Pharmacognostical and Antimicrobial Studies of the
Stem Barks of Prosopis cineraria (L) Druce
Stellaa Robertson1*and Narayanan N2
1Dept. of Pharmacognosy,
2College of Pharmacy,
Prosopis cineraria (L) Druce is one of the highly valued plant
in the Indigenous System of Medicine. Its bark is said to be a potent drug for
several ailments such as leprosy, dysentery, bronchitis, asthma, leucoderma, piles, muscular tremors, asthma, rheumatism and
inflammations. It is also known to possess anthelmintic, antibacterial,
antifungal, antiviral and anticancer activities. In
view of its medicinal importance, the present research was focused on the pharmacognostical and antimicrobial properties of stem bark
of P. cineraria by in vitro approach. Pharmacognostic
investigation of the fresh, powdered and anatomical sections of the stem barks
of P. cineraria was carried out to
determine its macroscopical and microscopical
characters along with the physico-chemical and
preliminary phytochemical analysis. The antimicrobial
activity of ethyl acetate and hydro alcoholic extracts of stem barks were
evaluated against two Gram positive (Staphylococcus aureus, Staphylococcus epidermidis), two Gram negative (Escherichia coli, Klebsiella pneumoniae) bacterial strains and two fungal strains (Aspergillus niger, Aspergillus fumigatus) by agar disc diffusion method. Minimum Inhibitory
Concentration (MIC) required for cessation of microbial growth was evaluated by
agar streak dilution method. Both the extracts showed dose dependent activity
against the microorganisms investigated. The hydroalcoholic
extract exhibited significant activity against the test organisms than the
ethyl acetate extract.
KEYWORDS: Prosopis cineraria, Antibacterial, Antifungal,
Minimum Inhibitory Concentration (MIC), Stem bark.
INTRODUCTION:
Prosopis cineraria (L) Druce
syn:
Prosopis spicigera
Linn., and Mimosa cineraria Linn1 belongs to
the family Leguminosae,
sub-family Mimosoideae
and Class Magnoliopsida – Dicotyledons,
is a large shrub up to 10m height with branches prickly, prickles curved and
compressed. The species is found throughout
Table
1: Analytical parameters of the stem bark of P. cineraria
|
S. no. |
Characteristics |
Results (%
w/w) |
|
I. |
Ash values |
|
|
1. |
Total
ash |
13.93 |
|
2. |
Water
soluble ash |
2.65 |
|
3. |
Acid
insoluble ash |
3.42 |
|
4. |
Sulphated
ash |
18.40 |
|
II. |
Extractive values |
|
|
1. |
Ethanol
soluble extractive value |
0.21 |
|
2. |
Water
soluble extractive value |
1.87 |
|
III. |
Loss on drying |
12.16 |
Table 2: Preliminary phytochemical
studies of extracts of P. cineraria
|
S. No. |
Tests |
EAPC |
HAPC |
|
1. |
Alkaloids |
+ |
+ |
|
2. |
Carbohydrates |
- |
- |
|
3. |
Glycosides |
- |
- |
|
4. |
Phenolic
compound |
+ |
+ |
|
5. |
Tannins |
- |
- |
|
6. |
Proteins
and amino acids |
- |
+ |
|
7. |
Saponins |
+ |
+ |
|
8. |
Gums
and mucilages |
- |
- |
|
9. |
Phytosterol |
- |
+ |
|
10. |
Fixed
oils and Fats |
- |
- |
|
11. |
Flavonoids |
+ |
+ |
+
= Present; - = Absent
chemical constants and preliminary phytochemical analysis of the different extracts of P. cineraria.
The study also includes the antimicrobial screening
of the extracts (ethyl acetate and hydroalcoholic
extracts) of P. cineraria which could
be useful for the development of new tools as antimicrobial agents for the
control of infectious diseases.
Fig 1 Stem bark of P.
cineraria
Plant
material:
The
plant specimens of P. cineraria were collected from the Mylapore, Thiruvallur district,
Tamil Nadu,
Macroscopic
and microscopic analysis:
Macroscopical features such as shape, size, fracture, colour, odour and taste were
carried out according to the method of Evans11. Microscopical
studies were also carried out using Nikon Labphot-2 microscope units. For
normal observations, bright field was used. For the study of crystals and
lignified cells, polarized light were employed. Since these structures have birefringent property under polarized light, they appear
bright dark background. The sections of the bark were taken through TS, TLS and
RLS with the help of rotary microtome. The average thickness of the sections
was 10-12 µm. Dewaxing of the sections was done and
the sections were stained out according to the methods outlined by Brain and
Turner12 and Johansen13.
Physico-chemical
analysis:
The
physico-chemical values such as the percentage of ash
values, loss on drying and extractive values were performed according to
official methods prescribed (Indian Pharmacopoeia)14 and the WHO
guidelines on quality control methods for medicinal plant materials (WHO/QCMMPM
guidelines)15.
Preparation of extracts:
The
stem barks were collected, shade dried and coarsely powdered by using pulvarizer. These coarse powders were then extracted with
ethyl acetate (EAPC) and 50% alcohol (HAPC) by cold percolation process to
yield the respective extracts. The extracts were reduced to a molten mass by
rotary vacuum evaporator and the respective yields of EAPC and HAPC were 0.87%
w/w and 1.16% w/w respectively.
Preliminary phytochemical
screening:
The
preliminary phytochemical screening was carried out
by using standard procedure described by Kokate16 and Harborne17.
Fig 2 T.S of bark through periderm
Antimicrobial activity study:
Bacterial
and fungal strains:
The
microbes used to determine the antimicrobial activity are two Gram positive (Staphylococcus aureus
ATCC 6538P, Staphylococcus epidermidis ATCC 155), two Gram negative (Escherichia coli ATCC 8739, Klebsiella pneumoniae
ATCC 29665) bacterial strains and two fungi (Aspergillus
Screening
of Antimicrobial Activity
The antimicrobial screening
was performed by agar diffusion method using a paper disc18,19. Nutrient agar and Saboraud’s
dextrose agar media were used for the antimicrobial screening. 1ml suspension
of the microorganisms (matched with McFarland barium sulphate
standard) was inoculated with 100ml of the
Table 3: Zone of Inhibition of stem bark of P. cineraria
|
S.
no. |
Organisms |
EAPC |
HAPC |
||||||
|
Standard (mm) |
50
µg |
100
µg |
200
µg |
Standard (mm) |
50
µg |
100
µg |
200
µg |
||
|
1. |
Staphylococcus aureus |
31 |
14 |
18 |
26 |
30 |
19 |
25 |
32 |
|
2. |
Staphylococcus epidermidis |
30 |
14 |
17 |
25 |
30 |
20 |
22 |
29 |
|
3. |
Escherichia coli |
31 |
15 |
18 |
20 |
30 |
16 |
20 |
29 |
|
4. |
Klebsiella pneumoniae |
29 |
12 |
15 |
19 |
31 |
15 |
20 |
27 |
|
5. |
Aspergillus |
32 |
12 |
19 |
21 |
28 |
14 |
16 |
18 |
|
6. |
Aspergillus fumigatus |
31 |
14 |
19 |
22 |
27 |
14 |
18 |
20 |
Fig 3 T.S of bark through Secondary phloem
Fig 4 Collapsed phloem- sclerotic bands & crystals
sterilized (autoclaved at 120˚C
for 30 min) medium (40-50˚C). The paper impregnated with the extracts (50,
100 and 200 µg/ml) was placed on the solidified medium. The plates were preincubated for 1h at room temperature and incubated at
37˚C for 24h and 48h for antibacterial and antifungal activities
respectively. Ciprofloxacin (50µg/disc) and Ketaconazole
(50µg/disc) was used as standard for antibacterial and antifungal activity
respectively.
Fig
5 TLS of Phloem- PLM view
The Minimum Inhibitory Concentration
(MIC) for the above organisms was found by agar streak dilution method20. Stock
solutions of the extracts (EAPC and HAPC) were mixed with the known quantity of
molten sterile agar media aseptically to provide the required concentrations.
About 20 ml of the media containing the extract was poured into each sterile petridish and allowed to solidify. Microorganisms were then
streaked one by one on the agar plate aseptically. After streaking, all the
plates were incubated at 37±1˚C for 24h and the plates were observed for
the growth of microorganism. The lowest concentration of the plant extract
required for inhibiting the growth was considered as the MIC of the extracts
against bacterial and fungal strains.
RESULTS:
Macroscopic
characters:
The outer surface of the stem
bark is pale grey and the inner surface is light brown. The stem bark exhibits
deeply fissured surface forming thick vertically oblong hard chunks.
Exfoliation through hard thick rectangular pieces; exposed surface is dark
brown and the taste is bitter (Fig1).
Microscopic
characters:
In transectional profile, the bark has deeper origin of periderm with wide irregular fissures. Periderm
region is wider and comprises of narrow tabular phellem
cells and equally developed inner phelloderm. Periderm is followed by very broad collapsed phloem. A
distinct cambial zone is seen between the secondary xylem and secondary phloem
(Fig2). Secondary phloem has regular radial files of sieve elements and fairly
wide, straight phloem rays. The phloem elements are rectangular and thick
walled comprising of sieve tube members, companion cells and tannin filled
phloem parenchyma (Fig3). Collapsed phloem consists of thick tangential bands
of sclerenchyma alternating with narrow bands of
collapsed phloem. When viewed under polarized light microscope, the segments of
phloem fibre are seen associated with prismatic
calcium oxalate crystals (Fig4). In TLS view of phloem, the axial parenchyma
exhibit dense vertical strands of prismatic crystals. These crystal strands are
associated with phloem parenchyma. The crystals vary from cuboidal,
rhomboidal and hexagonal morphological types. The phloem rays are mostly multiseriate, wide, high homocellular
and non-storied (Fig5). In RLS view, the periderm
exhibits wide zone of dark phellem and tannin free phelloderm. The secondary phloem shows wide horizontal
bands of phloem rays. The phloem rays consist of horizontal layers of oblong
homogenous cells with dense tannin contents. The vertical system shows phloem fibres, axial parenchyma with dense tannin accumulation
(Fig6).
Powder
microscopic observations
The bark powder is dark brown
in colour with bitter taste and odourless.
The microscopic study of powder revealed the presence of rhomboidal crystals of
calcium oxalate, libriform fibres,
thick masses of suberised phellem
cells and tannin filled parenchyma cells.
Physico-chemical studies
The percentage of total ash,
water-soluble ash, acid-insoluble ash, sulphated ash,
alcohol- water soluble extractive value and loss on drying with reference to
air dried powdered drug (Table 1).
Preliminary
phytochemical screening
The preliminary phytochemical
screening revealed the presence of alkaloids, phenolic compound, saponins and flavonoids in the EAPC extract while alkaloids, phenolic compound, saponins, flavonoids, phytosterol, proteins
and amino acids in the HAPC extract (Table 2).
Antimicrobial
activity
The
results of the antimicrobial activities of both the extracts EAPC and HAPC from
the stem bark of P. cineraria showed different
degree of activity against
the tested bacterial and fungal strains. The observed zones of inhibition and
the MIC values of each extract against the tested bacterial and fungal strains
were tabulated (Table 3 and 4).
Fig
6 RLS View
DISCUSSION:
Pharmacognostic study
In recent
years there has been a rapid increase in the standardization of selected
medicinal plants of potential therapeutic significance21,22.
Despite the modern techniques, identification of plant drugs by pharmacognostic studies is more reliable. According to
World Health Organization15 (WHO, 1998), the macroscopic and
microscopic description of a medicinal plant is the first step towards
establishing the identity and the degree of purity of such materials and should
be carried out before any tests are undertaken. Macroscopically, the stem bark
exhibits deeply fissured surface and rusty brown vertical patches. In transectional profile, deep seated first periderm and rhytidome type
sequent periderm, dense tangential cylinders of xylem
fibres, narrow, undilated, tanniniferous phloem rays and abundance of strand crystals
of prismatic type associated with the xylem fibres
are principal diagnostic characters of the bark. Further, the phloem rays
are non-storied, multiseriate and homocellular. All
these microscopic features can be employed to distinguish the bark samples of P. cineraria from its possible
adulterants/substitutes. The aforesaid characters are stable and reliable
features of the bark that are not influenced by environmental stress.
Fig 7a Antibacterial activity (Gram positive organisms)
Fig
7b Antibacterial (Gram negative organisms)
Fig
7c Antifungal activity
Fig
8a Antibacterial activity (Gram positive organisms)
Fig
8b Antibacterial activity (Gram negative organisms)
Fig
8c Antifungal activity
Table 4: MIC of stem bark of P.
cineraria
|
S.
No |
Organisms |
EAPC |
HAPC |
|
1. |
Staphylococcus aureus |
42 |
36 |
|
2. |
Staphylococcus epidermidis |
42 |
35 |
|
3. |
Escherichia coli |
41 |
39 |
|
4. |
Klebsiella pneumoniae |
44 |
40 |
|
5. |
Aspergillus |
44 |
41 |
|
6. |
Aspergillus fumigatus |
42 |
41 |
The physico-chemical evaluation of the drugs is an important
parameter in detecting adulteration or improper handling of drugs. Ash value of
a drug gives an idea of the earthy matter or the inorganic composition and
other impurties present along with the drug. The ash
values of the bark powder showed higher content of sulphated
ash followed by total ash. Extractive values are primarily useful for the
determination of exhausted or adulterated drugs. The water soluble extractive of
the bark powder was high. The preliminary phytochemical
screening of P. cineraria indicates
the presence of various secondary plant metabolites in the extracts such as alkaloids,
phenolic compound, saponins,
flavonoids, phytosterol,
proteins and amino acids that are known to possess various pharmacological effects and
may be responsible for the various actions of
P. cineraria.
Antimicrobial
activity
Infectious
diseases are a critical problem for health and they are the main cause of death
worldwide. Resistance of microbes to antibiotics and toxicities produced by
long term usage of antimicrobial compounds has initiated the search for safe
antimicrobials23. Though different types of antimicrobial agents are
available, there is an increase demand by people to use the natural products
and also researchers have identified a lot of plants with antimicrobial
activity24. Throughout the history of mankind, many infectious
diseases have been treated with plant extracts. The different concentrations
(50, 100, 150 µg/disc) of HAPC and EAPC extracts of P. cineraria were tested against Gram positive, Gram negative
bacterial strains and fungal strains. The higher concentrations of both
extracts had inhibitory effects towards the tested microorganisms. HAPC of P. cineraria stem bark (Fig7a, 7b, 7c)
was found to exhibit better inhibitory effects than EAPC (Fig8a, 8b, 8c)
against S. aureus,
S. epidermidis, E. coli and K. pneumonia but in case of
A. niger and A. fumigatus,
EAPC has shown better inhibitory effects and this effect was dose dependent.
The extracts showed antimicrobial activity were subjected to minimum inhibitory
concentration assay. In HAPC, the maximum inhibition against S. aureus
(32mm) and for both S. epidermidis and
E. coli (29mm) whereas in EAPC extract, the maximum inhibition against S. aureus
(26mm) and S. epidermidis
(29mm) at a concentration of 200 µg/ml.
The lowest MIC values were observed for HAPC (35 - 41 µg/ml) and EAPC
(41 - 44 µg/ml) against the bacteria and fungi tested.
CONCLUSION:
The plant P. cineraria is useful in traditional medicine
for the treatment of various ailments. So, it is important to standardize it
for the purpose of therapeutic use. The pharmacognostic
constants for the stem bark of this plant and the microscopic diagnostic features
reported in this work could be useful for the compilation of a suitable
monograph for its proper identification. The study of antimicrobial activity
supports the traditional usage of the plant P.
cineraria and suggests that the plant extracts possess compounds with
antimicrobial and antifungal properties which can be used as antimicrobial
agents in new drugs for the therapy of infectious diseases caused by pathogens.
The most active extracts can be subjected to isolation of the therapeutic
antimicrobials and carry out further pharmacological evaluation.
ACKNOWLEDGEMENT:
The
authors would like to thank Prof. P. Jayaraman,
Director of Plant Anatomy Research Centre for providing technical support
during the studies.
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Received on 08.05.2009
Accepted on 14.08.2009
© A&V Publication all right reserved
Research Journal of Pharmacognosy and Phytochemistry.
1(2): Sept. - Oct. 2009, 115-118