黄酮类抗氧化

IPCBEE vol.5 (2011) © (2011)IACSIT Press, Singapore

Antibacterial and Antioxidant potential of White and Pink Nelumbo Nucifera

Gaertn Flowers

Brindha Venkatesh

Department of Biochemistry PSG College of Arts and Science Coimbatore-1 014, TamilNadu, India E-mail: brindhavenkatesh@ymail.com

Arthi Dorai

Department of Biochemistry PSG College of Arts and Science Coimbatore-1 014, TamilNadu, India

E-mail: arthidorai@yahoo.com

as cancer, hypertension, diarrhea, fever, weakness, infection

Keywords-Nelumbo nucifera Gaertn; antibacterial activity; and body heat imbalance [8].

The major constituents isolated from the lotus plant are Minimum Inhibitory Concentration (MIC); antioxidant; agar

alkaloids (liensinine, neferine, nuciferine, remrefidine and disc diffusion.

isoliensinine) and flavonoids ((+)-1(R)-coclaurine, (-)-1(S)-norcoclaurine and quercetin 3-O-b-D-glucuronide). I. INTRODUCTION

Several previous reports suggested that seed could suppress

Medicinal plants are gifts of nature used to cure number cell cycle progression, cytokine genes expression and cell of human diseases. To promote the proper use and to proliferation in human peripheral blood mononuclear cells. determine their potential as sources for new drugs, it is Recently, the leaf of N.nucifera showed the hypotensive essential to study the medicinal plants [1]. Contrary to the effects that were mediated by vasodilatation via nitric oxide synthetic drugs, antimicrobials of plant origin are not [9] and betulinic acid isolated from rhizomes and used as associated with side effects and have an enormous anti-tumoral and melanoma specific cytotoxic agent. therapeutic potential to heal many infectious diseases. The

Abstract—The recent interest on alternative medicine has potential for developing antimicrobials from higher plants taken up great dimensions in changing the health care scenario appears rewarding as it will lead to the development of across the globe. The worldwide interest in medicinal plants phytomedicine to act against microbes [2]. Therefore, reflects recognition of the validity of studying the antimicrobial researchers are increasingly turning their attention to folk and antioxidant activity. Hence the present study was carried medicine to develop better drugs against microbial infections out to explore antibacterial and antioxidant potential of [3]. hydroethanolic extract of both white and pink flowers of A perusal of literature revealed that the flower parts of Nelumbo nucifera Gaertn (Nelumbonaceae) flower in vitro. Nelumbo nucifera had not been subjected to screening for The antibacterial activity was screened against different

antibacterial properties so far. From this viewpoint the

bacterial strains by detecting zone of inhibition and minimum

present study was carried out to evaluate the antibacterial inhibitory concentration (MIC). The zone of inhibition and

MIC values were compared with control compared with activity of hydroethanolic extract of white (HEWNN) and standard antibiotic disc suggesting their potential as pink (HEPNN) N. nucifera flowers.

Antioxidants act as a major defense against radical alternatives to orthodox antibiotics in the treatment of

mediated toxicity by protecting the damages caused by free infectious caused by these microorganisms. Total antioxidant

potential was evaluated in hydroethanolic extract of white radicals [4]. The natural antioxidant mechanism is inefficient Nelumbo nucifera (HEWNN) flower and hydroethanolic and hence dietary intake of antioxidant compounds is

important [5]. In recent years, there has been growing extract of pink Nelumbo nucifera (HEPNN) flower by: Ferric

reducing antioxidant power (FRAP), Hemoglobin interest in alternative therapies and the therapeutic use of glycosylation,Reducing power and Phosphomolybdenum and natural products, especially those derived from plants [6]. compared with the standard ascorbic acid in dose dependent Thus the present study was focused to further probe the new manner. Both HEWNN and HEPNN flower extracts showed safe and effective in vitro antioxidant agents in white and maximum activity 16.53 mg and 14.21 mg at 1000μg/ml pink N.nucifera flower extracts. concentration in FRAP assay. Significantly high antioxidant Nelumbo nucifera Gaertn (Family: Nelumbonaceae) activity (55.5% & 41.6%) was noticed in haemoglobin commonly known as Indian lotus, one of the oldest perennial glycosylation followed reducing power (0.52 & 0.45 Abs). The aquatic herb consumed throughout Asia. Pharmacological results suggest that alkaloids, phenols and flavonoids in flowers studies of the plant revealed that the whole plant possess provide considerable antioxidant activity. However, in

antidiabetic, antipyretic, anti-inflammatory, anticancerous,

comparison with HEPNN flower, HEWNN flower extract

antimicrobial, antiviral and anti-obesity properties [7]. exerted effective antibacterial and potent antioxidant activity

Furthermore, N.nucifera flower has considerable reputation which can be used as a lead compound for drug development

as a potent adjunct in the treatment of various ailments such in the future.

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However, scientific evidence on antioxidant potential in hydroethanolic extract of white and pink N.nucifera is still unknown. Therefore, our study has been focused to gain extensive knowledge regarding the power of antioxidants from both white and pink N.nucifera flowers and to tap their potential.

was stored in air-tight desiccator and used for further analysis.

F. Chemicals

Ammonium thiocyanate, ferrous chloride, ferric chloride, ammonium molybdate, potassium ferricyanide, tripyridyltriazine (TPTZ) and ascorbic acid were purchased

II. MATERIALS AND METHODS from Hi-media, Mumbai, India. All other chemicals and

reagents used were of analytical grade and were purchased

A. Plant material from Hi-media, Mumbai, India.

In FRAP assay, when a ferric tripyridyltriazine (FeIII -The flowers of N.nucifera were collected from different II

localities of Coimbatore District and authenticated by TPTZ) complex is reduced to the ferrous (Fe) form, an Botanical Survey of India (BSI) in “Tamil Nadu Agriculture intense blue colour with an absorption maximum at 593 nm University” Coimbatore, Tamil Nadu, India. A voucher develops and measured at low pH by the method of specimen (No.BSI/SC/5/23/09-10/Tech.279) has been Benzie.F.F. and J.J.Strain [12]. Degree of non-enzymatic deposited at the Herbarium of the Botany department of haemoglobin glycosylation was measured by Pal & Dutta “Tamil Nadu Agriculture University” for future reference. method [13]. The reducing power was measured according

to the method of Oyaizu [14]. Antioxidant capacity was

B. Plant Extraction

measured by phosphomolybdenum method [15].

The air-dried and powdered white and pink flowers

(100g of each) were cold macerated with 50% ethanol for 3 G. Statistical Analysis days, with occasional stirring. After 3 days, the suspension The mean zones of inhibition and MIC was calculated was filtered through a fine muslin cloth and was evaporated from the values of the three experiments for each isolates and to dryness at low temperature (< 40ºC) under reduced reported as final results. Antioxidant activity results are pressure in a rotary evaporator. Dark brown colored crystals expressed as mean ± SD. values. of approximately 8g was stored in air-tight desiccators and

III. RESULTS AND DISCUSSION used for further analysis.

The antibacterial activity of the hydroethanolic extract of C. Antibacterial Assay

both white and pink N.nucifera flower extracts were

Antibacterial activity was determined using the agar disc evaluated at two different concentrations (500 µg & 1000µg) diffusion method described by Parekh [10]. Each bacterial against five bacterial strains by the disk diffusion method inoculum was incubated in 2.5ml Mueller-Hinton broth at and the results were summarized in Table I. The antibacterial 37°C for 18 hours. Every inoculum was spread over plates activity of both N.nucifera flower extracts was found to be containing Mueller-Hinton agar. Five millimeter discs increased in dose dependent manner. The maximum zone of containing 500µg/ml and 1000µg/ml of extract were placed inhibition was exhibited by both white and pink N.nucifera on cultured pathogenic bacteria on agar plates and incubated flowers against Escherichia coli (16mm & 14mm), Bacillus at 37°C. The plates were checked for bacterial growth after a Subtilis (15mm & 13mm) and minimum of 16 hours and occasionally till 24 hours. The diameter of the zone of inhibition was then measured. TABLE I. ANTIBACTERIAL ACTIVITY OF BOTH WHITE AND PINK

ELUMBO UCIFERA FLOWERS BY DISC DIFFUSION METHOD NNCommercial disc of Chloramphenicol (30µg) was used as

positive control and experiment was done thrice for each Inhibition Zone (mm)*extract. MicroorganiWhite Pink Nelumbo ChlorampheniD. Determination of Minimum Inhibitory Concentration (MIC)

MICs are defined as the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. MIC of antibiotics was evaluated using standard microbroth dilution method [11].

E. Antioxidant activity

The air-dried and powdered flowers (100g of each) were cold macerated with 50% ethanol for 3 days, with occasional stirring. After 3 days, the suspension was filtered through a fine muslin cloth and was evaporated to dryness at low temperature (<40ºC) under reduced pressure in a rotary evaporator. Dark brown colored crystals of approximately 8g

sm

Nelumbo nucifera flower1000500µgµg/m/ml l

nucifera flower 1000µg/ml

500µg/ml

col (Control) 30µg/ml

30 22 29 26 27 Escherichia coli Klebsiella pneumonia Pseudomonas aeruginosa Bacillus Subtilis Staphylococcus aureus

16 11 14 9 12 6 10 8 9 7 8 5 15 9 13 5 13 10 11 7 * mean of three replicates

TABLE II.

ANTIOXIDANT ACTIVITY OF BOTH HEWNN AND HEPNN FLOWER EXTRACT BY FRAP ASSAY

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S.No Concentration White Nelumbo (µg/ml) nucifera flower

(mg)

1 200 2 400 3 600 4 800 5 1000 Pink Nelumbo nucifera flower

(mg)

0.5MINIMUM INHIBITORY CONCENTRATIONStaphylococcus aureus3.165 2.861 8.612 6.327 10.18 7.562 14.22 12.57 16.53 14.21 Opticaldensity0.40.30.20.10ggggggg0µ0µ0µ0µg0µ0µ0µ0µ0µg4243444547414849500µgwhite flowerpink flowerStaphylococcus aureus (13mm & 11mm). The moderate zone of inhibition was found in both white and pink flower extracts against Klebsiella pneumonia (12mm & 10mm) and Pseudomonas aeruginosa (9mm & 8mm).

Gram-negative bacteria were more susceptible to the N.nucifera flower extracts than gram-positive bacteria which contradict the previous reports that plant extracts are more active against gram-positive bacteria than gram-negative bacteria. However, the results revealed that the hydroethanolic extract of white N.nucifera flower showed effective antibacterial activity when compared to pink N.nucifera flower which may be due to its variation in phytochemical constituents like flavonoids, alkaloids and tannins which was also reported by Bose et al., [16] and these results were compared with the standard antibiotic chloramphenicol (30µg/ml). Similar work by Rogger et al., [17] showed that antibacterial effect of Tithonia diversifolia against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli suggesting that the plant can be used in the treatment of gastrointestinal infection and diarrhea in human.

The minimum inhibitory concentration for white N.nucifera flower extract against Escherichia coli and Staphylococcus aureus was found to be 430µg and 450µg respectively and pink flower showed 480µg and 490µg respectively (Fig. 1 and 2). The lowest MIC was exhibited by white N.nucifera flower extract against both the microorganisms when compared to pink N.nucifera flower. Ushimaru and Silva, [18] have reported that medicinal plants like Allium sativum, Zingiber officinale have antibacterial activity against Salmonella typhimurium, Staphylococcus aureus at minimum inhibitory concentration of 450µg/ml.The findings of this study confirmed the therapeutic potency of both white and pink N.nucifera flowers used in traditional medicine. These results offer promising lead for the discovery of potent antibacterial compounds in therapeutic and dietary use globally.

The antioxidative phytochemicals in grains, vegetables and fruits have received increasing attention recently for their potential role in prevention of human diseases as well as in food quality improvement [19]. Lotus has been used both as food and medicine in Asia, particularly in India [20]. The antioxidant activity of plant extracts vary with methods [21]. Therefore, a single assay

Concentration Figure 1. Minimum inhibitory concentration of both white and pink

Nelumbo nucifera Flowers against Escherichia coli

MINIMUM INHIBITORY CONCENTRATIONE.coli0.80.70.60.50.40.30.20.10ggggggggg0µ0µ0µ0µ0µ0µ0µ0µ4142434445490µ4748500µgOptical density white flowerpink flowerConcentration

Figure 2. Minimum inhibitory concentration of both white and pink

Nelumbo nucifera Flowers against Staphylococcus aureus

may be inadequate. For this reason, we cross checked antioxidant activities of both white and pink lotus flower extracts with different assays.

FRAP assay is presented as a novel method of assessing total antioxidant capacity and is considered as a useful indicator of the body's antioxidant status to counteract the oxidative damage due to ROS. The advantage of the FRAP assay is in being fast, easy to handle, with highly reproducible results [22]. Antioxidant activity by this method was evaluated on the basis of alkaloid and element content where ferric to ferrous ion reduction at low pH causes a ferrous-tripyridyl-triazine complex which has absorption at 593nm [23]. Both HEWNN and HEPNN flower extracts showed concentration dependent increase in their ferric reducing capacities. However, HEWNN flower extract was more potent than HEPNN flower which may be due to the presence of different levels of active constituents in both flowers (Table 2). The maximum antioxidant capacity of both HEWNN and HEPNN flower extract was found to be 16.53mg and 14.21mg at 1000μg/ml concentration in FRAP assay (Table 2).

High degree of haemoglobin glycosylation (Fig. 3) activity was noticed in HEWNN (55.5%) and HEPNN (41.6%) at 500μg/ml concentration. This was compared to that of standard ascorbic acid (62.6%) at 300μg/ml concentration. HbA1c, or glycosylated hemoglobin, is formed through the

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0.7Absorbance at 700 nm70EquivalentofAscorbicacid6050403020100100200300400500Concentration(µg/ml)HEWNNHEPNN0.60.50.40.30.20.10100200300400500Concentration (μg/mL)Ascorbic acidHEWNNHEPNN Figure 3. Antioxidant activity of HEWNN and HEPNN flowers by Haemoglobin glycosylation method. Values arthe average of duplicate

experiments and represented as mean ± standard deviationnon-

Figure 5. Antioxidant activity of HEWNN and HEPNN flowers Phosphomolybdenum method. Values are the average of duplicate

experiments and represented as mean ± standard deviation

enzymatic binding of circulating glucose to N terminal of beta chain of hemoglobin molecule (glycation) [24]. Degree of haemoglycosylation of HEWNN is much effective than HEPNN flower and exhibits increasing trend with the increasing concentration of plant extract. In figure 4, the reducing power increased with increasing concentration of plant extract and maximum reducing power of HEWNN, HEPNN and ascorbic acid at 700 nm were found to be 0.52, 0.45 and 0. absorbance respectively. These results clearly revealed that HEWNN flower extract have effective antioxidant activity than HEPNN flower extract, which may be due its phytochemical constituents.

The reducing ability of a compound generally depends on the presence of reductants (antioxidants) [25], which have been exhibited antioxidant potential by breaking the free radical chain by donating a hydrogen atom [26]. The presence of reductants in N.nucifera extract causes the reduction of the Fe3+/Ferricyanide complex to the ferrous form. The reducing power of HEWNN flower extract was very potent and had effective antioxidant activity than HEPNN extract. 10090%Scavenging80706050403020100100200300400500Concentration ( µg/ml)Ascorbic acidHEWNNHEPNNThe maximum values of both white and pink N.nucifera flower extract were found to be 62.5mg and 56.3mg ascorbic acid equivalent at 500µg/ml in Phosphomolybdenum assay. Our findings are in good accordance with the observation made by Nagarajan et al., 2008 [27] who reported that the alcoholic extract of Wrightia tomentosa showed antioxidant activity.

Phosphomolybdenum assay used to determine the total antioxidant capacity based on the reduction of Mo (VI) to Mo (V) by the antioxidant compound and the formation of a green phosphate/Mo(V) complex at acidic pH. This assay has been successful in the quantification of vitamin E antioxidant capacity and being simple and independent of other antioxidant measurements commonly employed, it was decided to extend its application to plant extracts [28]. Moreover, it is a quantitative one, since the antioxidant activity is expressed as the number of equivalents of ascorbic acid. The good antioxidant activity of HEWNN and HEPNN might be attributed due to the presence of phytochemical, such as Flavonoids and biflavones.

Hence, both HEWNN and HEPNN flowers were found to have the potential to be used as nutraceutical supplement due to its antioxidant activity as well as to exert potent supplement in prevention of degenerative diseases. However, in comparison with HEPNN flower, HEWNN flower extract exhibited significant antioxidant activity which might be due to the structural variation of antioxidant compounds such as number of phenolic hydroxyl or methoxyl groups, flavones hydroxyl, keto groups free carboxylic groups and other structural features. Therefore if a systematic investigation is initiated the traditional medicinal systems practiced in India can offer promising leads for the discovery of potent antioxidants that can have therapeutic and dietary use globally.

IV. CONCLUSION

The results of the study suggest that white flower of N.nucifera extract exert strong antibacterial and potent antioxidant activity when compared to pink flower of N.nucifera which might be due to the presence of rich phytochemical constituents. Future work is therefore under progress to identify and elucidate the bioactive principles

Figure 4. Reducing power of HEWNN and HEPNN flowers. Values are the average of duplicate experiments and represented as mean ± standard

deviation

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