Samples of Phoradendron californicum attached to Prosopis sp. were collected from the area known as “El Arenoso” (N 31^o02.18′, W 112^o02.58′; Lat: 31.038, Lon: -112.049) between the municipalities of Caborca and Altar in the state of Sonora, Mexico.

For extraction, stems were dried at room temperature and grounded, then mixed with methanol or chloroform in a proportion of 1:10 (sample:solvent) for three days. Samples were concentrated in a rotary evaporator and stored at - 20 °C until use.

Phytochemical screening

The phytochemical screening was determined following standard protocols previously reported (Savithramma et al., 2011). The qualitative assessment of the presence or absence of secondary metabolites was determined based on color change upon reaction (Savithramma et al., 2011; Uddin et al., 2011).

Total phenolic content

Total phenolic content was determined by the Folin-Ciocalteu method as previously described by Singleton and Rossy (Singleton and Rossy, 1965), with modifications. Briefly, 10 µL of sample (adjusted to 2.5 mg/mL) were mixed with 60 µL of sodium carbonate 7 % (w/v), 40 µL of Folin reagent (0.2 N) and 90 µL of milliQ water and placed in a 96-well microplate. The plate was incubated in the dark for 1 h, and then the absorbance measured at 750 nm by an ELISA plate reader (Multiskan EX, ThermoLabSystem, Waltham, MA, USA). A standard curve was prepared using different concentrations of gallic acid (0.8 - 0.006 mg/mL), and the results were expressed in terms of mg of gallic acid equivalent per gram of dry extract.

Total flavonoid content

Content of flavonoids was determined by the aluminum chloride method with modifications (Popova et al., 2004). Briefly, 10 μL of extract (adjusted to 2.5 mg/mL) were mixed with 130 μL of methanol and 10 μL of a 5 % (w/v) AlCl₃ solution in a 96-well microplate. The plate was incubated for 30 minutes in the dark and read at a wavelength of 412 nm using an ELISA plate reader (Multiskan EX, ThermoLabSystem, Waltham, MA, USA). A standard curve was prepared using different concentrations of quercetin (1 - 0.2 mg/mL), and the results were expressed in terms of mg of quercetin equivalent per gram of dry extract.

The antioxidant activity of P. californicum methanolic and chloroformic extracts was determined by the DPPH assay as previously reported (Usia et al., 2002) with modifications. The samples were prepared in methanol (100 µL) at different concentrations (3.12-400 µg/mL) and mixed with 300 µM of a DPPH solution (100 µL) in a 96-well plate (Costar, Corning, NY, USA), then the plates were incubated in the dark for 30 minutes, and the absorbance measured at 517 nm in an ELISA plate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). Ascorbic acid (70 µM) was used as antioxidant control. The antioxidant activity or free-radical scavenging activity is reported as percentage of decrease compared to ascorbic acid.

The human cervix (HeLa), human prostate (PC3) adenocarcinoma cell lines, and non-cancerous murine subcutaneous connective tissue (L-929) cell line were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). All cells were cultured in DMEM media supplemented with L-glutamine solution (200 mM), sodium pyruvate solution (100 mM), L-asparagine (98 %), L-arginine monohydrochloride (≥ 98 %), penicillin-streptomycin solution (1000 U/L U per mL) and 5% FBS (D5F) at 37 °C and 5 % of CO₂.

The antiproliferative activity was determined using the MTT assay (Mosmann, 1983). Initially, 50 µL of cells were seeded into a 96-well plate (1x10⁴ cells) (Costar, Corning, NY, USA) and incubated for 24 h at 37 °C and 5 % CO₂. Next, 50 µL of extract samples at different concentrations were added to each well, and plates were incubated for 48 h. During the last 4 h of treatment, 10 µL of an MTT solution (5 mg/mL) were added. The formed formazan crystals were dissolved using 100 µL of acidic isopropyl alcohol, and the absorbance of the plates were measured by an ELISA plate reader (Multiskan EX, ThermoLabSystem, Waltham, MA, USA) at 570 nm with a reference wavelength of 630 nm. DMSO was used as a solvent control.

The antibacterial activity was evaluated following the protocols established by the CLSI (Clinical and Laboratory Standards Institute, 2023). Briefly, the strains of E. coli ATCC 25922, S. enterica ATCC 14028 and L. monocytogenes ATCC 19115 were incubated for 18 h in LB media. Next, the strains were adjusted to the 0.5 McFarland scale (1x10⁸ UFC/mL) and then diluted 1:20 in LB media. Then, 10 µL of the inoculum were deposited in each well of a 96-well plate containing 100 µL of P. californicum extract at different concentrations (800 - 50 µg/mL). The plates were incubated for 24 h at 37 °C and measured by an ELISA plate reader (Multiskan EX, ThermoLabSystem, Waltham, MA, USA) at 620 nm.

The results shown were obtained by at least three independent experiments carried out in triplicate. Data was analyzed using GraphPad Prism 7.0 and statistical significance (p < 0.05; p < 0.001; p < 0.0001) was determined by two-way ANOVA with Bonferroni’s test.

The biological activities and antioxidant activity of natural extracts could be due to the presence of polyphenolic compounds such as flavonoids. Nevertheless, their presence can depend on the extraction procedure of the sample. Here, we determined the phytochemical profile and quantified the amount of total phenolics and flavonoids present in both P. californicum extracts by spectrophotometric techniques. Table 1 shows the phytochemical profile of methanolic and chloroformic extracts, finding that methanolic extract had presence of saponins, quinones, phenolics and free sugars, while the chloroformic extract was only positive to the presence of saponins. Table 2 shows the total phenolic and flavonoid content of both extracts. We observed that the methanolic extract has a higher amount of phenolic compounds (186.45 ± 4.58 mg EAG/g of extract) compared to the chloroformic extract (13.54 ± 1.57 mg EAG/g of extract) (p < 0.001). Nevertheless, the flavonoid content of both methanolic and chloroformic extracts is similar (19.92 ± 1.84 mgEQ/g of extract and 25.55 ± 0.73 mgEQ/g of extract, respectively) (p < 0.05).

Table 1.

	Phoradendron californicum extract
	Methanolic	Chloroformic
Terpenes	-	-
Flavonoids	-	-
Saponins	+	+
Quinones	+	-
Phenolics	+	-
Free aminoacids	-	-
Alkaloids	-	-
Free sugars	+	-

+ (presence); - (absence).

Table 2.

	Phoradendron californicum extract
	Methanolic	Chloroformic
*Total Phenolics	186.45 ± 4.58a	13.54 ± 1.57a
**Total Flavonoids	19.92 ± 1.84b	25.55 ± 0.73b
DPPH – IC50 (µg/mL)	47.62 ± 2.90c	˃ 400c

^*Expressed as mg equivalents of gallic acid/g of dry extract (mg EAG/g of extract) ^**Expressed as mg equivalents of quercetin/g of dry extract (mg EQ/g of extract) ^aValue of p < 0.001 of significance from the corresponding Total phenolics of methanolic to the Total phenolics of chloroformic extract. ^bValue of p < 0.05 of significance from the corresponding Total flavonoids of methanolic to the Total flavonoids of chloroformic extract. ^c Value of p < 0.001 of significance from the corresponding IC₅₀ of methanolic to the IC₅₀ of chloroformic extract.

Antioxidant activity of the methanolic and chloroformic extracts of P. californicum was evaluated by the DPPH assay. We can observe that the methanolic extract has potent antioxidant activity with an IC₅₀ of 47.62 ± 2.90 µg/mL (Table 2), and higher concentrations of 100 to 400 µg/mL are comparable to the antioxidant activity of ascorbic acid, which is a strong antioxidant compound. In comparison, the chloroformic extract presented a weak antioxidant activity even at the higher concentration of 400 µg/mL, where its antioxidant activity reaches a maximum of ~ 25 % (Figure 1).

Figure 1.

The antiproliferative activity of methanolic and chloroformic extracts of P. californicum was evaluated against HeLa and PC3 cancer cell lines, as well as an L929 normal cell line, by an MTT assay (Figure 2). A dose-response effect can be observed, especially for HeLa and PC3 cell lines treated with chloroformic extract. Phoradendron californicum chloroformic extract exhibited higher antiproliferative activity against all three tested cell lines in comparison to the methanolic extract. The most susceptible cell line was PC3 (IC₅₀: 167.67 ± 5.08 µg/mL), followed by HeLa (IC₅₀: 215.62 ± 14.70 µg/mL) and L929 (IC₅₀: 243.68 ± 10.54 µg/mL) (p < 0.001). For the methanolic extract, the IC₅₀ against PC3 and HeLa were similar (340 ± 11.58 µg/mL and 352.51 ± 9.87 µg/mL, respectively) with no statistical significance between them (p < 0.05), meanwhile, for L929 cell line the IC₅₀ was higher than 400 µg/mL (Table 3).

Figure 2.

Table 3.

Extract	IC50 (µg/mL)
	HeLa	PC-3	L-929
Methanolic	352.51 ± 9.87a	340 ± 11.58a	> 400a
Chloroformic	215.62 ± 14.70b,c	167.67 ± 5.08c	243.68 ± 10.54b,c

IC₅₀ values represent the mean of three independent experiments ± standard deviation. ^aValue of p ≤ 0.001 of significance between the IC₅₀ of L929 and the IC₅₀ of HeLa and PC3. ^bValue of p ≤ 0.05 of significance between the IC₅₀ of HeLa and L929. ^cValue of p < 0.001 of significance between the IC₅₀ of HeLa, PC-3 and L-929.

The antibacterial activity of methanolic and chloroformic extracts was evaluated against three different bacterial strains; Escherichia coli ATCC 25922, Salmonella enterica (serovar typhimurium) ATCC 14028 and Listeria monocytogenes ATCC 19115. As observed in Figure 3, the antibacterial activity depends on the strain and type of extract evaluated. S. enterica was more susceptible to the methanolic extract compared to the chloroformic extract, on the other hand, E. coli was more susceptible to the chloroformic extract compared to methanolic extract. For L. monocytogenes the chloroformic extract was more effective at the higher concentration evaluated (800 μg/mL) with a viability of around 60%.

Figure 3.