Identification and quantitation of the actual active components in bamboo juice and its oral liquid by NMR and UPLC-Q-TOF-MS

Bamboo juice (zhuli) is the sap from fresh poles of bamboo species such as Phyllostachys glauca and Phyllostachys edulis, traditionally consumed as an herbal medicine and natural beverage in China. Bamboo juice oral liquids (BJOLs) are widely used for treating cough and phlegm and are commonly prescribed, including to children and pregnant women. Despite their popularity, few studies have rigorously characterized bamboo juice components or identified bioactive compounds responsible for antitussive effects. Prior reports via GC/MS suggest the presence of phenols, amino acids, inorganic elements, and organic acids, with some claiming amino acids and guaiacol as main ingredients. This study aims to qualitatively and quantitatively analyze the main components of fresh bamboo juice and commercial BJOLs to identify potential active compounds for cough relief and assess product composition and safety.

Published work on bamboo juice composition is limited. GC/MS-based analyses have detected phenols, amino acids, inorganic elements, and organic acids, and some reports cited amino acids and guaiacol as primary constituents. Clinical and popular use in China maintains that BJOLs are effective against cough and phlegm, leading to routine prescriptions. However, definitive bioactive components remain unidentified, and prior chemical analyses have not reconciled claimed pharmacological effects with measured constituents. This study builds on earlier qualitative reports by isolating and structurally confirming 26 compounds and providing quantitative profiles, including an assessment of added preservatives and carbohydrate content.

- Sample collection: Liquid juice of Phyllostachys edulis was collected in August 2018 (Tonggu County, Jiangxi, China). Nine BJOL products were purchased from the Chinese market (with specified NMPA numbers). - Extraction and isolation: 100 kg of juice was concentrated (3.80 kg residue), sequentially extracted with ethyl acetate and water-saturated n-butanol to give respective fractions. Fractions underwent macroporous resin (HP-20) adsorption with gradient ethanol elution, Sephadex LH-20 gel filtration, and C18 reversed-phase preparative HPLC. The ethyl acetate fraction was further separated via silica gel chromatography (CH2Cl2/MeOH gradient) and C18 prep HPLC. Twenty-six compounds were isolated and identified by comparison of UV, NMR (1H/13C; Bruker AV-500), and HR-ESI-MS data with literature. - Quantitative analysis of bamboo juice components: UPLC-Q-TOF-MS (Agilent 6540; RRHD Eclipse Plus C18, 150×2.1 mm, 1.8 µm) using ACN (B)–0.1% formic acid in water (A) gradient (0–5 min 5% B; 5–35 min 5–55% B), 0.25 mL/min, 40°C, positive ESI mode. Purified compounds 1–26 (≥98% purity) served as external standards at defined stock concentrations. To avoid detector overloading, compounds were grouped for 10× or 100× dilution prior to injection. Calibration curves established across multiple concentrations; method validation (LOD/LOQ by S/N=3/10; precision via intra-/inter-day RSDs; accuracy via spike-recovery at two dilution levels) followed standard protocols. - Preservatives in BJOLs: Overloaded peaks absent in fresh juice but present in BJOLs were isolated by semi-preparative HPLC, identified by NMR/MS as benzoic acid, ethylparaben, and sorbic acid. Due to high levels, quantification used HPLC-UV (Waters 2695-2996; YMC-PACK ODS-AQ C18; MeOH vs water+0.5% acetic acid gradient 5–95% MeOH over 30 min; 1 mL/min; 25°C). Detection at 228 nm (benzoic acid), 255 nm (ethylparaben), 259 nm (sorbic acid). External calibration from mixed standards. - Carbohydrate analysis: Total and monosaccharide composition (arabinose, glucose, xylose, mannose) were measured by ion chromatography with amperometric detection after sulfuric acid hydrolysis (72% H2SO4, 105°C, 2 h), neutralization, and filtration. Mobile phase: water with 2.0 mM NaOH and 0.5 mM NaOAc, 1 mL/min, 60 min run. Calibration across six concentrations. - Data analysis: Results expressed as mean ± SD (n=3). SPSS 16.0 used for ANOVA. Method performance included LOD/LOQ, linearity, precision (intra-/inter-day), and recovery.

- Isolation/identification: Twenty-six compounds were isolated from fresh bamboo juice and structurally confirmed (including tachioside (12), 4-hydroxy-3,5-dimethoxyphenyl-β-D-glucopyranoside (13), and (+)-lyoniresinol-3α-O-β-D-glucopyranosyl (22)). - Main constituents in fresh bamboo juice (UPLC-Q-TOF-MS): Compounds 12, 13, and 22 were most abundant at 35.33 ± 0.10 mg/L, 20.71 ± 0.11 mg/L, and 47.15 ± 0.06 mg/L, respectively. Many other phenolics and lignan glycosides were present at lower levels. - Estimated intake: A standard adult daily dose of 60 mL fresh juice translates to ~2.8 mg or less per major compound, suggesting low likelihood of direct antitussive/anti-inflammatory efficacy at these doses. - BJOL vs fresh juice composition: BJOLs showed significantly lower concentrations of native bamboo constituents than fresh juice, consistent with dilution. Composition varied markedly among products. - Preservatives in BJOLs (HPLC-UV): Three preservatives were identified and quantified at high levels in BJOLs, but absent in fresh juice. Maximum measured concentrations: benzoic acid 2754.25 mg/L, ethylparaben 471.44 mg/L, sorbic acid 1797.48 mg/L. These levels greatly exceeded those of native bamboo compounds and surpass FAO/WHO guidelines (benzoic acid 0–5 mg/kg; ethylparaben 5–10 mg/kg; sorbic acid 0–25 mg/kg). Manufacturers confirmed preservatives were deliberately added. - Carbohydrates: Fresh bamboo juice contained very high total carbohydrates (191,129.25 mg/L), primarily glucose and arabinose; BJOLs contained 10,967.70–33,230.78 mg/L total carbohydrates with variable monosaccharide profiles, further supporting dilution in commercial products. - Absence of guaiacol: Guaiacol was not detected in fresh juice or BJOLs. - Method validation: LOD/LOQ suitable for all analytes; intra-/inter-day precision RSDs <3%; average recoveries >85%; calibration showed good linearity for target analytes.

The study set out to identify and quantify components in fresh bamboo juice and BJOLs to pinpoint potential antitussive agents. While 26 phenolic and lignan-type compounds were identified, their low concentrations in a typical consumption volume suggest insufficient dosing to account for clinically meaningful cough relief, and these compounds are not known for strong anti-inflammatory activity. In contrast, BJOLs contained high and potentially hazardous levels of antimicrobial preservatives (benzoic acid, ethylparaben, sorbic acid), which could plausibly contribute to perceived cough-relief effects through antimicrobial actions rather than intrinsic bamboo-derived actives. Marked compositional differences between fresh juice and BJOLs, together with high carbohydrate content as the predominant native components, suggest that traditional use may have provided nutritional (sugar) benefits rather than specific pharmacological effects. The findings raise important safety concerns for BJOLs in the Chinese market and imply that added preservatives, rather than bamboo constituents, may underlie observed efficacy, challenging assumptions about traditional formulations and highlighting the need for regulatory oversight.

This work provides the first comprehensive isolation and identification of 26 compounds from fresh bamboo juice and quantifies their levels alongside preservatives and carbohydrates in both fresh juice and nine BJOLs. Fresh bamboo juice’s major native constituents (compounds 12, 13, 22) occur at tens of mg/L, with total carbohydrate content far exceeding all other components. Commercial BJOLs are compositionally distinct, likely diluted, and frequently contain excessively high levels of benzoic acid, ethylparaben, and sorbic acid that surpass FAO/WHO limits, posing potential health risks. No evidence was found for specific trace actives with antitussive efficacy; the perceived benefits of BJOLs may relate to added preservatives’ antimicrobial action and carbohydrate content. Future research should systematically reevaluate traditional functional foods with modern analytical and pharmacological methods, establish safety and quality standards for natural product-based beverages/medicines, and investigate bioactivity at physiologically relevant doses.

- The study did not include direct pharmacological or clinical testing of antitussive or anti-inflammatory activity; conclusions about efficacy are inferential based on chemical content. - Only nine BJOL products were analyzed; while representative, broader market sampling would strengthen generalizability. - Quantification focused on identified compounds; uncharacterized constituents present at trace levels might have been missed. - Differences in raw material selection and processing among manufacturers were not controlled, contributing to variability and complicating attribution of effects to specific components.