Fetal bovine serum (FBS), minimum essential medium (MEM) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Life Technologies was obtained from Life Technologies Corporation (Carlsbad, CA). DMEM (without glucose and phenol red), adenosine triphosphate (ATP), adenosine diphosphate (ADP), RPMI-1640 medium (without phenol red), penicillin, streptomycin, dimethyl sulfoxide (DMSO), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), and lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO). The luciferase kit was obtained from Perkin Elmer (Boston, MA). Concanavalin A (Con A) and sodium pyruvate were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Bio-Rad protein assay dye reagent concentrate was obtained from Bio-Rad (Hercules, CA, USA). Human erythropoietin (EPO) ELISA kits were purchased from Wuhan Fine Biotech Co., Ltd. (FineTest) (Wuhan, HB, P.R. China). Phytochemical extracts were purchased from iHerb: Quercetin (Natural Factors, 500 mg quercetin per 700 mg powder); Curcumin (NOW Foods, containing turmeric root extract (Curcuma longa) with minimum 95% curcuminoids); Resveratrol (Natural Factors, containing 500 mg wine grape (Vitis vinifera) concentrate (fruit) plus resveratrol from Japanese knotweed and 250 mg natural trans-resveratrol per 750 mg powder); Catechins (DaVinci Laboratories, containing green tea (Camellia sinensis L.) leaf extract yielding 70% Epigallo-catechin-3-gallate (a polyphenol)). Chinese herbal extracts were purchased from PuraPharm International (H.K.) Ltd (Hong Kong SAR, China); the percentage yield from the crude herb of Cynomorii Songaricum Herba (Purapharm 1235), PanaxGinseng Radix (Purapharm 1304), and Ligustri Lucidum Fructus (Purapharm 1074) were 20%, and that of Angelica Sinensis Radix (Purapharm 1017) was 33%.

H9c2 cells, which are a subclone of rat heart myoblast cells, were purchased from the American Tissue Culture Centre (ATCC) (Rockville, MD, USA). Cells were cultured as monolayers in DMEM, supplemented with 10% (v/v) heat-inactivated FBS (HIFBS), 100 IU/mL of penicillin, 100 μg/mL of streptomycin, 1 mM sodium pyruvate, and 3.7 g/L NaHCO₃. All cells were grown under an atmosphere of 5% (v/v) CO₂ in air at 37˚C in a 90 mm culture plate.

HepG2 cell line originated from the human liver and was purchased from ATCC. Cells were cultured in MEM supplemented with 10% (v/v) FBS, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, 1 mM sodium pyruvate and 1.5 g/L NaHCO₃. All cells were cultured in an atmosphere of 5% CO₂ at 37˚C in a 90 mm culture plate.

1) Drug pre-incubation and ATP Generation

H9c2 cells were seeded in four 24-well plates (25,000 cells/0.5 mL). After stable attachment, the cells were pre-incubated for 4 h (for testing yang-invigorating effect) or 24 h (for testing Qi-invigorating effect) with a medium containing different concentrations (concentration ranges were listed in the result table) of phytochemical or herbal extracts dissolved in DMSO, 0.2% (v/v). Following the incubation, the cells were washed with phosphate-buffered saline (PBS-A, 200 μL/well; 136 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.2) and treated with 50 μg/mL digitonin at 37˚C for 3 min, except for the protein measurement plate, which was stored at 4˚C until protein determination. For the plate used for measuring basal ATP level, cells were incubated at 4˚C with ATP incubation buffer (120 mM KCl, 5 mM KH₂PO₄, 2 mM EGTA, 10 mM HEPES, 0.1 mM MgCl₂, 0.5% BSA, pH 7.4, 150 μL/well) and 30% (w/v) perchloric acid (PCA, 30 μL/well). For the plates used for ATP generation, cells were supplemented with 0.18 mM ADP, 5 mM pyruvate, and 15 mM L-malic acid (all dissolved in ATP incubation buffer, 100 μL for each reagent/well) and incubated at 37˚C for 7.5 and 15 min, respectively, followed by the addition of 30% PCA (60 μL/well). The plates (for measuring basal ATP and ATP generation) were then centrifuged at 540 ×g for 20 min at 4˚C. After centrifugation, aliquots (80 μL) of supernatants were neutralized with 60 μL 1.4 M KHCO₃ in 1.5 mL micro-centrifuge tubes, followed by vortex mixing and centrifugation at 2150 ×g for 10 min [17].

2) ATP assay

To account for ATP contamination in the ADP preparation, an ADP blank was prepared by aliquoting ADP, pyruvate, L-malic acid, and 30% PCA, as described earlier, to 1.5 mL in a micro-centrifuge tube, followed by vortex mixing and centrifugation at 20,000 ×g for 5 min. ATP standards (ranging from 0.0005 - 1.0 mM) were prepared by 10-fold serial dilutions. Aliquots (300 μL) of standard solutions were added to 60 μL 30% PCA, followed by vortex mixing, and 80 μL of the standard solutions, as well as the ADP blank, were neutralized with 60 μL 1.4 M KHCO₃, followed by vortex mixing and centrifugation at 20,000 ×g for 5 min. Fifty microliters of ATP standards (0.0005 - 1 mM) or tested samples were applied to 96-well white plates with 50 μL 10-fold diluted luciferase enzyme prepared in luciferase dilution buffer (250 mM Tricine, 5 mM EDTA (free acid), 5 mM dithiothreitol, 50 mM MgSO₄, pH 7.8 adjusted by 1 M KOH). The luminescence was read using a microplate reader. ATP content was estimated from an ATP standard calibration curve. ATP-GC was expressed in nmol/mg protein, and the area under the curve (AUC1) of the graph plotting ATP generation against incubation time was then computed. These AUC1 values were used to calculate the percentage of control (% Control-1), and then the AUC2 of the graph plotting % Control-1 against incubation time was computed. These AUC2 values were used to calculate the percentage of control (% Control-2) for comparison [17].

Proteins were extracted from the cultured cells using 0.1% Triton X in PBS-A, followed by 10-min of shaking at room temperature. Aliquots (10 μL) of samples, as well as BSA standards, were mixed with working protein-dye solution (200 μL/well), and the reaction mixtures were allowed to stand at room temperature for 5 min. The absorbance of the samples was then read at 595 nm [17].

HepG2 cells were seeded in 6-well plates at a cell concentration of 600,000 cells/2 mL. After stable attachment, the cells were rinsed with PBS-A and then pre-incubated for 5 h (HepG2 cells) with a DMEM (without glucose and phenol red) supplemented with 1mM sodium pyruvate, 25 mM glucose, 10% (v/v) FBS, 3.7 g/L NaHCO₃ containing different concentrations of phytochemical or herbal extracts (concentration ranges being listed in the result table) [dissolved in DMSO, 0.2% (v/v)]. After the incubation, supernatants of the culture medium were collected for the measurement of NO release, and the attached cells were rinsed with PBS-A and lysed with 0.1% (w/v) Triton-X in PBS-A. The lysates were used to measure protein concentrations by the Bradford method using BSA as the standard. The amount of NO released into the medium from phytochemical/herbal extract-incubated cells was measured using the Griess assay. The Griess assay is a spectrophotometric method based on a diazotization reaction for the detection of nitrite ( NO 2 − ) formed as a result of the oxidation of NO. Griess reagent solutions were prepared as follows, and sodium nitrite was dissolved in distilled deionized water (double-distilled (dd)H₂O) and used as a standard:

1) VCl₃ reduction solution: 80 mg VCl₃ was partly dissolved in a small amount of ddH₂O (around 2 mL), 0.84 mL hydrochloric acid (HCl, 37%) was added, and the solution was diluted to 10 mL with ddH₂O;

2) NED solution (0.1%): 1 mg NED was dissolved in 1 mL ddH₂O;

3) Sulfanilamide solution (2%): 0.02 g sulfanilamide was dissolved in 1 mL 10% (v/v) HCl;

4) Working Griess solution: 5 parts A + 1 part B + 1 part C.

One hundred and twenty microliters of supernatant/standard/medium blank were pipetted into a 96-well plate. Working Griess solution (100 μL) was applied to the corresponding wells. The sample mixtures were incubated at 45˚C for 60 min. The absorbance of sample mixtures was measured at 550 nm following incubation. The amount of NO released from the cells was expressed in nmol/mg protein, and this value was used to calculate the percentage of control (% control) for comparison [19].

HepG2 cells were seeded 24-well plates at a cell concentration of 150,000 cells/0.5 mL, and following stable attachment, cells were incubated for 48 h with a medium containing different concentrations (concentration ranges being listed in the result table) of phytochemical/herbal extracts [dissolved in DMSO, 0.2% (v/v)]. After incubation, cells were rinsed with PBS-A and lysed with lysis buffer (0.3 mL per well; 50 mM Tris, 0.9% NaCl, 0.1% SDS, pH 7.3). The lysates were used to measure protein concentration and EPO levels with the Bradford protein dye and ELISA kit, respectively. The EPO level was expressed in mIU/mg protein, and this value was used to estimate the percentage of control for comparison [19].

1) Animal care

Adult female ICR mice were maintained under a 12-hour dark/light cycle at an ambient temperature of approximately 22˚C with ad libitum access to food and water. Experimental protocols were approved by the Research Practice Committee at the Hong Kong University of Science and Technology (AEP-2023-0062).

2) Splenocyte isolation

Splenic tissue from mice was processed by pressing through a stainless-steel mesh sieve with a glass pestle in 25 mL RPMI-1640 medium to achieve a single-cell suspension. This suspension was centrifuged at 400 ×g for 5 min, and the resulting pellet was washed once with RPMI medium. Red blood cells in the pellet were lysed using 4.5 mL of water, and the lysis was halted by adding 0.5 mL of 10X Hank’s Balanced Salt Solution (HBSS: 1.4 M NaCl, 53 mM KCl, 4.4 mM KH₂PO₄, 55.6 mM glucose and 3.36 mM Na₂HPO₄) and 5 mL RPMI-medium supplemented with 5% (v/v) HIFBS. After another round of centrifugation, the pellet was resuspended in RPMI-1640 medium supplemented with 10% HIFBS for cell counting, using 0.4% trypan blue. The cells were then adjusted to 5 × 10⁶ cells/mL, and aliquots (80 μL) of the cell suspension were seeded in each well of a 96-well plate [18].

3) Drug incubation and Con A/LPS stimulation in vitro

Phytochemical/herbal extracts (dissolved in DMSO) were added at a range of concentrations in μg/mL (being listed in the result tables). Con A/LPS was added at final concentrations of 0.5, 1, 2, and 4 μg/mL. Splenocytes were then cultured for 72 h at 37˚C in a humidified atmosphere of 5% CO₂ in air. Thereafter, the extent of cell proliferation was assessed.

4) MTT-based cell proliferation assay

An aliquot (10 μL) of MTT (5 mg/mL in PBS-A) was added to each well. After 4 h of incubation, 100 μL of solubilization buffer (10% sodium dodecyl sulfate, 45% dimethylformamide, pH 4.7 adjusted by glacial acetic acid) was added, and the mixtures were incubated in 5% CO₂ at 37˚C overnight to dissolve the colored crystals. The extent of splenocyte proliferation was determined by measuring the absorbance at 600 nm using a microplate reader. The extent of Con A/LPS-stimulated proliferation of isolated splenocytes was estimated by computing the AUC (by GraphPad Prism) of a graph plotting the percentage of initial absorbance (mean absorbance of cells stimulated with mitogen/mean absorbance of cells not stimulated with mitogen × 100%) against mitogen concentrations. The extent of mitogen-stimulated proliferation of isolated splenocytes was estimated by comparison with the control [18].

Data from various pharmacological assays were analyzed by computing the area under the curve (AUC, estimated by GraphPad Prism) for graphs plotting percent control values against concentrations, expressed as 1-, 2-, or 4-fold of the lowest effective concentration (determined by response value significantly higher than the control). The activity index was determined by the ratio of AUCsample to AUCcontrol. The Yin index (i.e. composite index) was calculated by summing the T cell (Con-A stimulated) and B cell (LPS-stimulated) indices, while the Blood index (i.e. composite index) was a summation of the EPO and NO indices.

Data, which were expressed as mean ± SD, were analyzed by Student’s t-test to detect significant differences between groups when p < 0.05.