This laboratory-based qualitative phytochemical study examined medicinal plants used in traditional healing in Partido District, Camarines Sur. Plant selection was based on the ethnobotanical inventory of Cerio (2024), which documented 153 medicinal species used by Partido Albularyo and reported use value (UV), informant consensus factor, and fidelity level [14]. The 33 taxa included in the present analysis were selected from the published ethnobotanical list using their reported UVs to guide laboratory prioritization. Their published UVs ranged from 0.036 to 0.664. This ethnobotany-guided selection links the laboratory screening directly with the documented cultural importance of medicinal plants in the study area [6][10][14][26][27].

Plant materials were collected from locations within the fourth legislative district of Camarines Sur. Healthy and visibly undamaged plant parts were placed in individually labeled resealable bags and transported to the laboratory. Samples were rinsed with distilled water to remove adhering debris, shade-dried at room temperature, and ground to a fine powder. For extraction, powdered material was combined with analytical-grade ethanol at a plant-to-solvent ratio of 1:10 (w/v) and cold-macerated for 72 h with occasional stirring. The macerate was filtered through a Büchner funnel lined with filter paper, and the ethanolic filtrate was concentrated under reduced pressure using a rotary evaporator at 40˚C. Concentrated extracts were stored in closed containers until qualitative testing. Ethanol maceration and reduced-pressure concentration are widely used for broad recovery of plant secondary metabolites [17][18].

Nine phytochemical classes were screened using established pharmacognostic color, precipitation, layer, and foam reactions [15][16][28]-[34]. The procedures were applied to concentrated ethanolic extracts, except for the phlobatannin assay, for which an aqueous filtrate of powdered plant material was prepared. Reactions were read from the characteristic endpoint specified for each test and recorded on an ordinal scale as absent (−), trace (+), moderate (++), or abundant (+++).

2.3.1. Alkaloids: Mayer’s Test

Approximately 2 mL of crude ethanolic extract was mixed with 2 mL of 1% hydrochloric acid and gently warmed. After cooling, Mayer’s reagent (potassium mercuric iodide solution) was added dropwise along the wall of the test tube. Formation of a creamy white to pale-yellow precipitate or distinct turbidity was recorded as a positive alkaloid reaction [16][28][31][33].

2.3.2. Flavonoids: Shinoda Test

A 2 mL aliquot of crude extract was placed in a test tube and a few small fragments of magnesium ribbon or magnesium turnings were added. Concentrated hydrochloric acid was then added dropwise. Development of a pink, scarlet, red, orange-red, or magenta coloration within several minutes was recorded as a positive flavonoid reaction [15][29][32]-[34].

2.3.3. Glycosides: Liebermann Reaction

Crude extract (2 mL) was mixed with 2 mL of chloroform and 2 mL of glacial acetic acid. The mixture was cooled in an ice bath, after which concentrated sulfuric acid was carefully introduced along the side of the tube. A violet-to-blue-to-green color transition was recorded as a positive reaction for the glycosidic constituents screened by this procedure [16][32]-[34].

2.3.4. Phenolic Compounds: Ferric Chloride Test

Approximately 50 mg of concentrated extract was dissolved in 5 mL of distilled water. Three to four drops of neutral 5% ferric chloride solution were added and the mixture was observed immediately. Development of a dark green, bluish-green, or blue-black coloration was recorded as a positive reaction for phenolic compounds [15][16][28]-[30].

2.3.5. Phytosterols: Liebermann-Burchard Test

Approximately 50 mg of extract was dissolved in 2 mL of acetic anhydride. One to two drops of concentrated sulfuric acid were then added carefully along the inner wall of the test tube. Development of a blue-green to green coloration was recorded as a positive phytosterol reaction in the Liebermann-Burchard test [15][16][30][33].

2.3.6. Phlobatannins

An aqueous filtrate was prepared by dispersing powdered plant material in distilled water, shaking thoroughly, and filtering. Approximately 2 mL of the filtrate was treated with 1% aqueous hydrochloric acid and heated to boiling. Formation of a red precipitate was recorded as a positive phlobatannin reaction [28][31][32][34].

2.3.7. Steroids

Crude extract was mixed with 2 mL of chloroform, and concentrated sulfuric acid was carefully added along the side of the tube. Formation of a red coloration in the lower chloroform layer was recorded as a positive steroid reaction. A second color reaction was performed using a separate aliquot of crude extract mixed with 2 mL of chloroform followed by 2 mL each of concentrated sulfuric acid and acetic acid; development of a greenish coloration supported a positive steroid result [15][16][28][33][34].

2.3.8. Terpenoids

Crude extract was dissolved in 2 mL of chloroform and the solvent was evaporated to dryness. Concentrated sulfuric acid (2 mL) was added to the residue and the mixture was heated for approximately 2 min. Development of a greyish coloration was recorded as a positive terpenoid reaction [28][30][32]-[34].

2.3.9. Saponins: Foam Test

Approximately 50 mg of extract was diluted with distilled water to a final volume of 20 mL in a graduated cylinder and shaken vigorously for 15 min. Formation of a stable, persistent foam layer of approximately 2 cm was recorded as a positive saponin reaction [16][28]-[31][34].

Botanical nomenclature was standardized against the World Checklist of Vascular Plants and Plants of the World Online [35][36]. The names used for oregano, tubong usa, lemonsito, and rosas de hapon were Coleusamboinicus, Helleniaspeciosa, Citrus × microcarpa, and Chrysanthemum × morifolium, respectively. Pandan was retained at genus level as Pandanus sp.

Reaction categories were encoded for statistical summarization as − = 0, + = 1, ++ = 2, and +++ = 3. For each phytochemical class, the proportion of positive taxa (score > 0) was reported with a 95% Wilson score confidence interval [37]. The complete ordinal distribution was retained to summarize reaction intensity. Per-taxon phytochemical richness was defined as the number of positive classes (0 - 9), and an ordinal screening index was calculated as the sum of the nine reaction scores (0 - 27).

Cochran’s Q test was used as a global distributional test for binary presence/absence across the nine assay classes [38]. The Friedman test was applied to the ordinal reaction scores, with Kendall’s W reported as the corresponding concordance/effect-size measure [39]. Spearman rank correlation was used to examine the association of published UV with phytochemical richness and the ordinal screening index. Statistical significance was evaluated at α = 0.05.

No new interviews or interventions involving human or animal participants were conducted. Plant selection was based on the published ethnobotanical dataset of Cerio (2024) [14], with the local context of Partido Albularyo healing practices described in related studies [11][12]. The study workflow is shown in Figure 1Figure 1.

Figure 1. Study workflow from published ethnobotanical use values to plant preparation, ethanolic extraction, qualitative phytochemical screening, and statistical characterization.

The study included 33 medicinal taxa, all with use values reported by Cerio (2024) [14]. Leaves were the dominant sampled material (27/33, 81.8%); roots accounted for 3/33 (9.1%), and bark, fruit, and stem each accounted for one taxon (3.0%). Published UVs ranged from 0.036 to 0.664. Table 1 summarizes the plant material, published use value, phytochemical richness, and ordinal screening index.

Table 1. Medicinal plants selected using the ethnobotanical use values reported by Cerio (2024) [14] and their qualitative screening metrics.

Alkaloids, flavonoids, phenolics, and saponins were detected in all 33 ethanolic extracts. Phytosterols were detected in 31 taxa (93.9%; 95% CI 80.4 - 98.3%) and phlobatannins in 28 (84.8%; 95% CI 69.1 - 93.3%). Steroids were detected in 16 taxa (48.5%; 95% CI 32.5 - 64.8%), terpenoids in 9 (27.3%; 95% CI 15.1 - 44.2%), and glycosides in 6 (18.2%; 95% CI 8.6 - 34.4%). For each of the four universally detected classes, the 95% Wilson interval was 89.6 - 100.0%. Table 2 presents the complete rating distributions and Figure 2Figure 2 displays positive prevalence with confidence intervals.

Table 2. Phytochemical-class prevalence, 95% Wilson confidence intervals, and ordinal reaction distributions.

Figure 2. Positive prevalence of nine phytochemical classes across 33 medicinal plant extracts. Error bars show 95% Wilson score confidence intervals; labels show the number of positive taxa out of 33.

The qualitative screening matrix showed an uneven distribution of reactions across the nine assay classes. Cochran’s Q for binary presence/absence was 151.59 (df = 8, p < 0.001). For the ordinal reaction scores, Friedman χ²(8) = 184.02 (p < 0.001), with Kendall W = 0.70. Mean ordinal scores were 2.30/3 for phenolics, 2.27 for saponins, 2.03 for alkaloids and flavonoids, 1.67 for phlobatannins, 1.24 for phytosterols, 0.52 for steroids, 0.27 for terpenoids, and 0.18 for glycosides.

Phytochemical richness averaged 6.73 ± 0.98 classes per taxon, with a median of 7, interquartile range 6 - 7, and range 5 - 9. The ordinal screening index averaged 12.52 ± 2.71 (median 12; IQR 10 - 14; range 8 - 18). Oregano (Coleusamboinicus) reacted positively in all nine screened classes and had an index of 18/27. Moringa oleifera also scored 18/27 across eight positive classes, followed by Psidium guajava (17/27, eight classes) and Blumeabalsamifera (16/27, seven classes). Figure 3Figure 3 presents the complete score matrix.

Figure 3. Heatmap of qualitative phytochemical reaction scores across 33 medicinal taxa. Rows are identified by local names corresponding to the standardized scientific names in Table 1. Scores: 0 = absent, 1 = trace, 2 = moderate, 3 = abundant.

Across the 33 taxa, published UV was positively associated with phytochemical richness (Spearman ρ = 0.54, p = 0.0010) and with the ordinal screening index (ρ = 0.61, p < 0.001). Figure 4Figure 4 presents the relationship between published UV and the screening index.

Figure 4. Relationship between published ethnobotanical use value reported by Cerio (2024) [14] and the ordinal qualitative phytochemical screening index across the 33 medicinal taxa. Spearman ρ = 0.61, p < 0.001.