INTRODUCTION:

Human beings have been affected with microbial infections much longer than the first recorded documents written by mankind. According to WHO reports about 13.3 million death cases was recorded due to microbial infections¹. Antibiotics are used to eliminate some of these microbial diseases. Increase in the risk of microbial infections is because of the resistance developed by the microorganisms towards available antibiotics by the alteration in their gene structure and metabolism². The above reasons indicate that the use of other alternate medicine is very much important as only using antibiotics or synthetic drugs can’t eliminate these problems. Moreover the side effects caused by the use of synthetic drugs are sometime worst. So reducing these effects are necessary. Previous reports on the usage of herbal medicine reduced the side effects drastically and of low cost, hence it is easily available to common people³.

Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions and to defend against attack from predators. The use of plant as medicine is wide spread throughout the world⁴. Many of the pharmaceuticals currently available to physician have a long history of use as herbal remedies including Aspirin, Digitalis, Quinine and Opium. The bark of willow trees contain large amount of salicylic acid which is active compound of the aspirin. More than 100 plants were identified for the treatment of asthma, diabetics and hypertension⁵. Forty two plants species were identified from Ghana for the treatment of malaria⁶. High frequency of plant regeneration and anticancer alkaloid camptothecin production was developed⁷.

C. viscosa L belongs to family Capparaceae. It is commonly known as yellow spider flower in English and hurhur in Hindi. It is a small herb and is locally abundant as a weed in fallow lands, dry river beds or even in very poor soils. It is a native of tropical Africa, Malaysia, and South Arabia. The leaves of C. viscosa plant are arranged in alternate and distichous fashion. Leaf shape is obovate and elliptic, leaf base is cuneate, leaf apex is acute and leaf margin is ciliate. The macroscopic structure of C.viscosa reveals that the fresh leaves of C. viscosa is green in colour, 3-5 leaflets arranged in alternate fashion in a leaf stalk. Flowers are yellow in colour and bisexual. Flowering is throughout the year. Fruits are cylindrical, erect, glandular hairy. Even some contains reddish brown coloured seeds. Stems are subcutaneous, sticky in nature with hairs. Plants have long primary roots along with secondary branches⁸.

There are many medicinal uses of Cleome viscosa. The whole plant and its parts were widely used by local people for treatment of various diseases. It is used for preparation of ayurvedic medicines. It is used for treating fever, inflammation, liver diseases, etc. Leaves are used for treating fever and ulcers. When juice of the plant is mixed with water and is taken, then fever is cured. C. viscosa also possesses antidiarrhoeal, analgesic, antipyretic and antimicrobial activities. It is also used in Chinese medicines. The aqueous seed extract of the plant (200mg/kg) was administered orally to the animals with CCL4 (carbon tetrachloride) – induced hepatotoxicity and silymarin (200mg/kg) was given as a reference standard. The significant reductions in serum enzymes and increase in reduced glutathione suggested that the aqueous extract have membrane stabilizing activity. It is an effective medicinal plant for otitis media. Here plant leaves show antimicrobial activity against pathogenic bacteria causing otitis media⁹. Since there were no reports on the medicinal properties of the stems of C. viscosa, the present study was aimed to evaluate phytochemical composition, antimicrobial, hemolytic activity and HPLC analysis of ethanolic extract of C. viscosa Linn.

MATERIALS AND METHODS:

Chemicals:

Nutrient agar, Nutrient broth, Mueller Hinton agar, Vancomycin, Cotrimoxazole, Ciprofloxacin, Chloromphenicol, Piperacillin and ethanol were purchased from Himedia Pvt Ltd, Mumbai, India. All chemicals used were of analytical grade.

Preparation of plant extracts:

Fresh plant specimen, C. viscosa was collected from Ranipet, Vellore (12⁰58’12”N 79⁰9’15”E) in the month of August, 2013 and authenticated by Prof. J. Benjamin Prasad Kumar, Department of Plant Biology and Biotechnology, Voorhees College, Vellore. The voucher specimen was maintained in our laboratory with accession no. CV/VIT/MMRL/31.08.2013-56. The stems of C. viscosa were shade dried and powdered with a mechanical grinder. Powdered stems were extracted with ethanol using Soxhlet apparatus. The resultant extract was evaporated in Rota evaporator and stored in refrigerator at 4°C.

Preliminary Phytochemical screening:

The ethanolic extract was tested qualitatively for tannins, saponins, alkaloids, carbohydrates, phenolic compounds, proteins, phytosterols, flavonoids, glycosides, oil and fats by fallowing the methods of Harborne, 1973¹⁰.

Antimicrobial activity:

Microbial cultures:

The microorganisms used for the present study were Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 2063, Pseudomonas aeroginosa ATCC 27853, Escherichia coli ATCC 25922 and Klebsiella pneumonia ATCC 13883. The strains were grown and maintained in nutrient slants. From these slants, broth cultures were prepared and kept overnight which were further adjusted to obtain turbidity comparable to McFarland (0.5) standard as required.

Antibiotic discs:

Vancomycin (30µg/disc), Cotrimoxazole (23.75µg/disc), Ciprofloxacin (5µg/disc), Chloromphenicol (30µg/disc) and Piperacillin (100µg/disc) were used as positive controls for S. aureus, B. subtilis, P. aeroginosa, E. coli and K. pneumonia and sterilized distilled water was used as a negative control.

Antimicrobial activity by agar well diffusion method:

The test organisms were inoculated in nutrient broth (pH 7.4.) for 16 hours to obtain fresh culture. The bacterial isolates were swabbed on sterilized Mueller Hinton agar (MHA) petridishes using sterilized cotton swabs. Wells were made in each of these MHA plates using sterilized cork borer of 7 mm diameter. A volume of 100 µl of the ethanolic extracts of four different concentrations (200, 400, 800 and 1000 µg mL^-1) were prepared from the main stock and 100 µl of sterilized distilled water (negative control) were poured in the separate wells. The standard antibiotic discs were placed in each of the MHA plate as positive control. Then Plates were incubated at 37°C for 24 hours. Each experiment was performed in triplicates. After 24 hrs, the MHA plates were observed for the inhibition zone. The experiment was performed in triplicates¹¹.

Minimal inhibitory concentration (MIC):

MIC was assessed by broth dilution methods. The test microorganisms were inoculated into the Nutrient broth with different concentration of the extract and incubated for a definite period of time (16–20 h) and the lowest concentration of the extract that inhibited the visible growth of microorganisms was determined¹².

Total phenolic content:

Different concentrations of the extract ranging from 125 to 1000 µgmL^-1 was mixed with 2.5 mL of Folic- Ciocalteau reagent (1/10 dilution in purified water) and 2 mL of 7.5% Na₂CO₃ (w/v in purified water) fallowed by incubation at 45˚C for 15 min. The absorbance was measured at 765 nm using Na₂CO₃ solution (2 mL of 7.5% Na₂CO₃in 2.5 mL of distilled water) was used as blank. The results were expressed as Gallic acid equivalence (GAE) in µg¹³.

Total flavonoids content:

The diluted ethanolic extract was mixed with 1 mL of Aluminum chloride reagent (2gm of Al₂Cl₃ in 100mL ethanol). The mixture was incubated at room temperature for 1 hr. Al₂Cl₃ solution (3 mL of 2% Al₂Cl₃ in 3 mL of distilled water) was used as blank. The absorbance was measured at 420 nm using a UV–Visible spectrophotometer¹⁴.

Analysis of individual phenolic compounds by analytical RP-HPLC:

High performance liquid chromatography was carried out for the detection of Phenolic compounds in the extract. The HPLC system was equipped with dual λ detector and a waters1525 binary pump and consists of a column (C18). The gradient elution was carried out at 35ºC with the help of solution A (50mM sodium phosphate at pH-3.3 and 10% methanol) and B (70% methanol) at a flow rate of 1mL/min. First 100% solution A ,then for 15 min 70%A, again for 30 min 65%A, for next 20 min, 60%A, for another 5min, 50% and last 0% A for 25 min. The volume of the injection was 10µl of ethanolic extract dissolved in methanol. For analysis of phenolic compounds in the extract, the detected peaks at different wavelengths (250-510 nm) having different retention times were compared with respect to retention times of known standard compounds in the library¹⁵.

Hemolytic activity:

Hemolytic activity was performed fallowing the method of Gaurav et al., 2011¹⁶. Initially cell suspension was prepared as fallows. About five mL of heparinised blood from a healthy individual was centrifuged at 1500 rpm thrice and the plasma was discarded. The pellet was washed thrice with phosphate buffer saline (PBS) (pH 7.2). Thus obtained cells were resuspended in 0.5% normal saline. About 500 µl of cell suspension was mixed with 500 µl of extract in increasing concentrations (125, 250,500, 1000 µgmL-1 in PBS) and incubated at room temperature for 30 mints, then centrifuged at 1500 rpm for 10 mints. The supernatant containing free hemoglobin was measured using UV visible spectrophotometer at 540 nm with PBS and distilled water as minimal and maximal controls. The percentage of hemolytic activity was calculated by using the formula below.

% Hemolytic activity = [At – An / Ac – An] × 100

Where, At is the absorbance of test sample

An is absorbance of the control (saline control)

Ac is the absorbance of the control (water control)

Statistical analysis:

Graphpad Prism v.5.00 (La Jolla, CA, USA) and Microsoft Excel 2007 (Roselle, IL, USA) were used for the representation of the datas. All the experiments in this study were carried out in triplicates.

RESULTS:

Preliminary Phytochemical screening

The extract has shown the presence of alkaloids, carbohydrates, flavonoids, oils and fats and absence of sterols, saponins and tannins (Table 1).

Table 1 Phytochemical screening of ethanolic extract of C. viscosa Linn

Phytochemical Tests	Ethanolic extract of C. viscosa
Alkaloids	+
Carbohydrates	+
Flavonoids	+
Sterols	-
Oils and fats	+
Saponins	-
Tannins	-

Here, +ve : positive, -ve: negative for the phytochemical tests performed

Antimicrobial activity:

The ethanolic extract of C. viscosa has shown maximum zone of inhibition (18mm) against S. aureus at 200µgmL^-1 concentration. The result was expressed as mean± standard deviation in triplicates and was summarized in the table 2.

Minimum Inhibitory Concentration (MIC):

The minimum inhibitory concentration was carried out with the ethanolic extract of C. viscosa. The minimum inhibitory concentration observed was 100µg mL^-1. Growth inhibition was increased with the increasing in the range of concentration from 100-1000µg mL^-1.

Total phenolic content:

The total phenolic content of the ethanolic extract was represented as Gallic acid equivalence The ethanolic extract of C. viscosa had shown higher amount of phenolics in a concentration dependent manner. The total phenolic compounds in the extract were found to be 37.98 µg GAE/gm dry weight which was shown in the figure.

Figure.1. Quantitative analysis of total phenolic content in ethanolic extract of C. viscosa. The data was expressed as mean± standard deviation; n=3

Table 2 Antimicrobial activity of ethanolic extract of C. viscosa Linn.

Test organisms	*Inhibition zone diameter (mm) C. viscosa* stems**
Test organisms	Ethanolic extract	Positive Control	Negative Control
B. subtilis	8.0±0.64	18.0±1.0	0.0±0.0
E. coli	0.2±0.81	11.0±1.5	0.0±0.0
K. pneumoniae	1.0±0.32	9.0±0.57	0.0±0.0
P. aeroginosa	4.0±0.41	6.0±0.57	0.0±0.0
S. aureus	18.0±0.72	14.0±0.57	0.0±0.0

Values are expressed as mean ± standard deviation of the three replicates. Zone of inhibition not include the diameter of the well

Table 3 HPLC based phenolic compounds identification of ethanolic extract of C. viscosa Linn

Compound name	Sample retention time (min)	Reference retention time (min)	Wavelength (nm)
Myricetin	48.605	49.3	370
Kaempferol-7-O-neohesperidoside	53.387	53.2	370
Galocatechin	7.92	8.1	280
Gallic acid	5.367	5.8	250
protocatechuic acid	9.472	9.7	250
β – resorcylic acid	10.888	10.9	250

Total flavonoids content:

The quantitative determination of total flavonoids content in the extract resulted in 45.99 mg QE/gm of extract in a concentration dependent wise and was expressed as quercitin equivalence (QE) in µg. The result of quantitative determination of total flavonoids was shown in the figure 2.

Figure. 2. Quantitative analysis of total phenolic content in ethanolic extract of C. viscosa.The data was expressed as mean± standard deviation; n=3

RP-HPLC analysis:

HPLC analysis was carried out with the ethanolic extract of C. viscosa to detect the polyphenolic compounds present in the sample. Six poly phenols detected by the HPLC analysis were Myricetin (3,5,7,3’,4’,5’-OH), Kaempferol-7-O-neohesperidoside (-), galocatechin (R1=OH, R2=H, R3=OH), Gallic acid (3,4,5-OH) , Protocatechuic acid (3,4-OH) and β–resorcylic acid (2,4-OH) which were identified by comparing the sample retention times with reference retention times of the phenolic compounds library at different wavelengths ranging from 220-510 nm and were given in the table 3.

Fig. 3. Hemolytic activity of ethanolic extract of C. viscosa. The data was expressed as mean± standard deviation; n=3

Hemolytic activity:

The extract had shown very low hemolytic activity in dose dependent manner and the data was expressed as percentage hemolytic activity which is shown in the figure 3.

DISCUSSION:

In the present study the antimicrobial activity of the ethanolic extract of C. viscosa stem exhibited maximum zone of inhibition against S. aureus. The antibacterial activity of the stem extract was compared with the antibiotics. The extract had shown effective inhibition zone than the compared antibiotic, Ciproflaxocin. The extract also showed presence of high amount of flavanoids and phenolic compounds. In fact, a number of phenolic compounds from medicinal plant extracts have been reported for antimicrobial activity^17,18. Hence, HPLC analysis was performed to detect the presence of phenolic compounds in the extract. As a result HPLC analysis identified the presence of six phenolic compounds myricetin (3,5,7,3’,4’,5’-OH), Kaempferol-7-O-neohesperidoside (-) galocatechin (R1=OH, R2=H, R3=OH), Gallic acid (3,4,5-OH) , protocatechuic acid (3,4-OH), β–resorcylic acid(2,4-OH). To check the impact of the extract on human erythrocytes, hemolytic activity was performed that resulted in very minute hemolysis.

CONCLUSION:

It can be concluded that ethanolic extract of C. viscosa stems can be used as potent source of antimicrobial agent. Since there was no effective hemolytic activity observed, it can be used as a safe antimicrobial agent. Further phytochemical compounds which are responsible for the antimicrobial activity has to be isolated from C. viscosa stems and can be used as safe and potent antimicrobial drug.

ACKNOWLEDGEMENT:

Authors are grateful to the management and staff of VIT University, Vellore for providing all the necessary requirements to carry out this study successfully.

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Received on 25.07.2014 Modified on 13.08.2014

Research J. Pharm. and Tech. 7(10): Oct. 2014 Page 1140-1144