INTRODUCTION:

Medicinal plants have been identified and used throughout human history. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend against attack from predators such as insects, fungi and herbivorous mammals. At least 12,000 such compounds have been isolated so far; a number estimated to be less than 10% of the total. Chemical compounds in plants mediate their effects on the human body through processes identical to those already well understood for the chemical compounds in conventional drugs; thus herbal medicines do not differ greatly from conventional drugs in terms of how they work. This enables herbal medicines to be as effective as conventional medicines, but also gives them the same potential to cause harmful side effects (http://en.wikipedia.org/wiki/Medicinal plants).

Plant materials remain an important resource to combat serious diseases in the world. The traditional medicinal methods, especially the use of medicinal plants, still play a vital role to cover the basic health needs in the developing countries.

The medicinal value of these plants lies in some chemical active substances that produce a definite physiological action on the human body. The most important of these bioactive constituents of plants are alkaloids, tannin, flavonoid and phenolic compounds. Within the recent years, infections have increased to a great extent and antibiotics resistance effects become an ever-increasing therapeutic problem. Natural products of higher plants may possess a new source of antimicrobial agents with possibly novel mechanisms of action. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with synthetic antimicrobials. Therefore, it is of great interest to carry out a screening of these plants in order to validate their use in folk medicine and to reveal the active principle by isolation and characterization of their constituents. Systematic screening of them may result in the discovery of novel active compounds.

Clerodendrum infortunatum is a common shrub of 2-4 ft height of the family Verbeaceae. The species is native to tropical regions of Asia including India, Myanmar, Pakistan, Thailand and Sri Lanka. It is found along road side in North India and elsewhere, and flowers during February- May (Haines, 1925), identified by Linnaeus in 1753. In Ayurveda it is known by the Sanskrit names “Bharhi”, “Bhrigubhava”, “Padma”, “Fanji”, and “Brahman yastika” (Shasthri,1977). In Kerela, as “Peruvelum,” and in Hindi as “Bhant” or “Bharangi”. More than 500 species of the genus Clerodendrum are identified till now. Phyto-medical importance of various species of Clerodendrum genus has been reported in various indigenous systems of medicines.

This genus is being used specifically in Indian, Chinese, Thai, Korean, Japanese systems of medicine for the treatment of various diseases such as syphilis, typhoid, cancer, jaundice and hypertension. The beneficial medicinal effects of plant materials typically result from the combination of these secondary products (Tonthubthimthong et al., 2001). In 1985 Farnsworth et al., identified 119 secondary plant metabolites which were used as drugs. Phytochemicals are known to possess antioxidant (Wong et al., 2009), antibacterial (Nair et al., 2005), antifungal (Khan and Wassilew, 1987), antidiabetic (Singh and Gupta, 2007; Kumar et al., 2008), anti-inflammatory (Kumar et al., 2008), radio-protective activity (Jagetia et al., 2005) and due to these properties they are largely used for medicinal purpose. Therefore, qualitative and quantitative phytochemical screening of medicinal plants are necessary and the present study is designed to evaluate the bioactive chemical constituents of Clerodendrum infortunatum commonly used as medicine in India.

MATERIALS AND METHODS:

Collection of sample:

Clerodendrum infortunatum was collected from Golapbag area of the Burdwan District of West Bengal.

Preparation of extract:

The leaves and stem of the plant were collected and washed under running tap water to remove dust. The plant samples (2g each) were crushed in motor pestle and dissolved in four different solvents (water, methanol, ethanol, ethyl acetate). The solution then filtered and used for further phytochemical analysis.

Phytochemical analysis:

Qualitative and quantitative tests were performed for detection of phytochemical constituents of Clerodendrum infortunatum. Standard procedures as described by Kapoor et al., (1969), Harbone (1973, 1984), Smolenski et al., (1974), Boham and Kocipal- Abyazan (1974), Van-Burden and Robison (1981), Trease and Evans (1996), Sofowara (2008), Singleton et al., (1999), Edeoga, (2005) were used with some modifications to detect and estimate the phytochemicals present in the different extract fractions of the plant.

1. Qualitative tests:

Leaf extracts prepared in four different solvents (water, methanol, ethanol, ethyl acetate) were used for experimental purpose.

1.1. Test for flavonoids:

Leaf extract (500µl) was heated (80⁰-90⁰C) with 10 ml of ethyl acetate over a steam bath for 3 minutes. The mixture was filtered and 4 ml of filtrated was shaken with 1 ml of dilute ammonia solution and few drops of concentrated H₂SO₄was added. Yellow coloration of the solution shows positive result (Fig.1).

1.2. Test for alkaloids:

Leaf extract (500µl) was treated with 5 ml of aqueous 1% HCl solution on boiling water bath for 20 minutes. The solution was centrifuged for 10 minutes at 3000 rpm and 1 ml of supernatant was treated with few drops of WAGNER’S reagent (2 g of iodine and 6 g of KI dissolved in 100 ml of water).Reddish brown color of solution shows positive result.

1.3. Test for phenol:

Leaf extract (500µl) was boiled with 2 ml of distilled water on water bath and filtered. 10% of ferric chloride solution was added. Blue black color of solution shows positive results (Fig.2).

1.4. Test for anthraquinone:

Leaf extract (500 µl) was shaken with 10 ml of benzene and filtered. In filtrate 5 ml of 10% ammonia solution was added and mixed. Formation of violet and red color shows positive result (Fig.3).

1.5. Test for terpenoids:

Leaf extract (2 ml) was treated with 1 ml of 2, 4 dinitrophenyl hydrazine dissolved in 100 ml of 2M HCl. Yellow orange coloration of solution shows positive results (Fig.4).

1.6. Test for tannins:

Leaf extract (500µl) was boiled with 10 ml of distilled water and filtered. In the filtrate 1M Fecl₃ was added. Formation of blue black precipitate shows positive results (Fig.7).

1.7. Test for steroids:

Leaf extract (1ml) was treated with 2 ml of acetic anhydride and cooled on ice. The solution mixed with 0.5 ml of chloroform and 1 ml of concentrated H₂SO₄.Formation of reddish brown ring shows positive result (Fig.8).

1.8. Test for saponins:

Leaf extract (500µl) was boiled in 5 ml of distilled water and filtered. 2.5 ml of filtrate was mixed with 1.5 ml of distilled water and shake vigorously. Generation of stable and persistent froth shows positive result.

1.9. Test for carbohydrate:

Few drops of Molisch’s reagent was added to leaf extracts, shaken vigorously and then concentrated H₂SO₄was added. Formation of purple ring at the interface (Fig.5) .

1.10. Test for glycosides:

Leaf extract (500µl) was dissolved in pyridine solution. In this solution few drops of 20% sodium nitropruside and few drops of NaOH were added. Pink to red color shows positive result (Fig.6).

1.11. Test for proteins:

Leaf extract (500µl) was taken and 0.1 ml of Millions reagent was added. Brown coloration shows positive result (Fig.9)

2. Quantitative test:

Leaf extracts in four different solvents (water, methanol, ethanol, ethyl acetate) were used for experimental purpose.

2.1. Phenol test:

Leaf extract (1ml), 2ml of distilled water and 0.5 ml of Folin-ciocalteu’s reagent were taken in a test tube. After 3 minutes 2 ml of 20% sodium carbonate solution was added. The tubes were kept in boiling water bath for 1 minute and cooled and O.D was taken at 680 nm (Aruna P Jadhav et al., 2012) (Fig.10 and 11).

2.2. Flavonoids test:

Leaf extracts (1ml) was mixed with 4 ml of distilled water and 0.3 ml of 5% NaNo₂.After 5 minutes 0.3 ml of 10% Alcl₃ was added in the test solution and then 2ml of 1M NaOH also added. The solution was diluted and O.D measured at 520 nm (Fig.12 and 13).

3. Determination of antibacterial activity using agar cup method:

3.1. Antibacterial activity test by agar cup method:

The crude extracts were screened for antibacterial activity using agar well diffusion method described by Russel and Furr (1977).Agar cup method was followed in which culture of Bacillus subtilis and E coli were spreaded out on Muller Hinton agar plates. Wells were made on the plates with a cork borer (diameter 1.2 cm) to which different extracts were added in specific volume (1ml) along with controls (sterile water, methanol, ethanol, ethyl acetate). All the plates were incubated for 24 hours at 37⁰C.

3.2. Comparison of antibacterial activity of leaf and stem extracts of Clerodendrum infortunatum using agar cup assay:

The agar cup method was done to compare the antibacterial activity of ethanolic extracts of leaf and stem of Clerodendrum infortunatum.

4. Genomic DNA extraction of C. infortunatum:

The DNA was isolated from plant leaves by BARC (Bhaba Atomic Research Centre) method, developed by E. Nalini, N. Jawali and S.G. Bhagwat in 2003.

5. Partial purification of the leaf extract by column chromatography:

Column chromatography is the technique to separate the molecules on basis of their size, polarity, etc .The molecules were separated on the basis of polarity. For packing of the column, silica gel( 60-120 mesh ) was taken into the glass column and n-hexane was added slowly to saturate the gel. Next, leaf extract was passed through the column. The fractions were collected.

Fig 1: Test for flavonoids	Fig 2: Test for phenol	Fig3: Test for anthraquinone
Fig 4: Test for terpenoid	Fig 5: Test for carbohydrate	Fig 6: Test for glycosides
Fig 7: Test for tannins	Fig 8: Test for steroids	Fig9: Test for protein

Table1: PRESENCE OF PHYTOCHEMICAL CONSTITUENTS IN AQUEOUS EXTRACTION

Phytochemicals	*Clerodendrum* *infortunatum*	*Calotropis* *gigantea*	*Solanumnigrum*	*Ricinus* *communis*
Flavonoids	-ve	-ve	-ve	-ve
Alkaloids	+ve	+ve	+ve	+ve
Phenol	+ve	+ve	+ve	++ve
Anthraquinone	+ve	+ve	+ve	+ve
Terpenoids	+ve	+ve	+ve	++ve
Tannins	+ve	+ve	+ve	+ve
Steroids	+ve	+ve	-ve	+ve
Saponins	+ve	+ve	+ve	+ve
Carbohydrate	+ve	+ve	+ve	+ve
Glycosides	+ve	+ve	+ve	+ve
Protein	+ve	+ve	+ve	+ve

++ve: Strong positive, +ve: Positive, -ve: Negative

Table 2: PRESENCE OF PHYTOCHEMICAL CONSTITUENTS IN METHANOLIC EXTRACTION

Phytochemicals	*Clerodendrum* *infortunatum*	*Calotropis* *gigantea*	*Solanum* *nigrum*	*Ricinus* *communis*
Flavonoids	-ve	-ve	-ve	-ve
Alkaloids	+ve	+ve	+ve	+ve
Phenol	+ve	+ve	+ve	++ve
Anthraquinone	-ve	-ve	-ve	-ve
Terpenoids	+ve	+ve	+ve	++ve
Tannins	+ve	+ve	+ve	+ve
Steroids	+ve	+ve	+ve	+ve
Saponins	-ve	-ve	-ve	-ve
Carbohydrate	+ve	+ve	+ve	+ve
Glycosides	+ve	+ve	+ve	+ve
Proteins	+ve	+ve	+ve	+ve

++ve: Strong positive, +ve: Positive, -ve: Negative

Table 3: PRESENCE OF PHYTOCHEMICAL CONSTITUENTS IN ETHANOLIC EXTRACTION

Phytochemicals	*Clerodendrum* *infortunatum*	*Calotropis* *gigantea*	*Solanum* *nigrum*	*Ricinus* *communis*
Flavonoids	+ve	+ve	+ve	+ve
Alkaloids	+ve	+ve	+ve	+ve
Phenol	++ve	++ve	++ve	++ve
Anthraquinone	-ve	-ve	-ve	-ve
Terpenoids	++ve	++ve	++ve	++ve
Tannins	+ve	+ve	+ve	+ve
Steroids	+ve	-ve	+ve	+ve
Saponins	-ve	-ve	-ve	-ve
Carbohydrate	+ve	+ve	+ve	+ve
Glycosides	+ve	+ve	+ve	+ve
Proteins	+ve	+ve	+ve	+ve

++ve: Strong positive, +ve: Positive, -ve: Negative

Table 4: PRESENCE OF PHYTOCHEMICAL CONSTITUENTS IN ETHYL ACETATE EXTRACTION

Phytochemicals	*Clerodendrum* *infortunatum*	*Calotropis* *gigantea*	*Solanum* *nigrum*	*Ricinus* *Communis*
Flavonoids	-ve	-ve	-ve	-ve
Alkaloids	+ve	+ve	+ve	+ve
Phenol	+ve	+ve	+ve	++ve
Anthraquinone	-ve	-ve	-ve	-ve
Terpenoids	+ve	+ve	+ve	++ve
Tannins	-ve	-ve	-ve	-ve
Steroids	-ve	-ve	-ve	-ve
Saponins	+ve	+ve	+ve	+ve
Carbohydrate	+ve	+ve	+ve	+ve
Glycosides	+ve	+ve	+ve	+ve
Proteins	+ve	+ve	+ve	+ve

++ve: Strong positive. +ve: Positive. -ve: Negative

Fig13: Quantitative estimation of flavonoids compound of Four Clerodenrum different plants

Effect of leaf extract of Clerodendrum infortunatum on Bacillus subtilis and E. coli:

Fig 14: Zone of inhibition of leaf extract (ethanol)

Fig 15: Zone of inhibition of leaf extract (water)

Fig 16: Zone of inhibition of leaf extract (ethyl acetate)

Fig 17: Zone of inhibition of leaf extract (methanol).

Fig 18: Zone of inhibition of ethanolic leaf extract against E.coli.

Table7: COMPARISON OF QUANTITATIVE ESTIMATION OF FLAVONOIDS COMPOUNDS

EXTRACTION	O.D VALUE AT 520 nm
	*Clerodendrum* *infortunatum*	*Calotropis* *gigantea*	*Solanum* *nigrum*	*Ricinus* *Communis*
Aqueous	NIL	NIL	0.02	0.01
Methanolic	0.04	0.03	0.10	0.02
Ethanolic	0.17	0.03	0.2	0.07
Ethyl acetate	0.07	0.15	0.19	NIL

Table 8: CONCENTRATION OF FLAVONOIDS COMPOUNDS

Concentration of flavonoids(µg/ml)	O.D at 520nm
500	0.125
1000	0.236
1500	0.375
2000	0.449
Aqueous	NIL
Methanolic	0.04
Ethanolic	0.17
Ethyl acetate	0.07

Table 9: DETERMINATION OF ANTIBACTERIAL ACTIVITY OF LEAF EXTRACTS BY AGAR CUP METHOD

EXTRACTION	Bacterial culture	ZONE OF INHIBITION(cm)
	*Bacillus subtilis*	*Clerodendrum* *infortunatum*	*Calotropis* *Gigantean*	*Solanum* *Nigrum*	*Ricinus* *communis*
Aqueous		1.25	1.5	4	3.5
Methanolic		1.2	1.4	1.6	1.5
Ethanolic		2.2	2.0	1.3	2.5
Ethyl acetate		NIL	NIL	NIL	NIL
Ethanolic	*E. coli*	3.3	2.8	4.5	3

Fig19: Comparison of zone of inhibition of leaf and stem extract (ethanol) of Clerodendrum infortunatum.

Table 10: COMPARATIVE STUDY OF ETHANOLIC LEAF AND STEM EXTRACTS

Extraction of different plants	Bacterial culture	ZONE OF INHIBITION(cm)
Extraction of different plants	Bacterial culture	Ethanolic stem extract	Ethanolic leaf extract	Mixture of leaf and stem extract (1:1)
*Clerodendrum* *infortunatum*	*Bacillus subtilis*	1.5	1.7	2.2
*Calotropis* *Gigantean*		1.7	1.8	1.6
*Solanum* *Nigrum*		1.5	1.7	0.0
*Ricinus* *Communis*		2.1	2	2.8

Fig 20: 0.8% Agarose gel electrophoresis

RESULT AND DISCUSSION

The study has revealed that the presence of phytochemicals considered as active medicinal constituents. Important medicinal phytochemicals such as terpenoids, phenol, flavonoids, carbohydrate, protein, alkaloids and tannins were present in the studied plants. The result of the phytochemical analysis showed that the four plants(Clerodendrum infortunatum Calotropis gigantea, Solanum nigrum and Ricinus communis) are rich in at least one of alkaloids, flavonoids, terpenoids, phenol, steroids and tannins.The phytochemical screening and qualitative estimation of four medicinal plants (Clerodendrum infortunatum Calotropis gigantea, Solanum nigrum and Ricinus communis) showed that the leaves were rich in phenol, tannins, terpenoid, flavonoids, alkaloids etc. (Table no.1- 4).

Qualitative analysis of leaf extract of Clerodendrum infortunatum has shown that the extract contain flavonoids, phenol, anthraquinone, terpenoid, tannins, steroid, carbohydrate. But, all the crude extracts from leaf samples of Clerodendrum infortunatum Calotropis gigantea, Solanum nigrum and Ricinus communis. did not show any colour change for flavonoids test (Table no.1), only ethanolic leaf extracts of Clerodendrum infortunatum Calotropis gigantea, Solanum nigrum and Ricinus communis shown positive result for flavonoid test. However, methanol crude extracts from fresh leaves showed negative result for flavonoids, saponin and anthraquinone (Table no.2) but showed positive test for terpenoid, phenol, steroids, alkaloids(Table no.2).The ethanolic leaf extraction of Clerodendrum infortunatum showed positive results in flavonoids, phenol, steroids, terpenoids and carbohydrate test except anthraquinone test (Table no.3).

It was observed from the result that the ethanolic leaf extract contain higher amount of phenol compared with other extracts of Clerodendrum infortunatum Calotropis gigantea, Solanum nigrum and Ricinus communis. According to standard graph we can determine concentration of phenol in ethanolic leaf extract and it was near about 6000 µg/ml (Table no.6). In the quantitative test of flavonoids it was observed from the result that the ethanolic leaf extract contain higher amount of flavonoids compared with other solvent extracts of Clerodendrum infortunatum, Calotropis gigantea and Ricinus communis.According to the standard graph of flavonoids compounds we can determine the concentration of flavonoid in ethanolic leaf extract which was greater than 500 µg/ml (Table no.8).

From the above study it can be concluded that the studied plants showed strong antibacterial activity against some selective bacteria. The antibacterial activity of (water, ethanolic, methanolic, ethyl acetate) extracts of Clerodendrum infortunatum were given in table no. 9 (Fig no 14-18).From the results it was found that ethanolic extracts exhibited significant antibacterial activity compare to the other extraction of Clerodendrum infortunatum and Calotropis gigantea, Solanum nigram, Ricinus communis against Bacillus subtilis (Table no.9 and Fig no.14).Ethanolic extract of Clerodendrum infortunatum showed more inhibitory zone as compared to ethanolic extracts of Calotropis gigantea, Solanum nigrum (Table no.9). Ethanolic extract of Clerodendrum infortunatum also exhibited significant antibacterial activity against E.coli (Table no.9, Fig no.18).When the leaf and stem extracts were mixed together in equal concentration (1:1), it shows more inhibitory zone as compared to other individual extracts (Table no.11,Fig no.19). From these results we can conclude that some of the component from the mixtures of leaf and stem extracts exhibit the synergistic action against bacteria.

The genomic DNA was isolated and confirmed by agarose gel electrophoresis (Fig no.20). The extracted DNA sample were kept for further amplification and sequencing to check the homogeneity and phylogenetic relationship among the different species of the same plant from the Apocynaceae family. Different bioinformatical tools such as BLAST, FASTA, Clustal W will be used to find out phylogenetic relationship.

Partial purification of ethanolic leaf extract was done through column Chromatography and collected fraction was kept for further purification and mass determination through HPLC/ Gas Chromatography, Mass spectroscopy etc.

ACKNOWLEDGEMENTS:

The authors wish to acknowledge the Department of Biotechnology, Department of Environmental Science, The University Science Instrumentation Centre, The University of Burdwan for support and providing infrastructural facility. The authors also acknowledge DBT, Govt. of India for funding this work.

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Received on 25.11.2014 Accepted on 29.11.2014

Asian J. Res. Pharm. Sci. 4(4): Oct.-Dec. 2014; Page 187-195

Concentration of phenolic compound (µg/ml)	O.D at 680nm
1000	0.299
2000	0.439
3000	0.579
4000	0.719
5000	0.859
6000	0.999
7000	1.139
Aqueous	0.15
Methanolic	0.36
Ethanolic	1.01
Ethyl acetate	0.06

EXTRACTION	O.D VALUE AT 680 nm
	*Clerodendrum* *Infortunatum*	*Calotropis* *gigantea*	*Solanum* *nigrum*	*Ricinus* *Communis*
Aqueous	0.15	0.17	0.63	0.71
Methanolic	0.36	0.18	0.63	0.65
Ethanolic	1.01	0.12	0.23	0.77
Ethyl acetate	0.06	0.13	0.40	0.27