Antimicrobial evaluation and quantification of apigenin content by HPTLC in methanol stem extract of Cardiospermum halicacabum L

 

A. Rajasekaran*, R. Arivukkarasu, Joyce Mathew P, Raja Bonagiri, Ramadasu Partha Saradhi

KMCH College of Pharmacy, Coimbatore, Tamil Nadu

 

ABSTRACT:

The present study was aimed to evaluate the anti-microbial activity of methanolic stem extract of Cardiospermum halicacabum (MSECH) and to develop and validate a HPTLC method for quantification of apigenin in MSECH. The in vitro anti-microbial efficacy of MESCH was evaluated by disc diffusion method and minimum inhibitory concentration against six bacterial strains Staphylococcus aureus, Bacillus lentius, Bacillus subtilis, Klebsiella pneumonia, Pseudomonas aeruginosa, Escherichia coli and three fungal strains Aspergillus niger, Aspergillus fumigatus and Candida albicans. Gram positive organism Bacillus lentius and Gram negative organism Pseudomonas aeruginosa was found to be most susceptible bacterial strains. Significant antifungal activity was observed for MSECH against Candida albicans and Aspergillus fumigatus. The results of the present investigation indicated that MSECH possess anti-microbial activity which may be due to the presence of flavonoid apigenin. The linearity and correlation coefficient was found to be 100-1000 ng/spot and 0.9962 respectively for the quantification of apigenin in HPTLC method. Percentage recovery, LOD and LOQ found to be 98.64%, 140.46 ng/spot and 426.34 ng/spot respectively.

 

 

INTRODUCTION:

India has a rich heritage of 2000 medicinal plants used in Ayurveda, Siddha, which are called Alternative Systems of Medicine (ASM). The periodic use of plants as remedy for diseases may be traced from the ancient records like Rig Veda and works of Charaka and Sushrutha. Now herbal medicines can be also prepared in the form of pills, capsules or powders or in more concentrated liquid form called extracts and tinctures, they can also be applied topically in creams or ointments or applied directly to skin. Traditional medicine played an important role in the health care needs of India and other countries for thousands of years. Cardiospermum halicacabum L (Sapindaceae) is an herbaceous climber found throughout the plains of India, used widely in traditional medicine for curing various human ailments. The tender young shoots are used as a fodder, vegetable, diuretic, stomachic and rubefaciant¹. The juice of the herb is used to cure ear-ache and to reduce hardened tumours. It exhibits significant analgesic, anti-inflammatory and vasodepressant activity² Cardiospermum halicacabum extracts have been evaluated for their antipyretic activity against yeast-induced pyrexia in rats³.

 

The leaves and stems are used against common cold and angina. The roots are diuretic, diaphoretic, emetic, mucilaginous, and laxative. They are useful in treatment of arthritis, amenorrhea, lumbago, and rheumatism, stiffness of limbs, snake bite, piles, erysipelas and neuropathy^4-6. The plants have sedative action on CNS, inhibiting histamine release and nitric oxide production⁷. The herb is used in hair oils for treating dandruff, alopecia and for darkening hair^8,9. Though this plant is used in traditional practice for microbial treatments, there are no specific scientific reports for anti-microbial activity. Hence the present study was undertaken to ascertain scientific basis for the claim that it is used as an antimicrobial agent.

 

MATERIALS AND METHODS:

Instrumentation

Analysis was performed on a Camag HPTLC system equipped with a sample applicator Linomat V, twin trough development chamber (10x10) size, TLC Scanner III, Wincats integration software was used.

 

Reagents and Chemicals

Analytical grade Toluene, Ethyl acetate, Methanol, Formic acid, Chloroform, glacial acetic acid was obtained from Qualigens Ltd, Mumbai. Apigenin  were obtained from the Sigma Aldrich Ltd, Bombay. Precoated TLC aluminium sheets silica gel 60F254 (10 x10 cm, 0.2 mm thick) were obtained from E. Merck Ltd, Mumbai.

 

Test organisms

Gram positive bacteria, Staphylococcus aureus NCIM 2079,Micrococcus luteus NCIM 2169, Bacillus subtilis NCIM 2063 and Gram negative bacteria; Escherichia coli NCIM 2065, Salmonella paratyphi NCIM 2501, Pseudomonas aeroginosa NCIM 2200, Klebsiella pneumonia NCIM 2707, Vibrio cholrae were used as test organisms.The strains were obtained from Nation Collection of Industrial Micro organism, Pune . The fungal strains Aspergillus fumigates MTCC 1811, Aspergillus niger MTCC 1344 Monococcus purpura MTCC 1090 and the yeast Candida albicans MTCC 3100 were collected from Microbial type culture collection, Chandigarh.

 

Plant Collection Authentication and Extraction

The entire plant of Cardiospermum halicacabum L was collected during November 2010 from around Coimbatore, Tamilnadu, India. The plant was identified and authenticated by G.V.S. Murthy, Botanical Survey of India, Coimbatore. The entire plant was washed, cleaned with water; shade dried for 10 days under controlled temperature, the stem was separated from the collected plant and powdered, passed through a # 40 mesh sieve and stored in an air tight container for further use. Coarsely powdered dried stem (1.1 kg) was successively extracted in soxhlet apparatus using petroleum ether (60-80°C), chloroform, ethyl acetate and methanol for 72 h at room temperature respectively. The extracts were filtered and the solvents evaporated to dryness under reduced pressure in an Eyela rotary evaporator at 40 to 45ºC. The percentage yield for petroleum ether (60-80°C), chloroform, ethyl acetate and methanol was found to be 1.85, 3.24, 4.8 and 6.32% respectively. Methanol extract was used to quantify the flavonoid apigenin and to screen the anti microbial  activity.

 

Preparation of Standard Drug Solution

The standard drug of Chloramphenicol and fluconazole at the concentration of 10 mcg/ml and 25 mcg/ml using DMSO as a solvent and DMSO is used as a solvent control for this experiment.

 

Determination of antimicrobial activities of extract

The antimicrobial activities of petroleum ether (60-80°C), chloroform, ethyl acetate and methanol extracts were determined by disc diffusion method described by Omenka and Osuoha¹⁰ with slight modifications. The culture plates were each seeded with test organisms and allowed to solidify and there after punched with a sterile cork borer (5 mm diameter) to cut uniform wells. The open wells were filled with 0.05ml of the extract. The plates were then incubated at 37ºC for 24 h. For the fungi, the antimicrobial test was carried out using Saboraud dextrose agar (SDA) plates incubated at 30°C for 72 h. The zones of inhibition were then measured and recorded and compared with positive standards, ciprofloxacin (bacteria) and fluconazole (fungi) at the concentration of 5 mcg/ml and 10 mcg/ml using DMSO as a solvent and DMSO is used as a solvent control for this experiment.

 

Minimum inhibitory concentration (MIC)

Various concentrations of the stem methanol extracts of Cardiospermum halicacabum L were prepared 6.25 mg/ml, 12.5 mg/ml, 25 mg/ml, 50 mg/ml and 100 mg/ml. The culture plates were again seeded with test bacterial organisms and allowed to solidify and thereafter punched with a sterile cork borer (5 mm diameter) to cut uniform wells. The open wells were filled with 0.05 ml of the extract. The plates were then incubated at 37ºC for 24 h. The lowest concentration of the extract that showed inhibition of growth of the test organisms was read and taken as the minimum inhibitory concentration (MIC).

 

High performance thin layer chromatography

Preparation of Standard Apigenin solution and sample solution

Ten mg of apigenin were accurately weighed into 10 ml volumetric flask, and then the solution was made up to 10 ml with methanol. From the Stock solution of apigenin  1 ml was pipetted out and further diluted upto 10 ml to obtain the final concentration of 100 μg/ml. MSECH was diluted in methanol to produce a concentration of 10 mg/mL.

 

Chromatographic Condition

Standard Apigenin and MSECH were spotted on a Precoated TLC aluminium sheets silica gel60F₂₅₄ (10 x 10 cm, 0.2 mm thickness) as 6 mm wide band width by using automatic TLC applicator Linomat V, 10 mm from the bottom. Toluene: Ethyl acetate: Formic acid:methanol (3:6:1.4:0.4v/v) was used as mobile phase. The plates were then developed in twin trough chamber after for saturation for 15 min. After development the plates were dried in air and scanned at 254 nm for Apigenin by using CAMAG Scanner III. The plates were photographed at 254 nm by using CAMAG Reprostar instrument (Fig.3).

 

Calibration Curve for Standard Apigenin

The standard apigenin solutions (200-1000 ng/spot) were applied on TLC plate and developed and scanned as per the chromatographic conditions mentioned above. The peak areas were recorded. Calibration curve of apigenin was prepared by plotting peak area against concentration of apigenin applied.

 

Validation of the Proposed HPTLC Method

ICH guidelines were followed for the validation of the developed HPTLC method (precision, repeatability, accuracy, Ruggedness, Robustness, Plate efficiency and Flow constant).

 

RESULTS AND DISCUSSION:

MSECH was tested for antimicrobial activity against six bacterial and three fungal species where all of the tested concentration of exhibited antibacterial and antifungal activity (Table 1 and Fig.1). Excellent zone of Inhibition was produced by standard ciprofloxacin.

 

Table 1. Antibacterial activity of methanol stem extract of Cardiospermum halicacabum

 

 

Fig.1. Zone of inhibition produced by MSECH against tested bacterial strains

 

 

Fig.2. Zone of inhibition produced by MSECH against tested fungal strains

 

Fig.3. Photo documentation of Apigenin  at 254 nm

 

Table 2. Antifungal activity of methanol stem extract of Cardiospermum halicacabum

 

Table 3. Antibacterial activity of methanol stem extract of Cardiospermum halicacabum by MIC method

Concentration (µg/ml)	Minimum Inhibitory Concentration (µg/ml)
	Gram Positive Bacteria			Gram Negative Bacteria
	Staphylococcusaureus	Bacilluslentus	Bacillussubtilis	Klebsiellapneumonia	Pseudomonas aeruginosa	Escherichiacoli
250	62.5	125	62.5	125	62.5	125

 

 

Fig 4  Calibration curve for Apigenin

 

MSECH elicited better antibacterial activity against gram negative organism compared to gram positive organism. Inhibitory effect was found increase against gram negative organism with increase in concentration of MSECH.In MIC method, MSECH exhibited good activity against gram positive bacteria Bacillus subtilis and gram negative bacteria Pseudomonas aeruginosa. MSECH displayed antifungal activity against all the three fungal strains tested.

 

Table 4. Antifungal activity of methanol stem extract of Cardiospermum halicacabum by MIC method

Concentration (µg/ml)	Minimum Inhibitory Concentration(µg/ml)
	Aspergillus niger	Aspergillusfumigatus	Candida albicans
250	62.5	125	125

 

The antifungal activity was found be increase with increase in concentration of MSECH. The activity produced by MSECH at 250 and 100 mcg/ml was almost equal to the activity of standard drug fluconazole.

 

Quantification of apigenin in methanol extract of stem of Cardiospermum halicacabum L

Standard Apigenin (Rf: 0.91) showed single peaks in HPTLC chromatogram. The amount of apigenin present in the methanol extract was computed from the above calibration curve. The linearity of apigenin was found to be 100-1000 ng/spot (Fig.4). The correlation coefficient of apigenin was found to be 0.9962. The LOD and LOQ of Apigenin were found to be 140.46 ng/spot and 426.34 ng/spot respectively. Apigenin was to found contain in MSECH was well separated and showed similar

 

Validation of the Proposed HPTLC Method:

Linearity:

A representative calibration curve of apigenin was obtained by plotting the peak area of apigenin against the concentration of apigenin (100-1000 ng/spot). The correlation coefficient of apigenin was found to be 0.9967 and thus exhibits the good linearity between concentration and peak area.

 

Limit of Detection and Quantification: The LOD and LOQ of Apigenin were found to be 140.46 ng/spot and 426.34 ng/spot respectively.

 

Specificity:

It was observed that other constituent’s presents in the methanol extract did not interfere with the active constituent under study. The overlay spectrum of MSECH and standard apigenin found to be similar and hence method was found to be very specific. The peak purity of the apigenin acid was assessed by comparing the spectra at three different levels, viz. peak start, and peak apex and peak end positions of the spot.

 

Robustness of the method:

By introducing small changes in the mobile phase composition, mobile phase volume and duration of mobile phase saturation, the effects on the results were examined. Robustness of the method was done in triplicate at a concentration level of 2 μg/spot and the % R.S.D. of peak area was calculated.

Ruggedness of the method:

It expresses the precision within laboratories variations like different days, different analyst, and different equipment. Ruggedness of the method was assessed by spiking the standard 6 times in two different days with different analyst.

 

Plate efficiency (N):

Plate efficiency, also known as number of theoretical plates was calculated for the described method by the following equation:

16 x l x z

N = -------------

w2

Where, l is the distance (in mm) travelled by solvent front, z is the distance (in mm) travelled by the target spot from application point and w is the width of spot (in mm) in the direction of mobile phase ascending.

 

Flow constant:

The flow constant or velocity constant (k) is a measure of the migration rate of the solvent front. It is an important parameter for TLC users and can be used to calculate, for example, development times with different separation distances, provided that the sorbent, solvent system, chamber type and temperature remain constant. The flow constant is given by the following equation:

ZF²

k = ------------

t

Where,

Where, k is flow constant [mm2/s], ZF is distance between the solvent front and the solvent level [mm] and t is the development time [s]. The flow constant as calculated by this method was found to be 4.9 mm2 s-1 and 6.12 mm2 s-1.

 

Accuracy:

The accuracy was determined by standard addition method. To a fixed amount of preanalyzed sample of methanol extract of stem of Cardiospermum halicacabum L increasing amount of standard apigenin were added in all the levels of calibration curve. The Percentage recovery of apigenin were calculated at each level (n = 3) and shown in (Table 5).

 

Table 5.Summary of validation parameter

Parameters	Apigenin
Linearity
i)Range	100-1000 ng/spot
ii) Correlation Coefficient	0.9967
iii) Rf Value	0.91
Precision (%RSD)
i) Intra day	0.9012
ii) Inter day	0.7180
iii) Repeatability	0.9728
LOD	140.46 ng/spot
LOQ	426.34 ng/spot
Specificity	Specific
Ruggedness % RSD	0.9162
Robustness	Robust
Plate Efficiency	24.88 mm
Flow constant	4.9 mm2

 

 

Fig 5 Chromatogram of standard apigenin

 

Fig 6 Chromatogram of MSECH 

 

Fig 7 Overlay spectrum of Apigenin

 

 

CONCLUSION:

Gram positive organism Bacillus lentius and Gram negative organism Pseudomonas aeruginosa was found to be most susceptible bacterial strains. Among the fungal strains, Candida albicans and Aspergillus fumigatus was found to be more susceptible to MSECH. The overlay spectrums of standard apigenin and MSECH found to be similar and hence the method was found to be very specific (Fig.7). Since the proposed validated HPTLC method resolves and quantifies apigenin effectively, it can be used routinely to quantify the concentration of apigenin in the methanol stem extract of Cardiospermum halicacabum L.

 

REFERENCES:

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8.        Venkatesh Babu, K.C., Krishnakumari, S., 2006. Cardiospermum halicacabum suppresses the production of TNF-a and NO by human peripheral blood mononuclear cells. African Journal of Biomedical Research 9; 2006:95–99.

9.        Thabrew, I., Munasinghe, J., Chackrewarthi, S., Senarath, S. The effects of Cassia auriculata and Cardiospermum halicacabum teas on the steady state blood level and toxicity of carbamazepine. Journal of Ethnopharmacology 90; 2004: 145–150.

10.     10 Omenka, C. A. and J. O. Osuoha. Antimicrobial potency of Grapefruit seed extract on five selected pathogens. Nigerian Journal of Microbiology. 14 (2); 2000: 39-42.

 

 

 

 

 

 

 

Received on 01.03.2014          Modified on 30.03.2014

Accepted on 06.04.2014         © RJPT All right reserved