INTRODUCTION

Secondary metabolites are the natural phytochemicals which owe medicinal importance to the plants they belong (Justin et al., 2014). Plant secondary metabolites are a diverse group of molecules that are involved in the adaptation of plants to their environment but are not part of the primary biochemical pathways of cell growth and reproduction (Marinova et al., 2005). They

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Received 16th Feb, 2016

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act in defense purposes to protect a plant from any pos- sible harm in the ecological environment (Stamp et al., 2003). Phenolic compounds are aromatic secondary plant metabolites broadly distributed throughout the plant kingdom (Mamta et al., 2012). They confer unique taste and flavor to the plant and plant derived products (Omas-Barberan and Espin, 2001). Phenolic compounds are one of largest group of secondary metabolites syn- thesized by plants. They offer great deal of health ben-

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efits to the mankind which include their antioxidant, anti-inflammatory, anti-carcinogenic and other biologi- cal properties like reduction in blood cholesterol and lipid levels, delaying the development of chronic dis- eases such as cancer and Alzheimer’s disease (Park et al., (2001), Ali et al., (2011) and Bodeker, (2000). Flavonoids are a class of polyphenols which are water soluble pig- ments found in the vacuoles of plant cells (Justin et al., 2014). The role of flavonoids in flowers is to act as col- ouring agents for plant pollinators (Harborne, 1976) and in leaves, these compounds are increasingly believed to promote physiological survival of the plant, protecting it from fungal pathogens and UV-radiation (Harborne, 1993). They are anti-allergic, anti-cancer, antioxidant, anti-inflammatory and anti-viral. (Guardia et al., 2001).

Calotropis gigantea L. (family Asclepiadaceae), com- monly known as giant weed or milkweed, is a usual wasteland plant found along degraded roadside and overgrazed pastures (Caius 1986 and Sharma, 1954). Calotropis is drought impermeable and salt tolerant weed growing upto an altitude of 900 m asl throughout the country (Mueen et al., 2005). It prefers distressed sandy soils with mean annual rainfall: 300-400 mm. It has clusters of waxy flowers that are either white or lav- ender in colour. C. gigantea is a perennial herb with wide pharmacological significance in traditional and Unani systems of medicine. The flowers and bark with milky latex are reportedly known for various biological activi- ties including analgesic, antimicrobial, antioxidant, anti-pyretic, insecticidal, cytotoxic and hepatoprotective activity (Sarkar and Chakravarty, 2014). A wide range of chemical compounds including cardiac glycosides, fla- vonoids, terpenoids, alkaloids and resins have been iso- lated from this plant (Singh et al. 2014). Recent studies have shown that, C. asiatica accumulates major phyto- constituents in the months of summer season (Alqahtani et al., 2015). Moreover, studies on Ribes nigrum and ‘Zonouz' peel have shown that harvesting time and leaf position have a profound effect on their phenolic con- tents, (Hallman et al., 2013 and Michael et al., 2015).

Calotropis procera L. (family Asclepiadaceae), com- monly known as “Sodom apple” is well known for its high medicinal properties. C. procera is drought-resistant, salt-tolerant, animophilous or antomophilous plant. The plant grows along degraded road sides, lagoon edges and in overgrazed native pastures. It has a preference for and is often dominant in areas of abandoned cultivation, especially sandy soils in areas of low rainfall (Sharma et al., 2011). The leaves are useful in the treatment of paralysis, arthralegia, swelling and intermittent fevers. Methanolic and aqueous extracts of leaves of C. procera have been reported to have the potential of antioxidant and antibacterial activity (Patel et al., 2012). The present investigation was aimed to study the effet of collection of

Arshad Ahmad Najar, Swati Khare and Kirti Jain

two different tissues from two Calotropis species on their TPC and TFC contents. Quantitative measurement shows higher phenolic and flavonoid content in leaf and flower tissues of C. procera as compared to that in C. gigantea.

MATERIALS AND METHODS

Gallic acid and Quercetin (MERK®), Methanol (MERK®), Folin-ciocalteu Reagent (MERK®) (1:10 in deionised water), Sodium carbonate solution (7.5% w/v) ( MERK®), Sodium nitrate (MERK®), Alcl3 and NaOH (MERK®). Gallic acid and Quercetin (3 mg) were accurately weighed into a 10 ml volumetric flask, dissolved in small amount of distilled water and the solution was made up to 3 ml with the same solvent to make the final stock of 1 mg/ml.

The aerial parts (leaves and flowers) of the plants were collected in the months of March-April, 2014 from MPCST 23˚ 15´ 35. 760˝ N and 77˚ 24´ 45. 414˝ E and P&T Colony 23˚ 13´ 25. 582˝ N and77˚ 23´ 28. 294˝ E Bhopal, India. The collected plant samples were identi- fied by Dr Zia ul Hasan (Prof and Head, Faculty of Bot- any, Saifia Science College, Bhopal, India). The specimen samples of C. procera and C. gigantea were deposited in the departmental herbarium with respective voucher number 481/Bot/Saifia/2014 and 482/Bot/Saifia/2014.

The plant materials were collected at different inter- vals of time: (Dawn 6:30 am), (Noon 12:30 am) and (Dusk 6:30 pm) by hand plucking. The samples were washed thoroughly with the distilled water to remove the dirt and other contaminations followed by careful drying under shade at room temperature in order to pre- vent fungal infection and decomposition of active com- pounds. The dried leaves and flowers were powdered using a grinder and stored in airtight packing polythenes until required for use.

Extraction was carried out by employing the mac- eration method. Equal quantities (100 g) of powdered tissues of leaf and flowers were taken and put in 500 ml plastic container. These samples were macerated with 500 ml of 75% Methanol. Each was allowed to stand for two weeks with constant shaking at regular intervals under room temperature. After maceration samples were fine filtered through muslin cloth and then by Wattman filter paper and the solvents were evaporated to obtain the methanolic extracts of the leaves and flowers. These served as the stock solutions which were stored in a dry and cool place until needed for analysis.

The amount of total phenolics in the extracts was determined with the Folin- Ciocalteu reagent (Ainsworth and Gillespie, 2007). Gallic acid was used as a standard and the total phenolics were expressed as mg/g Gallic acid Equivalents (GAE). For this purpose, the calibra- tion curve of Gallic acid. 1 ml of a standard solution of concentration 0.01, 0.02, 0.03, 0.04 and 0.05 mg/ml of

Gallic acid was prepared in methanol. A concentration of 0.1 or 1 mg/ml of plant extract was also prepared in methanol and 0.5ml of each sample were introduced into test tubes and mixed with 2.5 ml of a 10 fold dilute Folin-Ciocalteu reagent and 2ml of 7.5% sodium car- bonate. The test tubes were allowed to stand for 30 min- utes at room temperature and the absorbance was read at 760 nm spectrometrically.

TOTAL FLAVONOID CONTENT

A double beam UV/Visible spectrophotometric method was used to estimate the flavonoid content (Zhishen et al., 999). A standard solution of quercetin was added to 75 μl of NaNO2 solution and mixed for 6 min before adding 0.15 ml of Alcl3 (100 g/l). After 6 min, 0.5 ml of NaOH was added. The final volume was adjusted to 5 ml with distilled water and thoroughly mixed. Absorb- ance of the mixture was taken at 510 nm against water as blank. Total flavonoid content was expressed as mg quercetin/g dry weight of methanolic extract.

RESULTS AND DISCUSSION

Calotropis sp (Ait.) R. Br., a wild growing plant of family Asclepiadaceae, is well known for its medicinal prop- erties. Different parts of this plant have been reported

to exhibit anti-inflammatory, analgesic, and antioxi- dant properties (Dwivedi et al., 2010). Plant second- ary metabolites are a diverse group of molecules that are involved in the adaptation of plants to their envi- ronment but are not part of the primary biochemical pathways of cell growth and reproduction. Secondary metabolites from plants have important biological and pharmacological activities, such as anti-oxidative, anti- allergic, hypoglycemic and anti-carcinogenic (Borneo and katalinic, 2011). Phenols are a class of secondary metabolites derived from the Shikimic acid pathway. They are believed to function in plant defense mecha- nisms against insect herbivores and fungi (Nikam et al., 2012).

Flavonoids as secondary metabolites are believed to act in defense related sinalling pathways in the plans against an array of biotic and abiotic stress related condi- tions (Marinova et al., 2005) . Effect of time of collection (seasonal variation) was evident in the content of TFC and TPC constituents of the two species of Calotropis. The amount of the total phenol was estimated with the Folin-Ciocalteu reagent. Gallic acid was used as a stand- ard compound and the total phenols were expressed as mg/g gallic acid equivalent (Fig. 1).

The highest phenolic content (TPC) was found in the methanolic extract (20.10 mg/gm) of C. the Quercetin reagent. Quercetin was used as a standard compound

and the Total falvanoids were expressed as mg/g Quer- cetin equivalent (Fig. 2). The highest falvanoid content (TFC) was found in the methanolic extract (36.755 mg/ gm) of C. procera flowers harvested at evening (Fig. 5 and Fig. 6). Our observations were in agreement with earlier such studies on Tecomella sp. wherein TPC and TFC where found to be higher in summer season than in winter or monsoon seasons (Patel and Patel, 2014).

Similar studies have been reported on C. asiatica wherein it was shown to accumulate major phytocon- stituents in the months of summer season (Alqahtani et al., 2015). Moreover, studies on Ribes nigrum and ‘Zonouz' peel have shown that harvesting time and leaf position have a profound effect on their phenolic contents and (Michael et al., 2015 and Hallman et al., 2013). The place of cultivation and the harvesting period affects the phenolic compounds in fruit tissues of two C. asiatica cultivars (Puttarak and Panchayupakaranant, 2012).

Here we have observed that time of collection has profound prospect in ascertaining the drug quality and further selection of elite chemovariant among the two species of Calotropis. However, the present study needs further investigation as to how the chemical composition of different tissues is affected by period of harvesting.

CONCLUSION

The present study revealed that time of collection has special effects on total phenolic content (TPC) and total flavonoid content (TFC) of C. gigantea and C. procera. Quantitative measurement shows higher phenol and flavonoid content in leaf and flower tissues of Calotropis procera in afternoon and evening sessions compared to Calotropis gigantea. Such studies have a prospect in deciding on proper timing of collection visavis selection of elite chemotype and further exploration for drug design and development.

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