Introduction
Persea americana Mill., commonly known as avocado, is a tropical fruit belonging to the family Lauraceae, and is native to South America, Mexico, and Central America [1, 4 ]. While the edible pulp of avocado is widely consumed fresh as well as processed into products like puree, guacamole, or dried forms, industrial processing generates significant amount of waste, including leaves, peels, and seeds. The peel constitutes approximately 11% and the seed 16% of the total fruit weight [20, 27].
Avocado pulp is rich in water, dietary fiber, B-complex vitamins, and fatty acids such as linoleic, palmitic, oleic, stearic, linolenic, capric, and myristic acids [12]. It also contains carotenoids including α-carotene, β-carotene, zeaxanthin, neoxanthin, and violaxanthin, as well as vitamin A [17]. Beyond the pulp, avocado seeds and leaves are sources of phenolic compounds and other bioactive molecules with therapeutic potential [27].
Avocado seeds contain all three macronutrients, carbohydrates, proteins, and lipids as well as substantial proportion of phytochemicals. Specifically, hexane extracts have revealed the presence of palmitic, palmitoleic, stearic, oleic, and linoleic acids, as well as sterols and triterpenoids [28].
Historically, avocado pulp and seeds have been used in traditional medicines for their antimicrobial, anti-inflammatory, antioxidant, and anticarcinogenic properties, mainly contributed by secondary metabolites like polyphenols, carotenoids, and tocopherols [15]. Avocado seeds have also shown antidiabetic and hypolipidemic potentials while enhancing glycogen storage in animal models [16, 30].
Gastric ulcer is the most common gastrointestinal disorder which is resulted from an imbalance between aggressive factors, such as hydrochloric acid, pepsin, free radicals, and protective mechanisms including bicarbonates and mucosal blood flow. Oxidative stress, alcohol consumption, H. pylori infection, and prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) are considered to be the main risk factors for gastric ulcer [14]. Researches have shown that avocado seeds aqueous extracts can alleviate NSAID-induced gastric ulcers in animal models by reducing the levels of malondialdehyde as well as increasing antioxidant enzyme activity like glutathione, catalase, and superoxide dismutase [26].
The present study aims to characterize the nutritional and bioactive composition of avocado seed and then to evaluate the therapeutic potentials of its aqueous extract on gastric ulcers in humans. This research mainly focusses on the the potential of avocado seed aqueous extract as a complementary therapy with conventional treatments, addressing gaps in current ulcer management approaches.
Materials and methods
Procurement of raw materials
Avocado fruits were purchased from super market in Lahore, Pakistan. The study did not include specific information about the origin of avocados, supplier, or harvest time, as the fruits were purchased from a local supermarket. However, the use of commercially available fruits reflects real-world consumer conditions, supporting the practical relevance of the study outcomes.
The seeds were separated from the fruits, properly cleaned and stored at 4 °C in zipper bags for further analysis. The raw material was analyzed at Food Science and Technology Lab 102, at University Institute of Diet and Nutritional Sciences, The University of Lahore. The study was also carried out in collaboration with the University of Lahore, Teaching Hospital.
This study was approved from the institutional review board of University of Lahore under IRB number: REC-UOL-/20 − 08/2022.
Sample collection procedures
48 patients were selected in each group. The sample size was calculated by the following formula.
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α = 5% [2].
Power = 0.84 [2].
Difference of SD = 0.879 [2].
n = 48.
60 patients were selected and 30 in each group (20% dropout have been added).
The sampling technique used for the study is nonprobability convenient sampling.
Inclusion criteria
Patients who were suffering from acute gastric ulcer stage 1 were included.Patients with gastric ulcer age from 25 to 60 years (male and female) were included.
Exclusion criteria
Patients who are already taking any supplement or medication other than gastric ulcer disease.Currently participants in any other intervention.Pregnant or lactating women.Patients having history of smoking.Patients suffering from other health disorders (Hypertension, diabetes, CKD).Patients suffering from gastritis and gastric cancer.
Sample preparation
Sample preparation was conducted following the method described by [31]. The avocado seeds were removed from the avocado, and air dried. They were then cut into small pieces and grinded in a blender.
1 kg of the avocado seed powder was mixed with 2000 mL of distilled water and the solution was kept for a duration of 24 h. Filtration of the mixture was then carried out by passing the mixture through a plug of cotton. Further filtration was carried out by the use of filter paper.
This aqueous extract of avocado seeds was then dried by freezing until the weight of the sample turned out to be constant upon weighing the sample repeatedly. The sample yielded an extract which was dark brown in color and gummy in nature. Storage of the sample was carried out at temperatures of 45 degrees Celsius for further use.
The process was conducted in the lab of the University of Lahore.
Analysis of chemicals
The composition of moisture, ash, crude protein, crude fat and crude fiber were conducted according to the AOAC methods. Nitrogen Free Extract (NFE) was measured by the difference method.
Moisture analysis
1.5 gram of the sample of avocado seed extract was taken for moisture analysis. Oven drying was the method used for the analysis. The amount of the sample was weighed in a dried and cleaned crucible and was then heated in the oven at a temperature of 105 °C for a period of 12 h.
Ash analysis
Ash content was determined by following the method of (AOAC, 2012) and AACC Method No. 08 − 01 was used to calculate the amount of ash. Muffle furnace (MF-1/02, PCSIR, Pakistan) was used.
Protein analysis
Kjeldahl method was used for protein analysis as explained by AOAC, 2012. For estimating the amount of nitrogen in the sample, the Kjeldahl Apparatus (Model: Serin-Nr: 808 − 100, Behr Dusseldort) was used.
Fat analysis
Soxhlet extraction was used for the analysis of crude fat in the sample. Petroleum ether was used as a solvent. The model of the Soxhlet Apparatus was (PCSIR, Model: SE-1 A/02, Pakistan).
AOAC method was used for the calculation of the amount of crude fat in a 3 g sample.
Fiber analysis
Fiber content was analyzed by following the protocol as demonstrated by [24]. Using the Soxhlet method, fat was extracted from the 2 g sample.
Nitrogen free extract
Nitrogen Free extract (NFE) was calculated by the subtraction of total ash, moisture, crude protein, crude fat and crude fiber from 100.
Phytochemical screening
Quantitative screening for tannins
Tannins were determined according to Ejikeme et al. [11].
Quantitative screening for alkaloids
Alkaloids were screened according to the method described by [13].
Determination of total phenolic content (TPC)
Total Phenolic Content was measured by the technique described by [3] by the Folin–Ciocalteu reagent assay methods. The standard used for calibration curve was gallic acid. The total phenolic content was measured and articulated as GAE (mg/g).
Determination of total flavonoid content
The calorimetric method was used for the determination of total flavonoid content by following the method as explained by [3].
Clinical evaluation of avocado seed extract in patients with gastric ulcers
Screening
To assess the therapeutic effect of avocado seeds extraction on gastric ulcer, 90 patients with an age ranging from 25 to 60 years were included in the study as per inclusion criteria mentioned. 90 people were assessed for eligibility. 12 were excluded for not meeting the inclusion criteria.
4 of the patients declined to participate, and 2 left for no reason. The randomized control trial, therefore consisted of 66 patients, which were equally divided randomly into 2 groups consisting of 33 patients each. All 33 patients in the controls and intervention groups were allocated to interventions, out of which 30 received the intervention in the control group and 28 patients received the allocated intervention in the experimental group.
Out of the 30 patients in the control group 2 patients discontinued the intervention without giving any reason. Similarly, 4 people discontinued the intervention in the experimental group. Final analysis consisted of 24 patients in both the groups as shown in Fig.
1.
In the control group, gastric ulcer patients were treated with conventional medicine which consisted of 20 mg Omeprazole and 500 mg Clarithromycin once a day before meal for a period of 12 weeks. The control group were also given modified diets to treat ulcers. The modified diet consisted of 1500–1600 kcals of which 52% were carbohydrates, 18% proteins and 30% fats.
The control group did not receive any intervention of avocado extract supplement.
On the other hand, the intervention group received the same amount of conventional medicine, along with the same modified diet plan, but were given the avocado seed extract supplementations at a dose of 100 mg/kg of the patients’ individual body weight, daily, two time a day before meal, for a period of 12 weeks.
Histopathological examination
For diagnosis of gastric ulcer, barium meal (barium sulphate) x-ray was performed with the help of a Radiologist. It involved no risks to the patient. Gastric ulcer diagnosis was further confirmed by endoscopy in these patients.
Blood samples were collected by the hospital lab assistant. The base line data was comprised of Helicobacter Pylori Antigen Test (Serum antigen) and Barium swallow. After that the patients were advised 3 g (Twice in a day) of avocado seeds extract supplements for 12 weeks before meal.
Patients were also advised to follow the given diet plan over the course of study.
Follow up
The follow up for patients was conducted once a week. Diet history was collected on food diaries. Patients were asked to face any constraints and barrier to follow the study procedure (Table 1).
After 12 weeks of study the same protocol of baseline visit was conducted. The baseline and post test data were compared to test the study hypothesis.
Table 1Treatment planControl groupIntervention groupTreatmentConventional medicine:• Omeprazole 20 mg + Clarithromycin 500 mg• Modified DietConventional medicine:• Omeprazole 20 mg + Clarithromycin 500 mg• Avocado seeds supplement• Modified dietDosage • 100 mg/kg body weight of avocado seed supplement Frequency• Daily (Twice a day)Duration• 12 weeks• 12 weeks
Fig. 1Flowchart illustrating the study design and participant allocation. Participants were screened for eligibility, randomized into control and intervention groups, and followed through treatment and outcome assessment phases
Statistical analysis
Statistical analysis of the efficacy trial was performed using two sample t-test (p ˂ 5). Results were presented as mean values ± standard deviation (SD), based on triplicate determinations for each parameter.
Results and discussion
Proximate analysis of avocado seeds
The results in Table 2 represent the mean percentage levels of moisture, ash, crude protein, crude fat, crude fiber, and nitrogen free extract (NFE) in avocado seeds, that were found to be in concentrations of 14.10 ± 0.15, 3.95 ± 0.02, 2.85 ± 0.02, 0.40 ± 0.01, 3.19 ± 0.02, and 82.85 ± 0.20 respectively.
Table 2Proximate analysis of avocado seedsCompositionMean concentration (%) ± SDMoisture14.10 ± 0.15Ash3.95 ± 0.02Crude protein2.85 ± 0.02Crude fat0.40 ± 0.01Crude fiber3.19 ± 0.02NFE82.85 ± 0.20 Values are expressed as mean ± Standard deviation (n = 3)
The proximate composition results indicate that avocado seeds contain a high proportion of nitrogen-free extract (82.85%), suggesting that carbohydrates are the predominant nutrient component. The relatively low fat (0.40%) and protein (2.85%) levels are consistent with earlier researches by Egbuonu et al. [8, 10], which also observed higher levels of carbohydrates and low concentrations of lipid and protein in avocado seeds. Nyakang’i et al. [24] observed the similar values of moisture content of avocado seeds which is also linked with good storage stability and a longer shelf life when properly stored.
Adequate levels of ash (3.95%) in this experiment reflects the presence of essential minerals such as calcium, potassium, and magnesium, which is almost similar as noted by Bangar et al. [5]. On the other hand, lower level of crude fiber (3.19%) than those reported earlier is contributed by certain factors like agronomic practices, varietal differences, maturity stage, and environmental factors during fruiting. The results of proximate composition of avocado seed confirm its nutritional potential as carbohydrate-rich, mineral-containing byproduct suitable for use in food formulations as well as a rich source of functional compounds.
Phytochemical analysis of avocado seeds
The levels different phytochemicals in avocado seeds like tannins and alkaloids have been presented in Table 3. The analysis showed that tannins were present at 0.81 ± 0.20 mg/100 g, while alkaloids were detected at 5.48 ± 0.50 mg/100 g.Table 3Phytochemical analysis of avocado seedsPhytochemicalsComposition (mg/100g)Tannins0.81±0.20Alkaloids5.48±0.50
The results showed potentials of avocado seeds with appreciable concentration of bioactive compounds having health-promoting properties. The low concentration of tannins suggests minimal astringency and low interference with protein digestibility, which make it suitable for food or nutraceutical applications. On the other hand, the relatively high concentration of alkaloid showed its prominent phytochemical activity as antimicrobial, anti-inflammatory, and gastroprotective agents.
The slight variations levels of phytochemicals of avocado seed that appeared in previous literature are mainly due to environmental conditions, extraction methods, and cultivar differences. In general, these results exhibited the presence of important secondary metabolites in avocado seeds, supporting their potential therapeutic relevance in managing oxidative stress and gastric disorders.
Total phenolic and flavonoid content of avocado seeds
The levels of phenolic and flavonoid contents of avocado seeds aqueous extract have been presented in Table 4. The TPC was recorded as 14.25 ± 1.50 mg GAE/g, while the TFC was 197.08 ± 7.60 µg RE/g. The high concentrations of phenolics highlights the antioxidant potentials which are mainly responsible for neutralizing free radicals and thus reducing oxidative damage.
On the other hand, comparatively smaller concentrations of flavonoids synergistically effect the overall antioxidant potential and play a vital role in protecting the gastric mucosa. These findings strengthen the knowledge about avocado seeds bioactive potential which serve as a valuable source of natural antioxidants for therapeutic applications.
Table 4Total flavonoid and phenolic contents of avocado seedsCompositionTotal flavonoid content - TFC (µg RE/g)197.08 ± 7.60Total phenolic content - TPC (mg GAE/g)14.25 ± 1.50Values are expressed as mean ± Standard deviation (n = 3)
Radiological and endoscopic findings
The radiological and endoscopic findings have been elaborated in Table 5; Fig. 2. Notable differences were observed between the control and intervention groups following treatment.
Even after twelve weeks of treatment most of the patients in control group showed clear ulcer craters with more than 2–3 mm in size surrounding edema, and prominent gastric folds radiating toward the lesion. These results are distinctive features of active gastric ulcer and persistent mucosal inflammation [23].
However, patients in intervention group showed a clear reduction in the number and size of mucosal defects. Similarly, the patients have less common edema rings and showed smoother gastric fold with less radiating patterns. The ulcer healing was more pronounced in lesser curvature of the stomach body suggesting accelerated mucosal recovery in intervention group.
These improvements indicated a positive effect of avocado seed aqueous extract in recovery when used with conventional therapy.
Phytochemicals present in avocado seed like flavonoids, polyphenols, and alkaloids exhibit properties such as antioxidative, anti-inflammatory, and cytoprotective which resulted in speedy recovery in gastric ulcer patients. Bioactive compounds such as phenolics promote gastric mucosal healing process by neutralizing reactive oxygen species and thus enhancing mucosal defense mechanisms [6, 22]. In the same way, plant-based flavonoids have also been reported to reduce oxidative damage and modulate inflammatory cytokines in gastric tissues [29].
Previous studies have also shown similar results with extracts of different other plants. Aloe vera and Carica papaya extracts have demonstrated notable gastroprotective activity by accelerating epithelial regeneration and buffering gastric acidity [7, 25]. Similarly, extract of licorice involves in suppression of gastric acid production and promoting mucus secretion resulted in enhancement of mucosal integrity and reducing the chances of recurrence of ulcer [18].
Similar mechanism involving antioxidant modulation and reinforcement of mucosal defenses have been observed in current research. The radiological and endoscopic investigation further strengthening the potential therapeutic role of avocado seed extract as an adjunctive agent in the management of gastric ulcer. Its bioactive compounds appear to facilitate mucosal restoration, reduce inflammation, and complement the effects of standard anti-ulcer medications.
Fig. 2Radiological findings (barium swallow followed by endoscopy)
Table 5Comparative barium meal X-ray and endoscopic findings in control and intervention groupsLocationGroupsNo. of patients with > 2–3 mm mucosal defect (crater)No. with ring of edema around craterNo. with gastric folds radiating towards craterControlInterventionControlInterventionControlInterventionLesser curvature of stomach body1310122103Lesser curvature of stomach antrum544121Posterior wall of stomach body372422Posterior wall of stomach antrum333111
Serological analysis using ELISA
The infection of Helicobacter pylori was confirmed by estimating the levels of antibodies like immunoglobulin G (IgG) and immunoglobulin M (IgM) using enzyme-linked immunosorbent assay (ELISA). Presence of IgM antibody is an indication of active or recent infection, while on the other hand, IgG values suggesting an ongoing or past exposure. The higher values of IgM (2.62) and IgG (28.5) revealed an active infection, indicating a positive immune response.
The results of IgM and Ig G indicate that participants were experiencing either an early or persistent phase of infection. These results also highlighting the role of H. pylori in the pathogenesis of gastric ulcers, where chronic infection leads to mucosal inflammation and ulceration [19, 21]. The presence of both classes of antibodies (IgM and IgG) authenticates the diagnostic reliability of ELISA for identifying H. pylori-associated gastric pathology (Table 6).
Table 6Serological detection of Helicobacter pylori by ELISATestReference valuesObserved valueResultHelicobacter pylori IgG (ELISA)Negative: < 12 Borderline Positive: 12.0–20.0 Positive: > 20.028.5PositiveHelicobacter pylori IgM (ELISA)Negative: < 0.9 Borderline Positive: 0.9–1.1 Positive: > 1.12.62Positive
The results from paired samples t-test (Table 7) exhibited improvements in the values of antibodies (IgG and IgM) following the intervention. A significant decrease in chronic infection markers was observed as estimated by the mean difference for IgG (18.19 ± 4.41), while a considerable reduction was also observed in acute-phase antibodies as evident by the mean difference for IgM (1.43 ± 0.51). These results suggested a significant reduction of Helicobacter pylori activity and improvement in mucosal recovery in patients received avocado seed extract in combination with standard treatment.
Table 7t-test of Helicobacter pylori IgG and IgM (ELISA)StatisticIgGIgMMean difference18.191.43Standard deviation4.410.51Standard error mean0.770.0995% Confidence interval (Lower)16.621.2495% Confidence interval (Upper)19.751.61t-value23.7115.96Degrees of freedom (df)3232p-value (2-tailed)0.0000.000
The observed antibody decline is consistent with the expected immune response during gastric healing, where reduced bacterial load leads to decreased antigenic stimulation. Previous studies have highlighted that bioactive compounds such as flavonoids, phenolics, and alkaloids in avocado seed extract possess anti-H. pylori and anti-inflammatory properties, contributing to mucosal protection and repair [2, 9]. These findings reinforce the potential role of avocado seed extract as a complementary therapy for H. pylori-associated gastric ulcers.
Conclusion
This study concludes that avocado seeds are a valuable source of macronutrients like protein, fiber, carbohydrates as well as phytochemicals such as phenolics and flavonoids. Avocado seed extract significantly (p < 0.05) improve the symptoms of gastric ulcers. Reductions in antibodies (IgG and IgM) levels and improvements in radiological findings (Barium Meal X-ray and endoscopic evaluations) were observed in participants of intervention group.
Faster and more pronounced gastric healing were observed compared with the control group receiving only conventional treatment and dietary modifications over the 12-week period of time. These findings suggest that avocado seed extract could serve as a natural adjunct to conventional ulcer therapy, warranting further clinical studies to confirm its long-term efficacy and safety.