INTRODUCTION:

The Decalepis hamiltonii Wight and Arn belongs to the family Asclepiadaceae. It is commonly called as Swallow root in Tamil Magali Kizhangu. It is an endemic and endangered medicinal plant ^[1]. According to Environmental information system (ENVIS), this species is endemic to Peninsular India and native to Deccan Peninsula and the forest areas of Western and Eastern Ghats of India. This has been recorded in the dry and moist deciduous forests of Tamilnadu, Karnataka and Andhra Pradesh^[2].

Roots of D. hamiltonii have traditionally been used as demulcent, diaphoretic, diuretic and tonic. It is used to treat loss of appetite, skin diseases, diarrhoea, nutritious disorders and as blood purifier ^[3-4]. Herbal plants are used medicinally in different countries and are a source of many potent and powerful drugs^[5]. A wide range of medicinal plant parts is used for extract as raw drugs and they possess varied medicinal properties. Herbs are safe, less toxic, economical and a reliable key natural resource of drugs all over the world^[6]. In recent years, antimicrobial and antioxidants have attracted a great deal of attention in the control of infectious diseases caused by pathogens and degenerative diseases in which oxidative damage has been implicated^[7]. Phenolic compounds are known to exhibit a range of biological activities, including anticancer, antibacterial, antioxidant and anti-inflammatory properties^[8]. The search for newer natural antimicrobial and antioxidants, especially of plant origin, has ever since increased. However, to the best of our knowledge, there has been no report about in vitro antimicrobial and antioxidant activity from the stem bark of D. hamiltonii. The aim of this study was to analyse the effectiveness of petroleum ether, methanol and aqueous for the extraction of antimicrobial and antioxidant phenolics from the stem bark of D. hamiltonii.

MATERIALS AND METHODS:

Plant material:

Well grown woody and healthy plant stem bark of Decalepis hamiltonii was collected from the Eastern Ghats of Kolli hills of Tribals inhabiting area. The voucher specimens were deposited at Department of Botany, Kongunadu Arts and Science College (Autonomous), Coimbatore, TamilNadu, India.

Chemicals and standards:

All the chemicals and solvents were analytical grade, obtained from HiMedia chemicals, Mumbai, India. Chemicals like Muller Hinton Agar, Saboraud’s Dextrose Agar, ampicillin, nystatin, DMSO, Petroleum ether, Methanol, 2, 2 - azinobis 3-ethylbenzo-thiozolin-6-sulphonate (ABTS) was obtained from Sigma chemicals, USA. The other chemicals used were Gallic acid, α-Tocoperol, Rutin, 2, 2-diphenylpicryl-1-picrylhydrazyl (DPPH), Tert-butylated hydroxytoluene (BHT), Ethylene diamine tetra acetic acid (EDTA), Trolox, Potassium persulphate.

Solvent extracts:

The stem bark were washed under running tap water and air dried for 15 days after that powdered and pulverized into fine powder using pestle and mortar. 50g of fine powder was packed with no.1 filter paper and placed in soxhlet apparatus along with solvents like petroleum ether followed by methanol. The residues were collected and dried in room temperature at 30˚C after which yield was weighed and used for further analysis.

Aqueous extract:

10 g of powdered sample was dissolved in 100 mL of distilled water and boiled for 2 h on slow heat. The residue was removed by filtering through 8 layers of muslin cloth; the filtrate was then centrifuged at 5000g for 10 min. The supernatant was collected and further boiled till the volume was reduced to one fourth of the original volume of the solvent used (that was 100 mL) giving the concentration of 400 mg/mL^[9]. It was then autoclaved at 121˚C and at 15 lbs pressure and stored at 4˚C^[10].

Preliminary Phytochemical analysis:

The extracts were screened for the presence of alkaloids, tannins, Phlobatannins, saponins, flavonoids, steroids, terpenoids, phenol, coumarin, glycosides, cardiac glycosides and reducing sugar by standard methods^[11-14].

Determination of total Phenolic Content:

Total polyphenol content was determined using Folin–Ciocalteu spectrophotometric method ^[15]. Plant extracts (100 µL) were mixed with 0.2 mL of Folin–Ciocalteu reagent and 2 mL of H₂O, and incubated at room temperature for 3 minutes. After that addition of 1 mL of 20% sodium carbonate can be done into the mixture, total polyphenols were determined after 1 h of incubation at room temperature. The absorbance of the resulting blue colour was measured in UV spectrophotometer at 765 nm against the blank. Quantification was done with respect to the standard curve of gallic acid. The results were expressed as gallic acid equivalents (GAE), mg per 100 g of dry weight. All determinations were performed in triplicate (n=3).

Culture media used:

Freshly prepared Muller Hinton Agar and Saboraud’s Dextrose Agar medium were used for bacteria and fungi respectively.

Microorganisms used:

In vitro antimicrobial activity was examined for the various solvent extracts of stem bark of the species D. hamiltonii against nine bacterial species which include the gram positive strains viz., Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 19433) and gram negative strains viz., Klebsiella pneumoniae (ATCC UC57), Escherichia coli (ATCC 15224), Proteus aeruginosa, P. mirabilis, Salmonella typhi (MTCC 733), Salmonella paratyphi A, Salmonella paratyphi B. The tested fungal were Candida albicans (ATCC 10231), Aspergillus niger (ATCC 16404) A. flavus and Fusarium oxysporum. The tested organisms were obtained from Division of Microbiology lab, PSG medical and Research Institute, Coimbatore, Tamil Nadu, India. The stock cultures were maintained on the respective broth at 4˚C.

In vitro antimicrobial activity:

The bark extracts were tested for their effect against the growth of pathogenic bacteria and fungus by well diffusion method^[16]. The bark extracts (petroleum ether, methanol and aqueous) of D. hamiltonii were employed for antimicrobial activity. The antibiotic discs, Ampicillin (10μg) and nystatin (10μg) served as positive control for bacteria and fungi respectively. The bacteria and fungi tested were inoculated into Muller Hinton Agar, Saboraud’s Dextrose Agar medium respectively. After the incubation period of 24 hours at a temperature of 35°C, three or four colonies isolated from these media were inoculated on 4mL of nutrient broth and incubated for 2 hrs at 35°C. The cultures were adjusted with sterile saline solution to obtain turbidity. Petri dishes containing Muller-Hinton agar medium were streaked separately with these microbial suspensions of bacteria and fungi. Well were made with flamed and cooled cork-borer (9mm) filled (0.02mL) with the extracts and the standard antibiotics ampicillin (50µL) and nystatin for bacteria and fungus respectively and DMSO (10%) used as negative control. After equilibrium at 4°C, the plates were incubated overnight at 37°C and the diameter of any resulting zones of inhibition was measured. Triplicates were maintained for all these experiments.

Antioxidant activity assays Free radical-scavenging activity on α,α-diphenyl-β-picrylhydrazyl (DPPH•):

DPPH radical-scavenging activity was determined according to the method ^[17]. An aliquot of each extract dissolved in DMSO and was mixed with 50 µL of 1 mM DPPH (Prepared with DMSO) was added, control was DPPH solution. The mixture was shaken followed by incubation at room temperature for 20 minutes in the dark. The absorbance against blank was measured at 517nm. The extract concentration that could scavenge 50% of the DPPH radicals (IC₅₀) was calculated from the plot of scavenging activity against the concentration of extract. Butylated hydroxy toluene (BHT) standard was used for comparison.

% of Inhibition

= {[Absorbance _Control− Absorbance _Sample]

Absorbance _Control × 100}

Antioxidant activity by the ABTS•+ assay:

The 2, 2'Azinobis (3-ethyl-benzothiozoline-6-sulfonic acid) disodium salt (ABTS) was dissolved in water to a 7mM concentration. ABTS radical cation (ABTS·+) was produced by reacting ABTS stock solution with 2.45 mM potassium per sulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. Prior to assay, the solution was diluted in ethanol (about 1:89 v/v) and equilibrated 30ºC to give an absorbance at 734 nm of 0.70±0.02 in a 1-cm cuvette ^[18]. The concentration of the extracts that produced between 20-80% inhibitions of the blank absorbance was determined and adapted. After the addition of 1 mL of diluted ABTS^·+ solution to 10μL of root extracts or Trolox standards (Final concentration 0-15 μM) in ethanol, optical density (OD) was taken at 30ºC exactly 30 minutes after the initial mixing. The unit of total antioxidant activity (TAA) is defined as the concentration of Trolox having equivalent antioxidant activity expressed as μmol/g sample extracts on dry matter.

Statistical analysis:

The data were subjected to a one-way analysis of variance (ANOVA) and the significance of the difference between mean (n=3) ±SD was determined by Duncan’s multiple range test (p<0.05).

RESULTS:

Extract yield and preliminary Phytochemical screening:

The result of the laboratory preparation of petroleum ether, methanol and aqueous extracts of bark of D. hamiltonii is presented in table 1. Among the various solvents highest percent yield (59.15) extract was achieved from methanol solvent and the colour of solvent was reddish. The Phytochemical analysis of various solvent extracts of stem bark is summarized in table 2. From this analysis, methanolic extract found to have more chemical constituents i.e., tannins, saponins, alkaloids, glycosides, flavonoids, steroids, terpenoids, phenol compared to other extracts. reducing sugar and phlobatannins were present only in aqueous extract.

Table 1: Percent yield of various extracts of stem bark of Decalepis hamiltonii

Solvent	Time of extraction	Colour of extract	Yield (%)
Petroleum ether	24 h	Yellowish	11.12
Methanol	24 h	Reddish	59.15
Aqueous	12 h	Reddish	6.8

Yield (%) = (Yield weight/Sample weight) x 100

Table 2: Preliminary phytochemical analysis of various stem bark extracts of Decalepis hamiltonii

Phytochemical constituents	Petroleum ether extract	Methanolic extract	Aqueous extract
Tannins	-	+	-
Saponins	-	+	+
Alkaloids	+	+	-
Glycosides	+	+	+
Reducing sugar	-	-	+
Flavonoids	-	+	+
Phlobatannins	-	-	+
Steroids	+	+	+
Terpenoids	+	+	-
Phenol	+	+	-
Cardio glycosides	+	+	+
Coumarin	-	+	+

Total phenolic Content:

Total phenolics of petroleum ether, methanol and aqueous extracts of stem bark of D. hamiltonii are shown in table 5. The total phenolics of the methanol extract (13.6 mg of GAE/g DW) of stem bark were found to be higher than in petroleum ether (8.6 mg of GAE/g DW) and aqueous (10.7 mg of GAE/g DW) extracts.

In vitro Antimicrobial Activity:

The antibacterial activity was demonstrated for susceptibility of bacterial pathogens to the petroleum ether methanol, and aqueous stem bark extract of Decalepis hamiltonii (Figure 1). The methanol extract was found to be highly active against Staphylococcus aureus (17.2±1.63), Proteus aeroginosa (16.3±1.22) and Klebsiella pneumonia (16.1±1.69) followed by aqueous extract shown higher zone of inhibition against P. aureginosa (15.3±0.81) and K. pneumonia (14.2±0.82) (Table 3). As far as the antifungal activity is concerned, the good activity against Candida albicans in methanol extract of Decalepis hamiltonii (Table 4).

Table 3: In vitro antibacterial activity of different stem bark extracts of Decalepis hamiltonii

Bacteria	Diameter of zone of inhibition (mm)
Bacteria	Petroleum ether	Methanolic extract	Aqueous extract	Ampicillin	DMSO (10%)
Enterococcus faecalis	9.5±0.61	12.4±1.24**	10.2±0.92**	26.1±0.93***	ND
Staphylococcus aureus	14.0±0.81***	17.2±1.63***	11.2±1.63**	29.2±1.24***	ND
E coli	9.6±0.67	11.1±1.67**	10.1±1.61**	21.3±1.69***	ND
Klebsiella pneumoniae	8.1±0.77	16.1±1.69***	14.2±0.82***	25.2±0.81***	ND
Proteus mirabilis	9.3±0.58	13.2±1.24**	12.3±0.88**	30.0±0.81***	ND
P. aureginosa	13.1±0.64***	16.3±1.22***	15.3±0.81***	33.7±0.72***	ND
Salmonella typhi	14.2±0.81***	15.3±1.24***	13.5±0.84**	24.4±1.52***	ND
S. paratyphi A	9.6±0.67	14.1±1.27***	9.4±0.64	22.3±1.34***	ND
S. paratyphi B	10.4±0.41**	12.0±1.33**	10.1±0.72**	28.2±0.82***	ND

Ampicillin- Positive control, DMSO- Negative control, ND-Not Detected

Table 4: In vitro antifungal activity of different stem bark extracts of Decalepis hamiltonii

Fungi	Diameter of Zone of inhibition (mm)
Fungi	Petroleum ether	Methanolic extract	Aqueous extract	Nystatin	DMSO (10%)
Candida albicans	10.2±0.73**	14.1±0.32***	11.5±0.12**	29.2±0.28***	ND
Aspergillus niger	9.5±0.53	11.3±0.16**	10.2±0.12**	31.1±0.12***	ND
A. flavus	10.6±0.71**	12.4±0.42**	12.1±0.081***	33.2±0.040***	ND
Fusarium oxysporum	9.9±0.56	12.2±0.53**	13.0±0.12**	39.3±0.18***	ND

Nystatin – Positive control, DMSO – Negative control, ND- Not detected

Figure 1. Antimicrobial activity of stem bark extract of Decalepis hamiltonii.

A-Staphylococcus aureus; B- Klebsiella pneumoniae; C- Proteus aureginosa; D- Salmonella typhi; E-Candida albicans. PC- Positive control; NC- Negative control; M-Methanol; A- Aqueous; P- Petroleum ether.

DISCUSSION:

In the present study the phytochemical screening, has revealed the presence of tannins, saponins, alkaloids, glycosides, flavonoids, steroids, terpenoids, phenol, reducing sugar and phlobatannins in the stem bark extracts of D. hamiltonii. Further the presence of different phytoconstituents in the three different extracts may be responsible for the therapeutic properties of stem bark of D. hamiltonii. Flavonoids and tannins are phenolic compounds and plant phenolics are a major group of compounds that act as primary antioxidants or free radical scavengers. Since these compounds were found to be present in the stem bark extracts, it might be responsible for the potent antioxidant capacity of D. hamiltonii. The total phenolic content of the methanol extract was found to be more than the other extracts studied. It could be due to the solubility of phenolics and other aroma compounds were higher in methanol solvent^[19]. The antioxidant activities of plant extracts are closely related to their total phenolic content^[20]. However, the methanol was found to be more efficient solvent for extracting the phenolic constituents in stem bark of D. hamiltonii than that of petroleum ether and aqueous solvents. The presence of phenolic content in root of D. hamiltonii has been reported in the study of Samydurai and Thangapandian^[21], and Muralidhar et al. ^[22].

The present study indicates antimicrobial property of the stem bark of D. hamiltonii against the selected strains of microorganism varies depends upon the solvents used for extraction. The petroleum ether, methanol and aqueous extracts were tested against 9 bacterial strains (both Gram^+ve and ^–ve) and 4 fungal strains. The result revealed that methanol extract showed highest inhibition capacity against tested pathogens compared with petroleum ether and aqueous extracts. Recently, a number of studies have been reported for antimicrobial activity of root of D. hamiltonii and were suggested that the most of the tested pathogens were susceptible for methanol extract^[23-26]. It should be supposed that phenolic substances might also be responsible for this activity. This is supported by the overall higher activity and higher content of phenol shown in the methanol extract, although in earlier study by Mohana et al.^[27]also reported that the effect of inhibitory substances. DPPH is usually used as a substrate to evaluate antioxidative activity of antioxidants^[28]. The method is based on the reduction of methanolic DPPH solution in the presence of a hydrogen donating antioxidant, due to the formation of the non-radical form DPPH-H by the reaction. The methanol solvent extract of stem bark of D. hamiltonii displayed the most favourable activity against DPPH•, and was significantly more active than the petroleum ether and aqueous solvents extracts. Many studies have been reported that root methanolic extract of D. hamiltonii showed high antioxidant activity measured as scavenging of DPPH^[29,
30]. ABTS•+ is a peroxidase substrate, which generates a metastable radical with blue-green color through oxidation in the presence of H₂O₂^[31]. Therefore, the decolorization assay of ABTS•+ is shown to be a very useful tool in expeditiously measuring the antioxidative activity of plant extracts. The results obtained clearly imply that methanol extract inhibit or scavenge the radical more than the petroleum ether and aqueous extract of D. hamiltonii. DPPH• and ABTS•+ radicals are the two most widely used chromogenic compounds to measure the antioxidant activity of biological materials. Both of them were characterized by excellent reproducibility and stability under certain assay conditions^[32].

CONCLUSION:

The result of present study provides information on the potential medicinal uses of this plant. Overall, methanol was the most effective solvent for extraction of antimicrobial and antioxidant phenolics from the stem bark of D. hamiltonii. At the same time the study provides data on the characteristics of the antimicrobial and antioxidant activity of the stem bark of the study plant. However, further investigation of individual compounds, from stem bark of D. hamiltonii could serve as antimicrobial agents for the therapy of infectious disease caused by pathogens and also potential natural antioxidants for the food industries.

REFERENCE

1. Jacob George, Ravishankar GA, John Pereira, Divakar S. Bio insecticide from swallow root (Decalepis hamiltonii) protects food grains against insect infestation. Current Science. 1999; 77: 501-502.

2. Vijai Kumar G, Maria GT, Anthonia OD, Mohtashim L. Biotechnology of Bioactive Compounds. John Wiley and Sons publishers, United States. 2015. p.736

3. Dey D, Das M, Sharma AK. Pharmacognosy of Indigenous Drugs. New Delhi: Central Council for Research in Ayurveda and Shiddha, 1999; 897.

4. Chopra RN, Nayar SL, Chopra K. Glossary of Indian medicinal plants. New Delhi; Central Institute of Scientific and Industrial Research, India; 1956; 35-96.

5. Srivastava J, Lambert J Vietmeyer N. Medicinal plants: An expanding role in development. World Bank Technical Paper. No. 320. 1996.

6. Sooad Al-Daihan, Manar Al-Faham, Nora Al-shawi, Rawan Almayman, Amal Brnawi, Seema zargar, Ramesa shafi Bhat. Antibacterial activity and phytochemical screening of some medicinal plants commonly used in Saudi Arabia against selected pathogenic microorganisms, Journal of King Saud University – Science. 2013; 25: 115–120.

7. Kumaran A, Joel Karunakaran R. Antioxidant and free radical scavenging activity of an aqueous extract of Coleus aromaticus. Food Chemistry. 2006; 97: 109–114.

8. Guy P, Kamatou P, Alvaro MV, Paul Steenkamp. Antioxidant, antiinflammatory activities and HPLC analysis of South African Salvia species, Food Chemistry. 2010; 119: 684–688.

9. Harborne JB. Phytochemical Methods, Chapman and Hall, Ltd., London, 1973; pp. 49 -188.

10. Jigna P, Sumitra VC, In vitro antimicrobial activity and phytochemical analysis of some Indian medicinal plants. Turkey Journal Biology. 2007; 31: 53–58.

11. Adetuyii AO, Popoola AV. Extraction and dye ability potential studies of the Colourant in Zanthoxylum Zanthoxyloides Plant on cotton Fabric. Journal of Science Engineering Technology. 2001; 8(2): 3291-99.

12. Trease, G.E., and W. C. Evans, 1989. Pharmacognosy. 13 Ed. Balliere Tindall, London, pp:176-180.

13. Sofowara A. Medicinal Plants and Traditional Medicine in West Africa John Wiley and Sons. New York, 1982; p. 256.

14. Harbone JB. Methods of extraction and isolation. In: phytochemical methods. 3^rd ed. Chapman and Hall: London. 1998; 42-98.

15. Gao X, Ohlander M, Jeppsson N, Bjork L, Trajkovski V. Changes in antioxidant effects and their relationship to phytonutrients in fruits of sea buckthorn (Hippophae rhamnoides L.) during maturation. Journal of Agri and Food Chemistry. 2000; 48: 1485–1490.

16. Collins CH, Lyne PM Grange JM. Microbiological Methods. 7^th Edition. Butterworth-Heinemann Ltd., Oxford. 1995; 512.

17. Blios M.S. Antioxidant determination by the use of a stable free radical. Nature. 1958; 26: 1199-1200.

18. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic BioMed. 1999; 26: 1231-37.

19. Sobhy M, Mohsen A, Ammar SM. Total phenolic contents and antioxidant activity of corn tassel extracts. Food Chemistry. 2009; 112(3): 595–98.

20. Rathee JS, Hassarajani SA, Chattopadhyay, S. Antioxidant activity of Nyctanthes arbor-tristis leaf extract. Food Chemistry. 2007; 103(4): 1350–1357.

21. Samydurai P, Thangapandian V. Antioxidant property and polyphenols evaluation of aqueous root extract of Decalepis hamiltonii Wight and Arn. International Current Pharmaceutical Journal. 2012; 1(4): 71-76.

22. Muralidhar TS, Sourav Acharya, Ramyashree C, Sowmya Reddy, Shruthi MR, Shantha SL. Study of Bioactive components in Decalepis hamiltonii in vitro. IOSR Journal of Pharmacy. 2014; 4(1): 62-66.

23. Samydurai P, Thangapandian V. Physico-phytochemical analysis and their minimum inhibitory concentrations of various extracts of Decalepis hamiltonii Wight and Arn against gastrointestinal disorder pathogens, International Journal of Pharmacy and Pharmaceutical Sciences. 2012; 4(3): 289 -292.

24. Devi M, Latha P. Antibacterial and phytochemical studies of various extracts of roots of Decalepis hamiltonii Wight and Arn. International Journal of Pharmacy and Pharmaceutical Sciences. 2012; 4 (2): 738- 740.

25. Poorna C, Madhu S, Manikanta Kumar A, Manjunath Shetty KS, Hemanth C. Antibacterial activity of various extracts of roots of Decalepis hamiltonii Wight and Arn. International Journal of Pharmacology Research. 2013; 3(2): 67-70.

26. Prakash P, Thiyagarajan G, Rengarajan M. Phytochemical screening and Antibacterial activity of root extracts of Decalepis hamiltonii Wight and Arn. International Journal of Pharma Research and Review. 2014; .3(11): 33-38.

27. Mohana DC, Raveesha KA, Rai KML. Herbal remedies for the management of seed-borne fungal pathogens by an edible plant Decalepis hamiltonii Wight and Arn. Arch. Phytopathol. Plant Protection. 2008; 41:38-49.

28. Oyaizu M. Studies on product of browning reaction prepared from glucose amine. Japanese Journal of Nutrition. 1986; 44: 307–315.

29. Srivastava A, Shereen RH, Shivanandappa T. Antioxidant activity of the roots of Decalepis hamiltonii (Wight and Arn.) LWT, 2006; 39: 1059-1065.

30. Harish R, Divakar S, Anup Srivastava, Shivanandappa T. Isolation of Antioxidant Compounds from the Methanolic Extract of the Roots of Decalepis hamiltonii (Wight and Arn.) Journal of Agric. Food Chemistry. 2005; 53: 7709-7714.

31. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine. 1999; 26(9–10): 1231–1237.

32. Arnao MB. Some methodological problems in the determination of antioxidant activity using chromogen radicals: A practical case. Trends in Food Science and Technology. 2000; 11(11): 419–421.

Received on 05.03.2016 Accepted on 25.03.2015

Asian J. Res. Pharm. Sci. 2016; 6(2): 129-134

DOI: 10.5958/2231-5659.2016.00018.7

S. No	Sample	Total Phenol Content (mg of GAE/g DW)	IC₅₀ % of DPPH (µg/ml)	TEAC (μmol TE/g extract)
1	Petroleum ether	8.6±0.29	84.4±1.79**	1790.12±47.72
2	Methanol	13.6±0.69***	56.5±2.31***	6771.44±49.24***
3	Aqueous	10.7±0.53**	127.8±2.53	2210.44±94.67**
4.	BHT		34.74±1.02***

ABSTRACT: