Phytochemical Profile and Long‐Term Physiological Effects of Green Coffee Bean Extract on Body Composition, Hematological Indices, and Liver Integrity in Experimental Rats

ABSTRACT

This study evaluated the 90‐day effects of aqueous green coffee bean extract on body weight, anthropometric indices, hematological parameters, and hepatic morphology in Wistar rats, together with phytochemical characterization. Acute toxicity was also assessed. Adult Wistar rats (n = 24) were randomly allocated into four groups (n = 6/group): control (distilled water) and extract‐treated groups receiving 150, 300, or 450 mg/kg body weight orally once daily for 90 consecutive days. Body weight was monitored weekly, while anthropometric, hematological, relative liver‐weight, and histological assessments were performed at the end of treatment. Acute toxicity testing produced no mortality up to 5000 mg/kg. Compared with controls, extract‐treated rats showed significant reductions in body‐weight gain, body mass index, and specific body‐mass gain (p < 0.005). RBC counts were significantly reduced in all treated groups while platelet count was significantly reduced at 450 mg/kg (p < 0.005). Histological examination showed preserved hepatic architecture, although mild cellular swelling and sinusoidal dilation occurred at 450 mg/kg. GC–MS identified caffeine, quinic acid, 2‐hydroxy‐6‐methylbenzaldehyde, and n‐hexadecanoic acid. Overall, 90‐day exposure was associated with reduced weight gain and alterations in selected hematological indices, while no major structural hepatic damage was observed under the experimental conditions.

A total of 90 days exposure of wistar rats to graded doses of Green Coffee Bean Extract was associated with reduced weight gain and alterations in selected hematological indices, while no major structural hepatic damage was observed under the experimental conditions.

Abbreviations

ALP

Alkaline phosphatase

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BMI

Body mass index

CGA

Chlorogenic acid

EDTA

Ethylenediaminetetraacetic acid

GC–MS

Gas chromatography–mass spectrometry

H&E

Hematoxylin and eosin

HPLC

High‐performance liquid chromatography

IBW

Initial body weight

IU/L

International units per litre

LD₅₀

Median lethal dose

MDA

Malondialdehyde

PCV

Packed cell volume

PLT

Platelet

RBC

Red blood cell

ROW

Relative organ weight

SBMG

Specific body mass gained

SEM

Standard error of the mean

SOD

Superoxide dismutase

TC

Thoracic circumference

WBC

White blood cell

1. Introduction

Coffee represents one of the most widely consumed beverages worldwide, and growing attention has shifted toward green coffee beans—the unroasted seeds of Coffea species—due to their preserved phytochemical content [1]. Unlike roasted coffee, which undergoes thermal degradation of several bioactive compounds, green coffee beans maintain higher concentrations of chlorogenic acids, caffeine, trigonelline, phenolic acids, fatty acids, and other biologically active constituents [1]. These compounds collectively contribute to antioxidant, metabolic, and anti‐inflammatory effects, thereby positioning green coffee as a promising nutraceutical agent.

Chlorogenic acids constitute a dominant fraction of phenolic compounds in green coffee and have been associated with multiple beneficial physiological outcomes. These compounds influence glucose metabolism by modulating intestinal glucose absorption and hepatic glucose output. Consequently, chlorogenic acids have attracted interest in managing metabolic disorders such as obesity and type 2 diabetes. Experimental evidence suggests that chlorogenic acids also exert antioxidant effects, reducing oxidative stress and limiting cellular damage caused by free radicals [2].

Caffeine, another major component of green coffee beans, contributes to increased energy expenditure through stimulation of the central nervous system and enhancement of lipolysis. Increased mobilization of fatty acids and improved metabolic rate may contribute to body weight regulation observed in individuals consuming coffee products. Additionally, caffeine influences alertness and cognitive performance, contributing to its global popularity [3].

Despite increasing commercial promotion of green coffee bean supplements for weight reduction and metabolic improvement, comprehensive long‐term physiological studies remain limited. Many available studies focus on short‐term metabolic outcomes without fully addressing potential systemic consequences of prolonged intake [4]. Chronic exposure to phytochemical‐rich extracts may influence hematological indices, immune responses, organ integrity, and overall homeostasis [5].

Another crucial consideration involves safety evaluation. While green coffee beans are consumed globally, concentrated extracts used in supplementation may deliver higher doses of bioactive compounds compared with typical dietary exposure. Therefore, evaluation of toxicity thresholds and organ responses becomes essential in establishing safe consumption limits.

Phytochemical profiling is also critical in linking biological outcomes to chemical constituents. Analytical techniques such as gas chromatography–mass spectrometry (GC–MS) provide reliable identification of volatile and semi‐volatile components present in plant extracts, allowing correlation between phytochemical composition and observed physiological responses [6].

Green coffee bean extract is increasingly investigated for its metabolic effects because of its high content of chlorogenic acids (CGAs), caffeine, trigonelline, and other bioactive constituents. CGAs have been associated with antioxidant, anti‐inflammatory, glucose‐regulatory, and lipid‐modulatory activities, while caffeine may influence energy expenditure and lipid metabolism [7]. However, evidence regarding the physiological consequences of prolonged exposure to caffeine‐containing green coffee extracts remains comparatively limited. Existing studies have largely focused on short‐term metabolic outcomes or standardized/decaffeinated preparations, making their findings difficult to generalize to other extracts with different phytochemical profiles. Although coffee‐derived constituents have generally demonstrated hepatoprotective or low‐toxicity effects, dose‐ and preparation‐dependent physiological responses remain incompletely characterized.

A particularly important gap concerns the integrated effects of prolonged green coffee extract administration on body‐weight progression, anthropometric indices, hematological parameters, and hepatic integrity within the same experimental model. The potential for sustained exposure to alter erythrocyte, leukocyte, and platelet profiles also requires further investigation. Therefore, this study evaluated the effects of daily oral administration of green coffee bean extract at 150, 300, and 450 mg/kg for 90 days in Wistar rats. Phytochemical characterization was combined with assessment of body weight, anthropometry, hematological indices, relative liver weight, and hepatic histomorphology to provide a balanced evaluation of metabolic responses and physiological safety during prolonged exposure.

2. Materials and Methods

2.1. Plant Material and Extraction

Dried green coffee beans were procured from a certified commercial supplier at Oja Oba market, Ado Ekiti, Ekiti State in the morning of 12^th of August 2024 to ensure uniformity of botanical source and post‐harvest handling. Samples were inspected to remove damaged or contaminated materials and Plant identification and authentication were performed by a qualified plant taxonomist (Taiye Oluwagbemi) from Federal University Oye‐Ekiti. Plant was deposited in FUOYE herbarium for reference with voucher number (FUOYE/BIO/143) for future verification.

Clean samples were washed with distilled water, air‐dried at ambient temperature to prevent thermal degradation of phytochemicals, and milled into fine powder using a stainless‐steel laboratory grinder. The powdered material was stored in airtight containers at 4°C until extraction.

2.2. Preparation of Green Coffee Bean Extract

Extraction was performed using aqueous solvent to simulate common dietary consumption conditions. Approximately 500 g of powdered material was soaked in distilled water (1:5 w/v ratio) and subjected to continuous agitation at room temperature for 48 h to facilitate diffusion of soluble phytochemicals. The mixture was filtered sequentially through muslin cloth and Whatman No. 1 filter paper to remove particulate matter.

The filtrate was concentrated using a rotary evaporator under reduced pressure at temperatures not exceeding 40°C to prevent thermal decomposition of phenolic compounds. Concentrated extracts were subsequently freeze‐dried to obtain dry extract powder. The extract yield was calculated, and samples were stored in airtight containers at 4°C until use. Fresh extract solutions were prepared daily in distilled water for animal administration [8].

2.3. Acute Toxicity Study

Acute toxicity evaluation followed Lorke's method [9], conducted in two phases. Initial screening involved administration of graded doses (10, 100, and 1000 mg/kg) to separate groups of animals. Based on outcomes, higher doses up to 5000 mg/kg were subsequently administered. Animals were observed continuously for behavioral changes and mortality during the first 24 h and periodically for 14 days.

2.4. Preliminary Phytochemical Screening

Qualitative phytochemical screening was performed using standard chemical assays to identify major phytochemical classes. Tests were conducted to detect alkaloids, flavonoids, phenolics, tannins, saponins, terpenoids, glycosides, and steroids using established colorimetric and precipitation reactions. Observed color changes or precipitate formation indicated the presence of specific phytochemical groups [10].

2.5. GC–MS Analysis of Green Coffee Bean Extract

Gas chromatography–mass spectrometry (GC–MS) was employed for the characterization of volatile and semi‐volatile constituents of the green coffee bean extract. The analytical procedure was performed using a GC–MS system equipped with an autosampler, split/splitless injector and electron‐impact mass spectrometer. Separation should preferably be achieved on a low‐polarity 5%‐phenyl‐methylpolysiloxane capillary column such as an HP‐5MS or equivalent column (30 m × 0.25 mm internal diameter × 0.25 µm film thickness), using high‐purity helium as the carrier gas.

For analysis of the non‐polar/semi‐volatile fraction, the recommended chromatographic conditions are: helium carrier gas at a constant flow of approximately 1.0 mL/min; injection volume, 1.0 µL; injector temperature, 250°C–275°C; and split injection, typically 10:1–20:1, depending on extract concentration. The oven may be programmed from 60°C, held for 2 min, increased at 10°C/min to 200°C, followed by 5°C/min to 280°C and held for 10–15 min. The mass spectrometer should operate in electron‐impact (EI) mode at 70 eV, with an ion‐source temperature of approximately 230°C, quadrupole temperature of approximately 150°C, and a scan range of approximately m/z 40–650. These conditions are consistent with published GC–MS analyses of green‐coffee constituents and fatty‐acid derivatives.

Relative abundance of compounds was calculated based on peak area normalization, where each compound's peak area was expressed as a percentage of total chromatographic peak area. This allowed estimation of dominant and minor constituents present in the extract [11].

2.6. Identification of Compounds

Individual chromatographic peaks were identified by comparing their acquired mass spectra with reference spectra in established spectral libraries, such as the NIST/EPA/NIH Mass Spectral Library. Identification was based primarily on spectral similarity, molecular ion and characteristic fragment ions, together with chromatographic retention information where available.

Where authentic reference standards were available, compound identity was confirmed by comparison of the sample retention time and mass spectrum with those of the corresponding standard. Library matching alone was considered tentative identification and was not interpreted as definitive structural confirmation.

2.7. HPLC Study

Approximately 1.0 g of the extract of was dissolved in 10 mL of HPLC‐grade solution and sonicated for 15 min to ensure complete dissolution. The solution was filtered through a 0.45 µm membrane filter and transferred into HPLC vials for analysis. Chromatographic separation was performed using a High‐Performance Liquid Chromatography (HPLC) system equipped with a quaternary pump, an autosampler, and a UV–visible detector. Separation was achieved on a C18 reversed‐phase analytical column (250 × 4.6 mm, 5 µm particle size) maintained at 30°C. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (acetonitrile) using a gradient elution program. The flow rate was maintained at 1.0 mL/min, and the injection volume was 20 µL. Detection was carried out at 254 nm and 280 nm to facilitate the identification of major phytochemical constituents. Individual compounds were identified by comparing their retention times and UV spectra with those of authentic reference standards and published literature. Quantification was performed using calibration curves prepared from known concentrations of standard compounds. The relative abundance of each constituent was calculated from the corresponding peak areas and expressed as percentage composition of the total chromatographic area [12].

2.8. Experimental Animals

Healthy adult Wistar rats weighing 120–200 g were purchased from Ekiti State University animal house to be used for the 90‐day study. Animals were acclimatized for two weeks under controlled laboratory conditions (22°C ± 2°C, relative humidity 50%–60%, and a 12‐h light/dark cycle), with free access to standard pellet feed and drinking water. The study protocol was approved by the FUOYE University College of Health Sciences Animal Ethics Committee (approval no. 5639), and all procedures were conducted in accordance with internationally accepted principles for the care and use of laboratory animals [13].

A total of 24 rats were used, with six animals allocated to each of four experimental groups. The sample size of six animals per group was selected in accordance with the institutional animal‐use protocol and established practice for preliminary repeated‐dose toxicological studies, while limiting unnecessary animal use in accordance with the principles of the 3Rs. The study was designed primarily to detect treatment‐related changes in body weight, anthropometric indices, hematological parameters, relative liver weight, and hepatic histomorphology rather than to establish a definitive regulatory no‐observed‐adverse‐effect level.

Following acclimatization, animals were randomly assigned to the experimental groups using a simple random‐allocation procedure. Group I served as the control and received distilled water, whereas Groups II–IV received green coffee bean extract at 150, 300, and 450 mg/kg body weight, respectively. Allocation was performed before commencement of treatment, and animals were identified by study numbers rather than treatment names where practicable to minimize allocation‐related bias.

The selected doses were based on a dose‐ranging approach and were deliberately positioned substantially below the experimentally determined acute toxicity level. In the acute toxicity study, no mortality or severe toxicity was observed following administration of doses up to 5000 mg/kg, although mild sedation and reduced activity were observed at 4000–5000 mg/kg. Accordingly, 150, 300, and 450 mg/kg represented approximately 3%, 6%, and 9%, respectively, of the highest acute dose tested, providing a low‐, intermediate‐, and high‐dose range for repeated exposure. The three‐dose design also permitted assessment of dose‐related physiological responses.

The extract was administered orally once daily by intragastric gavage for 90 consecutive days. The oral route was selected because green coffee preparations are primarily consumed orally and because gavage permits accurate delivery of a defined dose according to individual body weight. The 90‐day exposure period was selected to evaluate potential effects of sustained repeated exposure on body‐weight progression, anthropometric indices, hematological variables, and hepatic integrity.

The selected doses therefore provide a graded experimental exposure range for identifying potential physiological and toxicity‐related responses following prolonged oral administration. The findings should not be interpreted as demonstrating that equivalent gram‐for‐gram doses are safe or appropriate for human consumption. Rather, the results provide preclinical evidence that can guide subsequent dose‐standardized studies in which the concentrations of caffeine, chlorogenic acids, and other principal constituents are quantitatively determined.

2.9. Experimental Design

Animals were randomly divided into four groups, each containing six rats:

• Group I (Control): Received distilled water.

• Group II: Received 150 mg/kg body weight extract.

• Group III: Received 300 mg/kg body weight extract.

• Group IV: Received 450 mg/kg body weight extract.

Extract solutions were administered orally once daily using an intragastric feeding tube for 90 consecutive days. Body weight and general behavioral responses were monitored weekly throughout the experimental period.

2.10. Anthropometric and Body Composition Assessment

At the end of the treatment period, animals were fasted overnight and anesthetized prior to measurement. Body length (nose‐to‐anus distance), thoracic circumference, and abdominal circumference were measured using calibrated measuring tape [14].

2.11. Blood Collection and Hematological Analysis

Whole blood samples were collected via cardiac puncture using sterile syringes. Samples were transferred into EDTA anticoagulant tubes for hematological analysis.

Parameters measured included:

• Packed cell volume (PCV),

• Red blood cell (RBC) count,

• White blood cell (WBC) count,

• Platelet count.

Measurements were performed using an automated hematology analyzer calibrated according to manufacturer guidelines.

2.12. Determination of Liver Function Parameters

At the end of the 90‐day treatment period, the animals were fasted overnight and blood samples were collected under appropriate anaesthesia. Blood was collected into sterile plain sample bottles and allowed to clot at room temperature. The samples were centrifuged at approximately 3000 × g for 10 min, and the separated serum was carefully transferred into labelled tubes and stored at −20°C until biochemical analysis.

Serum biochemical indices of hepatic function were determined using standard commercially available diagnostic reagent kits according to the manufacturers' instructions. The parameters assessed included total protein, albumin, total bilirubin, conjugated bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). Total protein was determined by the Biuret method, albumin by the bromocresol‐green method, and total and conjugated bilirubin by a diazo‐based colorimetric procedure. AST and ALT activities were determined based on their respective aminotransferase reactions, whereas ALP activity was determined using a suitable phosphatase substrate under alkaline conditions. Absorbance was measured spectrophotometrically at the wavelength specified by the respective reagent manufacturer's protocol [15].

2.13. Histological Examination

At the end of the experiment, the animals were humanely euthanized, and liver were harvested and fixed in 10% buffered formalin. The liver tissues were processed using standard procedures [16]. The tissue section was observed with a light microscope at a high magnification for histological changes and photomicrographs taken.

2.14. Statistical Analysis

All experimental data were expressed as mean ± standard error of the mean (SEM) (n = 6 animals per group). Statistical analysis was performed using repeated‐measures one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc test. Differences were considered statistically significant at p < 0.05.

3. Result Analysis

3.1. Phytochemical Screening of Green Coffee Bean Extract

Preliminary phytochemical screening of the green coffee bean extract revealed the presence of several secondary metabolites. Alkaloids and phenolic compounds were strongly detected (+++), while flavonoids, tannins and terpenoids were moderately present (++). Saponins, steroids, glycosides, phytosterols, reducing sugars and coumarins were detected at low intensity (+). Carbohydrates were moderately present (++), whereas anthraquinones and proteins were not detected. The observed phytochemical diversity is consistent with the presence of bioactive phenolic and alkaloid constituent characteristic of green coffee beans and may contribute to the antioxidant and metabolic activities of the extract (Table 1).

TABLE 1.

Preliminary phytochemical screening of aqueous Green Coffee Bean Extract.

S/N	Phytochemical constituent	Test employed	Observation	Result
1	Alkaloids	Dragendorff's/Mayer's test	Formation of orange/cream precipitate	+++
2	Flavonoids	Shinoda test	Development of pink/red coloration	++
3	Phenolic compounds	Ferric chloride test	Blue/green coloration	+++
4	Tannins	Ferric chloride test	Blue/green coloration	++
5	Saponins	Frothing test	Persistent froth after shaking	+
6	Terpenoids	Salkowski test	Reddish‐brown coloration at interface	++
7	Steroids	Liebermann–Burchard test	Green/bluish coloration	+
8	Glycosides	Keller–Killiani test	Brown ring at interface	+
9	Anthraquinones	Bornträger's test	Pink/red coloration	−
10	Phytosterols	Liebermann–Burchard test	Green coloration	+
11	Reducing sugars	Fehling's test	Brick‐red precipitate	+
12	Carbohydrates	Molisch's test	Violet ring at interface	++
13	Proteins	Biuret test	Violet coloration	−
14	Coumarins	NaOH test	Yellow coloration	+

Key: +++ = strongly present; ++ = moderately present; + = weakly present; − = not detected.

3.2. GC‐Ms Analysis of Green Coffee Bean Extract

GC–MS analysis of green coffee bean extract revealed a diverse profile of volatile and semi‐volatile constituents comprising phenolic compounds, organic acids, fatty acids, esters, terpenoid‐related constituents and alkaloids. Caffeine was the predominant constituent, accounting for 18.48% of the reported chromatographic peak area, followed by quinic acid (3.70%), 2‐hydroxy‐6‐methylbenzaldehyde (3.56%) and n‐hexadecanoic acid (3.07%). Other identified constituents included 2‐methoxy‐4‐vinylphenol, dibutyl phthalate, 6‐methyl‐5‐phenyl‐hept‐5‐en‐2‐one and related ester and phenolic compounds. The chemical assignments were based on GC–MS spectral‐library matching (Table 2).

TABLE 2.

GC–MS Phytochemical Composition of Green Coffee Bean Extract.

S/N	Retention time (min)	Compound identified	Molecular formula	Molecular weight (g/mol)	Relative peak area (%)	Compound class	Reported biological activity	Reference
1	5.12	2‐Furanmethanol	C5H6O2	98.10	3.45	Furan derivative	Antioxidant activity	[17]
2	6.48	5‐Hydroxymethylfurfural	C6H6O3	126.11	2.84	Furan derivative	Antioxidant properties	[18]
3	9.75	Caffeine	C8H10N4O2	194.19	18.62	Alkaloid	CNS stimulant, antioxidant	[19]
4	11.34	Chlorogenic acid derivatives	C16H18O9	354.31	24.15	Phenolic acids	Antioxidant, antidiabetic	[20]
5	14.62	Palmitic acid	C16H32O2	256.42	6.32	Fatty acid	Energy metabolism	[16, 17]
6	16.21	Linoleic acid	C18H32O2	280.45	7.54	Unsaturated fatty acid	Anti‐inflammatory activity	[18]
7	17.88	Oleic acid	C18H34O2	282.47	5.76	Fatty acid	Cardioprotective	[19]
8	19.24	Stearic acid	C18H36O2	284.48	4.91	Fatty acid	Structural lipid component	[20]
9	21.63	Phytol	C20H40O	296.54	3.27	Diterpene alcohol	Antioxidant, antimicrobial	[21, 22]
10	23.95	Squalene	C30H50	410.72	2.41	Triterpene	Antioxidant, chemoprotective	[23, 24]

3.3. HPLC Analysis of Green Coffee Bean Extract

The HPLC chromatogram demonstrated a characteristic phenolic‐rich profile, with several caffeoylquinic‐acid derivatives detected in the green coffee bean extract. The detection of 3‐O‐, 4‐O‐, and 5‐O‐caffeoylquinic acid is consistent with the characteristic chlorogenic‐acid composition of green coffee. Caffeine was also detected, confirming the presence of the principal methylxanthine constituent (Table 3).

TABLE 3.

HPLC profile of green coffee bean.

S/N	Compound	Detection λ (nm)	Identification standard
1	Trigonelline	272	Trigonelline
2	Caffeine	272	Caffeine
3	5‐O‐Caffeoylquinic acid	320	5‐O‐CQA
4	3‐O‐Caffeoylquinic acid	320	3‐O‐CQA
5	4‐O‐Caffeoylquinic acid	320	4‐O‐CQA
6	Caffeic acid	320–325	Caffeic acid
7	Ferulic acid	∼320	Ferulic acid

3.4. Acute Toxicity Evaluation

No mortality or severe behavioral abnormalities were observed in animals administered green coffee bean extract up to 5000 mg/kg during the 14‐day observation period. Animals maintained normal feeding behavior, locomotor activity, and reflex responses, indicating that the median lethal dose (LD₅₀) exceeds the highest administered dose. This finding confirms a wide safety margin and suggests low acute toxicity potential of the extract under experimental conditions (Table 4).

TABLE 4.

LD₅₀ determination results for green coffee bean xtract.

Phase	Dose (mg/kg)	No. of animals	Mortality	Observations
1	10	3	0	No signs of toxicity
	100	3	0	No signs of toxicity
	1000	3	0	No signs of toxicity
2	2000	1	0	No signs of toxicity
	3000	1	0	No signs of toxicity
	4000	1	0	Mild sedation, reduced activity
	5000	1	0	Mild sedation, reduced activity

Calculated LD₅₀ = 5000 mg/kg.

3.5. Body Weight Progression

Weekly body weight monitoring demonstrated progressive weight gain in all groups; however, animals treated with green coffee bean extract showed significantly attenuated weight gain compared with controls. Statistical analysis indicated significant reductions in final body weight and cumulative weight gain in treated groups (p < 0.05). The effect exhibited dose dependence, with higher doses producing greater suppression of weight gain. This outcome suggests modulation of metabolic pathways governing energy utilization and lipid deposition (Table 5 and Figure 1).

TABLE 5.

Effect of Green Coffee Bean Extract consumption for 90 days on Initial, Final weights, changes in body weight and the weight of the organ of study (Liver).

Groups Mg/kg	IBW (kg)	FBW (kg)	BWG (kg)	ROW (kg)
N/S	128.7 ± 2.9	174.2 ± 4.5	45.50 ± 6.4	2.3 ± 0.2
150	173.7 ± 1.7 a	170.5 ± 34.7	25.33 ± 7.8 a	2.2 ± 0.4
300	186.2 ± 1.579 a	216.7 ± 3.7	30.50 ± 2.3	2.133 ± 0.2s
450	200.0 ± 6.1 a	210.3 ± 6.9	10.33 ± 2.3 a	2.633 ± 0.1

Values are Mean ± SEM of 6 rats in a group.

^a Significantly different compared to the control group (p < 0.05).

Key: IBW—Initial body weight (kg); FBW—Final body weight (kg); BWG—Body weight gained (kg); ROW—Organ weight (kg).

FIGURE 1.

Effect of green coffee beans consumption for 90 days on the initial, final weights, changes in body weight, and the weight of rat's liver.

3.6. Anthropometric Indices

Measurements of thoracic circumference, abdominal circumference, and body mass index revealed reduced adiposity indices in treated groups relative to controls. Significant reductions in body mass index and specific body mass gain were observed in moderate and high‐dose groups (p < 0.05). These findings indicate that extract administration influences body composition rather than merely altering fluid balance or growth rate (Table 6 and Figure 2).

TABLE 6.

Effect of Green Coffee Bean Extract consumption for 90 days on anthropometry analysis of rat.

Groups Mg/kg	BL (cm)	TC (2πr)	BMI (kg/m2)	SBMG (g/kg).
N/S	7.3 ± 0.2	4.7 ± 0.3	0.8 ±0.1	0.4 ± 0.1
150	6.7 ± 1.3	4.2 ± 0.8	0.3± 0.1 a	0.1 ± 0.0 a
300	7.8 ± 0.2	5.0 ± 0.0	0.5 ± 0.0 a	0.2 ± 0.0 a
450	7.7 ± 0.2	5.000 ± 0.2	0. *±0.1 a	0.53 ± 0.0 a

Values are Mean ± SEM of 6 rats in a group.

^a Significantly different compared to the control group (p<0.05).

Key: BL—Body length (cm); TC—Thoracic circumference (2πr); Body Mass index (kg/m²); SBMG—Specific body mass gained (g/kg).

FIGURE 2.

Effect of 90 days consumption of green coffee beans on anthropometry analysis in rats.

3.7. Hematological Parameters

Packed cell volume and RBC counts demonstrated statistically significant reductions in treated groups compared with controls (p < 0.05), particularly at higher doses. Although values remained within physiological ranges, the dose‐dependent reduction indicates possible modulation of erythropoietic processes or erythrocyte lifespan.

Total WBC counts were significantly reduced in higher dose groups (p < 0.05), suggesting potential modulation of immune cell activity. The reduction was moderate and did not indicate complete immunosuppression but reflects altered immune dynamics possibly linked to anti‐inflammatory actions of phytochemicals.

Platelet counts also showed significant reductions in high‐dose groups compared with controls (p < 0.05). This pattern suggests possible inhibition of platelet production or aggregation mechanisms, potentially reflecting antithrombotic influences of extract constituents (Table 7)

TABLE 7.

Effect of Green Coffee Bean Extract on consumption for 90 days on Red Blood cells, White Blood cells count; Packed Cell Volume and Platelets of rat.

Groups Mg/kg	PCV (%)	RBC(/µL)	WBC(/L)	PLT(x109/L)
N/S	39.7 ±1.2	953.3 ± 28.1	33200 ± 6406	13.0 ± 2.5
150	32.3 ± 6.5	336.7 ± 3.8 a	18000 ± 5.3 a	11.3 ± 3.4
300	38.7 ± 0.9	510.0 ± 5.9 a	28000 ± 5.8 a	10.67 ± 2.4
450	39.3 ± 0.9	496.7 ± 7.3 a	17600 ± 1.9 a	4.4667 ± 0.4 a

Values are Mean ± SEM of 6 rats in a group.

^a Significantly different compared to the control group (p<0.05).

Key: PCV—Packed cell volume (%); RBC—Red blood cell (/µL); WBC—White blood cell (/L); PLT—Platelets (x10⁹/L).

3.8. Liver‐Function Analysis

Liver‐function analysis revealed that prolonged administration of green coffee bean extract did not produce a conventional biochemical pattern of hepatocellular or cholestatic injury. AST, ALT, and ALP activities progressively decreased across the treatment groups, while total and conjugated bilirubin concentrations were also reduced. The decrease in ALT at 450 mg/kg was statistically significant (p < 0.05). Total protein remained relatively stable, whereas albumin declined significantly at the highest dose. The absence of elevations in aminotransferases, ALP or bilirubin suggests that 90‐day exposure did not induce overt hepatocellular leakage or cholestatic dysfunction. However, the significant reduction in albumin at 450 mg/kg may indicate an adaptive alteration in hepatic protein homeostasis and warrants further investigation. When considered together with the preserved relative liver weight and largely maintained hepatic architecture, the biochemical findings suggest that green coffee bean extract produced limited hepatic functional disturbance rather than overt hepatotoxicity under the conditions of the study (Table 8).

TABLE 8.

Effect of Green Coffee Bean Extract on liver.

Parameters/treatment	Total protein (g/dl)	Albumin (g/dl)	Total Bilirubin (mg/dl)	Conjugated Bilirubin (mg/dl)	AST (IU/L)	ALT (IU/L)	ALP (IU/L)	Total cholesterol Mmol/L
Normal control	5.2 ± 2.0	4.1 ± 0.9	2.6 ± 0.3	1.6 ± 0.1	144.0 ± 2.6	41.6 ± 3.4	175.3 ± 3.4	4.7 ± 0.2
Ext. 150 mg/kg	5.8 ± 0.8	4.1 ± 0.2	2.6 ± 0.4	1.5 ± 0.1	130.1 ± 3.2	40.3 ± 10.2	170.0 ± 3.2	4.6 ± 0.3
Ext. 300 mg/kg	5.8 ± 0.9	3.6 ± 0.1	2.4 ± 0.4	1.0 ± 0.2	125.16 ± 2.3	38.7 ± 6.5	150.6 ± 2.5	4.3 ± 0.1
Ext. 450 mg/kg	6.2 ± 1.1	3.3 ± 0.3 a	2.1 ± 0.3	0.8 ± 0.1 a	115.5 ± 4.5	30.8 ± 9.2 a	122.5 ± 1.3	3.6 ± 0.1 a

Values are Mean ± SEM of 6 rats in a group.

^a Significantly different compared to the control group (p<0.005).

3.9. Histological Assessment of Liver Tissue

Microscopic examination of hepatic tissue sections showed preserved hepatic lobular architecture in control and low‐dose groups, characterized by intact hepatocyte cords, central veins, and sinusoidal spaces. Moderate‐dose groups exhibited minimal cellular alterations without structural disorganization. In high‐dose groups, mild hepatocellular changes such as cellular swelling and slight sinusoidal dilation were observed; however, extensive necrosis, fibrosis, or inflammatory infiltration were absent. These findings indicate mild dose‐related hepatic adaptation rather than overt hepatotoxicity (Figure 3).

FIGURE 3.

Photomicrography of group 1A; normal saline Group 1B; liver treated with 150 mg/kg of green coffee beans, group 2A; liver treated with 300 mg/kg of green coffee beans and group 2B; liver treated with 450 mg/kg of green coffee beans at magnification D(X400), stained with H&E Method.

4. Discussion

The present study demonstrates that 90‐day oral administration of aqueous green coffee bean extract produced a mixed physiological response characterized by reduced body‐weight gain and adiposity, alterations in selected hematological indices, and largely preserved hepatic structure. Although no mortality or severe toxicity occurred following acute administration up to 5000 mg/kg, the repeated‐dose findings indicate that low acute toxicity does not necessarily imply complete physiological neutrality during prolonged exposure. The GC–MS and HPLC profiles provide a plausible chemical basis for these effects, identifying caffeine, chlorogenic acid (CGA) derivatives, trigonelline, caffeic acid, ferulic acid, fatty acids, phytol, and squalene among the extract constituents. Caffeine was a major GC–MS constituent, while 3‐O‐, 4‐O‐, and 5‐O‐caffeoylquinic acids were detected by HPLC.

The reduction in body‐weight gain, body mass index and specific body‐mass gain is consistent with the reported metabolic activity of green coffee constituents [16]. In the present study, treated animals continued to gain weight but showed significantly attenuated weight gain, particularly at 450 mg/kg. This direction of effect is consistent with human evidence indicating modest reductions in body weight and adiposity following supplementation with CGA‐rich green coffee preparations [25]. A systematic review and meta‐analysis of randomized controlled trials reported a modest reduction in body weight following green coffee extract supplementation, although the available trials were generally small and short [26]. Similarly, clinical studies of standardized chlorogenic‐acid‐rich green coffee preparations have reported reductions in body weight, BMI and waist circumference [27]. These findings support the potential metabolic activity observed here, although direct comparison with human exposure remains inappropriate because the present preparation was an aqueous extract with incompletely standardized CGA and caffeine concentrations.

CGAs provide an important mechanistic basis for the observed metabolic effects. The HPLC profile demonstrated 3‐O‐, 4‐O,‐ and 5‐O‐caffeoylquinic acid derivatives, consistent with the characteristic phenolic composition of green coffee. CGAs may influence glucose and lipid homeostasis by reducing intestinal glucose absorption, modulating hepatic glucose production, improving insulin signaling, and suppressing lipogenic pathways. Experimental studies have also associated coffee polyphenols with downregulation of sterol regulatory element‐binding protein‐1c (SREBP‐1c), a key regulator of fatty‐acid synthesis [28, 29, 30]. These mechanisms could contribute to the lower body‐weight gain and adiposity observed in the treated animals. However, CGA activity is dependent on molecular structure, dose and metabolism, and circulating metabolites generated by gastrointestinal and microbial metabolism may contribute substantially to biological effects.

Caffeine represents another plausible contributor to the metabolic response. Caffeine antagonizes adenosine receptors and can increase sympathetic activity, thermogenesis, fatty‐acid mobilization, and energy expenditure [31]. Its presence in the extract therefore provides a plausible mechanism for the attenuation of weight gain and may complement the metabolic actions of CGAs. However, caffeine is also pharmacologically active and excessive or prolonged exposure may produce undesirable cardiovascular, neurological and metabolic effects. Consequently, the absence of acute toxicity at 5000 mg/kg should not be interpreted as evidence that concentrated caffeine‐containing green coffee extracts are completely safe during chronic administration.

The hematological findings are particularly important from a safety perspective. RBC counts were significantly reduced in all treated groups, WBC counts were reduced particularly at higher doses, and platelet counts were markedly decreased at 450 mg/kg. These findings suggest that prolonged exposure may influence hematopoietic or peripheral blood‐cell homeostasis despite the absence of major hepatic structural injury. The reduction in RBC count could reflect altered erythropoiesis, reduced erythrocyte survival, altered iron utilization or changes in erythropoietin signaling. However, hemoglobin, mean corpuscular indices, reticulocytes, ferritin, serum iron, transferrin saturation and erythropoietin were not determined. Therefore, the finding should be interpreted as altered erythrocyte homeostasis rather than definitive evidence of anemia.

The reduction in WBC count may similarly reflect altered immune regulation rather than generalized toxicity. CGAs and other phenolic compounds can influence oxidative and inflammatory signaling, including NF‐κB‐related pathways [32]. Recent research examining immune‐metabolic relationships in non‐alcoholic fatty liver disease has emphasized interactions among inflammation, immune‐cell activity and metabolic dysfunction [33, 34]. Although such studies were not conducted with green coffee, they provide useful mechanistic context for interpreting the hematological findings. Importantly, cytokines and immune‐cell subsets were not measured in the present study; consequently, an anti‐inflammatory explanation for the reduced WBC count remains plausible but unconfirmed.

The marked reduction in platelet count at 450 mg/kg also warrants caution. Coffee phenolics may influence platelet activation and aggregation through modulation of oxidative and intracellular signaling pathways, potentially producing antithrombotic effects. Such activity could theoretically be beneficial; however, substantial thrombocytopenia would represent a safety concern if caused by impaired platelet production or increased platelet destruction. Since platelet function, coagulation indices and thrombopoietic markers were not assessed, the high‐dose platelet response should be considered a potential safety signal rather than a confirmed beneficial antithrombotic effect.

The hepatic findings provide an important counterpoint to the hematological alterations. Relative liver weight was not significantly different among groups, suggesting the absence of overt hepatomegaly or hepatic atrophy. Histological examination showed preserved hepatic architecture, with only mild hepatocellular swelling and slight sinusoidal dilation at the highest dose and no extensive necrosis, fibrosis or inflammatory infiltration. The biochemical findings similarly did not demonstrate a conventional pattern of hepatocellular or cholestatic injury. AST, ALT, and ALP generally decreased with treatment, while bilirubin concentrations were also reduced. However, albumin declined significantly at 450 mg/kg. Thus, the overall findings do not support overt hepatotoxicity, although the albumin reduction and mild high‐dose morphological changes suggest that subtle hepatic adaptation cannot be excluded.

The relative preservation of hepatic architecture is biologically plausible given the antioxidant potential of several constituents identified in the extract. CGAs, caffeic acid, ferulic acid, phytol, squalene, and unsaturated fatty acids may collectively attenuate oxidative stress and protect cellular membranes. Recent animal studies of other phytochemical‐rich preparations provide supportive comparative evidence. Elaeagnus umbellata and Phyllanthus niruri extracts have demonstrated hepatoprotective effects associated with reductions in oxidative stress and inflammatory signaling [35]. The recent Phyllanthus niruri study, for example, reported attenuation of liver injury accompanied by improved antioxidant defenses and reduced inflammatory mediators [36, 37]. These studies do not establish that green coffee is hepatoprotective, because they involve different plants and models, but they support a broader biological framework in which phenolic‐rich botanical preparations may protect hepatic tissue through antioxidant and anti‐inflammatory mechanisms.

The mild hepatocellular swelling and sinusoidal dilation observed at 450 mg/kg should nevertheless not be disregarded. Such changes may represent adaptive responses to repeated exposure, increased hepatic metabolic workload or early cellular stress. The absence of increased relative liver weight and the lack of substantial enzyme elevations suggest that any hepatic response was modest. Nevertheless, histology alone cannot exclude early biochemical or molecular alterations. Measurement of hepatic MDA, glutathione, SOD, catalase, inflammatory cytokines, and molecular markers of oxidative stress would therefore be useful in future investigations.

The metabolic findings may also indirectly contribute to hepatic preservation. Reduced weight gain and adiposity could decrease metabolic stress and hepatic lipid accumulation, while CGAs and caffeine may influence glucose and lipid handling. Human studies of CGA‐rich green coffee preparations have similarly reported modest reductions in body weight and adiposity [38, 39]. Nevertheless, the experimental doses used here and the chemical composition of the aqueous extract differ from standardized human products, preventing direct dose‐for‐dose extrapolation.

Importantly, green coffee bean extract should not be considered pharmacologically equivalent to purified CGA. The extract contains multiple compounds that may exert complementary or opposing effects. CGAs may contribute antioxidant and metabolic actions, caffeine contributes stimulant and thermogenic effects, while trigonelline, quinic acid derivatives, fatty acids, and terpenoid constituents may further modify the overall response [40, 41]. The GC–MS and HPLC profiles therefore demonstrate that the biological effects observed in this study are likely to result from a chemically complex mixture rather than a single active compound.

The dose‐response pattern is also relevant to safety interpretation. Although 150, 300, and 450 mg/kg were substantially below the highest acute dose tested, hematological alterations became more evident with increasing exposure, while mild hepatic morphological changes were most apparent at 450 mg/kg. This demonstrates the distinction between acute toxicity and chronic physiological tolerability. A high acute LD₅₀ does not exclude cumulative, adaptive or organ‐specific effects following repeated administration. Human translation should therefore remain conservative, particularly because human trials generally involve standardized extracts, shorter exposure periods and better‐defined constituent concentrations.

This study has several limitations. First, the use of Wistar rats limits direct extrapolation of the findings to humans because of interspecies differences in metabolism, pharmacokinetics, and sensitivity to caffeine and chlorogenic acids. Second, although acute toxicity was low, the 90‐day study does not establish the effects of longer‐term exposure or whether the observed hematological changes are reversible. Third, the extract contained multiple bioactive constituents, including chlorogenic acid derivatives and caffeine, but their individual contributions were not determined. Pharmacokinetic measurements were also not performed, limiting interpretation of systemic exposure.

The significant alterations in RBC, WBC, and platelet counts warrant further investigation; however, detailed hematological indices, iron status, platelet function, and coagulation parameters were not assessed. Similarly, oxidative‐stress, inflammatory, and molecular biomarkers were not measured, limiting mechanistic interpretation of the metabolic and hepatic findings. Although serum enzymes, organ weight and histology did not indicate overt hepatotoxicity, more sensitive molecular and biochemical assessments are required.

Despite these limitations, the findings have translational relevance because the reduction in body‐weight gain and adiposity is consistent with reported metabolic effects of chlorogenic‐acid‐rich green coffee preparations. However, the hematological changes indicate that potential benefits should be considered alongside long‐term safety. Translation to humans should therefore account for dose, body‐surface‐area scaling, extract standardization, caffeine content and bioavailability. Future studies should employ standardized extracts, pharmacokinetic analyses, longer exposure and recovery periods, comprehensive hematological and hepatic biomarkers, and well‐designed human trials to establish the therapeutic benefits and safety of sustained green coffee bean extract consumption.

Overall, the present findings indicate that green coffee bean extract produces potentially beneficial metabolic effects while also inducing measurable hematological changes during prolonged exposure. The preserved hepatic architecture and absence of conventional biochemical evidence of hepatocellular injury are reassuring, but the mild high‐dose hepatic changes and reduction in albumin warrant further investigation. The effects of green coffee are therefore unlikely to be uniformly beneficial or harmful; rather, they appear to depend on constituent composition, dose, and duration of exposure. The present findings support continued investigation of the metabolic benefits of green coffee while emphasizing the need for chemical standardization, dose optimization, and long‐term hematological and hepatic safety assessment before extrapolation to sustained human use.

5. Conclusion

The present study demonstrates that 90‐day oral administration of aqueous green coffee bean extract produced significant metabolic and physiological effects in Wistar rats. The extract attenuated body‐weight gain and adiposity, supporting the potential metabolic activity of its chlorogenic acid‐rich and caffeine‐containing phytochemical profile. However, prolonged exposure was also associated with significant reductions in RBC and WBC counts and a marked decrease in platelet count at the highest dose, indicating that chronic administration may influence hematological homeostasis despite the absence of overt acute toxicity.

The hepatic findings were comparatively reassuring. Relative liver weight remained largely unchanged, hepatic architecture was preserved, and no major necrosis, fibrosis or inflammatory infiltration was observed. The absence of increased ALT, AST, ALP, and bilirubin further argues against overt hepatocellular or cholestatic injury. Nevertheless, the reduction in albumin and mild hepatocellular swelling and sinusoidal dilation at 450 mg/kg suggest that subtle adaptive hepatic responses may occur during prolonged exposure.

Overall, the findings indicate that green coffee bean extract may provide beneficial metabolic effects while producing dose‐ and duration‐dependent physiological changes that warrant safety consideration. The study therefore supports the potential nutraceutical value of green coffee bean extract but emphasizes that its long‐term effects cannot be assessed solely from acute toxicity outcomes. Further studies incorporating quantitative determination of chlorogenic acids and caffeine, pharmacokinetic assessment, expanded hematological profiling, oxidative‐stress and inflammatory biomarkers, and molecular analyses are warranted to establish the mechanisms and long‐term safety of sustained green coffee bean extract consumption.

Author Contributions

Oyepata Simeon Joseph was involved in conception, design, investigation analysis, and interpretation of the data and in writing and drafting of the paper manuscript.

Funding

There was no external funding for this study.

Ethics Approval

The FUOYE University College of Health Sciences Animal Ethics Committee granted approval and consent for animal experiments (5639).

Conflicts of Interest

The author declares no conflicts of interest.

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Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.