STUDY OF ANTIMICROBIAL ACTIVITY AND PHYTOCHEMICAL EVALUATION OF JATROPHA GOSSYPIFOLIA, SAPIUM SEBIFERUM, KIRGANELIA RETICULATA, PHYLLANTHUS FRATERNUS AND PEDILANTHUS TITHYMALOIDES

Study of antimicrobial activity and phytochemical evaluation of Jatropha gossypifolia, Sapium sebiferum, Kirganelia reticulata, Phyllanthus fraternus and Pedilanthus tithymaloides

U. C. Bapat* and D. R. Mhapsekar

Department of Botany, St. Xavier’s College, Mahapalika Marg, Mumbai 400 001, Maharashtra State, India

ABSTRACT: Present investigation deals with the evaluation of the antimicrobial activity of extracts of Jatropha gossypifolia Linn., Sapium sebiferum (L.) Roxb., Kirganelia reticulata (Poir) Baill., Phyllanthus fraternus Webster and Pedilanthus tithymaloides (Linn.) Poit. against Escherichia coli, Proteus vulgaris, Staphylococcus aureus and Bacillus cereus and Trichophyton mentagrophytes and T. rubrum. The antibacterial activity was tested by the disc diffusion method and the antifungal (antidermatophytic) activity by the method of Abubacker et al. It was observed that only DMSO extracts of the leaves of K. reticulata and the flowers of P. tithymaloides inhibited the growth of P. vulgaris and S. aureus with MIC at 200 mg ml^-1. The extracts did not inhibit the growth of T. mentagrophytes and T. rubrum. The phytochemical analysis of these extracts indicated that the tannin and saponin contents were highest in the leaves of K. reticulata (6.0 and 12.04 % respectively) whereas, the flavonoid and steroid and triterpenoid contents were highest in theflowers of P. tithymaloides (41.20 % and 46.74 %). The MIC values of extracted tannins from the leaves and flowers of P. fraternus and P. tithymaloides respectively were found to be 40 mg ml^-1 (against S. aureus) whereas, that of extracted tannins and flavonoids from leaves of K. reticulata were 50 mg ml^-1 (against P. vulgaris and S. aureus). TLC of extracted tannins showed presence of 4 common components, the flavonoids showed 3 and the saponins showed 2 common components. TLC of steroids

Jatropha gossypifolia, Sapium sebiferum, Kirganelia reticulata, Phyllanthus fraternus, Pedilanthus tithymaloides, Trichophyton

Introduction: The use of medicinal plants plays a vital role in covering the basic health needs in developing countries and these plants may offer a new source of antibacterial, antifungal and antiviral agents with significant activity against infective microorganisms ¹.

Jatropha gossypifolia Linn. (= Jatropha gossypiifolia L. ²) is used in the traditional system of medicine for the treatment of various ailments, viz. arthritis, asthma, washing wounds, blood purifier, bronchitis, carbuncles, diarrhoea, dysentery, as an antidote for snake bite, in piles, eczema, fever, gum infections, inflammation, itching, leprosy, stomach ache and ulcer ³. Bark of root and leaf of Sapium sebiferum (L.) Roxb. [=Triadica sebifera (L.)Small ²] are used against eczema, wounds, edema and snake bites ⁴. The leaves of Kirganelia reticulata (Poir) Baill. (= Phyllanthus reticulatus Poir., family Phyllanthaceae ²) are diuretic and their juice is used in diarrhoea in infants. Powdered leaf is applied to sores, burns, suppurations and chafing of skin ⁵. Phyllanthus fraternus Webster (= Phyllanthus fraternus G.L. Webster, family Phyllanthaceae ²) is useful for treatment of hepatitis, tuberculosis, viral infections, liver diseases, anemia, dysentry and for bacterial infections such as cystitis, prostatitis, veneral diseases and urinary tract infections ⁶. The decoction of leaves of Pedilanthus tithymaloides (Linn.) Poit. (= Euphorbia tithymaloides L. ²) is used in abdominal disorder. The latex is used in the treatment of venereal diseases and to relieve sprain⁷.

The chemistry of the Euphorbiaceae is among the most diverse and interesting of flowering plant families and is comparable to the biological diversity of the family Euphorbiaceae ⁸. Various chemical constituents present in K. reticulata, P. fraternus and P. tithymaloides were earlier reported ^{6, 9, 10}.Hence, present study was undertaken to correlate the chemical constituents and their antibacterial activity of extracts of the different parts of K. reticulata, P. fraternus, P. tithymaloides, J. gossypifolia and S. sebiferum against Escherichia coli (NCIM No. 2931), Proteus vulgaris (NCIM No. 2813), Staphylococcus aureus (NCIM No. 5021) and Bacillus cereus (NCIM No. 2106)and antifungal activity against the dermatophytes, Trichophyton mentagrophytes and T. rubrum (causing superficial dermatophytoses).

Materials and Methods:

Collection of plant material:

The plant materials were collected from Sanjay Gandhi National Park, Borivili, Vasai, Malad and Matunga, Mumbai and authenticated by studying the morphological characters and by comparing with the Blatter Herbarium specimens. The materials were collected every year from 2009 to 2013 to check the reproducibility of the results. The plants were washed under running water, dried in the shade, powdered and sieved (mesh size 1mm).

Preparation of extracts:

20% extracts of dry and fresh parts of the plants were prepared in different solvents (distilled water, ethanol, methanol, petroleum ether and DMSO) by crushing the material and filtering it through Whatman No. 1 filter paper first and then through Millipore filter (0.45µm). The extracts were prepared in triplicates. For S. sebiferum, the extracts of dry material were prepared as the fresh material was not available at the time of study.

Antimicrobial activity Study:

Antibacterial activity assay and determination of MIC of the extracts:

The antibacterial activity was tested by the disc diffusion method ^{11, 12} with amoxycillin-10 mcg/disc as the antibiotic obtained from Hi-Media and nutrient agar plates were inoculated separately with E. coli, P. vulgaris, S. aureus and B. cereus. The MIC of extracts exhibiting inhibition of growth of organisms i.e. diameter of ZOI > 10 was determined by testing 5%, 10%, 15% and 20% extracts using pour plate method¹³ (with 6.0 mm wells each with 20µl of extract). The 2 µg of amoxycillin was used for S. aureus and 10 µg for E. coli, P. vulgaris and B. cereus. The antibacterial activity of 100% ethanolic extracts of tannins and 20% ethanolic extracts of flavonoids, saponins, steroids and triterpenoids extracted from the leaves of P. fratenus and K. reticulata and flowers of P. tithymaloides was determined by testing 5% extracts using pour plate method. The MIC of extracts of tannins and flavonoids was determined by testing 1%, 2%, 3%, 4% and 5% extracts using pour plate method. All the experiments were replicated thrice for the confirmation of results.

Antifungal (antidermatophytic) activity assay:

The antifungal activity was carried out by the method used by Abubacker et al (2008)and Websteret al (2008) ^{14, 15}. One ml of 20 % extracts of fresh and dry plant materials prepared in distilled water (DW) and ethanol (20 % EtOH) were added separately to four ml of SDA medium supplemented with chloramphenicol and cycloheximide ¹⁶. 0.2 ml (about 0.25 –0.4 x 10⁶spores/ml) of fungal spore suspension of T. mentagrophytes and T. rubrum were transferred separately on the plates and spread evenly. 10 mg/ml of Clotrimazole solution (Canesten obtained from Bayer Pharmaceuticals Pvt. Ltd.) was used as standard antifungal agent. The plates were incubated at 30+2 ⁰C for 21 days and the plates were checked every day for fungal growth (if any). The experiments were repeated thrice for confirmation of results.

Phytochemical screening:

Plant extracts exhibiting activity were screened for the presence of different classes of compounds including tannins, flavonoids, alkaloids, saponin glycosides, steroids and triterpenoids, carbohydrates and proteins^{17, 18}.

Extraction of tannins, flavonoids, saponins, steroids and triterpenoids:

The tannins were extracted using the method prescribed by Brindha et al ¹⁹, flavonoids by the method of Edeoga et al ²⁰, saponins by the method used by Malu et al ²¹ and steroids and triterpenoids by the method of Bai et al ²².These compounds were extracted and estimated in the leaves of P. fratenus and K. reticulata and flowers of P. tithymaloides showing antibacterial activity.

 Separation of compounds extracted by TLC:

The extracted tannins, saponins (ethanolic) and flavonoids, steroids and triterpenoids (20% aq. ethanolic) were applied on TLC plates of silica gel 60 F 254 of E. MerckKGaA with CAMAG Linomat IV HPTLC applicator (syringe). The pre-saturation time was 45 min for tannins, 20 min for saponins and 10 min for flavonoids and steroids and triterpenoids. The run distance was 80 mm. The plates were scanned at 200 nm, 254 nm and 366 nm before derivatization and at 366 nm and 540 nm after derivatization. The R_f values were measured by the CAMAG TLC scanner. The mobile phases and spraying reagents as well as the track positions and the volume of extracts used for TLC is tabulated (Tables 1 and 2 respectively) as follows:

Table 1: Mobile phases and spraying reagents for TLC

Table 2: Track positions and volume of extracts applied

Results and discussions:

Antimicrobial activity:

Antibacterial activity assay and determination of MIC of the extracts:

The antibacterial activity of the extracts of J. gossypifolia, P. fraternus and S. sebiferum did not inhibit the growth of bacteria  except the DMSO extract of the fresh leaves of P. fraternus that showed the inhibition of S. aureus (the diameter of ZOI = 13mm), the MIC being 150 mg ml^{-1 28}. Following are the results of the antibacterial activity of the fresh and dried parts of K. reticulata and P. tithymaloides and their MIC determination.

Table 3: Antibacterial activity of extracts of K. reticulata and P. tithymaloides expressed as DIAMETER OF Zone of Inhibition (ZOI IN MM)

 TABLE 4:  DETERMINATION OF MIC OF THE EXTRACTS OF LEAVES OF K. RETICULATA AND FLOWERS OF P. TITHYMALOIDES SHOWING INHIBITORY ACTIVITY AGAINST P. VULGARIS AND S. AUREUS (ZOI IN MM)]

The results indicate that the DMSO extracts of fresh leaves of K. reticulata inhibited the growth of P. vulgaris, whereas that of its dry leaves inhibited the growth of both P. vulgaris and S. aureus (Table 3)and their MIC values obtained (Table 4) were 200 mg ml^-1 (Fig. 1 to 3). The DMSO extracts of fresh flowers of P. tithymaloides inhibited the growth of P. vulgaris alone, whereas that of dry flowers inhibited the growth of both P. vulgaris and S. aureus with the diameter of ZOI (16.00 mm) being the highest against S. aureus and their MIC values were found to be 200 mg ml^-1(Fig. 4 to 6).

Eldeen et al(2011) and Shruthi et al (2010) obtained inhibitory activity of methanolic extract of dry leaves of K. reticulata against E. coli and S. aureus ^{29, 30}. The inhibition of growth of S. aureus by the ethanolic extract ³¹ and petroleum ether extract of the dried leaves ³² had also been reported. Mohd et al (2006). Adhikary et al (2013) reported inhibitory activity of the methanolic extracts of P. tithymaloides against E. coli and S. aureus ^{1, 33}. However, no inhibitory activity of the extracts of P. tithymaloides was observed against these organisms in the present study. These results are supported by the findings of Chaudhari et al (2012) ³⁴.

Antibacterial activity of the tannins, flavonoids, saponins and steroids and triterpenoids extracted from the leaves of P. fraternus and K. reticulata and flowers of P. tithymaloides and the determination of MIC of the same is tabulated in Tables 5 and 6.

   Table 5: Antibacterial activity of tannins, flavonoids, saponins and steroids and trierpenoids extracted from plants (diameter of ZOI in mm)

Table 6: Determination of MIC of extracted tannins and flavonoids (diameter of ZOI in mm)

It was observed that among the extracted compounds tested, the ethanolic extracts of tannins from the leaves of P. fraternus and flowers of P. tithymaloides inhibited the growth of S. aureus with the highest diameter of ZOI of 12.33mm for the latter and their MIC values were found to be 40 mg ml^-1(Fig 7 and 8). The ethanolic extracts of tannins and flavonoids from the leaves of K. reticulata showed inhibition of growth of P. vulgaris and S. aureus  (Table 5) and their MIC values were observed to be 50 mg ml^-1 (Table 6) (Fig 9 to 12).

Antifungal (antidermatophyte) activity assay:

It was observed that none of the extracts tested inhibited the growth of T. mentagrophytes and T. rubrum. Adejumo et al (2009) had reported the inhibitory activity of fresh leaves of J. gossypifolia against T. mentagrophytes and T. rubrum³⁵.

 Phytochemical screening:

The phytochemical tests of the DMSO extracts of fresh and dry leaves of K. reticulata and that of flowers of P. tithymaloides showed the presence of tannins, flavonoids, saponin glycosides, steroids and triterpenoids, carbohydrates and proteins in the present study. The same compouds were reported to be present in the DMSO extracts of fresh leaves of P. fraternus in the previous studies ²⁸. Presence of alkaloids, tannins, flavonoids, glycosides, saponins, steroids and triterpenoids in the dry leaves of K. reticulata had been reported [Narasimhudu et al and Gopinath et al (2012)] ^{36, 37}. Kantamreddi et al (2010)reported presence of steroid and triterpenoid in the methanolic extract of dry leaves of K. reticulata and absence of alkaloid and flavonoid of the same³⁸. The DMSO extract of leaves of K. reticulata in the present study exhibited presence of all the tested chemical constituents except alkaloids.

Raveen et al (2012)had shown presence of   flavonoids, alkaloids, steroids, tannins, saponins, proteins, carbohydrates and glycosides in the ethanolic extracts of P. tithymaloides ³⁹. Prakash et al (2013)observed the presence of tannins and absence of flavonoids, saponins, terpenoids and steroids in the aqueous extract of dry leavesof P. tithymaloides ⁴⁰. Present study showed the presence of all the tested phytoconstituents except alkaloids in the DMSO extracts of flowers of P. tithymaloides. There are no reports on the phytochemical analysis of flowers of P. tithymaloides.

 Estimation of tannins, flavonoids, saponins, steroids and triterpenoids:

Since the crude extracts of leaves of K. reticulata and P. fraternus and flowers of P. tithymaloides showed presence of tannins, flavonoids, saponins, steroids and triterpenoids, these compounds were extracted and their total content was determined (Table 7).

Table 7: Total tannins, flavonoids, saponins and steroids and triterpenoids in K. reticulata, P. fraternus P. tithymaloides.

It was observed that the tannin and saponin contents were highest in the leaves of K. reticulata whereas, the flavonoid and steroid and triterpenoid in the flowers of P. tithymaloides (Table 7).  Khatoon et al (2006) reported very low content of tannins in the whole plant of P. fraternus (less than 0.2%) ⁴¹, however its fresh leaves in the present study showed 2.82% tannins.

 Separation of extracted compounds by TLC:

Following results in Table 8 and 9 were obtained when the TLC of compounds was carried out.

Table 8: Number of bands observed in the TLC of compounds

Track 1                             2           3         4                1          2         3         4          5          6                  1         2           3         4           5         6

 FIG.13: Separation of tannins by TLC                FIG.14: Separation of flavonoids by TLC             FIG.15: Separation of saponins by TLC

1        2        3          4         5        6                       1       2         3          4         5        6

                         FIG. 16: Separation of steroids by TLC               FIG. 17: Separation of triterpenoids by TLC

FIG. 13: Track 1 - Gallic acid std.    Track 2 - K. reticulata leaves             Track 3 - P. fraternus leaves     Track 4 - Pedilanthus tithymaloides flowers]

FIG. 14, 15, 16 and 17:  Track 1 and 2 - K. reticulata leaves (2 and 5 µl respectively)

Track 3 and 4 - P. fraternus leaves (2 and 5 µl respectively)

Track 5 and 6 - Pedilanthus tithymaloides flowers (2 and 5 µl respectively)

 TABLE 9: COMMON COMPONENTS IN ALL THE ALL THE EXTRACT ON TLC PLATES

The leaves of K. reticulata and P. fraternus and flowersofof P. tithymaloides showed presence of

6, 8 and 6 components (bands) of tannins (Fig.13), 9, 8 and 11 of flavonoids (Fig.14) and 10, 8 and 11 components of saponins (Fig.15), 8, 11 and 6 of steroids (Fig.16) and 2, 1 and 1 bands of triterpenoids (Fig.17) respectively(Table 8)

Table 9 indicates that 4 components of tannins, 3 of flavonoids and 2 of saponins are present in all the extracts whereas TLC of steroids and triterpenoids did not show presence of any common components (Table 9) (Fig. 13 to 17). Sivasankar et al (2011) reported presence of rutin in the leaves of K. Reticulate ⁴² however present study did not show rutin in the TLC of flavonoids.

Conclusions: Present study shows that the DMSO extracts of the leaves of K. reticulata and the flowers of P. tithymaloides inhibited the growth of P. vulgaris and S. aureus and their MIC values obtained were 200 mg /ml. The extracts did not inhibit the growth of T. rubrum and T. mentagrophytes. The phytochemical analysis of these extracts indicated that the tannins and saponins contents were highest in the leaves of K. reticulata whereas, the flavonoids and steroids and triterpenoids contents were highest in the flowers of P. tithymaloides. TLC of extracted tannins showed presence of 4 common components, the flavonoids showed 3 and the saponins showed 2 common components in these plants. TLC of steroids and triterpenoids showed no common components. The chemical compounds present in these plants should be isolated and tested for their antibacterial activity

Acknowledgement: The authors gratefully acknowledge University Grants Commission for the financial assistance provided through the grant of Major Research Project to carry out this work. The authors express their sincere thanks to Principal, St. Xavier’s College for providing the facilities. The authors are grateful to Dr. Medhekar, Head of Skin and V. D. Department, G.T. hospital and Dermatologist, Dr. Miskeen of Dr. Miskeen’s Central Clinical Microbiology Laboratory, Thane for providing necessary facilities and help for the collection of dermatophyte samples.

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    How to cite this article:

    Bapat UC and Mhapsekar DR: Study of Antimicrobial Activity and Phytochemical Evaluation of Jatropha Gossypifolia, Sapium Sebiferum, Kirganelia Reticulata, Phyllanthus Fraternus and Pedilanthus Tithymaloides . Int J Pharm Sci Res2014; 5(11): 4933-41.doi: 10.13040/IJPSR.0975-8232.5 (11).4933-41.

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