Abstract
The present study describes chemical composition, antifungal activities, antioxidant assays and total phenolic content of essential oil and different extracts from leaves of Alpinia malaccensis. Sixty-eight components were identified by gas chromatography and gas chromatography–mass spectrometry with camphor (15.2%) and eucalyptol (10.2%) as the main component. The essential oil was found to possess remarkable fungicidal action against tested pathogens like Colletotricum falcatum, Rhizoctonia solani, Sclerotinia sclerotium and Sclerotium rolfsii followed by different extracts obtained in solvents like methanol, dichloromethane and hexane, respectively. The essential oil showed higher phenolic contents (69.51 μg/ml) than the extracts. The antioxidant assay of essential oil and extracts by different methods revealed good-to-moderate antioxidant potential with different IC50 values viz. (174.16–230.01 μg/ml) for Fe3+ reducing power, (111.74–158.23 μg/ml) for Fe2+ metal-chelating ability, (82.64–96.66 μg/ml) for DPPH, (94.28–189.41 μg/ml) for OH radical, (76.96–135.63 μg/ml), for NO radical and (107.30–199.75 μg/ml) in superoxide anion scavenging activities, respectively, compared to the standard antioxidants. Based on obtained results, the herb A. malaccensis can be a good source to develop a safe and sustainable natural antioxidant.
Public Interest Statement
The family Zingiberaceae, particularly known as Ginger family, is cosmopolitan in distribution. Herbs of this family are frequently used for various purposes like spices, dyes, perfumes, cosmetics, food flavour, ornamental, horticulture, etc. including traditional system of medicine. In the present scenario, there is an increasing demand for nutraceutical and natural food preservative to reduce the oxidative deterioration of food and food products over the synthetic compounds which may have devastating effect on human health and environment. The voluminous research on the Zingiberaceous plant is needed to explore its potential as antifungal and antioxidant agent so that it can be used for curing several diseases and for medicinal purposes. The present investigation reports the efficacy of A. malaccensis with antifungal and antioxidant potential so that this plant can be used as a safe and herbal food preservative because of its antifungal and antioxidant activity.
1. Introduction
Presently, the plants and plant-derived compounds are frequently employed in industries, food preservatives, pharmaceutical, cosmetics, perfumes, etc. because of their antifungal, antibacterial, antioxidant and bioregulatory properties (Ahmadi, Sadeghi, Modarresi, Abiri, & Mikaeli, Citation2010). They also possess a variety of phenolics, alkaloids and flavonoids that are often used to treat various diseases (Siddhuraju & Manian, Citation2007).
Synthetic compounds are used as antimicrobial agent in crop protection for the control of plant pathogen but have certain threats to environmental quality and human health (Tian et al., Citation2011). Antioxidants quench reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated in the body, thereby reducing oxidative stress (Al-Tawaha, Al-Karaki, & Massadeh, Citation2013). Though synthetic antioxidants like butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are employed in food products, they lead to liver damage (Valentão et al., Citation2002). Due to several limitations of synthetic compounds there is an urgent need for exploration and development of new, safe natural antifungal and antioxidants products (Pavela, Sajfrtová, Sovová, Bárnet, & Karban, Citation2010).
Zingiberaceae or the Ginger family is being explored worldwide due to its high medicinal value and versatile nature. It comprises 52 genera and about 1,587 species distributed throughout tropical Asia (The Plant List, Citation2013). India is rich in the diversity of Zingiberaceae and family being represented by 24 genera and 191 species (Karthikeyan, Citation2000). The zone of greatest concentration of this family is the north-east region of India comprising 19 genera and about 88 species (Prakash & Mehrotra, Citation1995).
A. malaccensis (Jangali adrak) is a perennial plant found abundantly and widely in the tropical and subtropical regions. It is a tall herb growing in forests whose rhizomes are fibrous in nature and cultivated as an ornamental plant (Nuntawong & Suksamrarn, Citation2008). Tuberpaste of A. malaccensis is applied as curative on sores (Abhyankar & Upadhyay, Citation2011). Fruits of this herb are applied on gastralgia (Bhuiyan, Chowdhury, Begum, & Nandi, Citation2010). There is no published report regarding the antioxidant activity and antifungal activity of the non polar extracts of Alpinia malaccensis leaves. In continuation to our research programme on Zingiberaceae, the present investigation was carried out to evaluate GC–MS analysis and evaluation of its antifungal and antioxidant activity. The total phenolic content of essential oils and extracts of leaves A. malaccensis were also evaluated.
2. Experimental
2.1. Collection of plant material
Fresh leaves of A. malaccensis were collected from the Tarai region of Kumaun hills in India. The plant was identified and authenticated by Dr D. S. Rawat (Plant taxonomist), Department of Biological Science, G. B. Pant University of Agriculture and Technology, Pantnagar, India. The voucher specimen has been deposited in the Department of Botany for future reference.
2.2. Extraction of essential oils
Fresh leaves (1 kg) were subjected to hydrodistillation in Clevenger’s apparatus for 8 h. Extraction of distillate with diethyl ether followed by drying over anhydrous Na2SO4 and removal of solvent yielded 0.08% of A. malaccensis leaves essential oil (AMLEO).
2.3. Preparation of the extracts
The leaves of A. malaccensis were cut into small pieces and shade-dried at room temperature. The material was then ground to fine powder. About 2 kg of the material was extracted through cold percolation method by successive soaking for 7 days each in different solvents of varying polarity like hexane, dichloromethane and methanol. The extracts were filtered using filter paper and muslin cloths. The filtrates so obtained were concentrated using a rotary evaporator. Yields of different extracts viz. A. malaccensis, leaves hexane extract (AMLHE), A. malaccensis leaves dichloromethane extract (AMLDE) and A. malaccensis, leaves methanol extract (AMLME) were found to be 0.82, 0.77 and 0.75%, respectively. The extracts were stored at 4°C for further analysis and evaluation of biological activities.
2.4. GC–FID analyses
Gas chromatographic analyses were carried using Thermoseries CERES 800 plus gas chromatograph with FID fitted detector using DB-5 capillary column (non-polar, 30 m × 0.32 mm id., 0.25 μm film thickness). Gas chromatography operation conditions: injection mode, split (40:1, v/v); injection volume1 μl, injector temperature: 220°C; detector temperature: 250°C; oven temperature programme: 60–246°C (3°C/min); carrier gas: helium (1.0 ml/min). Percentages of the individual components in the oil were obtained from the GC–FID peak area-% reports (Table ).
Table 1. Chemical composition of AMLEO
2.5. GC–MS analyses
Gas chromatography–mass spectrometry (GC–MS) analyses were carried out on Hewlett Packard 6890 gas chromatograph (Agilent Technologies, USA), fitted with an HP-5 (5% phenyl methylpolysiloxane, 30 m × 0.32 mm id., 0.25 μm film thickness) capillary column coupled with a model 5973 mass detector. GC–MS, injection mode, split (1:1 ratio, v/v); injection volume1 μl, injector temperature: 220°C; transfer line temperature: 240°C; oven temperature programme: 60–246°C (3°C/min); carrier gas: helium (1 ml/min); detector temperature: 250°C; mass spectra, electron impact (EI) mode, 70 eV; ion source temperature: 240°C. Individual components were identified by Wiley or NIST database comparison of KI values and mass spectra of constituents with Adams database (Adams, Citation2007) (Table ).
2.6. Antioxidant activity
The antioxidant activity of essential oil and extracts were studied using different methods.
2.6.1. DPPH radical scavenging activity
To evaluate scavenging ability of the antioxidants by this method, a standard protocol was followed (Sethi, Prakash, & Pant, Citation2015a). In brief, different amounts of the tested sample (50–250 μg/ml) were added to 5 ml of 0.004% methanol solution of DPPH. Finally, the absorbance was read against a blank at 515 nm after 30 min of incubation in the dark. All the observations were taken in triplicate. BHT, catechin and gallic acid were used as the standard antioxidant. Inhibition of free radical by DPPH in per cent (IC%) was calculated using the equation, IC% = (A0 − At/A0) × 100, where A0 and At are the absorbance values of the control and test sample, respectively. Per cent inhibition was plotted against concentrations and the equation for the line was used to obtain the IC50 value.
2.6.2. Hydroxyl radical scavenging activity
This activity was evaluated using the method described earlier (Olabinri et al., Citation2010). Sixty microlitres of FeSO4·7H2O (1 mM) was added to 90 μl of aqueous 1, 10 phenanthroline (1 mM), 2.4 ml of 0.2 M phosphate buffer (pH 7.8) was added to the above mixture, followed by addition of 150 μl of hydrogen peroxide (0.17 mM) and 1.5 ml of different concentrations of the sample (50–250 μg/ml). The absorbance of the mixture was read at 560 nm against blank after 5min. Ascorbic acid was used as standard. The % inhibition was calculated as, % hydroxyl radical scavenging capacity (IC%) = [(A0 − At)/A0] × 100, where A0 and At are the absorbance values of the control and the test sample, respectively. Lower IC50 value indicates greater hydroxyl radical scavenging ability.
2.6.3. NO radical scavenging activity
The nitric oxide scavenging activity of oil and extracts was determined using the previously reported method (Naskar et al., Citation2010). Two millilitres of sodium nitroprusside (SNP) (10 mM) in phosphate buffer saline (PBS) pH 7.4 were mixed with different concentration of sample (50–250 μg/ml) and incubated at 25°C for two and half hours. To the above sample, 1 ml of Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine dichloride and 2 ml orthophosphoric acid) was added. As a result pink colour was obtained and the absorbance was read at 546 nm. Ascorbic acid was used as standard. The % inhibition was calculated by the equation, % Nitric oxide scavenging capacity (IC%) = (A0 − At/A0) × 100, where A0 and At are the absorbance values of the control sample and the test sample, respectively. The per cent inhibition was plotted against concentration, and the equation for the line was used to obtain the IC50 value.
2.6.4. Superoxide anion scavenging activity
Superoxide anion scavenging activity of the oil and extracts was determined according to the reported method with slight modifications (Nishikimi, Rao, & Yagi, Citation1972). In brief, 1 ml of nitroblue tetrazolium (NBT) solution (100 μM of NBT in 100 mmol/l phosphate buffer, pH 7.4), 1 ml of NADH (468 μmol in 100 mM/l phosphate buffer, pH 7.4) solution and varying concentration of test samples (50–250 μg/ml) were mixed well. The reaction was started by the addition of 100 μl of Phenazine methosulfate solution (PMS) (60 mM of 100 mM/l phosphate buffer, pH 7.4). The reaction mixture was incubated at 30°C for 15 min. The absorbance was measured at 560 nm by spectrophotometer. All the readings were taken in triplicate and ascorbic acid was used as the standard. The % inhibition was calculated by the equation, % superoxide scavenging capacity (IC%) = (A0 − At/A0) × 100, where A0 and At are the absorbance values of the control sample and the test sample, respectively. Per cent inhibition was plotted against concentration, and the equation for the line was used to obtain the IC50 value.
2.6.5. Reducing power
The reducing power of essential oil and extracts was determined by the method reported earlier (Sethi et al., Citation2015a). Varying concentrations of tested sample (50–250 μg/ml) were mixed with 2.5 ml of the phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1% potassium ferricyanide K3Fe(CN)6. The mixtures were incubated at 50°C for 20 min. After incubation, 2.5 ml of 10% trichloroacetic acid was added to the mixtures, followed by centrifugation at 650 rpm for 10 min. The upper layer (5 ml) was mixed with 5 ml distilled H2O and 1 ml 0.1% ferric chloride and absorbance of the resulting solution was measured at 700 nm using spectrophotometer. All the readings were taken in triplicate and BHT (Butylated hydroxyl toluene), catechin and gallic acid were taken as the standard. The reducing power of samples was calculated by the following formula: RP (%) = (A0 − At) × 100, where A0 and At are the absorbance values of the control sample and the test sample, respectively. Per cent inhibition was plotted against concentration, and the equation for the line was used to obtain the RP50 value.
2.6.6. Metal chelating ability
The chelation of Fe2+ by essential oil and extracts was evaluated using the method developed earlier (Sethi et al., Citation2015a). Different concentrations of tested sample (50–250 μg/ml) were first mixed with 1 ml methanol and 3.7 ml deionized water. The resulting mixture was allowed to react with 0.1 ml of FeCl2 (2 mM) and 4.2 ml of ferrozine (5 mM) for 10 min at room temperature. The absorbance was measured at 562 nm. All the readings were taken in triplicate and EDTA (0.01 mM), citric acid (0.025 M) were taken as standard. The metal-chelating ability of the tested sample, expressed as percentage was calculated according to the formula IC (%) = [(A0 − At)/A0] × 100, where A0 and At are the absorbance values of the control sample and the test sample, respectively. The per cent of chelating ability was plotted against concentration, and the equation for the line was used to obtain the IC50 value.
2.7. Total phenols
The total phenolic content of oil and extracts was estimated by the method reported earlier (Singleton & Rossi, Citation1965). About 0.5 ml of the extract solutions was mixed with 1.0 ml of Folin–Ciocalteu reagent, 1.0 ml aqueous solution of 7% sodium carbonate and 5 ml distilled water. The reaction mixture was mixed thoroughly and was allowed to stand for 30 min. The absorbance was read at 765 nm. The same procedure was also adopted for the standard solutions of gallic acid. The calibration equation for gallic acid obtained was y = 0.011x + 0.031 (R2 = 0.998), where y is the absorbance and x is the concentration of gallic acid in μg/ml. All tests were carried out in triplicate and the obtained results were the mean values and the standard deviation.
2.8. Micro-organism
The pathogenic fungi viz. Colletotricum falcatum, Rhizoctonia solani, Sclerotinia sclerotium and Sclerotium rolfsii were maintained on potato dextrose agar (PDA).
2.9. Antifungal activity of AMLEO and extracts
The antifungal activity was determined using the disc diffusion method (Murray, Baron, Pfaller, Tenover, & Yolke, Citation1995). Initially, each sample was diluted with acetone: water (1:1) to obtain the final concentrations of 1,000, 500, 750, and 250 μg/ml, respectively. The pathogenic fungi were cultured on PDA media and incubated at 30°C for 3–4 days. A plug of one-week-old fungal culture (5 mm diameter) was placed on the centre of the sterilized plates containing PDA. About 10 μl of each concentration was injected to the sterile disc papers (6 mm diam.). Then the prepared discs were placed on the culture medium. Negative control was prepared using the same solvent employed to dissolve the oil and the extracts. The plates were then incubated at 30°C for 3–4 days in which fungal growth was monitored. The growth inhibition of each fungal strain was calculated as the percentage of inhibition of a radial growth relative to the control by the following equation:
Percentage of inhibition (%) = 100 × [1 − radial growth of treatment (mm)/radical growth of control (mm)]. All experiments were performed in triplicate.
3. Results and discussion
3.1. GC–MS of AMLEO
Sixty-nine components comprising over 81.0% were identified in AMLEO. The oil was dominated by camphor (15.2%), eucalyptol (10.2%), carotol (7.9%), methyl (E)-cinnamate (7.7%), α-terpineol (6.6%), borneol (2.8%), daucol (2.6%), bornyl acetate (2.2%), caryophyllene oxide (2.1%), widdrol (1.7%), β-eudesmol (1.5%), myrtenal (1.3%), fokienol (1.2%) and α-thujopsan-2-ol (1.2%), respectively (Table ). Among the identified components, 0.8% were monoterpene hydrocarbons, 44.5% oxygenated monoterpene. Among sesquiterpenoids 2% were hydrocarbons, and 21.5% were oxygenated sesquiterpenes, respectively. In the oil, 0.9% were oxygenated diterpenes and 11.3% aliphatic compounds could be identified.
Comparing the present investigation the report with Sahoo, Singh, and Nayak, (Citation2014) (21) from Southern part of India, it was observed that there was a vast chemical diversity in the qualitative and quantitative make-up of essential oil constituents in leaves of A. malaccensis. The oil from Southern part revealed the presence of α-phellandrene (43.9%), β-cymene (31.7%), β-pinene (4.6%), β-caryophyllene (3.3%), caryophyllene oxide (1.7%), α-pinene (1.5%) as the major compound. Muchtaridi et al. (Citation2014) (12) from Indonesia reported α-pinene (30.57%) as the major compound in oil. The analysis of oils reported from Indian habitat by us and Sahoo et al. (Citation2014) (21) were different in qualitative and quantitative make-up beside some common molecules in all the oils viz. methyl (E)-cinnamate, trans carveol, α-terpineol, pinocarvone, linalool oxide, linalool reported by us and Indonesian oils were missing in the oil reported from Southern India. In Indian oils, the compounds reported by us and missing in the oil reported by Sahoo et al. (Citation2014) (21) were carotol (7.9%), daucol (2.6%), β-eudesmol (1.5%), fokienol (1.2%), widdrol (1.7%), α-thujopsan-2-ol (1.2%), myrtenal (1.3%), borneol (2.8%), eucalyptol (10.2%), etc. Similarly, the compounds reported by Sahoo et al. (Citation2014) (21) and missing in our findings were δ-cadinene (0.8%), β-caryophyllene (3.3%), α-phellandrene (43.9%), β-cymene (31.7%), pinocarveol (2.2%) and α-selinene (0.7%). In other study Bhuiyan et al. (Citation2010) (5) from Bangladesh reported α-phellandrene (31.80%), eucalyptol (13.76%), o-cymene (11.45%), β-pinene (11.34%) and limonene (6.44%) in A. malaccensis leaves essential oil. From this study, it can be concluded that the essential oils from different origins have different molecular diversities in terms of terpenoids, in spite of having same morphological and taxonomical characters, hence are different chemotypes of A. malaccensis. This molecular diversity of chemotypes could be possibly due to the climatic, altitudinal and edaphic factors.
3.2. Antioxidant activity
AMLEO and the extracts exhibited strong to good DPPH radical scavenging activity (Table ). The radical scavenging potential in the form of their IC50 values revealed the order AMLEO (IC50 = 82.64 μg/ml) > AMLME (IC50 = 83.28 μg/ml) > AMLDE (IC50 = 86.39 μg/ml) > AMLHE (IC50 = 96.66 μg/ml). The order of IC50 for standard antioxidant was ascorbic acid (IC50 = 32.46 μg/ml) > BHT (IC50 = 33.81 μg/ml) > catechin (IC50 = 42.99 μg/ml) > gallic acid (IC50 = 49.27 μg/ml). DPPH exhibiting absorbance at 517 nm disappeared after accepting a hydrogen radical from an antioxidant compound (Matthäus, Citation2002). The antioxidant power of AMLEO and extracts might be attributed to their hydrogen-donating ability to DPPH free radical.
Table 2. Antioxidant potential in term of IC50 values for AMLEO and different extracts of A. malaccensis along with their total phenolic content
AMLEO and extracts scavenge OH free radical as function of amounts in a selected dose levels in comparison to the standard antioxidant ascorbic acid. The results obtained for their good antioxidant potential in terms of IC50 (μg/ml) values were 189.41, 162.22, 135.46 and 94.28, for AMLHE, AMLDE, AMLME and AMLEO, respectively. IC50 of ascorbic acid was obtained as 44.36 μg/ml.
Through their nitric oxide scavenging ability, AMLEO and all the extracts exhibited varying degrees of antioxidant potential as indicated by the IC50 values obtained from these samples. However, the order of nitric oxide scavenging activity was AMLEO (IC50 = 76.96 μg/ml) > AMLME (IC50 = 86.74 μg/ml) > AMLDE (IC50 = 108.44 μg/ml) > AMLHE (IC50 = 135.63 μg/ml), whereas the IC50 of ascorbic acid was 62.03 μg/ml lower than the samples.
AMLEO and different extracts were found to scavenge the superoxides with different rates in terms of different IC50 values. AMLHE, AMLDE, AMLME and AMLEO were able to scavenge the superoxide anion with an IC50 of 199.75, 173.87, 74.20 and 107.30 μg/ml, respectively. IC50 ascorbic acid, the standard antioxidant was obtained as 32.28 μg/ml.
Reducing power acts as an important indicator of testing antioxidant potential. In this assay, ferric ions (Fe3+) are reduced to ferrous ions (Fe2+) with change in colour whose intensity depends on the reducing potential of the compounds available in the reaction medium, subsequently, related to the antioxidant activity (Siddhuraju, Mohan, & Beaker, Citation2002). In the present study, the reducing activity of Fe3+ to Fe2+ to exhibit the antioxidant ability in terms of their RP50 values was obtained in the order of AMLEO (RP50 = 174.16 μg/ml) > AMLME (RP50 = 194.46 μg/ml) > AMLDE (RP50 = 225.17 μg/ml) > AMLHE (RP50 = 230.01 μg/ml). However the RP50 of the entire standard was obtained as BHT (RP50 = 117.88 μg/ml) > catechin (RP50 = 143.91 μg/ml) > gallic acid (RP50 = 151.47 μg/ml).
Fe2+ ion forms complex with ferrozine in the presence of competitor complexing agent the equilibrium between Fe2+ ion and ferrozine is disturbed and indicated by decrease in colour intensity, thus showing its antioxidant activity. The IC50 values for AMLEO and various extracts for their antioxidant potentiality in terms of chelating ability were obtained as AMLEO (IC50 = 111.74 μg/ml) > AMLME (IC50 = 110.37 μg/ml) > AMLDE (IC50 = 144.77 μg/ml) > AMLHE (IC50 = 158.23 μg/ml). However, the IC50 for standard viz. EDTA and citric acid was obtained IC50 = 47.72 and 61.76 μg/ml, respectively. The IC50 values of AMLEO and all the extracts assayed for the antioxidant activity by various methods along with their phenolic content are reported in Table .
3.3. Total phenols
The total phenols in AMLEO and all the extracts were obtained in the range of 31.51–69.51 μg/ml. The highest phenolic content was observed in AMLEO the followed by AMLME, AMLDE and AMLHE. The antioxidant activity of AMLEO and different extracts might be possibly due to the presence of phenols in them. A direct correlation has been observed between phenols and antioxidant activity (Sethi et al., Citation2015a). The total phenols were correlated with IC50/RP50 values of different methods used for the determination of antioxidant assay in essential oil and extracts. The results obtained showed negative correlation with DPPH radical scavenging, metal-chelating ability, superoxide anion scavenging activity, ·OH radical scavenging activity and nitric oxide radical scavenging activity and at α = 0.01 or 0.05 (level of significance) (Table )
Table 3. Correlation of total phenols with IC50 values
3.4. Antifungal activity of AMLEO and extracts
Antifungal activity of AMLEO and all the extracts exhibited inhibitory effect on the growth of all fungi in a dose-dependent manner (Table ). S. rolfsii was most suppressed as its growth was mostly reduced by all tested doses followed by S. sclerotium, R. solani and C. falcatum. AMLEO exhibited strong inhibitory effect against fungus R. solani (77.50%), S. sclerotium (83.33%) and S. rolfsii (90.83%) even at a lower concentration of 250 μg/ml. However, it could not suppress the C. falcatum at this concentration.
Table 4. Antifungal activity of AMLEO and different extracts of A. malaccensis
Almost all the extracts and AMLEO were fungicidal (100% inhibition) towards C. falcatum and R. solani at a concentration of 750 μg/ml or 1,000 μg/ml. AMLME exhibited strongest fungicidal action by completely suppressing all the fungus at a concentration of 1,000 μg/ml. At the same concentration, AMLEO and AMLDE exhibited strong antifungal activity against all the plant pathogenic fungi by suppressing them completely. However, the suppression of AMLEO towards C. falcatum was found to be weak (43.33%). AMLHE exhibited moderate antifungal activity against all the plant pathogenic fungi ranging from 80.83 to 100%. The present study indicated that AMLME, AMLDE and AMLEO were found to be the significant, effective and remarkable fungicides in this study in accordance to their inhibition action against all tested pathogenic fungi followed by other extracts.
The fungal toxicity of AMLEO might be possibly due to the presence of mono and sesquiterpenoids or might also be due to the synergistic and antagonist effects of the compounds (Cakir, Kordali, Zengin, Izumi, & Hirata, Citation2004; Deba, Xuan, Yasuda, & Tawata, Citation2008). Camphor (15.2%), the major constituent beside eucalyptol, α-terpineol, methyl (E)-cinnamate, and carotol in AMLEO might be responsible for the antifungal activity of this plant, as the antifungal activity of these compounds have already been reported (Carson & Riley, Citation1995; Gupta & Saxena, Citation2010; Huang et al., Citation2009; Misiak et al., Citation2004; Morcia, Malnati, & Terzi, Citation2012). The antifungal activity of extracts might be due to the presence of diverse group of phytoconstituents. The properties and antifungal action of flavonoids, phenolics have been reported earlier (Orhan, Özçelik, Özgen, & Ergun, Citation2010; Winkelhausen, Pospiech, & Laufenberg, Citation2005). In our previous investigation, we have reported the antifungal activity of AMLEO (Sethi, Prakash, & Pant, Citation2015b). The fungicidal action of AMLME might be due to high polarity of methanol which can extract polar compounds, thus inhibiting the fungus. Thus, it can be concluded that AMLEO and AMLME of this traditional herb can be developed as alternative natural fungicides, thereby replacing synthetic fungicide after proper screening. However, further clinical studies are needed to obtain proper information regarding the practical effectiveness.
Funding
The work was supported by the DST INSPIRE [grant number IF-10376] and University Grants Commission (UGC) [grant number 41-234/2012(SR)], New Delhi.
Supplementary material
The supplementary material for this paper is available online at http://dx.doi.org/10.1080/23312009.2016.1223781.
Supplementary_material.doc
Download MS Word (84.2 MB)Acknowledgement
Authors are thankful to DST-INSPIRE and University grant commission (UGC), New Delhi, Govt. of India for financial assistance. JNU, New Delhi is highly acknowledged for providing GC-MS facility. The antifungal activity was carried out at the Department of Pathology, G. B. Pant University of Agriculture and Technology, Pantnagar, India.
Additional information
Notes on contributors
Sonali Sethi
Sonali Sethi, the first author, obtained her PhD from G. B. Pant University of Agriculture and Technology, Pantnagar in 2015. Her research focuses on the plants of family Zingiberaceae for several biological activities. She had pursued her PhD under the supervision of Om Prakash.
Om Prakash
Prakash has 16 years of experience in the field of natural product and he has handled many national projects funded by University Grants Commission and DST, New Delhi. His work in the field can be evidenced by many published national and international papers in referred journals. His current research interest focuses mainly on analysis of several natural compounds by chromatographic techniques such as GC and HPLC, mass spectrometry, FTIR, NMR, and determination of various biological activities. The present study deals with chemical analysis and biological activity of Alpinia malaccensis leaves. The study will be helpful in the preparation of database, so that the herb can be explored scientifically and judiciously.
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