Phytochemical Screening and Antimicrobial activity of Solanum erianthum D. Don

Vol. 4, No. 2 (2014): 507-511 Research Article

Open Access

I

ISSSSNN:: 22332200--66881100

Phytochemical Screening and Antimicrobial activity

of

Solanum erianthum

D. Don

Radhika Mahadev

, Ramakrishnaiah Hanumanthaiah

*, Krishna Venkatarangaiah

,

Deepalakshmi Arjunan Paranthaman

1

Department of P. G. Studies and Research in Biotechnology, Government Science College, Bangalore -560001, India

2

Department of P. G. Studies and Research in Biotechnology and Bioinformatics, Kuvempu University, Shankaraghatta - 577451, India

* Corresponding author: R. Hanumanthaiah; e-mail: hramabt@yahoo.com

ABSTRACT

The present study was carried out for phytochemical screening of principle bioactive compounds in Solanum erianthum. Phytochemical analysis revealed the presence of tannin, saponin, terpenoid, steroid, flavonoid and alkaloid. The petroleum ether, chloroform, methanol and aqueous extracts were subjected for antimicrobial activity against twelve bacteria and three fungal strains using agar well diffusion method. Leaf extracts inhibited B. subtilis, P. aeruginosa, K. pneumoniae, S. typhi, C. albicans and C. tropicalis. Fruit extracts possessed antimicrobial activity against B. subtilis, P. aeruginosa, S. typhi, C. albicans and C. tropicalis. P. aeruginosa showed significant sensitivity to stem extract. The Minimal Inhibition Concentration (MIC) values ranged from 1.6 - 1.92 mgmL-1_{. Among the} different solvent extracts used for the study, the extract prepared in methanol and water proved to have greater antimicrobial activity. The results suggested that methanolic and aqueous leaf, fruit and stem extracts were highly potent against P. aeruginosa and can be used in treatment of nosocomial infections such as pneumonia, urinary tract infections (UTIs) and bacteremia. The study revealed the high medicinal property of Solanum erianthum.

Keywords:

Concentration.

INTRODUCTION

Higher plants constitute a major reservoir of raw materials used in pharmaceuticals, cosmetics and traditional Indian medicine. Indeed much of the pharmacopoeia of scientific medicine was derived from the herbal lore of native people and majority of the drugs commonly used today are derived from plants [1].

Solanum erianthum is known to be medicinally useful.

The flavanoid rich extract of S. erianthum leaves possesses antibacterial and antifungal activity. It is effective against Gram positive bacteria and fungi such

as Aspergillus flavus and some Candida species. Leaves

are considered to be laxative and diuretic, and are used in treating leprosy, sexually-transmitted diseases and malaria [2]. Leaves possess expectorant property and used to treat hemorrhoids, scrofula and leucorrhea. Heated leaves are applied to the forehead as an analgesic for headaches while the leaf decoction is used for vertigo. An extract of root is used to treat dysentery,

fever, diarrhea, digestive problems and body pains [3]. Further, root bark is used as an anti - inflammatory and used in the treatment of arthritis. Fruits are said to be poisonous due to the presence of glyco - alkaloids. Steroidal alkaloids from S. erianthum are useful in industries as steroidal precursors. Solasodine present in fruit is useful for the production of medicinal steroids like anti - inflammatory corticosteroids, contraceptive steroids and anabolic steroids [4, 2].

Literatures are available on the phytochemical analysis and antimicrobial activity of various Solanum species. Tannins, saponins, alkaloids, flavonoids, steroid and terpenoids were found to be present in extracts of S.

xanthocarpum, S. surattense, S. nigrum and S. trilobatum

[5-8]. The antibacterial property of S. torvum against B.

subtilis and P. aeruginosa is well established [9]. Similar

results were also reported in S. surattense, S.

xanthocarpum and S. trilobatum on P. aeruginosa, B.

subtilis and S. typhi [10-12].

The present study was undertaken to evaluate phytochemical analysis and antimicrobial activity of sequential extracts of S. erianthum.

MATERIALS AND METHODS

Plant material

The specimen was collected from outskirts of Bangalore city and was identified as Solanum

erianthum D. Don. (Syn S. verbascifolium non L).

Subsequent authentication was done at Regional Research Institute, Central Council for Research in Siddha and Ayurveda with the voucher specimen no RRCBI - 4865. Voucher specimen is also maintained in the herbarium of the Research Centre (VN 510).

Extraction of plant material

Fresh field grown plants were collected and washed with running tap water followed by distilled water to remove the adhered dust particles. After blotting, the sample was air dried in shade, ground to fine powder and stored in clean air tight containers. About 30 g of each sample was placed in the soxhlet extractor for 48 h in batches and the sample was sequentially extracted separately using the solvents petroleum ether, chloroform, methanol and water. All the extracts were dried in vacuum rotary evaporator at 40 º_{C under} reduced pressure, weighed and stored at 4 º_{C in amber} bottles.

Phytochemical Screening

Phytochemical analysis was carried out for identification of tannins, saponins, steroid, cardiac glycoside, alkaloids, terpenes, flavonoids, anthraquinone and phlobatannin according to standard methods [13, 14].

Antimicrobial activity Test microorganisms

Antibacterial assay was carried out on twelve bacteria listed in Table 2, were procured from American Type Culture Collection (ATCC), National Collection of Industrial Microorganisms (NCIM), National Collection of Type Culture (NCTC), and Microbial Type Culture Collection (MTCC) Institutes. All the organisms were sub cultured and maintained on nutrient media at 37 º_C.

Antifungal assay was conducted on Candida tropicalis MTCC 184, Candida albicans MTCC 183 and Candida

glabrata MTCC 3018. Fungal cultures were maintained

on Sabouraud dextrose agar at 30 º_C.

Antibacterial activity

Antibacterial activity of sequential extracts of S.

erianthum was carried out by agar well diffusion

method using the Clinical and Laboratory Standards Institute (CLSI) and National Committee for Clinical Laboratory Standards (NCCLS) [15, 16]. Pure culture from the plates were inoculated into Mueller-Hinton Agar (MHA) plate and sub cultured at 37 ˚C for 24 h.

Inoculum was prepared by aseptically adding the culture into fresh media and the cell density was adjusted to 0.5 McFarland turbidity standard to yield a bacterial suspension of 1.5×108 cfumL-1_{. Standardized} inoculum was transferred and spread evenly on a MHA plate to yield a lawn culture. Wells of 6 mm diameter were cut on the agar plate and 100 µL of extracts were tested with a concentration of 10 mgmL-1 _{of DMSO.} Extracts were allowed to diffuse for half an hour at 4 ˚C and incubated at 37 ˚C for 24 h. Well containing 5 % DMSO served as negative control and gentamicin (10 μg) was used as positive control. The plates were observed for inhibition of bacterial growth that was indicated by the clear zone around the well. The size of zone of inhibition (including well) was measured in millimeters. The absence of zone inhibition was interpreted as the absence of activity. All experiments were carried out in triplicates and repeated four times under strict aseptic conditions. The activities were expressed as resistant, if the zone of inhibition was less than 7 mm, intermediate (8 - 10 mm) and sensitive if more than 11 mm [15, 16].

Anti-fungal activity

Sequential extracts of S. erianthum were used for testing as described for antibacterial activity. Sabouraud Dextrose Agar (SDA) medium was taken as the assay medium and amphotericin B (100 µg) as standard antifungal agent [17].

Minimum Inhibitory Concentrations (MIC)

MIC test was carried out by agar dilution method [18]. Different concentrations of sequential extracts of S.

erianthum from 10 to 2 mg/5 mL were prepared in

DMSO and filtered using membrane filter. The extracts were mixed thoroughly with 5 mL of autoclaved MHA medium for bacterial and SDA medium for fungal strains and poured into sterile petriplates and were allowed to solidify. Inoculum suspensions (10 μL) of various microbial isolates were seeded on individual agar plates. Growth control was prepared by inoculating 10 μL of each culture suspension on respective medium without any extract or solvent (drug-free medium). Solvent control was prepared by pouring 0.5 mL of DMSO to 5 mL of MHA medium and SDA medium followed by seeding of cultures. The plates were allowed to diffuse for 1 h and incubated at 37 ˚C for 24 h for bacterial cultures and 30 ˚_{C for fungal} cultures. Minimum Inhibitory Concentration was determined as the lowest concentration of extract that inhibited the visible growth of a microorganism after overnight incubation.

RESULTS AND DISCUSSION

Phytochemical analysis

Table 1. Phytochemical analysis of Solanum erianthum

Secondary Metabolites

Leaf Stem Fruit Root

PE CE ME AE PE CE ME AE PE CE ME AE PE CE ME AE

Tannin - + + + - - - -

Steroid - - + + - - + + - - + + - - - -

Flavonoid - - + + - - + + - - + + - - - -

Saponin + + + + + + + + + + + + + + + +

Phlobatanin - - - -

Cardiac glycoside - - - -

Anthraquinone - - - -

Terpenoid - - + + - - + + - - + + - - - -

Alkaloid

Dragendroff - + + + - + + + - - + + - + - +

Mayer - + + + - - + + - - + + - - - -

Hager + - + + - - + + - + + + - - + -

Wagner + + + + - + + + + - + + + + + -

Note: PE - petroleum extract, CE - chloroform extract, ME - Methanolic extract, AE - Aqueous extract, + - Present, - - Absent

Steroid, terpenoid, flavanoid, alkaloid, saponin and tannin were present in methanolic and aqueous leaf extracts. Stem and fruit aqueous and methanolic extracts showed the presence of steroid, terpenoid, saponin, alkaloid, flavonoid while tannin was absent. Root showed the presence of saponin and tannin in all the extracts. Considering all the four parts studied, methanolic and aqueous extracts revealed more number of compounds. This suggests that methanol and water were able to extract majority of the compounds. Studies conducted in S. xanthocarpum, S.

surattense, S. nigrum and S. trilobatum also exhibited

the presence of saponin, tannin, alkaloid, flavonoid and steroid [5-8].

Antimicrobial activity and Minimum Inhibitory Concentration

Antimicrobial activity of methanol, aqueous, petroleum ether and chloroform extracts of S. erianthum were analyzed against twelve bacterial and three fungal strains using agar diffusion method and the results are presented in Tables 2 and 3. MIC was determined by agar dilution method for the extracts which showed positive result with antimicrobial susceptibility test (Table 4). Of all the extracts tested only methanol and aqueous extract possessed antimicrobial activity.

Table 2. Antibacterial activity of Solanum erianthum

Microorganism Zone of inhibition in mm

Bacteria Gen Leaf Stem Fruit Root

PE CE ME AE PE CE ME AE PE CE ME AE PE CE ME AE

E. coli ATCC 25922 20±0.62 - - - -

E. coli NCIM 2931 22±0.22 - - - -

E. coli NCTC 12923 21±0.13 - - - -

S. aureus ATCC 25923 23±0.41 - - - -

S. aureus NCIM 5022 25±0.37 - - - -

S. aureus NCTC 10788 22±0.51 - - - -

P. aeruginosa ATCC

27853 25±0.08 - -

*_20±

0.08

*_16±

0.43 - -

*_23±

0.86

*_20±

0.74 - -

*_22±

0.36

*_19±

0.83 - - - -

B. subtilis NCIM 2480 28±0.29 - - *26 ± 0.40

*₁₈

±0.65 - - - -

*₂₀

±0.67

*_{19 ±}

0.77 - - - -

B. subtilis NCTC

10400 25±0.36 - - - -

K. pneumoniae NCIM

2957 28±0.65 - -

*_{20 ±}

0.58 - - - -

S. abony ACM 5080 19±0.31 - - - -

S. abony 21±0.23 - - - -

S. typhi 27±0.60 - - *20 ±

0.61

*₁₆

±0.24 - - - -

*_{12 ±}

0.09 - - - - -

n = 12, *P < 0.01. Gen - Gentamicin, PE - Petroleum ether Extract; CE - Chloroform Extract; ME - Methanol Extract; AE - Aqueous Extract. + - present, - - absent

Table 3. Antifungal activity of sequential extracts of Solanum erianthum

Fungi Amp

Leaf Stem Fruit Root

PE CE ME AE PE CE ME AE PE CE ME AE PE CE ME AE

C. albicans

MTCC 183 17±0.09 - -

*_14±

0.35

*_13±

0.17 - -

*_15±

0.63

*_14±

0.71 - -

*_14±

0.08

*_13±

0.76 - - - -

C. tropicalis

MTCC 184 16±0.54 - -

*_15±

0.28

*_14±

0.85 - -

*_16±

0.04

*_15±

0.18 - -

*_15±

0.09

*_14±

0.24 - - - -

C. glabrata

MTCC 3018 18±0.43 - - - -

Table 4. Minimal Inhibition Concentration (MIC) of various extracts of Solanum erianthum

Leaf Stem Fruit

ME AE ME AE ME AE

Bacteria

P. aeruginosa ATCC 27853 *_1.6±0.17 *_1.6±0.34 *_1.84±0.12 *_1.84±0.19 *_1.84±0.61 *_1.68±0.43

B. subtilis NCIM 2480 *_1.6±0.09 *_{1.6±0.78 ND ND} *_1.92±0.32 *_1.84±0.98

K. pneumoniae NCIM2957 *_{1.92±0.35 ND ND ND ND ND}

S. typhi *_1.84±0.31 *_{1.92±0.01 ND ND} *_{1.92±0.35 ND}

Fungi

C. albicans MTCC 183 *_1.84±0.75 *_1.84±0.52 *_1.76±0.23 *_1.92±0.34 *_1.76±0.43 *_1.84±0.13

C. tropicalis MTCC 184 *_1.76±0.09 *_1.92±0.56 *_1.84±0.76 *_1.92±0.54 *_1.76±0.26 *_1.84±0.89

n = 12, *P < 0.01. ME - Methanol Extract; AE – Aqueous Extract, ND - not determined

Organisms such as B. subtilis NCIM 2480, P. aeruginosa ATCC 27853, K. pneumoniae NCIM 2957, Candida

albicans MTCC 183 and Candida tropicalis MTCC 184

were susceptible to methanolic leaf extract. The aqueous leaf extract showed inhibitory action against S.

typhi, P. aeruginosa ATCC 27853 and B. subtilis NCIM

2480. The results showed that the methanolic and aqueous extract of leaf have broad spectrum antibacterial activity against Gram positive and Gram negative bacteria. Amongst the test microorganisms, B.

subtilis was found to be most sensitive. Whilst E. coli

ATCC 25922, E. coli NCIM 2931, E. coli NCTC 12923, S.

aureus ATCC 25923, NCTC 10788 and NCIM 5022, S.

abony and Candida glabrata MTCC 3018 were

comparatively resistant. MIC values ranged from 1.92 - 1.6 mgmL-1 _{for methanol and aqueous leaf extracts.} Methanolic leaf extract showed MIC of 1.6 mgmL-1 against P. aeruginosa and B.subtilis. Growth of K.

pneumoniae and S. typhi were inhibited at a MIC of 1.92

mgmL-1_{. MIC values against Candida albicans and}

Candida tropicalis were 1.84 - 1.92 mgmL-1_.

The methanolic fruit extract inhibited S. typhi, B. subtilis NCIM 2480 and P. aeruginosa ATCC 27853, while its aqueous extract was effective against B. subtilis NCIM 2480 and P. aeruginosa ATCC 27853. P. aeruginosa was highly susceptible. Rest of the organisms tested were found to be resistant to fruit extracts. P. aeruginosa inhibited growth at 1.84 mgmL-1 _{for methanolic extract} and 1.68 mgmL-1 _{for aqueous extract. MIC values} against Candida albicans and Candida tropicalis were 1.76 - 1.84 mgmL-1 _{for aqueous and methanolic extracts} respectively.

Stem extracts were effective only against P. aeruginosa ATCC 27853, Candida albicans MTCC 183 and Candida

tropicalis MTCC 184. Other organisms tested showed

no inhibition and therefore did not show broad spectrum antimicrobial activity. Further MIC values were relatively low ranging between 1.76 mgmL-1 _{- 1.92}

mgmL-1 _{for P. aeruginosa, Candida albicans and Candida}

tropicalis. However, root extracts did not show

significant antimicrobial activity on the tested organisms.

Plants belonging to the Solanum genus have been reported to have remarkable pharmacological activity [19]. Wiart et al reported the antimicrobial activity of S.

torvum against B. subtilis and P. aeruginosa [9]. Similar

results were also reported in S. surattense, S.

xanthocarpum and S. trilobatum on P. aeruginosa, B.

subtilis and S. typhi [10-12].

The antimicrobial activity of methanol extracts in S.

erianthum was more pronounced than the aqueous

extract against all the organisms tested. The lower antimicrobial activities of the petroleum ether and chloroform extracts were noted against most microbial strains in the present study. Similar results were reported by Koduru et al in Solanum aculeastrum [20]. Since petroleum ether and chloroform can dissolve nonpolar compounds, due to the insolubility of the active compounds and denaturation of the active compounds during extraction process are regarded as reason for the lower activity as opined by Igbinosa et al [21].

It was noteworthy that abundance of phytochemicals such as terpenoid, alkaloid, flavonoid, steroid, saponin and tannin in S. erianthum constitutes the main antimicrobial principle as suggested by Reynolds and Dweck, Tambekar and Khante,Thambiraj and Paulsamy, Subashini et al [22-25].

In this study, the leaf and fruit extracts showed broad spectrum antimicrobial activity against Gram positive and Gram negative bacteria as well as fungal strains. The activity of the extracts against the Gram negative bacteria is significant as these bacteria are known to exhibit high degree of resistance to conventional antibiotics [26]. Similarly, fungal strains Candida

albicans MTCC 183 and Candida tropicalis MTCC 184

were inhibited by leaf, stem and fruit extracts which suggests the antifungal potential of S. erianthum. This was further corroborated by low MIC ranges for most of the organisms.

Inhibition of P. aeruginosa showed that S. erianthum can be effectively used for cure of urinary tract infection which corroborates the traditional claim of use of S. erianthum for the treatment of UTI and leucorrhoea [27]. The high inhibitory property of S.

erianthum on S. typhi and K. pneumoniae suggests that

it can be used in treatment of typhoid and pneumonia.

Candida infections of the mouth, skin, or vagina occur

for no apparent reason [28]. Since S. erianthum forbids

Candida species it can be used to reduce their

CONCLUSION

The plant S. erianthum exhibited the presence of many secondary metabolites and revealed broad spectrum antimicrobial activity on the tested microorganisms. This investigation strongly suggests the possibility of this plant as an important source of antimicrobial drug development.

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