estimation of flavonoid lantana camara linn verbenaceae

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_________________________________* Corresponding author: Vedavathi.TE-mail address: [email protected]

Available online at www.ijrpp.comPrint ISSN: 2278 – 2648Online ISSN: 2278 - 2656 IJRPP | Volume 2 | Issue 1 | 2013 Research article

Estimation of flavonoid, phenolic content and free radical scavenging activity of fresh unripe fruits of lantana camara linn (verbenaceae)

*Vedavathi T, Bhargavi K, Swetha G, Mythri K.CMR College of Pharmacy, Kandlakoya, Medchal Road, Hyderabad-501 401.

ABSTRACTThe Lantana camara Linn (verbenaceae) is available throughout world as a weed. The genus Lantana includes 2500 species worldwide and is known for its bioactive secondary metabolites and essential oils. It is used for the treatment of various disorders. The therapeutic effects of tannins and flavonoids can be largely attributed to their free radical scavenging property and it is responsible for many pharmacological activities. The present study is designed for the extraction of fresh unripe fruits using alcohol (cold, hot and micro wave techniques). The extracts were investigated for phytochemicals, total phenolic components, flavonoidal content and in-vitro free radical scavenging property (DPPH–RSA method). The preliminary phytochemical investigations revealed that presence of glycosides, alkaloids, carbohydrates, flavonoids, inuline, tannins (phenolic compounds). The total Phenolic content of alcoholic extracts (cold, hot and micro wave) showed the content values of 4.67±0.22% w/w, 6.92±0.41% w/w and 7.291±0.13%w/w and total flavonoids estimation of alcoholic extracts (cold, hot and micro wave methods) showed the content values of 5.12±0.08% w/w, 6.623±0.32%w/w and 7.458±0.24%w/w for Quercetin respectively. Further investigation were carried out for In-vitro free radical scavenging assay by calculating its % inhibition by means of IC50 values, all the extracts concentration has been adjusted to come under the linearity range and here many reference standards like Tannic acid, Quercetin, Ascorbic acid have been taken for the method suitability. The results revealed that fresh unripe fruits of this plant have free radical scavenging potential. Among these results alcoholic extract by micro wave technique has more yield and more potent than other extraction methods. In conclusion that the Lantana camara Linn. (Verbenaceae) fresh unripe fruits possesses the antioxidant substance which may be potential responsible for the treatment of tumors and rheumatism and other oxidative stress related diseases.

Keywords: Lantana camara Linn. (Verbenaceae), Total phenolic content (TPC), Total flavonoid content (TFC), radical scavenging assay (DPPH – RSA)

INTRODUCTIONThe plant Lantana camara Linn, family Verbenaceae is also known as wild sage, red sage, Spanish flag and is also available throughout central

and south India in most dry stony hills and black soil[1]. Fruits are small, 5 mm diameter, greenish-

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blue, blackish, drupaceous, shinning with two nut lets almost throughout the year and dispersed by birds. Seeds germinate very easily. The chemical constituents of lantana camara are caryophyllene, 1-α-phellandrene, lantadene-A, lantadene-B[1], flavoniods[2], saponins, tannins, γ- gurjunene[3] , lancamarone quinine, lantanine etc.The plant is vulnerary, diaphoretic, carminative, antispasmodic and tonic. In many parts of the world the plant has been used to treat a wide variety of disorders like anti-inflammatory[4], anti-malarial[5], fungicidal[6], eczema[7], rheumatism[8], asthma[9], inhibitor of acetyl cholinesterase[10], abortificient[11], skin diseases[12], high blood pressure and biological control[13]. In the folk medicine, it is used especially for tumors and cancer [1]. A tea prepared from the leaves and flowers is taken against fever, influenza and stomach ache. In Central and South America the leaves were made into a poultice to treat sores, chicken pox and measles. In Ghana infusions of the whole plant is used against bronchitis. The powdered root in milk was given to children for stomach ache. In Asian countries leaves are used for cuts, rheumatism, ulcers and as a vermifuge. Decoctions are applied externally against leprosy and scabies [14]. In India the leaves of the plant are boiled for tea and the decoction is a remedy against cough. The decoction of the whole plant is given as treatment against tetanus, rheumatism, malaria and ataxia of abdominal viscera. It is used as a lotion for wounds. Leaf powder is applied to cuts, ulcers and swellings[15].There are some of the reports related to antioxidant activity, total phenolic, total flavonoidal content estimations on total plant, leaves, ripe fruits etc. But, there is no proper scientific studies have been reported for the fresh unripe fruits of this plant. Due to this considerable savings in the time, energy and active constituents (degradation is also less), the novel microwave method of extraction is used to compare with the traditional methods [16]. So the aim of the study is to compare the yield value of the extract and to investigate the total phenolic content, total flavonoidal content and in-vitro free radical scavenging activity of alcoholic extracts (cold, hot and micro wave techniques) of fresh unripe fruits of this plant. An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules.

Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates and inhibit other oxidation reactions by being oxidized themselves. As a result, antioxidants are often reducing agents such as ascorbic acid or polyphenols [17]. However, it is unknown whether oxidative stress is the cause or the consequence of disease. Antioxidants are also widely used as ingredients in dietary supplements in the hope of maintaining health and preventing diseases such as cancer and tumors. Although initial studies suggested that antioxidant supplements might promote health and suggested instead that excess supplementation may be harmful. In addition to these uses of natural antioxidants in medicine, these compounds have many industrial uses, such as preservatives in food and cosmetics[18].So that we got an interest to study by means of preliminary in-vitroantioxidant work which we have carried out in fresh unripe fruits of Lantana camara.

MATERIAL AND METHODSChemicals and reagents Chemicals used in this study were 1, 1-diphenyl-2-picryl hydrazyl (DPPH) obtained from John Baker Inc Colorado, U.S.A, Quercitin, tannic acid, ascorbic acid, aluminium chloride, sodium hydroxide, folin-denis reagent (Phosphomolybdic acid, sodium tungustate, phosphoric acid), sodium carbonate and sodium nitrite are obtained from SD Fine Chemicals Ltd, India. All other reagents and solvents used in the study are of analytical grade.

Plant materialFresh unripe fruits of Lantana camara Linn family Verbenaceae were collected from surrounding CMR College of Pharmacy, Medchal, Rangareddy district, India during the month of June. The fresh unripe fruits were crushed for extraction purpose.

Preparation of crude extracts Alcoholic extract100 g of fresh unripe fruits of Lantana camara Linn (Verbenaceae) were weighed and crushed in an electronic mixer and were soaked in 200 ml of alcohol for about 2 days in 500ml borosilicate glass

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jars for each method (cold, hot and microwave) of extraction processes.

Cold extractionThe imbibed material after two days was filtered through cotton bag and squeezed.

Hot extractionThe imbibed material was packed in Soxhlet apparatus with the help of cotton bag and extracted at 50 - 55ºc and continued until the complete exhausting of crude drug (no color in the siphon tube). The cotton bag was removed from extractor, pressed the marc and added the contents to RBF to distill the solvent [19].

Micro wave extractionThe imbibed material was taken as suspension (50ml) in solvent in a pyrex cylindrical tubes. Tubes are irradiated in a Microwave oven (450W) and the extracts were not allowed to boil, therefore, after 30 s they were cooled to room temperature for few min; the irradiation step was repeated for 20 times in the same way, it gives corresponding extract [16]. After cooling, it is filtered through a cotton bag.

To prepare dry powder form of extractThe dry powder of this extract was prepared by concentrating the resultant filtrates by distillation of

the solvent and the resultant concentrate was dried by using the simple saloon water sprayer, by spraying the extract on the glass plates, after the predetermined flow conditioned consistency thick solution was poured into the sprayer (here the above concentrated extract solution varies to nature of plant material), by which it was air dried. The clumpy dry powder obtained was scraped by the knife from the plate and packed in air tight plastic container and stored in vacuum desiccators as such. The preconditioned set method can be optimized by evaluating the quantitative test of any existed constituents like phenols, flavonoids by suitable validated methods. This present study was undertaken by the spectrophotometric method.

Yields of various extractsCold extraction process – 3.46 GmsContinuous hot extraction by Soxhlet apparatus -4.92 GmsMicrowave process – 6.12 Gms

Qualitative Preliminary PhytochemicalscreeningThe extracts thus obtained were subjected to preliminary phytochemical screening [20]. The results obtained in the present investigation of fresh unripe fruits of Lantana camara Linn showed in the table 1.

TABLE-1: Phytochemical analysis of various extracts of fresh unripe fruits of Lantana camara Linn.

Test of extract Hot extract Cold extract Microwave Extract

AlkaloidsAminoacidsCarbohydratesFlavanoidsGlycosidesInulinTanninsProteinsStarchNapthoquinones

+-++-++---

+-+++++---

+-++-++---

Note: ‘+’ Positive, ‘-’ Negative


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Estimation of TPC by UV – Visible spectrophotometerBy Folin – Denis MethodThis method is based on the oxidation of molecule containing a –OH groups. The tannin and tannin like compound reduce Phosphotungustomolybdic acid in alkaline solution to produce a highly blue colored solution [21, 22]. 100 mg of the each powder of extract was dissolved and diluted with distilled water and that has adjusted to come under the linearity range i.e. (50 μg/ml) of the drug was withdrawn in 10ml volumetric flask separately. To each flask 0.5 ml of Folin-Denis reagent and 1ml of Sodium carbonate

was added and volume is made up to 10 ml with distilled water. The absorbance was measured at absorption maxima 700 nm within 30 minute of reaction against the blank. The total phenolic content was determined by using calibration curve (5 to 25 μg/ml). Three readings were taken for each and every solution for checking the reproducibility and to get accurate result. Results are provided in (Table-2 and Figure-1). The intensity of the solution is proportional to the amount of tannins and can be estimated against standard tannic acid, the total phenolic content, expressed as mg tannic acid equivalents per 100g dry weight of sample

.

FIGURE 1: Results of Total Phenolic content

R2 values represented mean data set of n=3

Table 2: Results of Total phenolic content

No Concentration of extract % w/w of total tannin1 Alcoholic cold 4.67± 0.222 Alcoholic hot 6.92± 0.413 Alcoholic microwave 7.291±0.13

Values are mean ± S.E.M, n=3

Total Flavonoid Content by Spectrophotometer Aluminum chloride visible Spectrophotometric assay methodTotal flavonoid contents were measured with the

aluminum chloride visible Spectrophotometric assay [23]. Alcoholic extracts that has been adjusted to come under the linearity range i.e. (400 μg/ml) and

different dilution of standard solution of Quercetin(10-50 μg/ml) were added to 10ml volumetric flask containing 4ml of water. To the above mixture, 0.3ml of 5% NaNO2 was added. After 5 minutes, 0.3ml of 10% AlCl3 was added. After 6 min, 2ml of 1 M NaOH was added and the total volume was made up to 10ml with distill water. Then the solution was mixed well and the absorbance was measured against

y = 0.006x + 0.001R² = 0.998

00.020.040.060.08

0.10.120.140.160.18

0 5 10 15 20 25 30

Standard Graph for Tannic acid (TPC)

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a freshly prepared reagent blank at 510 nm. Results are provided in (Table 3 and Figure 2) Total flavonoid content of the extracts was expressed as

percentage of Quercetin equivalent per 100 g dry weight of sample.

FIGURE 2: Results of Flavonoid content


Table 3: Results of Flavonoids content

No Concentration of extract %w/w of total flavonoid

1 Alcoholic cold 5.12 ± 0.08

2 Alcoholic hot 6.623± 0.32

3 Alcoholic microwave 7.458±0.24

Values are mean ± S.E.M, n=3

DPPH –RSA methodThe free radical scavenging activity of each of alcoholic extracts and the standard L-Ascorbic Acid (Vitamin C) was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH[24,25]. Here, 0.1mM solution of DPPH in alcohol was prepared and it must be protected from light influence by maintaining the dark condition and also fold by aluminum foil and 3 ml of this solution was added to 1ml various conc. (10-50 µg/ml) of

extracts or standard solution of (10-50 µg/ml). Absorbance was taken after 30 min at 517nm. Results are provided in (Table 4 and Figure 3-6).

% inhibition activity = A0 – A1 X 100 A0

Where, A0 is the absorbance of the control,A1 is the absorbance of extract/standard taken as

Ascorbic acid.

y = 0.001x + 0.001R² = 0.998

00.010.020.030.040.050.060.070.080.09

0.1

0 10 20 30 40 50 60

Standard Graph for Quercetin


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FIGURE 3: Results of DPPH radical scavenging assay






y = 1.649x + 1.318R² = 0.998

0

20

40

60

80

100

0 10 20 30 40 50 60

STD Ascorbic Acid

y = 1.643x + 0.669R² = 0.996

0

20

40

60

80

100

0 10 20 30 40 50 60

Alcoholic Cold Extract

y = 1.492x + 0.511R² = 0.998

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60

Alcoholic Hot Extract

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RESULTSEffect of TPC & Flavonoid content The quantitative determination of the total phenolic content, expressed as mg tannic acid equivalents and per 100g weight of sample. TPC of alcoholic extracts (cold, hot and microwave) showed the content values of 4.67µg/ml, 6.92µg/ml and 7.29µg/ml and total flavonoid content of the extracts was expressed as percentage of Quercetin equivalent per 100 g dryweight of sample. TFC estimation of alcoholic extracts (cold, hot and microwave) showed the content values of 5 µg/ml, 6.625µg/ml and 7.5µg/ml The above results showed that alcoholic microwave extraction contain more tannins and flavonoid content than the alcoholic hot and cold extract.

Capacity of DPPH –RSA The antioxidant reacts with stable free radical, DPPH and converts it to 1, 1- Diphenyl-2- Picryl Hydrazine. The ability to scavenge the free radical, DPPH was measured at an absorbance of 517 nm. So the DPPH – RSA and its %inhibition of alcoholic extracts (cold, hot and microwave) showed that IC50 values 27.81μg/ml (R2=0.996), 33.76 μg/ml (R2=0.997) and 36.87μg/ml (R2=0.998) respectively. Ascorbic acid has taken as reference which showed 37.96μg/ml. (R2=0.998) among these results microwave extract has more potent than hot and cold extracts. The overall results of % inhibition as shown in the (Table 4) respective to IC50 values and regression R2 is the mean value of (n=3).

Table 4: % Inhibition by DPPH-RSA

Samples Equation * R2 values IC 50 valuesStandard Ascorbic Acid y = 1.649x + 1.318 0.998 37.96μg/ml

Alcoholic Cold Extract y = 1.643x + 0.669 0.996 27.81μg/mlAlcoholic Hot Extract y = 1.492x + 0.511 0.998 33.76μg/mlAlcoholic Microwave Extract y = 1.627x + 1.300 0.998 36.87.μg/ml

*Data set of n=3 and mean R2 values obtained from the graphs

DISCUSSIONThe fresh unripe fruits Lantana camara Linn family Verbenaceae possesses the antioxidant property

substance which may be potential responsible for the treatment of cancer and tumors there is much scopefor fresh unripe fruit portion and more number of studies can be undertaken like oxidative stress,

y = 1.627x + 1.300R² = 0.998

0102030405060708090

0 10 20 30 40 50 60

Alcoholic Microwave Extract


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hepatoprotective, nephroprotective, anticancer, skin disorders, rheumatism, asthma etc. In future we look

forward to check the potency of the fruits by means of in-vivo anti-oxidant studies.

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estimation of flavonoid lantana camara linn verbenaceae

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