INTRODUCTION:

Flowers are the important parts of the plants and many edible flowers have been used in various parts of the world for centuries as food, medicine, for food garnishing etc. Flowers are consumed fresh or dried, in cocktails, canned in sugar, preserved in distillates etc. Many natural antioxidants such as phenolic acids, flavonoids, anthocyanins and other phenolic compounds are found in flowers. Flowers are reported to possess several biological activities such as antimicrobial, acaricidal, antioxidant, anti-HIV, cytotoxic, antidepressant, anti-inflammatory etc. The high antioxidant activity of flowers may be attributed to the level of polyphenol compounds including flavonoid.

They are also promising sources of various minerals which have key role in the normal health of an individual ^[1-10].

Humboldtia brunonis Wall (Caesalpiniaceae) is distributed in S. India and is a 5-6 m tall tree. It is distributed in S. Canara, Coorg, Mysore, Malabar, W. Nilgiris, Hassan, Chikmagalur ^[11]. The leaf and bark of H. brunonis are used as medicine by Kurichia and Kuttanaika tribes in Mananthavady of Wayanad district, Kerala ^[12]. Antimicrobial, cardioprotective, nephroprotective, hepatoprotective and antioxidant activities of H. brunonis have been investigated ^[13,14]. Kydia calycina Roxb (Malvaceae) is distributed in India and Burma and is a small tree ^[11]. The paste of leaves is applied in rheumatism in the Sewa river catchment area in Northwest Himalaya ^[15]. The plant K. calycina has shown antifungal, analgesic and anti-inflammatory, hepatoprotective activity ^[16,17,18]. Calycopteris floribunda (Roxb.) Poiret belonging to Combretaceae is a straggling shrub distributed in Indo-Malaysia ^[19]. The plant C. floribunda has got several ethnomedicinal uses ^[20-25]. Cytotoxic, antioxidant and β-glucuronidase inhibitory, antimicrobial, hypoglycaemic, hepatoprotective activity of C. floribunda have been studied ^[26-32]. In the present study, we have estimated the content of major and minor elements by ICP-OES technique and determined anticariogenic potential of flowers of Calycopteris floribunda (Roxb.) Poiret, Humboldtia brunonis Wall and Kydia calycina Roxb.

MATERIALS AND METHODS:

Collection and identification of plant materials:

Table 1 presents the place of collection and voucher number of plants selected. The plants were authenticated by Dr. Vinayaka K.S. The voucher specimens were deposited in the department herbaria, Department of Microbiology, SRNMN College of Applied Sciences, Shimoga, Karnataka. The flowers were shade dried and powdered using blender.

Table 1: Plants selected for the study

Plant name	Place of collection	Voucher number
K.calicyna (Kc)	Hanagerekatte, Shivamogga (D)	SRNMN/PK/Kc-006
H.brunonis (Hb)	Hulikal, Hosanagara (T), Shivamogga (D)	SRNMN/PK/Hb-020
C.floribunda (Cf)	Hosur, Sagara (T), Shivamogga (D)	SRNMN/PK/Cf-035

Elemental analysis of flowers:

A known quantity of each of the powdered flower material (1gm) was digested in 10ml of ultrapure metal free nitric acid in a microwave digester (CEM). After complete digestion, the content was diluted to 25ml with distilled water. Elemental analysis was performed using Inductively Coupled Plasma with Optical Emission Spectroscope (ICP-OES, Agilent Technologies 700series, US). The microwave digested sample was aspirated into ICP-OES to estimate macroelements viz., Calcium (Ca), Potassium (K), Sodium (Na) and Magnesium (Mg) and microelements viz., Manganese (Mn), Iron (Fe), Zinc (Zn), Nickel (Ni), Chromium (Cr), Lithium (Li) and Copper (Cu) present in the flower material. The calibration standards were prepared by diluting the stock multi-elemental standard solution (1000 mg/L) in nitric acid ^[33]. Instrument configuration and experimental conditions are summarized in Table 2.

Extraction of powdered flowers:

50 grams of each of the powdered flower material was subjected to soxhlet extraction and extracted using solvents viz., petroleum ether (P), chloroform (C) and methanol (M) on polarity basis. The extracts were filtered through 4-fold muslin cloth followed by Whatman No. 1 filter paper, concentrated in vacuum under reduced pressure and dried in the desiccator ^[34]. Methanol extract of the flowers was subjected to preliminary phytochemical analysis to screen the presence of various phytoconstituents ^[35].

Anticariogenic activity of flower extracts:

The inhibitory effect of flower extracts against cariogenic bacteria was tested against 4 isolates of Streptococcus mutans (Sm-01 to Sm-04) which were recovered previously from carious teeth by Agar well diffusion assay. The 24 hours old Brain heart infusion broth (HiMedia, Mumbai) cultures of these bacteria were swabbed aseptically on sterile Brain heart infusion agar (HiMedia, Mumbai) plates using sterile cotton swabs. With the help of sterile cork borer, wells of 6mm diameter were punched in the inoculated plates and 200µl of solvent extracts of flowers (50mg/ml of 25% Dimethyl sulfoxide (DMSO)), standard (Streptomycin, 1mg/ml) and DMSO (25%) were transferred into labeled wells. The plates were incubated at 37^oC for 24 hours in upright position and the zone of inhibition formed was measured ^[34]. The experiment was repeated two times and the average value was recorded.

Table 2: ICP-OES Operation conditions:

Parameter	Value
Power (kW)	1.2
Plasma flow (L/min)	15.0
Auxiliary flow (L/min)	1.50
Nebulizer flow (L/min)	0.75
Sample flow rate (L/min)	1.5
Replicate read time (s)	3.00
Instrument stabilization delay (s)	15.0
Sample uptake delay (s)	10.0
Pump rate (rpm)	15.0
Rinse time (s)	10.0
Spray chamber	Cyclonic type
Elements, wavelengths (nm)	Ca (422.673), Cu (327.395), Na (589.592) Cr (267.716), Fe (238.204), K (766.491), Mg(279.553), Mn (257.610), Ni (231.604), Zn (213.857), Li (670.783)

RESULTS:

In the present study, we have estimated the elements by ICP-OES. Estimation of four major elements viz., calcium, magnesium, sodium and potassium and seven minor elements viz., iron, manganese, zinc, copper, chromium, nickel and lithium in microwave digested flower specimens was carried out. Among flowers, K. calycina was found to contain high quantity of most of the elements estimated. Among major elements, the content of potassium was highest followed by calcium, magnesium and sodium. Among the minor elements estimated, the content of iron and chromium was high and low respectively (Table 3).

Table 3: Contents of various elements in flowers:

Elements	Content (ppm)
Elements	*K. calycina*	*H. brunonis*	*C. floribunda*
Calcium	9220.06	3404.53	6977.93
Magnesium	3032.73	1666.26	2638.79
Sodium	71.62	75.59	103.30
Potassium	12696.65	9914.64	8829.19
Iron	362.46	152.95	378.86
Manganese	24.82	14.71	340.52
Zinc	20.32	28.14	13.57
Copper	19.23	17.48	12.95
Chromium	1.85	0.43	1.03
Lithium	5.26	6.41	5.39
Nickel	7.25	1.09	2.96

Preliminary phytochemical analysis of methanol extract of flowers revealed the presence of saponins, tannins, triterpenoids and flavonoids in C. floribunda; saponins and flavonoids in K. calycina and tannins and flavonoids in H. brunonis. Anticariogenic activity of solvent extracts of selected flowers was tested against 4 isolates of S. mutans. The zone of inhibition formed around the well was taken as positive for antibacterial activity. All the 4 isolates were found to be susceptible to solvent extracts of flowers with zone of inhibition ranging 1.3 to 3.3cm. Among flowers, extracts of C. floribunda exhibited highest inhibition of test bacteria followed by K. calycina and H. brunonis. Overall, among different flower extracts, chloroform extract of C. floribunda and K. calycina and methanol extract of H. brunonis exhibited highest inhibition of test bacteria. Inhibition caused by standard antibiotic was higher than that of flower extracts. There was no inhibition of test bacteria in case of DMSO (Table 4).

Table 4: Anticariogenic activity of solvent extracts of flowers:

Extract	Zone of inhibition in cm
Extract	Sm-01	Sm-02	Sm-03	Sm-04
Cf-P	2.5	2.4	3.3	2.4
Cf-C	2.8	3.2	2.8	2.6
Cf-M	2.6	3.0	2.7	2.5
Kc-P	1.5	1.5	1.4	1.5
Kc-C	2.0	2.7	2.1	2.6
Kc-M	2.1	1.7	2.1	1.7
Hb-P	1.5	1.5	1.3	1.6
Hb-C	1.7	1.6	1.5	1.6
Hb-M	2.0	1.9	1.8	2.1
Streptomycin	3.2	3.6	3.9	2.9
DMSO	0.0	0.0	0.0	0.0

DISCUSSION:

Every individual require a huge number of organic and inorganic compounds in order to meet the requirements for daily activities. Plants constitute a major portion of diet and hence, their nutritive value is important. Nutrients such as carbohydrates, fats and proteins are consumed in large quantity and form the major portion of the diet. However, mineral elements and vitamins form comparatively smaller part and are consumed in much smaller amounts. Elements are categorized into major (macro) and minor elements based on their requirements. The elements such as magnesium, potassium, sodium, calcium, iron, manganese, zinc, copper and others are essential micronutrients and these elements perform a variety of biochemical functions. These elements serve as components of enzymes, regulate cellular energy transduction, gas transport, antioxidant defense, membrane receptor functions, second-messenger systems and integration of physiological functions. Some 25 elements have been identified as important for maintenance of human health; therefore, the study of elements in food and plants is of great interest ^[36-39].

The analytical techniques used for determination of elemental content are mainly based on atomic spectrometry (for e.g., flame atomic absorption spectrometry, graphite furnace atomic absorption spectrometry) with single element detection. In this regard, ICP-OES is advantageous as multi-elemental determination is possible. Because of this, ICP-OES has become one of the most widely used analytical techniques for elemental determination and many studies have been conducted to validate this method for metal analysis in different sample types including plants ^[39,40,41].

Sodium, the major cation present in the extracellular fluid, is important in regulation of osmotic pressure, acid base balance and cellular membrane potential. It is also important for the transportation of substances actively through the cell membrane ^[42]. The content of sodium was high in C. floribunda followed by H. brunonis and K. calycina. Potassium is an important intracellular cation required for transmission of nerve impulses, muscle contraction and the maintenance of blood pressure ^[37,42]. The quantity of potassium was highest in K. calycina followed by H. brunonis and C. floribunda. Calcium is found in bones and teeth. It has several regulatory functions such as blood clotting, hormone secretion, muscle contraction, enzyme activation and maintenance of normal cardiac rhythm ^[37,42]. The content of calcium was found higher in K. calycina followed by C. floribunda and H. brunonis. Magnesium is important in metabolism of proteins and nucleic acids, neuromuscular transmission and muscle contraction, bone growth and regulation of blood pressure. It serves as a co-factor of several enzymes ^[36,42]. The quantity of magnesium was higher in K. calycina followed by C. floribunda and H. brunonis.

Iron is a key component of proteins such as haemoglobin, myoglobin, cytochromes and others and it plays an important role in the transportation, storage and utilization of oxygen. It also serves as co-factor of many enzymes ^[36,42,43]. The content of iron was found to be maximum in C. floribunda followed by K. calycina and H. brunonis. Zinc is very important for growth, sexual development, wound healing, immune system functioning and other physiological processes. It is a component of insulin and it assist in functioning of several other hormones which are important for reproduction and synthesis of DNA, RNA and protein. It also serves as cofactor of many enzymes ^[42,43]. H. brunonis was found to contain high content of zinc followed by K. calycina and C. floribunda. Copper is important for absorption of iron, synthesis of haemoglobin, phospholipids and is a co-factor of many enzymes. It is also necessary for the development of connective tissues, nerve coverings, and bone and it participates in energy metabolism ^[42,43]. The content of copper was highest in K. calycina followed by H. brunonis and C. floribunda. Manganese serves as a co-factor of many enzymes. It is also associated with bone development and metabolism of amino acid, lipid and carbohydrates ^[42,43]. C. floribunda contained high quantity of manganese followed by K. calycina and H. brunonis. Nickel is a nutritionally essential trace metal for at least several animals, plants and microbes and therefore deficiency or toxicity symptoms occurs when too little or too much is taken up by the organism ^[44]. The content of nickel was highest in K. calycina followed by C. floribunda and H. brunonis. Chromium is an essential element involved in action of insulin as demonstrated in the studies of chromium deficiency. Its deficiency may lead to altered glucose metabolism ^[45]. K. calycina contained high content of chromium followed by C. floribunda and H. brunonis. Lithium as lithium carbonate was used to treat gout and to dissolve urate bladder stones. Lithium carbonate was found to be beneficial in manic depressive illness. Today, lithium carbonate is one of the most widely prescribed psychiatric drugs ^[46]. H. brunonis was found to contain high lithium content whereas K. calycina and C. floribunda contained more or less similar quantity of lithium.

Dental caries is a transmissible infectious disease of oral cavity affecting people of all age groups. Microorganisms play a significant role in dental caries and the microflora of dental caries is highly complex and varies among individuals. The composition of microflora may depend on diet, saliva, and the chronicity of the lesion. Mutans group streptococci, such as S. mutans and S. sobrinus, and lactobacilli are important bacteria being implicated in the initiation and progression of disease. Mutans Streptococci are considered as the principal etiological agents of dental caries. These organisms are acidogenic and cause demineralization of enamel and dentin by fermenting dietary carbohydrates. They are also aciduric, which gives them the competitive survival advantage ^[47,48,49]. Antibiotics are best suited for the treatment of infection in modern dentistry. However, cariogenic bacteria developing resistance to commonly used antibiotics makes it necessary to select agents useful for successful therapy of dental caries. It is observed that herbal products used against oral infections have marked inhibitory effect on dental pathogens. Various plants and formulations containing the plant extracts are used traditionally for dental care. Plants such as babul, neem, clove and many others are used in India for brushing teeth and traditional dental care in India ^[50,51].

In the present study, solvent extracts of flowers exhibited marked anticariogenic activity against 4 isolates of S. mutans recovered from carious teeth. Studies have been carried out on the efficacy of extracts of flowers of certain plants against cariogenic bacteria. Aqueous extract of flowers of Clitoria ternatea was found to possess inhibitory activity against S. mutans and Lactobacillus casei isolated from oral cavity ^[52]. Methanol and hexane extracts of flowers of Psidium guajava were shown to exhibit inhibition of S. mutans ^[53]. It has been found that phytochemicals such as flavonoids, saponins, tannins, terpenoids have anticariogenic effects on oral bacteria ^[54-57]. In our study, the observed anticariogenic activity of flower extracts might be related to the presence of secondary metabolites such as flavonoids, saponins, tannins and terpenoids present in the solvent extracts.

CONCLUSION:

The flowers of C. floribunda, H. brunonis and K. calycina are shown to be good source of various major and minor elements having profound effect on health of an individual. Also, the flowers exhibited marked inhibition of cariogenic bacteria indicating their possible use in the treatment of dental caries. Further, isolation of active components possessing anticariogenic activity is under progress.

ACKNOWLEDGEMENTS

The authors express thanks to Head, Department of Microbiology, Principal, SRNMN College of Applied Sciences, Shivamogga and NES, Shivamogga for giving support to conduct work. Authors are thankful to Mr. Vijayananda BN for help rendered.

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Received on 16.05.2013 Modified on 12.06.2013

Asian J. Research Chem. 6(7): July 2013; Page 623-627