Kingdom Saudi Medicinal Plants of Asir region as well

Kingdom of Saudi Arabia (KSA) is characterized with a wide variety of flora,
consisting of different species of herbs, shrubs and trees. It contains
different types of edible and medicinal plants. Asir province is full of
natural and ancient herbal resources that inspire ones senses with beauty and
fragrance. Here potential medicinal and aromatic plants present a genetic
diversity that must not be ignored (Leipzig, 1996). Asir region, KSA is located
at the southwest of Saudi Arabia. The hazy high ground gives an attractive
bionetwork for the coniferous trees to grow in plenty. The big cities of the
province are Khamis Mushayt, Bishah and Abha. There are total 261 plant species
found in this region out of which 165 species have medicinal importance but
there is lack of full information covering the active constituents of medicinal
plants in this region or documented medicinal uses (Yassin et al., 2013).

 

A meticulous, literature survey on Saudi 
Medicinal Plants of Asir region as well as on the quality
standardization of various other medicinal plants have been carried out from various
web portals, national and international journals, herbal database from other
published research materials. Through the literature, it has also been clear
that a large number of medicinal plants in the flora of Saudi Arabia have also
been used intensively in indigenous medicine from ancient time (Abbas et al., 1992; Alsherif et al., 2012;
Al-Sherif et al., 2013; Hegazy et al., 2014;
Mossa et al., 2000; Al-Yahya
et al., 1990;  Kuete, et al., 2013). Pharmacognostic standardization of
medicinal plants is extremely essential and crucial to maintain the identity,
quality, purity and safety of crude drugs. (Anonymous,
1998). From the documentation, it also becomes evident that
despite the extensive traditional use of these plants their standardization and
pharmacognostical constants have not been investigated. On the basis of
literature survey, Datura stramonium
was selected for its pharmacognostic quality standardization studies.

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D. stramonium is commonly known as
Jimsonweed belonging to
nightshade family. This plant was originated in
Mexico, but has now become naturalized in many
parts of the world. It is an annual, erect, branching herb and forms a bush up to 0.6 to 1.5 m
tall. The leaves of the plant are about 8 to 25 cm in length, smooth
and margin is toothed, and irregularly undulated. The lower surface is a week
green while the upper surface of the leaves is dark green in color and. The
taste of the leaves is bitter nauseating. The
white to violet colored flowers are trumpet-shape, and grow on short stems. The
egg-shaped
capsule of seed is covered with spines or bald (Stace, 1997; Henkel, 1911; Grieve, 1971).

 

All parts of D. stramonium plants contain tropane alkaloids which includes atropine, dl hyoscyamine, and hyoscine, which are known as anticholinergics. Due to ignorance
in villages, many people get hospitalized after ingestion of the plant due to
its psychoactive effects (Preissel, and Preissel, 2002; Giannini, 1986).
 D. stramonium intoxication typically produces hyperthermia, tachycardia, delirium,
bizarre behavior, and severe mydriasis with resultant
painful photophobia which takes to last many
days (Freye, 2009). The
symptoms generally start around 30 minutes to 1 hour after ingesting the herb
and last from 24 to 48 hours (Pennachio
et al., 2010). In this intoxication, physostigmine can be injected in as an antidote ( Goldfrank and  Neil, 2006).

 

In
Ayurveda, D. stramonium has been used
for long in asthma symptoms. In 18th century, the British Physician,
James Anderson, seek the knowledge of this practice and made it famous in
whole Europe (Barceloux, 2008;
Pennachio et al., 2010). The extract of D. stramonium, boiled with specific grease is
reported to cure all type of inflammations, burnings and scaldings (Maud, 1971). The native American Pueblo people once used D. stramonium  as an anaesthetic to make patients unconscious
while broken bones were fixed (Turner, 2009). The Chinese also
utilized it in the form of anesthesia during medical surgery (Nellis, 1997). Thus plant D. stramonium was
found much of ethno-medicinal importance. Therefore this article aimed
to investigate for the first time the pharmacognostic
quality parameters and DNA fingerprint of D. stramonium plants growing in Asir region, KSA.

 

MATERIALS AND METHODS

Collection and
Authentication

 

The fresh leaves of the plant Datura
stramonium were collected from the roadside area of Bani-Malik of Abha city,
KSA at the altitude of 2200m, in March, 2017.
The plant material was identified by Dr. Mahadevan Ninjaian, Senior Professor of Pharmacognosy Department, College of Pharmacy,
King Khalid University, Abha, KSA. The fresh leaves were utilized for the study
of macroscopic and microscopic characters as well as DNA fingerprinting whereas
other part of collected plant material was shade-dried and coarsely powdered by
using grinder. This coarse powder was used for physico-chemical parameters and preliminary
phytochemical investigation, as per standard methods.

 

Macroscopic and Microscopic
Studies

The macroscopy of the leaves was
done accord­ing to standard methods (Brain and Turner, 1975; Trease and
Evans, 2002). Hand
section of the fresh leaves and dried coarse powder was stained and mounted for
micro-techniques (Johansen, 1940). The photographs of
microscopy were taken with the help of inverted microscope for photo-documen­tation.

 

Physicochemical
Analysis

Physicochemical
parameters includes Moister content analysis, Foreign matter analysis, Total
ash value, Acid-insoluble ash value, Extractive values, Fluorescent analysis,  Powdered drug reaction with reagents and pH
value was performed according to standard procedures given in the literature (Harborne, 1998; Ansari, 2008)

 

Phytochemical
Analysis

100 g coarse powder
of air dried leaves of D. stramonium were packed in muslin cloth and
subjected to soxhelet extractor for continuous hot extraction with hexane, chloroform,
ethanol and distilled water for 8 hours respectively. Then extracts were
filtered and filtrate was then evaporated till dryness. Preliminary
phytochemical screening was done for the identification of various chemical
constituents in the various extracts as described by Harborne and Khandelwal (Harborne, 1998; Khandelwal, 2005)

 

DNA fingerprint

Forty nine milligram from young leaves of D.
stramonium was grinded using mortar and pestle. DNA from leaves was
isolated by using DNeasy plant mini kit (QIAGEN-USA) as described previously.
Oligo349, (5’– GGA GCC CCC T–3′) wad applied for D. stramonium identification
(Zhao et al., 1999; Mahmood et al., 2016) 49 ng of extracted
genomic DNA of the leaves, 200 ?M each dNTP, 25 mM MgCl2; 7 pmol of
Oligo349 and 0.2 ?L of Taq DNA polymerase (3U/?L) were applied  in 25 ?L of Eppendorf  PCR tubes. Thermal cycler Bio-Rad initiated
at 94°C for 5 min for denaturation, followed by forty nine cycles at 94°C for 1
minute for denaturation, 30°C for 1 minute for annealing, 72°C for 2 minutes
for extension and concluded with 72°C for 7 minutes for final extension. By
using 1.4% gel electrophoresis, ethidinium bromide for staining and wide range
molecular size all amplified bands were analyzed and recoded. No genomic DNA
template was examined in PCR amplification reactions as negative control and
PCR reaction steps was repeated three times to examine the reproducibility (Aljibouri
et al., 2013)

 

RESULTS

Macroscopic characteristics:

The macroscopic study showed that
the leaves of D.
stramonium are
simple, glabrous, triangular ovate with acuminate apex and asymmetric wedged base. These
are grayish green in color with slight faint characteristic odor and generally
5-20 cm long and 4-15 cm wide. Margin is unequal dentate
lobed and irregular serrate type. Petiole is grooved on lower side and
slightly bent. Midrib is prominent
on both upper and lower surfaces. Reticulate is pinnate
venation type and secondary veins
generally 5-7, arise on each side, leaving midrib at the angle of about 45o
(Fig. 1).

Fig. 1: Leaf
morphology of D. stramonium

 

Microscopic characteristics:

The leaf sample was studied microscopically by transverse
section through midrib. Fig.
2 shows epidermis, spongy
parenchyma, collenchyma, central non-lignified phloem, lignified xylem and
parenchymatous pericycle in midrib
region.

 

Fig. 2: Transverse section of D. stramonium fresh leaf in midrib region

 

 

Microscopy of Powdered leaf

The crude powder
of leaf was dark green in color with characteristic odor and astringent taste.
The powdered leaf of D.
stramonium
under microscopic investigation showed epidermal cells with anisocytic and
anomocytic stomata, longitudinal upper and lower palisade cells, matrix showing
Ca-oxalate crystals of cluster type, xylem
vessels pitted annular and reticulate type (Fig. 3).

 

Epidermal
Cells with Anisocytic and Anomocytic Stomata

Longitudinal
Upper and lower Palisade Cells

Matrix showing Ca-oxalate Crystals of Cluster Type
 

Xylem Vessels of annular Type

Fig.
3: Microscopical characters of D.
stramonium leaf powder

 

Phytochemical analysis:

The result of qualitative phytochemical analysis of various
extracts of the crude powder of
D. stramonium
leaf is shown in Table 1. The leaf had maximum amount of alkaloid and sterols
followed by tannin. The other phyto-constituents like glycosides and flavonoids
were present in trace amounts in the leaf while saponins were absent

 

 

 

 

 

 

Table 1: Phytochemicals
in D. stramonium
leaf extracts

Test

Test
type

Hexane
Extract

Chloroform
Extract

Ethanol
extract

Aqueous
extract

 
Sterols

1-Salkowaski
test
2-
Libermann- burchards test


+

+
+

+

+

 
Alkaloids

1-
Alkaloids 
2-Dragandorff
3-Wagners
1-Mayers




+
+

+
+
+

+
+
+

Glycosides

2-Bontrager’s
test

+

Tannins
test

1-Ferric
chloride test

+

+

Flavonoids

1-Shinoda
test

+

+

Carbohydrate

1-Fehling
test
2-Molish
test
3-Barfoed’s
test





+
+
+

+
+
+

 

Physicochemical analysis:

The
physiochemical characterizations of leaf are shown in Table 2. The moisture
content of leaf was 9.8%. The ash values were determined by two different ways viz.,
total ash and acid insoluble ash. The total ash in leaf was 15.5%, while acid insoluble
ash was 3.1%. The maximum extractive value was found in aqueous solution and minimum
was in hexane.

 

Table 2: Physicochemical
parameters of D.
stramonium leaf

S.
No.

Parameters

%
Value  (w/w*) leaf

1

Moisture
Content Analysis

9.80±0.25

2

Foreign
Matter Analysis

4.17±0.0

3

Total
Ash Value

15.5±0.31

4

Acid
Insoluble Ash Value

3.1±0.0

5

Hexane
Soluble

2.28±0.01

6

Chloroform
Soluble

2.30±0.02

7

Ethanol
Soluble

11.37±0.11

8

Aqueous
Soluble

15.30±0.19

Average of three
readings ± SEM

Fluorescence analysis:

The
fluorescent analysis under visible light and UV light by treatment of different
chemical reagents showed different color. The fluorescence characteristics of
leaf are summarized in Table 3.

 

 

 

 

 

 

 

 

 

Table
3: Fluorescence
analysis of leaves of D. stramonium

Solvent
used

Day
Light

UV
light (254nm)

UV
light (366nm)

1N
HCl

Greenish

Light
green

Blackish
green

50
% HCl

Greenish
yellow

Blackish
green

Grayish
green

50
% HNO3

Orange
brown

Dark
green

Greenish
black

50
% H2SO4

Brown

Light
green

Dark
brownish green

1N
NaOH

Reddish
brown

Dark
brown

Brown

Alcoholic
NaOH

Blackish
brown

Green

Greenish
brown

Methanol

Blackish
green

Light
orange

Dark
brown

1%
KOH

Blackish
orange

Light
green

Blackish
green

Lead
acetate

Yellowish
brown

Black

Greenish
black

 

Powdered
drug reaction with different reagent:

The dried powder of D. stramonium was reacted with different organic and inorganic
reagents and color appeared in the test-tube was noted as given in the table 5.

 

Table
5:  Powdered drug reaction with different
reagent

Treatment

Observation
(Color)

Conc.
HCl

Dark
green

Conc.
HNO3

Orange
Brown

Conc.
H2SO4

Blackish
brown

Glacial
Acetic acid

Light
greenish yellow

Iodine
solution

Dark
brown

NaOH
in Methanol

Brownish
green

 

Determination of pH drug

The pH of
1% and 10% aqueous solution of D.
stramonium dried
leaf
powder was observed using pH meter and the
results found are as follows:

 

Table
6: pH

Sample

pH

pH of 1 % solution

6.2

pH of 10 % solution

5.8

 

 

DNA fingerprinting
of D. stramonium leaf

Applying the
Oligo349, (5’– GGA GCC CCC T–3′) primer on D. stramonium plant,
generated 3 scorable bands, ranged from 730 pb to 370 pb (Fig. 4). The most
prominent bands was 730 pb followed by 370 pb while the 470 pb was found to be
the least one (Fig. 4).

 

 

 

Fig.4: Assessment of
DNA finger print of D. stramonium leaf fragments obtained with primer
sequence (5’– GGA GCC CCC T–3′); Marker: Wide Range DNA Marker

 

DISCUSSION:

Pharmacognostic
standardization is extremely essential and crucial to maintain the identity,
quality, purity and safety of crude drugs. According to World Health
Organization (Anonymous, 1998), the macroscopic and
microscopic information of a crude drug is the first step towards fixing its
identity and purity. Macroscopical and microscopic studies are reliable, simple
and cheapest in establishing the identity of source materials (Anonymous, 2000).
Macroscopic evaluation is a method of qualitative analysis based on
morphological characters of plant parts or organoleptic profiles of drugs (Thomas, 2008).  The macroscopic and microscopic characters of D. stramonium
noted in this study, serve as analytical characters of a particular plant
species which are helpful in recognizing the plant at species level and also in
prevention of adulteration or substitution. Though, an additional difficulty
takes places when the drug is in dried powder form. Even if the plant is
identified correctly, it can be misused or rather substitution can occur in
powder form. Hence it is essential to have some microscopic characteristics of
the particular plant parts in powder form also. Hence, the coarse powder of the
leaf of D.
stramonium was analyzed for some diagnostic
characters like type of stomata, crystals, xylem vessels, etc.

 

The
powder was also examined for various phytochemical constituents and this
analysis revealed more amount of alkaloids in leaf in comparison to other
phytoconstituents. The phytochemical screening of various extracts of leaf
powder reveals the presence of various classes of phytochemical which already
have shown medicinal potential in other plants
(Jasmeet et al., 2017). The
physicochemical parameters like moister content, ash values and extractive
values were also evaluated and may assist in preparing quality standard
parameters for monograph of the plant. In leaf, the extractive value was
highest in water followed by methanol. However, it indicates that polar
compounds are more. The extractive values are helpful to identify the type of phytoconstituents
present in the crude drug based on polarity of solvents (Patel and
Dhanabal, 2013). Thus, preliminary analysis of
phytochemical along with physicochemical analysis ensures purity, identity and
quality of the drug and also gives an idea about the phytoconstituents present
in D.
stramonium especially for the future studies of
pharmacological type at molecular level (Mukherjee, 2002).

 

The
fluorescent analysis helps in the qualitative evaluation of crude drugs which
can be used as a reference data for the identification of adulterants. The fluorescence
study of the drugs under UV light and visible light by treatment of different analytical
grade chemical reagents showed different color. This is attributed to the ultra
violet light which produces fluorescence in many phytoconstituents present in
the drug that do not fluoresce in daylight (Nagani, 2011).
Thus fluorescence is used for qualitative assessment of D. stramonium.
The microscopic characters, the physiochemical studies and fluorescence
analysis collectively can be helpful in the quality control of the crude drug
and are prime stem for evaluation (Chanda,
2014).

 

DNA fingerprint
is an important tool in authenticating and characterizing a particular plant
for its correct identification (Showkat et al., 2015). The earlier
reporters recommended that RAPD-PCR fingerprints should be applied to evaluate the
molecular variability among individuals with specific fingerprints (Chowda-Reddy
et al., 2012). It was also reported that the numbers of amplified bands
by using specific primer could identify the plants species precisely (Aljibouri
et al., 2013). The obtained results were in agreement with the outcome
of the number of RAPD fragments of Acer
negundo plant whereas 3 scorable bands identified
male and female plants (Zhao et al., 1999). By using the sequence
(5’CAACAATGGCTACCACCC3′) of RAPD primer for the endangered species due to
habitat loss of Pittosporum
meriocarpum plants characterized
also by three amplified bands (Thakur, 2016). Investigating the plant species using
molecular techniques play an important role in exploring their taxonomic status
Therefore, D. stramonium a medicinally important taxon growing in Abha
area, KSA could easily be identified by the random amplified polymorphic DNA
(RAPD) primer for the plant conservation and to meet the demand of herbal
plants formulations at the molecular level.

 

Pharmaognostic studies on different
plants and their parts have already been reported (Bhide et al.,
2011; Sandhya et al., 2011; Baravalia et al., 2011). D. stramonium
is reported as the traditional plant of Saudi Arabian flora (Alfarhan,
1999). Therefore this study is an addition to the wealth of knowledge of Saudi
Arabian flora of medicinal type, especially concerning its quality
standardization. As we know, fixing standards is an integral part of fixing the
quality and correct identity of a crude drug and D. stramonium is traditionally used to treat many important ailments
and illness as mentioned above hence it was important to standardize it for its
inclusion as an official drug in the national list of medicinal plants.

 

CONCLUSIONS:

It
can be stated that the pharmacognostic studies resulted in quality standards of
the plant which could be useful for checking the authenticity of D. stramonium of Saudi Arabian
geographical origin. These standards can ensure in preserving the quality of the
crude drug and can also be helpful for the preparation of a monograph as well
as contribution in the medicinal plant literature of national wealth.