Introduction

In recent years, herbal medicines have received a great attention and acceptance worldwide as
a primary source of healthcare due to their low cost, promising efficiency and lesser side
effects Furthermore, plant based active biomolecules serve as one of the most promising
resources of new nutraceutical and dietary supplements due to their varied structures and
mode of action and thus act as a template for drug discovery programs. Among the age-
related neurological disorders, Alzheimer’s disease (AD) is the most prevalent and chronic
form of dementia, estimated to affect 66 million by 2030 .The most accepted therapeutic
approach for the treatment of AD is cholinesterase inhibitors, based on cholinergic hypothesis
in order to improve the cholinergic neurotransmission in the cortical or hippocampal regions
Recently, AD has been proposed as Type III diabetes due to shared molecular and cellular
pathophysiology between diabetes and cognitive decline with increased cholinesterase
activities. Diabetes mellitus or type II diabetes (T2DM), a chronic metabolic disorder, is
characterized by postprandial and fasting hyperglycaemia, lipoedema, oxidative stress
resulting from the defects in insulin secretion and action or both An effective approach
involving the inhibition of carbohydrate hydrolysing enzymes to control postprandial
hyperglycaemia and improving insulin signalling is the most conventional therapeutic
strategy for the treatment of diabetes mellitus. Moreover, T2DM is strongly associated with
the hypertension leading to the development of cardiovascular diseases which is the major
cause of death in many countries. Tyrosinase is a key enzyme involved in neuro-melanin
formation in mammalian brain which causes oxidative damage leading to the neuro-
degeneration associated with AD. As the synthetic drugs developed for AD and T2DM (e.g.,
galanthamine, taurine, dopenzil and acarbose) are reported to show many adverse effects
including gastrointestinal diseases, hepatotoxicity and renal tumors, natural enzyme inhibitors
from medicinal and aromatic plants have received great attention in recent years

Aim of the study:

To consider the increasing demand and tremendous medicinal importance of threatened

plant species, a detailed study was undertaken to evaluate its antioxidant potential, secondary
metabolite profiling, cytotoxicity, anti-inflammatory potential and in vitro enzyme inhibitory
activities on key enzymes linked to hyperglycaemia, hypertension and cognitive disorders in
different plant parts of wild and in vitro-raised plants with respect to different solvent systems
for its sustainable utilization.
Materials and methods:

Anti-cholinesterase activity of leaves and rhizome of wild and cultured plant extracts was
investigated against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
enzymes. In vitro anti-hyperglycaemic (α-amylase and PTP1B),anti-hypertensive
(angiotensin-converting enzyme), anti-tyrosine and anti-inflammatory potential (5-
lipoxygenase and hyaluronidase) of different plant parts of wild and in vitro raised plants
with respect to different solvent systems were also evaluated. In vitro cytotoxic effect of
rootstock extracts of wild and in vitro-derived plants were against cancer and two normal
(HEK and MEF) cell lines. Secondary metabolite profiling of rhizome segments of wild and
in vitro-derived plants was carried out by quantitative gas chromatography-mass
spectrometry (GC-MS).

Extract preparation

Various plant parts (leaf and rootstock) of wild as well as in vitro-raised plants were washed
under running tap water, blotted with tissue paper and shed dried at 25 °C. Briefly, 1 g of
shed dried powders were homogenized in 100 ml of respective solvents (methanol, acetone,
chloroform, acetonitrile and water) and extractions were carried out on an orbital shaker
(REMI, India) with constant shaking at 180 rpm for 24 h. The resulting extracts were
concentrated under rotary evaporator and stored in the dark at 4 °C until use.

Evaluation of bioactive secondary metabolites

Plant extracts were analysed for quantification of total phenolic content (TPC), total
flavonoid content (TFC), total alkaloid content (TAC) and total tannin content (TTC)
according to the methods described by Bose et al. (2016). The estimation of TPC, TFC, TAC
and TTC were expressed as mg standard Gallic acid equivalent (GAE), quercetin equivalent
(QE), atropine equivalent (AE) and tannic acid equivalent (TAE) per g of dry tissue
respectively.

Determination of antioxidant activity

The radical scavenging activities of the plant extracts (mother and in vitro-derived) were
determined using ABTS•+ scavenging assay, lipid peroxidation inhibition method and total
antioxidant capacity. The antioxidant potential of plant extracts in various solvent systems for
ABTS•+ and lipid peroxidation (LPO˙) radical scavenging assay was expressed as IC50 value
and compared against potent antioxidants, Trolox and Rutin (1-100 µg/ml), respectively. The
total antioxidant capacity was expressed as ascorbic acid equivalents.

In vitro acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities

In vitro AChE and BuChE inhibitory activities were evaluated according to the by modifying
the colorimetric method previously developed. The results for enzyme inhibition were
expressed as IC50 value, determined by dose–effect curves by linear regression and
compared to standard inhibitor galanthamine (0-10 µg/ml).

In vitro anti-hyperglycaemic activity

The α-amylase enzyme inhibition study was carried out spectrophotometrically using the
procedure reported by Wang. Protein tyrosine phosphatase 1B (PTP1B) inhibitory activity
was evaluated using p-nitrophenyl phosphate according to the procedure reported by
Acarbose (20-300 µg/ml) and ursolic acid (5-50 µg/ml) were used as standards.

In vitro anti-hypertensive activity

In vitro angiotensin converting enzyme (ACE) inhibition of the plant extracts from wild and
in vitro-derived was determined according to the method previously described. The method is
based on enzymatic cleavage of labelled dansyltriglycine (50-350 µg/ml) by the enzyme ACE
into dansylglycine which is further quantified by High Pressure Liquid Chromatography
(HPLC) with UV detection at 250 nm. Captopril was used as standard inhibitor (20-250
µg/ml).