Pharmaceutical

Communication

Biosci. Biotech. Res. Comm. 10(4): 601-611 (2017)

Evaluation of some biological properties of

Saussurea

costus

crude root extract

Emad M. Abdallah,

* Kamal A. Qureshi,

Ahmed M. H. Ali

1,3

and Gamal O. Elhassan

Department of Laboratory Sciences, College of Sciences and Arts at Al-Rass, Qassim University,

Al-Rass, Saudi Arabia

Department of Pharmaceutics, Unaizah College of Pharmacy, Qassim University, Saudi Arabia

Department of Zoology and Entomology, Faculty of Science, Assiut University, Assiut, Egypt

ABSTRACT

The current study aimed to evaluate some biological activities of Saussurea costus(S. costus) such as phytochemi-

cal constituents, antimicrobial activity and antifeedant potential on the larvae of Spodoptera littoralis. The results

revealed that the methanol extract of roots of S. costus are rich in some bioactive phytochemical compounds such as

alkaloids, phenols/polyphenols,  avonoids, terpenoids, tannins, coumarins, quinines, steroids, cardiac glycosides and

resins. The antimicrobial screening revealed that, among 12 referenced microbial strains (10 bacteria and 2 fungi),

4 Gram-positive bacteria exhibited high susceptibility with the methanol and ethanol extracts of S. costus, namely

Bacillus cereus ATCC 10876 (IZ 16.0±0.0, 15.5±0.5 mm, MIC100, 50 mg/ml, MBC 200, 100 mg/ml), Staphylococcus

saprophyticus ATCC 43867 (IZ 14.5± 0.5, 15.5±0.5 mm, MIC50, 50 mg/ml, MBC100, 100 mg/ml), Staphylococcus

epidermidis ATCC 12228 (IZ 13.5±0.5, 14.5±0.5mm, MIC50, 50 mg/ml, MBC 200, 100 mg/ml) and Staphylococcus

aureus ATCC 29213 (IZ 11.0±0.0, 11.5±0.5 mm, MIC 100, 50 mg/ml, MBC 200, 100 mg/ml), respectively. Also, 1 fungal

strain (Aspergillus niger ATCC 6275) revealed high susceptibility with the extracts (IZ 26.0±1.0 mm, MIC and MFC

50mg/ml). Other microorganisms recorded weak or no effect. Furthermore, the ethanolic extract of S. costus pro-

vided an anitfeedant effect toward Spodoptera littoralis larvae at different concentrations. In conclusion, the current

 ndings provide evidence that roots of Saussurea costus is rich in bioactive phytochemical compounds and it might

be a promising source of antimicrobial compounds as well as antifeedant activity against the larvae of Spodoptera

littoralis.

KEY WORDS: ANTIBACTERIAL, ANTIFUNGAL, PHYTOCHEMICAL,

SAUSSUREA COSTUS

, ANTIFEEDANT, INSECTICIDAL,

SPODOPTERA

LITTORALIS

, TRADITIONAL MEDICINE

601

ARTICLE INFORMATION:

*Corresponding Author: emad100sdl@yahoo.com

Received 21

Oct, 2017

Accepted after revision 24

Dec, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF: 4.006

reserved.

Online Contents Available at:

http//www.bbrc.in/

DOI: 10.21786/bbrc/10.4/2

602 BIOLOGICAL PROPERTIES OF

SAUSSUREA COSTUS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Emad M. Abdallah et al.

INTRODUCTION

Medicinal plants are used for healing purposes through-

out the human history; even in the current era, there

are up to 80% of the world population most of them are

living in the developing countries, rely on traditional

herbal medicine on their primary health care systems,

many of these herbal drugs prescribed in traditional

medicine have inadequate knowledge and untested by

scienti c methods (Ekor 2013 Qazi and Molvi 2016).

On the other side, Modern medicine stands helpless in

the front of the growing phenomenon of antimicrobial

resistance to antibiotics, which considered as a major

health problem and required prompt attention. This cri-

sis encouraged scholars and researchers to develop the

current antibiotics, synthesize new antibiotics or to  nd

new alternatives. The latter option is preferable because,

in nature, plants arise as one of the largest pharmaceuti-

cal factories ever known and plants were the main source

of drugs for humankind since antiquity. Many medicinal

plants produce diverse groups of secondary metabolites

known as phytochemical compounds, which may sup-

press the microbial growth by different modes of action

such as interference with cellular metabolic processes,

cellular membrane perturbations or by modulating the

signal transduction or gene expression pathways (Omo-

jate et al., 2014 Mohamed et al. 2017).

Saussurea costus (Falc.) Lipschitz, synonymous with

Saussurea lappa C.B. Clarke, belongs to family Aster-

aceae, this family includes about 1000 genera and

30,000 species, widely distributed in different regions

in the world; However, numerous species are found in

India (Pandey et al., 2007). It is also distributed in Paki-

stan and some parts of Himalayas (Shah 2006). Saus-

surea costus(S. costus) is well known in Islamic medi-

cine, which enlisted in the Holy Ahadith said by Prophet

Muhammad (Peace be upon him) (Ahmad et al., 2009).

It is known in Arab countries as “Al-Kost Al-Hindi” and

used by traditional healers since the era of the Islamic

civilization. For example but not limited to, S. costusis

traditionally used as stimulant, antiseptic, carminative,

sedative, bronchodilator and astringent agent (Wani

et al., 2011). In the scienti c literature, the biological

activities of the roots of S. costus (synonymous with

S. lappa) are widely investigated. Scienti c investi-

gations revealed that it has anti-trypanosomal activ-

ity (Julianti et al, 2011), it has “complement-inhibitor”

substances helpful in the treatment of some diseases

related to excessive activation of the complement sys-

tem, like rheumatoid arthritis, respiratory distress and

systemic lupus erythematosus (Fan et al., 2014). It was

published that S. costus has a good anticancer activity

on the tested cell lines (Robinson et al., 2008). The etha-

nol extract of S. lappa (synonymous S. costus) recorded

a wide spectrum antimicrobial activity against some

human pathogens (Hasson et al., 2013). In addition,

many investigations reported other bioactive properties

of S. costus roots such as anti-ulcer, anti-in ammatory,

hepatoprotective, immunomodulator, hypoglycaemic,

spasmolytic, anticonvulsant, antidiarrheal and antiviral

activity (Zahra et al., 2014 Ghasham et al., 2017).

Egyptian cotton leafworm, Spodoptera littoralis (Lep-

idoptera: Noctuidae), is responsible for causing devastat-

ing damage for numerous vegetables and crops (Kandil

et al., 2003; Adham, et al., 2009). This polyphagous pest

is widely distributed in Africa and Middle Eastern coun-

tries (Abdel-Rahim and Azab 2008; Rizk et al. 2010; El-

Zoghby et al. 2011). Several synthetic pesticides have

been used to manage the agriculture crops from insect

infestation. These synthetic insecticides cause seri-

ous hazard to the environment due to residual toxicity

(White, 1995; El- Torkey 2008; Rizk et al. 2010). There-

fore, scientists developed safe alternative insecticides

with no residual activity. In this regard, various phyto-

chemical plant extracts from several botanical sources

against the speci c pest have been evaluated (Kamaraj

et al., 2010). The current study aimed to investigate some

biological properties of the methanol extract of the roots

of S. costus, including the phytochemical constituents,

antimicrobial activity and antifeedant potential on the

larvae of Spodoptera littoralis.

MATERIALS AND METHODS

The dry roots of S. costus(Figure 1) were purchased from

a herbal market at Qassim region, Saudi Arabia. The

herbal seller showed the trademark of the package and it

has been con rmed that it was exported from India. The

authentication of plant material was con rmed at the

department of Laboratory Sciences, College of Sciences

and Arts, Al Rass, Saudi Arabia.

12 standard pathogenic test organisms were used in

this study; Bacillus cereus ATCC

10876

, Staphylococ-

cus epidermidis ATCC

12228

, Staphylococcus aureus

ATCC

29213

, Staphylococcus saprophyticus ATCC

FIGURE 1. The dried roots of

Saussurea costus

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOLOGICAL PROPERTIES OF

SAUSSUREA COSTUS

603

Emad M. Abdallah et al.

43867

and Streptococcus pneumonia ATCC

49619

Escherichia coli ATCC

25922

, Proteus vulgaris ATCC

6380

, Klebsiella pneumonia ATCC

27736

, Pseu-

domonas aeruginosa ATCC

9027

, Shigella  exneri

ATCC

12022

, Candida albicans ATCC

10231

and

Aspergillus niger ATCC

6275

The extraction of plant material was carried out by

following the maceration method. The roots of the plant

were crushed into small particles and then  ne powder

was obtained by using electrical mixer grinder. The mac-

eration of plant material was carried out by mixing of

 ne powder into 70 % ethanol and 80 % methanol sol-

vents separately. Then, all mixtures were incubated in a

shaker incubator at 40

C temp., 50 r.p.m. shaking speed

for up to 3 days in a well-tighten dark container. After

incubation, the mixtures were centrifuged at 5000 r.p.m.

for 15 minutes and then  ltered through Whatman  lter

paper No.1. The  ltrates were subjected to evaporate the

solvents by using a rotary evaporator to get a semi-solid

mushy crude extract, which was dried in hot air oven at

C for 48 hrs. The dried crude extracts were kept in a

refrigerator until used (Ghasham et al., 2017). Methanol

crude extract was analyzed for potential phytochemi-

cal molecules, methanol and ethanol extracts were used

in the antimicrobial investigation, while ethanol crude

extract was employed in the antifeedant examination

against the larvae of Spodoptera littoralis.

In order to detect the various phytochemical constitu-

ents, the aqueous methanolic extract (100 mg/mL) was

used. The colourimetric tests listed below were used as

reported by (Ghasham et al. 2017, Sasidharan et al., 2011).

1 mL methanolic extract was mixed well with 1 mL of

1 % hydrochloric acid solution, followed by slight heat-

ing till the steaming. After that, 06 drops of Wagner’s

reagent were added into 1 mL of acidi ed extract. The

formation of a brownish-red precipitate was observed

for a positive test.

Carboxylic acid

2 mL of sodium bicarbonate solution was added to 1 mL

of methanolic extract. The formation of effervescence

was observed for a positive test.

Cardiac glycosides

1 mL methanolic extract was dissolved in 1 mL of chlo-

roform, followed by addition of 2-3 drops of the sul-

phuric acid solution at the side of the test tube to form

a layer. The formation of a brown ring at interphase was

observed for a positive test.

Coumarins

1 mL of methanolic extract was mixed with 1 mL of 10

% sodium hydroxide solution. The formation of yellow

colouration was observed for a positive test.

Emodins

1 mL of ammonia and 1.5 mL of benzene solutions were

added to 1 mL of methanolic extract. The formation of

red colouration was observed for a positive test.

Flavonoids

1 mL methanolic extract was added to 1 mL of 10 % lead

acetate solution. The formation of a yellow coloured

precipitate was observed for a positive test.

Leucoanthocyanins

1 mL of isoamyl alcohol was taken into a test tube, fol-

lowed by slow addition of 1 mL of methanolic extract.

The formation of red colouration at upper layer was

observed for a positive test.

Lipids

0.5 mL of methanolic extract was mixed with 5 ml of

ether. This mixture was allowed for evaporation on  lter

paper and dried the  lter paper. The formation of an

appearance of spot-on  lter paper was observed for a

positive test.

Phenols/Polyphenols

1 mL methanolic extract was added into 0.5 mL of 10

% ethanolic ferric chloride solution. The formation of

blue-green to dark blue colouration was observed for a

positive test.

Phlobatannins

1 mL methanolic extract was added to 1 mL of 1 %

hydrochloric acid solution, followed by boiling the mix-

ture. The formation of a red precipitate was observed for

a positive test.

Quinones

1 mL of concentrated sulphuric acid was taken into a

test tube, followed by addition of 1 mL of methanolic

extract. The formation of red colouration was observed

for a positive test.

Resins

Few drops of acetic anhydride solution were added to 1

mL of methanolic extract, followed by addition of 1 mL

of concentrated sulphuric acid. The formation of orange

to yellow colouration was observed for a positive test.

Saponins

5 mL of puri ed distilled water was taken into a test

tube, followed by addition of 1 mL of methanolic extract

and the whole mixture was well stirred. The formation

of continuous effervescence was observed for a positive

test.

Emad M. Abdallah et al.

604 BIOLOGICAL PROPERTIES OF

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BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Steroids

1 mL methanolic extract was mixed with 1 mL of chlo-

roform, followed by addition of 2 mL of acetic anhy-

dride and then few drops of concentrated sulphuric acid

solution. The formation of dark green colouration was

observed for a positive test.

Tannins

2-3 drops of 1 % lead acetate solution were added to 1

mL methanolic extract. The formation of dark blue or

greenish grey colouration was observed for a positive

test.

Terpenoids

2.5 mL of methanolic extract was mixed with 1 mL of

chloroform and then 1.5 mL of concentrated sulphuric

acid solution was added. The formation of reddish brown

colour at the interface was observed for a positive test.

Volatile oil

0.5 mL of diluted sodium hydroxide and 0.5 mL of

diluted hydrochloric acid were added to 2 mL of metha-

nolic extract and mixed well. The formation of a white

precipitate was observed for a positive test.

ANTIMICROBIAL TESTING

The antimicrobial potential of S. costus root extract was

evaluated using agar-well diffusion method as described

by (Abdallah, 2014) with some modi cations. Before to

the experimental phase, all identi ed microbial isolates

were sub-cultured in a tighten bottles containing either

Mueller-Hinton broth (18-24 hours, 35

C) for bacte-

ria or Sabouraud dextrose broth (48 hours, 25

C) for

fungi. After incubation, all turbid bottles- as a result of

growth-were transferred and kept in the fridge (4

C) to

keep the microbial growth at the exponential phase until

used. Autoclaved Bottles containing 20 ml of Mueller-

Hinton agar or Sabouraud dextrose agar was poured hot

on sterile Petri-dishes (90 mm in diameter) and left at

room temperature until solidi ed. Working microbial

strains were taken from the broth cultures (previously

prepared) and adjusted as McFarland standard, then 100

µl from each microbial strain was put over Mueller-Hin-

ton or Sabouraud dextrose agar plates (depending on the

type of microorganism) and distributed above the agar

using sterile cotton swabs. Wells were punched into the

agar with a sterile cork borer (6 mm in diameter). Then,

100 l from each extract (500 mg/ml) was dropped into

the wells, extracts were previously reconstituted in 10%

di-methyl-sulphoxide (DMSO) to make a concentration

500 mg/ml. 10 % DMSO did not show any inhibitory

effect on microorganisms. Another well (in the centre)

was loaded with 100 l of 5 mg/ml Chloramphenicol for

bacteria or 10 mg/ml clotrimazol for fungi. Plates were

incubated at 35°C for 24 hours for bacteria or at 25

for up to 48 hours for fungi. The antimicrobial activities

of the tested extracts were determined by measuring the

clear zone of inhibition in millimetre (mm) ± standard

error of the mean.

MIC, MBC AND MFC ASSAY

Only microorganisms that showed high antimicrobial

activity was tested for MIC, MBC and MFC. The mini-

mum inhibitory concentration (MIC) was determined

using microdilution method as described by Hassan

et al. (2009) with slight modi cation. Brie y, in a set of

sterile test tubes, serial two-fold dilutions were made to

get 6 tubes containing 1 ml of 200, 100, 50, 25, 12.5 and

6.25 mg/ml of the extract, respectively. Additional two

tubes were also used, one tube containing 1 ml of 10%

DMSO to serve as negative control and the other tube

containing 1ml of 5mg/ml chloramphenicol to serve as

positive control. Then, 1 ml of sterile Mueller-Hinton

broth and 100µl of the adjusted microbial strain were

added to each tube (8 tubes). Tubes were gently shake

and placed in the incubator at 35°C for 24 hours for

bacteria or at 25oC for up to 48 hours for fungi. The

lowest concentration (highest dilution) of the extract

that showed no visible microbial growth (no turbidity)

compared with the control tubes was considered as MIC.

The minimum bactericidal concentration (MBC) and

minimum fungicidal concentration (MFC) was evaluated

by determined by sub-culturing the test dilution on to

unseeded plates of Mueller-Hinton agar for bacteria or

Sabouraud dextrose agar for fungi and incubated further

for 18-24 h for bacteria or 48 h for fungi. The highest

dilution that revealed no single bacterial colony on the

plates was taken as MBC or MFC.

ANTIFEEDANT ASSAY AND STARVATION

PERCENTAGE

A strain of S. littoralis was reared in the laboratory.

Larvae were fed on fresh castor leaves, Ricinus commu-

nius. Adults were provided with 10% sugar solution. All

the bioassays were conducted at 26± 2° C and 65±5 %

R.H., with 8:16 L:D h photoperiod. The experiments were

carried out on the 4th instar larvae. Serious of ascend-

ing crude concentrations were prepared (0.6 %, 1.25 %,

2.5 %, 5%, 10 % and 20%) by dilution in 70 % etha-

nol. Control discs were sprayed with the solvent alone.

400 larvae were starved overnight, then divided into 8

groups of 50 larvae each, six different concentrations of

plant extract (S. costus), one group for the control and

one group as starved larvae. Equal discs of castor bean

leaves were rinsed in each treatment and in the control,

the treated and untreated leaves were shad-dried. All

Emad M. Abdallah et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOLOGICAL PROPERTIES OF

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605

larvae control and treated leaves were weighted before

and after treatment for 3 days. The dried leaves were

placed individually in plastic Petri dishes. Ten larvae

were transferred into each cup and allowed to feed on

the treated and untreated leaves, the starved larvae were

left without feeding for 24h. Five replicates for each

treatment were carried out. The starvation percentages

of tested larvae were calculated (Mostafa 1969 ; Abdel-

Mageed et al. 1975).

Starvation (%) = C – E/C – S X 100

Where:

C = Mean weight gain of untreated larvae after 24 h;

E = Mean weight gain of treated larvae for each concen-

tration after 24 h; and

S = Mean weight gain of starved untreated larvae after

24 h.

The antifeedant index (AFI) was calculated according

to Sadek (2003).

AFI (%)= [(C-T) / (C + T)] X 100

Where:

C: the amount of food consumed (leaves) in the control;

and

T: the amount of food consumed (leaves) in the treat-

ment.

STATISTICAL ANALYSIS

Quantitative data were expressed as a mean ± standard

error of means. One-way analysis of variance ANOVA

was used and P < 0.05 was used in testing the statisti-

cal signi cance. Paired-Samples T-test was employed to

determine any signi cant differences between methanol

and ethanol extracts of the antimicrobial assay. The pro-

gram used was SPSS-Statistical Package, version 11.

RESULTS AND DISCUSSION

PHYTOCHEMICAL SCREENING

The results of phytochemical screening suggest that,

Saussurea costus roots are rich source of various bioac-

tive constituents such as alkaloids, cardiac glycosides,

coumarins,  avonoids, phenols, quinones, resins, ster-

oids, tannins and terpenoids. These results are summa-

rized in Table 1.

These phytochemical constituents are important

for the use of health care. The  ndings of the present

study agreed with previous studies; Chaudhary (2015)

has reported that S. lappa (synonymous S. costus), is a

rich source of alkaloids, steroids,  avonoids and resins.

Moreover, Pandey et al., 2007 have reported that many

bioactive molecules were identi ed and isolated from S.

costus, such as sesquiterpene lactones, costunolide, iso-

dehydrocostus, isozaluzanin-C, guiainolide, cynaropic-

rin, reynosin, santamarine and many more. Undoubt-

edly, the diverse biological activities of S. costus are

attributed to its richness in phytochemical compounds.

Accordingly, it is recommended that more studies may

lead to the understanding which molecules are respon-

sible for the antibacterial, antifungal and antifeedant

activities against Spodoptera littoralis larvae.

ANTIMICROBIL SCREENING

In the current study, 6 mm inhibition zone (IZ) means

that there is no antimicrobial activity of the extract (the

zone of the hole on the agar plate is 6 mm), above 6 mm

to less than 10 mm means that there is a weak antimi-

crobial activity, from 10 mm to 12 mm means that there

is a moderate antimicrobial activity (the double of the

hole diameter), above 12 mm is noticeable or good anti-

microbial activity. Philip et al. (2009) considered that

IZ above 10 mm is good antimicrobial activity. Unlike

the antibiotics, there is no standard criterion in explain-

ing the IZ for the crude plant extracts. The results of

the antimicrobial activity are demonstrated in (Table 2)

and (Figures 2-6). The results of the antimicrobial ef -

cacy of methanolic and ethanolic extracts of S. costus

roots have shown that Gram-positive bacteria were more

susceptible. Bacillus cereus ATCC 10876 has recorded

the highest susceptibility (16.0±0.0, 15.5±0.5 mm), fol-

Table 1. phytochemical analysis of Saussurea

costus roots

Phytochemical constituents Test results

Alkaloids +

Cardiac glycosides +

Coumarins +

Flavonoids +

Phenol/Polyphenols +

Quinones +

Resins +

Steroids +

Tannins +

Terpenoids +

Carboxylic acid -

Leucoanthocyanins -

Lipids -

Emodins -

Phlobatannins -

Saponins -

Volatile oil -

+ = test positive, - = test negative

Emad M. Abdallah et al.

606 BIOLOGICAL PROPERTIES OF

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Table 2. The antimicrobial activity of the methanol and ethanol extracts of S. costus roots against

different microorganisms

Tested

Compound

Mean inhibition zones (IZ) in millimetre

Gram-positive bacteria Gram-negative bacteria Fungi

Sa Se Ss Sp Bc Ec Pa Pv Kp Sf As Ca

MeOH of S. costus

(500 mg/ml)

11.0

±0.0

13.5

±0.5

14.5

±0.5

6. 5

±0. 5

16.0

±0. 0

8.5

±0.5

9.5

±0.5

9.0

±0.0

6.0

±0.0

6.0

±0.0

26.0

±1.0

6.0

±0.0

EtOH of S. costus

(500 mg/ml)

11.5

±0.5

14.5

±0.5

15.5

±0.5

6. 5

±0. 5

15.5

±0. 5

8.5

±0.5

7.5

±0.5

9.0

±0.0

6.0

±0.0

6.0

±0.0

27.5

±0.5

7.5

±0.5

Chloramphenicol

(5 mg/ml)

34.0

±0.5

38.0

±3.0

36.5

±1.5

20.5

±1.0

34.0

±2.0

34.0

±1.0

19.5

±0.5

33.0

±1.0

33.5

±0.5

34.5

±0.5

Clotrimazole (10 mg/ml) - - - - - - - - - - 40.5

±1.5

36.0

±2.0

10% DMSO 6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

6.0

±0.0

*MeOH= methanol extract, EtOH=Ethanol extract, Sa= Staphylococcus aureus ATCC 29213, Se=Staphylococcus epidermidis ATCC

12228, Ss=Staphylococcus saprophyticus ATCC 43867, Sp=Streptococcus pneumonia ATCC 49619, Bc= Bacillus cereus ATCC 10876,

Ec=Escherichia coli ATCC 25922, Pa=Pseudomonas aeruginosa ATCC 9027 Pv=Proteus vulgaris ATCC 6380, Kp=Klebsiella pneumonia

ATCC 27736, Sf=Shigella  exsneri ATCC 12022, As= Aspergillus niger ATCC 6275, Ca= Candida albicans ATCC 10231.

FIGURE 2. Mean zone of inhibitions of different bacterial strains due to the effect methanol and ethanol

extracts of S. costus compared with chloramphenicol*

*Abbreviations of the names of microorganisms are detiled under (Table2).

lowed by Staphylococcus saprophyticus ATCC 43867

(14.5±0.0, 15.5±0.5 mm), Staphylococcus epidermidis

ATCC 12228 (13.5±0.5, 15.5±0.5 mm) and Staphylococ-

cus aureus ATCC 29213 (11.0±0.0, 11.5±0.5 mm), respec-

tively. While, Streptococcus pneumonia ATCC 49619

has shown very weak susceptibility, which was 6.5±0.5

mm for methanolic and ethanolic extracts. On the other

side, the Gram-negative bacteria exhibited weak or no

susceptibility at all. Weak susceptibility was found with

Pseudomonas aeruginosa ATCC 9027 (9.5±0.5, 7.5±0.5

mm), Proteus vulgaris ATCC 6380 (9.0±0.0, 9.0±0.0 mm)

and Escherichia coli ATCC 25922 (8.5±0.5, 8.5±0.5 mm)

for methanolic and ethanolic extracts, respectively.

While, Klebsiella pneumonia ATCC 27736 and Shi-

gella  exneri ATCC 12022 revealed no susceptibility

against the tested extract, which agrees with the results

of Mohamed et al. (2017) who stated that, methanolic

extract of S. costus roots has signi cant level of anti-

bacterial activity against Staphylococcus aureus and

Bacillus subtilis (Gram-positive) and showed no effect

against Escherichia coli and Pseudomonas aeruginosa

(Gram-negative). Interestingly, these results are in

Emad M. Abdallah et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOLOGICAL PROPERTIES OF

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607

FIGURE 3. Susceptibility of gram-positive bacteria to methanol and eth-

anol extracts of S. costus roots compared with chloramphenicol

FIGURE 4. Susceptibility of gram-negative bacteria to methanol and

ethanol extracts of S. costus roots compared with chloramphenicol

agreement in-partial with the  ndings of Hasson et al.

(2013), in their study they have reported that S. lappa

(synonymous S.costus) has exhibited signi cant level

of antibacterial activity against different Gram-posi-

tive and Gram-negative pathogenic bacteria, includ-

ing Staphylococcus aureus, Pseudomonas aeruginosa,

Acinetobacter baumanii, Escherichia coli and Klebsiella

pneumonia.

After comparing the results of our  ndings with the

results of Hasson et al., 2013, we have concluded that,

the former study has used 99.9 % ethanol as a solvent to

extract the crude and in our study, we used 70 % Ethanol

and used 80 % methanol. It is well known that absolute

ethanol can collect non-polar constituents better than

70 % ethanol. Whereas, methanol can collect some non-

polar and polar constituents from the plant materials.

The statistical analysis (Paired-Samples T test) showed

that, there was no signi cant difference between anti-

bacterial activity of 70 % ethanolic and 80 % methanolic

extracts, which means that the effective antibacterial

compounds are present in the non-polar fraction. This

consumption is supported with the  ndings of Pandey et

al. (2008), mentioned that, the essential oil of S. costus

roots has exhibited better antibacterial effects as com-

pared with the methanolic extract. In addition, S. costus

has showed high signi cant level of antifungal activity

Emad M. Abdallah et al.

608 BIOLOGICAL PROPERTIES OF

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BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 5. Mean zone of inhibitions of different fungal strains due to the effect methanol and

ethanol extracts of S. costus compared with clotrimazole*

*An=Aspergillus niger, Ca= Candida albicans

FIGURE 6. Susceptibility of fungalstrains to methanol and ethanol

extracts of S. costus roots compared with clotrimazole

Table 3. MIC and MBC of the methanolic and ethanolic extracts of the S. costus roots

Bacterial strain MIC mg/ml MBC mg/ml MBC/MIC

MeOH EtOH MeOH EtOH MeOH EtOH

Staphylococcus saprophyticus ATCC 43867 50 50 100 100 2 2

Staphylococcus epidermidis ATCC 12228 50 50 200 100 4 2

Bacillus cereus ATCC 10876 100 50 200 100 2 2

Staphylococcus aureus ATCC 29213 100 50 200 100 2 2

*MeOH= methanol extract, EtOH=Ethanol extract.

against Aspergillus niger ATCC 6275 and weak effect

against Candida albicans ATCC 10231.

It disagrees with the  ndings of Mohamed et al.

(2017) who reported good antifungal activity of S. lappa

against Candida albicans. This contradiction is related

to the solvent used in extraction, as reported by Patil et

al. (2009) that, diethyl ether fraction has showed promi-

nent fungicidal activity against Candida albicans. How-

ever, the antimicrobial activity resulted from the current

investigation was not competitor to chloramphenicol or

clotrimazole. These referenced antibiotics are present in

a pure form (single compound), while the extracts are

investigated as a crude. Therefore, the antimicrobial ef -

cacy of S. costus roots could be competitor to antibiot-

Emad M. Abdallah et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS BIOLOGICAL PROPERTIES OF

SAUSSUREA COSTUS

609

Table 4. MIC and MFC of the methanolic and ethanolic extracts of the S. costus roots

Fungal strain MIC mg/ml MFC mg/ml MFC/MIC

MeOH EtOH MeOH EtOH MeOH EtOH

Aspergillus niger ATCC 6275 50 50 50 50 1 1

*MeOH= methanol extract, EtOH=Ethanol extract.

Table 5. Antifeedant activity of ethanolic extract of S.

costus against 4th instar larvae of S. littoralis.

Concentration

Antifeedant index (%) ±SE

Mean*

Days post-treatment

1st 2nd

0.6% 28.39± 4.22e 26.01± 1.64d 27.20 %

1.25% 39.54± 3.27cd 39.75± 5.50c 39.64%

2.5% 35.16± 3.36de 38.34± 3.21c 36.75%

5% 46.58± 1.96c 43.03± 2.52bc 44.80%

10% 58.34± 4.75b 50.90± 2.67b 54.60%

20% 85.36± 2.24a 70.16± 3.23a 77.76%

*Data are expressed as mean ± SE (n=5), total mean of each treatment

at different time intervals, values were analyzed by one-way ANOVA,

where means within each column followed by different letters are

signi cantly different (P< 0.05 by LSD).

Table 6. Starvation percentage (%) of the 4

instar larvae of S. littoralis treated with

the ethanolic extract of S. costus

Treatments Time Average weight

(mg/larva)

Difference*

(mg/larva)

Starvation (%) Average

0.6

0 min

24h

48h

63.13

74.30

93.62

--------

+11.17

+30.49

-------

25.09

24.18

24.63%

1.25

0 min

24h

48h

69.10

76.24

84.12

--------

+7.14

+15.02

-------

42.38

52.05

47.21%

2.50

0 min

24h

48h

60.11

70.00

70.73

--------

+9.89

+10.62

-------

30.58

59.98

45.28%

0 min

24h

48h

60.83

64.20

72.31

--------

+3.37

+11.48

-------

58.55

58.43

58.49%

0 min

24h

48h

68.23

64.75

77.80

--------

-3.48

+9.57

-------

87.94

61.87

74.90%

0 min

24h

48h

68.00

63.25

65.78

--------

-4.75

-2.22

-------

93.39

83.11

88.25%

Control

0 min

24h

48h

68.10

85.12

112.01

--------

+17.02

+43.91

-------

Starved larvae 0 min

24h

48h

61.68

55.39

50.09

--------

-6.29

-11.59

-------

ics if the bioactive compound (s) isolated and studied

in a future studies. This hypothesis is boosted by the

results of MIC, MBC and MFC as shown in (Tables 3

and 4), which revealed that, the methanolic extract was

bacteriostatic to Staphylococcus saprophyticus, Staphy-

lococcus epidermidis, Bacillus cereus and Staphylococ-

cus aureus at 50, 50, 100 and 100 mg/ml, respectively;

and the bactericidal activity was at 100, 200, 200, 200

mg/ml, respectively. As well, the ethanolic extract was

bacteriostatic to Staphylococcus saprophyticus, Staphy-

lococcus epidermidis, Bacillus cereus and Staphylococ-

cus aureus at 50, 50, 50 and 50 mg/ml, respectively; and

the bactericidal activity was detected at 100, 100, 100,

100 mg/ml, respectively. In addition, the methanolic and

ethanolic extracts were bacteriostatic and bactericidal

to Aspergillus niger at 50 mg/ml. The values of MBC/

MIC were ranging between 2-4 for bacterial strains and

1 for fungal strain. Djeussi (2013) has stated that, the

Emad M. Abdallah et al.

610 BIOLOGICAL PROPERTIES OF

SAUSSUREA COSTUS

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

plant extract is a bactericidal when the ratio of MBC/

MIC equals 4 and bacteriostatic when MBC/MIC ratio is

>4. Accordingly, S. costus roots may possess new natu-

ral antimicrobial agents that require isolation of these

novel and natural bioactive molecules.

ANTIFEEDANT PROPERTIES

The antifeedant potential results of the ethanolic extract

of S. costus on the larvae of Spodoptera littoralis are

presented in (Table 5), the crude methanol extract

exhibited antifeedant effect on the 4

instar larvae of

S. littoralis. The antifeedant activity are varying from

27.2%, 39.64%, 36.75%, 44.8%, 54.6 to 77.76% at 0.6,

1.25, 2.5, 5, 10 and 20% concentrations, respectively. It

was noticed that the antifeedant activity on the larvae

increased by days in all concentrations after treatment.

Data in (Table 6) shows the starvation percentage of the

instar larvae of S. littoralis treated with the ethanolic

extract of S. costus. The starvation percentage same as

antifeedant activity which increased with the increas-

ing of the concentration during 48 hours. The root of

S. lappa which have the essential oil and the alkaloid

considered as insect repellent (Kapoor, 2001). The Costu-

nolide that isolated from root extract of S. lappa showed

80% antifeedant activity to citrus pest Papilio demoleus

(Vattikonda et al., 2014). The plant extract of of S. cos-

tus may be useful for effective control of S. littoralis at

larval stages.

CONCLUSION

Roots of Saussurea costus(S. costus) are widely used

in the traditional medicine; it is frequently mentioned

in the Islamic medicine as well as ancient Indian and

Chinese medicine. the current investigation revealed the

presence of many bioactive phytochemical molecules,

antimicrobial activity, and antifeedant effect against

Spodoptera littoralis larvae, which offers a scienti c

basis for traditional uses of S. costus roots as antimicro-

bial and insect repellent. We recommend further future

studies using different solvents and extraction systems

as we assume that there are perhaps more bioactive

compounds in the non-polar or aromatic fraction. More-

over, it is worthy to separate and identify these bioactive

compounds from the roots of S. costus in order to get

new natural and effective drugs.

SOURCE OF SUPPORT

Nil

CONFLICT OF INTEREST

None declared

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