Morphology, Phenolic Content, and Antioxidant Activity of Etlingera fimbriobracteata (K.Schum.) R.M.Sm.
and E. philippinensis (Ridl.) R.M.Sm. (Zingiberaceae)
Noe P. Mendez1*, Rainear A. Mendez2, Angie Rose Villafranca-Tuba3, Romeo M. Tubongbanua Jr.1, and Florfe M. Acma1,4
1Department of Biology, College of Arts and Sciences,Central Mindanao University, University Town, Maramag, 8714 Bukidnon, Philippines
2Soil and Plant Analysis Laboratory, College of Agriculture,
Central Mindanao University, University Town, Maramag, 8714 Bukidnon, Philippines
3Natural Science Research Center, Central Mindanao University, University Town, Maramag, 8714 Bukidnon, Philippines
4Center for Biodiversity Research and Extension in Mindanao (CEBREM), Central Mindanao University, University Town,
Maramag 8714 Bukidnon, Philippines
Two of the most studied Etlingera species of the ginger family (Zingiberaceae) in the Philippines were Etlingera fimbriobracteata (K.Schum.) R.M.Sm. and E. philippinensis (Ridl.) R.M.Sm.
However, little is known about the biochemical analyses of these species to support their potential as ethnomedicinal plants in the country. Thus, this study was carried out to describe the morphology and determine the phenolic content and antioxidant activity of the ethanolic extracts of the two Etlingera species. Data revealed that the two species are correctly identified and their full description, updated distribution, phenology, and habitat and ecology are provided in this paper. The two species are recognizable in both vegetative and reproductive parts. E.
fimbriobracteata has reddish color towards the base of the pseudo stem, bright yellow flowers incurved upon maturity, and green sterile bracts that form reddish upon maturity, whereas E. philippinensis has mandarin to deep red inflorescence and has a long outward labellum.
The total phenolic content (TPC) expressed as milligram gallic acid equivalent per gram dried sample (mg GAE/g dried sample) revealed the highest phenolics in the leaves (13.20 ± 0.35 in E. fimbriobracteata and 7.21 ± 0.33 in E. philippinensis) than rhizomes (1.44 ± 0.04 in E.
fimbriobracteata and 0.46 ± 0.30 in E. philippinensis). Further, total antioxidant activity (TAA) expressed as milligram ascorbic acid equivalent per gram dried sample (mg AAE/g dried sample) was also observed highest in leaves (12.69 ± 0.36 in E. fimbriobracteata and 7.22 ± 0.26 in E. philippinensis) compared to the rhizomes (1.82 ± 0.01 in E. fimbriobracteata and 1.38 ± 0.07 in E. philippinensis). The reducing power (RP) expressed as milligram gallic RP equivalent per gram dried sample (mg GPRE/g dried sample) also revealed higher for the leaves (10.16 ± 2.18 in E. fimbriobracteata and 7.53 ± 0.80 in E. philippinensis) than rhizomes (0.97 ± 0.18 in E.
fimbriobracteata and 0.09 ± 0.09 in E. philippinensis). The high contents of phenolic compounds
*Corresponding author: [email protected]
ISSN 0031 - 7683
Date Received: 30 Oct 2022
contribute to the antioxidant activity of extracts from the two species. Based on the correlation analysis, a perfect positive linear relationship was observed among the TPC, TAA, and RP (r
= 1, p < 0.001). These imply that E. fimbriobracteata and E. philippinensis could potentially be used as new sources of natural antioxidants.
Keywords: Etlingera, Morphology, Philippine endemic species, reducing power, total antioxidant activity, total phenolic content
INTRODUCTION
Etlingera fimbriobracteata (K.Schum.) R.M.Sm. was formerly known as E. pandanicarpa (Elmer) A.D.Poulsen in the Philippines. However, the possibility that E.
pandanicarpa is a synonym of E. fimbriobracteata was raised by Poulsen (2006) revising the genus in Borneo and was confirmed by the study of Poulsen and Docot (2018).
Since then, the accepted name was E. fimbriobracteata following the principle of priority. The inflorescences of E. fimbriobracteata arise from its rhizomes near the base and are embedded in the soil with numerous yellow flowers (Shahid-Ud-Daula et al. 2019). On the other hand, Etlingera philippinensis (Ridl.) R.M.Sm. is a Philippine endemic ginger species and was formerly known as Amomum philippinensis and Hornstedtia philippinensis in 1915 and 1925, respectively.
The essential oils of E. fimbriobracteata were observed from the leaves, aerial stems, basal stems, and rhizomes of E. fimbriobracteata by Ud-Daulaal et al. (2016). The bioactive potential of its methanolic extracts from the leaves, stems, and rhizomes was also investigated by Shahid-Ud-Daula et al. (2019). The pollen materials of E. philippinensis were studied by Mendez et al. (2017) and Acma and Mendez (2018a) by determining the pollen morphology, pollen viability and tube growth, and elemental composition. The phytochemical screening and antioxidant activity of E. philippinensis were done by Barbosa et al. (2016) and gave positive results. In addition, Mabini and Barbosa (2018) conducted a study on the total phenolic content (TPC) and antioxidant activity of the methanolic extracts of Etlingera philippinensis. To the extent of the authors’ knowledge, these are the only laboratory analyses of these species in the Philippines and no studies were conducted on ethanolic extracts of these species using Philippine materials.
This study was carried out to describe the morphology and determine the phenolic content and antioxidant activity of E. fimbriobracteata and E. philippinensis, which are reported by Mendez et al. (2017) and Acma et al. (2020) as alternative medicines used by local people in Mindanao.
The leaves and rhizomes were studied, as these plant parts were used by the indigenous people. Thus, this study is noteworthy since this is the first report of these
species using ethanolic extracts of leaves and rhizomes, which could be potentially used as new sources of natural antioxidants. The local people in Mindanao will benefit from the information gathered in this study and the other researchers will be given insights to expand studies on these species to create products for application, such as for food, tea, condiments, and medicines.
MATERIAL AND METHODS
Entry Protocol
Prior to the conduct of the study, prior informed consent and communication letter were personally submitted by NPM at the office of the Barangay Captain of Datu Salumay in Marilog District to collect samples from Mt.
Malambo. A gratuitous permit with WGP number XI2017- 03 issued by the Department of Environment and Natural Resources (DENR) – Region XI on March 2017 to Dr.
Victor B. Amoroso of the Center for Biodiversity Research and Extension in Mindanao (CEBREM) was used as the collection permit in this study.
Place and Duration of the Study
The leaf and rhizome samples together with the voucher specimens were scrupulously collected from Mt. Malambo in Marilog District, Davao City. These samples were transported to Central Mindanao University (CMU), province of Bukidnon for processing. The determination of Total Phenolic Content (TPC), total antioxidant activity (TAA), and reducing power (RP) of dry-weight leaves and rhizomes of E. fimbriobracteata and E. philippinensis was conducted at the Natural Science Laboratory of Natural Science Research Center (NSRC) in CMU from September 2021–January 2022 after necessary permits were obtained from the concerned laboratory authorities.
Collection, Measurement, and Description of Plant Materials
The vegetative and reproductive parts of the two species were collected for voucher purposes. The vegetative materials were freshly collected from the wild using
clippers and bolo. These materials were measured, described and documented, and placed inside the plastic cellophane bags. Wet tissue papers were also added inside the cellophane bags to prevent dehydration. The reproductive materials were also collected, described, and photographed. The floral dissection was made in the field to describe and take photographs of the detailed parts since ginger flowers are ephemeral. Then after, the dissected parts were placed inside small containers for further morphological examination. All specimens were transported to CMU for further processing.
Using the materials placed inside the small containers, a stereo microscope at the NSRC Plant Tissue and Spore Culture Laboratory was used to view the dissected parts for description. After the microscopic examination, these floral parts were placed inside the prepared pickled collection and preserved in 80% ethanol as a pickled collection along with its inflorescence inside. The herbarium specimens were also dried and processed.
Herbarium labels were attached along with the specimens, including the pickled collection. These specimens were then deposited at the CMU Herbarium.
Sample Preparation and Extraction
The leaves and rhizomes of two species, which were collected and placed separately inside labeled plastic bags, were processed at CMU. Leaf and rhizome samples were washed and the earthy matters were removed prior to air- drying. Drying was done on the samples since this process removes water from the materials to produce a dry product.
Dried samples were then powdered and placed inside zip- lock cellophane bags. These samples were weighed, labeled with substantial information, and stored until used.
Extracts were prepared following the method of Padda and Picha (2008) with some minor modifications. The dried leaf and rhizome powder were extracted with absolute ethanol with a ratio of 1 g: 25.0 mL at room temperature (25 °C). The mixtures were shaken for 1 h in an orbital shaker at 300 rpm and centrifuged for 5 min at 5,000 rpm. The supernatants were collected in separate 15 mL conical tubes. Extracts were stored at 2–8 °C and used in the succeeding analyses. The ethanolic leaf and rhizome extracts of the two species were subjected to the TPC, TAA, and RP determination in 96-well plate format colorimetric assays. Samples were analyzed in triplicates with three trials for each replicate.
Total Phenolic Content (TPC)
The TPC of the extracts was determined using the method described by Ainsworth and Gillespie (2007) with some modifications. Briefly, 200 µL of the extracts and 200 µL of 10% Folin-Ciocalteu reagent were transferred in a 2-mL
centrifuge tube. The reaction mixture was set aside for 5 min and added with 800 µL of 10% sodium carbonate.
The mixture was set aside at room temperature (25 °C) for 30 min and centrifuged at 11,000 rpm for three min.
A 200 µL of the resulting solution was then transferred to the assigned microplate wells. The absorbance was determined at 750 nm using a microplate reader (Molecular Devices Spectramax® 250). Likewise, the same method was done to prepare a standard calibration curve by using 200 µL of standard solutions with a concentration range of 0–100 ppm gallic acid (GA) in 10-ppm increments from a stock solution of 100 ppm GA in absolute ethanol. The TPC was determined and expressed as milligram gallic acid equivalent per gram dried sample (mg GAE/g dried sample) by interpolating sample absorbance against the standard calibration curve using the formula below:
(1) where: A = gallic acid concentration of the sample solution determined from the calibration curve (mg GAE/L)
B = concentration of test solution (g/L, gram dried sample per L solution)
Total Antioxidant Activity (TAA)
The TAA was determined using the phosphomolybdenum method of Prieto et al. (1999) with slight modifications.
Briefly, 50 µL of the extracts were placed in centrifuge tubes and diluted with 200 µL (1:1 ethanol: water). The solution was added with 600 µL of reagent solution (prepared by mixing equal amounts of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate) and incubated at 95 °C for 90 min. The samples were drawn and allowed to cool at room temperature (25
°C) and centrifuged for another three min at 11,000 rpm.
The absorbance of the supernatant was measured at 695 nm against a blank using a microplate reader (Molecular Devices Spectramax® 250). Also, the same method was done to prepare a standard calibration curve by using 200 µL of standard solutions with a concentration range of 0–150 ppm ascorbic acid (AA) in 15-ppm increments from a stock solution of 300 ppm AA in absolute ethanol.
TAA was determined by interpolating sample absorbance against the standard calibration curve. The TAA was calculated using the equation as follows:
(2) where: A = ascorbic acid concentration of the solution determined from the calibration curve (mg AAE/L)
B = concentration of the test solution (g/L, gram dried sample per L solution)
Reducing Power (RP)
The RP was determined by adapting the method described by Murugan and Iyer (2011) with minimal modifications.
In a centrifuge tube containing 1 mL of the extracts, 200 µL of 0.2 M phosphate buffer (pH 6.6) and 200 µL of 1% (w/v) solution of potassium ferricyanide were added.
Subsequently, the mixture was incubated at 50 °C for 30 min. After cooling to room temperature (25 °C), a 200 µL of 1% (w/v) trichloroacetic acid was added. The mixture was centrifuged for three min at 11,000 rpm. An aliquot of 200 µL of the supernatant was transferred to a 96-well plate, and 20 µL of 1% (w/v) solution of ferric chloride was added. The absorbance was measured at 620 nm using a microplate spectrophotometer (Molecular Devices Spectramax® 250). Likewise, the same method was done to prepare a standard calibration curve by using 1000 µL of standard solutions with a concentration range of 0–100 ppm GA in 10-ppm increments from a stock solution of 100 ppm GA in absolute ethanol. The sample concentration was determined by interpolating sample absorbance against the standard curve. The RP was expressed as milligram gallic acid RP equivalent per gram dried sample (mg GRPE/g dried sample) and calculated as follows:
(3) where: A = gallic acid concentration of the test solution determined from the calibration curve (mg GRPE/L)
B = concentration of the test solution (g/L, gram dried sample per L solution)
Statistical Analysis
The TPC, TAA, and RP analyses were done in three replicates, and the determination for each assay was carried out in three trials per replicate. The data gathered among the TPC, TAA, and RP of the two species were correlated using Pearson’s correlation at a 0.001 level of significance. The results of absorption measurement were put into Microsoft Excel to obtain a calibration curve of standard gallic acid solution (TPC and RP) and ascorbic acid solution (TAA) in the form of a graph of concentration versus absorption curve. All results were expressed as mean values ± SD (standard deviation).
RESULTS AND DISCUSSION
Gross Morphology of Etlingera fimbriobracteata (K.Schum.) R.M.Sm. and E. philippinensis (Ridl.) R.M.Sm. Etlingera fimbriobracteata (K.Schum.) R.M.Sm. (Figure 1).
Plant description. Terrestrial herb, 2.5–3 m tall. Rhizome submerged in the soil, 3–6 cm in diam., creamy white to reddish, extending to 20 cm in the ground. Leafy shoots 1.8–2.1 m long, 7–10 cm apart; base swollen, 4–7 cm in diam., red to brownish, glabrous to pubescent; leaf sheath pale green, glabrous. Ligule entire, oblong, 14–18 mm × 4–7 mm, brown, glabrous. Petiole 4–7 mm × 2–3 mm, greenish to brown. Lamina narrowly ovate to lanceolate, 47–65 cm × 12–25 cm, adaxially green, glabrous; midrib adaxially yellowish to brownish, ridged, glabrous; margin entire, brown, glabrous; base rounded with fine hairs; apex acuminate, recurved. Flowering shoot radical, 11.5–11.9 cm
× 4.5–5.3 cm, lateral to ascending from base of leafy shoot;
peduncle 4.3–4.7 cm × 3.9–4.2 cm, pinkish when young, red when mature. Inflorescence is obconic, measuring 8–10 cm
× 6–8 cm, 20–26 flowers in one inflorescence, 7–10 flowers open at a time during anthesis or when an inflorescence is
Figure 1. Etlingera fimbriobracteata (K.Schum.) R.M.Sm.: [A]
habit; [B] leafy shoot (inset: ligule); [C] floral parts (infl – inflorescence; fl – flower; sb – sterile bracts; fb – fertile bracts; bt – bracteole; cx – calyx; cl – corolla lobes;
lb – labellum; as – anther sacs; st – style, sg – stigma, eg – epigynous gland). Scale bars = 2 cm. Photographs:
N.P. Mendez.
in full bloom which forms a truncate top. Bracts oblong, apex notched and hairy, pink towards the top and white towards the bottom, larger ones measuring 4 cm × 1 cm.
Bracteoles tubular, hairy at the tip, pink towards the upper portion while white at the base, 3 cm long. Calyx elongated, fused, tubular, 3-tipped, pinkish, 3.5 mm × 0.3 mm. Flower 4.3–5.5 cm long × 2–2.3 cm wide. Corolla tube white in color. Corolla lobes oblanceolate, 3 cm × 1 cm, yellow;
dorsal corolla lobe 20–23 mm × 4–6 mm, bright yellow, apex rounded to calculate; lateral corolla lobes 20–22 mm × 4–5 mm, bright yellow, apex rounded to calculate; staminal tube 7–8 mm, creamy white, glabrous. Labellum obovate, 12–14 mm × 7–9 mm, lateral lobes folded over the stamen, reaching 5–6 mm longer than apex of corolla lobes, bright yellow, glabrous, margin entire, apex rounded. Stamen 4.9–5.1 cm long; anther 6–7 mm × 2–3 mm, creamy yellow, with fine hairs, thecae dehiscent almost the entire length c.
3–4 mm; filament 5 cm × 0.7–0.9 cm, white, glabrous. Pistil 5.9–6.1 cm × 0.1 cm; stigma club-shaped, 2 mm × 2 mm, white, hairy on top; ostiole 1 mm, pubescent; style 5.3–5.5 cm × 0.1 cm wide, white; ovary 1–2 mm × 0.4–0.5 mm.
Infructescence clumped, 9–12.5 cm × 8–10 cm, brownish to black, grooved; fruits elongated, 3–4 cm in diam., calyx absent, brownish to black, grooved; seeds black, 0.3–0.4 mm across including the aril.
Etlingera philippinensis (Ridl.) R.M.Sm. (Figure 2) Plant description. Terrestrial herb, 2–2.5 m tall. Rhizome long-creeping, 1.5–2.5 cm in diam., pinkish to red, creeping up to 35–43 cm. Leafy shoots 1.9–2.2 cm long, 6–9 cm apart; base swollen, 4.5–4.9 cm in diam., reddish to brown, glabrous; leaf sheath green with violet marks, grooved. Ligule entire, oblong, 12–14 mm × 3–6 mm, green to brownish, with purplish color, glabrous.
Petiole 3–6 mm × 1–2 mm, greenish to brown. Lamina narrowly lanceolate, 32–41 cm × 9–11 cm, adaxially green, glabrous, abaxially paler green, glabrous; midrib adaxially yellowish to brown, ridged, glabrous; margin entire, green to yellow; base oblique; apex acuminate, recurved. Flowering shoot radical, 8.5–9.4 cm × 4.5–5 cm, lateral from base of the leafy shoot; peduncle 3.5–4.4 cm × 1.3–1.5 cm, pale red when young, deep red when mature. Inflorescence cone-shaped, 9 × 2 cm, 5–7 flowers in one inflorescence with 1–2 flowers open at a time during anthesis. Bracts elliptic-lanceolate, reddish but white at base, 3.5–3.8 cm × 2.3–2.9 cm. Bracteoles tubular, about 3.5 cm long. Calyx elongated, fused, tubular, 3-tipped, red in color except basal part which is white, 5 cm long. Flower 5–7 cm long × 4–6 cm wide white.
Lobes lanceolate, measuring 3.7 cm × 0.7 cm, bright red in color; dorsal corolla lobe 35–39 mm × 6–9 mm, bright red, apex rounded, entire; lateral corolla lobes 34–37 mm
× 5–8 mm, bright red, apex rounded, entire; staminal tube 3–4 cm, creamy white, glabrous. Labellum bright red or
scarlet, ovate, 2.5 cm × 1 cm. Stamen 6.5–7.6 cm long;
anther 4–6 mm × 2–3 mm, creamy yellow, with fine hairs, thecae dehiscent almost the entire length c. 2–3 mm; filament 5.2–5.4 cm × 0.7–0.9 cm, white, glabrous, slightly pubescent at the middle part. Pistil 6.7–7.3 cm × 0.1 cm; stigma triangular-shaped, 2 mm × 1.5 mm, white, hairy on top; ostiole 1 mm, pubescent; style 5.7–6.1 cm
× 0.1 cm wide, white; ovary 1–2 mm × 0.3–0.4 mm.
Infructescence not observed.
Total Phenolic Content (TPC)
The results of the TPC were derived from a calibration curve (y = 0.0546 x 0.0658, R2 = 0.9946) of gallic acid (0–200 mg/mL). In the absorbance measurement which is indicated by the correlation coefficients ® of 0.9946, this val®® is close to 1 which indicated that the regression equation is linear and would be used to determine the TPC of the ethanolic leaf and rhizome extracts of E.
fimbriobracteata and E. philippinensis. The ethanolic extracts of the two Etlingera species revealed that the leaves (13.20 ± 0.35 in E. fimbriobracteata and 7.21 ± 0.33 in E. philippinensis) had greater amounts of phenolics than the rhizomes (1.44 ± 0.04 in E. fimbriobracteata and 0.46 ± 0.30 in E. philippinensis) (Table 1).
Figure 2. Etlingera philippinensis (Ridl.) R.M.Sm.: [A] habit;
[B] leafy shoot (inset: ligule); [C] floral parts (infl – inflorescence; fl – flower; sb – sterile bract; fb – fertile bract; bt – bracteole; cx – calyx, cl – corolla lobes; lb – labellum; as – anther sacs; st – style, sg – stigma). Scale bars = 2 cm. Photographs: N.P. Mendez.
The mean value of the TPC in leaves of E. fimbriobracteata (13.20 ± 0.35 mg GAE/g sample) in this study is relatively higher than the studies of Mabini and Barbosa (2018) with 0.55 mg GAE/g sample on methanolic extracts of Etlingera philippinensis (Mabini and Barbosa 2018), 1.67 mg GAE/g sample on methanolic extracts of Hornstedtia conoidea Ridl (Barbosa and Nueva 2019), and 7.21 mg GAE/g sample of E. philippinensis (present study). In terms of E. philippinensis leaves (7.21 ± 0.33 mg GAE/g sample), it is also higher than E. philippinensis (Mabini and Barbosa 2018) and H. conoidea (Nueva and Barbosa 2019) but lower in E. fimbriobracteata (present study).
The mean value of TPC in E. fimbriobracteata rhizomes (1.44 ± 0.04 mg GAE/g sample) is higher than E.
philippinensis with 0.35 mg GAE/g sample (Mabini and Barbosa 2018), H. conoidea with 1.28 mg GAE/g sample (Barbosa and Nueva 2019), and E. philippinensis (present study). The TPC of E. philippinensis rhizomes (0.46 ± 0.30 mg GAE/g sample) is higher on E. philippinensis (Mabini and Barbosa 2018), but lower on H. conoidea (Barbosa and Nueva 2019) and E. fimbriobracteata (present study). These slight variations in the mean values of TPC might be due to the different amounts of sugars, ascorbic acid, duration, or methods of extraction that may change the amount of phenolics (Burri et al. 2017).
The total quantity of phenolic compounds present in the samples is proportional to intensity of blue color produced (Abdel-Hameed 2009).
Phenolic compounds are important plant constituents with redox properties responsible for antioxidant activity (Soobrattee et al. 2005). The total phenolic compounds also play an effective role in stabilizing lipid peroxidation (Yen et al. 1993). These phenolic compounds contribute to antioxidant activity due to the arrangement of functional groups (hydroxyl) about its nuclear structure for hydrogen donation in order to stabilize radical molecules (Soobrattee et al. 2008; Alam et al. 2018). In previous studies, phenolic compounds were known to have various biological effects as antioxidants, protecting cell structures, anti- inflammatory, antiseptic (Prihardini and Wiyono 2015), antimutagenic, antibacterial properties, and these activities might be related to the antioxidant activity (Shui and Leong 2002).
The phenolic compounds have recently received considerable attention because of their physiological functions, including antioxidant and free-radical scavenging abilities that affected the quality and nutritional value (Govindarajan et al. 2007). The TPC can be used to conduct rapid screening of antioxidant activity (Baba and Malik 2015). The quantification of TPC in ethanolic leaf and rhizome extracts of the two species employing the Folin-Ciocalteu method, is convenient, simple, and reproducible (Cirillo and Lemma 2012; Danciu et al. 2015). This method involves electron transfer in an alkaline medium from phenolic compound to phosphomolybdic/phosphotungstic acid complexes to form blue complexes that are determined spectroscopically (Singleton et al. 1999).
Total Antioxidant Activity (TAA) and Reducing Power (RP)
The results for the TAA of the ethanolic extracts of leaves and rhizomes of the two species were calculated from a calibration graph, which was linear over the calibration range with an r2 value of 0.9963 (y = 0.0149 x 0.0465) of L-ascorbic acid (0–100 mg/mL). In the absorbance measurement, which is indicated by the correlation coefficients ® of 0.9963, this val®(r) is close to 1, which indicated that the regression equation is linear and would be used to determine the TAA of the ethanolic leaf and rhizome extracts of E. fimbriobracteata and E.
philippinensis. The ethanolic extracts revealed that the leaves (12.69 ± 0.36 in E. fimbriobracteata and 7.22
± 0.26 in E. philippinensis) had a higher number of antioxidants compared to the rhizomes (1.82 ± 0.01 in E.
fimbriobracteata and 1.38 ± 0.07 in E. philippinensis).
The data for the RP was derived from a calibration curve (y
= 0.0505 x 0.5037, r2 = 0.9975) of gallic acid (0–1000 mg/
mL). In the absorbance measurement which is indicated by the correlation coefficients (r) of 0.9975, this value (r) is close to 1, which indicated that the regression equation is linear and would be used to determine the RP of the ethanolic leaf and rhizome extracts of E. fimbriobracteata and E. philippinensis. The ethanolic extracts revealed that the leaves (10.16 ± 2.18 in E. fimbriobracteata and 7.53
± 0.80 in E. philippinensis) are higher compared to the rhizomes (0.97 ± 0.18 in E. fimbriobracteata and 0.09 ± 0.09 in E. philippinensis) (Table 2).
The mean value of TAA of the E. fimbriobracteata leaves (12.69 ± 0.36 mg TAA/g sample) is higher compared to the studies of Mabini and Barbosa (2018) on E. philippinensis (0.79 mg AAE/g sample) and Barbosa and Nueva (2019) on H. conoidea (4.67 mg AAE/g sample). For the TAA of the E. philippinensis leaves (7.22 ± 0.26 mg TAA/g sample), it is also higher than E. philippinensis (Mabini and Barbosa 2018) and H. conoidea (Barbosa and Nueva
Table 1. Mean total phenolic content and flavonoid content extraction yield of leaves and rhizomes of E. fimbriobracteata and E.
philippinensis.
Species TPC (mg GAE/g dried sample)
Leaves Rhizomes
E. fimbriobracteata 13.20 ± 0.35 1.44 ± 0.04 E. philippinensis 7.21 ± 0.33 0.46 ± 0.30
2019) but lower compared to E. fimbriobracteata in this study. For the mean values of TAA of the rhizomes of E.
fimbriobracteata (1.82 ± 0.01 mg TAA/g sample) and E.
philippinensis (1.38 ± 0.07 mg TAA/g sample), these are higher than the studies of Mabini and Barbosa (2018) on E. philippinensis (0.55 mg AAE/g sample) but lower than the study of Barbosa and Nueva (2019) on H. conoidea (with 2.03 mg AAE/g sample).
Antioxidant activity correlated with their chemical structure and the content of phenolic flavonoid compounds (Andzi Barhé and Feuya Tchonya 2016). The antioxidant activity of this extract is probably caused by the presence of phenolic compounds processed by extracts (Sembring et al. 2018). The reducing activities of E. fimbriobracteata and E. philippinensis are significant indicators of their potential antioxidant activity. It is because the RP of plant extracts was associated with their antioxidant activity (Pan et al. 2008).
Recently, there has been an increase in research works on the potential phytochemicals from plants for therapeutic uses because many phytochemicals have been demonstrated to have antioxidant activities (Kairupan et al. 2019). Populations of the two species were found in the montane forests of Mt. Malambo in partially open forest, which supported Frankel and Berenbaum (1999) that foliage of tropical forest plants produced more antioxidants when exposed to elevated light conditions.
Yashin et al. (2017) reported that most biologically active compounds are contained in various spices and herbs, in which these two studied herbaceous species were used as spice agents by the local people in Mindanao.
Correlation among Total Phenolic Content (TPC), Total Antioxidant Activity (TAA), and Reducing Power (RP)
The contribution of the phenolic compounds in the ethanolic extracts of the two species to the antioxidant activity was determined by Pearson’s correlation coefficient. A perfect positive linear relationship was observed among the TPC, TAA, and RP (r = 1, p < 0.001) (Table 3).
By comparing the correlation coefficients (R value), it is possible that phenolic compounds significantly contribute
to the antioxidant activities of the plants’ extracts. The strong correlation between the results using the two methods of measuring TPC and antioxidant activity showed that phenol compounds largely contribute to the antioxidant activities of these plants and, therefore, could play an important role in the beneficial effects of E. fimbriobracteata and E. philippinensis. Several reports also showed a close relationship between TPC and antioxidant activity since phenolic compounds serve as hydrogen-donating agents (Li and Jiang 2007; Prasad et al. 2005; Yang et al. 2014). With the data of this paper, this study supported the claim of Stanković (2011) that the higher the phenolic content, the higher the antioxidant activity.
CONCLUSIONS AND RECOMMENDATIONS
The two studied species belong to the same genus, but their morphology is recognizable on both vegetative and reproductive parts. E. fimbriobracteata is unique among all Philippine Etlingera species by having reddish color towards the base of the pseudo stem, bright yellow flowers which incurved upon maturity, and green sterile bracts that form reddish upon maturity. On the other hand, E.
philippinensis has mandarin to deep red inflorescence and is unique among other Philippine Etlingera species by having long outward labellum. In terms of the vegetative parts, the petiole of E. philippinensis is violet in color and has an obvious oblique leaf base. The highest phenolic content (mg GAE/g dried sample) was observed in the leaves (13.20 ± 0.35 in E. fimbriobracteata and 7.21 ± 0.33 in E. philippinensis) than the rhizomes (1.44 ± 0.04 in E. fimbriobracteata and 0.46 ± 0.30 in E. philippinensis).
Table 2. Mean total antioxidant activity (TAA) and reducing power extraction yield of leaves and rhizomes of E.
fimbriobracteata and E. philippinensis.
Species TAA (mg AAE/g dried sample) RP (mg GRPE/g dried sample)
Leaves Rhizomes Leaves Rhizomes
E. fimbriobracteata 12.69 ± 0.36 1.82 ± 0.01 10.16 ± 2.18 0.97 ± 0.18
E. philippinensis 7.22 ± 0.26 1.38 ± 0.07 7.53 ± 0.80 0.09 ± 0.09
Table 3. Pearson’s correlation coefficients among total phenolic content, total antioxidant activity, and reducing power.
Assays TPC TAA RP
TPC 1 1* 1*
TAA 1* 1 1*
RP 1* 1* 1
*Correlation is significant at 0.001 level
Further, TAA (mg AAE/g dried sample) was observed higher in leaves (12.69 ± 0.36 in E. fimbriobracteata and 7.22 ± 0.26 in E. philippinensis) than rhizomes (1.82 ± 0.01 in E. fimbriobracteata and 1.38 ± 0.07 in E. philippinensis). The RP (mg GPRE/g dried sample) revealed higher for the leaves (10.16 ± 2.18 in E.
fimbriobracteata and 7.53 ± 0.80 in E. philippinensis) than the rhizomes (0.97 ± 0.18 in E. fimbriobracteata and 0.09 ± 0.09 in E. philippinensis). Overall, the highest phenolic content was obtained for the extracts of leaves than rhizomes. A perfect positive linear relationship was observed among the TPC, TAA, and RP (r = 1, p < 0.001).
These imply that the high contents of phenolic compounds contribute to the antioxidant activity of extracts of the two species. This paper calls for thorough phytochemical analyses to be done to identify the active phenolic and antioxidant components of these Philippine ginger species.
ACKNOWLEDGMENTS
The authors would like to thank the CMU, the CMU Department of Biology, and CEBREM for the opportunity to conduct this study. We thank CHED (Commission on Higher Education) under the DARE-TO (Discovery- Applied Research and Extension for Trans/Inter- disciplinary Opportunities) for partially funding this research which has facilitated the completion of this study.
We thank the DENR (Department of Environment and Natural Resources) Region XI Office for the issuance of the gratuitous permit to Dr. Victor B. Amoroso and the barangay officials of Datu Salumay in Marilog District for allowing NPM to conduct fieldwork at Mt. Malambo. We thank Prof. Hannah P. Lumista, Chris Rey M. Lituañas, Dr. Fulgent P. Coritico, and Dr. Merced G. Melencion for helping the first author in the finalization of the manuscript. Due acknowledgments are also given to the concerned authorities of CMU NSRC Natural Science Laboratory for the analysis of samples and the Plant Tissue and Spore Culture Laboratory where the microscopy of floral parts was conducted.
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APPENDICES
Etlingera fimbriobracteata (K.Schum.) R.M.Sm.
Notes Roy. Bot. Gard. Edinburgh 43 (1986) 245; --Poulsen
& Docot, Edinburgh J. Bot. 76 (2019) 37; --Amomum fimbriobracteatum K.Schum., Bot. Jahrb. Syst. 27 (1899) 317; --Type: from Borneo. --Etlingera pandanicarpa (Elmer) A.D.Poulsen, Blumea 48 (2003) 525; --Amomum pandanicarpum (Elmer) Elmer, LPB 8 (1915) 2899;
--Merr., EPFP 1 (1922) 240; --Hornstedtia pandanicarpa Elmer, LPB 8 (1915) 2899; --Type: Elmer 10508 (DS, lecto;
BISH, BM, BO, F, FI, G, GH, K, L, MO, NY, P, U, US, Z, isolecto), Mindanao: Davao, Mt Apo, Todaya (Fig. 1).
Phenology. Flowering from March to June, while fruiting was observed between June and October.
Local name and use. This species was known as “tagbak”
in Bisaya (Acma 2010) and bag-ang bag-ang in Davao Oriental (Acma and Mendez 2018b) (reported as E.
dalican). Rhizomes of E. fimbriobracteata were used as spice agents (Jason Batawan, pers. comm.). The vegetative parts of the plant are aromatic and the seeds are edible when ripe (Acma 2010). Some of the vegetative parts of the plant were also used to cure fever as claimed by the local people in Mindanao (Acma and Mendez 2018b).
Distribution. Borneo and Philippines: CEBU, PANAY, LUZON (Bulacan), and MINDANAO (Bukidnon, Davao, and Davao Oriental).
Habitat and ecology. Several populations of E.
fimbriobracteata were found near the forest trails of Mt.
Malambo at elevation 1,220 masl. Another population was found along the road near the Bemwa Farm at 1,210 masl.
In Mt. Hamiguitan, Davao Oriental, E. fimbriobracteata (reported as E. dalican) was found on a slope, shady places where there was no direct sunlight. The species was found along the trail in primary lowland forest with tall trees and leaf litter at 364 masl (Acma and Mendez 2018b).
Specimens examined. PHILIPPINES. Mindanao: Davao City, Marilog District, Brgy. Datu Salumay, Mt. Malambo, 1220 masl, 17 December 2021, NPM008, N.P. Mendez with R.M. Tubongbanua.
Other specimens examined. PHILIPPINES. Mindanao:
Davao City, Marilog District, Brgy. Datu Salumay, Mt.
Malambo, 1200 masl, flowering and fruiting 7 May 2018, VBA10771, F.M. Acma with N.P. Mendez and V.B.
Amoroso (CMUH 00012088).
PHILIPPINES. Mindanao: Davao City, Marilog District, Brgy. Datu Salumay, Mt. Malambo, 1200 masl, flowering and fruiting 7 May 2018, VBA10259, F.M. Acma with N.P. Mendez & V.B. Amoroso (CMUH 00011170).
Notes. Etlingera fimbriobracteata was formally known as E. pandanicarpa in the Philippines and was considered as a Philippine endemic species. However, Poulsen and Docot (2018) synonymized this species to E.
fimbriobracteata which originated in Borneo. Since then, E. fimbriobracteata was the accepted species name following the Principle of Priority. This species is unique among all Philippine Etlingera species by having reddish color towards the base of the pseudo stem, bright yellow flowers which incurved upon maturity and green sterile bracts that form to reddish upon maturity. In terms of its the infructescence, a dark green to brown color and later turns to black molar-like structure of fruits is a diagnostic character of the species.
Etlingera philippinensis (Ridl.) R.M.Sm.
Not. Roy. Bot. Gard. Edinburgh 43 (1986) 248;
--Poulsen & Docot, Edinburgh J. Bot. 76 (2019) 39;
--Amomum philippinense (Ridl.) Merr., EPFP 1 (1922) 240; --Hornstedtia philippinensis Ridl., Govt. Lab. Publ.
(Philip.) 35 (1906) 86, excl. fruit description; LPB 2 (1909) 605; PJS 4 c (1909) Bot. 175; --Elmer, LPB 8 (1915) 2905; LPB 8 (1919) 2981; --Type: E.B. Copeland 416 (SING, lecto; G, K, P, isolecto), Mindanao: Davao, Mar-1904 (Fig. 2).
Phenology. Flowering all year round.
Local name and use. This species is locally known as
“tagbak” (Elmer 1915; Acma 2010), pinoon (Barbosa et al. 2016), and “tapay-tapay” (Mendez et al. 2017). Some parts of the leaves and rhizomes were boiled with water and used by the local people to cure fever.
Distribution. Endemic to the Philippines. LUZON (Laguna, Quezon, and Sorsogon), MINDORO, PALAWAN, PANAY, NEGROS, BILIRAN, LEYTE, and MINDANAO (Bukidnon and Davao).
Habitat and ecology. Etlingera philippinensis is the most widespread endemic Etlingera species in the Philippines.
Populations of this species were recorded in an open area which was also the same with the earlier reports of Acma (2010), Barbosa et al. (2016), and Mendez et al. (2017).
This species prefers area where there is a partial sunlight.
Some populations were also recorded to occur near the streams and creeks. In this study, this species is quiet interesting because most of its populations could be found near the edge of the forest patches where there is partial to open area where sunlight penetrates. There were also inflorescences (in anthesis) in three forest edges but no vegetative parts on the adjacent areas found. This might be because the rhizomes of E. philippinensis is long- creeping, making the rhizomes still bearing flowers even the vegetative parts are decayed or cut due to its location.
Specimens examined. PHILIPPINES. Mindanao: Davao City, Marilog District, Brgy. Datu Salumay, Mt. Malambo, 1220 masl, 17 December 2021, NPM009, N.P. Mendez with R.M. Tubongbanua.
Other specimens examined. PHILIPPINES. Mindanao:
Davao City, Marilog District, Brgy. Datu Salumay, Mt. Malambo, 1200 masl, flowering 7 May 2018, VBA00010561, F.M. Acma with N.P. Mendez & V.B.
Amoroso (Pickled collection).
PHILIPPINES. Mindanao: Davao City, Marilog District, Brgy. Datu Salumay, Mt. Malambo, flowering, nd, F.M.
Acma (Pickled collection) (CMUH 00010860).
PHILIPPINES. Mindanao: Bukidnon, Kibawe, flowering, nd, G.B. Barbosa (Pickled collection) (CMUH 00010852).
Notes. Recently, E. philippinensis had been recorded in the different provinces of the Philippines, making it a widespread endemic species in the country. Its flowers fall under the Achasma-type Etlingera with its smooth and elongated labellum. The flowers of this species also closely resemble to E. coccinea except for the inward enfolded yellow margin in the labellum of the latter species. In terms of the vegetative parts, this species could be distinguished if the petiole has purplish to violet color and an obvious oblique leaf base.
The work of Acma and Mendez (2018a) was the first report regarding the fruiting of E. philippinensis which was not observed by Elmer (1915). The fruiting of E.
philippinensis was hard to observe in both wild and cultivated populations, since the flowers last up to 1–2 days only, then a new flower emerged. A week after its first flowering, the inflorescence became dried.
