Medicinal plants has a potential source of therapeutic aid has attended a significant role

in health system all over the world for both human and animals not only in the diseased
condition but also has potential material for maintaining proper health. Plants have
been used for centuries in traditional medicine as they contain components of
therapeutic values. according to World Health Organization (WHO) more than 80% of
the world's population relies on traditional medicines for health care needs plants are
natural source of antimicrobial agents. They contain a wide range of metabolites that
can be extracted fromthem and used to treat infectious and chronic diseases.

The total population of world utilizes plants as drugs mainly in the developing
countries. There are more than 85,000 plant species that have been documented for
medicinal use globally. So plant derived natural products hold great promise for
discovery and development of new pharmaceuticals in diverse human ailments. The
WHO estimates the therapeutic use of natural products was perhaps the oldest medical
practices. Interestingly, 64% of that almost 75% of the world population had
therapeutic experience with herbal remedies more than 6000plants in India including
endemic are in use in traditional folk and herbal medicine.

World health organization has listed over 21000 plant species used around the
world for medicinal purpose. In India, about 2500 plant species are being used
indigenous system of medicine. The red data book lists 427 Indian medicinal plant
entries on endangered species, of which 28 are considered extinct, 124 endangered, 81
are rare and 34 in sufficiently known (Sreenivasulu, 2015).

Among the medicinal plants that are threatened and endemic, Boswellia serrata
and Boswellia ovalifoliolata are very prominent and valuable. These two plants have
been taken up for the present study.Chetty et al., 2002) reported that both B.
serrata(threatened) and B. ovalifoliolata (endemic and endangered) occur at the
foothills of Seshachalam hill ranges of Eastern Ghats of Andhra Pradesh.

2.1 Origin and distribution of Boswellia serrata Roxb.and Boswellia

ovalifoliolata Bal. & Henry
The genus Boswellia belongs to the Burseraceae family and is widely
distributed in the dry regions of tropical Africa, Arabia and India. In Africa it is

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distributed in Somalia, Ethiopia, Eritrea, Kenya, Sudan, Tanzania, Madagascar and
some other countries. In Arabia, it is mainly restricted to Yemen, Oman and Socotra. In
India, it is distributed in a few regions such as Rajasthan, South East Punjab, Danwara,
Madras, etc.

There are about 18 species of Boswellia which are shrubs or trees with outer
bark often flaking. they include B. sacra, B. frereana, B. neglecta, B. microphylla, B.
papyrifera, B. ogadensis, B. pirottae, B. rivae, B. madagascariensis, B. socotrana, B.
popoviana, B. nana, B. ameera, B. bullata, B. dioscoridis, B. elongata, B. serrata and
B. ovalifoliolata. Only the last two species have been reported to be distributed in India
(Arabia, 2005; Latheef et al., 2008). Sunnichan et al. (2005) mentioned that B. serrata
is the only species found in India. But other workers reported the B. ovalifoliolata
occurs onthe foothills of Seshachalam hill ranges of Eastern Ghats in Chittoor, Kadapa
and Kurnool Districts of Andhra Pradesh up to an altitude of about 600-900 m.

Boswellia serrata is a moderate sized frankincence oleo gum resin is obtained

by incision of the bark of the several species of Boswellia belongs to Burseraceae. It is
known as olibanum and salai guggul in Ayurvedic medicine (Wallis, 1967; Evans,
1996). The plant is native to India, Arabian peninsular (Yemen, Sultanate of Oman),
Red sea region of North- East Africa (Somalia, Eritrea). B. serrata Roxb. is a deciduous
middle sized tree which is mostly concentrated in tropical parts of Asia and Africa and
widely grows in dry hilly forests of India especially, Rajasthan, Madhya Pradesh,
Gujarat, Bihar, Assam, Orissa as well as central peninsular regions of Andhra Pradesh
and Assam. The generic name Boswellia is given after Dr. James Boswell of Edinbergh
botanical garden and friend of William Roxburgh, Director of Indian botanical garden,
Calcutta. The specific name, serrata comes from serra (a saw) referring to the toothed
leaf margins.

2.2 Threatened status of Boswellia serrata and Boswellia

ovalifoliolata
Boswellia serrata has been listed in the threatened species of India (Sharma,
1983). Indiscriminate exploitation for its high economic value, the tree has been
identified by the government of India for its genetic improvement. There are several

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constrains in the cultivation of Boswellia serrata under natural conditions. Seed
viability is very poor and germination percentage is very low (10-12%). the seeds
require high humid conditions for germination. conventional method of propagation of
Boswellia serrata is by stem and root cuttings. These methods are season dependent
and time consuming.

B. ovalifoliolata is globally endangered strict endemic medium sized deciduous

medicinally important tree species and listed in CITES Red Data book under medicinal
plants (Rani and Pullaiah 2002; Reddy et al., 2002).

2.3 Vegetative morphology of Boswellia serrata

Boswellia serrata is a medium to large sized deciduous tree up to 18 m in height
and 2.4 m in the grith (normally 1.5 m) the bark of this plant is thin, greenish grey,
yellow or reddish and finally turning to ash colour, peeling off in smooth, exfoliating
papery flakes, blaze pinkish and exuding small drops of resin (Saxena and Brahmam,
1994). The leaves alternate imparipinnate, 30-45 cm long, exstipulate and crowded at
the end of the branches. The leaflets are 2.5-6.3 ×1.2× 30 cm, ovate or ovate lanceolate,
8-15 in number, nearly sessile with short toothed mostly pubescent. The flowers are
bisexual, small white in axillary racemes or panicles at the tip of the branches. The
calyx is small cupular and 5-6 lobed. The petals are 0.5- 0.8 cm oblong - ovate with
basal disk. (Dymock et al., 1972; Anonymous,1988). The fruits are cotyledonous,
trifed, 1.25 cm long, trigonous, splitting in to three valves. Seeds are heart shaped and
attached to the inner angle of the fruit, compressed, pendulous.

2.4 Vegetative morphology of Boswellia ovalifoliolata

Boswellia ovalifoliolata is a deciduous medium sized tree. Leaves are
imparipinnate, alternate or crowded at the ends of branches, leaflets sessile, ovate-
oblong unequal at base, glabrous beneath, veins reddish. The leaves are generally
alternate, spiral and odd pinnately compound with opposite, frequently long petiolate
entire to serrate, pinnately veined leaflets whose symmetry is distinctive in some
genera. Some members are known to have trifoliate (or) unifoliate leaves. The leaf and
leaflet stalks and axis may be brown and scurfy, while the leaf base is swollen and
maybe concave adaxially. The family members tend to be without stipules. The flowers
may have 4-5 faintly connate but imbricate sepals with an equal number of distinct,

14
imbricate petals, the stamens may contain nectar discs, have distinct glabrous filaments
that come in 1-2 whorls and in numbers equal lying or twice the number of petals . The
pollen is contained within two locules of the anthers that open longitudinaliy along
slits. The gynoecium contains 3-5 connate carpels, one style, one stigma that is head
like to be lobed. Flowers in axillary panicles. Calyx is 5-toothed. Petals are 5 and
imbricate.

2.5 Economic importance of Boswellia serrata and Boswellia

ovalifoliolata
Boswellia serrata commonly known as salai is a commercially important tree
which exudes volatile oil, resin and gum. The oil obtained from salai is very similar to
turpentine oil. The oil has been found suitable for paint making, varnish, lacquers and
used as printing ink in textile industries and in making distempers. It has been reported
as a unique non coniferous source of turpentine employed for the manufacture of paints
and varnishes.The resin obtained from salai is used for preparation of varnishes. The
tree exudes of B. serrata resembles Canada balsam in colour and consistency. B.
serrata is also a timber yielding tree. The timber which is of sap wood is soft and
moderately value. Bark is used against diahhroea,, dysentery, pulmonary and cutaneous
troubles and also used in incense (Wealth of India, 1988).The extract of this plant is
used for rheumatic pains by folklore and wood of this species is specifically used in toy
making industry besides its commercial importance as aromatic resin yielding plant.
Most commonly it is used in rheumatism and urinary disorders (Nadkarni, 1954). In
recent years, the gum resin has been used extensively in pharmaceutical formulations
for relieving aches and pain, particularly associated with arthritis (Singh and Atal,
1986; Chikamai, 2002). It is also reported to be useful in the treatment of bronchitis,
asthma, cough, bad throat and various intestinal problems. It has diaphoretic and
astringent properties and is prescribed in various syphilitic and pulmonary diseases. It
acts as both internal and external stimulant, expectorant, diuretic and for treating
stomach ache. The gum is also prescribed in cases of jaundice, diahhroea, dysentery,
dyspepsia and haemorrhoids.

B. ovalifoliolata gum has been used as an alternative source of incense of B.

serrata gum, by the tribal people inhabiting in and around the forest areas of

15
Seshachalam and Nallamala forest region (Ahmedullah and Nayar, 1986). It has been
used in traditional medicine to cure different ailments like, stomach ache, rheumatic
pains, ulcers, amoebic dysentery and as immunostimulant by adivasi tribes of
Seshachalam hills (Savitramma and Sulochana, 1998). The fresh leaf juice of B.
ovalifoliolata is used to prevent throat ulcers (Savithramma and Sulochana, 1998).
Decoction of the stem bark 10 - 25 ml per day reduces rheumatic pains (Nagaraju and
Rao, 1990). The gum obtained from the trunk is highly medicated and is sold in the
local market by the native Tribals as Konda sambrani.

2.6 Chemical composition of Boswellia serrata and Boswellia

ovalifoliolata
Gum resin of B. serrata is reported to contain monoterpenes, diterpenes and
triterpenes (Gayathri et al., 2007). Boswellic acids are considered to be the ingredients
responsible for inflammatory activity, since these compounds inhibit luekotriene
biosynthesis by impairing the lipoxygenase activity (Wildfeuer et al., 1998; Safayhi et
al., 2000). Moreover, 12-ursene-2-di-ketone which was isolated from this crude extract
can block specific cellular targets that are responsible for dopaminergic and cholinergic
effects (Gayathri et al., 2007; Shah et al., 2007). There are numerous active constituents
in the gum resin of B. serrata. It contains the fatty acids palmitic, stearic, oleic, and
linoleic. It also contains the sugars glucose, arabinose, rhamnose, galactose, fructose,
glucuronic acid, and idose. The gum resin also contains triterpene alcohol, serratol, and
triterpenoids. The volatile oil contains p-cymene, d-limonene, terpinolene, bornyl
acetates, and methyl chavicol.

2.7 Medicinal uses of Boswellia serrata and Boswellia ovalifoliolata

The oleo gum resin of B. serrata is used in various Unani and Ayurvedic
preparations. It is reported to be useful in the treatment of bronchitis, asthma, cough,
bad throat and various intestinal problems. It is a diaphoretic and astringent prescribed
in various syphilitic and pulmonary diseases. It acts as both internal and external
stimulant. The gum is also prescribed in cases of jaundice, diarrhoea, dysentery,
dyspepsia and hemorrhoids. It is also recommended in weak and unhealthy kind of
ulceration (Aman and Balu, 2009). The plant also possesses potent analgesic and anti-
inflammatory activity (Sharma et al., 2010). Gum resin is used for treating chronic

16
laryngitis, fevers, urethrorrhea, jaundice, diaphoresis, convulsions and ring worm. It
possesses diuretic, expectorant, anti inflammatory and antiseptic.The bark is used for
diahhroea, jaundice, pita diseases and considered as a tonic, sweet and cooling.

The fresh leaf juice of B. ovalfoliolata used to prevent throat ulcers

(Savithramma and Sulochana, 1998). Decoction of the stem bark 10-25 ml per day
reduces rheumatic pains (Nagaraju and Rao, 1990). The gum obtained from the trunk
which is highly medicated. Gum is used to cure amoebic dysentery (Sudhakar, 1998).
In recent years, the gum resin has been used extensively in pharmaceutical formulations
for relieving aches and pain, particularly associated with arthritis (Singh and Atal,
1986; Chikamai, 2002). It is also reported to be useful in the treatment of brnchitis,
asthma, cough, bad throat and various intestinal problems. It has diaphoretic and
astringent properties and is prescribed in various syphilitic and pulmonary diseases. It
acts as both internal and external stimulant, expectorant, diuretic and for treating
stomach ache. The gum is also prescribed in cases of jaundice, diahhroea, dysentery,
dyspepsia and haemorrhoids. Boswellia serrata gum resin is used for treating chronic
laryngitis, fevers, urethrorrhea, jaundice, diaphoresis, convulsions and ringworm. It is
possesses diuretic, expectorant, anti-inflammatory and antiseptic. The bark is used for
diarrhea, jaundice, Pita diseases, and considered as a tonic, sweet and cooling. In Indian
Ayurvedic systems, it is known among herbalists as a treatment for arthritis, very
beneficial in helping inflammation and joint health. Extracts of Boswellia serrata have
natural anti-inflammatory activity at sites where chronic inflammation is present by
switching off pro-inflammatory cytokines and mediators, which initiate the process.
Analysis of the ingredients of these extracts revealed that the pentacyclic triterpenes
boswellic acids possess biological activities and appear to be responsible for the
respective pharmacological actions. Boswella serrata safety is approved, it does not
have any side-effect on blood pressure, heart rate of the gastric irritation and ulcers
associated with many anti-inflammatory and antiarthritic drugs present in the market
today. In India, it is considered the best for osteoarthritis, rheumatoid arthritis, soft
tissue rheumatism, low back pain, myositis and fibrositis.

Olibanum
Olibanum is a natural ole gum resin that exudes from tappings in the bark of
Boswellia trees. Frankincense is a complex mixture composed of about 5-9% highly

17
aromatic essential oil (mono and sesquiterpenes) 65- 85% alcohol- soluble resins
(diterpenes, triterpenes) and the remaining water soluble gums (polysaccharides)
(Tucker, 1986, Khan and Farooqui, 1991) mono and sesquiterpenes are highly volatile
compounds, diterpenes exhibit low volatility, triterpenes very low volatility and poly
saccharides are not volatile. The constituents of oil are α- pinenedipentene,
phellandrene, cadinene, camphene, p-cymene, α- borneol, verbenone and verbenol.

2.8 Micropropagation studies in Boswellia serrata and Boswellia

ovalifoliolata
B. serrata is an endangered, dry deciduous, moderate branched species. The rate
of natural propagation of B. serrata is too slow where older trees get propagated
through root suckers. B. ovalifoliolata is an endemic species. It is a medium sized tree
and differs morphologically from the widely occurring B. serrata (Balkrishnan and
Henry, 1961; Gamble, 1967). Tissue culture technique is one of the efficient tool to
achieve the goals of conservation of rare, endangered and threatened plants (Ahire et
al., 2011). In vitro culture of medicinal plants have the ability to synthesize the
chemicals de novo under controlled conditions and can be utilized to produce the
medicinally important compounds similar to that of mother plants. Purohit et al.,
(1995); Suthar et al., (2011) reported on in vitro propagation of B. serrata by using
cotyledonary nodal segments and Ghorpade et al., (2010) reported in vitro zygotic
embryo germination studies in B. serrata. Nikam et al., (2012) reported on
micropropagation and non- steroidal anti inflammatory and arthritic agent boswellic
acid production in callus cultures of B. serrata. Chandrashekar et al., (2005) reported
on in vitro micropropagation of B. ovalifoliolata.

2.9Need for in vitro culture

The rate of seed germination in B. serrata and B. ovalifoliolata is very low and
in vitro zygotic embryo culture plays an important role in in vitro propagation of
endangered forest tree species to overcome physical biotic interference (Raghavan,
2003; Rambabu et al., 2006). Further, there is a lack of suitable vegetative propagation
methods for multiplication of the plant. Hence, a micropropagation of B. serrata was
attempted by Purohit et al., (1995), wherein plant regeneration was achieved with a
long duration of organogenesis and the plantlets showed high rate of mortality during

18
their hardening, acclimatization and soil establishment phases. Hence, there is an
essential need to develop a suitable micropropagation protocol for efficient and high
frequency mass multiplication of B. serrata and B. ovalifoliolata

2.10 Review on phytochemical analysis of B. serrata and B.

ovalifoliolata
Several workers reported on Preliminary phytochemical analysis of the
ethanolic gum extract of B. ovalifoliolata was carried out by Geetha and Sereu, (2013).
Aman et al., 2010 reported anti microbial and phytochemical screening of B. serrata.
Phytochemical evaluation and anti inflammatory activity of B. ovalifoliolata was
reported by Selvan et al., (2013). Singh et al (2012) reported the presence of alkaloids,
carbohydrates, phytosterols, terpenoids, phenolic compounds, flavonoids and tannins.
Salai guggal contains essential oil, gum and resin. Its essential oil is a mixture of
monoterpenes, diterpenes and sesquiterpenes. In addition phenolic compounds and a
diterpene alcohol (serratol) is also found in essential oil. Ganzera et al., (2001); Tawab
et al., (2001); Schweizer et al., (2000); Krohn et al., (2001); Rajendra et al., (2010).
Several workers reported on HPLC analysis of B. serrata. This method provides
differentiation and standardization of gum resin of different origin and gum resin
phytopharmaceuticals. Kumar and Saxena, (1979) reported on TLC of essential oil
from B. serrata leaves.

Wahab et al., (1987) reported on extract of Salai guggal. Pearson and

Singh,(1918) reported α and β-pinene as the main constituents of oil. Winterstein et al.,
(1932) reported separation of essential oil, resin and gum. Girgune et al., (1979) have
reported the presence of α-thujene (50%), α-pinene (6.2%), d-limonene (4.5%), p-
cymene (14%), cadinene 4%, geraniol (0.8%) and elemol1.3% as the main constituents
of the essential oil. Gupta et al., (1984) developed the non-aqueous titrimetric method
for the estimation of total triterpene acids present in different forms of B. serrata on the
basis of β-boswellic acid which constitutes more than 30% of the total triterpene acids..

Pozharitskaya et al., (2006) reported on separation and quantification of

terpinoids of B. serrata by planar chromatography techniques (TLC and AMD).
Column chromatography experiments were reported in ethanolic extract of oleo gum
resin of B. ovalifoliolata by Chib et al., (2013). Hamm et al., (2005) reported on

19
chemical investigation by headspace SPME and GC-MS of volatile and semi volatile
terpenes in various olibanum samples. Chemical composition and biological activities
of essential oils from the oleogum resins of three endemic Soqotraen Boswellia species
was reported by Ali et al., (2008).

2.11Molecular Genetic studies of B. serrata and B. ovalifoliolata

The use of highly discriminatory methods for the identification and
characterization of genotypes is essential for plant protection and appropriate use.
Among the polymerase chain reaction (PCR)- based molecular techniques, random
amplified polymorphic DNA (RAPD) is convenient in performance and does not
require any information about the DNA sequence to be amplified (Weder, 2002). Due
to its procedural simplicity, the use of RAPD as molecular markers for taxonomic and
systematic analysis of plants (Bartish et al., 2000). Recently, RAPD has been used for
the estimation of genetic diversity in various endangered plant species (Wang et al.,
2005). Sharma and Purohit (2011) developed a DNA isolation protocol from
polysaccharide rich leaves of Boswellia serrata.

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