Medicinal living organisms, including human beings. These plants are

Medicinal plants can be defined as herbaceous plants which
are used as such or a part thereof, in its natural form or pharmacologically
processed to protect from diseases, maintain health or cure ailments. In one
form or another, they benefit virtually everyone on Earth through nutrition,
toiletry, bodily care, incense and ritual healing (Medicinal and Aromatic Plant
Working Group, 2010).

 Medicinal plants can
also be described as fallow and/or cultivated plants that can be directly or
indirectly used for medical purpose based on the fact that these  plants contain  so 
called  active  ingredients 
(active  principles  or 
biologically active 
principles)  that  affect 
physiological  (metabolic)  processes 
of living organisms, including human beings.  These plants are composed of dynamic chemical ingredients
in their stem, root, leaf, bark, fruits and seeds that produces a physiological
response that ensures treatment of various human and animal diseases. In the
body tissues, these chemicals function together in harmony to produce instant
healing effects in the body of the diseased (Hamayun et al., 2006).

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Aromatic plants consist of fragrant volatile constituents that
exist in the form of essential oils, gum exudates, balsam and oleoresin in one
or more parts i.e. namely, root, wood, bark, stem, foliage, flower and fruit.
The specific fragrance results from the multitude of complex chemical compounds
present in the plant body. Moreover, aromatic plants produce essential fragrant
oils that are oily in nature and characterize the essence or the active
constituents of the plants. They are termed as volatile or ethereal oils owing
to their property of evaporating when exposed to air at ordinary temperatures
(Joy et al., 2001)

Since antiquity plants have served as a source of medicinal
compounds and been playing an overriding role in maintaining the human health
ever since. The World Health Organization estimates that 80% of world’s
population uses traditional therapies that involves folk medicines composed of
plant extracts of their active constituents. Over half of the modern medical preparations
are of plant derivatives (Kirbag et al., 2009).

 Many medicinal plants
contain phytochemicals like vitamins (A, C, E and K), carotenoids, terpenoids,
flavonoids, polyphenols, alkaloids, tannins, saponins, pigments, enzymes and
minerals that possess antimicrobial and antioxidant activity that helps in
healing wounds and  different ailments (Madhuri
and Pandey, 2009 ). They hold special immunomodulatory and antioxidant
characteristics that induces antibacterial affects as well as stimulates both
non-specific and specific immunity in the body which be of great significance
in therapeutic treatments. (Pandey and Chowdhry, 2006).


1.3 Pre-extraction preparation and Extraction Techniques
of Medicinal and Aromatic Plants

The qualitative and quantitative investigations of bioactive
mixes from plant materials for the most part depend on the determination of
extraction technique (Smith, 2003; Sasidharan et al., 2011). But the extraction techniques needs to be employed
after proper sample preparation from the medicinal plant species.

1.3.1 Pre-extraction preparation of plant samples

In order to keep the biomolecules in the plants intact the
first step is the preparation of plant samples. Plants samples include leaves,
barks, roots, fruits and flowers which can be obtained from both fresh and
dried plant materials. Grinding and drying are essential pre-preparation
techniques that ensures the preservation of phytochemicals in the final
extracts (Azwanida, 2015). The
pre-extraction sample preparation techniques will be discussed below in detail.

a)  Fresh
vs. dried samples

In medicinal plant studies both fresh and dried samples can be
used but mostly dried samples are preferred keeping in view the time available
for experimental design. The ideal time interval between the harvest and
experimental procedure for fresh sample should be limited to 3 hours only in
order to maintain freshness of the plant samples since fresh samples are
fragile and more prone to deterioration as compared to dry samples (Sulaiman et al., 2011). For example, when fresh
and dried Moringa oliefera leaves were put to test, it showed no
significant difference in total phenolics but with upper flavonoids content in
dried sample (Wang et al., 2010).



b)  Grinded
vs. powdered sample

By lowering the particle size the surface contact between the
sample and the extraction solvents can be elevated. For this grinding can be
done which results in rough smaller whereas powdering the samples result in a
more homogenized and smaller particle, leading to better surface interaction
with extraction solvents. Powdering is considered to be more efficient since
interaction between the extraction solvent and the target analytes is essential
for proper extraction. For proficient extraction particle size smaller than 0.5
mm is considered to be ideal (Zhang et
al., 2013).

For reduction of particle size of the samples conventional
mortar and pestle or electric blenders and mills are commonly used (Borhan et al., 2013)

c)  Air-drying,
microwave-drying, oven-drying and freeze-drying (lyophilisation) of plants

 Air-drying can
take from 3-7 days to months and up to a year depending upon the type of sample
being dried (e.g. leaves or seed). Plant leaves with stem are tied together and
exposed to air in ambient temperature for air drying. This procedure ensures
the preservation of heat liable compounds since no plant material is projected
to high temperatures that could alter its natural composition. Although it’s an
efficient drying process, but it can be more time intensive as compared to
microwave drying or freeze drying also it’s more prone to contamination due to
varying temperature conditions(Wang et
al., 2010).

In Microwave-drying the
plant samples are subjected to electromagnetic radiation that holds both
electric and magnetic fields. The electric field sources instantaneous heating via
dipolar rotation; accompanied by placement on the electric field of the molecules
possessing a permanent or induced dipole moment (e.g. solvents or samples), and
ionic induction, that produce oscillation of the molecules which results in
collisions between molecules leading to  instant
heating of the samples concurrently (Kaufman et al., 2002).. Although microwave
drying can reduce the drying time but at times it can lead to degradation of
phytochemicals present in the plant samples (Wang et al., 2010).

In Oven-drying thermal
energy is used to eliminate moisture from the samples. This sample preparation
is thought to be one of the simplest and hasty thermal processing that can
preserve phytochemicals in the plant samples (Mediani et al., 2010).

Freeze-drying uses sublimation
process as its basic principal. It is a process when a solid is converted into
gaseous state without entering the liquid phase. Sample is frozen at -80°C to
-20°C prior to lyophilisation to solidify any liquid (e.g. solvent, moisture)
in the samples. The sample is instantly lyophilized after freezing it overnight
for 12 hours to prevent the frozen liquid in the sample from melting Mouth of
the test tube or any container holding the sample is wrapped with
needle-poked-parafilm to avoid loss of sample during the process. Freeze-drying
produced much increased levels of phenolic contents as compared to air-dying
since much of the phytochemicals are preserved during this technique. Although
efficient freeze-drying is considered to be an intensive and costly procedure
for drying as compared to regular air drying and microwave-drying. Thus, this
technique is only limited to fragile and heat sensitive materials of high value
(Wang et al., 2010).

1.3.2 Extraction Techniques of Medicinal and
Aromatic Plants

Extraction can be defined as the segregation of
pharmaceutically vigorous parts of plant using relevant solvents using standard
procedures (Handa et al., 2008). The
purpose of all extraction is to separate the soluble plant metabolites, leaving
behind the insoluble cellular marc (residue).The initial unpolished extracts
obtained using extraction procedures include composite combinations of many
plant metabolites, such as alkaloids, glycosides, phenolics, terpenoids and
flavonoids. The initial plant extracts so obtained may be readily used as
therapeutic agents as tinctures and fluid extracts while others might need
further processing (Wang et al.,
2010). The commonly employed extraction methods will be discussed in detail

a.   Maceration,
infusion, percolation

is a procedure widely adapted in medicinal plant research and is commonly used
in wine making. It involves soaking plant materials (coarse or powdered) in a closed
container with a solvent and let to stay at room temperature for a period of
minimum 3 days with frequent stirring(Handa et
al., 2008). The purpose of this process is to soften and break the plant’s
cell wall to discharge the soluble phytochemicals. After 3 days, the mixture is
pressed or strained by filtration. In this method, conduction and convention
are used for heat transfer through convection and conduction and type of
compound so obtained from the sample depends upon the type of solvent involved (Wang
et al., 2010).

Using the same principle as maceration, in Infusion and decoction the plant material is soaked in cold or boiled water with
the only difference being the time of soaking. In infusion, the maceration
period is shorter and the sample is boiled in specified volume of water (e.g.
1:4 or 1:16) for a defined time for decoction (Handa et al., 2008).

is another method that uses the same principal as maceration in which a
special equipment called percolator is used. Dried powdered samples are filled
in the percolator, accompanied by boiling water and macerated for 2 hours. The
percolation process is usually done at moderate rate (e.g. 6 drops /min) until
the extraction is completed before evaporation to get a concentrated extracts (Rathi
et al., 2006)

b.  Water

Decoction is the extraction procedure in
which the herbal or plant material is boiled to dissolve the chemicals present
in the plant material like roots, stem, shoots, leaf, rhizomes or bark. In this
process the plant material is first mashed to ensure maximum dissolution and
then boiled in water to extract oils, volatile organic compounds and other
various chemical compounds.  The boiling
water extracts (BWE) so obtained contains bioactive compounds present in the
plant material which are used for pharmacological procedures afterwards. Lower
Molecular weight compounds like steroids, terpenes, alkaloids and phenolic
compounds will together with Higher Molecular Weight substances, like
polysaccharides, be isolated by the boiling water. In Mali, traditional healers
mostly use water decoction techniques for the extraction of plant constituents
for medicinal purposes (Inngjerdingen et
al., 2004; Togola et al., 2005;
Grønhaug et al., 2008;Pham et al., 2011a).

c.    Soxhlet extraction or hot continuous extraction 

The plant material is pre-extracted by
Soxhlet with organic solvents to remove low molecular weight and lipophilic
compounds (Austarheim et al., 2012;
Inngjerdingen et al., 2012, 2013). In
Soxhlet extraction, a porous bag or thimble (made from a powerful cellulose) is
filled with finely ground which is placed in the thimble chamber of the Soxhlet
apparatus. In the bottom of the flask the extraction solvents is heated which vaporizes
into the sample thimble, resulting in condensation in the condenser and drip
back. As soon as the liquid content extends to the siphon arm the liquid
contents are emptied into the bottom flask again and the process is repeated in
order to isolate polysaccharides (Wang et
al., 2010).

d.  Microwave
assisted extraction (MAE)

For the partition of analytes from the sample matrix into
the solvent microwave energy is employed (Trusheva et al., 2007). Microwave radiation act together with dipoles of
polar and polarizable materials (e.g. solvents and sample) producing heat close
to the material surface. Here, heat is transferred through conduction. Dipole
rotation of the atoms instigated by microwave electromagnetic disturbs hydrogen
holding; upgrading the relocation of broke up particles and advances
dissolvable entrance into the network (Kaufmann et al., 2002).

Ultrasound-assisted extraction (UAE) or
sonication extraction

Ultrasounds ranging from 20 kHz to 2000 kHz are used in
UAE (Handa et al., 2008). The
mechanic impact of acoustic cavitation from the ultrasound builds the surface
contact between solvents and samples and penetrability of cell walls. When the
plant cell wall disrupts when subjected to UAE since the physical and chemical
properties of the material are transformed, promoting the discharge important
plant compounds and enhancing mass transport of the solvents into the plant
cells (Dhanani et al., 2013). This
technology can be used on both small scale and large scale as its relatively
low cost phytochemical extraction technique (Wang et al., 2010).

Accelerated solvent extraction (ASE)

Since this method used minimal amount of
solvent it’s considered to be more efficient as compared maceration and Soxhlet
extraction. In a stainless steel extraction cell the pre-prepared plant sample
is packed with inert material such as sand to prevent sample from aggregating
and block the system tubing (Dhanani et
al., 2013). In a Packed ASE cell multiple layers are formed which includes
layers of sand-sample mixture in between cellulose filter paper and sand layers.
This automated extraction technology can control temperature and pressure for
each discrete sample and takes less than 60 minutes for extraction(Tan et al., 2014)


1.4 Potentials of Medicinal plants in Pakistan

Pakistan is rich in biodiversity
of medicinal and aromatic plants owing to the varied ecological zones and diversity
of favorable climatic conditions it possess (khan et al., 2007). Out of a total of 5700 medicinal plant species
approximately 400-600 are said to be found in Pakistan alone. Until early 70’s
almost 80% of the total population of Pakistan depended on folk medicines for
the treatment of diseases, even till date 80% of rural population have faith in
traditional medication for their principal health care (Hocking et al., 1958).

According to survey reports,
about 2000 medicinal and aromatic plant species grow in Pakistan using which
approximately 40000 Hakims (herbal drug practitioners) extract and prepare
their drugs (Sher and Hussain, 2009; Sher et
al., 2005).However, due to lack of proper knowledge and techniques only a
small amount of them is harvested and 90% of medicinal herbs for medicines are
imported (Sher et al., 2004, 2010c;
Sher and Alyemeni, 2011).

 The abundance of medicinal plants is
comparatively higher in mountainous region than in plain areas .Plain areas
also produce certain medicinally essential plant species like Calotropis procera which can be used to
cure around 50 various diseases including snake sting, skin, feet ear and eye
diseases but the mode of injection needs to be carefully administered since
it’s a poisonous plant and if taken without proper technique, it might act lethal
(khan et al., 2007).

Various medicinal and aromatic
plant species are reportedly found throughout Pakistan, Himalayan Regions,
Northern Areas, Swat District, Chitral District, Murree, Galliyat, Hazara
Division and some parts of Sindh being the major hotspots.

Ecologically Pakistan is divided
into 9 zones comprising of approximately 6000 plant species. Amongst the 6000
plant species, 410 are reported to be endemic to Pakistan while about 200
species are reportedly threatened owing to various reasons. The richest region
from floral perspective is the Hindukush-Himalayan region where so far 2500
plant species have been recorded which includes 90% of all the endemic plants,
reported from Pakistan (Adnan et al.,
2006; Sher & Hussain, 2009; Sher et
al., 2004).

In the Himalayan Region, at least
70-80% population depends upon wild plant species for therapeutic purposes (Pie
et al., 1987). In Pakistan,
especially Northern region, after proper negotiation between the locals and the
end users, medicinal plants can generate a handsome income source for the
indigenous people by exporting the tradable medicinal plants on sustainable
basis (Shinwari et al., 2003).

The Hindukush-Himalayan tract of
Pakistan spreads over the Malakand and Hazara administrative divisions of the
Khyber Pakhtunkhwa. This zone hosts a diverse flora and fauna. The flora in
specific is comprised of a variety of progenitors of economically useful crops
and a multitude of medicinal and aromatic plants that holds a strong potential
of important pharmaceutical applications. Presently the medicinal and aromatic
plant species in these regions are at a serious risk and its habitats are being
altered by demographic, economic and technological changes.

Pakistan exports medicinal plants
worth 5.45 million dollars per year and is ranked among the top eight exporting
countries of medicinal and aromatic plants in the world. Over 60% of the total
exports are harvested from the Hindukush-Himalayas regions of the country
(Champion et al., 1965; Sher et al., 2005; Sher & Hussain, 2009).
These plants are exported throughout the world including Germany, USA, Middle
East, India, Iran, etc. Despite being this rich in plant diversity Pakistan
still imports medicinal and aromatic plants worth US$ 130 million, from the
above countries (Sher & Hussain, 2009).

In 2006 Shinwari et al., published a “Pictorial guide of
medicinal plants of Pakistan” in which he reported more than 500 species of
flowering plants, being used a medicine in Pakistan.

Haq and Hussein in 1993
identified 70 species of medicinal plants in Mansehra, NWFP province.
Ethnobotanical studies on Ayubia National Park, Nathiagali, and Swat have been
conducted in detail by Shinwari et al.,
in 2002 and 2003. In 2009, Ali and Qaisar found 83 taxa that were used
locally in Chitral district of Hindukush range. In 1992 Goodman and Ghafoor identified
114 species with local ethnobotanical usage from Baluchistan province. Shah in
1996 reported 171 medicinal plant species used by locals in Kharan district,

1.5 The need for conservation of economically
important plants

Currently Pakistan is facing serious problems in terms of loss
of floral richness and diversity. For commercial purposes and chemical analysis
these economically important medicinal and aromatic plants are harvested which
are threatening there their abundance, and even existence (Gilani et al., 2009).  Unsustainable harvesting and uses of different
forms and deforestation, followed by heavy grazing/browsing by domestic livestock
are the major factors behind the instant loss of floral biodiversity (Scakali,
2008). These major factors have been reported to affect about 10% of all medicinal
plant species found in Pakistan. (Larsen & Smith, 2004; Hameed et al., 2011).

Studies reveal that commercial collectors use non selective
harvesting techniques which leads to alterations in population size and
structure of plant diversity. Another reason leading to endangerment of these
economically important plant species are lack of appropriate knowledge about
the part used and time of harvesting. The proper time of harvesting, plant age,
and the part to be collected, ensures the concentration of therapeutically
active biochemical ingredients and their yield. Secondly, mismanagement and
minimum profitable exploitation not only by local community but also by
visiting collectors results due to lack of knowledge concerning economic value
of medicinal and aromatic plants (Sher et
al., 2010a; 2010b).

To protect the biodiversity of plant species, sustainable
use of plant resources needs to be ensured. Although plants are renewable
resources, we still need to adopt proper harvesting practices in order protect
and conserve the biological diversity, and also meet the demands for treatment
of severe ailments.

For this purpose an area in the Changla Gali area was
declared as Medicinal and Aromatic Plants conservation Area in order to
conserve the rich diversity present in the area. But a few problems prevailed
in the area which inhibited the growth of these medicinally important plants.

 The local community
selected for present study were found to have little knowledge about the
uses,trade, cultivation and ecology of MAPs. Medicinal and aromatic plants were
been collected and sold in the local market by few local collectors.
Conservation of natural bio-resources and ecosystem providing habitat to medicinal
and aromatic plants was reported to be under constant pressure due to human
population flux, overgrazing, over harvesting of such plants and terrace
cultivation in the forest area. Hence fencing of the conservation area was
thought to be a wise solution to the problem of over harvesting.

The MAPs in the research area were at a severe risk due to
overgrazing, deforestation, and large quantities of fuel and fodder collection.
Moreover, over-harvesting of these MAPs by local collectors to be sold in local
market at low prices would inhibit their regeneration capacity thus posing a
threat to the livelihood of these commercially important plants.

In such social and ecological constraints, conserving the
area was considered to be the need of the time in order to keep a sustainable
quantity of the plants for the coming generations also it can ensure both
Ex-Situ and In-Situ Conservation by encouraging the regeneration of plants
and protecting them from overgrazing or overharvesting.

            For this matter a total of 4 Acres
(16187.4 m2) area was fenced in June,2015 by Non Timber Forest Products (NTFP)
Directorate, Forest Department, Khyber Pukhtun Khwa which was later on expanded
to a total of  19 Acres in May,2017.

The conservation area under study is of utmost biodiversity
importance owing to the presence of commercially important MAPs (MAPs).
Although pre-conservation data is available but since this area has recently
been declared therefore no recent work regarding this area is available.

The current study is intended to generate
baseline information about:

Types and amount of medicinally and aromatically
important plant species growing in the area.

Various factors affecting the growth and decline
of these economically important plants that will help biodiversity
conversationalists for taking appropriate steps for managing the ecosystem for
both present and future generations.

To analyze the Phytosociological characteristics
along with the diversity patterns of Medicinal and Aromatic Plants in Changla
Gali Conservation area.

To conduct a comparative analysis of the
pre-post conservation of the area with special reference to Medicinal and
Aromatic Plants

To evaluate the conservation status of the
economically important Medicinal and Aromatic Plants (MAP’s).