Pak. J. Bot., 43(5): 2613-2617, 2011.

IN VITRO BACTERICIDAL AND BACTERIOSTATIC POTENTIAL OF INGRIDIENTS

OF TRADITIONAL MEDICINE OBTAINED FROM KACHA AREA (RIVER INDUS)
DISTRICT D.I.KHAN, KPK, AGAINST HUMAN BACTERIAL PATHOGENS
ADNAN AMIN* AND M. AYAZ KHAN

Gomal Center of Biochemistry and Biotechnology (GCBB), Gomal University, D.I. Khan,KPK, Pakistan.

Abstract

The aim of this study was to analyze and evaluate antimicrobial potential of medicinal plants obtained from kacha area
of river indus, that are used as ingredients of traditional medicine for treatment of multiple infectious diseases. The
antimicrobial activities of methanol and aqueous extracts of 5 medicinal plants of a traditional medicine were evaluated
against 6 human gram positive (Staphylococcus aureus, Micrococcos luteus) and gram negative (Escherichia coli,
Pseudomonas aeruginosa, Enterobacter, Klebsiella pneumoniae) pathogens. The disc diffusion and broth macro dilution
assay was used to determine the zone of inhibitions and the minimum inhibitory concentration respectively. The
ciprofloxacin and streptomycin were used as standard agents. Both aqueous and methanol fractions of all 5 tested plants
exhibited antimicrobial activity against one or more species of microorganisms. The most active extract found was
Azadirachta indica leaves which represented widest zone of inhibition of 16(±0.05) mm and minimum inhibitory
concentration 0.19mg/ml against Klebsiella pneumoniae. Calotropis procera leaves was found least active representing
lowest Zones of inhibition 3.13(±0.05) mm and highest minimum inhibitory concentration value (20mg/ml) against test
microorganisms. Over all methanol fractions of medicinal plants represented stronger biological activity against test
microorganisms than aqueous extracts. A good majority of extracts were bactericidal. These results afford the ground
information for potential use of crude extracts with high MIC and MBC values. Moreover a synergistic effect is expected
when used in combination. For this further attempt are in progress to investigate antimicrobial potential of combination
medicine.

Introduction 31.49°N and the longitudes70.55°E, at the elevation of

173m, 568'. The Indus River is a major river which flows
Inappropriate use of antibiotics has led to an alarming through Pakistan. The total length of the river is 3,180
increase (Hart & Karriuri, 1998) in multiple drugs kilometers (1,976 miles) and is Pakistan's longest river.
resistance (MDRS) thus necessitating the need to search The river has a total drainage area exceeding 1,165,000
for new biologically active molecules (Sieradski et al., square kilometers (450,000 square miles). The river's
1999) from different sources (microorganism, animals) estimated annual flow stands at around 207 cubic
including plants (Tomoko et al., 2002). Today the kilometers, making it the twenty-first largest river in the
researchers believe that "green medicine" irrespective of world in terms of annual flow. The Indus River Delta
synthetic drugs, are not only free of side effects but have consists of clay and other fertile soils, and is very
and have excellent therapeutic efficacy (Iwu et al., 1999). swampy. The delta receives between 10 and 20 inches of
Use of plants as drugs and remedies for multiple rainfall in a normal year.
human diseases is centuries old (Farnsworth & Loub,
1983; Gulfraz et al., 2011) and even today sciences of Plant Material: Collection was based on information
traditional medicinal plants is practiced successfully given by local inhabitants during ethno botanical surveys.
worldwide (Qin & Xu, 1998) and are relied upon by 80% Identification of plant species was performed at the
of the world’s population, as they represent a vast department of Pharmacognosy, Faculty of Pharmacy
untapped source for medicines.(Iwu et al., 1999). There Gomal University D.I.Khan, Khyber Pakhtoon Khawa,
are more than 35,000 plant species being used in various Pakistan. The identified species were Azadirachta indica
human cultures around the world for medicinal purpose (Meliaceae), Cuseuta reflexa (Meliaceae), Rhazya Stricta
(Srinivasan et al., 2001), While more than 2000 species (Apocynaceae), Calotropis procera (Apocynaceae) and
are reported with therapeutic value and are being used Melia azedarach (Meliaceae) (Table 1).
through generations in various traditional health care
systems (Gajera et al., 2005; Arora, 1998). As a Preparation of plant extracts: Whole plant material
significant majority of world’s population believe in folk (excluding the root and fruit portion) collected in the
medicine, the present study was designed to determine month of August-September 2009 at early morning hours
antimicrobial spectrum of traditional medicine. Five was subjected to sun shade drying(15-20 days) followed
representative plants used as ingredients of folk medicine by powdering using Wiley Mill (mesh size 300). About
were evaluated for antimicrobial activity. 200 g of dry powdered leaves were extracted with 300ml
of 95% ethanol using rotary shaker (190-200 rpm)
Materials and Methods overnight, filtering it with filter paper and concentrating it
to one-fifth of the volume. Crude aqueous extract was
Study area: Plant species were collected from Kacha area prepared by subjecting plant material (10 g) to slow heat
(River Indus delta) district D.I.Khan, Khyber Pakhtoon for 6 hours and filtered through filter paper and
Khawa, Pakistan. The district D.I.Khan lies in the South concentrated to one-fifth of the total volume (Vlietinck &
Zone of Khyber Pakhtoon Khawa between the latitudes Vanden Berghe, 1991)
*Corresponding author: [email protected], Ph. +92(966)750424-9 ext 3058, Fax +92(966)750131
2614 ADNAN AMIN & M. AYAZ KHAN.

Table 1. Plants material and traditional claims.

Botanical name Family Common name Traditional claim Part used
Azadirachta indica Meliaceae Indian Lilac / Neem Antimicrobial Whole Plant
Melia azedarach Meliaceae Bakain / Beed tree Antimicrobial Leaves/Stem
Calotropis procera Apocynaceae Milkweed / Aak Antimicrobial Milk/ Leaves
Cuseuta reflexa Convolulacae Dodder / Amerbel Antimicrobial Whole Plant
Rhazya Stricta Apocynaceae Daryai booti / Veena Antimicrobial Leaves/Stem

Microorganisms: Six bacterial species viz., Escherichia phenol red. For susceptibility testing, in a first step
coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, Mueller Hinton broth (1ml) was distributed from the first
Pseudomonas aeruginosa (clinical strain/PIMS), to the twelfth test tubes. Dry extracts and pure compounds
Enterobacter (clinical strain/PIMS), Staphylococcus areus were initially dissolved in DMSO (l ml) and subsequently
(MRSA, clinical strain/PIMS), Micrococcus luteus in Mueller Hinton broth, to reach a final concentration of
(clinical strain/PIMS) were used in antimicrobial assay. 20mg/ml. These solutions (1 ml) were added to the first
Strains were obtained from microbiology research lab test tube. Successive dilutions were done by transferring
(MRL), Microbiology department, Quaid-e-Azam the mixture/solution (1 ml) from the first to the eleventh
University, Islamabad, Pakistan, where these were tube. An aliquot (l ml) was discarded from the eleventh
identified and characterized. These strains were tube. The twelfth tube served as growth control as no
maintained on agar slants at 4°C in Gomal Center of sample (extract, pure compounds, or reference antibiotics)
Biochemistry and Biotechnology (GCBB) for was added. A microbial suspension (1 ml, 105 colony
antimicrobial tests. The microorganisms were incubated forming units), obtained from an overnight growth at 37◦C
overnight at 37°C in Mueller-Hinton Broth (Oxoid) at pH was added to each test tube. The final concentration of the
7.4. The reference antibiotics used were ciprofloxacin extracts adopted to evaluate the antimicrobial activity
(10µg) and streptomycin (10µg) (Oxoid) (Table 1). ranged from 20 mg/ml to 0.095mg/ml. Test tubes were
incubated aerobically at 37°C for 24 h before being read.
Inoculum preparation: A loopful of isolated colonies The MIC was considered as the lowest concentration of
was inoculated into 4 ml peptone water and incubated at the sample that prevented visible growth or changed in
37°C for 4 h. The turbidity of actively growing bacterial color from red to yellow due to the formation of acidic
suspension was adjusted to match the turbidity standard of metabolites corresponding to microbial growth.
0.5 McFarland units prepared by mixing 0.5 ml of 1.75%
(w/v) Barium chloride dihydrate with 99.5 ml 1% (v/v) Minimum inhibitory concentration (MBC): Minimum
Sulphuric acid The concentration of suspension was Inhibitory Concentration (MBC) of the selected plant
standardized by adjusting optical density to 0.1 at 600nm parts was measured by the viable cell count method (Toda
wavelength (Shimadzu UV 1700) (TereSchuk et al., 1997) et al., 1989), and the results were expressed as number of
This turbidity is equivalent to approximately 1–2 108 viable cells as a percentage of the control.
colony-forming units per milliliter (cfu/ml). This 24 h
grown suspension was used for further testing. Results and Discussions

Antibacterial activity A total of 20 organic and aqueous extracts from 5

different plants species were investigated. The
Disc diffusion method: The antimicrobial test was antimicrobial activities (zones of inhibition mm) of
performed by the disc diffusion method as described by standard drugs are listed (Table 2).The disc diffusion
Bauer et al., (1966) modified by Vlietinck & Vanden method was employed for determination of zone of
Berghe, (1991) using a cell suspension of about 1.5×106 inhibition between the edge of the filter paper and the
CFU/ml obtained following a 0.5 McFarland turbidity edge of the inhibition area. The results of the in vitro
standard. The concentration of the suspension was antibacterial activity brought to light interesting facts.
standardized by adjusting the optical density to 0.1 at Organic leaf extracts of Azadirachta indica were found
600nm wavelength (SHIMADZU UV-1700 highly active with widest zones of inhibition range
spectrophotometer) (Tereschuck et al., 1997). Petri dishes 6.02(±0.1) to 16(±0.05) mm, while Calotropis procera
were filled with Agar Mueller Hinton agar and inoculated (white exudates) was found inactive against test strains
with the test microorganism. Filter paper discs (6mm (Table 3, 4). Almost all crude extracts (aqueous and
diameter) were prepared at the concentration of 50mg/disc organic) were found active against gram negative than
for crude extracts. gram positive bacteria. The MIC and MBC values are
listed in Table 5 and Table 6. It was observed that plants
Macrobroth dilution method: The minimum inhibitory differ significantly in their activities against tested
concentration (MIC), considered as the lowest microorganisms; most of the plants were found active
concentration of the sample, that inhibits the visible against clinical isolate of Enterobacter, followed by
growth of a microbe, was determined by the macrobroth Escherichia coli, Klebsiella pneumoniae, Pseudomonas
dilution method (Carbonnelle et al., 1987), in Muller aeroginosa, Micrococcus luteus and Staphylococcus
Hinton broth supplemented with 10% glucose and 0.5% aureus.
BACTERICIDAL POTENTIAL OF MEDICINE AGAINST HUMAN PATHOGENS 2615

Table 2. Zone of inhibitions of reference antibiotic standards.

Microorganism (mm)
Antibiotic
Ec Kp Ent Ps Ml Sta
Ciprofloxacin 14 16 16 15 5 6
Streptomycin 15 14 13 12 5 7
Ec = E.Coli, Kp = Klebsiella pneumoniae, Ent = Enterobacter, Ps = Pseudomonas aeroginosa,
Ml = Micrococcus luteus, Sta = Staphylococcus areus (methicilline resistant)

Table 3. Zones of inhibitions of aqueous fractions.

Microorganism (mm)
Plant
EC KP Ent PS ML Sta
Azadirachta indica (Stem) 8.06(±0.05) na 7.09(±0.5) na na na
Azadirachta indica (Leaves) na na 6.02(±0.1) na na na
Melia azedarach (Stem) na 9.03(±0.5) na na na na
Melia azedarach (Leaves) 8.02(±0.1) na na na na na
Calotropis procera (Stem) na na 11.03(±0.05) na na na
Calotropis procera (Leaves) na na 3.13(±0.05) na na na
Calotropis procera (White exudate) na na na na na na
Cuseuta reflexa (Stem) 12.09(±0.1) 4.06(±0.5 na na na na
Rhazya stricta (Stem) na na 6.03(±0.05) na na na
Rhazya stricta (Leaves) na na na na na na
Ec = E.Coli, Kp = Klebsiella pneumoniae, Ent = Enterobacter, Ps = Pseudomonas aeroginosa, Ml = Micrococcus luteus,
Sta = Staphylococcus areus (methicilline resistant), na = Not active

Table 4. Zones of inhibitions of organic fractions.

Microorganism (mm)
Plant
EC KP Ent PS ML Sta
Azadirachta indica (Stem) na na 6.99(±0.1) na na na
Azadirachta indica (Leaves) 14.03(±0.5) 16.0(±0.0) 14.09(±0.1) 9.06(±0.5) 11.0(±0.05) 9.0(±0.05)
Melia azedarach (Stem) na na na na na Na
Melia azedarach (Leaves) na na na na na 9.06(±0.05)
Calotropis procera (Stem) na na na na na na
Calotropis procera (Leaves) na na na 12.0(±0.1) 16(±0.5) na
Calotropis procera (White exudate) na na na na na na
Cuseuta reflexa (Stem) 6.03(±0.05) na na na na na
Rhazya stricta (Stem) 6.0(±0.5) 7.0(±0.5) 7.0(±0.05) na na na
Rhazya stricta (Leaves) 6.1(±0.5) 5.06(±0.05) na na na na
Ec = E.Coli, Kp = Klebsiella pneumoniae, Ent = Enterobacter, Ps = Pseudomonas aeroginosa, Ml = Micrococcus luteus, Sta =
Staphylococcus areus (methicilline resistant), na = Not active

Table 5. Minimum inhibitory concentration of medicinal plants.

Minimum inhibitory concentration (mg/ml)
Plant Aqueous fraction Organic fraction
EC Kp Ent Ps Ml Sta Ec Kp Ent Ps Ml Sta
Azadirachta indica (Stem) 0.78 - 1.25 - - - - - 1.25 - - -
Azadirachta indica (Leaves) - - 1.25 - - - 0.19 0.19 0.19 0.78 0.19 0.156
Melia azedarach (Stem) - 0.19 - - - - - - - - -
Melia azedarach (Leaves) 0.78 - - - - - - - - - - 0.78
Calotropis procera (Stem) - - 0.19 - - - - - - - - -
Calotropis procera (Leaves) - - 20 - - - - - - 0.312 0.78 -
Calotropis procera (White exudate) - - - - - - - - - - - -
Cuseuta reflexa (Stem) 2.5 >20 - - - - - - - - - -
Rhazya stricta (Stem) - - >20 - - - >20 >20 - - - -
Rhazya stricta (Leaves) - - - - - - >20 >20 - - - -
Ec = E.Coli, Kp = Klebsiella pneumoniae, Ent = Enterobacter, Ps = Pseudomonas aeroginosa, Ml = Micrococcus luteus, Sta =
Staphylococcus areus (methicilline resistant), - Not determined
2616 ADNAN AMIN & M. AYAZ KHAN.

Table 6. Minimum bactericidal concentration (MBC) of medicinal plants.

Minimum bactericidal concentration (mg/ml)
Plant Aqueous fraction Organic fraction
EC Kp Ent Ps Ml Sta Ec Kp Ent Ps Ml Sta
Azadirachta indica (Stem) 0.625 - 1.25 - - - - - - - - -
Azadirachta indica (Leaves) - - 5 - - - 0.156 0.156 0.39 0.78 0.19 0.156
Melia azedarach (Stem) - 0.312 - - - - - - - - -
Melia azedarach (Leaves) - - - - - - - - - - - 1.25
Calotropis procera (Stem) - - 0.156 - - - - - - - - -
Calotropis procera (Leaves) - - 20 - - - - - - 1.25 0.78 -
Cuseuta reflexa (Stem) 2.5 - - - - - - - - - - -
Ec = E.Coli, Kp = Klebsiella pneumoniae, Ent = Enterobacter, Ps = Pseudomonas aeroginosa, Ml = Micrococcus luteus, Sta =
Staphylococcus areus (methicilline resistant), - = Not determined

Almost all plants used in the traditional medicine References

exhibited moderate antimicrobial activities when tested
individually as reported earlier (Wafaa et al., 2007,
Arora, D.S. 1998. Antimicrobial activity of tea (Camellia
Takazawa 1982, Parrotta, 2001) except Rhyzea stricta sinensis). Antibio Chemother, 2: 4-5.
and cuscuta reflexa. Little is known regarding Bauer, A.W., W.M.M. Kirby, J.C. Sherris and M. Turck. 1966.
antimicrobial activities of Rhyzea stricta and Cuscuta Antibiotic susceptibility testing by a standardized single
reflexa (Pal et al., 2006). However present study confirms disk method. Amer. J. Clin. Pathol., 45: 493-496.
their antimicrobial (bacteriostatic) potential. Although Carbonnelle, B., F. Denis, A. Marmonier, G. Pignon and R.
their antibacterial activities were found less (Paul et al., Vargue. 1987. Bactériologie Médicale. In: Techniques
Usuelles. S.I.M.E.P., 130-198.
2006, Rios & Rico, 2005) compared to other plant Eloff, J.N. 1998. Which extractant should be used for the
extracts. Most of the crude extracts exhibited bactericidal screening and isolation of antimicrobial components from
activities than bacteriostatic. The bactericidal activities plants? J. Ethnopharmacol. 60: 1-8.
were recorded as three to four times higher than static Farnsworth, N.R. and W.D. Loub, 1983. In Plants: The
activities (Gnanamani et al., 2003, Kharea et al., 2005) potentials for extracting protein, medicines, and other
which simply highlights efftiveness of traditional useful chemicals. Workshop Proceedings, Congress office
of Technology Assessment, Washington, D.C. 178.
medicine. It is worthy to note that MIC and MBC values Gajera, H.P., S.V. Patel and B.A. Golakiya. 2005. Antioxidant
of assayed plant extracts are comparatively higher as properties of some therapeutically active medicinal plants,
compared to reported elsewhere (Kareem et al., 2008, an overview. J. Med. All Profess., 27: 91-100.
Subapriya & Nagini, 2005). It is suggested that individual Gnanamani, K., P. Shanmuga, N. Radhakrishnan and B. Mary.
plants may be contributing to the effectiveness of 2003. Antibacterial activity of two plant extracts on eight
traditional medicine. Moreover the dose used for burn pathogens. J. Ethnopharmacol, 86: 59-61.
treatment of infections reported was high (2 gram thrice a Gulfraz, M., A. Sadiq, H. Tariq, M. Imran, R. Qureshi and A.
day) which obviates the study findings. Organic fractions Zeenat. 2011. Phytochemical analysis and antibacterial
were found highly active than aqueous extracts, this activity of Eruca sativa seed. Pak. J. Bot., 43(2): 1351-
proves the fact that majority of active constituents are 1359.
Hart, C.A. and S. Karriuri. 1998. Antimicrobial resistance in
soluble in organic solvents. Eloff, (1998) Spectrum of
developing countries. B. Med J., 317: 421-452.
activity of plants tested was limited to gram negative Iwu, M.W., A.R. Duncan and C.O. Okunji. 1999. New
strains than gram positive strains. This finding reflects antimicrobials of plant origin. In: Janick J. (Eds.):
limited use of medicinal plants against mixed infections Perspectives on new crops and new uses. ASHS press.
(Sieradski et al., 1999). Alexandria, VA: 457-462.
Kareem, S., O.I. Akpan and O.P. Ojo. 2008. Antimicrobial
Conclusion activities of Calotropis procera on selected pathogenic
microorganisms. Afri. J. Biomed Res., 11: 105-110.
Kharea, S.K., S. Rajni, J. Astha , P. Shikha and G. Deepti. 2005.
Almost all crude extracts are already known for less Antimicrobial activity of some natural dyes. J. Dye Pigm.,
or high antimicrobial activities but during present study 66: 99-102.
only a few crude extracts proved good antimicrobial Parrotta, J.A. 2001. Healing plants of peninsular India. A.B.
activities when tested individually against selected International Wallingford, UK. p. 944.
microorganisms. Rest of the extracts were although found Paul, C., J. Arnold, L. Vlietinck, V.B.D. Vanden and M. Louis.
2006. Anti-infective potential of natural products: How to
biologically active (except one) but exhibited high develop a stronger In vitro proof-of-concept. J.
bactericidal and bacteristatic activities. Therefore it is Ethnopharmacol., 106: 290-302.
concluded that crude extracts possess good potential for Qin, G.W. and R.S. Xu. 1998. Recent advances on bioactive
antimicrobial activity. Further investigations are in natural products from Chinese medicinal plants. Med. Res.
progress to compare antimicrobial activities of Rev., 18: 375-382.
individually tested extracts with folk medicine. Rios, J.L. and M.C. Recio. 2005. Medicinal plants and
antimicrobial activity J. Ethnopharmacol., 100: 80-84.
BACTERICIDAL POTENTIAL OF MEDICINE AGAINST HUMAN PATHOGENS 2617

Sieradski, K., R.B. Roberts, S.W. Haber and A. Tomasz. 1999. Tomoko, N., A. Takashi, T. Hirumo, I. Yuka, M. Hiroko, I.
The development of vancomycin resistance in a patient Munikazo, T. Totshiyuki, I. Tetsuro, A. Fujio, I. Iria, N.
with methicilline staphylococcus aureus infection. N Eng. J. Tsotumo and W. Kazuhito. 2002. Antibacterial activity of
Med., 340: 517-523. extracts prepared from tropical and subtropical plants on
Srinivasan, D., S. Nathan, T. Suresh and P. Lakshmana. 2001. methicillin-resistant Staphylococcus aureus. J. Heal. Sci.,
Antimicrobial activity of certain Indian medicinal plants 48: 273-276.
used in folkloric medicine. J. Ethnopharmacol., 74: 217- Vlietinck, A.J. and D.A. Vanden Berghe. 1991. Can
220. Ethnopharmacology contribute to the development of
Subapriya, R. and S. Nagini. 2005. Medicinal properties of neem antiviral drugs? J. Ethnopharmacol., 32: 141-153.
leaves: A review. Curr. Med. Chem. Anticancer. Agent., Wafaa, A. H., A. Hassan and M.A. Nefisa. 2007. Biological and
5(2): 146-9. anti-microbial activities of aqueous extracts from Neem
Takazawa, H., F. Tajima and C. Miyashifa. 1982. An antifungal
Tree (Azadirachta indica A. Juss., Meliaceae), J. Appl. Sci.
compound from shitake (Lentinus edodes) Yaku Zass
(Japanese), 102: 489-491. Res., 3: 1050- 1055.
Toda, M., S. Okubo, R. Hiyoshi and T. Shimamura. 1989. The
bactericidal activity of tea and coffee. Leter Appl
Microbiol, 8: 123-125.

(Received for publication 14 May 2010)