aluminum chloride colorimetric method, CEDAR, gingers, folin-Ciocalteu method, Philippine endemic, phosphomolybdenum method.

Phenolics are secondary metabolites in plants (Alara et al., 2021). These plant metabolites are essential in the physiological defense response of the body, such as anti-aging, anti-inflammatory, antioxidant, and anti-proliferative activities (Lin et al. 2016). One of the many observed compounds in plant phenolics is antioxidants (Sroka, 2005). Antioxidants are substances that delay or prevent oxidation, while antioxidant activity is the reaction of antioxidants and oxidants at a constant rate (Santos-Sanchez et al., 2019).

Zingiberaceae are flowering plants common in the tropics and subtropical parts of the mountains (Panchareon et al., 2000). This family consists of at least 53 genera and over 1200 species (Kress et al., 2002). Meistera is one of the genera of Zingiberaceae that formerly shared with the genus Amomum Roxb. After the recent morphological revision of de Boer et al. (2018), it was established as a distinct genus and was reinstated. Meistera muricarpa (Elmer) Škorničk and M.F. Newman is a Philippine endemic species and was recorded in Mt. Hamiguitan Range Wildlife Sanctuary in Davao Oriental (Acero et al., 2019), Cinchona Forest Reserve in Bukidnon (Jayme et al. 2019), and Mt. Musuan in Bukidnon (Mendez et al., 2023a). This species is distinct from other species of Meistera by having a spatulate labellum and echinate fruits (Acero et al., 2019).

 In ethnomedicinal practices in Bukidnon, the seeds from the ripe fruits of this species are consumed by the locals as it is affirmed to cure stomach-related disorders (Acma, 2010). The qualitative phytochemical screening of leaves, rhizomes, and fruits of M. muricarpa revealed the presence of various phytochemical compounds, including alkaloids, carbohydrates, glycosides, fixed oils, fats, saponins, tannins, and proteins (Acma 2013). However, the quantitative phytochemical tests of this species have not been investigated, making this paper the first report on the TPC, TFC, and TAA, which can be used for future applications of this species. Thus, this study was conducted to investigate the gross morphological characteristics of M. muricarpa and determine its total phenolic content (TPC), total flavonoid content (TFC), and total antioxidant activity (TAA) of the ethanolic leaf and fruit extracts.

Entry Protocol. Before conducting the study, the necessary permits and approvals were secured. A formal letter was personally submitted to the office of the Municipal Mayor of Impasug-ong, while a prior informed consent (PIC) was obtained from the Barangay Captain of Barangay Impalutao, Municipality of Impasug-ong. Additionally, a Wildlife Gratuitous Permit (WGP) with permit number R10-2024-14 was obtained from the Department of Environment and Natural Resources (DENR) - Region X, located in Cagayan de Oro City, Misamis Oriental. This permit is a requirement for researching wildlife, as outlined in the DENR Administrative Order No. 2004-55, which streamlines the procedures for wildlife research and conservation 

Place and Duration of the Study. The leaf and fruit samples and voucher specimens of M. muricarpa were collected from the Center for Ecological Development and Recreation (CEDAR), Impalutao, Impasug-ong, Bukidnon, Philippines (8.254137ºN, 125.036881ºE) (Fig. 1). The morphological dissection, measurements, and description of M. muricarpa were done in the field. The samples for laboratory analyses and voucher specimens were transported to Central Mindanao University for processing. The analyses of TPC, TFC, and TAA were carried out at the Natural Science Laboratory of the Natural Science Research Center (NSRC), Central Mindanao University, Musuan, Bukidnon, from December 2023 to April 2024.

Fig. 1. Study site. A) Map of the Philippines; B) Map of CEDAR (red circle indicates the location of species; C) Habitat of M. muricarpa (A & B: ©2024 Google Earth).

Sample Preparation and Extraction. To prevent dehydration, the collected leaf and fruit samples were placed in plastic cellophane bags with moist tissue paper. The samples were then washed to remove any soil or debris and air-dried at room temperature (25°C) for 14 days. The dried samples were subsequently homogenized using a household blender and stored in zip-lock cellophane bags until further analysis. Ethanol extracts were prepared using a modified version of the method described by Padda and Picha (2008). Specifically, 1 g of dried leaf or fruit powder was extracted with 25.0 mL of absolute ethanol at room temperature (25°C ± 2°C). The mixtures were shaken at 300 rpm for 1 hour using an orbital shaker, followed by centrifugation at 5,000 rpm for 5 minutes. The resulting extracts were collected in 15 mL conical tubes and stored at 2–8°C.

The stored ethanolic leaf and fruit extracts were used to determine the total phenolic content (TPC), total flavonoid content (TFC), and total antioxidant activity (TAA) using 96-well plate format colorimetric assays.

Determination of Total Phenolic Content. The total phenolic content (TPC) of the plant extracts was determined using a modified version of the Folin-Ciocalteu assay described by Ainsworth and Gillespie (2007). The assay involved mixing 200 μL of plant extract with 200 μL of 10% Folin-Ciocalteu reagent in a 2-mL centrifuge tube. After a 5-minute incubation, 800 μL of 10% sodium carbonate was added, and the mixture was incubated for an additional 30 minutes at room temperature. The mixture was then centrifuged at 11,000 rpm for 3 minutes, and 200 μL of the resulting solution was transferred to a microplate well. The absorbance was measured at 750 nm using a microplate reader. A standard calibration curve was prepared using gallic acid (GA) standards with concentrations ranging from 0 to 100 ppm, in increments of 10 ppm, from a 100 ppm GA stock solution in absolute alcohol. The TPC was calculated and expressed as milligram gallic acid equivalent per gram dried sample (mg GAE/g sample) by comparing the sample absorbance to the standard calibration curve using the following formula:

      (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)

Determination of Total Flavonoid Content. The TFC was assessed using the aluminum chloride colorimetric method, as described by Nurcholis et al. (2021). Briefly, 50 µL of plant extract was added to a 96-well plate, followed by the addition of 10 µL of 10% aluminum chloride, 130 µL of 96% ethanol, and 10 µL of 1M sodium acetate. The mixture was incubated in the dark for 40 minutes at room temperature. The absorbance was measured at 415 nm using a microplate reader. The TFC was expressed as milligram quercetin equivalent per gram dried sample (mg QE/g dried sample) by comparing the sample absorbance to a standard calibration curve. The TFC was calculated using the following formula:

    (2)

where  A = quercetin concentration of the sample solution determined from the calibration curve (mg QE/L)

B = concentration of test solution (g/L, gram dried sample per L solution)

Determination of Total Antioxidant Activity. The TAA was assessed using the phosphomolybdenum method described by Prieto et al. (1999), with minor modifications to the reagent solution volume. Briefly, 50 μL of the extracts were diluted with 200 μL of a 1:1 ethanol-water solution in centrifuge tubes. Then, 600 μL of the reagent solution, comprising equal parts of 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate, was added. The mixture was incubated at 95°C for 1 hour and 30 minutes and cooled to room temperature (25°C). After centrifugation at 11,000 rpm for 3 minutes, the absorbance of the supernatant was measured at 695 nm against a blank using a microplate reader. A standard calibration curve was generated using ascorbic acid (AA) standards with concentrations ranging from 0 to 150 ppm, in increments of 15 ppm, prepared from a 300 ppm AA stock solution in absolute alcohol. The TAA was determined by comparing the sample absorbance to the standard calibration curve. The TAA was calculated using the following equation:

       (3)

where  A = ascorbic acid concentration of the sample solution determined from the calibration curve (mg AAE/L)

B = concentration of test solution (g/L, gram dried sample per L solution)

Statistical Analysis. All TPC, TFC, and TAA analyses were performed in triplicate, with three trials conducted per replicate to ensure reliability and accuracy. The data obtained from the leaf and fruit samples of the species were then subjected to statistical analysis. A t-test was performed at a 0.001 level of significance to determine significant differences between the means of the two groups. Additionally, Pearson's correlation analysis was conducted at a 0.001 level of significance to examine the relationships between TPC, TFC, and TAA. This comprehensive statistical approach allowed for a thorough understanding of the differences and correlations between the phytochemical parameters of the leaf and fruit samples.

Plant Description. Terrestrial perennial herb, arching, reaching as high as 2–2.5 m tall. Stilt roots absent. Rhizomes below ground, pinkish, sometimes deep red, 2.4–2.8 × 3.6–3.9 cm diam., brownish. Leaves subsessile, distichous, oblong to lanceolate, 24–41 × 5.5–5.9 cm, adaxially dark green and glabrous, abaxially pale green and glabrous; midrib adaxially greenish to yellowish, continuous, abaxially greenish to yellowish, glabrous; margin entire, wavy, brownish; base acute; apex acuminate, slightly recurved. Leaf sheath green with prominent hairs, grooved, green. Leaf internodes 5 cm distance from each other, glabrous, green. Petiole short, green, 0.9–1.2 cm, pubescent. Ligule greenish, pubescent, 0.6–0.8 × 0.7 cm, yellowish to green. Swollen base brownish, glabrous, 7–7.2 cm. Inflorescence short, lateral, bearing 1–3 flowers, 7.5 × 1.5–2 cm. Bracts delicate, ovate with acute apex, reddish, margin entire, apex acute, 0.8 × 0.3 cm, glabrous. Bracteoles tubular, pale red, acute apex, 2 × 0.3 cm, glabrous. Calyx tubular, fused, pinkish in color, 2–2.5 × 0.3–0.5 cm wide, glabrous. Flower 6 × 2.5 cm. Lateral corolla lobes oblong to lanceolate, glabrous, trilobed, pinkish to red, with visible veins, entire margin, rounded apex, rounded apex, dorsal lobe pale red, 1.5 cm × 0.2–0.3 cm. Dorsal corolla lobe reddish, rounded apex, 2.0–2.5 × 0.3–0.4 cm. Labellum spatulate, frilly, lower lobes tucked, stamen, glabrous, bright yellow, orange-red tinge, entire margin, acute apex, 3.5–4.0 × 0.2–0.3 cm. Style white, 1.7 × 1–2 cm; anther sac 0.5–0.7 cm long × 1–2 cm wide. Fruit capsule, indehiscent with dense prickles, echinate. Seeds globose, creamy white when young, black when mature, and white, transparent aril, 0.1–0.2 cm (Fig. 2).

Local name and Uses: Locally called “tugis” (Govaerts, 2005). The seeds are edible and used as a relief for stomach illnesses (Acma, 2010).

Distribution: LUZON: Bataan, Mariveles. Isabela and Quezon. MINDANAO: Bukidnon, Cotabato, Davao Oriental, Davao del Sur, Misamis Oriental, and Zamboanga. Endemic to the Philippines (Pelser et al., 2011 onwards).

Habitat and Ecology: Meistera muricarpa is found in the Mindanao mountains, including Mt. Musuan (Mendez et al. 2023a), Cinchona Forest Reserve (Jayme et al., 2020), and Mt. Hamiguitan Expansion Site (Acero et al., 2019). This species is mostly found in wet tropics (Govaerts, 2005), lowland, and montane evergreen forests at elevations of 216–1112 masl (Laxmay & Newman 2012).

Fig. 2. Meistera muricarpa (Elmer) Škorničk. & M.F.Newman. A) Habit, B) Mid foliage, C) Inflorescence, D) Infructescence, E) Longitudinal section of the fruit, F) Floral dissection: infl–inflorescence; fl–flower; br–bracts; b–bracteoles; cx–calyx; cl–corolla lobes; lm–labellum; s–stamen; as–anther sacs; o–ovary (Scale bar: 1 cm).

Total Phenolic Content. The TPC of the ethanolic extracts of leaves and fruits of M. muricarpa is presented in Table 1. The results were derived from a calibration curve (equation: y = 0.0671x + 0.0267; R² = 0.9951) of gallic acid (0–200 mg/mL). The TPC of the leaves of M. muricarpa in this is study with 2.45 ± 0.04 mg GAE/g sample is lower compared to Etlingera fimbriobracteata (K.Schum) R.M.Sm. with 13.20 ± 0.35 mg GAE/g sample and E. philippinensis (Ridl.) R.M.Sm. with 7.21 ± 0.33 mg GAE/g sample (Mendez et al., 2023b), and E. dostseiana Naive, Demayo & Alejandro with 15.44 ± 0.80 mg GAE/g sample (Mendez et al., 2023c). 

Table 1: Mean TPC of the ethanolic extract of the leaves and fruits of M. muricarpa.

In plant parts, fruits exhibit a good source of compounds with phenolic relevance, such as phenols, lignins, lignans, coumarins, tannins, phenolic acids, and flavonoids (Xu et al. 2017). These phenolics are also called secondary plant metabolites. The group of compounds with single or more aromatic rings that are attached to hydroxyl groups with more than 8000 known structures (Balasundram et al., 2006). These compounds hold a significant role in protecting plants from ultraviolet (UV) rays, and diseases, as well as deterring parasites, insects, and predators (Caleja et al. 2017; Durazzo et al. 2019). 

The fruit of M. muricarpa has been traditionally used to alleviate stomachaches, and locals often consume the ripe seeds from its fruits (Acma 2010). Previous studies have reported that M. muricarpa exhibits high radical scavenging activity in its rhizomes (Barbosa et al., 2016). The rhizomes of ginger species are known to accumulate high levels of antioxidants and secondary metabolites compared to other plant parts (Jitoe et al., 1992).

Phytochemical analysis of M. muricarpa has revealed the presence of various bioactive compounds, including phenolic compounds such as alkaloids, flavonoids, saponins, and tannins, as well as non-phenolic compounds like steroids, in its leaves and rhizomes (Barbosa et al. 2016). Fruits are known to be rich in phenolic compounds, particularly flavonoids, which are responsible for their yellow, red, and blue colors (Belitz et al., 2009; Lampila et al., 2009). To date, over 5,000 flavonoids have been identified, and these compounds play essential roles in protecting plants against pathogens and excessive UV light (Winkel-Shirley, 2002).

Hence, the high phenolic content of the fruits of M. muricarpa might be due to flavonoids as well as flavonoid derivatives, anthocyanins, which are mainly responsible for the pigment of fruits, pollination, and absorption of light as well as guard plants from the damage of UV rays (Castañeda-Ovando et al., 2009; Ahmed et al., 2016). Other factors that might contribute to the high phenolic content of M. muricarpa are the degree of ripeness, variety, climate, soil composition, geographic location, and storage conditions (Belitz et al., 2009).

Total Flavonoid Content. The TFC of the ethanolic extracts of leaves and fruits of M. muricarpa are presented in Table 2. The results were derived from the calibration curve (equation: y=0.0394–0.003; R² 0.9996) of quercetin (0–300 mg/mL).

Table 2: Mean total flavonoid content (TFC) of the ethanolic extract of the leaves and fruits of M. muricarpa.

Flavonoids are a diverse group of secondary metabolites that are ubiquitous in plants, fruits, and seeds. They are responsible for the biological colors, fragrances, and flavors of plants, and play crucial physiological roles in regulating cell growth, attracting pollinators, and protecting plants from biotic and abiotic stresses (De Luna et al., 2020). Additionally, flavonoids function as signal molecules, ultraviolet filters, and scavengers of reactive oxygen species (Di Ferdinando et al., 2011; Panche et al., 2016; Dias et al., 2021).

Flavonoids are distributed throughout plant tissues, both within cells and on the surfaces of various plant organs (Ferreira et al., 2006). The concentration of bioactive compounds, including flavonoids, is typically higher in sunlight-exposed plant parts, such as leaves, due to photosynthesis (Kumar et al., 2019). This may explain why the leaves of M. muricarpa contain higher.

The amount of bioactive compounds in fruits can be influenced by various factors, including the degree of ripeness, variety, climate, soil composition, geographic location, and storage conditions (Belitz et al., 2009). These factors can contribute to variations in the phytochemical composition of fruits, highlighting the importance of considering these factors when evaluating the nutritional and medicinal properties of plant-based materials.

Total Antioxidant Activity. The total antioxidant activity of the ethanolic extracts of leaves and fruits of M. muricarpa is presented in Table 3. The results were derived from the calibration curve (equation: y=0.0164–0.0092; R² = 0.9996) of ascorbic acid (0–150 mg/mL). The TAA of the leaves of M. muricarpa in this is study with 8.28 ± 0.15 mg AAE/g sample is lower compared to E. fimbriobracteata with 12.69 ± 0.36 mg AAE/g sample and E. philippinensis with 7.22 ± 0.26 mg AAE/g sample (Mendez et al., 2023b) and E. dostseiana with 14.24 ± 0.25 mg AAE/g sample (Mendez et al., 2023c).

Table 3: Mean TAA of the ethanolic extract of the leaves and fruits of M. muricarpa.

Antioxidants, as molecules, possess the ability to neutralize the activity of free radicals. Their action involves the inactivation of these harmful molecules (Halliwell, 1996; Devasagayam et al., 2004). There are a variety of secondary metabolites produced by plants through the normal metabolic pathways. These secondary plant metabolites include flavonoids, essential oils, alkaloids, lignans, terpenes, terpenoids, tocopherols, phenolic acids, peptides, and polyfunctional organic acids (Shahidi and Ambigaipalan 2015). Among these various compounds, phenolic acids are considered the primary natural sources of antioxidants (Chanwitheesuk et al., 2005; Maisuthisakul et al., 2007).

The high antioxidant activity of the fruits of M. muricarpa may also be due to its contained flavonoids (Barbosa et al., 2016) that are recognized as natural antioxidants and other bioactive compounds. These exhibited the high antioxidant activity of fruits agree with the antioxidant activity of other Meistera species. M. chinensis ethanolic fruit extracts in the DPPH assay recorded 47.62 ± 2.93 mg/L in IC50, which suggests a high potency of antioxidants in fruits (Musdalipah et al., 2021). Hence, M. muricarpa is also a great source of natural antioxidants, and this study provides additional new scientific knowledge about the plant’s potential.

Correlation of Total Phenolic Content, Total Flavonoid Content, and Total Antioxidant Activity. The relationship between TPC, TFC, and TAA of M. muricarpa leaves and fruits was assessed using Pearson's Correlation analysis. This statistical method measures the strength and direction of linear correlations, with correlation coefficients (r) ranging from -1 to 1. A correlation coefficient close to 1 or -1 indicates a strong positive or negative linear relationship, respectively, while a value near zero suggests no significant linear correlation (Table 4).

Table 4: Results of the correlation analysis between the TPC, TFC, and TAA of the ethanolic leaf and fruit extracts of M. muricarpa.

A strong positive linear correlation was observed between total phenolic content (TPC) and total flavonoid content (TFC) (r=0.99, p<0.001), TPC and total antioxidant activity (TAA) (r=0.99, p<0.001), and TAA and TFC (r=0.99, p<0.001). These findings suggest that plant parts with higher phenolic content exhibit significantly greater flavonoid and antioxidant activities. This implies that the phenolic compounds in M. muricarpa fruits are primarily responsible for their high antioxidant activity.

The presence of polyphenols in plant extracts is likely a major contributor to their antioxidant properties. The structure of phenolic compounds plays a crucial role in determining their antioxidant activity, particularly the electron delocalization over an aromatic nucleus (Jing et al. 2010). When phenolic compounds react with free radicals, the gained electron is delocalized over the phenolic antioxidant, stabilized by the resonance effect of the aromatic nucleus, thereby blocking the continuation of the free radical chain reaction (Tsao and Deng 2004).

This study revealed that M. muricarpa is characterized by its bright yellow flowers, orange-red tinged indehiscent echinate capsule fruits, and dense prickles. Phytochemical analyses showed that the fruits exhibited higher total phenolic content (TPC) and total antioxidant activity (TAA) compared to the leaves. Conversely, the leaves had higher total flavonoid content (TFC) than the fruits. Specifically, the fruits contained 6.99 ± 0.19 mg GAE/g dry weight sample of phenolics, surpassing the leaves with 2.45 ± 0.04 mg GAE/g dry weight sample. The leaves yielded 5.11 ± 0.06 mg QE/g dry weight sample of flavonoids, exceeding the fruits with 0.29 ± 0.01 mg QE/g dry weight sample. Furthermore, the fruits demonstrated higher antioxidant activity with 21.13 ± 0.87 mg AAE/g dry weight sample, compared to the leaves with 8.28 ± 0.16 mg AAE/g dry weight sample.

This study suggests that the leaves and fruits of M. muricarpa could serve as a valuable source of natural antioxidants, offering potential applications in the development of novel antioxidant agents. There is also a need to explore quantitative phytochemical screening for other Zingiberaceae species in the Philippines that have been reported and used for ethnomedicine.