# Phytochemical, Antimicrobial, and Toxicological Evaluation of Traditional Herbs Used to Treat Sore Throat

Authors: Arifa Mehreen; Muzzamil Waheed; Iram Liaqat; Najma Arshad
Journal: BioMed Research International
 (2016)

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## Abstract

Thein vitro antibacterial activities of 29 traditional medicinal plants used in respiratory ailments were assessed on multidrug resistant Gram-positive and Gram-negative bacteria isolated from the sore throat patients and two reference strains. The methanolic, n-hexane, and aqueous extracts were screened by the agar well diffusion assay. Bioactive fractions of effective extracts were identified on TLC coupled with bioautography, while their toxicity was determined using haemolytic assay against human erythrocytes. Qualitative and quantitative phytochemical analysis of effective extracts was also performed. Methanolic extract of 18 plants showed antimicrobial activity against test strains.Adhatoda vasica (ZI = 17–21 mm, MIC: 7.12–62.5 μg/mL),Althaea officinalis (ZI = 16–20 mm, MIC: 15.62–31.25 μg/mL),Cordia latifolia (ZI = 16–20 mm, MIC: 12.62–62.5 μg/mL),Origanum vulgare (ZI = 20–22 mm, MIC: 3–15.62 μg/mL),Thymus vulgaris (ZI = 21–25 mm, MIC: 7.81–31.25 μg/mL), andZiziphus jujuba (ZI = 14–20 mm, MIC: 7.81–31.25 μg/mL) showed significant antibacterial activity. Alkaloid fractions ofAdhatoda vasica,Cordia latifolia, andOriganum vulgare and flavonoid fraction of theAlthaea officinalis,Origanum vulgare,Thymus Vulgaris, andZiziphus jujuba exhibited antimicrobial activity. Effective plant extracts show 0.93–0.7% erythrocyte haemolysis. The results obtained from this study provide a scientific rationale for the traditional use of these herbs and laid the basis for future studies to explore novel antimicrobial compounds.


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## Body

## 1. Introduction

Infectious diseases are a major cause of death and disability in humans as they are responsible for about 22% of the disease burden globally [1]. Sore throat is the most prevalent illness known to mankind. It has been reported that almost 25% of population experienced 2-3 episodes of infection every year [2].Staphylococcus aureus (S. aureus) is an opportunistic pathogen carried asymptomatically on the human body, mainly the anterior nares and throat [3]. Carriage ofS. aureus plays a key role in the epidemiology and pathogenesis of infection and is associated with an increased risk of infectious complications after surgery in patients, intravascular devices, and superinfections [4]. Approximately 80% of invasive nosocomial infections are caused by strains of endogenous origin of nasal and throat [5, 6]. Antibiotics have saved lives of millions of people and contributed to improving quality and expectancy of life over the last century [7]. Since the discovery of penicillin in 1940s, antibiotics have been the best choice for the treatment of infectious diseases. However, the clinical efficacy of many presently used antibiotics is being threatened by the emergence of multidrug resistant (MDR) pathogens, as well as the use and misuse of existing antibiotics in humans, animals, and agriculture [8]. Several other factors like poverty, overcrowding, and lack of education all make this wonderful weapon useless. A number of multidrug resistant bacteria are increasingly observed in the normal community and hospital setting [9]. Methicillin-resistantS. aureus (MRSA) strains have acquired a gene that makes them resistant to all beta-lactam antibiotics. It is estimated that more people have died of MRSA infection than of other life threating diseases like HIV/AIDS and tuberculosis [10]. Hence, search for novel antimicrobial compounds or alternative therapy for these resistant infectious agents is inevitable.Traditional herbal medicines have received much attention as a source of novel antibacterial drugs since they are considered as safe for human use [11]. Plant based medicines are widely used for primary health care in many developing countries [12]. As a result, it is found that about 60–80% of the world population relies on traditional treatment [13]. Plants have also been explored to get crude natural extracts for testing and developing potent and new antimicrobial drugs. A large number of secondary metabolites such as alkaloids, tannins, and flavonoids extracted from different medicinal plants have shown antimicrobial potential [14]. This finding has raised hope of obtaining novel antibiotics that can aid in fighting against multiple drug resistant bacteria [15].For tracking and characterization of the active components thin layer chromatography (TLC) coupled with direct bioautography has gained the popularity as an effective technique [16]. Although TLC has a lower separation power than HPLC and GC, it offers several benefits such as limited samples preparation and ability to run many samples in parallel [17]. It has been proven to be effective, time-saving, and economical tool for bioassay-guided isolation of antibacterial components [18].For the use of these herbs or their active part in human consumption, their safety towards human must be evaluated. Plants may contain such compounds that may have haemolytic or antihaemolytic effect on human erythrocytes. Therefore, many of the traditional plants need to be evaluated for their potential haemolytic activity to be categorised as a safer remedy to cure diseases.The aims of the present study were (i) to screen the selected traditional herbs, chosen for the fact that they have been used in treating respiratory ailments or their symptoms, for potential antibacterial activity, (ii) to isolate active fraction(s), showing antibacterial activity by TLC direct bioautography, (iii) to carry out qualitative and quantitative phytochemical studies on active fractions, and (iv) to do toxicity analysis by determining the haemolysis percentage of the extract against human blood cells.


## 2. Experimental

### 2.1. Preparation of Plant Extract and Antimicrobial Efficacy

The fresh plant materials of selected plant (Table1) were dried in open air protected from direct exposure to sunlight. Dried plant materials were separately powdered and extracted with 95% methanol or n-hexane using Soxhlet method, while aqueous extracts were prepared by soaking plant material in deionized water for 02-03 hours at 70°C. The extract so obtained was concentrated on rotary evaporator. The resulting material was dried to maximum possible level in freeze dryer and stored in glass vials at 4°C until used. For antimicrobial testing, 100 mg of the dry residue was dissolved in respective solvents or DMSO and evaluated through well diffusion assay against the six clinical and standard strains. Zone of inhibition was measured in mm and the breakpoint for susceptibility was taken as zone of inhibition >11 mm. Vancomycin and gentamicin were used as positive control, while DMSO was employed as a negative control.Table 1
List of plants used in herbal medicine for the treatment of sore throat.
Serial number
Plant name
Family name
Common name
Part of plant used
Voucher number
1
Adhatoda vasica(L.)
Acanthaceae
Nees
Leaves
ZD-IM-01
2
Aloe vera(L.)
Xanthorrhoeaceae
Alovera
Leaves
ZD-IM-02
3
Althaea officinalis(L.)
Malvaceae
Marshmallow
Flowers, leaves
ZD-IM-03
4
Cinnamomum zeylanicum(L.)
Lauraceae
Cinnamon
Bark
ZD-IM-04
5
Cordia latifolia(Roxb.)
Boraginaceae
Sapistan, lasori
Fruits
ZD-IM-05
6
Emblica officinalis(Gaertn.)
Euphorbiaceae
Indian gooseberry
Fruits
ZD-IM-06
7
Eugenia caryophyllata(L.)
Myrtaceae
Clove
Fruit
ZD-IM-07
8
Glycyrrhiza glabra(L.)
Leguminosae
Liquorice
Roots
ZD-IM-08
9
Hyssopus officinalis(L.)
Lamiaceae
Hyssop
Leaves, flowers
ZD-IM-09
10
Lawsonia inermis(L.)
Lythraceae
Henna
Leaves
ZD-IM-10
11
Linum usitatissimum(L.)
Linaceae
Flax seed
Seed
ZD-IM-11
12
Malva sylvestris(L.)
Malvaceae
Mallow
Flowers
ZD-IM-12
13
Matricaria chamomilla(L.)
Asteraceae
Chamomile
Flowers
ZD-IM-13
14
Mentha piperita(L.)
Lamiaceae
Mint
Leaves
ZD-IM-14
15
Moringa oleifera(Lam.)
Moringaceae
Moringa, drumstick tree
Leaves
ZD-IM-15
16
Morus alba(L.)
Moraceae
White mulberry
Fruits, leaves
ZD-IM-16
17
Ocimum basilicum(L.)
Labiatae
Basil
Leaves
ZD-IM-17
18
Onosma bracteatum(L.)
Boraginaceae
Gao zuban
Leaves, flowers
ZD-IM-18
19
Origanum vulgare(L.)
Labiatae
Oregano
Leaves
ZD-IM-19
20
Pimpinella anisum(L.)
Umbelliferae
Anise
Fruit
ZD-IM-20
21
Piper longum(L.)
Piperaceae
Long pepper
Fruit
ZD-IM-21
22
Saussurea lappa(L.)
Compositae
Kuth
Root
ZD-IM-22
23
Sisymbrium irio(L.)
Brassicaceae
London rocket
Seeds
ZD-IM-23
24
Syzygium cumini(L.)
Myrtaceae
Jambolan
Fruit, leaves
ZD-IM-24
25
Thymus vulgaris(L.)
Lamiaceae
Thyme
Leaves
ZD-IM-25
26
Viola odorata(L.)
Violaceae
Sweet violet
Leaves, flowers
ZD-IM-26
27
Vitis vinifera(L.)
Vitaceae
Grapes
Leaves, fruits
ZD-IM-27
28
Zingiber officinale(Roscoe)
Zingiberaceae
Ginger
Root
ZD-IM-28
29
Ziziphus jujuba(mill.)
Rhamnaceae
Red date
Fruit
ZD-IM-29
The plants materials were dried in shades, identified by the help of Dr. Muhammad Nasir (Taxonomist, Department of Botany, University of the Punjab), and kept in Microbiology and Immunology Laboratory (Department of Zoology, University of the Punjab) under the voucher numbers listed above.


### 2.2. Bacterial Strains

Methicillin-resistant and methicillin-susceptibleS. aureus andPseudomonas aeruginosa (P. aeruginosa) strains were isolated from sore throat patients from tertiary care hospitals at Faisalabad. ATCC strains ofEscherichia coli (E. coli) andS. aureus were obtained from lab stock. All strains were routinely grown and identified. For further use they were preserved at −20°C.


### 2.3. Antimicrobial Susceptibility of Clinical Isolates

Antibiotic sensitivity of the isolatedS. aureus strains was determined by Standard Disc Diffusion Method [19]. Different antibiotics (Oxoid) were used in the present work: tetracycline (30 μg), vancomycin (30 μg), clindamycin (2 μg), methicillin (5 μg), cefixime (5 μg), erythromycin (15 μg), oxacillin (1 μg), gentamicin (10 μg), streptomycin (10 μg), chloramphenicol (30 μg), linezolid (10 μg), ampicillin-sulbactam (20 μg), and penicillin (10 μg) [20].


### 2.4. Minimum Inhibitory Concentration (MIC)

Minimum inhibitory concentrations (MIC) of antibiotics and plant extracts were determined by the microdilution method as described by Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards, NCCLS) [20]. The antibiotic was serially diluted in Mueller-Hinton broth in 96-well plates. The plant extracts solutions were separately added to wells in a final concentration of 1 mg/mL; then bacterial inoculum size of 105 CFU/mL was added to each well. Controls without plant extracts, without bacterial inoculum, or with plant extracts only were also included in the experiment. Each plant extract was run in duplicate. The test plates were incubated at 37°C for 24 h. The MIC was taken as the minimum concentration of the dilutions that inhibited the growth of the test microorganism. The growth was recorded in terms of button formation in wells; it was confirmed by adding tetrazolium chloride.


### 2.5. Toxicity of Extracts to Human Erythrocytes

The cellular toxicity of the extracts from different plants was determined [21]. Erythrocytes were isolated from human blood by removing plasma and buffy coat and suspended in phosphate buffered saline (10 mM phosphate, 150 mM sodium chloride) making a final concentration of 2% erythrocytes. The dilutions of plant extract (100 mg/mL) were made and mixed with erythrocytes keeping the final volume up to 1 mL. The cells were incubated for 1 hr at 37°C and finally centrifuged at 1500 rpm for 10 minutes [22]. The lysis of the cells was observed by determining absorbance at 540 nm using spectrometer. Cell in distilled water was taken as negative and cells suspended in normal saline were taken as positive control. The mixture was centrifuged at 1500 rpm for 1 minute in a laboratory centrifuge. The free haemoglobin in the supernatant was measured at 540 nm.The haemolysis percentage was calculated by the following formula:(1)
%
haemolysis
=
A
t
-
A
n
A
c
-
A
n
∗
100
.At is absorbance of test sample, An is absorbance of the control (saline), and Ac is absorbance of the control (water).


### 2.6. Screening and Estimation of Some Phytochemical Components

Phytochemical screening of the effective extract for the detection of alkaloids, flavonoids, steroids, terpenoid, and other secondary metabolites was carried out by following the procedure of Trease and Evans [23]. Total alkaloids, flavonoids, and saponin fraction were also separated and their antimicrobial activity was carried out [24].


### 2.7. Thin Layer Chromatography, Bioautography, and Identification of the Chemical Nature of the Bioactive Compound

For TLC, 10–20μL of plant extract was loaded on silica gel sheet (Merck, Darmstadt, Germany). Different solvent systems, chloroform : methanol (80 : 20), n-butanol : acetic acid : water (4 : 1 : 1), and acetone : methanol (1 : 1), were used as mobile phase. The TLC plates (a–f) were run in triplicate. Plate “a” was used to determine the spots by visualizing in UV light to see if the separated spot was UV active after which it was sprayed with vanillin sulphuric acid reagent (2%) to detect all the separated spots. Plate “b” was used for autobiography. Agar overlay method [25] was used for direct bioautographic assay, with little modification, to detect antimicrobial fractions in plant extracts which were active against clinical sample of MRSA andE. coli. Soft nutrient agar (1%) was seeded with 106 CFU/mL of MRSA/E. coli and overlaid on TLC plate. It was then incubated overnight in a controlled environment at 37°C. Subsequently, the plates were sprayed with 2,3,5-triphenyltetrazolium chloride and further incubated for three hours at 37°C. Microbial growth inhibition appeared as clear zone against a pink background. The Rf value of the bioactive spots was measured as the ratio of mobility of centre of a bioactive spot against the total distance travelled by the solvent front. The bioactive spots were scraped from the TLC plate “c” and eluted in 500 μL ethanol. The solution was filtered using Whatman filter paper number 1 and concentrated and again bioassay was performed to recheck the activity. Plates “d, e, and f” were sprayed separately with TLC reagents including FeCl3 (10%), Dragendorff’s reagent, and KOH (5N) to detect the presence of phenolic compounds, alkaloids, and flavonoids, respectively, in bioactive spots.


## 2.1. Preparation of Plant Extract and Antimicrobial Efficacy

The fresh plant materials of selected plant (Table1) were dried in open air protected from direct exposure to sunlight. Dried plant materials were separately powdered and extracted with 95% methanol or n-hexane using Soxhlet method, while aqueous extracts were prepared by soaking plant material in deionized water for 02-03 hours at 70°C. The extract so obtained was concentrated on rotary evaporator. The resulting material was dried to maximum possible level in freeze dryer and stored in glass vials at 4°C until used. For antimicrobial testing, 100 mg of the dry residue was dissolved in respective solvents or DMSO and evaluated through well diffusion assay against the six clinical and standard strains. Zone of inhibition was measured in mm and the breakpoint for susceptibility was taken as zone of inhibition >11 mm. Vancomycin and gentamicin were used as positive control, while DMSO was employed as a negative control.Table 1
List of plants used in herbal medicine for the treatment of sore throat.
Serial number
Plant name
Family name
Common name
Part of plant used
Voucher number
1
Adhatoda vasica(L.)
Acanthaceae
Nees
Leaves
ZD-IM-01
2
Aloe vera(L.)
Xanthorrhoeaceae
Alovera
Leaves
ZD-IM-02
3
Althaea officinalis(L.)
Malvaceae
Marshmallow
Flowers, leaves
ZD-IM-03
4
Cinnamomum zeylanicum(L.)
Lauraceae
Cinnamon
Bark
ZD-IM-04
5
Cordia latifolia(Roxb.)
Boraginaceae
Sapistan, lasori
Fruits
ZD-IM-05
6
Emblica officinalis(Gaertn.)
Euphorbiaceae
Indian gooseberry
Fruits
ZD-IM-06
7
Eugenia caryophyllata(L.)
Myrtaceae
Clove
Fruit
ZD-IM-07
8
Glycyrrhiza glabra(L.)
Leguminosae
Liquorice
Roots
ZD-IM-08
9
Hyssopus officinalis(L.)
Lamiaceae
Hyssop
Leaves, flowers
ZD-IM-09
10
Lawsonia inermis(L.)
Lythraceae
Henna
Leaves
ZD-IM-10
11
Linum usitatissimum(L.)
Linaceae
Flax seed
Seed
ZD-IM-11
12
Malva sylvestris(L.)
Malvaceae
Mallow
Flowers
ZD-IM-12
13
Matricaria chamomilla(L.)
Asteraceae
Chamomile
Flowers
ZD-IM-13
14
Mentha piperita(L.)
Lamiaceae
Mint
Leaves
ZD-IM-14
15
Moringa oleifera(Lam.)
Moringaceae
Moringa, drumstick tree
Leaves
ZD-IM-15
16
Morus alba(L.)
Moraceae
White mulberry
Fruits, leaves
ZD-IM-16
17
Ocimum basilicum(L.)
Labiatae
Basil
Leaves
ZD-IM-17
18
Onosma bracteatum(L.)
Boraginaceae
Gao zuban
Leaves, flowers
ZD-IM-18
19
Origanum vulgare(L.)
Labiatae
Oregano
Leaves
ZD-IM-19
20
Pimpinella anisum(L.)
Umbelliferae
Anise
Fruit
ZD-IM-20
21
Piper longum(L.)
Piperaceae
Long pepper
Fruit
ZD-IM-21
22
Saussurea lappa(L.)
Compositae
Kuth
Root
ZD-IM-22
23
Sisymbrium irio(L.)
Brassicaceae
London rocket
Seeds
ZD-IM-23
24
Syzygium cumini(L.)
Myrtaceae
Jambolan
Fruit, leaves
ZD-IM-24
25
Thymus vulgaris(L.)
Lamiaceae
Thyme
Leaves
ZD-IM-25
26
Viola odorata(L.)
Violaceae
Sweet violet
Leaves, flowers
ZD-IM-26
27
Vitis vinifera(L.)
Vitaceae
Grapes
Leaves, fruits
ZD-IM-27
28
Zingiber officinale(Roscoe)
Zingiberaceae
Ginger
Root
ZD-IM-28
29
Ziziphus jujuba(mill.)
Rhamnaceae
Red date
Fruit
ZD-IM-29
The plants materials were dried in shades, identified by the help of Dr. Muhammad Nasir (Taxonomist, Department of Botany, University of the Punjab), and kept in Microbiology and Immunology Laboratory (Department of Zoology, University of the Punjab) under the voucher numbers listed above.


## 2.2. Bacterial Strains

Methicillin-resistant and methicillin-susceptibleS. aureus andPseudomonas aeruginosa (P. aeruginosa) strains were isolated from sore throat patients from tertiary care hospitals at Faisalabad. ATCC strains ofEscherichia coli (E. coli) andS. aureus were obtained from lab stock. All strains were routinely grown and identified. For further use they were preserved at −20°C.


## 2.3. Antimicrobial Susceptibility of Clinical Isolates

Antibiotic sensitivity of the isolatedS. aureus strains was determined by Standard Disc Diffusion Method [19]. Different antibiotics (Oxoid) were used in the present work: tetracycline (30 μg), vancomycin (30 μg), clindamycin (2 μg), methicillin (5 μg), cefixime (5 μg), erythromycin (15 μg), oxacillin (1 μg), gentamicin (10 μg), streptomycin (10 μg), chloramphenicol (30 μg), linezolid (10 μg), ampicillin-sulbactam (20 μg), and penicillin (10 μg) [20].


## 2.4. Minimum Inhibitory Concentration (MIC)

Minimum inhibitory concentrations (MIC) of antibiotics and plant extracts were determined by the microdilution method as described by Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards, NCCLS) [20]. The antibiotic was serially diluted in Mueller-Hinton broth in 96-well plates. The plant extracts solutions were separately added to wells in a final concentration of 1 mg/mL; then bacterial inoculum size of 105 CFU/mL was added to each well. Controls without plant extracts, without bacterial inoculum, or with plant extracts only were also included in the experiment. Each plant extract was run in duplicate. The test plates were incubated at 37°C for 24 h. The MIC was taken as the minimum concentration of the dilutions that inhibited the growth of the test microorganism. The growth was recorded in terms of button formation in wells; it was confirmed by adding tetrazolium chloride.


## 2.5. Toxicity of Extracts to Human Erythrocytes

The cellular toxicity of the extracts from different plants was determined [21]. Erythrocytes were isolated from human blood by removing plasma and buffy coat and suspended in phosphate buffered saline (10 mM phosphate, 150 mM sodium chloride) making a final concentration of 2% erythrocytes. The dilutions of plant extract (100 mg/mL) were made and mixed with erythrocytes keeping the final volume up to 1 mL. The cells were incubated for 1 hr at 37°C and finally centrifuged at 1500 rpm for 10 minutes [22]. The lysis of the cells was observed by determining absorbance at 540 nm using spectrometer. Cell in distilled water was taken as negative and cells suspended in normal saline were taken as positive control. The mixture was centrifuged at 1500 rpm for 1 minute in a laboratory centrifuge. The free haemoglobin in the supernatant was measured at 540 nm.The haemolysis percentage was calculated by the following formula:(1)
%
haemolysis
=
A
t
-
A
n
A
c
-
A
n
∗
100
.At is absorbance of test sample, An is absorbance of the control (saline), and Ac is absorbance of the control (water).


## 2.6. Screening and Estimation of Some Phytochemical Components

Phytochemical screening of the effective extract for the detection of alkaloids, flavonoids, steroids, terpenoid, and other secondary metabolites was carried out by following the procedure of Trease and Evans [23]. Total alkaloids, flavonoids, and saponin fraction were also separated and their antimicrobial activity was carried out [24].


## 2.7. Thin Layer Chromatography, Bioautography, and Identification of the Chemical Nature of the Bioactive Compound

For TLC, 10–20μL of plant extract was loaded on silica gel sheet (Merck, Darmstadt, Germany). Different solvent systems, chloroform : methanol (80 : 20), n-butanol : acetic acid : water (4 : 1 : 1), and acetone : methanol (1 : 1), were used as mobile phase. The TLC plates (a–f) were run in triplicate. Plate “a” was used to determine the spots by visualizing in UV light to see if the separated spot was UV active after which it was sprayed with vanillin sulphuric acid reagent (2%) to detect all the separated spots. Plate “b” was used for autobiography. Agar overlay method [25] was used for direct bioautographic assay, with little modification, to detect antimicrobial fractions in plant extracts which were active against clinical sample of MRSA andE. coli. Soft nutrient agar (1%) was seeded with 106 CFU/mL of MRSA/E. coli and overlaid on TLC plate. It was then incubated overnight in a controlled environment at 37°C. Subsequently, the plates were sprayed with 2,3,5-triphenyltetrazolium chloride and further incubated for three hours at 37°C. Microbial growth inhibition appeared as clear zone against a pink background. The Rf value of the bioactive spots was measured as the ratio of mobility of centre of a bioactive spot against the total distance travelled by the solvent front. The bioactive spots were scraped from the TLC plate “c” and eluted in 500 μL ethanol. The solution was filtered using Whatman filter paper number 1 and concentrated and again bioassay was performed to recheck the activity. Plates “d, e, and f” were sprayed separately with TLC reagents including FeCl3 (10%), Dragendorff’s reagent, and KOH (5N) to detect the presence of phenolic compounds, alkaloids, and flavonoids, respectively, in bioactive spots.


## 3. Results

### 3.1. Antibiotic Susceptibility and Responses to Plant Extracts

All clinical isolates and ATCCE. coli were resistant to beta-lactam group, whereas ATCC 25923 was sensitive to all antibiotics used in the study. The clinical isolates were also resistant to diverse groups of antibiotics (Table 2). Aqueous and n-hexane extracts displayed antimicrobial activity with ZI < 11 mm, while methanolic extracts of 18/29 plants were found to be active and showed varying activity with a zone of inhibition of 11–25 mm for all the clinical and reference strains. The MIC of the extracts ranged from 3.90 to 250 μg/mL (Table 3).Table 2
Antibiogram of the clinical and standard bacteria.
Clinical sample
Resistant
Intermediate
Sensitive
S1
Me, cef, eryth, oxa, strep, amp, pen
No
Tet, vanco, clind, genta, chlo, lin
S2
Tet, clin, me, cef, ery, oxa, chlo, amp, pen
None
Van, gen, strep, lin
S3
Tet, me, cef, oxa, gen, pen
None
Van, clin, ery, strep, chlo, lin, amp
P. aeruginosa
me, cef, oxa, gen, pen, ery
Tet, clin, amp
Van, clin, strep, chlo, lin
E. coliATCC 8739
Me, pen
None
Tet, cef, oxa, gen, Van, clin, ery, strep, chlo, lin, amp
S. aureusATCC 25923
None
None
Tet, me, cef, oxa, gen, pen, Van, clin, ery, strep, chlo, lin, ampTable 3
MIC/MBC of plant extracts against selected bacteria.
Serial number
Plant name
S1
S2
S3
P. aeruginosa
S. aureus (25293)
E. coli (8739)
ZI
MIC
µg/mL
ZI
MIC
ZI
MIC
ZI
MIC
ZI
MIC
ZI
MIC
1
Adhatoda vasica
17
31.25
18
31.25
21
31.25
19
62.5
21
7.81
18
31.25
2
Althaea officinalis
16
31.25
18
31.25
20
31.25
20
31.25
20
15.63
20
31.25
3
Cinnamomum zeylanicum
20
250
22
125
25
7.81
20
125
25
125
22
250
4
Cordia latifolia
16
31.25
18
62.5
20
31.25
17
125
20
12.63
19
62.50
5
Emblica officinalis
11
125
12
250
13
125
13
125
13
62.50
12
250
6
Glycyrrhiza glabra
18
125
20
250
22
125
17
62.50
22
31.25
19
125
7
Hyssopus officinalis
18
125
19
250
22
125
18
31.25
22
31.25
18
62.50
8
Lawsonia inermis
12
250
13
250
15
250
14
125
15
125
13
250
9
Linum usitatissimum
21
250
22
250
22
250
19
125
22
125
20
250
10
Malva sylvestris
15
125
16
250
18
125
15
62.5
18
62.50
16
125
11
Matricaria chamomilla
12
125
13
250
14
125
13
62.5
13
62.50
15
125
12
Moringa oleifera
13
250
14
500
15
250
15
62.5
15
62.50
14
250
13
Morus alba
13
125
12
250
13
125
13
125
13
31.25
13
125
14
Origanum vulgare
22
7.81
20
15.62
22
7.81
20
7.81
22
3.91
21
15.63
15
Sisymbrium irio
20
125
22
250
25
125
21
62.5
25
62.50
20
125
16
Syzygium cumini
13
62.5
13
125
15
62.50
13
31.25
15
15.63
13
62.50
17
Thymus vulgaris
22
15.63
22
31.25
25
15.63
22
7.81
25
7.81
21
31.25
18
Ziziphus jujuba
14
15.63
14
31.25
12
15.63
21
7.81
14
7.81
16
31.25


### 3.2. Phytochemical Analysis

The plant extracts which showed promising antimicrobial activity were investigated further for the presence of flavonoids, terpenoids, steroids, and glycosides, and other phytochemicals results are given in Table4. Alkaloids, flavonoids, and saponin fraction were separated. MIC and their bioactivity were checked out (Table 5). Alkaloids fraction ofAdhatoda vasica,Cordia latifolia, andOriganum vulgare and flavonoid fraction ofAlthaea officinalis,Origanum vulgare,Thymus vulgaris, andZiziphus jujuba displayed antimicrobial activity against MRSA. MIC of the alkaloids and flavonoids ranged from 3.25 to 12.5 (μg/mL) and zone of inhibition ranged from 13 to 18 mm.Table 4
Phytochemical analysis of the methanolic extracts of plants possessing antibacterial activity.
Serial number
Plant name
Yield%
Alk.
Flv.
Ter.
Ste.
Sap.
Tan.
Carb.
Pro.
Car. Gly.
Phe.
Red. Sug.
1
Adhatoda vasica
18.2
+
+
+
−∗
∗
+
+
−
+
+
+
+
2
Althaea officinalis
22
−
+
−
++∗
−
++
++
+
++
++
++
3
Cordia latifolia
29.9
+
−
−
−
−
++
++
++
−
−
−
4
Origanum vulgare
35.6
+
+
−
+
+
−
−
+
+
+
+
5
Thymus vulgaris
22.9
−
+
−
−
−
−
−
+
−
+
−
6
Ziziphus jujuba
22.5
−
+
+
+
−
−
+
−
−
+
+
Tan., tannins; Alk., alkaloids; Flv., flavonoids; Ter., terpenoids; Ste., steroids; Sap., saponins; Carb., carbohydrate; Pro., proteins; Car. Gly., cardiac glycosides; Phe., phenols; Red. Sug., reducing sugar.
++∗: high concentration.
-
∗
∗: absence.Table 5
Bioactivity of secondary metabolites rich fraction.
Serial number
Plant name
Alkaloid
Flavonoid
Saponin
ZI (mm)
MIC (µg/mL)
% age estimation
ZI (mm)
MIC (µg/mL)
% age estimation
ZI (mm)
MIC (µg/mL)
% age estimation
1
Adhatoda vasica
13
6.25
2
0
>100
—
0
>100
—
2
Althaea officinalis
—
N.D∗
N.D
14
12.5
1.03
0
>100
2.7
3
Cordia latifolia
14
12.5
0.8
0
N.D
—
0
N.D
—
4
Origanum vulgare
18
6.25
1.5
15
3.25
2.5
—
N.D
—
5
Thymus vulgaris
—
N.D
—
16
6.25
2.3
—
N.D
—
6
Ziziphus jujuba
—
N.D
—
13
6.25
1.33
—
N.D
—
∗N.D: activity not determined.


### 3.3. Thin Layer Chromatography and Autobiography

Different solvent systems comprising water, methanol, acetic acid, chloroform, n-butanol, toluene, ethyl acetate, and formic acid in different combination were used to separate the active part. The best solvent systems were chloroform and methanol (80 : 20), n-butanol, acetic acid, and water (4 : 1 : 1). Active parts of all the plant extract were not separated in a single solvent system which gives us the information that the active fractions vary in their chemical nature. Antimicrobial fractions of the crude extract were separated by TLC followed by autobiography method. The Rf value of active fraction is shown in Table6. When TLC plate was visualized under UV different coloured spots were seen and marked and their antimicrobial activity was checked out. The chemical nature of the antimicrobial compound was detected by spraying with Dragendorff’s reagent for alkaloids and 10% FeCl3 for phenolic compound and 5N KOH solution for flavonoids.Table 6
Retention factor of the antibacterial fraction of the methanolic crude extract.
Serial number
Plant name
Rf∗ valueCm
Mobile phase
1
Adhatoda vasica
0.4 (alkaloids)0.860.92
Chloroform : methanol (80 : 20)
2
Ziziphus jujuba
0.15 (phenol)0.18 (flavonoids)0.65 (phenol)0.71 (phenol)0.85
Chloroform : methanol (80 : 20)
3
Althaea officinalis
0.720.45 (flavonoids)
Chloroform : methanol (80 : 20)
4
Cordia latifolia
0.670.84 (alkaloids)0.35
Acetone : methanol (1 : 1)
5
Origanum vulgare
0.750.79 (flavonoids)0.82 (alkaloids)
Butanol : acetic acid : water (4 : 1 : 1)
6
Thymus vulgaris
0.72 (phenol)0.8
Butanol : acetic acid : water (4 : 1 : 1)
∗Retention factor.Thin layer chromatography plates after the development show various components in light and when visualized under UV other parts were also visualized. Direct autobiography on TLC plates revealed presence of active antimicrobial components in all six plants with different Rf values (Table6).


### 3.4. Haemolysis Percentage of the Crude Methanolic Extract

In vitro evaluation of the toxicity of the extract by comparing the haemolysis percentages is shown in Figure 1. It has been shown in the graph that the maximum haemolytic activity was shown by methanolic extract of theCordia latifolia and the minimum haemolytic activity was shown byOriganum vulgare. On the basis of haemolytic potential the selected plants could be arranged as follows:Cordia latifolia >Ziziphus jujuba >Althaea officinalis
=
Adhatoda vasica >Thymus vulgaris >Origanum vulgare.Figure 1
Toxicity of methanolic extracts of selected plants against human erythrocytes.


## 3.1. Antibiotic Susceptibility and Responses to Plant Extracts

All clinical isolates and ATCCE. coli were resistant to beta-lactam group, whereas ATCC 25923 was sensitive to all antibiotics used in the study. The clinical isolates were also resistant to diverse groups of antibiotics (Table 2). Aqueous and n-hexane extracts displayed antimicrobial activity with ZI < 11 mm, while methanolic extracts of 18/29 plants were found to be active and showed varying activity with a zone of inhibition of 11–25 mm for all the clinical and reference strains. The MIC of the extracts ranged from 3.90 to 250 μg/mL (Table 3).Table 2
Antibiogram of the clinical and standard bacteria.
Clinical sample
Resistant
Intermediate
Sensitive
S1
Me, cef, eryth, oxa, strep, amp, pen
No
Tet, vanco, clind, genta, chlo, lin
S2
Tet, clin, me, cef, ery, oxa, chlo, amp, pen
None
Van, gen, strep, lin
S3
Tet, me, cef, oxa, gen, pen
None
Van, clin, ery, strep, chlo, lin, amp
P. aeruginosa
me, cef, oxa, gen, pen, ery
Tet, clin, amp
Van, clin, strep, chlo, lin
E. coliATCC 8739
Me, pen
None
Tet, cef, oxa, gen, Van, clin, ery, strep, chlo, lin, amp
S. aureusATCC 25923
None
None
Tet, me, cef, oxa, gen, pen, Van, clin, ery, strep, chlo, lin, ampTable 3
MIC/MBC of plant extracts against selected bacteria.
Serial number
Plant name
S1
S2
S3
P. aeruginosa
S. aureus (25293)
E. coli (8739)
ZI
MIC
µg/mL
ZI
MIC
ZI
MIC
ZI
MIC
ZI
MIC
ZI
MIC
1
Adhatoda vasica
17
31.25
18
31.25
21
31.25
19
62.5
21
7.81
18
31.25
2
Althaea officinalis
16
31.25
18
31.25
20
31.25
20
31.25
20
15.63
20
31.25
3
Cinnamomum zeylanicum
20
250
22
125
25
7.81
20
125
25
125
22
250
4
Cordia latifolia
16
31.25
18
62.5
20
31.25
17
125
20
12.63
19
62.50
5
Emblica officinalis
11
125
12
250
13
125
13
125
13
62.50
12
250
6
Glycyrrhiza glabra
18
125
20
250
22
125
17
62.50
22
31.25
19
125
7
Hyssopus officinalis
18
125
19
250
22
125
18
31.25
22
31.25
18
62.50
8
Lawsonia inermis
12
250
13
250
15
250
14
125
15
125
13
250
9
Linum usitatissimum
21
250
22
250
22
250
19
125
22
125
20
250
10
Malva sylvestris
15
125
16
250
18
125
15
62.5
18
62.50
16
125
11
Matricaria chamomilla
12
125
13
250
14
125
13
62.5
13
62.50
15
125
12
Moringa oleifera
13
250
14
500
15
250
15
62.5
15
62.50
14
250
13
Morus alba
13
125
12
250
13
125
13
125
13
31.25
13
125
14
Origanum vulgare
22
7.81
20
15.62
22
7.81
20
7.81
22
3.91
21
15.63
15
Sisymbrium irio
20
125
22
250
25
125
21
62.5
25
62.50
20
125
16
Syzygium cumini
13
62.5
13
125
15
62.50
13
31.25
15
15.63
13
62.50
17
Thymus vulgaris
22
15.63
22
31.25
25
15.63
22
7.81
25
7.81
21
31.25
18
Ziziphus jujuba
14
15.63
14
31.25
12
15.63
21
7.81
14
7.81
16
31.25


## 3.2. Phytochemical Analysis

The plant extracts which showed promising antimicrobial activity were investigated further for the presence of flavonoids, terpenoids, steroids, and glycosides, and other phytochemicals results are given in Table4. Alkaloids, flavonoids, and saponin fraction were separated. MIC and their bioactivity were checked out (Table 5). Alkaloids fraction ofAdhatoda vasica,Cordia latifolia, andOriganum vulgare and flavonoid fraction ofAlthaea officinalis,Origanum vulgare,Thymus vulgaris, andZiziphus jujuba displayed antimicrobial activity against MRSA. MIC of the alkaloids and flavonoids ranged from 3.25 to 12.5 (μg/mL) and zone of inhibition ranged from 13 to 18 mm.Table 4
Phytochemical analysis of the methanolic extracts of plants possessing antibacterial activity.
Serial number
Plant name
Yield%
Alk.
Flv.
Ter.
Ste.
Sap.
Tan.
Carb.
Pro.
Car. Gly.
Phe.
Red. Sug.
1
Adhatoda vasica
18.2
+
+
+
−∗
∗
+
+
−
+
+
+
+
2
Althaea officinalis
22
−
+
−
++∗
−
++
++
+
++
++
++
3
Cordia latifolia
29.9
+
−
−
−
−
++
++
++
−
−
−
4
Origanum vulgare
35.6
+
+
−
+
+
−
−
+
+
+
+
5
Thymus vulgaris
22.9
−
+
−
−
−
−
−
+
−
+
−
6
Ziziphus jujuba
22.5
−
+
+
+
−
−
+
−
−
+
+
Tan., tannins; Alk., alkaloids; Flv., flavonoids; Ter., terpenoids; Ste., steroids; Sap., saponins; Carb., carbohydrate; Pro., proteins; Car. Gly., cardiac glycosides; Phe., phenols; Red. Sug., reducing sugar.
++∗: high concentration.
-
∗
∗: absence.Table 5
Bioactivity of secondary metabolites rich fraction.
Serial number
Plant name
Alkaloid
Flavonoid
Saponin
ZI (mm)
MIC (µg/mL)
% age estimation
ZI (mm)
MIC (µg/mL)
% age estimation
ZI (mm)
MIC (µg/mL)
% age estimation
1
Adhatoda vasica
13
6.25
2
0
>100
—
0
>100
—
2
Althaea officinalis
—
N.D∗
N.D
14
12.5
1.03
0
>100
2.7
3
Cordia latifolia
14
12.5
0.8
0
N.D
—
0
N.D
—
4
Origanum vulgare
18
6.25
1.5
15
3.25
2.5
—
N.D
—
5
Thymus vulgaris
—
N.D
—
16
6.25
2.3
—
N.D
—
6
Ziziphus jujuba
—
N.D
—
13
6.25
1.33
—
N.D
—
∗N.D: activity not determined.


## 3.3. Thin Layer Chromatography and Autobiography

Different solvent systems comprising water, methanol, acetic acid, chloroform, n-butanol, toluene, ethyl acetate, and formic acid in different combination were used to separate the active part. The best solvent systems were chloroform and methanol (80 : 20), n-butanol, acetic acid, and water (4 : 1 : 1). Active parts of all the plant extract were not separated in a single solvent system which gives us the information that the active fractions vary in their chemical nature. Antimicrobial fractions of the crude extract were separated by TLC followed by autobiography method. The Rf value of active fraction is shown in Table6. When TLC plate was visualized under UV different coloured spots were seen and marked and their antimicrobial activity was checked out. The chemical nature of the antimicrobial compound was detected by spraying with Dragendorff’s reagent for alkaloids and 10% FeCl3 for phenolic compound and 5N KOH solution for flavonoids.Table 6
Retention factor of the antibacterial fraction of the methanolic crude extract.
Serial number
Plant name
Rf∗ valueCm
Mobile phase
1
Adhatoda vasica
0.4 (alkaloids)0.860.92
Chloroform : methanol (80 : 20)
2
Ziziphus jujuba
0.15 (phenol)0.18 (flavonoids)0.65 (phenol)0.71 (phenol)0.85
Chloroform : methanol (80 : 20)
3
Althaea officinalis
0.720.45 (flavonoids)
Chloroform : methanol (80 : 20)
4
Cordia latifolia
0.670.84 (alkaloids)0.35
Acetone : methanol (1 : 1)
5
Origanum vulgare
0.750.79 (flavonoids)0.82 (alkaloids)
Butanol : acetic acid : water (4 : 1 : 1)
6
Thymus vulgaris
0.72 (phenol)0.8
Butanol : acetic acid : water (4 : 1 : 1)
∗Retention factor.Thin layer chromatography plates after the development show various components in light and when visualized under UV other parts were also visualized. Direct autobiography on TLC plates revealed presence of active antimicrobial components in all six plants with different Rf values (Table6).


## 3.4. Haemolysis Percentage of the Crude Methanolic Extract

In vitro evaluation of the toxicity of the extract by comparing the haemolysis percentages is shown in Figure 1. It has been shown in the graph that the maximum haemolytic activity was shown by methanolic extract of theCordia latifolia and the minimum haemolytic activity was shown byOriganum vulgare. On the basis of haemolytic potential the selected plants could be arranged as follows:Cordia latifolia >Ziziphus jujuba >Althaea officinalis
=
Adhatoda vasica >Thymus vulgaris >Origanum vulgare.Figure 1
Toxicity of methanolic extracts of selected plants against human erythrocytes.


## 4. Discussion

The interest in research on medicinal plants has increased over the last few decades due to the emergence of MDR strains of important pathogens likeS. aureus,P. aeruginosa, andE. coli [26]. Some of the antibiotics against which resistance has been developed include penicillin G, macrolides, lincosamides, tetracycline, and gentamicin [27]. Investigations are needed for antibacterial agents that offer broad-spectrum activity against resistant strains of microorganisms. Toxicity profiling of the plant extract also needs scientific validation.In the present study, twenty-nine plants that were recommended by herbal practitioners for treatment of diseases related to respiratory tract were evaluated for their antibacterial activity against MDR strains ofS. aureus,E. coli, andP. aeruginosa isolated from sore throat patients. Although the antibacterial effect of these plants has already been reported by many workers, their activities against MDR strains are less evaluated [28–31]. Moreover, the current study focused on the phytochemical nature of active fraction. Three types of extracts, n-hexane, water, and methanol, were tested for their possible antibacterial activity against ATCC bacterial cultures of Gram-positive and Gram-negative bacteria and clinical samples of MDRS. aureus andP. aeruginosa isolated from the sore throat patients. A zone of inhibition of >11 mm was considered as a standard for effectiveness of extract against bacteria. The n-hexane extracts did not show any zone of inhibition, while aqueous extract inhibits microbial growth with a zone of inhibition of less than 11 mm. Although the aqueous extract preparation mimics the traditional method of use, very little antimicrobial activity was observed in it, which might be due to less solubility of active components in water [32, 33]. On the other hand methanolic extract of 18/29 plants exhibits ZI > 11 mm and MIC of 3.91–500 μg/mL, indicating that methanolic extract contains a variety and better quantity of bioactive compounds. Such observation has also been noticed by some workers [34, 35].Plants extracts having MIC ≤ 100μg/mL against all test strains were selected for detailed study including phytochemical analysis (qualitative and quantitative), TLC, autobiography, andin vitro toxicity. Qualitative analysis revealed the presence of alkaloids, flavonoids, terpenoids, steroids, saponin, tannins, carbohydrates, proteins, cardiac glycosides, phenols, and reducing sugars. The saponin, alkaloids, and flavonoids are documented as the most active ingredients to which the antimicrobial activities of many plant species can be attributed [36]. Therefore, quantitative analysis was carried out for these. Moreover, their rich fractions were verified to be responsible for antibacterial activity against test strains.Adhatoda vasica contained bioactive alkaloid (2%) fraction. On TLC plates, three spots were noticed by bioautography which separated at 0.4, 0.86, and 0.92 Rf values in chloroform : methanol (80 : 20) solvent system. One of the spots was identified as alkaloid, while chemical nature of other 2 spots could not be determined.Adhatoda vasica has also been recognized for its diverse health benefits along with antimicrobial potential. Kaur et al. [37] and Sawant et al. [38] have recently reported antimicrobial activity against respiratory pathogensS. aureus andE.coli. The differences may be due to strain specificity or geographical variations in plant spp. Alkaloids l-vasicinone, deoxyvasicine, maiontone, vasicinolone, and vasicinol were reported inAdhatoda vasica by Jain and Sharma [39]. The current study indicates that it is not only alkaloid that shows antimicrobial activity; some other compounds of different nature are also bioactive and further studies are required for their separation and identification.Origanum vulgare contained bioactive alkaloid (1.5%) and flavonoid (2.5%) fraction. On TLC plates, three spots were noticed by following autobiography which separated at Rf 0.75, 0.79, and 0.82 values in butanol : acetic acid : water (4 : 1 : 1) solvent system. One of the spots with Rf value of 0.79 was identified as alkaloid and another one with Rf value of 0.82 was identified as flavonoid, while chemical nature of the third spot could not be determined.Origanum vulgare and its EO have also been recognized for its antimicrobial and antioxidant nature [40]. The current study indicates that alkaloid and flavonoids as well as other compounds with antimicrobial potential can be purified fromOriganum vulgare and used against MDR.Thymus vulgaris leaves and essential oil are used in cosmetic and food additives [41, 42]. Antimicrobial potential of thymus was also reported [43, 44], but nature of component was not known. In this study, phenolic compound with Rf value of 0.72 was found responsible for antibiotic activity, while another of unknown nature was also bioactive.Flavonoid ofZiziphus displayed antimicrobial activity. Recently published data on antimicrobial properties against Gram-positive and Gram-negative pathogenic bacteria have been reported [45], whereas the presence of alkaloids, flavonoids, and saponin inZiziphus jujuba extracts was reported by [46]. The current study confirms antimicrobial nature of its flavonoid. TLC plate separation leads to identification of 5 spots with bioautography of Rf value of 0.15, 0.18, 0.65, 0.71, and 0.85 through autobiography. Among them 3 were phenolic; one of them was flavonoid, whereas chemical nature of one spot with Rf of 0.85 remained unclear. The current study indicates that the flavonoids with Rf of 0.45 and one compound with Rf of 0.72 have this activity. In a review presence of a variety of flavonoids in the methanolic extract was reported by Gao et al. [47].TLC separation of methanolic extract ofCordia latifolia showed 03 bioactive spots with Rf value of 0.84, 0.67, and 0.35. The alkaloid fraction of the methanolic extract with Rf value of 0.84 and other two spots with Rf value of 0.67 and 0.35 of unknown nature show antibacterial activity.Scientific validations of the safety of extracts are mandatory for recommendation to human. The erythrocyte haemolysis has been widely used as an indicator of toxicity of injectable formulations as well as a general indication of membrane toxicity. Haemolysis is due to red blood cells destruction which resulted from lysis of membrane lipid bilayer. This haemolysis relates to concentration and chemical composition of extract. Data about the safety ofCordia latifolia andAlthaea officinalis has been provided by May and Willuhn [48] and Caparroz-Assef et al. [49]. Extracts ofAdhatoda vasica,Althaea officinalis,Oregano vulgaris,Cordia latifolia,Althaea officinalis, andThymus vulgaris were used in toxicity study and revealed 0.93–6.48% haemolytic activity and are found to be safe for human use.


## 5. Conclusion

In conclusion 29 plants included in the study were already known for their antimicrobial nature; the current study found that 18/29 possess these characteristics against MDR strains and 6/18 are good candidates for isolation of effective components.Cordia latifolia,Ziziphus jujuba,Althaea officinalis,Adhatoda vasica,Thymus vulgaris, andOriganum vulgare have good antimicrobial activity and their haemolysis assay declared them safe candidate for the treatment of sore throat in human. Alkaloids ofAdhatoda vasica,Cordia latifolia, andOriganum vulgare and flavonoids ofAlthaea officinalis,Origanum vulgare,Thymus vulgaris, andZiziphus jujuba may be utilized for the treatment of MRSA.



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*Source: 8503426-2016-06-26.xml*