20
Bioactive profile of Holostemma adakodien extracts through
various bioassays
Introduction
For the last few decades, plants have served as an important source of several
novel biomolecules with medicinal potential. Therapeutic efficacy of plant crude
extracts and isolated compounds have been evolved in course of time and generated a
number of popular modern day medicines (Ansari and Inamdar, 2010). Novel drug
delivery systems have been utilized in the modern herbal formulations (Ajazuddin,
2010). In several instances, safety and efficacy of herbal medicines have been
investigated (Hasani 2010) and the World Health Organization (WHO) has estimated
more than 4000 million people of the world is dependent on traditional medicine
(Farnsworth 1985).
The therapeutic efficacy of many indigenous plants, for various diseases has
been described by traditional herbal medicinal practitioners (Gami Bharat and Parabia,
2010). Natural products are the sources of synthetic and traditional herbal medicine.
They are still the primary health care system in some parts of the world (Shaukat
Mahmud 2010). In India, local empirical knowledge about medicinal properties of
plants is the basis for their use as home remedies. It is generally accepted by many
Indians and elsewhere in the world that beneficial medicinal effects can be obtained by
ingesting plant products. Plants have basis of many traditional medicines throughout
the world for thousands of years and continue to provide new remedies to mankind
(Patel, 2009).
21
Natural drug from the plants are gaining popularity because of several
advantages such as often fewer side effect, better patient tolerance, relatively less
expensive and acceptance due to a long history of use, especially herbal medicines
provide rational means for the treatment of many diseases that are obstinate and
incurable in other system of medicine. Increasing interest by multinational
pharmaceutical companies and domestic manufacturers of herbal-based medicines is
contributing significant economic growth of the global medicinal plants sector. Among
these natural (70 medicinal plants) analog, Holostemma adakodien is one of the
potential, endemic medicinal plant distributed throughout the southern region of India
especially in tropical forests (Ved and Goraya, 2007). The demand for the H.
adakodien root tubers is very high because it was one of the major ingredients in
Ayurvedic drug Jivanti (Kolammal, 1979). Moreover, It was calculated that more than
150 metric tons of H. adakodien has been utilized per year only in South Indian
Pharmacies (Karmarker 2001), which leads the commercial exploitation. According to
Pushpangadan (1996), more than 1.6 tons of Holostemma root tubers ere utilized per
annum by the four districts of Southern Kerala. Roots of H. adakodien are reported to
possess cooling, alterative tonic and laxative properties. Root paste is applied in
ophthalmic and orchitis. They are also used in diabetes, gonorrhea, coughs and
stomachache (Pulliah, 2002). Common ailments such as fever, dysentery, night
blindness, poisonous affections and tuberculosis are also treated using the roots
(Ravikumar and Ved, 2002).
The screening of plants species especially for phytochemicals of ethno
pharmacological importance will provide essential information in the search of new
pharmaceuticals. Less than 10% of the plant species have been screened for their
22
bioactive compounds (Meyer et al., 1996). Scientist with the aim of establishing the
antimicrobial activity of medicinal plants and to identify those compounds which
possess antimicrobial properties, get themselves involved in the screening of those
medicinal plants (Abinu 2007, Ndukwe 2007). Human pathogens are resistant to
several available antibiotics and this is supported by many researchers (Mathias 2000;
Ganguly 2001; Martino 2002).
Therapeutically potential antimicrobial of plant origin are highly efficient in the
treatment of infectious diseases, simultaneously they mitigate many of the side effects
that are linked with synthetic microbials (Iwu 1999). Advantage of using
antimicrobial compounds of plant origin in better patient tolerance, relatively less
expensive, acceptance due to long history of use and being renewable in nature
(Vermani and Garg, 2002).
Besides small molecules from medicinal chemistry, natural products are still
major sources of innovative therapeutic agents for various conditions including
infectious diseases (Clardy and Walsh, 2004). The antimicrobial compounds produced
by plants are active against plant and human pathogenic micro organisms (Mitscher
1987). Guidelines to study the herbal compounds are lacking and a very meager
portion of this tremendous potential drug repertoire has been screened scientifically till
date (Kamboj, 2000).
Now a days 25-50% of all synthetic drugs prescribed in USA are plant based
although 1% of the higher plant species has been screened for active compounds
(Sheldon et al, 1997). According to Posey and Dutfield (1996) this percentage will
23
continue to grow. These advancements further strengthen our knowledge and microbes
in the field of ethno pharmacology (Barbour 2004).
With an ever increasing momentum in the quest for newer antimicrobial agents
to counteract the bacterial drug resistance, plants are being increasingly explored
in many parts of the world. Plants offer new source of potential activity against
infectious micro organism. The screening of plant extracts and natural products for
antimicrobial activity has shown that higher plants represent a potential source of
new anti – infective agents (Press, 1996) as well an serve in drug discovery from
natural products for primary lead compounds. So the present study indented to study
the bioactive potential of H. adakodien with special reference to antibacterial,
antifungal, icthio toxicity and brine shrimp lethality assays and their validation of these
agents.
Based on this need, the present chapter in intended to evaluate the bioactive
potential especially antibacterial, antifungal, Icthiotoxicity, larvicidal and brine shrimp
leathality of H.adakodien collected from south Travancore region.
24
Materials and Methods
Biology of test plant
H. adakodien is a handsome laticiferous twiner, provided with a conspicuous
flowers (Warrier, 2004), and tuberous roots. Roots are about 3cm across, whitish
inside, thick, white and on drying turn into an elastic residue. The leaves opposite and
base deeply heart shaped with blunt acuminate apex, margin-entire, hairless, lateral
nerves about 5, lower pairs arise from the base of the leaves. Leaves petiolate, petioles
up to 3cm long, Large purple bisexual flowers are seen in axillary cymose
inflorescence. An inflorescence bears about 2-20 flowers. Fruits follicles, 1-2,
lanceolate, the second one often suppressed (Plate 1).
Plate-1 Experimental plant Holostemma adakodien with flower
25
Flowering takes place from September to November and March to April
(Ravikumar and Ved, 2002). After 25 to 28 days of bud initiation flowers open.
Anthesis is noticed from 8.30 to 10hr. and is maximum between 9.00 to 9.30 a.m. On
the first day of flower opening the stigma receptivity was at its maximum (Manju and
Kurian, 1999). Fruiting takes place from April to October.
Collection of plants
Fresh plants or parts of the plant were collected from Mananvilai village of
Kappiyari Panchayat in Kalkulam Taluk. Polythene bags were used to keep the
collected material in fresh condition for short periods. The plant was identified and
authenticated by Dr. Santhoshkumar, Tropical Botanical Garden and Research Institute,
Palode, Thiruvananthapuram. Lenses were used to have spot identification and to record
their morphological characters.
Extract preparation
The collected plant materials were shade dried with in a temperature range of 28-
350C, until the moisture level reached less than 14%. Before feeding into a grinder,
the plant materials were minced with wooden knife and then they were made
into powder using teeth milk and sieved. Then the powder was stored in airtight-
container and kept at room temperature until further use. The powder was
subjected to extraction with different solvent systems.
About 10g of each finely powdered plant material ( leaf, stem, root) was
mixed with 10 ml of different solvents such as methanol, n- butyl alcohol and
acetone in a conical flask, plugged with cotton and kept for 30 days with
26
periodical shaking. The solvent along with powder was filtered through four layers
of muslin cloth. The residue was discarded. Filtrate was refluxed at appropriate
boiling point (650C) and the extract was concentrated to about one fourth of the
original volume. The concentrate thus obtained was stored at 400C in airtight bottles
for further studies.
In vitro antibacterial screening of the isolates
Antibacterial studies were carried out using the bacterial type cultures obtained
form Microbial Type Culture Collections (MTCC), Chandigarh (Table 1). The MTCC
type cultures were initially activated in nutrient broth and subsequently purified by agar
streak plate method. Different techniques of agar diffusion method were evaluated
for the antibacterial susceptibility test
Table-1, Bacterial strains used for antibacterial screening
No Test Organism Strain
1 Escherichia coli MTCC1678
2 Klebsiella pneumonia MTCC7028
3 Pseudomonas aeruginosa MTCC7083
4 Salmonella paratyphi MTCC3220
5 Staphylococcus aureus MTCC9011
6 Bacillus subtilis MTCC1790
7 Enterococcus faecalis MTCC459
8 Steptococcus pyrogens MTCC*
* Received from Biotech Research Laboratory, Thiru. Vi. Ka. Govt. Arts College,
Tiruvarur.
27
Well Diffusion Technique
The principles of antibiotic diffusion assays and specific solid and liquid
medium were prepared according to Perez (1990). Initially, nutrient agar spread plates
were prepared using 0.1 ml of inoculum containing appropriate bacteria of 18 h culture.
The plates were kept as such for 15 minutes for the adhesion of bacteria on the
medium. Wells were cut in each plate using a sterile cork borer of 7 mm diameter (The
Himedia manual, 1998). Each well was bottom sealed with 1% agar solution and filled
with MeOH dissolved extract up to the brim. Methanol was used as the positive
control. After 24 h of incubation at 30 2 C or 20 C in a B.O.D incubator, the
diameter of inhibition zones were measured. The area of the inhibition zone was
calculated as follows:
Cross diameter of the inhibition zone = m
Net diameter of the well = n
Net diameter of the inhibition zone, x = m-n
Net radius of the inhibition zone, r = x/2
Area of the inhibition zone = r2 ( = 3.14)
Biotoxicity studies
Brine Shrimp Cytotoxicity
About 0.1gm of Artemia salina cysts was aerated in 1 lit capacity glass cylinder
(jar) containing filtered seawater. The air stone was placed in bottom of the jar to
ensure complete hydration of the cysts. After 24 hr the newly hatched free-swimming
pink-coloured nauplii were harvested from the bottom outlet. As the cyst capsules
floated on the surface, this collection method ensured pure harvest of nauplii.
28
The freshly hatched free-swimming nauplii were used for bioassay. The essay
system was prepared with 2 ml of filtered seawater containing chosen concentration of
methanolic extract in cavity blocks (Embryo cup). Parallel vehicle control (using 2%
methanol) and negative control wells also kept. In each, 20 nauplii were transferred and
the setup was allowed to remain for 24 hr, under constant illumination. After 24hr, the
dead nauplli were counted with a hand lens. Based on the percent mortality, the LD50
of the test compound was determined using probit scale.
Larvicidal activity
As the largest of mosquitoes (Culex sp).were more accessible for control, the
early second instar larvae were chosen for the experiments. The susceptibility or
resistance of the mosquito larvae to the selected concentration of the extracts was
carried out by adopting standard bioassay protocols (WHO 1975) Observations were
made after 24hr of treatment for larvicidal activity.
Icthyotoxicity assay
Fingerlings (1.5-2.0cm) of marine acclimated Oreochromis mossambicus were
used for evaluating the ichthyotoxic potential. Five fingerlings were introduced in each
experimental and control. Glass bowls containing 1000ml seawater dissolved with
chosen concentration of the extract. Immediate reflex changes and mortality were
observed continuously for six hours at 1 hr interval for the next 12 hr. After 12 hr of
exposure, the number dead and live fish were counted. The acute toxicological reflexes
were observed and recorded.
29
The median lethal dose of the highly active secondary metabolites, which taken
up for the on captivity disease control experiments, were evaluated using large
O.mossambicus. The chosen doses of plant extracts were prepared in NS and injected
intra-peritoneally to the fish. The mortality percentage was converted into probit scale
to determine the LD50 values
Antifungal Assay
Tube dilution technique was adopted for evaluating potent antifungal effects of
the N-butyl alcoholic extract from H.adakodien against Aspergillus, Candida albicans
and Candida sps. Sabouraud broth (Hi Media - M063) was prepared according to
MTCC manual. In this technique, stock solution of the drug was prepared in normal
saline (NS) and added to the diluent (Sabouraud broth), at an increasing dilution. The
fungal hyphae taken from the plate culture were inoculated and incubated for 72 h at
35 2 C. After 72 h of incubation, the growth/inhibition was observed.
30
Results
Antibacterial screening
The results of initial antibacterial screening on different parts of H. adakodian
with different solvent systems are displayed in Plate 2. The antibacterial activity of H.
adakodien against potential human pathogen E. coli, K. pneumoniae, P. aeruginosa, S.
paratyphi, S. aureus, B. subtilis, E. faecalis and S. pyrogens, the herbal extracts were
made with there different solvents like methanol, acetone and n- butyl alcohol are
presented in Table 2. It is noteworthy that no major variation was established on the
anti-bacterial activity by different solvents.
Table-2. Antibacterial activity of the different parts of H. adakodien using various
solvents
Plant part Extract 1 2 3 4 5 6 7 8
Leaf
Methanol - - - - 9 12 13.7 10
Acetone - - - - - 11 8.7 8.2
N-butyl alcohol 10 - 8.5 8.9 13.4 11 7 9
Stem
Methanol 9 - - 7 8 15.2 7 -
Acetone - - - - - 10 10 -
N-butyl alcohol 12.3 9 10 8 13.8 9 12.3 6
Root
Methanol 7 - - - - 9 9 9
Acetone 9 - 7
N-butyl alcohol - - 11 - 9 15 9 7
(- No zone of inhibition) 1. E.coli 2.K.pneumoniae 3. P.aeruginosa 4.
S.paratyphi 5. S.aureus 6. B.subtilis 7. E.faecalis 8. S.pyrogens
31
The result also reveals that among the methanol, acetone and n-butyl alcoholic
extracts of the stem, maximum antibacterial activity was shown by the methanolic
extract on B. subtilis with a zone of inhibition of 15.2mm. The growth of E. faecalis
was inhibited up to a zone of 12.3mm by the butyl alcoholic extracts. Equal action was
shown by the n-butyl alcoholic extracts on P. aeruginosa with a zone of 10mm and the
acetone extracts on B. subtilis and Enterococcus faecalis (inhibition zone 10mm)
(Fig.1) .
Fig.1 Antibacterial activity of H.adakodien stem
Least antibacterial activity was shown by the methanolic extract on
S. paratyphi and E. faecalis with an inhibiting zone of 7mm each. Moderate response
was shown by n-butyl alcoholic extract on B.vsubtilis (9mm), S. paratyphi (8mm),
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8
inhibitionzone(m
m)
Organism
Methanol
Acetone
N butyl alcohol
32
K. pneumoniae (9mm) and methanolic extracts on S.paratyphi, (9mm) and on S. aureus
(8mm).
Leaf extracts of H.adakodien with there different solvents showed much
variation in their activity. Methanol extracts showed maximum activity against E.
faecalis and the zone of inhibition was 13.7mm followed by the n-butyl alcoholic
extract on Staphylococcus aureus (13.4mm) (Fig.2).
Fig.2 Antibacterial activity of H.adakodien Leaf
Among the three solvent extracts, acetone extract did not inhibit the growth of
S. paratyph,i S. aureus, P. aeruginosa, K. pneumoniae and E. coli. E. coli K.
pneumoniae, P. aeruginosa and S. paratyphi were highly susceptible to the methanolic
extracts. Among the three solvent extracts n-butyl alcoholic extract was highly
efficient in its antibacterial action, inhibiting all the eight bacterial strains. Acetone
extract showed least response by inhibiting the growth of only three strains.
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8
inhibitionzone(m
m)
Organism
Methanol
Acetone
N butyl alcohol
33
Among the three extract of the root n-butyl alcoholic extract of the root showed
the maximum growth inhibiting action on B. subtilis (inhibition zone 15mm) (Fig.3) .
Likewise B. subtilis was inhibited up to a zone of 14.2 mm by the methanolic extract.
In contrast to that the acetone extract is highly inefficient and showed poor antibacterial
activity by inhibiting B. subtilis and S. pyrogens with zones of 9mm and 7mm
respectively.
Fig .3 Antibacterial activity of H.adakodien root
Antibacterial activity of the various plant parts such as stem, leaf and root of H.
adakodien against four gram negative and gram positive bacteria were studied.
Methanol, acetone and n-butyl alcohol were used as solvents. Both methanol and n-
butyl alcohol extracts showed comparatively higher antibacterial property against both
gram positive and gram negative strains. However n-butyl alcoholic extract is more
antibacterial. Lesser action is shown by the methanolic extract. Least activity is shown
0
2
4
6
8
10
12
14
16
1 2 3 4 5 6 7 8
Inhibitionzone(m
m)
Organism
Methanol
Acetone
N butyl alcohol
34
by acetone extract. N-butyl alcoholic extract of the stem, leaf as well as root showed
the maximum antibacterial activities.
Biotoxicity Studies
Brineshrimp cytotoxicity assay
The results of Artemia cytotoxicity bioassay are depicted in Table-3. The
various solvent extracts of the endemic medicinal plant H.vadakodien exhibited high
toxicity against Artemia nauplii. N- butyl alcoholic extract of the leaf exhibited high
toxicity against Artemia nauplii followed by methanolic extract of the leaf.
Table-3, Artemia cytotoxicity profile of H.adakodien at 300 C
Plant part Extract Concentration(%) Mortality
Leaf
Methanol 4 51.0±4.58
10 85.00±2.54
Acetone 4 20.2±3.6
10 60.2 ±7.0
N-butyl alcohol 4 20.0±4.14
10 90.6±3.2
Methanol 4 21.2±2.5
35
Stem
10
23.8±6.25
Acetone 4 10.0±1.26
10 48.4±0.89
N-butyl alcohol 4 11.4±3.2
10 60.0±7.37
Root
Methanol 4 20.6±3.0
10 80.2±4.74
Acetone 4 21.2±2.5
10 40.2±1.32
N-butyl alcohol 4 40.0±5.6
10 67.22±0.83
Larvicidal activity
The results of the mortality profile of second instar larva of culex on
H. adakodien extracts with various solvents are shown in Table-4. The results
indicated that the methanolic extract showed varied results. The extract of the root
showed more potent larvicidal activity than the leaf. The root extract effectively killed
all the second instar larvae at 5.2%, whereas the stem extract produced same degree of
activity at 3.6% level (Fig.4)
36
Table-4, Larvicidal profile of methanolic extract of H.adakodien
Plant part Concentrationmg/l Mortality(%)
Leaf 6 100
2 80.4±3.05
Stem 6 100
2 60.2±2.6
Root 6 100
2 80.0±2.89
Icthyotoxicity studies
Icthyotoxicity profile of N-butyl alcoholic extract of the various parts of
H. adakodien are presented in Table -5 . The root extract was extremely toxic and
killed all the fingerlings of O. mossambicus within a short time of exposure of 56 min
at 4% level, 1hr at 2% level. The leaf extract was toxic upto 4mg/l and 2mg/l was less
toxic and did not influence the mortality rate within 6hrs. Stem extract was least toxic
and 20% mortality was recorded only at 2mg/l.
37
0
20
40
60
80
100
120
Leaf Stem Root
Mortality
%
Table-5 Icthyotoxicity profile of N-butyl alcoholic extract of H. adakodien
Plant part Concentration(%) Mortality(%) Timeof death(h)
Leaf 4 100 1
2 60.6±5.6 6
Stem 4 70.2±4.26 6
2 20.0±2.89 6
Root 4 100 56min
2 100 1
Fig.4 Icthyotoxicity profile of N-butyl alcoholic extract of H.adakodien at 4%
concentration
38
Antifungal activity
Antifungal activity of the N-butyl alcoholic extract of H.adakodien stem was
assayed and the data on the effect of plant extracts on the growth of A. niger,C.
albicans and Candida sp. are presented in the Table-6 diagrammatically displayed in
Fig.5 .
Table-6, Antifungal activity of H. adakodien stem extract
Sl.No Organism Zone of inhibition in mm
Control Test
1 Aspergillus niger 18.2 10.6
2 C.albicans 20 10
3 Candida sp. 19.2 9.8
Fig.5 Antifungal activity of H. adakodien stem extract
The data revealed that significant reduction in the growth of A.niger was observed
with the N-butyl alcoholic extract of the plant (Plate-3).
9.4
9.6
9.8
10
10.2
10.4
10.6
10.8
Aspergillus niger C.albicans Candida sp.
Inhibitionzone(m
m)
organism
39
Plate-3
Antifungal activity of N-butyl alcoholic extract of H.adakodien leaf
Aspergillus niger Candida albicans
c-control
Candida sp.
40
Discussion
Medicinal plants represent a rich source of antimicrobial agents (Mahesh and
Satish, 2008). Plants are used medicinally indifferent countries and are a source of
many potent and powerful drugs. Plants is important source of potentially useful
structures for the development of new chemotherapeutic agents. Many reports are
available on the antiviral, antibacterial, antifungal, anthelmintic, antimolluscal and anti-
inflammatory properties of plants(Samy and Ignacimuthu 2000;Palombo and Semple
2001; Kumaraswamy 2002; Stepanovic 2003; Bylka 2004; Behera 2005; Govindarajan
et al .,2006). Many of the plant materials used in traditional medicine are readily
available in rural areas at relatively cheaper than modern medicine (Mann 2008). The
effects of plant extracts on bacteria have been studied by a very large number of
researchers in different parts of the world (Reddy 2001; Ateb and ErdoUrul, 2003).
Plants are sources of natural pesticides that make excellent leads for new pesticide
development(Arokiyaraj 2008; Gangadevi 2008; Satish 2008; Brinda 2009; Jagdish
2009; Milind Pande 2009;Shanmugavalli 2009; Swarna Latha and Neelakanta and
Vetrivel Rajan 2009).The crude extracts of Viscum album L.subsp.abietis(Wiesb)
dissolved in methanol inhibited the growth of gram positive and gram negative bacteria
and fungus (Omer Erturk 2003).
Researchers are being carried out tediously to find new and promising
antimicrobial from plants. Plants provide us with an enormous array of chemicals
which inhibit the growth and multiplication of microbes. Among them, the secondary
metabolites are more specialized and are usually peculiar to are plant or species. Some
41
of these metabolites are defensive compounds designed to deter or kill disease causing
microorganisms, potential predators or competitors. The use of medicinal plants play a
vital role in providing the basic health needs in developing countries and these plants
may offer a new source of antimicrobial with significant activity against, infective
microorganism (Coethodesouza 2004). Many plant extracts have been used to as a
source of medicine to cure urinary tract infections, respiratory disorders, cutaneous
affections, inflammations etc.
H.adakodien is native to India, Burma and Sri Lanka (Huber, 1983). This plant
is reported as vulnerable in Karnataka and endangered in Kerala by the Foundation for
Revitalization of Local Health Tradition (Ravikumar and Ved, 2002). Many authorities
explained this species as rare, endemic and endangered medicinal plant (Purohit 1994;
Krishnan 1995; Sudha and Seeni, 1996). It is a vulnerable medicinal plant of Munnar
forest region ( Bhat and Padmaja, 1991) and threatened in Kerala (Sasidharan, 1991).
It is in the vulnerable status in India (Nayar and Sastry, 1987 and Rajasekharan and
Ganeshan, 2002). In Kanyakumari District, it in present in the upper Kodayar region of
Kalkulam Taluk, and its distribution status in Endemic, Endangered and Rare in the
sacred groves (Sukumaran, 2002). In the present study the antibacterial activity of the
various organs of the plant H. adakodien was studied.
Antibacterial activity
Meena Thomas Irimpan (2011) reported that the methanolic, ethanolic and
hydro alcoholic extracts of H. adakodien leaf showed antibacterial activity against
gram positive bacteria S. aureus and B. subtilis also methanolic extracts showed greater
activity than the antibiotic gentamycin against gram negative S. typhymurium. The
42
concentration of an antimicrobial agent is of prime importance. Agents may produce
many changes in microorganism and different changes may be related to varying
concentration of the agent present. In the present investigation the agar spread plate
were filled with the extract of different plants parts with different solvents and their
respective antibacterial activity against the test organisms were noticed. Visible zones
of inhibition of bacterial growth were seen around the wells which contained extracts
that possess antibacterial activity.
In the present investigation a variety of gram positive and gram negative stains
were selected for the screening of antimicrobial effect of the methanol, acetone and n-
butyl alcoholic extracts of various plant parts such as to stem, leaf and root of H.
adakodien to perceive the antimicrobial spectrum as well as to authenticate ethno
medicinal claims. Maximum antibacterial activity was shown by the methanolic extract
of the stem followed by the n-butyl alcoholic extract of the leaf.
Influence of solvents
Among the three solvents methanol, acetone and n-butyl alcohol, used in the
present work for the extraction of leaf, stem and root of H. adakodien, n-butyl alcohol
was highly efficient and the extracts showed inhibiting activity on the eight bacterial
strains under study. This was followed by that of methanolic extracts.
Yogesh and Mohan (2007), showed that the methanolic extracts of Cryptolepis
buchanani and Pergularia daemia inhibited the growth of gram positive bacteria such
as Staphylococcus epidermidis, Staphylococcus aureus and gram negative bacteria such
as Salmonella typhi and Salmonella paratyphi where as the methanolic extract of
Leptadenia pyrotechnica could inhibit the growth of the above mentioned gram
43
positive bacteria and could not inhibit the growth of the gram negative bacteria such as
S.typhi and S.paratyphi. Methanolic extract of the leaf of H.adakodien showed greater
antibacterial activity against gram negative Salmonella typhimurium than the antibiotic
gentamycin(Meena Thomas Irimpan 2011)
This work proves that the N-butyl alcoholic extact of H.adakodien showed
maximum antibacterial activity, than that the stem. Successfull prediction of botanical
compounds from plant material is largely dependent on the type of solvent used in the
extraction procedure. Traditional medical practitioners use water as the solvent
(Ahmed 1998). The relatively high potency of n-butyl alocohol may be attributed to
the dissolving power n-butyl alcohol over acetone and methanol. The potential
antibacterial effects of the plants could be enhanced by extracting with n-butyl alcohol
instead of other solvents, including water as applied. The medicinal value of a plant has
in some chemical substances that produce a definite physiological action on the human
body. The most important of these bioactive constituents of plants are alkaloids,
tannins, flavonoids and phenolic compounds (Edeoga 2005).
The leaf and stem extract of H.adakodien shows the presence of flavonoids,
phenols, saponins, sterols, tannins and terpenoids. Both methanolic and water
extracts leaf and stem of H.adakodien contains phenol. (Meena Thomas Irimpan
2011). It is thought that enzyme inhibition by the phenolic compounds is the
mechanism for the micro organism inhibition. This is possibly done by the oxidized
compounds through reaction with sulfhydryl groups or through more non specific
interactions with the proteins (Coman 1999).
44
Water extract of H. adakodien possess saponins (Meena Thomas Irimpan
2011). Saponins by nature are detergents with steroidal structure, which might be
expected to affects biomembranes. Certain saponins are found to be apoptotic also
(Hsu et al, 2001). Many plants contain non – toxic glycosides which can get
hydrolysed to release phenolics which are toxic to microbial pathogens (Aboaba and
Efuwape, 2001). Plants produce a diverse array of secondary metabolites, many of
which have antimicrobial activity. The anti bacterial activity of H.adakodien might be
due to the presence of phenols, saponins, sterols, tannins and terpenoids.
The present work aims at studying the antibacterial effect of H.adakodien
extracts (viz, leaf, stem and root) with methanol, acetone and n-butyl alcohol on eight
bacterial strains. Among these eight stains four are gram positive. They are
Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis and Streptococcus
pyrogens. The other four E.coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and
Salmonella typhi are gram negative bacteria.
Higher resistance of Gram negative bacteria to external substances had been
reported (Negi 2005). The selective bacterial activity against gram negative bacteria
of the methanol, acetone and n-butyl alcoholic extracts of the leaf, stem and root
extracts of Holostemma adakodien could be due to the fact that a number of
antimicrobial compounds could not inhibit the growth of gram negative bacteria due to
a failure of outer membrane penetration. The resistance of gram negative bacterial to
the antimicrobial substances is concerned with the lipopolysaccharides in their outer
membrane (Gao 1999). The changes in membrane fatty acids composition of
microbial cells in the presence of sub lethal concentration of antimicrobial compounds
(eg.thymol, eugenol, carvacrol etc) in response to a stress condition was studied. It was
45
found that gram negative bacteria did not show substantial changes in its fatty acid
composition (Pasqua 2006). This is an indication of the high resistance of gram
negative bacteria to the tested compounds.
The gram negative bacterial cell wall outer membrane appears to act as a barrier
to many substances including antibiotics (Tortora 2001). Gram positive test organisms
(Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis and Streptococcus
pyrogens) showed higher sensitivity against the methanolic, acetone and n-butyl
alcoholic extracts of H.adakodien than the gram negative bacteria. The reason may be
due to the difference between the cell wall compositions. Gram-positive bacteria
contain an outer peptidoglycone layer, which is an infection permeability barrier
(Scherrer and Gerhardt, 1971).
The greater susceptibility of Gram positive bacteria has been previousy reported
for South American (Paz 1995), African (Kudi et al, 1999; Vlientinck 1995) and
Australian (Palombo and Semple, 2001) plant extracts.
Antifungal activity
Various publications have documented the antimicrobial activity of plant
extracts and essential oils (Hili, 1997, Lis-Balchin, and Deans, 1997). The growth of
all the three fungal strains were inhibited by the N-butyl alcoholic extracts the plant.
There was maximum reduction in the growth of Aspergillus niger followed by Candida
sp.
46
Brine Shrimp Lethality Studies
In the present study the Brine Shrimp Lethality of the various solvent extracts
of H. adakodien was determined. N-butyl alcoholic extracts of the leaf showed more
significant activity than the other extracts. The degree of lethality was directly
proportional to the concentration of the extract. Maximum mortality was observed at a
concentration of 4% in the case of N-butyl alcoholic extract followed by methanolic
extract of the leaf. The brine Shrimp activity of certain medicinal plants were reported
by Ahmed Taha and Hasim Alsayed,(2000); Krishnaraju,(2000); Padmaja (2002);
Moshi (2009)
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