, #adrenal gland weight, #level of plasma glucose, AST, CK

[15]

Mice

–

#Abeta 1–40 and 1–42 levels

[16]

BME

Mice

Diazepam

#MAP kinase, #pCREB, #iNOS

[17]

BME

Mice

Nω-Nitro-L-arginine

"CaM, "pCREB/CREB levels

[18]

BME

Mice

Scopolamine

"NMDAR GluN2B subunit expression, "spatial memory, #AchE activity

[19]

CDRI-08

Mice

Scopolamine

"Neuronal and glial plasticity markers

[20]

Anti-arthritic

BME

Rat

Collagen

#Cyclooxygenase, #lipoxygenase, #neutrophil infiltration, #seru anticollagen

[21]

Anti-autism

BME

Rat

VPA

#Oxidative stress

[22]

Anti-cancer

BME

EAT cell line

Dose-dependent toxicity

"Bax, # Bcl-2

[23]

Ethanolic BME

MCF-7 and MDA-MB 231 cell lines

Dose-dependent toxicity

"Cytotoxicity, #cell proliferation

[24]

male Swiss mice

Scopolamine

"Cognition

[25]

Swiss mice

Scopolamine

"TLT, "NTR

[25]

BME

Rat

Colchicine

"Antioxidant enzymes, #memory impairment

[26]

BME

Mice

oxygen- and glucosedeprivation-

#Hippocampal cell damage

[27]

Methanolic BME

Rodent

Imipramine

"Memory retention

[28]

Bacopaside-I

Mice

Reserpine

"Brain antioxidant activity

[29]

Methanolic BME

Mice

Morphine

"Body weight

[30]

Methanolic BME

Mice

Morphine

#Locomotion, #rearing, #defecation effects

[31]

Anti-diabetes

CDRI-08

Mice

STZ

"Spatial memory, "AMPA receptor GluR2 subunit gene expression, #oxidative stress

[32]

Anti-dopaminergic/ serotonergic

n-butanol extract

Mice

Morphine

#Locomotor activity

[33]

BME

Mice

3-NPA

#Oxidative stress, #cell death

[34]

Anti-emetic

BME

Suncus murinus

Cisplatin

#Retching, #vomiting

[35]

BME (n-butanol fraction)

Pigeon

Cisplatin; BHT

#Emetogenesis

[36]

Anti-epilectic

Bacosade

C. elegans

Pilocarpine

#convulsion

[37]

Anti-Helicobacter pylori

BME

Human colonic mucosal incubates

–

#Mucin secretion, #acid-pepsin secretion

[38]

Anti-cholinesterase

BME, G. biloba extract

Anti-dementic

BME, G. biloba extract

Anti-depressant

Continued

TABLE 15.2 Pharmacological properties of extracts and bioactive compounds.—Cont’d

Bioactivity

Crude powder/extract/ fraction/formulation

Animal model and/or cell line

Anti-hyperglycemic

BME

Male Wister rat

Bacosine

Inductive chemical an/or mechanism; standard drug(s) (if any)

Mode of action

References

Nicotinamide, streptozotocin

#Lipid peroxidation

[39]

Rat

Alloxan

"MDA, "GSH, "SOD and "CAT

[40]

Ethanolic BME

Mice and rat

Prostaglandin

#Edema

[41]

Methanolic BME

Rat

Carrageenan, BHT

"Paw edema inhibition

[42]

The triterpenoid fraction

Peripheral blood mononuclear and peritoneal exudate cells

–

#Inflammatory cytokines, #TNF-α and interleukin-6

[43]

BME

Mice

Naloxone

#Tail flick latency

[44]

Methanolic whole plant extract

Swiss albino mice

Acetic acid; aspirin

"Serum glucose concentrations

[45]

Methanolic BME

Rat

ccI; Gabapentin

"Pain threshold,#allodynia, #hyperalgesia

[46]

Anti-muscarinic

BME

PC12 cell line

SNP

"Neurotropic factor, "muscarinic muscarinic-1 receptor expression #acetylcholine esterase, #membrane damage

[47]

Anti-oxidant

BME

Human nonimmortalized fibroblasts

H2O2

#DNA damage

[48]

Anti-inflammatory

Anti-nociceptive

Anti-parkinsons

BME

Fibrosarcoma bearing rat

3-methylcholanthrene

"Antioxidant enzyme,#lipid peroxidation, #tumor markers

[49]

DHC-1

Rat

Cisplatin

"SOD, " CAT, "reduced glutathione

[50]

BME

Rat

STZ; Glibenclamide

"SOD, "CAT, "GPx, "GSH level

[51]

BME

Rat

–

#Oxidative stress in brain tissue

[52]

BME

Rat

Morphine

"Total reduced glutathione, "SOD, "CAT, "GPx, "membrane bound ATP-ases activities

[53]

Leaf powder

N27 cell lines

3-NPA

#Malondialdehyde, #ROS, #hydroperoxide levels, #protein carbonyls

[34]

BME

Rat

Lead

#ROS, #LPP, #TPCC

[54]

BME

Female mice

Stress

#Decabromodiphenyl ether induced toxicity, #oxidative stress

[55]

Aqueous BME

C. elegans

Paraquat

"Upregulate of hsp-16.2, "ROS scavenging, "longevity

[56]

CDRI-08

Mice

PBDE-209

#Oxidative stress, #memory impairment

[57]

BME

C. elegans

6-OHDA

#Alpha synuclein, #dopaminergic neurodegeneration

[58]

Alcoholic BME

Rat

6-OHDA

"Glutathione-S-transferase, "glutathione reductase, "glutathione peroxidase, "SOD and " CAT

[59]

BME

SK-N-SH cell line

MPP

"Keap1 expression, "endogenous GSH synthesis, " Akt phosphorylation

[60]

Continued

TABLE 15.2 Pharmacological properties of extracts and bioactive compounds.—Cont’d

Bioactivity

Crude powder/extract/ fraction/formulation

Animal model and/or cell line

Inductive chemical an/or mechanism; standard drug(s) (if any)

Antipsychotic

BME

Mice

Ketamine

#Monoamine oxidase activity

[61]

Anti- schizophrenic

BME

Rat

Phencyclidine

"Neuronal density

[62,63]

Calcium antagonistic

Ethanolic BME

Guinea-pig trachea

Histamine, serotonin, bradykinin, prostaglandin E(2) and arachidonic acid

"Calcium channel blocking activity

[41]

Cardioprotection

BME

Rat

Myocardial antioxidants

#Myocardial apoptosis, #caspase 3, #Bax protein

[64]

Protandim

HL-1 cardiomyocytes

H2O2

"Activation of Nrf2, #oxidative heart cell apoptosis

[65]

Cytoprotection

BME

HaCaT cell

Benzopyrene

#Benzo [a] pyrene induced apoptosis, "Beclin-1-dependent autophagy

[66]

Cytotoxicity

BME

S-180 cell line

Dose dependent toxicity

#Cell viability

[67]

Emetogenesis

Methanolic and n-butanolic fractions

Pigeon

Cisplatin

#Dopamine upsurge,

[68]

Aqueous BME

Swiss mice

Genoprotection

Mode of action

References

#intestinal 5-HT Nicotine

#Micronucleated polychromatic erythrocytes, #Hepatic GSH, #ALP, #GST

[69]

Hepatoprotection

Bacoside A

Albino male rats

DEN

" SOD, "catalase, "GP, "GR, " GST, " glutathione # AkT, LDH, AP, Gamma-GT

[70]

Bacoside A

Rat

D-GalN

# Vit-C, Vit-E

[71]

BME

Rat

Nitrobenzene

" SOD, "CAT, "GPx

[72]

Ethanolic leaf extract

Sprague-Dawley rats

Hypobaric hypoxia

Cytochrome c oxidase activity

[73]

BME

Neonatal rat

Dose-dependent assay

" Cognitive learning

[74]

Ethanolic leaf extract

Postnatal rat

" Serotonin, "memory retention # dopamine

[75]

BESEB CDRI-08

Rat

D-galactose

" Antioxidant response element, " activity of antioxidant enzymes, # serum advance glycation end product, "nuclear transcription factor NF-E2-related factor 2, # serotonin

[76]

Alcoholic BME

Sprague-Dawley rat

–

# CYP3A and Pgp activity, # testosterone 6β-hydroxylation

[77]

BME

Rat

Sodium butyrate

" Kinase ERK/CREB signaling cascade, "upregulation of p300, Ac-H3 and Ac-H4 levels, #protein phosphatases (PP1α, PP2A)

[78]

BME

Mice

Sodium fluoride

# Depression, # memory impairment

[79]

Membrane stabilization

Bacoside A

Rat

Smoke

" Lipid peroxides, "cholesterol, " C/P ratio, #phospholipids, # mitochondrial enzymes

[80]

MWS

Whole plant extract

Guinea-pig

Naloxone

# Contraction

[81]

Memory enhancer

Continued

TABLE 15.2 Pharmacological properties of extracts and bioactive compounds.—Cont’d

Bioactivity

Crude powder/extract/ fraction/formulation

Animal model and/or cell line

Inductive chemical an/or mechanism; standard drug(s) (if any)

Neuroprotection

BME

Rat

BME and RS extract

Mode of action

References

Scopolamine

# Memory dysfunction, # oxidative stress

[82]

Human glial (U-87 MG) and embryonic mouse hypothalamus cell line

–

" Amyloid precursor protein synthesis, "neurotrophic factors

[83]

BME

PC12 cells

SNP

" BDNF,#mitochondrial and plasma membrane damage, #HOand iNOS expression

[84]

Whole plant BME

Male Wistar rats

Aluminum

# SOD, #CAT, #GPx, #TBARS

[85]

BME

PC12 cells

Scopolamine

# NO generation, #iNOS expression, #Bax, #Bcl-2, # cytochrome-c, vcaspase-3

[86]

CDRI-08

Mice

CoCl2

" Fmr-1 gene expression, #Hif-1α

[87]

BME

Male Wistar rats

Streptozotocin

" Learning and memory retention, " antioxidant enzymes, "reduced glutathione, #TBARS

[88]

Bacopaside I

APP/PS1 transgenic mice

–

" Amyloid clearance by immune stimulation, " phagocytosis

[89]

BME

Mice

Rotenone

" Oxidative homeostasis, # mitochondrial dysfunction

[90]

Neuroinflammation

Bacoside

Rat brain

Age

# Cytokines, #iNOS protein expression, #total nitrite, # lipofuscin

[91]

Neuropharmacological

Bacoside A

Rat

Smoke

# Oxidative damage

[92]

Bacoside

Rat

Smoke

# HO-1 expression,

[86]

#nitric oxide, #inos expression Neurotoxicity

BME

Rat

Aluminum

# Lipid and protein damage, # oxidative stress

[93]

Oxido-nitrosative

BME

L132 cell line

SNP

# Nitric oxide synthase expression, # proteolytic membrane damage

[94]

Renoprotective

Alcoholic BME

Rat

HCD

" Antioxidant enzymes "eNOS, # iNOS

[95]

TLE antagonism

BME

Rat

Carbamazepine

" 5-HT(2C) receptor upregulation, " inositol triphosphate

[96]

Tumor-preventive

Ethanolic plant extract

Dalton’s lymphoma ascites tumor cells (Dla)

Dose dependent cytotoxicity

# Tumor formation

[96a]

Wound healing

Methanolic BME, Bacoside A

Swiss albino rat

Incision wound; Nitrofurazone

" Wound epithelialization, " collagen fiber cross-linking, " granuloma weight

[97]

Alcoholic BME

Rat

Incision wound

# Myeloperoxidase, #free radical induced tissue damage

[98]

" Prevented loss of/induced/enhanced/improved/increased/upregulated/elicited/promoted/restored/activated/inhibited depletion/protected. # Downregulated/attenuated/decreased/declined/terminated/blocked/prevented/inhibited.

502 Studies in Natural Products Chemistry H O H O

O O

H O

O

O

H H

O O

H

H O

H

H O

O

H O

H

O

O

H

O H

O H

O O

O H

O

O

HH H

O

H

O

Bacopasaponin A

Bacopasaponin B

O

O H

H

O O

O

H H

H

H

H

O H

H H O

H

O

O O H

H O

O O

O H

O H

O

O

O

H O

H

H

H

O H

Bacopasaponin C

O H

O

O

O

O O H

O

O O

H

O H

H

Bacopasaponin D

FIG. 15.1 Chemical structures of bioactive phytochemicals from Bacopa monnieri.

15 503

Advances in dammarane-type triterpenoid saponins Chapter

O

O O

O

H H

O O H

H

H

H

H O

O

O

H O O H

O O H

O O

H

O OH

H

H

O

H

O

O O

O

H O

H H

H

OH

O

O HO O

H O

O H

O

Bacopasaponin E

H

H O

O O

O

H H

H

H

H

HH H

O O O

H O

Bacopasaponin F

O

HO

O

O H

H

H O

H

O

O

O O

O H

H O

H O

O

H

O

O O

O

H

H H

O H

O O s O O

FIG. 15.1—Cont’d

O O

O H

Bacopasaponin G

O

O

H O O

O

O OH

O

O

H

O H

Bacopaside I

H

504 Studies in Natural Products Chemistry

O O

O

H

H

O O

H

H H

H

O

O O

O H

H

O HO O

H

H

H O O H

O

H H

O

O S

O

O H

Bacopaside II

H

O H

O O

O

O

O H

HO

O

O

O

O

O

O

H H

H H

O O

H

H

Bacopaside III

O H

O

O

H H

O O

O

H H

H

H H

O

O H H

O

O

O H

O O

H

H H

H

H

Bacopaside IV FIG. 15.1—Cont’d

O

O

O

O

O

H

O

O O

O

O O H

O O

H

H

Bacopaside V

H

15 505

Advances in dammarane-type triterpenoid saponins Chapter

H

O

O O

O

O

O

H H

H H

O

O S

H

O

O H

O

O

H

O

H

O

H

O

O

O H

O

O

O

O

H

H

O

H

Bacopaside XII

Bacopaside XI

H O H

O

O O

H H O

O

O H

H O

O

O

O

O H

O H H

H

H O

O

O

H

H

H

O H

O O

H O

O

H H

H

H H

H

H H

H

O O O

H

H

O

O

O

O

O O

H

H

O

O

H

O H

H

Bacoside A1 FIG. 15.1—Cont’d

O O

H O H

O

O O

O

H O

O O

O H

O

O H

O

H

H

Bacoside A3

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In B. monnieri, a very small number of multiplied shoots were derived from Murashige and Skoog (MS) media supplemented with different concentrations of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA) [111]. In another study, the highest number (18.85) of multiplied shoots was noted in differentially supplemented MS media with the maximum number of roots per explant reported to be nine [116]. Multiple shoot bud initiation were reported from leaf and stem explants which was reported on the basis of percentage of the explant’s surface area covered by shoot buds [115]. MS media supplemented with IAA (0.2 mg/L) and BAP (1.5 mg/L) in combination with adenine sulfate (60 mg/L) produced only 18 shoots/explants [117].

Pharmacological properties Adaptogenic/anxiolytic activity The adaptogenic property of a B. monniera extract (BME) was assessed against acute stress (AS) and chronic stress (CS) models in male SpragueDawley (SD) rats using root powder of Panax quinquefolium (PGR) as standard. The rats (180–200 g), were pretreated for 45 min before immobilization stress with these components for 3 days in AS and 7 days in CS. Histological and biochemical analysis indicated low dosage of BME (40 mg/kg p.o.) reversed AS-induced enhancement of ulcer index, adrenal gland weight, level of plasma glucose, aspartate aminotransferase (AST) and creatine kinase (CK) whereas its higher dosage (80 mg/kg p.o.) and PGR (100 mg/kg p.o.) reversed CS induced anomalies [15]. A randomized, double-blind, placebo-controlled cross-over study registered with the Australian and New Zealand Clinical Trials Registry (ACTRN12612000834853) showed the anxiolytic and adaptogenic activity of standardized BME (KeenMind®—CDRI 08), based on the completion of a multitasking framework (MTF) completed by 17 healthy participants at baseline. They were treated with 320 mg and 640 mg of BME 1 h and 2 h after consuming a placebo. The results focused on cognitive measures, mood effects, salivary cortisol and some adaptogenic and nootropic effects of BME supplementation. The neurocognitive properties of BME were also determined by the Cognitive Demand Battery (CDB) completed by 24 volunteers in a cross-over design which provided cardiovascular and mood assessments before and after treatment [118,119].

Anti-Alzheimer’s activity BME (40 or 160 mg/kg/day for 2/8 months) used to study the Abeta pathology in PSAPP mice, significantly lowered (60%) Abeta 1–40 and 1–42 levels, reversed open field hyperlocomotion behavioral changes and Y-maze performance. The results indicated its prospect in AD therapeutics [16]. Pretreatment of the human neuroblastoma cell line SK-N-SH with BME provided protection against H2O2 and acrolein induced cellular damage. Generation

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of intracellular reactive oxygen species (ROS) preserved the mitochondrial membrane potential (MMP), prevented the modifications of several redox regulated proteins like NF-kappaB, Sirt1, ERK1/2, and p66Shc. Acrolein, byproducts of lipid peroxidation, was reported to accumulate in higher amount in vulnerable brain region and primary hippocampal culture in Alzheimer’s patient. On the other hand, H2O2 mediated oxidative modifications of proteins, lipids, and DNA were prevented by BME pretreatment; thus rendering protection against AD [120]. Free radical scavenging mechanisms of BME prevented cellular damage in areas of prefrontal cortex, hippocampus, and striatum, reduced lipoxygenase activity, increased glutathione peroxidase and iron chelating, protected the cholinergic neurons, reduced anticholinesterase activity like standard anti-AD drugs donepezil, rivastigmine, and galantamine. It decreased hippocampal β-amyloid deposition, stress-induced hippocampal damage and induced nitric oxide-mediated cerebral vasodilation. In experimental model, it improved the total memory score, logical memory associate learning and cognitive impairment which indicated BME as a potent neuroprotective agent against neurodegenerative disorders involving oxidative stress conditions [121].

Anti-amnesic activity BME reversed both diazepam and scopolamine induced amnesia in mice by antagonizing MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist and N(omega)-nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor. In L-NNA induced amnesia, pretreatment with plant extract significantly increased calmodulin (CaM) and pCREB/CREB levels [18]. Pre- and postnatal administration of specialized BME (CDRI-08) acted upon the expression of key neuronal markers (BDNF and Arc) and glial (GFAP) plasticity markers in the cerebrum of scopolamine induced amnesic mice and thus decreased acetyl cholinesterase (AChE) activity [20].

Anti-cancer activity Apoptosis inductive property of ethanolic BME was assessed on mouse S-180 cell line for cell viability in a dose- and time-dependent manner using dye exclusion method. Morphological differences between BME-treated and untreated cells were studied by transmission electron microscopy (TEM), quantification of glutathione (GSH) levels and the percentage of apoptotic cells was studied using annexin V-fluorescein isothiocyante (FITC) assay. The study indicated maximum cytotoxicity obtained with 90.2% cell death at 550 μg/mL concentration [67]. The herbal product targeted anti-cancer therapy mostly trigger the cellular apoptotic pathway through caspase proteolysis. BME has already been reported for prevention of malignant ascites fluid accumulation in tumor cell and in Ehrlich ascites tumor (EAT) cell line. Water extract (BMWE) caused constitutive expression of a 15-KDa endogenous

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endonuclease called caspase-activated DNase (CAD). It activated caspase-3 expression in cytoplasm and its nuclear translocation caused in vivo DNA fragmentation leading to cell death. Upregulation of the pro-apoptotic gene Bax and downregulation of the anti-apoptotic gene Bcl-2 were caused by BMWE treatment [23]. To evaluate the in vitro cytotoxic activity of dichloromethane (DCM) fraction of ethanolic BME on cell lines, two different cell lines (MCF-7 and MDA-MB 231) were selected and anti-tumorigenic and anti-proliferative activities were studied using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay and the resultant IC50 values for MCF-7 and MDA-MB 231 cell lines were found to be 72.0 and 75.0 μg/mL, respectively. The cytotoxicity was possibly due to the presence of cucurbitacins and betulinic acid in DCM fraction [24].

Anti-cholinesterase activity Standardized BME and the extract of Ginkgo biloba possessed antidementic and anticholinesterase activities in adult male Swiss mice. Dosage dependent (10–1000 μg) in vitro AChE activity in scopolamine-dementia groups using spectrophotometric study revealed IC50 of G. biloba was 268.33 μg, whereas in case of BME the value was 30 and 60 μg [25].

Anti-dementia activity Dementia or mild short term memory deficit can be the first stages of Alzheimer’s causing complete cognitive impairment. BME (30 mg/kg) and extract of G. biloba (15, 30 and 60 mg/kg) showed antidementic activity in passive avoidance test against scopolamine (3 mg/kg ip)-induced memory deficits in mice and caused 40–80% increase in transfer latency time (TLT) and no transfer response (NTR) [25]. In rats, cognitive decline and associated depletion in antioxidant enzyme level were induced by intracerebroventricular application of colchicine (15 μg/5 μL) and were assessed by elevated plus maze. BME (50 mg/kg b.w.) actively attenuated oxidative damage, restored LPO and protein carbonyl levels, altered activity of membrane bound enzymes [Na(+)K(+) ATPase and AChE] and reversed memory impairment in colchicine treated rats [26,122].

Anti-depressant activity Alcoholic whole plant extract (100–1000 μg/mL) reduced in vitro morphine withdrawal syndrome (MWS) in guinea-pig ileum. The plant extract based pretreatment, 15 min before morphine exposure caused dose-dependent reduction in the naloxone-induced contraction [81]. Effect of methanolic BME on morphine withdrawal induced depression in mice was evaluated using forced swimming test (FST) 3 days after last morphine injection. Chronic morphine treatments were done by injecting mice with a dose of 20–65 mg/kg b.w. twice

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daily for eight consecutive days. The experimental dosage (10, 20, and 30 mg/kg b.w.) significantly inhibited opioid withdrawal induced depression, increased body weight which was tentatively attributed to bacoside A3 due to its major presence (4 μg/mg) in extract quantified via high performance liquid chromatography (HPLC) technique [30].

Anti-diabetic activity In diabetes pathogenesis, altered level of immunoglobulins (Igs) play important role. All diabetic drug can revert serum serum IgG levels in diabetics. In a clinical trial involving three Rasayana drugs, i.e., Asparagus recemosus, B. monnieri and Centella asiatica similar results were obtained with increased level of serum IgA, IgM and serum proteins [123]. Mice with diabetes mellitus type-2, treated with CDRI-08 (50 or 100 mg/kg b.w. oral), showed enhanced spatial memory associated with reduced oxidative stress and upregulation of the AMPA receptor GluR2 subunit gene expression in the hippocampus whereas higher dose (150 mg/kg b.w.) exhibited anti-diabetic effect in streptozotocin (STZ)-induced diabetes [32].

Anti-epileptic activity Generation of pilocarpine-induced temporal lobe epileptogenesis is associated with the hippocampus region where acetylcholine esterase and malate dehydrogenase activity are increased in the muscle but decreased in the heart whereas insulin and T3 content are increased significantly and among the different gamma- aminobutyric activity GABA(A) receptor subunits such as GABA(Aά1), GABA(Aά5) GABA(Aδ), and glutamate decarboxylase (GAD) are downregulated whereas GABA(Aγ) subunit is upregulated. Increased metabolism and excitability in epileptic rats was associated with repetitive seizures which was prevented by B. monnieri and bacoside A treatment causing reduction of the impairment in the peripheral nervous system indicating their possible therapeutic application against epilepsy associated behavioral deficit [124,125].

Anti-fertility activity BME (250 mg/kg b.w./day, for 28 and 56 days) when administered orally in Parkes (P) strain mice, it caused reduction in motility, viability, morphology and number of spermatozoa in cauda epididymidis. Histologically, alterations in the seminiferous tubules were observed in testes in mice with intraepithelial vacuolation, loosening of germinal epithelium, germ cells exfoliation and occurrence of giant cells. The libido was retained but the fertility was compromised in male mice [126].

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Anti-Helicobacter pylori activity Standardized BME is known to possess prophylactic and curative effects exhibited by alteration of various defensive mucosal factors like mucin secretion enhancement, longer life span of mucosal cells and higher gastric antioxidant effect. BME at 1000 μg/mL dose showed anti-Helicobacter pylori activity and a dose of 10 μg/mL increased prostaglandins (PGE and PGI2) in human colonic mucosal incubates which could probably be implicated to the reduced the risk of ulcerogenesis [38].

Anti-hypercholesterolemic/renoprotective activity Alcoholic BME (ABME) acted as a renoprotective agent in hypercholesterolemic rats. In a study where hypercholesterolemic diet (HCD) fed rats were sacrificed after 45 days and lipid, antioxidant status, and histological analysis from blood and kidney samples revealed that the levels of total cholesterol (TC), triacylglycerol (TG), phospholipids (PLs), renal functional parameters (urea, creatinine, and uric acid), and lipid peroxidation (LPO) products were significantly attenuated (P < 0.01) in ABME-treated hypercholesterolemic rats. The mRNA levels of Endothelial nitric oxide synthase (eNOS) and Inducible nitric oxide synthase (iNOS) genes were significantly upregulated and downregulated (P < 0.01) respectively with a rise in the levels of the antioxidant enzymes. Histological analysis indicated that the application of ABME protected the kidney from hypercholesterolemia-mediated oxidolipidemic damage [95]. The alcoholic and hydroalcoholic extracts of BME acted against hyperglycemia induced oxidative stress, resulted into the diabetic nephropathy (DN). In male Wistar rats, diabetes was induced by application of nicotinamide (230 mg/kg, i.p.) and streptozotocin (65 mg/kg i.p.). The rats were treated with extracts (100, 200, and 400 mg/kg) and stigmasterol (5 and 10 mg/kg) and on 30th to 45th days their nephroprotective efficacy was evaluated. Significant attenuation in the serum lipid, glucose, uric acid and creatinine levels were observed. Moreover, the level of superoxide dismutase (SOD) and GSH were altered and a decrease in lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) was also observed [39].

Anti-inflammatory activity Ethanolic BME was applied against histamine, serotonin, bradykinin, prostaglandin E(2) and arachidonic acid-induced edema and prostaglandin E(2)-induced inflammation in rats [41]. Methanolic BME caused 82% edema inhibition (100 mg/kg i.p.) in carrageenan-induced rat paw edema, when compared to indomethacin (INDO) (3 mg/kg) with 70% edema inhibition. It also inhibited 5-lipoxygenase (5-LOX), 15-LOX and cyclooxygenase-2 (COX-2) activities significantly in rat monocytes in vivo. Ethyl acetate fraction showed higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity with a

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IC(50) value of 30 μg/mL when compared to butylated hydroxyl toluene (BHT) with a IC(50) value of 13 μg/mL) as well as hydroxyl radical scavenging activity with IC(50) value of 25 μg/mL. This fraction also inhibited LOX and COX activities in Ca-A23187 stimulated rat mononuclear cells with 5-LOX IC(50) value of 25 μg/mL and COX-2 IC(50) value of 1.32 μg/mL. It was also reported for reduction of ex-vivo TNF-alpha release [42]. The triterpenoid and bacoside-enriched fractions of BME inhibited production of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 in lipopolysaccharide (LPS)-activated peripheral blood mononuclear cells and peritoneal exudate cells in vitro. They also possessed anti-oedematogenic effect but anti-arthritic potential was only exerted by triterpenoid rich fraction via modulating the pro-inflammatory mediator release [43].

Anti-nociceptive activity Anti-nociceptive effects of aqueous BME (80, 120, 160 mg/kg; oral) was studied with selective α2 receptor blocker yohimbine, selective β1 receptor blocker atenolol, serotonin receptor antagonist cyproheptadine and a nonselective opioid receptor antagonist naloxone in experimental mice and rats. Plant extract treated writhes ranged from 14.50 to 282.35 whereas application of yohimbine, β1 atenolol receptor blocker and cyproheptadine pretreatment (1 mg/kg i.p.) caused writhes 31.00s, 113.50s and 88.17 s, respectively. The tail flick latency enhancement was inhibited by prior treatment with naloxone (2 mg/kg, i.p) writhes 282.35 and 107.35 in the ABME-treated and naloxonetreated groups, respectively [44]. The standardized methanolic BME (40 and 80 mg/kg, p.o.) showed marked anti-nociceptive effect in the chronic constriction injury (CCI) model of neuropathic pain induced by partial denervation of the hindpaw of experimental rat due to placement ligatures around the sciatic nerve. CCI generated static- (days 3–21), dynamic- (days 14–21) and coldallodynia (days 3–21) plus heat- and mechano-hyperalgesia (days 3–21) were attenuated by BME and standard gabapentin (75 mg/kg, i.p) [46].

Anti-muscarinic activity Scopolamine, the competitive antagonist of muscarinic acetylcholine receptors is considered as an anti-muscarinic and anti-cholinergic drug. BME exhibited neuro-modulatory and antioxidant properties by alleviating scopolamine (3 μg/mL)-induced cytotoxicity in PC12 cells; restoring the levels of antioxidant enzymes and lipid peroxidation. BME (100 μg/mL) pre-treatment ameliorated mitochondrial and plasma membrane damage caused by scopolamine up to 54.83% and 30.30%, determined by MTT and lactate dehydrogenase (LDH) assays respectively. The acetylcholine esterase activity was downregulated and brain-derived neurotropic factor and muscarinic muscarinic-1 receptor expression was upregulated [47].

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Anti-oxidant activity Methanol BME with free radical scavenging capacity ameliorated hydrogen peroxide (H2O2) and/or UV-photolysis induced DNA cleavage in radical induced cytotoxicity in human non-immortalized fibroblasts [48]. DHC-1, (herbal formulation of B. monniera, Emblica officinalis, Glycyrrhiza glabra, Mangifera indica and Syzygium aromaticum) exerted protection against isoproterenol-induced myocardial infarction and cisplatin-induced renal damage following lowered level of serum markers and lipid peroxidation with higher level of SOD, catalase (CAT) and reduced glutathione [50]. BME ameliorated lead-induced oxidative stress in different regions of male rat brain which was associated with increase in ROS peroxidation products (LPP) and total protein carbonyl content (TPCC) which was lowered by BME and combined meso-2,3-dimercaptosuccinic acid (DMSA) therapy [54].

Anti-Parkinson’s activity The protein alpha synuclein aggregation, dopaminergic neurodegeneration and cognitive and motor impairment are associated with the Parkinson’s patients. The model organism, Caenorhabditis elegans strains; transgenic model expressing “human” alpha-synuclein [NL5901 (P(unc-54)::alphasynuclein::YFP + unc-119)], and a pharmacological model expressing green fluorescent protein (GFP) specifically in the dopaminergic neurons [BZ555 (P(dat-1)::GFP)] were selected to check neuroprotective and cognition enhancing effects of BME against catecholaminergic neurotoxin 6-hydroxy dopamine (6-OHDA) induced aberrations [58]. The neurodegenerative disorder was reported to be associated with oxidative stress damage in which herbicide paraquat (PQ) plays a significant role for disease onset. Alcoholic BME (20 and 40 mg/kg b.w.) slowed down the process of neuronal injury in 6-OHDA-rat model of Parkinson’s as determined by the neurobehavioral activity (rotarod, locomotor activity, grip test, forced swim test, radial arm maze) and biochemical estimation. After infusion of 6-OHDA (12 μg in 0.01% in ascorbic acid-saline) on 21st day following BME-pretreatment, lesioning percentage and dose-dependent restoration of depleted activities of glutathione-S-transferase, glutathione reductase, glutathione peroxidase, SOD, and CAT were noticed [59]. BME (50 μg/mL) acted against PQ-induced and 1-methyl-4-phenyl-pyridinium iodide (MPP(+))-induced toxicities by preventing ROS generation, decreasing mitochondrial superoxide level, activating the nuclear factor erythroid 2-related factor 2 pathway by modulating Keap1 expression and by upregulating the endogenous GSH synthesis and thus preserving the cellular redox homeostasis [60].

Antipsychotic activity Standardized BME (40, 80, and 120 mg/kg, p.o.) possessed antipsychotic potential in ketamine-induced psychosis mice model and reduced

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hyperactivity with an EC50 value of 76.60 mg/kg. The 80 mg/kg dose showed higher TLT in passive avoidance task and reversed ketamine-induced enhanced monoamine oxidase activity in cortex and striatum and normalized the acetylcholinesterase activity and the glutamate levels in the hippocampus [61].

Anti-rheumatic activity BME mediated cure of rheumatoid arthritis was studied using a type II collagen-induced arthritis rat model where male Wistar rats were immunized with bovine type II collagen in complete Freund’s adjuvant. The categorical changes in inflammatory mediators (cyclooxygenase, lipoxygenase, myeloperoxidase) and serum anti-collagen IgG and IgM levels, paw swelling, arthritic index analyzed in control and experimental rats and plant extract mediated inhibition of cyclooxygenase and lipoxygenase activities were observed. Decreased neutrophil infiltration and reduced footpad swelling and arthritic symptoms indicated its anti-arthritic property [21].

Anti-schizophrenic activity Application of BME (500 mg/day for a month) caused reduction in psychopathology without any treatment directed side effect in schizophrenia [127]. The effect was also examined on cerebral glutamate/N-methyl-D-aspartate receptor subtype 1 (NMDAR1) immunodensity in sub-chronic phencyclidine (PCP) rat model of schizophrenia and was measured in prefrontal cortex, striatum, cornu ammonis fields I (CA 1) and 2/3 (CA2/3) and in dentate gyrus (DG) using immunohistochemistry. Downregulation of NMDAR1 in CA2/3 and DG indicated underlying mechanism for possible schizophrenia treatment by the extract [62].

Anti-thyroid activity BME (200 mg/kg) played a potential role in regulating thyroid hormone concentrations in male mice by increasing T4 concentration up to 41% in serum without enhancing hepatic LPO. It also reduced the level of SOD and CAT activities and acted as a potent agent against regulation of hypothyroidism [128].

Cardioprotection Isolated rodent hearts were perfused in a Langendorff model of ischaemiareperfusion (I-R) injury to study the cardioprotective effects of BME on cardiomyocyte apoptosis and antioxidant activity. The extract (75 mg/kg oral for 3 weeks) enhanced the levels of myocardial antioxidants (superoxide dismutase, catalase and glutathione), inducted heat shock protein 72 (HSP72) and reduced myocardial apoptosis, caspase 3 and Bax protein expression

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[64]. The phytochemical composition, named Protandim, prepared from botanicals such as B. monniera, Silybum marianum, Withania somnifera, Camellia sinensis and Curcuma longa was screened for its role in HL-1 cardiomycetes treatment through nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation, transcriptional regulation of phase II antioxidant enzymes and protection from oxidative damage caused by hydrogen peroxide. The alleviating mechanism of Nrf2 was silenced by shRNA as the protective mechanism that was found to follow Nrf2 pathway [65].

Cognition enhancement The nootropic action of clinically available B. monniera extract was evaluated on human recombinant monoamine oxidase-A (MAO-A) and MAO-B enzymes using MAO-Glo(TM) assay kit (Promega Corporation, USA) [77]. BacoMind, the phytochemical-rich standardized extract of B. monnieri is commercially used as a potent memory enhancing agent. To check its in vitro toxicity level and possible mutagenic attributes, clastogenicity assays were performed at 31.2, 62.5 and 125 μg/mL, and Salmonella reverse mutation assay (Ames test) was performed at doses of 61.72, 185.18, 555.55, 1666.67 and 5000.00 μg/plate where these doses were chosen on the basis of mitotic index (MI) and cytokinesis-block proliferation index (CBPI). In vitro antioxidant and anticlastogenic studies revealed dose-dependent protection against the clastogens and no mutagenic effect on the tested strains in the Ames test [129]. B. monnieri has been in use to cure cognitive disorder and behavioral defects from ancient period. Recently meta-analysis using MEDLINE, EMBASE, CINAHL, AMED, Cochrane Central of clinical trial, WHO registry, Thai Medical Index, Index Medicus Siriraj library and www. clinicaltrial.gov database to June 2013 generated information regarding the use of BME in cognitive performance or memory has revealed improved cognition among 437 eligible subjects showed by shortened Trail B test and decreased choice reaction time. [130]. In a 16-week long randomized, placebo-controlled, double-blind, parallel group trial of CDRI 08 among 6–14 years old male children, BME enriched extract was found to improve memory, attention and cured behavioral abnormalities. The level of hyperactivity and inattention in children were measured by electroencephalography (EEG), Conners’ Parent Rating Scale (CPRS), quality of cognition, mood and sleep [131].

Emetogenic activity Cisplatin (7.0 mg/kg, i.v.) is known to induce reproducible emesis in healthy pigeons exhibited by dopamine upsurge and intestinal 5-hydroxytryptamine (5-HT) concentration. The methanolic (10–40 mg/kg), the bacoside-rich n-butanolic fractions (5–20 mg/kg), the N-(2-mercaptopropionyl) glycine

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(10 mg/kg) and the standard metoclopramide (30 mg/kg) up to 66.3%, 71.6%, 76.5% and 48.9%, respectively, acted as potent emetogenic chemotherapy [68]. In another study, same concentration of plant extract reduced emetic retching and/or vomiting over a 2 day period in Suncus murinus (house musk shrew) up to 59.4% and 78.9%, respectively, which were comparable to 5-HT3 receptor antagonist, palonosetron (0.5 mg/kg, s.c.) in which 71% reduction was achieved. The study indicated that BME alone or in combination with standard drugs reduced the effects of chemotherapy-induced emesis [35]. The emetogenic effect of synthetic N-(2-mercaptopropionyl) glycine (MPG) (10, 20, 30 mg/kg), vitamin C (100, 200, 300 mg/kg), grape seed proanthocyanidin (GP) (50, 100, 150 mg/kg) and n-butanolic fraction of BME (5, 10, 20 mg/kg) were examined against cisplatin (7.0 mg/kg, i.v.) induced emesis in pigeons during a 24 h study period and the antioxidant activity of the same was evaluated by DPPH free radical scavenging assay (FRSA) which indicated low EC50 values for MPG (67.66 μg/mL), vit-C (69.42 μg/mL), GP (6.498 μg/mL) and butanol fraction of BME (BME-ButFr) (55.61 μg/mL) when compared to butylated hydroxytoluene (BHT) standard (98.17 μg/mL) [36].

Hepatoprotection Oral consumption of alcoholic extract of B. monnieri displayed hepatoprotective role in experimental rats with acute liver toxicity [132]. A randomized interventional study including 66 patients with Child-Pugh B class liver cirrhosis and hepatic encephalopathy (0–2nd stages) showed curative effect of the medicine when standard treatment was offered to 36 patients and 30 patients received CognoBlend capsules (4/day) for 5 weeks along with the standard treatment. Congoblend contained Gingko biloba, Cat’s Claw, Gotu Kola, Rosemary and B. monnieri extract and the group under combined treatment exhibited higher percentage of clinical advancement in psychometric tests, EEG and serum biochemistry when compared to the other group [133]. The herbal product Intellan, composed of Centella asiatica, B. monnieri, Coriandrum sativum, Amomum subulatum and Emblica officinalis, was screened as nootropic agent with Cytacon (cyanocobalamine) on animal models for 6–8 weeks. There was significant rise in the hematological parameters including hemoglobin/HCT, total leucocyte count, the lymphocytes count by intellan; whereas RBC counts, platelet counts, monocyte counts and hematocrit were increased by cyanocobalamine [134]. The nephrotoxicity induced by morphine and/or street heroin (20 mg/kg for 14 and 21 days) was attenuated by pretreatment with methanolic BME (40 mg/kg) or ascorbic acid (50 mg/kg) 2 h before the opioid application. The active fraction, enriched with bacoside-A3 (37.5 μg/mg), bacopaside-II (4.62 μg/mg) and bacopasaponin-C (1.91 μg/mg) were found to be potent antioxidant and hepatoprotective agents [135].

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Mast cell stabilizing activity Methanolic fraction of BME exhibited highest mast cell stabilizing efficacy (in vitro) than petroleum ether, chloroform, and water extracts and the activity was comparable to the potent mast cell stabilizer, disodium cromoglycate [136].

Memory enhancement BME enhanced learning and memory process in 10 days old neonatal rat pups. After administration of different doses of BME for different time interval they were subjected to spatial learning (T- Maze) and passive avoidance tests along with the age matched normal and gum acacia control rats. Significant improvement in spatial learning performance and enhanced memory retention in neonatal rats were observed [74]. The standardized BME (BESEB CDRI08) acted on serotonergic receptors and other neurotransmitters on Wistar rat pups. A single dose of BESEB CDRI-08 during postnatal days, 15–29 rats showed higher latency during hippocampal-dependent learning associated with enhanced 5HT(3A) receptor expression, hippocampal serotonin and acetylcholine levels, 1-(m-chlorophenyl)-biguanide (mCPBG) induced behavioral and histochemical aberrations [137]. The effect of BME leaf ethanolic extract (40 mg/kg b.w. + 0.5% gum acacia; oral) on the serotonergic system of rats postnatal day (PND), 15–29 rats were evaluated with reference to learning (PND 32–37) and retention of memory (PND-47-53). The level of 5-HT was significantly elevated up to PND-37 and was then restored back to normal PND-53 and simultaneous upregulation of mRNA expression of serotonin synthesizing enzyme tryptophan hydroxylase-2 (TPH2) and serotonin transporter (SERT) were observed on PND-29 and PND-37 [75]. The cellular mechanisms of hippocampus-dependent memory were investigated via contextual fear conditioning (CFC). Standardized BME (CDRI-08) improved hippocampus-dependent learning in postnatal rats by elevating serotonin, 5-hydroxytryptamine (5-HT) level, activating 5-HT3A receptors and cyclic adenosine monophosphate (cAMP) response element binding (CREB) protein. The Wistar rat pups were subjected to CFC following daily (postnatal days 15–29) administration of vehicle solution (0.5% gum acacia +0.9% saline)/ CDRI-08 (80 mg/kg, p.o.)/sodium butyrate (NaB) (1.2 g/kg in PBS, i.p.). CDRI-08/NaB treated group showed enhanced freezing behavior, activation of extracellular signal-regulated kinase ERK/CREB signaling cascade and upregulation of p300, Ac-H3 and Ac-H4 levels and downregulation of HDACs (1, 2) and protein phosphatases (PP1α, PP2A), increased brain-derived neurotrophic factor (BDNF) (exon IV) mRNA in hippocampus following CFC when compared to the control group [78].

Neuropharmacological properties BME rendered protection against crackers’ smoke induced neuronal and physiological changes via iNOS modulation and hemeoxygenase-1 (HO-1)

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expression in rats. The 3 weeks long exposure of rats to smoke for 1 h with different doses of BME (i.e., 10, 20 and 40 mg/kg b.w.) replenished oxidative stress induced depletion of antioxidant enzyme status and monoamine oxidase activity [86]. Chronic exposure to passive cigarette smoking (CS) altered the mitochondrial functions, neurotransmitter level, lipid peroxidation, membrane histology, and apoptotic expression hazardously in rat brain which was found to be reversed by neuroactive agent bacoside-A [138].

Neuroprotection Methanolic BME ameliorated S-nitroso-N-acetyl-penicillamine (SNAP) induced toxicity in culture of purified rat astrocytes. After 18 h of treatment, SNAP generated reactive species but rupture of cellular membrane was not induced. BME inhibited ROS production and dose dependent reversal of DNA damage [139]. Aluminum (AlCl3; 50 mg/kg/day/month) induced neurotoxicity in the cerebral cortex of male Wistar rats (8 months old) was inhibited by BME via alleviating associated oxidative damage [93]. BME rendered neuroprotection in primary cortical cultured neurons against the beta-amyloid protein (25–35) induced neurotoxicity by suppressing cellular acetylcholinesterase activity and by inhibiting lipid peroxidation. In culture, BME promoted higher percentage of cell survival when compared to neuronal cells growing in regular culture medium but it was not able to alter glutamate-induced excitotoxicity evaluated by monitoring cellular oxidative stress [140]. Neuroprotective properties of BME against rotenone induced oxidative damage and neurotoxicity were studied in flies (Oregon K strain, adult males). The flies were exposed to a standardized BME powder for 7 days and reduced rotenone (500 μM) induced oxidative stress (measured via paraquat oxidative stress bioassay) and lower mortality when flies were exposed to rotenone + BME [141]. BME (120, 160 and 240 mg/kg p.o.) was applied as a curative treatment against sudden brain stroke as it could alleviate the transient intracarotid artery (ICA) occlusion induced ischemia and cognitive dysfunction in Wistar rats with improvement in neurodeficit score and fore limb muscle grip strength. The infarct size in the ischemic brain was reduced. The levels of nitrite, nitrate and lipid peroxidation were decreased and significant rise in catalase activity was observed [142]. B. monnieri leaf powder modulated endogenous levels of oxidative stress markers in the prepubertal mice brain. Alcoholic extract of BME provided neuroprotection against 3-nitropropionic acid (3-NPA)-induced oxidative stress and cytotoxicity in dopaminergic (N27) cell lines by reducing the level of oxidative markers (malondialdehyde, hydroperoxide, protein carbonyls) in cytosol of brain regions. It also enhanced the levels of reduced glutathione and thiol depleted by 3-NPA [34]. The anti-oxidative neuroprotection (via redox and enzyme induction), acetylcholinesterase inhibition and/or choline acetyltransferase activation, β-amyloid reduction, increased cerebral blood flow, and neurotransmitter (acetylcholine [ACh], 5-HT, dopamine [DA]) modulation were attributed to the neuroprotective efficacy of BME

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[143]. Supercritical extract of BME and rosemary antioxidant extract obtained from Rosmarinus officinalis were found to be non-toxic at 1:1 ration on human glial (U-87 MG) and embryonic mouse hypothalamus cell line up to a dose of 200μg/mL. The combined form was found to be more potent to enhance amyloid precursor protein synthesis and neurotrophic factor production in hypothalamus cells when compared to the single agent but phospho tau expression inhibition pattern was similar in both cases. The result suggested neuroprotective and antioxidant activities of these extracts were exhibited in the combined form [83]. The alteration of cytochrome P450 (CYP) enzymes potentiated herb-drug interactions. Effect of BME was analyzed in vitro on five major CYP isoforms using a luminescent CYP recombinant human enzyme assay where non-competitive inhibition of CYP2C19 (IC50/Ki ¼ 23.67/9.5μg/mL), CYP2C9 (36.49/12.5 μg/mL), CYP1A2 (52.20/25.1μg/mL); competitive inhibition of CYP3A4 (83.95/14.5 μg/mL) and weak inhibition of CYP2D6 (IC50¼ 2061.50 μg/mL) were observed. Due to oral consumption of this extract stronger inhibition of intestinal CYPs were observed than hepatic CYPs. At 600 μg/mL estimated gut concentration, based on a daily dosage of 300 mg/day, BME reduced CYP3A4, CYP2C9 and CYP2C19 catalysis to <10% compared to the total activity (without inhibitor ¼ 100%) which suggested that BME can be co-administered with CYP1A2, CYP3A4, CYP2C9 and CYP2C19 metabolized drugs [144]. Neuroprotective property of BME (BMEE) was assessed in pretreated PC12 cells under SNP-induced neuronal damage and oxido-nitrative stress. BMEE modulated iNOS upregulated by oxidative stress of sodium nitroprusside (SNP); HSPs and apoptotic markers. ROS estimation, comet assay and mitochondrial membrane potential assays determined that BMEE ameliorated mitochondrial and plasma membrane damage, caused restoration of cellular, nuclear and mitochondrial integrity and replenished the antioxidant enzyme level depleted by SNP. The proteolytic damage of the cell caused by SNP-induced apoptotic protein biomarkers like Bax, Bcl-2, cytochrome-c and caspase-3 was efficiently attenuated and expression of neuronal cell stress markers was downregulated with upregulation of BDNF. Free radical scavenging activity actively worked as neuroprotective mechanism against SNP-induced toxicity [84]. Neurodegenerative diseases caused impairment of antioxidant system and structural changes in hippocampus. The whole plant BME acted against aluminum (Al)induced oxidative stress and hippocampus damage in male Wistar rats. After 1 month of oral administration biochemical and microscopic studies revealed the reduction of oxidative damage in the hippocampus. TBARS concentration was decreased significantly. Al-induced vacuolation, lipofuscin deposition and pyramidal cell degeneration in the hippocampus were also attenuated by BME [85]. The standardized BME, CDRI-08, altered the expression pattern of the GluN2B subunit of NMDAR in scopolamine-induced amnesic mice. Scopalamine treatment caused marked downregulation of the NMDAR GluN2B subunit expression in prefrontal cortex and hippocampus with higher acetylcholinesterase activity which has been restored by CDRI-08 administration and improved

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spatial memory [19]. It was also found to affect gene expression pattern of Fmr-1 in the cobalt chloride (CoCl2)-induced hypoxia mimetic mice hippocampus. CoCl2 toxicity is known to hamper spatial memory at a higher degree via upregulating the Hif-1α and downregulating the Fmr-1 gene expression which were reversed by CDRI-08 treatment. These findings may lead to the treatment of many hypoxia led memory impairment [87].

Nootropic effect Standardized BME (120 mg/kg; oral) exerted anti-amnesic effects against diazepam (1.75 mg/kg i.p.) induced amnesia in mice using Morris water maze test [145]. According to the molecular studies, diazepam upregulated mitogen activated protein kinase (MAPK), phosphorylated CREB (pCREB) and iNOS which were suppressed by BME. Further downregulated nitrite level was attenuated but no changes were observed in CREB expression, phosphodiesterase (PDE), cAMP, nitrate and total nitrite level [17]. In other experiment, the methanolic BME (MBME), Ayurvedic Ghrita (AGBME) and lipid extracts (LBME) prepared and standardized by high-performance thin-layer chromatography (HPTLC) showed nootropic activity in rats. The extracts (100, 200 and 400 mg/kg) were orally administered daily and were evaluated using the twotrial Y-maze test against scopolamine (1 mg/kg i.p.) induced anterograde amnesia. The conditioned avoidance response was found to be higher in the groups treated with high doses of LBME and MBME than that of treated with AGBME. Brain noradrenaline and 5-hydroxytryptamine were significantly decreased and an increase in dopamine levels in the LBME-treated groups was observed [146]. The polyherbal formulation called Brahmi Ghrta com˙ pluriposed of B. monnieri, Acorus calamus, Sassurea lappa and Convolvulos caulis showed anti-amnesic activity at doses of 400 and 800 mg/kg, p.o. during scopolamine (1 mg/kg, s.c.) induced amnesia in Charles Foster rats using elevated plus maze, passive avoidance and active avoidance tests where Piracetam (500mg/kg p.o.) was used as control [147].

TLE antagonism Temporal lobe epilepsy (TLE) is associated with emotional outburst, depression, anxiety, aggression and impaired memory which is known to cause upregulation of 5-HT(2C) receptors, 5-HT(2C) gene expression, with inositol triphosphate content and decreased affinity in hippocampus in pilocarpine-induced epileptic rats. These alteration were found to be reverted by carbamazepine and BMEs confirmed by the forced swim test of depressive behavior pattern during epilepsy [96].

Wound healing activity Wound healing effects of ethanol whole plant extract (50%) of B. monniera (25 mg/kg oral for 10 days) was studied on incision and dead space wound

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models in rats on the basis of in vitro antimicrobial and in vivo wound breaking strength, WBS (incision model), rate of contraction, period of epithelization, histology of skin, antioxidants (catalase, superoxide dismutase, and reduced glutathione), acute inflammatory marker (myeloperoxidase), connective tissue markers (hydroxyproline, hexosamine, and hexuronic acid) and deep connective tissue histology. Besides its antioxidant and antimicrobial efficacy, BME treatment promoted early epithelization, skin collagen tissue formation and increased collagen formation in skin and deeper wound [98].

Activity of the active compounds Anti-amyloid activity Bacosides, class of compounds extracted from the B. monnieri plant, exhibit interesting therapeutic properties, particularly anti-amyloid activity. The spectroscopic and microscopic study has revealed bacoside-A significantly acted on fibrillation in the presence of lipid bilayers and membrane interactions of the amyloidogenic fragment of the prion protein PrP (106–126) that therapeutically enhanced cognitive functions and reduction of amyloid protein toxicity by inhibiting bilayer interactions of the peptide aggregates [148].

Anti-anxiolytic activity Bacoside-A (25.5%) rich standardized BME (5, 10 and 20 mg/kg, p.o.) exerted anti-anxiolytic effect in an open-field, elevated plus maze, social interaction and novelty-suppressed feeding latency test in rats and the efficacy was compared with that of lorazepam [149]. The stress stimuli enhanced the Hsp70 expression, lowered the expression of P450, activity of SOD, cytochrome P450 (P450) dependent 7-pentoxyresorufin-o-dealkylase (PROD) and 7-ethoxyresorufin-o-deethylase (EROD) in hippocampus which were found to be attenuated with aqueous BME (20 and 40 mg/kg) when administered orally for seven consecutive days [150].

Anti-autism activity The ameliorative effect of BME on early prenatal or postnatal autism induced by valproic acid (VPA) exposure was studied on rats. The female pregnant rats were administered saline/VPA (600 mg/kg, i.p.) prenatally after 12.5 day of gestation and on postnatal day (PND) 300 mg/kg/p.o. extract was applied on the experimental group. Analysis of behavioral tests (nociception, locomotor activity, exploratory activity, anxiety and social behavior), histopathological study and biochemical estimations (serotonin, glutathione, catalase and nitric oxide) revealed the effects of BME in alleviating oxidative stress and in restoring cerebellum histoarchitecture and also in improving behavioral alterations via providing anti-autistic protection [22].

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Anti-cancer activity Protandim is a polyherbal formulation prepared from the extracts of B. monnieri, Silybum marianum, Withania somnifera, Camellia sinensis, and Curcuma longa. It exhibited potent in vivo antioxidant effects and induced three- to ninefold heme oxygenase-1 (HO-1) expression in cultured MIN6 cells, mouse beta-cell line and in human neuroblastoma cell line (SK-NMC) in a synergistic way [151]. The metastasis of hepatocellular carcinoma (HC) is a multi-factorial complex process that involves MMP-2 and MMP-9 for tumor invasion. Bacoside-A co-treatment significantly lowered metastatic activity of MMP-2 and MMP-9 in DEN-induced HC [70]. Bacoside-A mediated cure of glioblastoma multiforme (GBME) included excessive phosphorylation of calcium/calmodulin-dependent protein kinase IIA (CaMKIIA/ CaMK2A) enzyme associated with calcium release from the smooth endoplasmic reticular (SER) networks. High intracellular calcium content stimulated cell hypertrophy and organellar congestion, cell swelling, and membrane rupture by huge extracellular macropinocytotic fluid intake that led to the glioblastoma tumor cell death [152].

Anti-depressant activity Triterpene glycosides, bacopasides VI–VIII, together with three known analogs, bacopaside I, II and bacopasaponsin C, isolated from B monnieri possessed anti-depressant activity under forced swimming test and tail suspension assay in mice model [153]. Application of the bacopaside I (50, 15 and 5 mg/kg for 7 days) significantly decreased the immobility time in mouse models; reversed reserpine-induced depressive-like behaviors, including low temperature and ptosis but enhanced brain antioxidantion and noradrenergic activation [29].

Anti-dopaminergic/serotonergic effect The n-butanol extract of the plant (nBt-ext BME) containing bacoside A (bacoside A3, bacopaside II and bacopasaponin C) successfully attenuated morphine-induced hyperactivity and acted on dopamine and serotonin turnover in the striatum, parameters of opioid sensitivity and dependence. The mice were pretreated orally with saline or nBt-ext BME (5, 10 and 15 mg/kg), 1 h before morphine administration. The locomotor activity was recorded, striatal tissues were removed to analyze DA, serotonin (5HT) and other metabolites through HPLC coupled with electrochemical detection. The nBt-ext BME markedly reduced locomotor activity in both groups but lowered morphine-induced dopamine, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-H1AA) upsurges in the striatum. The effect on DA, 5-HT and their metabolites were found to be null in saline treated group indicating an anti-dopaminergic/serotonergic effect of nBt-ext BME [33].

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Anti-hyperglycemic activity Ethyl acetate fraction (EAF) of the ethanolic BME rendered antihyperglycemic effect by increasing peripheral glucose consumption in diaphragm and oxidative damage protection in alloxanized diabetic rats due to the presence of higher amount of bacosine having insulin-like activity. Bacosine significantly decreased blood glucose and glycosylated hemoglobin content and enhanced anti-oxidative enzyme level in diabetic rat liver when compared to the drug α-tocopherol [40].

Anti-inflammatory activity Bacoside caused inhibition of LOX and COX activities in Ca-A23187 stimulated rat mononuclear cells with 5-LOX IC(50) value of 68 μg/mL and with COX-2 IC(50) value of 0.23 μg/mL [42].

Anti-nematode properties Bacoside A significantly reduced the seizure/convulsion in T-type Ca(2+) channel cca-1 mutant C. elegans at higher temperatures (26–28 1 °C), with 1 buffer (100 mM NaCl, 50 mM MgCl2). Abnormal neuronal cell burst was reportedly suppressed by bacoside-A [37].

Anti-oxidant activity Radical scavenging property of Bacoside-A rendered protection against dichlorvos intoxication in mice which involved normalization of reduced levels of TBARS and propionyl-coA carboxylase (PCC), CAT, SOD, GPx and GSH and ROS generation during serum pathogenesis by bromelain and bacoside A co-administration [154].

Cognition enhancement The efficacy of bacoside-A, aglycones (jujubogenin and pseudojujubogenin) and their derivatives (ebelin lactone and bacogenin A1) were compared in silico and in vitro for cognitive efficacy. Firstly, they were docked at 5-HT1A, 5-HT2A, D1, D2, M1 receptors and AChE by AutoDock and their central nervous system (CNS) drug-like properties were determined using Discovery Studio molecular properties and ADMET descriptors. In vitro studies were performed by radio ligand receptor binding and AChE inhibition assays. The aglycones and their derivatives showed better affinity, higher blood brain barrier (BBB) penetration and more CNS drug-like properties. The binding affinity of ebelin lactone was higher toward M1 (Ki ¼ 0.45 μM) and 5-HT2A (4.21 μM) receptors whereas bacoside A and bacopaside X

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(9.06 μM) showed binding affinity toward the D1 receptor but none has shown any inhibitory activity against AChE. The chemical transformation of active constituents of BME potentiated their pharmacological activity [155]. The P-glycoprotein (P-gp) and ATP-dependent drug efflux transporter are known to be responsible to potentiate the development of herb-drug interactions and in case of herbal drug induced memory enhance, functional alteration of membrane transporters play a major role. The effect of BME and its active constituents (bacopaside I, bacopaside II and bacopasaponin C, bacoside A and bacoside A3) were studied on human MDR1 gene transfected LLCGA5-COL150 cell line. To determine P-gp function luminescent P-gp ATPase assay and Rh123 transport assay were used. All of them were able to inhibit the rhodamine 123 (Rh123) transport across LLC-GA5-COL150 cell monolayer but in case of bacopaside II the reduced P-gp efflux ratio of Rh123 was fourfold [156]. In APP/PS1 transgenic mice bacopaside I treatment remarkably ameliorated learning deficits, improved long-term spatial memory and reduced plaque load by providing amyloid clearance and thus stimulating innate immune response [89].

Cytogenetic effect In vitro cytogenetic effect of the active saponins, bacosides A and B were evaluated on human peripheral blood lymphocytes by chromosomal aberration (CA) assay and sister chromatid exchange (SCE) assay. The rate of chromatid type aberrations and sister chromatid reciprocal interchanges in the treated cells was scored with untreated control. Bacoside A (30 μg/mL) significantly increased both CA and SCE frequency whereas bacoside B showed an increase only in SCE [157].

Hepatoprotection The carcinogen N-nitrosodiethylamine (DEN) induced hepatotoxicity includes ROS generation, oxidative stress and cellular injury which was found to be altered by the BME marker compound, bacoside A. The 14 days oral pretreatment with the compound (15 mg/kg b.w./day) played protective role against carcinogen-induced liver damage in adult albino male rats; intoxicated with DEN (200 mg/kg b.w., i.p.). After 1 week of maintenance of the experimental rats, the liver weight, activity of serum marker enzymes, LPO and activities of antioxidant enzymes were studied. Elevated level of the aspartate transaminases (Akt), alanine transaminases (AT), LDH, alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (gamma-GT) were markedly decreased and level of SOD, CAT, GP, glutathione reductase (GR), glutathione-Stransferase (GST), and reduced glutathione were enhanced to normal level in bacoside A pretreated rats. [158]. Oral administration of bacoside–A (10 mg/kg of b.w. daily) altered d-GalN induced liver injury in rats in similar

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methods after 21 days of pretreatment followed by application of d-GalN (300 mg/kg of b.w). It has also restored the decreased levels of vit-C, and vit-E induced by d-GalN [71].

Neuropharmacological properties Bacopaside I protected against transient focal ischemia induced by middle cerebral artery occlusion (MCAO) as evidenced by experimentation in adult male SD rats. Assessment of cerebral infarct volume, edema, cerebral energy metabolism, relative enzyme activities, MDA content, nitric oxide (NO) level, and antioxidant enzyme activities performed after 70 h of MCAO reperfusion indicated neuroprotective effect of the compound against cerebral ischemia induced injury. Increase in the brain ATP content, energy charge (EC), total adenine nucleotides (TAN), NO level, Na+ K+ ATPase and Ca2+ Mg2+ ATPase activities were associated with neuroprotection [29]. Bacoside A was found to influence neonatal hypoglycemia where alterations in dopaminergic functions were caused by the dopamine D1 and D2 receptor subtypes. Significant decrease in dopamine D1 receptor number induced hypoglycemic condition as depicted by the receptor-binding study. The hypoglycemic neonatal rats treated with BME alone and bacoside A ameliorated the dopaminergic and cAMP imbalance and significantly reversed the altered gene expression parameters of Bax and SOD [159].

Nootropic effect The nootropic effect of six active phytochemicals of B. monnieri, namely bacoside A3, bacopaside I, bacopaside II, bacosaponin C, bacosine, and bacoside A was evaluated on human recombinant MAO-A and MAO-B enzymes using MAO-Glo(TM) assay kit. Bacopaside I inhibited the MAO-A enzyme most selectively with IC50 and Ki values of 17.08 and 42.5 μg/mL, respectively [77].

Oxido-nitrosative stress related activity BME attenuated sodium nitroprusside (SNP)-induced apoptosis in a human embryonic lung epithelial cell line (L132) determined via MTT and LDH leakage assays and reversal of mitochondrial and plasma membrane damage achieved with downregulation of inducible nitric oxide synthase expression. Pretreatment with active BME attenuated Bax, cytochrome-c and caspase-3 mediated proteolytic cell damage confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays [94].

LDH-stabilizing activity The potent antioxidant, bacoside A exerted protection against cigarette smoking-induced toxicity via modulating LDH and its isoenzymes. The

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exposure to cigarette smoke and simultaneous administration of bacoside A was done for 12 weeks. Bacoside-A treatment stabilized cell membrane lipid peroxidation and leakage of LDH into blood circulation possibly through free radical scavenging activity [80].

Wound healing activity Alcoholic extract of BME and isolated compound bacoside A (100–200 μg/mL) has shown faster rate (18.30 0.01 days) of excised wound epithelialization along with enhanced tensile strength (538.47  0.14 g) of the incision wound. Moreover, increased granuloma weight in the bacoside-A treated experimental Swiss albino rats was observed where screening was done using nitrofurazone as standard [97].

Concluding remarks B. monnieri is a potent candidate in amelioration of cognitive problem and is a potential candidate in drug discovery against poor memory and many neurological and psychological disorders such as epilepsy, PD, AD associated dementia, ADHD in children and anxiety mostly via reducing oxidative damage, NT modulation, and cognitive enhancement. In this present review, we have compiled and summarized multifaceted applications and recent reports on traditional uses, pharmacological properties, phytochemistry, toxicity and quality control aspects of various extracts, fractions, isolated compounds and polyherbal and Ayurvedic formulations containing B. monnieri which serve as connecting links between the scientific investigations performed on the plant related to herb based drug discovery and the traditional use of botanicals or as ethno-therapeutics. Ethnobotanical studies have indicated its use in the treatment of an array of diseases. A number of pharmacological investigations have been carried out in either with crude extracts, semi-purified fractions or with isolated phyto-constituents.

Abbreviations 3-NPA 5-HIAA 5-HT 6-OHDA ABME ACh AChE AD ADHD AGBME Akt

3-nitropropionic acid 5-hydroxyindole acetic acid 5-hydroxytryptamine 6-hydroxy dopamine alcoholic Bacopa monnieri extract acetylcholine acetyl cholinesterase Alzheimer’s disease attention-deficit hyperactivity disorder ayurvedic ghrita Bacopa monnieri extract aspartate transaminases

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AP AS AST AT BAP BBB BHT CaMKIIA/CaMK2A BME BMWE CA CAD CAM cAMP CaM CAT CBPI CCI CDB CFC CK CNS CoCl2 COX CPRS CREB CS CS CYP DA DCM DEN DG DMSA DN DOPAC DPPH EAF EC EEG EAT eNOS EROD FITC

alkaline phosphatase acute stress aspartate aminotransferase alanine transaminases 6-benzylaminopurine blood brain barrier butylated hydroxyl toluene calcium/calmodulin-dependent protein kinase IIA Bacopa monnieri extract Bacopa monnieri water extract chromosomal aberration caspase-activated DNase complementary and alternative medicine cyclic adenosine monophosphate calmodulin catalase cytokinesis-block proliferation index chronic constriction injury cognitive demand battery contextual fear conditioning creatine kinase central nervous system cobalt chloride cyclooxygenase Conners’ parent rating scale cAMP response element binding protein chronic stress cigarette smoking cytochrome P450 dopamine dichloromethane N-nitrosodiethylamine dentate gyrus meso-2,3-dimercaptosuccinic acid diabetic nephropathy dihydroxyphenylacetic acid 1,1-diphenyl-2-picrylhydrazyl ethyl acetate fraction energy charge electroencephalography ehrlich ascites tumor endothelial nitric oxide synthase 7-ethoxyresorufin-o-deethylase fluorescein isothiocyante

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FRSA FST GABA GAD Gamma-GT GBME GFP GP GPx GR GSH GST HC HCD HO-1 HPLC HPTLC HSP HVA IAA IgS INDO iNOS IUCN LBME LDH L-NNA LOX LPO LPP LPS MAO MAP MBME MCAO mCPBG MDA MI MMP MO MPG MPP MS

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free radical scavenging assay forced swimming test gamma-aminobutyric activity glutamate decarboxylase gamma-glutamyl transpeptidase glioblastoma multiforme green fluorescent protein grape seed proanthocyanidin glutathione peroxidase glutathione reductase glutathione glutathione-S-transferase hepatocellular carcinoma hypercholesterolemic diet heme oxygenase-1 (HO-1) high performance liquid chromatography high-performance thin-layer chromatography heat shock protein homovanillic acid indole-3-acetic acid immunoglobulins indomethacin inducible nitric oxide synthase international union for conservation of nature and national resources lipid Bacopa monnieri extract lactate dehydrogenase N(omega)-nitro-L-arginine lipoxygenase lipid peroxidation lipid peroxidation products lipopolysaccharide monoamine oxidase mitogen activated protein methanolic Bacopa monnieri extract middle cerebral artery occlusion 1-(m-chlorophenyl)-biguanide malondialdehyde mitotic index mitochondrial membrane potential monoamine oxidase N-(2-mercaptopropionyl) glycine 1-methyl-4-phenyl-pyridinium iodide Murashige and Skoog

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MTF MTT MWS n-Bt Ext BME NF-E2RF2 NIH NMDA NMDAR1 NO NTR OSD PCC PCP pCREB PD PDE P-gp PGR PLs PND PP PQ PROD ROS SCE SER SERT SD SNAP SNP SOD STZ TAN TBARS TC TEM TG TLE TLT TNF-α TPCC TPH2 VPA

multitasking framework 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide morphine withdrawal syndrome n-butanol Bacopa monnieri extract nuclear factor erythroid 2-related factor 2 National Institutes of Health N-methyl-D-aspartate N-methyl-D-aspartate receptor subtype 1 nitric oxide no transfer response oxidative stress damage propionyl-CoA carboxylase phencyclidine phosphorylated CREB Parkinson’s disease phosphodiesterase P-glycoprotein root powder of Panax quinquefolium phospholipids post natal day protein phosphatases paraquat 7-pentoxyresorufin-o-dealkylase reactive oxygen species sister chromatid exchange smooth endoplasmic reticular serotonin transporter Sprague-Dawley S-nitroso-N-acetyl-penicillamine sodium nitroprusside superoxide dismutase streptozotocin total adenine nucleotides thiobarbituric acid reactive substances total cholesterol transmission electron microscopy triacylglycerol temporal lobe epilepsy transfer latency time tumor necrosis factor-α total protein carbonyl content tryptophan hydroxylase-2 valproic acid

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