Influence of conventional and modern scheme of extraction and solvents on phytochemicals and biological activities of Sphaeranthus indicus

Present study was based upon the phytochemical screening, antimicrobial and antithrombotic potential of a traditional herb Spharenthus indicus (commonly called East Indian globe thistle). A range of solvents from non-polar to polar (n-hexane, ethyl acetate, methanol) were used to attained the extract by employing conventional and modern methods of extraction like orbital shaker assisted extraction, decoction and microwave assisted extraction in order to check their effect on concentration of phytoconstituent and biological activities. Percentage yield was varied from 2.01 to 30.05 % of dry weight. Phytochemical screening provides a detailed profile in plant matrix like tannins are present in methanol and ethyl acetate extracts while absent in n-hexane extracts. Flavonoids, phenols and saponins are present in methanol, ethyl acetate and n-hexane extracts in decreasing order. Cumarins are present in high amount in methanol extracts. The antibacterial test was applied on all extracts of plant and the larger zone of inhibition was found against S. typhi 18.9±0.4 (mm) for microwave extract of methanol. The thrombolytic activity of S. indicus showed greater activity of 80 % for methanol extract (microwave assisted). This work emphasizes the consideration of microwave assisted extraction on the basis of scientific evidence in order to utilize S. indicus for the basic and applied research of biology.


Introduction
The man curiosity for search of natural drug has long history, of which there is plenty of evidence from many sources: preserved monuments, written documents, and even original plant medicines.The struggles of many years against illness gave us in result the awareness of medicinal plants that is how man learned to hunt drugs in, seeds, barks, fruit, and additional parts of the plants.Usually plant based medicine are functional by self-medication or recommended by general practitioner or pharmacist.These medicines are considered safe for public because these are obtained by natural sources [1].The synthetic drugs and antibiotics have some limitations: First of all, these medicines are of high cost and due to which out of range from a common patient of developing countries.Secondly, the antibiotics become ineffectual by the time because microorganisms gain resistance in opposition to antibiotics [2,3].Moreover the antibiotics also involve unpleasant effects on the host, for example, immune suppression, allergic reactions and hypersensitivity.Natural products make his way via friendliness nature to discover new antibiotics [4,5].Sphaeranthus indicus Linn belongs to family Asteraceae.The plant is distributed all over the wet lands and also in plain areas of Pakistan, Sri Lanka, India and Australia [6].The plant is involved to treat epileptic convulsions, mental illnesses, leprosy, hemicranias and as nervine tonic [7].It is used to treat conditions of jaundice, piles, hepatitis, hepatopathy, diabetes, fever, pectoralgia, cough, gastropathy, hernia, hemorrhoids, helminthiasis, dyspepsia and skin diseases such as pruritus and edema.Plant root oil reported to be useful in curing scrofula.

Saponins
This test also called foam test.Extract was mixed in water and subjected to continous shaking form is produced and persist for 10 min, shows the presence of saponins.Tannins Extract was diluted by adding 1.00 mL of water in 0.5 mL of sample.Few drops of ferric chloride added to the test tube in test mixture.Blue colour was appeared to confirm the presence of Gallic tannins and catecholic greenish black color confirmed the presence of catecholic tannins.

Alkaloids:
Small amount of Meyer's reagent (Creamish white precipitate)/ Wagner reagent (reddish brown precipitate) was poured in 1.00 mL of extract that confirmed the alkaloids.Terpenoids (Salkowski test): Chloroform containing 5.00 mL extract was mixed with 3.00 mL of cocn.H2SO4.Terpenoids were confirmed by reddish brown color.Flavonoids Small amount of extract was mixed with NaOH solution.Intense yellow color which became colorless that pointed out flavonoids presence.Fatty Acids Solution of 0.5 mL extract in 5.00 mL ether was poured on filter paper and allowed to evaporate.Transparent spots indicated occurrence of fatty acids.Anthocyanins Extract solution was mixed with ammonia and few drops of 2 N HCl.Pink-red/ blue violet color indicated anthocyanins.

Anthraquinones
To check anthraquinones benzene (10.00mL) was mixed with (0.5 gm) of each plant extract and shaken well and then (5.00 mL) of 10 % ammonia was added after filtration.Red/pink to violet colors showed presence of anthraquinones.Coumarins Measured 3.00 mL of 10% NaOH was added in 3.00 mL of extract, yellow color indicates the presence of cumarins.Emodins NH4OH (2.00 mL) was added in 3.00 mL of benzene along with small amount of extract.Red color confirmed the emodins in samples.

Biological activities Antithrombotic activity
For evaluation of thrombolytic activity 5.00 mL of distilled water was shaken carefully with commercially available Streptokinase.This stock solution was used for in vitro antithrombotic activity.Extracts were soaked in distilled water for overnight.Supernatants were separated by filtration using 0.22 micron syringe filter paper.Blood samples were collected from healthy volunteer and stored in pre-weighted sterilized and labeled vials.All sample vials were incubated for 45 min at 37 o C for clotting.Vials were again weighted after removing serum carefully from clot.Weight of clot was calculated by following formula.Weight of clot = weight clot carrying tubeweight of empty tube About 100 µL of each extract was added to clots, Streptokinase (positive control) and distilled water (negative control).All mixtures were incubated at 37 ᵒ C for 90 min.Fluid was removed and samples were weighted again.Percentage lysis was determined with respect to control [17].Antibacterial activity Antimicrobial susceptibility test was done by disc diffusion method [18].For growing strains Muller Hinton agar media was used.Apparently agar was transparent and light yellowish-brown in color.For the preparation of media, 34.00 gm of Muller Hinton ground agar was weighed and dissolved in 1 L of distilled water and mixture was allowed to sterilize by using autoclave at 121°C for 20 min.The media was shifted instantly in sterile Petri plates, allowed to cool and was kept at the room temperature in order to solidify.Every bacterial culture was streaked on nutrient agar to obtain single colonies and incubated overnight at 37 °C.After incubation, one or two single colonies and inoculate in 0.85% saline solution and adjusted the turbidity to meet the 0.5 McFarland turbidity standards.The 10 µL bacterial suspensions were spread on agar medium with the help of sterilized micropipette.Petri plates were revolved to distribute bacteria evenly.The extracts of sample are diluted to estimate the antibacterial activity.The sterilized discs of filter paper (5.00 mm) were cut and loaded with diluted extracts and ciprofloxacin as standard antibacterial drug.The extract soaked filter paper discs were put on the agar surface.The plates were incubated for 24 h at 37 o C. Antibacterial activity was measured in terms of inhibition zones in mm.    1.  2).The relation of extraction scheme and solvents with the antithombotic activity was also studied by the Pearson correlation that indicate that amount of phyto constituent in term of percent yield of specific scheme is strongly correlated with the biological activity.The results obtained were r 2 = 0.999 r 2 = 0.997and r 2 = 0.754 in case of orbital shaker extraction, decoction and microwave assisted extraction respectively.These findings were in agreements that solvent type and extraction scheme strongly influence the amount of phytochemicals and in turn biological activities.Ethyl acetate and N-hexane extracts of orbital shaker following the same trend and gave batter zones then decoction treated extracts.As a whole all methanolic extracts exhibited larger zone while smaller zone in case of n-Hexane.A comparative view for activities of all sample extracts to Ciprofloxacin in terms of percentage inhibition with respect to standard against all strains of bacteria is given in Table 2 and Figure 3 and 4.  in different extraction procedures.Nine experimental runs in which the effect two independent variables (3 levels of each) on the dependent variables (percent yield and total constituents were performed.All experiments were performed in triplicate.Interestingly all the best results in term of % yield, phytochemicals concentration and their biological activities were displayed by aqueous methanolic extract of MAE method.The results specified that microwave assisted extraction can be practicable alternatives for conventional extraction method.Origin of this resistance is multifactorial which may be due to specific nature of the association between bacteria and antibiotics, the dose of antibacterial agent, environmental factors and host characteristics.These circumstances has forced researchers to explore new antimicrobial agents of variable origins as novel chemotherapeutical agents against bacteria but manufacturing cost for these synthetic drugs is very high and they may be cause of adverse effects as compared to herbal antimicrobial drugs.This entire scenario has made medicinal plants as a safe and cheap source of such type of medicines.

A B
heat that has strong impact on heating kinetics that exert pressure on cell wall and cell membrane structures thus enhancing the diffusion/partition rate of the solute onto plant matrix [20, 21].While in conventional methods the shift of heat is either through convection or conduction from the surface of material.Solvent ability of extraction depends upon phytochemical solubility, mass transferring kinetic and solute strength [22, 23].

Figure 3 .Figure 4 .
Figure 3. Zone of inhibition (antimicrobial effect) by different extracts of S. indicus (A) on E. coli (B) on S. typhi (C) on S. aureus; (D) on B. subtilis