In Vitro Bioactive Potential of an Ethnomedicinal Mangrove Plant, (Heritiera Fomes Buch

Indian Journal of Geo-Marine Sciences Vol. 44(5), May 2015, pp. 704-713

In Vitro bioactive potential of an ethnomedicinal mangrove plant, (Heritiera fomes Buch. Ham.) from Odisha Coast, India

Jayanta Kumar Patra1,2 & Hrudayanath Thatoi1*

1Department of Biotechnology, College of Engineering and Technology, Biju Patnaik University of Technology, Bhubaneswar-751003, Odisha, India 2School of Biotechnology, Yeungnam University, Gyeongsan-712-749, Republic of Korea *[E-mail: [email protected]]

Received 05 February 2014; revised 05 March 2014

Heritiera fomes, a mangrove species occurring in mangrove forest along Odisha coast, India is known for its ethnomedicinal uses for the treatment of gastrointestinal, hepatic and skin diseases etc. All the four solvent (acetone, ethanol, methanol and aqueous) extracts of leaf and stem powder showed antibacterial activity (11 mm to 22 mm) at 50 mg/ml concentration. Leaf extracts possess better antioxidant properties (90% scavenging) as compared to stem extracts with dose dependency. Phytochemical screening of extracts showed the presence of phenols, cardiac glycosides, terpenoids etc. Overall results indicated that extracts of H. fomes might be applicable in natural medicine and pharmaceutical industries for drug discovery.

[Keywords: Bioactivity, Heritiera fomes, mangrove plants, phytochemicals]

Introduction “Sundari” is one of the most extensively occurring Mangroves are salt tolerant forest ecosystems true mangrove species along Odisha coast, India and found mainly in the tropical and subtropical the leaves, stem and bark have been traditionally used intertidal regions of the world largely confined to the for the treatment of various diseases such as region between 30º North and South of the equator1. gastrointestinal disorders (including dysentery, They are good source of herbal medicine because they diarrhoea, indigestion, colic, acidity, constipation, can grow under stressful conditions such as water bloating, lack of appetite, stomach ache), hepatic logging, fluctuating tidal and salinity etc. where no disorders (including jaundice, hepatitis); skin diseases vascular plants can survive and thus they produce (including eczema, abscess, acne, boils, scabies, several secondary metabolites which are of medicinal itch, infections, dermatitis, rash, sores, scar, importance2. Many mangrove plants are used in warts), diabetes and goiter9,10. Although H. fomes has traditional medicine being important source of shown some ethnomedicinal properties, there are no phytochemicals, natural antioxidants and studies focusing on its antioxidant and antimicrobial antimicrobials agents as reported by several authors3-6. activities of solvent extracts. Consequently, the Such bioactivity of the mangrove plants can be objective of the present study is to determine the assigned to phytochemicals present in the extracts7. antimicrobial and antioxidant activities of H. fomes. It Since mangrove plants possess bioactive is also aimed to screen the phytochemicals with a antimicrobial substances, which are safer to use view to explore their possible application in without any side effects, there is need to screen out pharmaceutics. more mangrove plants with potent antimicrobial activities. Apart from being a source of Materials and Methods antimicrobial compounds, mangroves are also Fresh, young and tender leaves and stem of potential sources of natural antioxidants and some of Heritiera fomes Buch.-Ham. (Sterculiaceae) were them are used as food8. collected from the mangrove forests along Odisha The Heritiera fomes commonly known as coast, India (Figure 1). The specimens were identified PATRA AND THATOI: IN VITRO BIOACTIVE POTENTIAL OF MANGROVE PLANT, HERITIERA FOMES 705

at Department of Natural Products, Institute of the method of Oyaizu17. The DPPH (2,2-diphenyl -1- Minerals and Materials Technology, Bhubaneswar picryl hydrazyl) radical scavenging effect was (RRL-B), Odisha, India and voucher specimen (VS determined by following modified methods of Hatano No. RRL-B-12568) was deposited. The extracts of the et al.18. The ferrous ion chelating activity was dried powder of the leaves and stem of the plant were assessed as described by Zhao et al.19. Nitric Oxide prepared using solvents (acetone, ethanol, methanol scavenging activity was assessed by Griess reaction and water). Liquid extracts were evaporated to method20. dryness by vacuum distillation and stored at 4°C for A qualitative phytochemical test to detect the further analysis. presence of alkaloid, tannin, saponin, flavonoid, cardiac glycosides, sterols, anthroquinone glycosides, carbohydrates and protein was carried out using standard procedures21 and quantitative phytochemical test for determination of proteins, carbohydrates, flavonoid, alkaloids, riboflavin, thiamine, and tannins21 and nitrogen, potassium, phosphorous, potassium pentoxide, potassium dioxide was carried out using standard procedures22.

Statistical Analysis Experiments were carried out in triplicates and the data was expressed as mean value ± standard deviation. Where applicable, the means of all the parameters were examined for significance by two Fig. 1— Map showing study sites way analysis of variance (ANOVA) and the differences between samples were determined by Duncan’s Multiple Range test using GenStat In vitro antimicrobial activity of leaf and stem discovery (edition 3) statistical software package. extracts of H. fomes were carried out against nine Differences were considered significant at a human pathogenic bacteria and three fungi strains. probability level of P<0.05. Correlation analysis Bacteria used in the study were obtained from between different antioxidant assays and between Institute of Microbial Technology, Chandigarh and TAC, PC and ASA were carried out using the others including the fungi were lab isolates. Agar cup correlation programme in MINITAB Software. The plate method of Khalid et al.11 was carried out to regression analysis between the DPPH radical establish the antibacterial activity of all the four scavenging and Nitric oxide scavenging assay were solvent extracts against the test pathogens. The also carried out using MINITAB Software. antifungal activity study was carried out by agar well diffusion method of Perez et al.12. Minimal inhibitory concentration (MIC) was determined by two fold Results microdilution method13. Two antibiotics viz. The results of the antimicrobial screening of the Neomycin (30µg/disc) and Norfloxacin (10µg/disc) solvent extracts of leaf and stem of H. fomes against for bacteria and antifungal agent, clotrimazole (100 ten pathogenic bacteria and three fungal strains are μg/ml) for fungus were used as standard. summarized in Table 1 and Figure 2 (antimicrobial); Total phenolic content (PA) was estimated Table 2 (MIC and MBC) and Table 3 (antifungal) according to the methods of Slinkard and Singleton14 respectively. Different extracts of leaf and stem of H. using catechol as standard phenolic compound. fomes was found to be active against all the Ascorbic acid content (ASA) was estimated following pathogenic bacteria tested except the aqueous stem the methods of Barros et al.15 with slight extract which was inactive against Bacillus brevis. modifications. Total antioxidant capacity (TAC) of Zone of inhibition of the H. fomes extract against plant extracts was determined by Prieto et al.16. The different pathogenic bacteria varied between 11 mm reducing power of solvent extracts was determined by to 22 mm (Table 1 and Figure 2). 706 INDIAN J. MAR. SCI., VOL. 44, NO. 5 MAY 2015

Table 1: Antibacterial activity of solvent extracts of leaf and stem of Heritiera fomes and standard antibiotics Leaf Stem Antibiotic Name Neomycin Norfloxacin Strains Acetone Ethanol Methanol Aqueous Acetone Ethanol Methanol Aqueous (30µg/disc) (10µg/disc) S. aureus 21a±1.4 18cd±0.49 18cd±0.21 16de±0.35 19no±1.4 20n±0.71 19no±0.21 18o±0.49 17± 0.70 33± 0.35 S. flexneri 19b±0.71 17d±0.49 14ef±0.21 15e±0.21 18o±0.21 19no±0.21 17op±0.49 15pq±0.21 18± 0.35 30± 1.4 B. licheniformis 18b±0.35 17d±0.49 15e±0.71 12f±0.49 15pq±0.49 17op±0.71 16p±0.35 17op±0.71 21± 1.4 30± 0.35 B. brevis 20ab±0.71 16de±0.21 17d±0.49 15e±0.71 14q±0.35 13q±0.49 13q±0.21 0±0 27± 0.35 33± 0.70 V. cholerae 21a±0.49 17d±0.71 15e±0.35 14ef±0.35 13q±0.49 14q±0.21 13q±0.35 14q±0.49 18± 0.70 36± 0.70 P. aeruginosa 21a±0.21 19c±0.71 17d±0.49 13f±0.49 18o±0.49 17op±0.71 16p±0.49 18o±0.49 18± 0.35 40± 0.35 S. epidermidis 19b±0.49 17d±0.49 16de±0.71 12f±0.49 16p±0.71 17o±0.21 15pq±0.71 22m±1.4 15± 0.07 35± 0.70 B. subtilis 15d±0.49 16de±0.49 14ef±0.21 13f±0.35 17op±0.49 16p±0.71 16p±0.49 18o±0.49 16± 1.4 36± 0.07 E. coli 18b±0.35 18cd±0.21 16d±0.49 13f±0.35 16p±0.21 18o±0.21 16p±0.21 16p±0.35 14± 0.07 35± 0.35

useful antimicrobial compounds as the MIC values ranged in between 1.25-5.0 mg/ml (Table 2). The results of the antifungal activity of H. fomes screened against three pathogenic fungi along with a standard antifungal agent (Clotrimazole, 100 µg/ml) are given in Table 3. Both the methanol and aqueous leaf extracts of H. fomes were active against the fungal strains (7 mm to 14 mm) (Table 3). However, the activity of the plant extract is comparatively less than that of the standard antifungal agent. Methanol extract of leaf of H. fomes showed significantly higher antifungal activity against C. kruzii with zone of inhibition of 14 mm, in comparison to other extracts and fungal strains at p<0.05 (LSD-0.506). Several antioxidant assays were performed on

different solvent extracts of H. fomes to study the Fig. 2— Antibacterial activity of extracts of H. fomes. antioxidant potential of the plant and its parts. The results of the seven antioxidant activity of all the The figure 2 showed the diameter of the zone of four solvent extracts of H. fomes mentioned above inhibition which confirms the antibacterial property of and the standard butylated hydroxyl toluene (BHT) the plant extract. Highest antibacterial activity in the were summarized in Table 4 and Figure-3A, B, C, D leaves was found in the acetone extract against S. and E. Higher DPPH radical scavenging activity were aureus with zone of inhibition of 21 mm, which is shown by the acetone leaf extract (88.35±0.71 %) and significantly different from other extracts and other the ethanol stem extract (91.59±0.12%) which pathogenic strains at p<0.05 (LSD-1.652). But in case increased with increase in concentration of the of the stem, the aqueous extract showed highest plant extract (Figure 3A). The DPPH free radical antibacterial activity against S. epidermidis with zone scavenging capacities of H. fomes showed of inhibition of 22 mm, which is significantly comparatively higher activity than BHT with dose different from other solvent extracts and other dependency. The acetone leaf extracts showed pathogenic strains at p<0.05 (LSD-1.076). significantly higher DPPH radical scavenging activity Minimum inhibitory concentration (MIC) values in all at p<0.05 (LSD-1.667) at 100 μg/ml concentration the solvent extracts of both leaf and stem of H. fomes followed by 75 μg/ml and 50 μg/ml concentration ranged from 1.25 mg/ml to 5.0 mg/ml (Table 2) and whereas the ethanol extract of the stem showed the minimum bactericidal concentration (MBC) significantly higher DPPH radical scavenging at 100 varied from 2.5 mg/ml upto values greater than 5.0 μg/ml concentration in comparison to other solvent mg/ml (Table 2). Results of the present study extracts at p<0.05 (LSD-0.680). revealed that the plant extracts would be potentially

PATRA AND THATOI: IN VITRO BIOACTIVE POTENTIAL OF MANGROVE PLANT, HERITIERA FOMES 707

Table 2: MIC and MBC of solvent extracts of leaf of H. fomes against nine bacterial pathogens Strains Acetone extract Ethanol extract Methanol extract Aqueous extract Leaf Stem Leaf Stem Leaf Stem Leaf Stem MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC S. aureus 2.5* 5 1.25 2.5 2.5 5 2.5 5 2.5 5 1.25 2.5 2.5 5 2.5 5 S. flexneri 2.5 5 2.5 5 5 >5 2.5 5 5 >5 2.5 5 2.5 5 5 5 B. licheniformis 5 >5 2.5 5 2.5 5 2.5 5 5 >5 2.5 5 >5 >5 2.5 >5 B. brevis 2.5 5 2.5 5 5 >5 5 >5 2.5 5 5 >5 2.5 5 - - V. cholerae 2.5 5 1.25 2.5 2.5 5 2.5 5 5 >5 5 >5 5 >5 5 >5 P. aeruginosa 2.5 5 1.25 2.5 2.5 5 1.25 2.5 2.5 5 2.5 5 5 >5 2.5 5 S. epidermidis 2.5 5 2.5 5 2.5 5 2.5 5 2.5 5 5 >5 >5 >5 1.25 2.5 B. subtilis 5 >5 5 >5 2.5 5 5 >5 5 >5 2.5 5 5 >5 2.5 5 E. coli 2.5 5 2.5 5 2.5 5 2.5 5 2.5 5 2.5 5 5 >5 2.5 5 *Values in (mg/ml) , “-” - Not detected, “>’- value is greater than

Table 3: Antifungal activity of solvent extracts of leaf and stem of H. fomes against three fungal strain Antifungal Strains Leaf Stem agent Clotrimazole Acetone Ethanol Methanol Aqueous Acetone Ethanol Methanol Aqueous (100 μg/ml) Candida kruzii 0±0 0±0 14d ±0.34 13a ±0.05 0±0 0±0 0±0 0±0 20±0.49 Fusarium sp. 0±0 0±0 8b ±0.05 9b ±0.34 0±0 0±0 9b ±0.21 0±0 17±0.21 Butyodiploda sp. 0±0 0±0 0±0 8b ±0.21 0±0 0±0 0±0 0±0 19±0.21

Table 4: Total phenol content, ascorbic acid content and total antioxidant capacity in different solvent extracts of H. fomes Leaf Stem Strains Acetone Ethanol Methanol Aqueous Acetone Ethanol Methanol Aqueous Total phenol content* 3.10±0.002 2.95±0.004 0.75±0.003 2.47±0.003 2.625±0.001 1.952±0.01 0.617±0.007 2.275±0.035 Ascorbic acid* 2.52±0.002 5.68±0.002 3.44±0.028 0.669±0.028 2.687±0.013 1.845±0.021 1.108±0.025 2.942±0.004 Total antioxidant 168.75±8.8 187.5±17.7 41.31±2.65 142.76±3.91 122.5±0.0 100±0.0 68.78±2.99 175.89±3.56 capacity* *All data expressed in % dry weight

Table 5a: Comparison between different antioxidant assays as represented by correlation coefficient (r value) leaf of H. fomes

TAC PC ASA DPPH NOS REDUCING METAL CHELETING TAC - 0.925 0.255 -0.477 -0.972 0.581 0.860 PC - 0.042 0.470 0.963 -0.837 -0.662 ASA - -0.601 -0.229 -0.368 0.306 DPPH - 0.611 -0.457 -0.099 NOS - -0.709 -0.737 REDUCING - 0.153 METAL CHELETING -

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Table 5b: Comparison between different antioxidant assays as represented by correlation coefficient (r value) bark of H. fomes METAL TAC(B) PC(B) ASA(B) DPPH(B) NOS(B) REDUCING(B) CHELETING(B) TAC(B) - -0.932 0.998 0.272 -0.995 0.187 -0.467 PC(B) - -0.920 -0.507 0.900 -0.265 0.267 ASA(B) - 0.218 -0.997 0.224 -0.446 DPPH(B) - -0.210 -0.395 -0.288 NOS(B) - -0.173 0.498 REDUCING(B) - 0.740 METAL - CHELETING(B)

Table 6: Screening of phytochemicals of leaf and Tests stem samples of different solventS extractsolvent e xoftr aH.ct Acetone fomesE thanol Methanol Aqueous Leaf Stem Leaf Stem Leaf Stem Leaf Stem /Bark /Bark /Bark /Bark Alkaloid + + + + + + + + Cardiac glycoside + + + + + + + + Anthraquinone glycoside + + + + + + + + Tannin + + + + + + + + Steroids - - + + + + + + Saponins + - - - - - + + Flavonoids + + + + + + + + Gums and mucilages ------+ + Carbohydrates + + + + + + + + Proteins and amino acids - + - + - + - + Terpinoid + + + - + - + +

Extracts of H. fomes showed strong nitric oxide than the standard BHT (Figure 3C). scavenging potential which increases with increase in The reducing capacity of the solvent extracts concentration of the sample (Figure 3B). The nitric increased with increasing concentration of the plant oxide scavenging potential of the standard BHT extract. Methanol leaf and ethanol stem extracts of studied at 50, 75 and 100 µg/ ml concentrations, this plant showed significantly higher reducing power showed activity of 34.33±1.05, 41.42±0.53, at 100 μg/ml concentration in comparison to other 55.22±1.06 % respectively which are comparatively solvent extracts and concentrations at p<0.05 (LSD- lower than that of plant extracts (Figure 3E). The 0.0229 and 0.0227) respectively (Figure 3D). Extracts acetone, ethanol and aqueous extracts of both leaf and of H. fomes showed higher amount of phenol stem of H. fomes showed significantly higher activity content (Table 4). The extracts of H. fomes are good at p<0.05 (LSD-0.960 and 1.11) than the methanol sources of ascorbic acids (Table 4) which proved that extracts respectively. Results showed significant plants are the rich source of ascorbic acid. metal chelating activity in different extracts and Total antioxidant capacities of the solvent extracts different concentrations at p<0.05 (LSD is 0.338 and are summarized in Table 4. The total antioxidant 0.351) for leaf and stem respectively. The metal capacity of H. fomes showed that many phenolic chelating activity of all the four extracts showed compounds contribute significantly to the total dose dependency which was comparatively higher antioxidant activity of medicinal plants. PATRA AND THATOI: IN VITRO BIOACTIVE POTENTIAL OF MANGROVE PLANT, HERITIERA FOMES 709

Figure 3B: Nitric oxide scavenging activity Figure 3A: DPPH free radical scavenging activity Difference in superscript letters indicate Difference in superscript letters indicate significance level at p < 0.05 sig nificance level at p < 0.05

Comparative antioxidant potential of the plant conditions such as violent environments, high extracts with the standard BHT revealed that the concentration of moisture, high and low tides of water extracts possess comparatively higher antioxidant etc. and thus are endowed with certain chemical potential than the standard BHT (Figure 3E). The compounds which can protect them from these biotic relationship between various antioxidant assays were and abiotic stress. H. fomes is reported to be used for correlated statistically and presented in Table 5A and treatment of various ailments. Phytochemicals from B and Figure 4A and B. this plant is expected to contain bioactive compounds All four extracts of both leaf and stem of H. fomes that can be used for medicinal purpose. Thus, the showed the presence of almost all the phytochemicals crude extracts of leaf and stem of H. fomes were such as alkaloid, cardiac glycoside, anthraquinone prepared by successive solvent extraction procedure glycoside, tannin, steroids, flavonoids, terpenoids, by using four different solvents for their bioactivity carbohydrates, protein and amino acids except evaluation. Percentage yield of different extracts of saponins and gums and mucilage which are not leaf and stem powders were determined which ranged detected in all the extracts (Table 6). The quantitative between 7.5±0.012 % to 17.4±0.065 % dry weight phytochemical analysis of the dry plant powder of H. (for leaf powder) and 1.095±0.005% to 2.55 ±0.008 % fomes also showed the presence of primary and dry weight (for stem powder) (data not shown). secondary metabolites as well as vitamins and Antimicrobial herbal medicines is often minerals (Table 7). considered important in preventing and managing wound infections and being natural in origin, they are Discussion ideal to use. Active components present in these Mangroves are exceptional among the herbal medicines usually interfere with growth and tropical forest types which exist under stressful metabolism of microorganisms and prevent them 710 INDIAN J. MAR. SCI., VOL. 44, NO. 5 MAY 2015

from contamination. According to Fabry et al.23 for crude solvent extracts of plants to be considered as potentially useful therapeutics, they must have MIC values <8mg/ml24. Antibacterial activity of the ethanol extract of bark of H. fomes from Sundarbans, West Bengal, was reported by Wangensteen et al.25. The present finding supports the ethnomedicinal use of this plant against diseases related to microbial infections such as skin diseases and gastrointestinal disorders. The use of traditional medicine as source of natural antioxidants is widespread and serves as leads for the development of novel drugs26. Mangrove plants are potent source of natural antioxidant as they survive in high environmental stress conditions. Due to the multifaceted aspects of antioxidants and their reactivity, several antioxidant assays were applied for evaluation of antioxidant properties of plants and their parts, since result of a single method can give only a reductive view of the 8 Figure 3C: Metal chelating activity antioxidant properties of the extracts . Wangensteen 25 Difference in superscript letters indicate et al. has also reported the DPPH scavenging significance level at p < 0.05 potential of the bark extracts of H. fomes collected from mangrove forests of the Sundarbans which corroborates with the present findings. Many researchers have reported positive correlation between free radical scavenging activity and total phenolic compound8 which is also corroborated with the present finding. The free radical scavenging capacity of compounds has been attributed to various mechanisms such as prevention of chain inhibition, chelating metals, reductive capacity and radical scavenging8. The positive chelating and nitric

oxide scavenging activity of the mangrove extracts might be due to the presence of secondary metabolites in the plant. H. fomes showed strong reducing power. Same trend has also been reported by Banerjee et al.26 in methanol extracts of higher plants. Antioxidant activity of both the parts of the plant might be due to the reduction of superoxide anion, inactivation of free radicals or complexion with metal ions or combination of the three. The reducing capacity of a compound may serve as indicator of its potential antioxidant capacity27. Antioxidant activity is system -dependent. Moreover, it depends on the method adopted6. Figure 3D: Reducing power Difference in superscript letters indicate significance level at p < 0.05

PATRA AND THATOI: IN VITRO BIOACTIVE POTENTIAL OF MANGROVE PLANT, HERITIERA FOMES 711

strong antioxidant properties as evidenced from the present results.

A

Figure 3E: Antioxidant activity of standard BHT

Phenolic extracts contribute directly to the antioxidative action of plant extracts because of their radical scavenging ability due to their hydroxyl groups. The studied plant, H. fomes and its parts are rich in phenols and polyphenolic compounds which is the indication of its strong antioxidant capacity. B Ascorbic acid is an important natural antioxidant present in plants. The results showed that there is a strong linear and positive correlation between the total antioxidant activity and phenol content (r=0.925), phenol content and nitric oxide scavenging (r=0.963), total antioxidant capacity and metal chelating (r=0.860) in the leaf extract whereas in case of the stem extracts, linear and positive correlation was found out in the total antioxidant capacity and ascorbic acid content (r=0.998), phenol content and nitric oxide scavenging (r=0.900), reducing and metal chelating

(r=0.740). Interestingly, when DPPH scavenging activity of the acetone leaf and stem extracts are Figure 4: Correlation curve between DPPH and plotted (regression analysis) against nitric oxide NOS (%age scavenging) of acetone extracts of H. fomes, A: leaf extract; B: stem/bark extract scavenging, a positive trend was observed, which implies that acetone extract of H. fomes might be playing an important role in protecting cells against reactive oxygen species. Table 7: Quantitative phytochemical contents of crude plant The differences in correlation coefficient among Ppowdersaramete ofrs* H. fomes Leaf Stem/Bark different antioxidant methods indicate that a single Primary metabolites assay or experiment cannot be used to assess the total antioxidant activity of any plant sample. Results from Carbohydrate 6.45±0.001 10.38±0.169 the antioxidant assays showed that all extracts might content act as radical scavengers to a certain extent. The Protein content 8.312±0.004 9.13±0.028 distinct scavenging activities of different extracts Secondary metabolites could be due to the diverse chemical nature of various Phenol 2. 47±0.007 2.25±0.056 phytochemicals that react with different types of free Flavonoid 1.88±0.18 1.48±0.09 28 radicals in unique way . Ethnomedicinal use of this Tannin 2.4±0.28 2.13±0.05 plant by the local healers against diseases like Alkaloid 1.88±0.14 1.26±0.07 diabetes and hepatic disorders are well validated by its Saponin 15.2±0.14 10.8±0.07 712 INDIAN J. MAR. SCI., VOL. 44, NO. 5 MAY 2015

Vitamins References Thiamine 0.184±0.002 0.203±0.001

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