review of literature - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/44794/9/review of... ·...

Review of Literature 2014

8

The conifers form a unique distinctive group among the gymnosperms and are largely

confined to the hills and mountains of the tropics, subtropics, temperate and alpine

climatic zones. Among the conifers, pines or the genus Pinus forms the largest and

traditionally important group. For centuries pines have held economic, aesthetic and

ecological importance. They provide forestation in areas where deciduous trees are

unable to grow due to altitude and latitude. Pines also leach a sap called “Resin” that

hosts different uses including sealant, glue and varnish. These resins are remedy for

ulcer, smallpox and syphilis. Overall, Pinus species hold distinctive characters among

gymnosperms with tremendous antioxidant potential that are explored herein.


9

PINUS

Systematic Position of the Genus (India Biodiversity portal) Kingdom: Plantae

Phylum: Pinophyta Class: Pinopsida

Order: Pinales Family: Pinaceae

Genus: Pinus

Figure 1: Vegetation of Pinus roxburghii Sarg. in Kangra district of Himachal Pradesh


10

The Genus Pinus forms an important group among the members of conifers. They are conical

trees when young and becomes rounded or flat topped with age. The branches are dimorphic

and occur in pseudo whorls at the upper region of the trunk. The branches are of two types

namely the long and the dwarf shoots.The branches end in buds which are generally ovoid to

cylindrical and are covered by bud scales. The buds are resinous. The trees attain a maximum

length of 55 metres and a diameter of over 100 cm. Figure 2 shows the pictures of Pinus

roxburghii from different Himalayan regions i.e. Uttarakhand, Sikkim and Palampur in

Himachal Pradesh.

Pinus is native to all the continents along the globe and forms the dominant vegetation of the

boreal and temperate forests of the northern hemisphere (Figure 3). Its southern most

distribution can be noted in Sumatra just before the equator in South East Asia. In the

southern hemisphere some species of Pinus are largely introduced as ornamental or timber

trees (Mirov 1967, Kral 1993). In India, six indigenous Pinus species are encountered out of

which four of them are confined to the Himalayan regions. These species include Pinus

roxburghii Sarg. , Pinus wallichiana A.B. Jacks, Pinus armandii Franch and Pinus

gerardiana Wall ex Lamb. Pinus armandii Franch grows in the extreme northern region of

Arunachal Pradesh bordering China. While Pinus merkusii Jungh and de Vries grows in hills

of Mayanmar and Andaman and Nicobar Islands. (Bhatnagar et al.,1996). In addition to it,

Pinus bhutanica is also identified in some pockets of Arunachal Pradesh (Farjon et al., 2013).


11

(9 (A)

(B)

(C)

Figure 2: Whole plant of Pinus roxburghii Sarg. from (A) Uttarakhand, (B) and Sikkim (C) Collection site at Palampur (Himachal Pradesh)


12

Figure 3: Worldwide distribution of the genus Pinus across the globe. (Source: Farjon et al., 2013) Pinus insularis Endl grows in north eastern India in the state of Meghalaya. The overall

distribution of the various species of Pinus across the globe is given in Figure 3 and the

distribution in India is given in Figure 4.

In India, Pinus species are found in Jammu & Kashmir, Himachal Pradesh, Uttarakhand, West

Kameng district of Arunachal Pradesh, Tenga valley of Arunachal Pradesh, North eastern

regions of the country including borders of Tibet, Meghalaya, Indo-Burma border and West

Bengal. In all these regions, occurrence of the plant is at different altitudes and climatic

conditions. While near Indo-Tibet border in the north eastern region it is found at 1500-3600

m and prefers high moisture conditions to grow; in Jammu & Kashmir it grows in drier sunny

slopes at an altitude of 2000-3350 meter.


13

Figure 4: Distribution of Pinus species in the Indian subcontinent

Table 1 summarizes distribution of the Pinus species in India, its altitude and preferred

climatic conditions.


14

Table 1: Distribution of species of Pinus in Indian subcontinent

Species Distribution Altitude Climate Reference

Pinus gerardiana

Jammu and Kashmir, Himacal Pradesh

2000m-3350m Grows in drier sunny slopes

Farjon et al., 2013

Pinus roxburghii

Jammu and Kashmir to West Kameng district of Arunachal Pradesh

400m-2300m Grows in drier and outer Himalayan valleys

Farjon et al., 2013

Pinus wallichiana

Jammu and Kashmir to West Kameng district of Arunachal Pradesh

Till 2700m Occurs in drier region of Himalayas along with other species

Farjon et al., 2013

Pinus bhutanica Tenga valley in Arunachal Pradesh

1000m Drier and inner valleys along with other species

Farjon et al., 2013

Pinus insularis North east India, Meghalaya

Upto 3000m Prefer high moisture

Pinus armandii Extreme north eastern India bordering Tibet

1500-3600m Prefers high moisture

Critchfield et al.,1996

Pinus mekusii Indo Burma border

1500 m Prefers high moisture

Bhatnagar et al., 1996

Morphology of Pinus

Pinus is a tree with a pyramidal appearance. Its stem is woody and has prominent bark. The

bark peels of at maturity in most of the species. The plant has two types of root systems

namely the main root which has an unlimited growth and the branch root which grows to a

limited length .Often mycorrhizal association are found in the root system of Pinus. Two

types of branching system have been observed in Pinus namely the long shoots and the dwarf


15

shoots. The long shoots are the branches which have the potentiality of unlimited growth.

These branches bear an apical bud enclosed within bud scales. The second type of shoots is

the dwarf shoot or brachyblasts. They arise at the axil of scale leaf on the long shoots. The

dwarf shoots have two opposite scale leaves called prophylls. They are followed by 5-13

cataphylls of spiral orientation. They finally enclose the functional leaves or needles whose

number may vary from 1 to 5 depending upon the species (Bhatnagar et al., 1996).

The leaves of Pinus are of four types and are present at various stages of their development.

The first formed leaves are the cotyledons that emerge out of the germinating seeds. They

vary from 4 to 24 in numbers. Primary leaves are the first formed leaves of the young

seedling. They are acicular, alternate in arrangement and produced only in the first year of

development. They are also produced in matured plants in response to wound. The cataphylls

are non-chlorophyllous primary leaves produced on shoots. They are subulate and lanceolate

with erose hyaline to ciliate margins and leave a distinctive pattern on the branch when they

fall off. The needles are the photosynthetic leaves of Pinus and occur in fascicles of one to

five. They generally remain bounded by a sheath like structure which may be persistent or fall

off during the course of development. The needles are sessile, and ovoid in abaxial surface

and sheathed at the base by 12-15 overlapping scale leaves or cataphylls. The scale leaves or

cataphylls are firm and compactly arranged at the base of needles fascicles .The epidermal

surface of the needle are interrupted by the presence of stomata. The stomata occur both on

adaxial and abaxial surface of the needle. The stomata are in general sunken and are arranged

in specific rows along a pit that lines all along the epidermal surface (Figure 5).


16

Figure 5: Vertical section of the needle of Pinus roxburghii Sarg. showing sunken stomata

The stem is stout and erect and is covered by bark. At maturity the barks become furrowed in

various patterns. The morphology of the bark changes with the age of the plant .The bark

morphology is often used for the identification of species (Figures 6 A & B). The

reproductive structure of Pinus occurs in the form of cone on the short branches. The plants

are monoecious i.e. male and female cones occur separately in same plant. The staminate

cones are numerous and small and occur in cluster. Each cone consists of a central axis with

spirally arranged microsporophyll. Each microsporophyll bears a pair of microsporangia at its

abaxial surface. The distal portion of the microsporophyll points upward. The female

reproductive organs are organised in the form of cones which are larger than the male

counterpart (Figure 7). They generally take 2-3 years to mature and are often hard and woody

structures. Each cone has a central stalk with spirally arranged megasporophyll (Figure 8).

Each megasporophyll is comprised of a paper like bract scale and a more hard ovuliferous

scale.


17

Figure 6: The stem bark of Pinus roxburghii Sarg. (A) Showing characteristic pattern of scales (B) Enlarged picture of the stem bark of Pinus roxburghii Sarg.

Both these scales are firmly attached to each other at maturity. Each ovuliferous scale bears a

pair of anatropous ovule in its adaxial surface. The distal portion of ovuliferous scale is

expanded to form a structure called umbo. They may be characterised by the presence of

spines or bristles and takes the form of a pyramidal structure called apophysis.

The seeds of species of Pinus are dispersed both by air and by animals. Around 20 species of

Pinus have wingless seeds and are dispersed by birds and most of them belong to the

subgenus strobus (Tomback et al., 1990). It has also been noted that the seeds of the Pine

species weighing less than 100mg are wind dispersed while the heavier seeds are dispersed by

animals(Benkman, 1995). It has also been observed that the shift from wind to animal


18

dispersal of the seeds has also been accompanied by a shift in the morphology of cone

structure (Strauss et al.,1990). The principal birds involved in the dispersal of wingless to

nearly wingless seeds of Pines are nutcrackers (Nucifraga caryocatactes and Nucifraga

columbiana) and pinyon jay (Gymnorhinus cyanocephalus) (Ligon, 1978). The pine species

whose seeds are dispersed by birds have the following adaptive features:

a) Seeds lack most or the entire thin membranous wing

b) Seeds are heavier as compared to the seeds of wind dispersed pines

c) There is a tendency of retaining of ripe seeds within the cones till withering due to the

process of indehiscence or restraining flanges after cone dehisce.

Thus, the possibility of wind dispersal gets reduced due to the wingless nature of seeds and

indehiscence of cones. In addition to that the pines whose seeds are dispersed by birds have a

shrubby habit with broad and multi branched canopies and upward oriented branches

supporting horizontally directed cones at the branch tips. This results in direct visibility and

accessibility of the cones to nutcrackers (Hutchins et al.,1982). Four species of seed dispersed

pines namely P. cembra, P. albicaulis, P. flexilis, and P. armandii show multitrunk growth

forms. Though they don’t facilitate the seed harvest but may be a consequence of seed

catching by the nutcracker (Lanner, 1980).


19

Figure 7: Shoot of Pinus roxburghii Sarg. with a female cone.

Figure 8: Characteristic young female cones of Pinus roxburghii Sarg.


20

Classification of genus Pinus:

The genus Pinus is the largest among in the family Pinaceae and consists of 114 species.

Three main categories of pine trees are found i.e subgenus Strobus or the soft pines, the

subsection Ducampopinus or the piniyon or foxtail pine and the subgenus Pinus or the hard

pines. The classification is based on the different morphological characteristics. The three

subgeneras are distinguished from one another by the following characters: (Michael Frankis ,

2002)

a) Subgenus: Strobus: Scales without a sealing band, umbo terminal, seedwings adnate with

one fibrovascular bundle per leaf.

b) Subgenus: Ducampopinus: Scale without a sealing band, umbo dorsal, seedwings

articulate with one fibrovascular bundle per leaf.

c) Subgenus: Pinus: Scale with a sealing band, umbo dorsal, seedwings articulate with two

fibrovascular bundles per leaf.

Intrageneric classification of Pinus. based on cone characters and reproductive structures are

shown in Figure 9.

Traditional uses of gymnosperms

Gymnosperms have been traditionally used by the indigenous people throughout the globe.

They have a wide spectrum of usage starting from domestic house hold to traditional

medicine. It has been reported that the Cedrus deodara oil is used as an insect repellent, for

protection against ticks of cattle and also for seed treatment by the local people of Doda

district of Jammu and Kashmir (Slathia et al., 2007). The wood of Cedrus deodara is also


21

Pinus

Subgenus: StrobusCharacters: 1. Scale without sealing band

2. Umbo terminal3. One vascular bundle per leaf

Subgenus: DucampopinusCharacters: 1. Scale without sealing band

2. Umbo dorsal3. One vascular bundle per leaf

Subgenus: PinusCharacters: 1. Scale with sealing band

2. Umbo dorsal3. two vascular bundle per leaf

Section: QuinquefoliaeSubsection: Strobistrobus, monticola, flexilis, reflexa, ayachuitestrobiformis,, chiapensis, peuce, walliachana,bhutanica,armandii, dabeshanensis,lambertiana, amamiana, fenzeliana, morrisonicola,wangii, parviflora, pumila,

Subsection: Cembraecembra, sibirica, koraiensis, albicaulis

Section: ParryaSubsection: Nelsonianaenelsonii

Subsection: Krempfianaekrempfii

Subsection: Gerardianaegerardiana, bungeana, squamata

Subsection: Rzedowskianaepinceana, maximartinezii, rzedowskiiSubsection: Cembroidesmonophylla, edulis, remota, cembroides,quadrifolia, discolor, johannis, culminicola, orizabensis

Subsection: Balfourianaebalfouriana, longaeva, aristata

Section: PinusSubsection: Pinussylvestris, densiflora, tabuliformis, densata, taiwanensis, mugo, nigra, heldreichii, thunbergii, luchuensis,hwangshanensis, massoniana, resinosa, tropicalis, yunnanensis, kesiya

Section: PineaSubsection: Pineae pinea

Subsection: Pinasterpinaster, canariensis, brutia, latteri, merkusii roxburghii, halepensis,

Section: TrifoliaeSubsection: Leiophyllaeleiophylla, lumholtziiSubsection: Australespalustris, echinata, glabra,serotina, rigida, virginiana, clausa, pungens, taeda, hondurensis, elliottii,caribea, occidentalis, cubensisSubsection: Contortaecontorta, banksianaSubsection: Oocarpaeattenuata, muricata, radiata, patula, teocote, herrerae, lawsonii,tecunumanii, pringlei, greggii,jaliscana, praetermissa, oocarpaSubsection: Ponderosaetorreyana, sabiniana, coulteri,jeffreyi, ponderosa, arizonica,engelmanii, durangensis, hartwegii,cooperi, estevezii, montezumae, devoniana, pseudostrobus,apulcensis, maximinoi, gordoniana

Vegetation of Pinus roxburghii Sarg. at Uttarakhand

Figure 9: Intrageneric classification of Pinus (Michael Frankis, 2002)


22

used in Ayurveda for the treatment of rheumatoid arthritis (Chandur et al., 2011). The resin of

Cedrus deodara is used for skin treatment (Jan et al., 2011). Apart from these, various plant

parts of Cedrus deodara is also reported to have antioxidant, anti-diabetic, anti-tubercular

and anti-spasmodic activities (Gupta et al., 2011).

Another plant of tremendous popularity among the ethnic people is Taxus. In ayurvedic

medicine Taxus wallichiana finds its use for the treatment of headache, diarrhoea, hysteria

and epilepsy. It is also used as an anti-fertility agent (Rahman et al., 2013). The bright red

berries of Taxus wallichiana are consumed as food by the villagers in Nepal. It has also been

reported that the bark of Taxus baccata is used for the treatment of breast infection in the

Garhwal Himalayan region (Bhatt et al., 2013). Taxus baccata is also used as an antiparasitic

agent by the local people of north western Spain (González-Hernández et al., 2004). Taxus

baccata is also used as purgative, antirheumatic and antispasmodic agent. It is also used in

renal disorders and digestive disorders by the local people of Pakistan (Ummara et al., 2013).

The local people around Nanda Devi biosphere reserves use Taxus bark and leaves for curing

cold and cough (Rahman et al., 2013). It has been reported that the traditional healers in

Manipur use tablets made from Cephalotaxus griffithi for the cure of cancer (Moirangthem et

al., 2012). The potentiality of Taxus as an anticancer agent is also well established. Tamofixen

is a drug which is obtained from the leaves of Taxus baccata is used for the treatment of

breast cancer. Docetaxel is another semisynthetic agent isolated from Taxus baccata and is

used as a curative agent for breast cancer (Pant et al., 2008).

The seeds and oil of Juniperus communis are used as a diuretic, carminative and stimulant. It

is also used for the treatment of skin diseases and also as fuel wood by the people of


23

Pakhtoonkhwa province in Pakistan (Jan et al., 2011). The plant juice of Juniperus indica is

used by local people of Nepal as appetizer. It is used for the treatment of diarrhoea and

abdominal pain, diseases of spleen, tumors, bronchitis, piles and vaginal diseases. Its berries

are also used for the treatment of dental ailments and piles (Malla et al., 2009). In Italy, the

fruits of Juniperus communis, Juniperus oxycedrus and Juniperus sabina are used as

condiments for roasted meat. They are also macerated with alcohol to make various types of

liquors. The fruits are macerated and eaten for the treatment of cough and sore throat (Idolo

et al., 2010). The gum of Juniperus excelsa is used for the treatment of stomach ache, back

ache and rheumatism. Leaf infusion is used for cardiac and nervous problems and also as a

dye (Pirani et al., 2011). Juniperus communis is also used for seasoning cheese by the people

of Italy (Mattalia et al., 2012). Juniperus excelsa is used in folk medicine for the treatment of

diarrhoea, abdominal spasm, asthma, leucorrhoea, fever, gonorrhoea and numerous other

ailments (Khan et al., 2012). The decoction of Juniperus oxycedrus is used for healing of

wounds, diabetes, asthma, bronchitis and animal diseases (Demirci et al., 2012)

The leaves of Abies pindrow is also used as a substitute of tea. The resin of Cedrus deodara

is used for skin treatment by the localites of the same region (Jan et al., 2011). Various plant

parts of Abies pindrow is used as a curative agent for fever, asthma, bronchitis and diabetes

(Majeed et al., 2013) .The seeds of Ginkgo biloba are eaten by the Chinese people (Dar et al.,

2006). The whole plant of Picea smithiana is used for the treatment of skin disease, eye

disease and renal disorders by the indigenous people of Shogran valley in Pakistan (Ummara

et al., 2013).


24

Traditional usage of Pinus

Pinus have its habitat largely on the subtropical to temperate region and dominates in the hilly

terrain. They have a close association with the common people and have been used

extensively for various purposes. The plant produces copious amount of hard wood by its

extensive secondary growth. The value of the wood is enhanced by the presence of resin

canals filled with resins which are resistant to microbe and insect attack. This makes the wood

of Pinus extremely resistant to microbial decay. Pinus often finds wide application in folk and

rural medicines. The resin extracted from Pinus wallichiana is used for the cure of cracked

heels (Kumar et al., 2012). In Haramosh and Bugrote valleys of the northern Giligit region of

Pakistan, the resins obtained from Pinus gerardiana and Pinus wallichiana is used for the

cure of wounds. Burnt woods locally called “Kaalo” are used as antiseptic (Khan et al., 2007).

Young shoot and fresh cone of Pinus sylvestris is also used for the treatment of cough and

chronic bronchitis (Kizilarslan et al., 2013). The young shoots of Pinus pinaster is used as an

antidiuretic and antitussive (González-Hernández et al., 2004). It has been reported that Pinus

ponderosa is used indigenously by the native Americans for the treatment of dermatological

and gynaecological disorders (Wennerberg, 2004). The inner bark of Pinus resinosa is used

for the treatment of wound, sore and ulcer (Moore, 2003). The seed of Pinus pinea is used as

a tonic by the local population of Israel (Lev, 2006). Pinus brutia resin finds various

applications among the indigenous people of Turkey. The resin is processed into a chewing

gum which prevents bad breath and cleans teeth. Resin administered with honey is used for

the cure of stomach ache, ulcer and as an antidiabetes. Resin with hot water is externally

applied on cut wounds (Satil et al., 2011).


25

Therapeutical use of Pinus roxburghii Sarg.

In ayurveda Pinus roxburghii Sarg. is used as an intestinal antiseptic , antidyslipidemic,

spasmolytic and antioxidant. It is also used for the treatment of diseases of eyes, ears, throat,

blood and skin, diaphoresis, ulcer, inflammations and itching (Kaushik et al., 2013). It has

also been reported that the resin extracted from Pinus roxburghii Sarg. is used in the treatment

of snake bite and other skin diseases (Mughal, 2013).The resin of Pinus roxburghii Sarg. and

Pinus wallichiana is used as stomachic and for the treatment of gonorrhoea. The resin is also

externally applied as a plaster to buboes and abscesses. The resin is also used as hair remover

and is also applied to cattles for healing of wounds. The leaf extract is used in burning of

body, fainting and ulceration. The oil is used as antiflatulent (Ishtiaq et al., 2013).

In Nepal, Pinus roxburghii Sarg. finds extensive indigenous use. The resin obtained from the

plant is used to relive cough and gastric troubles. Wood oil is used as a nerve tonic,

haemostatic, expectorant and diuretic. It is also used in skin diseases, burns and cracks.

Turpentine oil obtained from the plant is used as an antiseptic and also used as an expectorant

in bronchitis (Kunwar et al., 2009). The local people of Chakrata forest division of

Uttarakhand use Pinus roxburghii saw dust with honey for the treatment of bronchitis and

asthma (Dobhal et al., 2007). The resin of Pinus roxburghii is used by the people of Mornaula

reserve forest in Uttarakhand for the cure of pimples (Pant et al., 2009). The wood of Pinus

roxburghii Sarg. is aromatic and often used as a deodorant by the people of Indian Himalayas.

The leaves are used for increasing the flow of urine and decoction of leaves are locally

applied for the treatment of sprains (Kaushik et al., 2010). In Badgaun and Gulmi regions of

Nepal, the resin of Pinus roxburghii Sarg. is used in the treatment of boils. It is also used as


26

stimulant, stomachic and diuretic (Acharya, 2012).The gum of Pinus roxburghii Sarg. is bitter

and is used as carminative, emmenagogue, expectorant, aphrodisiac, fattening, diuretic,

anthelmintic and analgesic. It is also used in the treatment of vagina, uterus and eye and is

good in dyspepsia, diaphoresis, scabies, asthma, ear discharge, tooth ache, piles and liver

diseases (Khan et al., 2012).

Information on Antioxidant capacity of Gymnosperms

The gymnosperms are among the most explored plants for their antioxidant activity. They are

the group of comparatively primitive plants and have substantial quantities of phenolics,

flavonoids and other natural products. This led scientist to explore this group of plants as a

source of antioxidant.

Aerial parts of Abies georgei is rich in terpenes, lignans and flavonoids and exhibited strong

inhibitory activity against lipopolysaccharide (LPS)-induced NO production in RAW 264.7

macrophages (Yang et al. 2011). The bark extracts of Abies spectabilis possess significant

polyphenol content and antioxidant activity. Oligomeric C-type proanthocyanidins, mainly

trimeric gallocatechin derivatives, were found to be the most abundant compound in the bark

of Abies spectabilis (Dall'acqua et al. 2012). The hydroalcoholic bark extract of Araucaria

angustifolia exhibited protective action against hydrogen peroxide induced stress in culture.

An Afzelechin derivative, isolated from the plant also acts as a strong antioxidant (Seccon et

al. 2010). The leaf extract of Araucaria excelsa is rich in phenolics and also possess

antioxidant activity (Michael et al. 2010).


27

Exploration for flavonoid glycosides have been carried on Cephalotaxus korean (Bae et. al,

2007). A glycoside, apigenin 5-O-alpha-L-rhamnopyranosyl-(1-->3)-beta-D-glucopyranoside

along with four known flavonol glycosides were isolated from the leaf of Cephalotaxus

koreana. All of the extracted glycosides were effective against DPPH free radical and

superoxide ion scavenging activity. Lignans isolated from Taxus baccata showed inhibition

against butyrylcholinesterase and lipoxygenase activity in addition to strong scavenging

activity against DPPH and ferric reducing antioxidant power (Kucukboyaci et al. 2010).

The berries of two varieties of Juniperus communis namely communis and saxtilis were

evaluated for their antioxidant potential by Miceli et al. (2009). The results indicated that the

variety communis had higher phenolic and flavonoid content and also showed stronger

antioxidant potential than the other variety. Ennajar et al. (2009) reported that the extract of

Juniperus phoenicea is a rich source of phenolics, tannins, anthocyanins and flavonoids. It

was found that the antioxidant potential of the extracts had correlations with the chemical

constituents. The ethyl acetate fractions of extracts of Juniperus excelsa also possess

antioxidant property (Moein et al., 2010). The essential oil isolated from the two sub species

of Juniperus excesla namely subspecies excelsa and subspecies polycarpos exhibited

antioxidant property (Emami et al., 2007). The acetone extracts of fruits of several species of

Juniperus inhibited linoleic acid oxidation. They also exhibited acetylcholine esterase activity

(Öztürk et al., 2011). Similar results were also obtained by Orhan et al. (2011) while

analysing species of Juniperus. Species of Cupressus has also been reported to possess

antioxidant activity (Emami et al., 2007).


28

The chloroform extract of dried heartwood of Cedrus deodara showed strong antioxidant

activity on DPPH free radical (Tiwari et al., 2001). The stems of Podocarpus latifolius

exhibited DPPH radical scavenging and Cox-2 inhibitory activity. Tyrosinase and Cox-1

inhibition has also been reported by the extracts of Podocarpus elongatus. (Abdillahi et al.,

2011).

Ginkgo biloba, a living fossil among gymnosperm also possesses antioxidant property. A

novel polysaccharide (GBP50S2) with antioxidant activity has been isolated from Ginkgo

biloba which was found to possess 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging

and hydroxyl ion scavenging activity (Yuan et al., 2010). Prodelphinidins, proanthocyanidins,

catechin, epigallocatechin and gallocatechin have also been isolated from the leaves of

Ginkgo biloba with strong DPPH scavenging activitiy (Qadan et al., 2011).

Mourya et al. (2011) also reported antioxidant and antimicrobial activity from the leaf extract

of Cycas revoluta. Three phenolic compounds namely 3, 4-dimethoxychlorogenic acid,

resveratrol, and 3-methoxyresveratrol have also been isolated from stem Gnetum gnemon

(Atun et al., 2007). All the compounds isolated showed hydroxyl ion scavenging activity. Six

stilbenoids namely gnetin L, gnetin C, gnemonosides A, C, and D, and resveratrol have been

isolated from dried endosperm extracts of Gnetum gnemon L (Kato E. et al., 2009). All the

stilbenoids isolated showed 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging

activity similar to that of ascorbic acid and dl-alpha-tocopherol. The biological activities of

Gnetum gnemon was also analysed by Wazir et al (2011). The results indicated that the

methanolic twig extract showed strong reducing activity (FRAP). 5, 7, 2-trihydroxy-5'-

methoxyflavone have been isolated from the liana Gnetum macrostachyum (Saisin et al.


29

2009), which showed activity against DPPH radical. Parsaeimehr et al. (2010) reported the

antioxidant potential of several species of Ephedra. Similar results were also obtained by

Song et al. (2010) from Ephedra sinica. Torreya grandis cv. Merrillii, is a gymnosperm

endemic to China. The seeds are used as a popular snack, possessing beneficial effects on

preventing angiosclerosis and coronary heart diseases. The extract of the plant also possess

antioxidant property (Shi et al. 2009).

Reports on Antioxidant potential of different species of Pine

Popularity of the genus Pinus as a plant having strong antioxidant potential is quite high.

Numerous groups of researchers from various corners of globe reported its antioxidant nature

in relation to its medicinal property. The antioxidant potential from the needles of Pinus

densiflora was explored by Jung et al. (2003). The antioxidant activity of Pinus densiflora

needle was explored by Kwak and coworkers (2006). Their reports suggested that the

methanolic extract of the plant showed strong antioxidant activity. (+)-isolarisiresinol

xylopyranoside as well as two active flavonoids [kaempferol 3-O-beta-galactopyranoside and

its 6"-acetyl derivative], were isolated from the plant. Jung et al. (2009) reported diterpene

glucoside and flavonoid glucoside from the ethyl acetate extracts of the needles of Pinus

densiflora. Park et al (2011) reported that the essential oils of the needles of Pinus densiflora

and Pinus thunbergii exhibited antioxidant and antimicrobial properties.

The antioxidant property of Pinus massoniana was investigated by Cui et al. (2005). The bark

extract of Pinus massoniana is also found to be inhibitory to cancer cells (Limei et al., 2008).

The bark extract was found to be a scavenger of Superoxide ion and DPPH radical. The bark

extracts of Pinus massoniana also have hepatoprotective effect (Wang et al., 2010). The barks


30

of Pinus pinaster and Pinus radiata was also studied by Jerez et al. (2007). Their report

suggests the aqueous extracts of both the species have potent antiradical activity.

Ku et al (2007) estimated the phenolic composition from the bark of a variety of pine species.

Their results confirmed that among the Pine species explored for the quantification, Pinus

rigida showed highest polyphenol content and antioxidant potential. Chen et al. (2011)

worked on the antioxidant potential of oil extracted from the nuts of Pinus koraiensis. The

result on antioxidant activity showed that the oil improve the activities of SOD, glutathione

peroxidase and total antioxidant capacity, and reduce the content of MDA. The antioxidant

activity of pine pollen extract was investigated by Lee et al. (2009). It was found that pine

pollen extract significantly reduced the amount of malondialdehyde and protein carbonyls

formed and also showed strong free radical scavenging activity on 1, 1-diphenyl-2-

picrylhydrazyl radical and hydrogen peroxide. Antioxidant activity from various species of

pinus occurring in natural forests of Greece has also been worked out by Guri et al. (2006).

Their studies indicated that ethanol extracts of the needle significantly inhibited Fe2+-induced

lipid peroxidation and scavenged DPPH radical. Hsu et al (2005) reported that the needle

extracts of Pinus morrosinicola had strong superoxide anion scavenging activity along with

inhibition of leukemia cell line U937. The antioxidant property of the needle extracts of Pinus

morrosinicola has also been worked out by Gow-Chin et al. (2008). It protects LDL oxidation

and also possesses anti-inflammatory action. Pinus halepensis has also been reported to

possess antioxidant activity and bioactive compounds (Dhibi et al.,2012).

It has been reported by Grassmann et al. (2003) that the essential oil of Pinus mugo possesses

antioxidant property when tested in lipophilic medium. The antioxidant activity of Pinus


31

nigra was explored by Gulcin et al. (2003). According to their findings, the antioxidant

activity increased with the increase in concentration of the extracts. The potential of volatile

compounds isolated from Bosnian pine (Pinus heldreichii) was also highlighted by Maric et

al. (2007). Zhang et al. (2009) reported the strong free radical scavenging and inhibitory

activity on adenocarcinoma cells by the extracts from the barks of Pinus caribaea. Akbulut et

al. (2009) reported the antioxidant activity of pine honey collected from Turkey. The results

showed that a correlation exists between the total antioxidant activity and total phenolic

contents of the samples. Sato et al (2009) showed beneficial effect of pine bark extracts on

atherosclerosis development in male mice. It has also been shown by Jeong et al. (2009) that

the water extracts of pine needles exhibited strong scavenging action on hydroxyl radical and

intracellular ROS, and chelating action of Fe2+ ion. Lantto et al. (2009) explored the

antioxidant activity of Siberian pine, Pinus siberica. The extract of Pinus siberica was found

to scavenge the DPPH and hydroxyl free radicals and decreased the viability of tumourigenic

neuroblastoma cell line. Ince et al. (2009) reported that the extracts of Pinus brutia act as an

anti-inflammatory agent in rat model.

The chemical constituents and antioxidant potential of Pinus armandii was worked upon by

Yang et al. (2010). According to their reports the oil extracted from Pinus armandii is a rich

source of terpenoids and exhibits strong DPPH scavenging activity higher than that of BHT

and ascorbic acid. Shen et al. (2010) isolated catechin, epicatechin, quercetin and ferulic acid

from the ethyl acetate fraction of the bark extracts of Pinus thunbergiana. Their work also

suggested antioxidant potential of the plant. Apetrei et al. (2011) reported the antioxidant

potential of Pinus cembra bark and needles. According to their reports, the bark extract had

the higher concentrations of total phenolics, flavonoids and proanthocyanidins than the needle


32

extracts and was more active as a free radical scavenger and reducing agent. Maimoona et al.

(2011) estimated the total phenolics and flavonoids in the bark extracts of Pinus roxburghii

and Pinus wallichiana. Puri et al., (2010) reported the antidyslipidemic activity of the needles

of Pinus roxburghii. Their reports suggested that the alcoholic extract has the highest capacity

of scavenging DPPH and ABTS•+ free radicals. The extracts also decreased the plasma

triglyceride, total cholesterol, glycerol and low density lipoproteins-cholesterol.

Oxidative stress and Reactive oxygen species

Reactive oxygen species (ROS) denote a collection of oxygen radicals (O2-, OH•) and some

other derivatives of oxygen radical like H2O2. These are generated during normal metabolism

of cells involving the mitochondrial electron transport, microsomal Cyt P450 and other

systems.

However, excessive generation of ROS induced by various stimuli (mainly biotic and abiotic

stress) sometimes exceed the normal antioxidant capacity of the living organelles leading to

oxidative damage of macromolecules like DNA (Cui et al., 2012), lipids (Pizzimenti et al.,

2010), proteins (Lobo et al., 2010) and play an important role in various diseases such as

cancer (Nogueira et al., 2013), rheumatoid arthritis (Harty et al., 2011) , degenerative

processes of aging (Zeng et al., 2014) and cardiovascular diseases (Csányi et al., 2014).

Superoxide anion: Superoxide anion is a reactive oxygen species and is generated by

sequential reduction of molecular oxygen initially resulting in the formation of superoxide

anion and ultimately resulting in the formation of hydrogen peroxide and water. Within the


33

respiratory chain the mitochondria Complex I and Complex III are the major source of

superoxide anions (Drose et al., 2012)

Figure 10: Mitochondrial ROS generation (Kakkar & Singh ,2007)

Hydroxyl Radical: This is one of the important reactive oxygen species and is considered to

be most reactive among all of them. Having a very short half-life of 10-9 seconds (Valko et al.,

2007), they are formed by successive reduction of molecular oxygen during the cell

metabolism and are responsible for cytotoxic effects in the living system. It is assumed that

hydroxyl radical is generated within the living system by the Fenton and Haber Weiss

reactions (Thomas et al., 2009).

Fe2+ + H2O2 Fe3+ + •OH + HO–


34

It is reported that hydroxyl radical can cause damage to the biomolecules resulting in the

occurrence of disorders such as cancer, cardiovascular diseases and neurodegenrative

disorders (Ayala et al., 2014).

Hydrogen peroxide: Hydrogen peroxide is a dismutation product of superoxide anion and is

formed by the action of enzyme superoxide dismutase. Some of the enzymes such as xanthine

oxidase also generate hydrogen peroxide from superoxide ion. Hydrogen peroxide has much

less reactivity as compared to the other reactive oxygen species. However, it is found to react

with heme proteins resulting in physiological disorders (Vallelian et al., 2008).

2 O2- + 2H+ H2O2 + O2

2 H2O2 2 H2O+ O2

Peroxyl and alkoxyl radical: Peroxyl radicals are analogues of hydroperoxyl radicals in

which the hydrogen atom is replaced by an organic group. They are stable than the respective

hydrogen analogues due to the presence of a stable carbon-oxygen bond. The peroxyl radicals

are primarily formed by the oxidation of lipid molecules primarily in the membranes (Eboh,

2014). They are involved in autoxidation of lipids, inactivation of certain enzymes resulting in

physiological imbalance and initiation of certain diseases such as cancers, neurodisorders and

ischaemia (Masood et al., 2014). Alkoxyl radicals are produced by the decomposition of alkyl

peroxides (El-Bahr, 2013). The alkoxyl radicals are reported to be cytotoxic and cause cell

damage (Tewari et al., 2014).

Nitric oxide: Nitric oxide may be considered as both a free radical and a molecule with a

single unpaired electron. They are formed from L-Arginine by the enzyme nitric oxide


35

synthase (Tennyson et al., 2011). Over production of nitric oxide leads to nitrosative stress.

This happens when the production of reactive nitrogen species in the cells exceeds the cell’s

ability to nullify them. This leads to nitrosylation and nitrotyrosination reaction that can alter

the structure of biomolecules and also inhibit their normal function (Valko et al., 2007). Nitric

oxide plays an important role in the onset of certain neurodegenerative diseases such as

Parkinson’s Disease, Alzheimer’s Disease, multiple sclerosis (Steinert , 2010) , Hungtington’s

disease (Carrizzo et al., 2014) and stroke (Knott et al., 2009). They are also responsible for

chronic inflammatory diseases such as rheumatoid arthritis (Nagy et al., 2010) and

inflammatory bowel disease (Dhillon et al., 2014).

Figure 11: Pathway leading to nitric oxide generation within a cell.

Peroxynitrite: Peroxynitrite is one of the reactive radicals that is generated by the reaction of

nitric oxide and superoxide radical.

2 O2- + •NO ONO2

-

Peroxynitrite is a strong oxidant and reacts directly with electron rich groups such as

sulphydryls , iron-sulphur centres, zinc-thiolates and active site sulphydryls in tyrosine

phosphatases.


36

Figure 12: Oxidative stress (ROS/RNS) generation and antioxidant machinery (Kakkar & Singh, 2007)

Peroxynitrite modifies protein containing heme prosthetic groups such as haemoglobin,

myoglobin , cytochrome c and oxidises its ferrous ion into corresponding ferric form (Pacher

et al., 2007). They also alter the protein structure and function by oxidizing cysteine and

nitration of tyrosine in the enzyme tyrosine hydroxylase resulting in the loss of function of

that enzyme (Blanchard-Fillion et al., 2001). Peroxynitrite is reported to damage human and

bovine surfactants leading to impaired pulmonary defence (Szabó et al., 2007).

Free radicals in diseases

Reactive oxygen species are constantly generated in the living system. Their direct deleterious

effect includes oxidative damage to various biomolecules including DNA, lipids and proteins.

Once they are in excess, these reactive oxygen species bring about lots of physiological


37

imbalance which ultimately result in occurrence of a number of diseases. It has been reported

that reactive oxygen induced damage of eyelens mediate the formation of cataract

(Babizhayev et al., 2010). Imbalance of the reactive oxygen species also results in the

occurrence of glaucoma (Szaflik et al., 2010). It has also been reported that the oxidative

stress in conjugation with an imbalance of iron radicals play a vital role in progression of

Parkinson’s disease (Friedlich et al., 2009). It has also been reported that the mitochondrial

dysfunction through generation of reactive oxygen species also result in the occurrence of

Parkinson’s disease (Keane et al., 2011). Reactive oxygen species also creates an oxidative

stress in striatal and cortical neurons resulting in their degeneration. This eventually results in

the onset of Huntington’s disease (Ribeiro et al., 2013). It has also been reported that

excessive ROS are generated due to mitochondrial dysfunction which eventually lead to

neurotoxicity and onset of Alzheimer’s disease (Zhao et al., 2013).

Involvements of reactive oxygen species in genetic disorders are also well marked. Trisomy

of chromosome number 21 in humans leads to the occurrence of Down’s syndrome which is

manifested as mental retardation. It has been observed that the Down’s syndrome patients are

more vulnerable to oxidative damage which ultimately leads to occurrence of Alzheimer’s

disease (Di Domenico et al., 2014).

Reactive oxygen species is also responsible for the occurrence of Prion’s disease. It has been

reported that increase in ROS is related to the onset of Prion’s disease (Haigh et al., 2011).

Rheumatoid arthritis is also a common disorder of bone which is of wide spread occurrence. It

has been reported that patients having rheumatoid arthritis have significantly higher


38

malondialdehyde and low levels of endogenous antioxidants indicating the possible role of

reactive oxygen species in the onset of the disease (El-barbary et al., 2011).

Among the infectious diseases, involvement of reactive oxygen species and hepatitis C is well

marked. It has been observed that individuals with chronic hepatitis C show an oxidative

stress and increased concentration of MDA (El-Kannishy et al., 2012). It has also been

proposed that HCV infection restrains antioxidant defence system within the human system

leading to the generation of reactive oxygen and reactive nitrogen species. This leads to the

damage of biomolecules and also increases the severity of the infection (Paracha et al., 2013).

Onset of influenza has also been reported to have relation with reactive oxygen species. It has

been shown that influenza virus M2 protein increases the concentration of reactive oxygen

species and inhibits epithelial sodium channels (Lazrak et al., 2009). It has also been shown

that influenza A virus infection results in a rapid influx of inflammatory cells, increased

reactive oxygen species production and acute lung injury. Proinflammatory stimuli induce

intracellular ROS generation by activating NADPH oxidase (Ye et al., 2013).

AIDS is a deadly disease which has affected over 1 million people in United States alone and

33 Million people worldwide. It has been observed that there is an increased oxidative stress

condition in chronically infected HIV1 patients manifested by an increased reactive oxygen

species generation. In addition to it, these patients also have a reduced glutathione and

thioredoxin concentration coupled with a disturbance in mitochondrial membrane potential

(Salmen et al., 2012). It has also been reported that HIV infection increases the levels of

superoxide anion and peroxynitrite, the latter of which promotes HIV replication in

macrophages (Dayton, 2008).


39

Diabetes is another disease of wide spread occurrence and also has an intricate relationship

with reactive oxygen species. Diabetic nephropathy is an end stage renal failure and is

characterized by a loss in podocytes during early onset of the disease. It has been reported that

the podocyte apoptosis is sharply increased by the onset of hyperglycemia. It has been found

that increased extracellular glucose rapidly stimulated production of reactive oxygen species

within the cell through NADPH oxidase and mitochondrial pathways. This leads to the

activation of pro-apoptotic p38 mitogen-activated protein kinase, caspase 3 and apoptosis of

podocytes (Susztak et al., 2006).

Oxidation Balance

Figure 13: Oxidative stress due to imbalance in ROS generation and antioxidant status is reported to play a major role in diabetes and associated metabolic syndrome.

NADPH Oxidase

Xanthine oxidase

Cycloxygenase, lipoxygenase

Mitochondrial Electron Transport

eNOS, Myeloperoxidase etc.

Superoxide dismutase

Catalase

Glutathione peroxidase

Alpha‐tocopherol

Glutathione etc.

Metabolic syndrome


40

It has also been reported that hyperglycemia induces ROS generation through activation of

glycation reaction and electron transport chain in mitochondria. Under diabetic condition

coupled with an increase in ROS activity, deterioration of β-cell function and increased

insulin resistance take place. This results in aggravation of type 2 diabetes (Kaneto et al.,

2010). Involvement of reactive oxygen species in atherosclerosis has also been reported. In

atherosclerosis the reactive oxygen species oxidises the LDL which are then easily taken up

by the macrophages initiating the pathology of the process (Vogiatzi et al., 2009).

The involvement of reactive oxygen species in the onset of cancer is well marked (Costa et

al., 2014). Cancers arise primarily in the regions of irritation, infection or inflamations all of

which have a high reactive oxygen species levels (Liou et al., 2010). The deleterious effect

of reactive oxygen species is oxidation of biomeolecules including DNA. Once DNA is

oxidised, the defect is likely to be carried over to the next generation of cells with certain

degree of structural and physiological anomaly. This ultimately results in the occurrence of

cancer in due course. It is known that reactive oxygen species leads to unpaired or mispaired

damage of DNA. This ultimately leads to mutations involving transitions and transversions

(Rubin et al., 2009). In case the mutation is centered on some critical gene including

oncogenes or tumour suppressor genes, carcinogenesis takes place. Reactive oxygen species is

involved both in initiation and progression of cancer (Waris et al., 2006). Asthma is another

important pathophysiologic disorder that mainly occurs due to allergens and pollutants. Its

main manifestation is inflammation of airways. It has been reported that the allergens present

in the atmosphere induce increased generation of reactive oxygen species in the living system.

These ROS then induce cellular signalling which ultimately leads to a cascade of events

resulting in respiratory inflammation (Zuo et al., 2013).


41

Natural antioxidants

The living system has got an intricate antioxidant defence system in the form of various

antioxidant enzymes. However, the antioxidant defence system weakens with time. Thus,

supplementation with some external antioxidants is extremely necessary in order to combat

the deleterious effects of the free radicals. The polyphenols are a group of natural antioxidants

which are the secondary metabolites produced by the plants. These phytochemicals by the

virtue of their chemical structure have got the property of scavenging free radicals and

reactive oxygen species. Bioactive non-nutrients found in plants are generally aromatic

phenolics compounds called as ‘polyphenols’. They form the secondary metabolites of the

plants and can be broadly organized into a) Phenolic acids, which contains derivatives of

benzoic acid and derivatives of cinnamic acid b) Flavonoids c) Lignans and d) Stilbenes etc

(D’Archivo et. al., 2007). Most of these compounds possess one or more hydroxyl group

bonded to an aromatic ring. Compounds that have several hydroxyl groups are called as

polyphenols. Due to their aromatic ringed structure these polyphenols have the ability to form

a phenoxide ion by losing a proton and consequent delocalization of the extra electron

through the aromatic ring. Thus, they behave more as an acid than an alcohol. The phenoxide

ion has the ability to further loose an electron to form the corresponding radical which can

also delocalize. (Maestri et. al., 2006). This property contributes the radical scavenging

activity and antioxidant activity of the polyphenols. (Figure 14) shows various categories of

polyphenols.


42

Polyphenols

Polyphenols are a group of mostly naturally occurring compounds which contain a large

number of phenol moieties as their structural unit. They form the secondary metabolites and

are abundantly distributed in various parts of the plants. They confer resistance to the plants

from attack of pathogen and also protect them from harmful ultraviolet radiations. Presently

more than 8000 phenolic compounds are known among which nearly 4000 of them belong to

the group of flavonoids (Tsao, 2010).

Biological significance of polyphenols

Polyphenols have always been associated with the prevention of a wide array of diseases.

Their unique molecular configuration helps them to accept electrons from other radicals

formed in the biological system resulting in the termination of the chain reactions within the

cell (Pandey et al., 2009). In this process they themselves get oxidised and form a

comparatively less reactive radical that is less injurious to the living system. This chemical

property of flavonoid is responsible for a wide number of diseases. It has been reported that

flavonoids inhibit the DNA damage in human lymphocytes in a dose dependent manner.

(Boligon et al., 2012). Polyphenols also confer protection against oxidative damage of LDL

by various agents. Studies have found that polyphenols are inhibitors of LDL oxidation

(Costa-Mugica et al., 2012). It has also been found that polyphenols present in red wine

effectively reduce LDL oxidation (Arranz et al., 2012). They do so by scavenging reactive

oxygen species by their unique electron accepting ability. In addition to that, flavonoids have

also been reported to protect against endothelial dysfunction by induction of heme oxygenase

gene (Zheng et al., 2009). These might ultimately inhibit the development of atherosclerosis.


43

The inhibitory effect of flavonoids against atherosclerosis has also been described by

Salvamani et al., (2014).

Figure 14: Schematic representation of various typs of polyphenols (D’Archivio et al., 2007)

Table 5: Sources of Polyphenols in food (Manach et al. 2004)


44

Polyphenols Source Hydroxybenzoic Acids (2,6) Black Berry Protocatechuic Acid Raspberry Gallic Acid Black currant p‐Hydroxybenzoic Acid Strawberry Hydroxycinnamic Acid (2,5‐7) Blueberry Caffeic Acid Kiwi Chlorogenic Acid Cherry Coumaric Acid Plum Ferulic Acid Aubergine Sinapic Acid Pear, Chicory, Artichoke, Potato, Corn Flour, Cider,

Coffee Anthocyanins (8‐10) Aubergine Cyanidin Blackberry Pelargonidin Blackcurrant Peonidin Blueberry Delphinidin Black Grape Malvidin Cherry, Strawberry, Red Wine, Plum, Red Cabbage Flavonols Yellow onion Quercetin Curly kale Kaempferol Leek Myricetin Cherry tomato, Broccoli, Blue berry, Blackcurrant,

Apricot Apple, Beans, Black grape, Tomato, Tea Flavones Parsley Apigenin Celery Luteolin Capsicum pepper Flavanones Orange Juice Heserpetin Grapefruit juice Naringenin Lemon juice Isoflavones Soy flour Diadzen Soybeans Genistein Miso Glycetin Tofu, tempeh, Soy milk Monomeric flavanols Chocolate Catechin Beans Epicatechin Apricot, Cherry, Peach, Grape, Blackberry, Apple,

Green tea, Black tea, Red wine, Cider


45

The role of polyphenolic compounds in cardioprotective activity is also immense (Lecour et

al., 2011). Quercetin and its derivative prevent oxidative stress and prevent the progression of

atherosclerosis (Loke et al., 2010). It has been found by Park et al., (2013) that luteolin

inhibited the lipopolysaccharide induced NO production in RAW 264.7 macrophages and

production of prostaglandin E2, all of which are linked to inflammatory responses. This was

also accompanied by reduced expression of inducible Nitric Oxide Synthase in the cell lines.

Chen et al., (2011) reported that quercetin pentaacetate and rutin strongly inhibited PGE2

production and expression of COX2 protein all of which are directly related to inflammatory

responses.

The cancer preventive activity of the plant polyphenol is also well marked. They also

influence the metabolism of procarcinogens by altering the expression of cytochrome P450

enzymes or by induction of UDP-glucuronyl transferase, quinone reductase and glutathione S-

transferase which are responsible for detoxification of carcinogens (Batra et al., 2013). The

polyphenols also inhibit carcinogensis by initiation of the process of DNA repair (Das et al.,

2013). Secondly polyphenols act as inhibitors of cell proliferation (Liang et al., 2013). It has

been reported that apigenin inhibits cell proliferation and induces apoptosis in human

myeloma cells (Zhao et al., 2011). Luteolin is also reported to have inhibitory effect on

proliferation of prostate carcinoma cells (Tsui et al., 2012). It has been reported that tea

polyphenols and curcumin inhibit key signal transduction protein kinase such as mitogen

activated protein kinase, IκB and certain cyclin dependent kinases. These result in inhibition

of cell growth and transformation, promote apoptosis and inhibit angiogenesis. These

phenomena have been noted in the tissues of skin, prostate, colon and lungs. (Lambert et al.,

2005). It has also been reported that the flavonoid (-) – epigallocatechin gallate (EGCG)


46

inhibits the chymotrypsin like activity of proteasome which plays an active role in the onset

of tumour cell proliferation and drug resistance development during carcinogenesis. It has

also been reported that synthetic (-)-EGCG amides and (-)-EGCG analogues with

modifications in A and C rings or ester bond result in inhibition of chymotrypsin like activity

of the proteasome of purified 20S proteasome and resulted in growth arrest in the G1 phase of

the cell cycle in leukemia Jurkat T cells and suppressed colony formation in human prostate

cancer LNCaP cells (Chen et al., 2008). The flavonoids apigenin and quercetin are also potent

inhibitors of proteasome and induce apoptosis in human tumour cells (Chen et al., 2005). It is

also assumed that flavonoids with hydroxylated B ring and/or unsaturated C ring are potent

inhibitors of tumour cells and also strong inducers of apoptosis (Chen et al., 2007). The

isoflavone genistein is also inhibitory to carcinogensis. It has been found by De Assis et al., et

al. (2011) that geinstein significantly inhibits tumourogenesis . It is reported that the

compound confers protection from mammary (Wang et al., 2014) and prostate cancers

(Pavese et al., 2014). They do so by modulating specific sex steroid receptors and growth

factor signalling pathways (Lamartiniere et al., 2002).

The polyphenols are also well marked for their diabetes controlling ability. A lot of plants

have been used for the treatment of diabetes. The active constituents from the plants including

phenolics and flavonoids are responsible for their antidiabetic activity (Jung et al. 2006).

Mention may be made of Sarcopoterium spinosum whose roots contains catechin and

epicatechin and possess antidiabetic potential. Menezes et al. (2007) reported that the aqueous

extracts of Bauhinia forficate and Bauhinia monandra exhibit hypoglycemic activity. This

activity was largely due to the presence of glucosyl flavonoids as an active constituent.

Similar antidiabetic effects have also been observed in plnats like Gymnema sylvestre, Ricinus


47

communis, Swertia punicea, Combretum micranthum, Parinari exelsa, Elephantopus scaber

and lots more (Rao et al., 2010). It has been reported by Ong et al., (2012) that chlorogenic

acid is an inhibitor of gluconeogenesis and thus has a beneficial effect. It has been found by

Liu et al. (2000) that the polyphenol isoferulic acid inhibits gluconeogenesis and increases the

utilization of glucose by the peripheral tissue. This results in lowering of plasma glucose

concentrations in diabetic rats. It has also been found that isoferulic acid rises the levels of

GLUT4 mRNA whose translated product mediate the transport of glucose across the

membranes. It has been reported by Collins et al. (2007) that epigallactocatechin gallate, the

main constituent of green tea is an inhibitor of gluconeogenesis in primary hepatocytes.

Apart from these, polyphenols also protect from a wide array of other diseases such as the

neurodegenerative disease including the Parkinson’s disease, Alzheimer’s disease. It also

confers protection from osteoporosis (Hagiwara et al., 2011). The onset of osteoporosis is

intricately related to oxidative stress in humans. It has been suggested that oxidative stress is

associated with an increase in bone resorption and reduced bone mass in women which

ultimately leads to osteoporotic condition in women (Baek et al., 2010). It has been found

that the condition of osteoporosis can be minimised by the consumption or administration of

polyphenolic compounds. It has been reported that the flavonoid naringenin enhance the

proliferation of bone mesenchymal stem cells (Zhang et al., 2009). Luteolin effectively

decreased the differentiation of both bone marrow mono nuclear cells and Raw 264.7 cells

into osteoclasts . It also inhibits the bone resorptive capacity of differentiated osteoclasts (Kim

et al., 2011) all of which leads to the prevention of bone loss and decreased occurrence of

osteoporosis. It has been shown by Wattel et al. (2004) that quercetin decreased

osteoclastogenesis in a dose dependent manner. They also inhibit NFκB and activator protein


48

1 which are the transcription factors responsible for osteoclast development. Thus, it is

noteworthy that the polyphenols play a crucial role in prevention of various diseases in

humans. Most of these polyphenols are present in plants and need to be extracted, isolated and

purified for the benefit of the human society. In this aspect proper identification of plants are

very important. Table 6 lists some of the pharmacologically active constituents from plants

belonging to Pinus genera..

Table 6: Selected chemical constituents isolated from some Pinus genera

S.No. Name of Pinus species

Compounds reported

References

Caryophyllene oxide Thunbergol

Abi-Ayad et al., 2011

1. Pinus halepensis

α-Pinene Fekih et al., 2014 2. Pinus brutia Gallic acid, Protocatechuic acid,

Vanillin, Caffeic acid, Myrecetin, Resveratrol,Kaempferol, Catechin hydrate

Kivrak et al., 2013

3. Pinus caribaea α-Pinene, β-caryophyllene Sonibare et al., 2008 4. Pinus cembra α-Pinene Apetrei et al., 2013 5. α-pinene, β-pinene, camphene,

carvacrol, tetracosanol, hexacosanol, octacosanol, β-sitosterol and stigmasterol

Cheng et al.,2013

6. Pinus merkusii α-Pinene, β-Pinene Wiyono et al., 2006 7. Pinus nigra α-Pinene, β-Pinene Sezik et al., 2010 8. Pinus peuce α-Pinene, β-Pinene Karapandzova et al.,

2011 9. Pinus pinaster α-Pinene, z-caryophyllene Amri et al., 2013 10. Pinus maritime (Pycnogenol)-Catechin,

Taxifolin, Vanillic acid, protocatechuic acid, ferulic acid

Rohdewald, 2005

Pycnogenol and Pinus maritima:


49

The genus Pinus has been the focus of attention so far as research on antioxidants is

concerned. The genus is rich in polyphenols and flavonoids which add up to their antioxidant

property. Great emphasis has been given to the biological activities of plant extracts which are

obtained from the bark of the French maritime pine Pinus maritima. (syn Pinus pinaster).

Pycnogenol is a standardized extract composed of a mixture of flavonoids, mainly

procyanidins and phenolic acids obtained from the outer bark of Pinus pinaster. Ait. Subsp.

atlantica. and presently is a registered trademark of Horphag Research Ltd. (Gulati et al.,

2005). Jerez et al. (2005) worked on the procyanidin rich fractions from Pinus pinaster barks.

Their results suggested that the gallate free procyanidin mixtures from Pinus pinaster barks

are active free radical scavengers against ABTS and DPPH radicals. Pycnogenol possess a

wide array of beneficial activity which includes antioxidant, free radical scavenging and NO

quenching activity (Packer et al., 1999), inhibition of lipid peroxidation (Kim et al., 2000),

inhibition of proinflammatory cytokine actions (Cho et al., 2000), protection of nerve cells

against β-amyloid, or glutamate induced toxicity (Peng et al., 2002), increased antioxidant

capacity/activity in human (Devaraj et al., 2002), inhibition of histamine release from mast

cells (Sharma et. al, 2003) and lots of other health beneficiary activities.

The in-vivo antioxidant activity of procyanidin rich extracts of Pinus maritima was studied by

Busserolles et al. (2006). According to their findings the treatment of rats with the bark

extracts of Pinus maritima in higher total antioxidant capacity in plasma. Pycnogenol, which

contains flavonoids enhance endothelial nitric oxide synthase expression and release of NO

from endothelial cells helping in vasodialation (Nishioka K et al. (2007). Krizkova et al

(2008) reported that Pycnogenol effectively reduces the mutagenic activity of ofloxacin and


50

acridine orange in flagellate Euglena gracilis model.. It was found by Feng et al (2008) that

Pycnogenol inhibits intracellular replication of HIV by inducing expression of Mn-SOD. It

also inhibits binding of the virus to the host cell. It has also been reported by Choi et al (2009)

that Pycnogenol reduces histamine release from rat peritoneal mast cells triggered by anti-

DNP IgE. It also inhibited protein expression and secretion of tumor necrosis factor-alpha and

interleukin-6 in anti-DNP IgE-stimulated rat peritoneal mast cells and also suppressed nuclear

factor kappa B activation indicating the clinical importance of Pycnogenol in the mast cell

mediated immediate-type allergic diseases. Pycnogenol also exhibit antidiabetic effects in a

rat model of type 2 DM by potentiating the antioxidant defense system (Parveen et al. 2010).

Pycnogenol have also been reported to increase the levels of Cu-Zn SOD in the brains of

diabetic rats (Kolacek et al. 2010). It has been reported by Lee et al. (2010) that Pycnogenol

increased glucose uptake in fully differentiated 3T3-L1 adipocytes and increased the relative

abundance of both GLUT4 and Akt mRNAs through the PI3K pathway in a dose dependent

manner. Frontela-Saseta et al. (2011) reported that fruit juices enriched with Pycnogenol led

to high anti-proliferative effect on a colon carcinoma cell line Caco-2. Errichi et al. (2011)

reported that intake of Pycnogenol by women significantly reduced symptoms associated with

menopausal transitions. Pycnogenol is also reported to reduce the sign and symptoms of

allergic asthma (Belcaro et al. 2011). Enseleit et al. (2012) reported that Pycnogenol improves

endothelial function in patients with CAD by reducing oxidative stress. Fruit juices enriched

with Pycnogenol also confer protection against colon carcinoma cells.

Pycnogenol also counteracts toxicity in animal model system. Kim et al. (2012) found that

administration of Pycnogenol has beneficial effects in cyclophosphamide induced embryo-


51

fetal developmental toxicity in rats. The protective effects may be attributed to the fact that

Pycnogenol inhibits both lipid peroxidation and increased antioxidant activity. Mei et al

(2012) reported that Pycnogenol possess hepatoprotective property and decreased the levels of

liver triglyceride and serum alanine amino transferase. Peng et al. (2012) reported inhibitory

activity of Pycnogenol on COX-2 and iNOS and indicated anti-inflammatory and anti-

arthritic activity of the product. Studies have shown that Pycnogenol improves endothelial

function in patients with coronary artery disease. It reduces oxidative stress by reducing the

level of isoprostanes (Enseleit et al., 2012). It has also been reported that Pycnogenol is

effective for the treatment of oral mucositis in children (Khurana et al., 2013). Studies also

reveal that daily intake of Pycnogenol reduces reactive oxygen species in the plasma of

healthy smokers conferring protection from the deleterious effects of free radicals (Belcaro et

al., 2013). Pycnogenol also reportedly confers protection against allergic asthma and can be

used as a curative agent for the treatment of the same (Shin et al., 2013). Traumatic brain

injury is the condition which involves primary and secondary injury cascades and delayed

neuronal dysfunction and death. It has been observed that Pycnogenol is beneficial for the

treatment of traumatic brain injury (Scheff et al., 2013).

Flavangenol: Flavangenol is another product obtained from the bark of French maritime

pine. The product is rich in procyanidin B1 and has shown to possess a wide array of

beneficial activities. It has been shown by Kimura et al. (2010) that flavangenol significantly

inhibited increases in skin thickness, and the formation of wrinkles and melanin granules, as

well as increase diameter and length of skin blood vessels. It is also reported to prevent

increase in the number of apoptotic, Ki-67-positive and 8-hydroxy-2'-deoxyguanosine

positive cells, and the expression of skin vascular endothelial growth factor induced by


52

chronic UVB irradiation. Shimada et al. (2011) reported the beneficial effect of flavangenol

on collagen-induced arthritis in rats by inhibiting the acute and chronic inflammatory

reactions. Sugaya et al (2011) reported that flavangenol is a potent antioxidant and exerts anti-

atherosclerotic action. According to Ohkita et al. (2011) flavangenol has a strong antioxidant

effect and exerts ameliorative effects on cardiovascular, skin, cognitive, and menstrual

disorders, as well as in the context of other diseases and disease processes such as diabetes

and inflammation. Shimada et al. (2011) reported that flavangenol had a suppressive effect on

increase in body weight and accumulation of visceral and subcutaneous fat. The same group

also reported (2012) antioxidant properties anti-obesity effect of flavangenol. According to

their findings, flavangenol significantly the mRNA expression levels of fatty acid oxidative

enzymes (peroxisomal proliferator-activated receptor α, acyl-CoA oxidase, carnitine

palmitoyl transferase) and suppressed intracellular fat accumulation.

Thus, it is evident from the literature reports that Pinus genus has a wide array of chemical

constituents and most of them contribute to medicinal property. Thus in our present study an

attempt has been made to identify some of the chemical constituents present in this genus and

also explore its antioxidant potential.

Studies related to genetic diversity of Pinus

Random amplified polymorphic DNA is a molecular PCR based technique used to evaluate

the genetic diversity of plants where DNA sequences are amplified randomly (Senthilkumar

et al., 2011). This technique finds its application for genetic diversity analysis for both plants

and animal species. The technique has been applied in order to evaluate the genotypic

variations in the genus Pinus. Kurt et al., (2011) worked on the genotypic variations among


53

the populations of Pinus brutia using RAPD markers in order to find a better conservational

and propagative approach. The genetic variability of Pinus gerardiana was also worked out

by Kant et al., (2006). Their results indicated high degree of variability among the samples

studied indicating cross pollination as the possible mode of reproduction. The genetic

variations of Pinus nigra was studied by Rubio-Moraga et al., (2012) using simple sequence

repeats. Microsatellite gene based markers were also used to assess the genetic linkage maps

of Pinus pinaster (De Miguel et al., 2012). Thus based on the mentioned study, it is clear that

the species of Pinus have a high degree of intraspecific variability. Thus in our study, the

genetic variability among the different populations of Pinus roxburghii has been studied

which might give a better preservation and propagations of a plant as it is supposed to have a

strong antioxidant potential and needs a better conservation if it is bio prospected in future as

a source of antioxidant for management of free radical induced disorders.

Popularity of natural antioxidants:

Presently thrust is given on the exploration of natural antioxidants and also augmentation of

natural antioxidants within food and as dietary supplement. The natural antioxidants have

proven to be more effective than their artificial counterparts because they hardly have any

side effects upon intake. Moreover they are widely available in nature, the major source being

the plants. Thus the sentiment of common people is gradually getting focussed towards nature

and its resources. Instead of relying on the artificial compounds as a source of their

antioxidant they are presently getting more inclined towards natures’ wealth. This concept is

gradually getting transmitted throughout the length and breadth of the globe and industries are

cropping up to meet the growing demand of natural antioxidants. Presently apart from direct


54

consumption of natural antioxidant as food or food supplement, its mode of action is

exploited for the storage of food and increase the shelf life.

The antioxidants increase the shelf life of foods by a variety of ways. Antioxidants can delay

or inhibit the first steps of the oxidation process as a primary antioxidant, or slow down the

rate of oxidation as secondary antioxidants. Some antioxidants such as ascorbates increase the

shelf life of the food by removing oxygen, itself getting oxidised. Others like tocopherols

interfere with the process of oxidation. Antioxidant activities of some compounds are further

enhanced by the chelating agents such as citric acid and lecithin. Presently the natural food

market including sensory and textural food additives and functional ingredients have a total

valuation of 4 billion dollars and is growing at a significant rate of 6-7 percent in the year

2012. This increase is largely due to an increase in health conscious consumers throughout the

globe.

According to Food and Marketing Institute’s 2011 survey titled “ Shopping for Health”

antioxidants are ranked among the top five health components the citizens of United states

want in their daily food uptake. According to the survey by Mintel, that new antioxidant

launched for both food and food supplement increased by 10% between 2010 and 2011. It has

been reported by Packaged Facts, Rockville, MD that there has been a significant increase in

the launch of food products which claim of having antioxidant activity.

The popularity of antioxidants has not only made an impact on human mindset but also they

are rapidly trying to introduce antioxidants in the animal feed. According to the report of

Transparency Market Research animal feed antioxidant demand was over USD 162.0 million

in 2011 and is likely to reach 216.8 million USD by the year 2018.


55

A group of carotenoids which has gained prominence in global market are the carotenoids.

The global market of carotenoids had a total valuation of 766 million US dollars in the year

2007 and is expected to reach 919 million dollars in the year 2015 with a compound annual

growth rate of 2.3%. Amongst the carotenoids, β-carotene has the largest share of market with

a total value of 247 million US dollar in the year 2007 and is likely to reach 285 million US

dollars by the year 2015 with a compound annual growth rate of 1.8% (BCC Market Research

Report,2011,Code FOD025D). The global carotenoid market can reach US$1.3 Billion by

2017 (Yakob et al., 2014). It has also been reported that United States and Europe dominate

the global carotenoid markets. Apart from consumption of carotenoids for health benefits

some companies have also explored the use carotenoids as colouring agents.

Green tea is currently emerging as a very popular health drink across the globe. Green tea is

prepared using the leaves of Camellia sinensis undergoing minimum oxidation process.

Presently green tea is the fastest growing segment in overall tea industry due to its health

benefits. China is the largest producer and exporter of green tea and over the entire Asia-

Pacific region leads the green tea market globally. The green tea has got numerous health

benefits including fat reduction. This has resulted in the growing demands of green tea

throughout the world. Sri Lanka is one of the fore runnerin green tea production in the world

(Basu Majumder et al., 2010). Grand View Research, Inc. reported that the global market for

tea polyphenols is likely to reach a total valuation of USD 367.7 million by the year 2020. It

has also been reported that the green tea polyphenols is the leading product segment that is

consumed worldwide and accounts for the 70% of the total market globally.


56

Presently a lot of research is in progress in relation to the plants and its antioxidant properties.

However not much has been done in the group of gymnosperm in the Indian subcontinent.

Thus, in this study Pinus roxburghii Sarg. has been undertaken as a model to explore its

antioxidant potential. This is based on the extensive literature survey which indicates that the

plant and its related species are extensively used by the people as folk medicine and for

therapeutic purpose.

review of literature - inflibnetshodhganga.inflibnet.ac.in/bitstream/10603/44794/9/review of... ·...

Documents