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)