Introduction The world average stands at 12.5% while

Introduction

 

Plant
has been used for medicinal purpose for a long before recorded history.
Medicinal Plants have been used throughout ancient civilization; there has been
many description of medicinal value of plants in ancient text and records. Much
of the medicinal plants have developed through observation of wild animals and
by trial and error.

Many conventional drugs
are derived from plants. Salicylic acid a precursor of aspirin was originally
derived from white willow bark and the meadowsweet. Cinchona bark is the source
of malaria fighting quinine. Opium poppy yields morphine, codeine and
paregoric, a remedy for diarrhea. Other therapeutic area were natural products
are have had an impact is in the longevity of life and cancer treatment, most
of the anti-cancer drugs are derived from plants and micro-organism. Natural
products have been proven to be the richest source of medicinal compounds, screening
the flora and fauna, soil samples, fungus and bacteria is often done to
discover a new drug or its lead structure.

                                                                                                       

Globally, there has been a growth in the
plant derived medically useful formulations, drugs and health-care products. It
has a market covering more than 60 % products derived from plant origin. India exhibits
remarkable outlook in modern medicines that are based on natural products
besides traditional system of Indian medicines. Almost, 70% of the modern
medicines in India are derived from natural products.

 

India has an area of 2.4 % on the world
with 8% global biodiversity. It is one of the 12 mega diversity hot spot
regions of the world, other countries being Brazil, Columbia, China, South
Africa, Mexico, Venezuela, Indonesia, Ecuador, Peru, USA and Bolivia.
Throughout the country forest of India harbor 90% of India’s medicinal plants
diversity. Only approximately 10% of the known medicinal plants of India are
restricted to non-forest area/habitats. According to (Schippmann, 2002), one fifth of all the plants found in India are
used for medicinal purpose. The world average stands at 12.5% while India has
20% plant species of medicinal value. India has about 44% of flora, which is
used medicinally (Hamilton, 2003). India
is one of the richest in plant medical traditions, it is estimated that around
25,000 effective plant based formulations are used in folk medicine, which are
known to rural communities in India. It is estimated that there are over 7800
medicinal drug manufacturing units in India, which consume about 2000 tons of
herbs annually (Ramakrishnappa, 2002);
India and China are the largest users of medicinal plants (Wang, 20002). 

 

Picrorhiza kurroa
Royle ex Benth (family Scrophulariaceae) is an important perennial medicinal
herb endemic to North-Western Himalayan region, found between 3000 and 5000 m
altitudes (Pandit et al. 2012). Picroside-I and picroside-II- the main bioactive
constituents of P. kurroa are used in
herbal formulations such as Picroliv, Katuki, Arogya, Kutaki, Livocare,
Livomap, Livomyn, Livplus, Pravekliv and Vimliv for the treatment of liver
disorders, fever, asthma, malaria, jaundice, inflammation, allergy,
hepatitis-B, etc. (Sah and Varshney 2013;
Bhandari et al. 2009). P. kurroa
has been listed as an endangered medicinal plant species by the International
Union for Conservation of Nature and Natural Resources (Nayar and Sastri 1990). Global supply (excluding China and
Pakistan) of P. kurroa is around 375 tons,
with India contributing around 70 tons. Today price of plant material varies
from Rs. 250 to 770 per kg (Shitiz et al. 2013). Owing to its economic
importance and depleting population in natural habitat, immediate thrust has to
be given for its conservation, micropropagation and in vitro production of
secondary metabolites. Different in vitro culture techniques have been employed
for micropropagation, conservation and secondary metabolite production in P. kurroa (Rawat et al. 2013; Patial et al. 2012; Sood and Chauhan 2010)
but limited progress has been made so far with respect to development of rapid
and cost effective approach for enhancing secondary metabolite production in
this plant species.

 

Many
fungi are found to be associated with plants as endophytes, living in
association with almost every plant (Wilson
1995; Rodriguez and Redman 2008). Endophytes play a very important role in
nature which helps the plants to adapt to different environmental conditions,
and they also influence plant nutrition, growth rate, survival, and
distribution (Nagabhyru et al. 2013; Wani et al. 2015). Endophytic microorganism are known to produce
wide range of bioactive secondary metabolites due to the intimate plant-microbe
interactions leading to a molecular cross-talk between the two symbiotic
partners during various stages of development of the host (Tan et al. 2001; Brader et al. 2014). Therefore they are
metabolically more dynamic than their counterparts living in less challenging
environmental conditions.

“Several
species of endophytes produce secondary metabolites called volatile organic
compounds (VOCs) that vaporize at ambient temperatures (Schalchli et al. 2014; Hung et al. 2015). These compounds act as
signaling molecules to facilitate interactions with other microorganisms and
plants, thus influencing the dynamics of the ecosystem (Wenke et al. 2010; Schalchli
et al. 2014; Ditengou et al. 2015). VOCs of microorganisms
influence plant growth and development through various mechanisms, for example
by inhibiting the plant pathogens, by favorably modulating the plant gene
expression, and by programming the plant root architecture (Minerdi et al. 2011;
Schalchli et al. 2014; Ditengou et al. 2015). In addition to their
ecological functions, microbial VOCs may serve as potential antimicrobials,
biofuel molecules, commodity chemicals or their precursors (Fortman et al. 2008, Alpha et al. 2015; Strobel 2015). Among
the terpenes, fungi usually produce sesquiterpenes and diterpenes; however,
some recent reports have revealed that they produce monoterpenoids as well (Tomsheck et al. 2010; Strobel et al.
2011; Riyaz-Ul-Hassan et al. 2013;
Shaw et al. 2015). Monoterpene
biosynthetic pathways may remain silent under normal culture conditions (Riyaz-Ul- Hassan et al. 2012). Thus, these compounds are relatively unknown from
the microbial sources.”

 

Objective

 

Ø  Maintenance of culture of Picrorhiza kurroa Royle ex Benth obtained from leaf explant established in tissue culture facility of
the College of Basic Sciences and Humanities.

Ø  Qualitative/Quantitative analysis of picrosides
content by inducing endophyte as an elicitor(s) for Picrorhiza kurroa Royle ex Benth established in vitro.

Ø  Expression
analysis of Mevalonic Acid (MVA) and Methyl Erythritol Phosphate (MEP) pathway
genes of Picrosides biosynthesis.

 

 

 

 

 

Review
of Literature

 

Picrorhiza
kurroa Royle ex Benth. Commonly known as Kutki, belongs to
family Scrophulariaceae. It is found in the Himalayan regions of China,
Pakistan, India, Bhutan and Nepal. It is considered as an important medicinal
plant which is mostly used in the traditional medicinal system for asthma,
jaundice, fever, malaria, snake bite and liver disorders Different
pharmacological activities of P. kurroa include anti-microbial, anti-oxidant,
anti-bacterial, ant mutagenic, cardio-protective, hepato-protective,
anti-malarial, anti-diabetic, anti-inflammatory, anti-cancer, anti-ulcer and
nephro-protective activities were recorded from this plant.

 

History of Picrorhiza kurroa Royle ex Benth

 Picrorhiza
is small genera of the family Scrophulariaceae and belongs to the tribe
Veroniceae. This family is placed in the order Scrophularials, subclass
Asteridae and Dicotyledonae class of Angiosperm, according to the taxonomical
system of cronquist. The genus Picrorhiza
was supposed to be monotypic with only species of P. kurroa, until Pennell in the year 1943 distinguished another
species: P.scrophulariiflora. Hong (1984)
brought this new species under a separate genus and named it as Neopicrorhiza scrophulariiflora. This
name has been accepted as the official name of this species (Brummit, 1992). The genus Picrorhiza and the species Picrorhiza kurroa appeared for the first
time on a drawing published by Royle on August 24, 1835 in his “Illustrations
of Botany” (Royle, 1835-1840a),
Bentham described the genus and the species in his “Scrophulctrineae
Indicae” which was published on November 17, 1835 (Bentham, 1835). The
name of Picrorhiza is derived from
the bitter root, where “Picros” means bitter, while “rhiza” means root. The
specific name Kutki is derived from “Karu”, the Punjabi name of the plant,
which means bitter as well (Aswal et al. 1994).

Synonyms of Picrorhiza kurroa

The
complete botanical name is Picrorhiza kurroa Royle ex Bentham. It has got two
synonyms i.e. Picrorhiza lindleyana
(Wall.) Steud. And Veronica lindleyana
Wall.

Hindi:
Kardi, Karoi, Karwi, Kutki

Urdu:
Kutki

Nepali:
Kurki

Malayalam:
Katukhurohani

Sanskrit:
Anjani, Arishta, Katumbhara

Telugu:
Katuka-rogani, Katukarogani, Katukkurohini

Tamil:
Acokarokini, Akutam, Amakkini

 

Classification
of P. kurroa

Kingdom:    Plantae

Class        :    Dicotiledonea

Subclass:    Asteride

Order
    :     Scrophulariales

Family    :    Scrophulariaceae

Genus     :    Picrorhiza

Species    :    Picrorhiza
kurroa

 

Habitat

 

Kutki is a perennial
herb found in the Himalayan region (Garhwal to Bhutan), West China, South-East
Tibet and North Burma. It grows in wild form in alpine regions on rock crevices
and also in organic soils. In Nepal, Kutki is found in abundance in alpine
Himalayan region between altitudes of 3500m and 4800m and also in the western
regions of Nepal where it grows on the rock’s crevices on the north facing
slopes, cliffs and the turf of glacial flats. P. kurroa Royle ex Benth.is
present in wild form in the north-western Himalayan region from Kashmir to
Sikkim. It is found in the North-Western Himalayan region from Kashmir to
Kumaun and Nepal and Garhwal regions in India.

 

 

 

 

Botanical Description

Scrophulariaceae is a
large plant family, having around 200 genera and 3000 species, mostly found in
temperate regions of the world. The family includes plant such as popular
garden plants (including tiny alpines) and some other plants used for aesthetic
value, like Penstemon, Mimulus and Calceolaria.

It is a perennial herb
with elongated rhizome; the leaves are basal and alternate with a length of
5-10 cm long. Terminal spikes are present. Calyx is divided into 5 parts equally;
corolla has 4 or 5 lobes, 4-5 mm long, bilobate with lobes. Stamens are 4 in
number, which are inserted on corolla tube, slightly didynamous. Stigma is
capitate.

Fruit is small acute
capsule, tapered at top, dehiscing into 4 valves and is 12 mm long.

Seeds are ellipsoid
many in number, seed coat is very thick, transparent.

Pollen grains are
round, tri-colpate, with incomplete or perforate tectum, the partial tectum is micro
reticulate, colpus membrane is smooth or occasionally coarse.

Rhizome of the plant is
2.5-12 cm long and 0.3-1 cm thick, sub cylindrical, straight or curved to some extent,
it is externally greyish-brown, external surface with longitudinal furrows and
spherical scars of the roots make it coarse, growing bud is enclosed by crown
of leaves. Rhizomes has 20-25 layers of cork consisting of tangentially
extended suberized cells, 1-2 layered cork cambian, cortex single layered or
not present, main cortex continues in some cases, 1 or 2 small sized vascular
bundles (xylem and phloem) present in the cortex. Vascular bundles surrounded
by fibrous bundle sheath. Secondary phloem consists of parenchyma cells and few
dispersed fibers. Cambian is thick with 2-4 layers. Secondary xylem made up of
tracheids, vessels, fibers and parenchyma cells. Vessels are of different size
and shape, tracheids are long, thick walled, lignified, more or less
cylindrical with blunt pointed ends. 25-105 µm in diameter starch grains are
present abundantly.

Roots when young show
single layered epidermis, some epidermal cells stretch forming unicellular
hairs. Single-layered hypodermis, cortex is 8-14 layered, with ovoid to
polygonal thick walled parenchymatous cells. Roots show 4-15 layers of cork,
1-2 layers of cork cambium after maturation. Vessel are of different size and
shape, tubular with tail-like and tapered ends while some are barrel shaped
with perforate on end walls or adjacent walls. Tracheids are cylindrical with
tapered sharp ends.

 

Phytochemistry of P. kurroa

P. kurroa
accumulates various types of glycosides such as iridoid glycosides,
cucurbitacin glycosides, phenylethanoid glycosides, and acetophenone
glycosides. Iridoidglycosides are the main constitutents of P. kurroa and are commonly known as
picrosides.

                  Chemical Structure

          References

Basic structure of
Picroside

Kitagawa et
al., 1971

R1=OH, R2=OH, R3=OH
                  Catapol

Wang et al.,
1993

R1=OH, R2=OH,
R3=

                  Picroside I

Kitagawa et
al., 1971

R2=OH,R3=OH,
R1=

                  Picroside II

Weinges et al.,
1972
 Wang et al., 1993
 

R1=OH,R2=OH,
R3=
                  Picroside III

Weinges and Kunstlet,1977

R1=OH, R2=OH, R3=

                 Picroside IV

Li et al.,
1998

R1=OH, R2=OH, R3=

                Picroside V

Simon, 1989

 
R1=OH, R3=OH, R2=

                  Kutkoside

Singh and Rastogi, 1972

R2=OH, R3=OH, R1=

                  Veonicoside

Stuppner and Wanger, 1989

R2=OH, R3=OH, R1=

                  Specioside

Li et al.,
1998

R2=OH, R3=OH, R1=

                  Verminoside

Li et al.,
1998

R2=OH, R3=OH, R1=

                 6-Feruloylcatapol
 

Stuppner and Wanger,1989; Simon, 1989

R2=OH, R3=OH, R1=

                    Minecoside
 

Stuppner and Wanger1989; Simon, 1989

                    Aucubin

Wang et al.,
1993

                    
 
 
                   Pikuroside

Jia et al.,
1999

 

 

 

 

 

 

Pharmacological
profile of Picrorhiza kurroa

 

Traditional uses of
kutki have been anaylsed by various pharmacological studies. Biological
activities that have been assessed include hepatoprotective, antioxidant,
immunomodulatory, anticancer, anti-inflammatory, antimicrobial, antidiabetic, nephroprotective,
analgesic, cardioprotective.

 

 

 

 

 

Fig.:
Pharmacological
use of P. kurroa (in chronological
order and alphabetical order within each year). (Salma et al., 2017)

Plant part

Pharmacological activity

Reference

Not specified

Bronchial asthma

Dorsch et al., (1983)

Not specified

Hepatoprotective, anticholestatic, antioxidant and immune modulating
activity

Atal et al., (1986)
 

Not specified

Potentiate photo
chemotherapy
in vitiligo

Bedi et al., (1989)

Root

Anti-inflammatory properties or anti-thrombic drugs

Engels et al., (1992)

Rhizome and root

Human viral hepatitis

Kapahi et al., (1993)

Not specified

Hepatic and upper respiratory tract infection, fever, dyspepsia and
scorpion sting

Dong et al., (1995)

Rhizome

High blood pressure, intestinal pain, eye disease gastritis, bile
disease, sore throats, blood, and lung fever.

Lama et al., (2001)

Rhizome

Abdominal ulcer in mice

Ray et al., (2002)

Not specified

Treating coughs and colds

Ghimire et al., (2005)

Not specified

Immunomodulatory effects

Gupta et al., (2006)

Not specified

Diabetic nephropathy

Lee et al., (2006)

Rhizome and root

Cardiomyopathy

Rajaprabh et al., (2007)

Not specified

Hepatoprotective

Chander et al., 1990,
Jeyakumar et al., 2008

Not specified

Anti-diabetic,

Hussain et al., (2009)

Not specified

Anti-cholestatic

Verna et al., (2009)

Rhizome

Antibacterial activity against B. cereus, E. coli,
K. pneumoniae, S. aureus, S. pyogens and S. typhi

Kumar et al., (2010)

Not specified

Potential drug against NAFLD

Shetty et al., (2010)

Not specified

Immunomodulator

Sidiq et al., (2010)

Not specified

Antineoplastic

Rajkumar et al., (2010)

Rhizome

Management of alcohol-induced liver damage

Sinha et al., (2011)

Rhizome

Anti-microbial activity

Rathee et al., (2012)

Root

Anti-hyper Lipidemic activity

Singh et al., (2012)

Not specified

Anti-oxidant

Zhang et al., (2012)

Not specified

Anti-inflammatory

Zhang et al., (2012)

Leaf

Antioxidant

Kant et al., (2013)

Root

Cardioprotective activity

Nandave et al (2013)

Root

Analgesic activity

Rupali et al. (2013)

Rhizome

Antiasthmatic activity

Sehgal et al. (2013)

Rhizome

Antidiabetic activity

Husain et al. (2014)

Rhizome

Anticancer and cytotoxic potential

Kumar and Ramesh (2014)

Root

Anticonvulsant activity

Pathan and Ambavade (2014)

Rhizome

Alcohol abuse and cirrhosis of liver

Deshpande et al. (2015)

Stolon

Anti-microbial activity

Laxmi and preeti (2015)

Glycosidal extract of Plant

Nephroprotective and nephrocurative activity

Siddiqi et al. (2015)

Rhizome

Clastogenic effect

Balkrishna et al. (2016)

 

 

Antioxidant
activity

 

Antioxidant agents work as radical
scavengers that prevent the human body from various diseases (Kalaivani and Mathew, 2010). Deshpande et al., (2015) reported that activities of liver enzymes are
reduced among the liver cirrhosis patients following the treatment with the P. Kurroa
plant extract. The effectiveness of plant extracts as an antioxidant were
reported by Rajkumar et al., (2011) employing radical
scavenging assays, ferric reducing antioxidant property and thiobarbituric acid
assays for analyzing inhibition of lipid peroxidation. Kant et al. (2013) used
diverse antioxidant testing methods to corroborate the antioxidant efficacy of
the leaf fractions of P. kurroa. The
rhizome ethanol extract of P. kurroa
at the dose of 20 mg/ kg body weight, healed rapidly the stomach wall of
indomethacin induced gastric ulcerated rats by an in vivo free radical
scavenging action (Ray et al., 2002).