Research Proposal: Synergistic Effect of Lemon and Cucumber Juices on Calcium Oxalate Crystal Inhibition

**"The Synergistic Effect of *Citrus limon* (Lemon) and *Cucumis sativus* (Cucumber) Juices on Calcium Oxalate Crystal Inhibition in an In Vitro Model"** Abila, Jay-Ann P. Gomez, Huey F. Magtibay, Reuel Anthony B. Occiano, Samantha Faye F. Rivera, Rheiren C. Salvo, Janna Margaret, E. CHAPTER...

**"The Synergistic Effect of *Citrus limon* (Lemon) and *Cucumis sativus* (Cucumber) Juices on Calcium Oxalate Crystal Inhibition in an In Vitro Model"** Abila, Jay-Ann P. Gomez, Huey F. Magtibay, Reuel Anthony B. Occiano, Samantha Faye F. Rivera, Rheiren C. Salvo, Janna Margaret, E. CHAPTER 1 ========= **INTRODUCTION** ---------------- ### Background of the Study Calcium oxalate crystals are the most prevalent component of kidney stones (nephrolithiasis), accounting for approximately 80% of cases worldwide (Preminger *et al*., 2024). These crystals form due to the supersaturation of calcium and oxalate ions in the urine, which initiates nucleation, growth, and aggregation processes that can lead to stone formation (Allam, 2024; Shastri, 2023; Tamborino, 2024). According to the Philippine Health Research Registry (with a target completion date of 2022), nephrolithiasis affects 2.3% of the Philippine population, with a 5-year recurrence rate of kidney stones ranging from 35% to 50%. Therefore, effective prevention and management strategies, such as the use of natural remedies like lemon and cucumber juices, are essential for reducing the incidence and recurrence of kidney stones. Citrate - a substance found in citrus fruits, can inhibit calcium oxalate's spontaneous nucleation and agglomeration. Consequently, citrate plays a significant role in lowering urine calcium levels, which, when elevated, poses a major risk of kidney stone formation. The urine calcium level is reduced by citrate-calcium complexes -- which sequestrate the Ca^+2^ ions (Barghouthy & Somani, 2021). Moreover, one of the factors that enables citrate to inhibit calcium oxalate crystals is its ability to provide alkalinity to urine pH due to the production of bicarbonate. With the addition of potassium consumption, urine alkalinization is more achieved as potassium increases the tubular secretion of potassium ions in exchange for sodium ions. Therefore, the synergistic effect of the two substances is essential for the prevention and inhibition of the formation of calcium oxalate crystals (Barghouthy & Somani, 2021). The use of plant-based treatments for managing calcium oxalate crystals has gained attention due to their potential to inhibit crystal formation naturally. These remedies are often more cost-effective and carry fewer side effects compared to synthetic treatments. A promising combination is *Citrus limon* (lemon) and *Cucumis sativus* (cucumber) juices. As a natural source of dietary citrate, citrus fruits and juices have been used as alternatives to potassium citrate (Gopala *et al*., 2023). Due to its high concentration of citrate, lemon juice is the most commonly recommended citrus fruit juice for patients (Gopala *et al*., 2023). Additionally, cucumber (*Cucumis sativus*) contains high levels of potassium, which contributes to its effectiveness in dissolving calcium oxalate kidney stones. The study highlights that cucumber juice serves as a potent natural remedy due to its diuretic properties and ability to reduce the formation of calcium oxalate crystals (Sariyanto, 2020). Together, the combination of lemon and cucumber juices provides a complementary approach to enhancing the dissolution and prevention of kidney stone formation. Investigating plant-based alternatives is a critical first step in developing innovative treatments for calcium oxalate-related conditions. This in vitro study seeks to provide foundational evidence of the efficacy of lemon and cucumber juices in inhibiting calcium oxalate crystals. By exploring their synergistic potential, this research aims to contribute to the development of cost-effective, natural interventions for managing kidney stones. The study specifically aims to assess the inhibitory effects of combined lemon and cucumber juices on calcium oxalate crystal formation, providing insights for future therapeutic applications. ### Statement of the problem ### Kidney stones, primarily composed of calcium oxalate, affect a significant portion of the population, with high recurrence rates. Current treatment methods are often invasive and costly. While Citrus limon (lemon) and Cucumis sativus (cucumber) juices have shown individual potential in inhibiting calcium oxalate crystal formation, their synergistic effect remains unexplored. This study aims to investigate the combined inhibitory effect of lemon and cucumber juices on calcium oxalate crystal formation, providing a natural and cost-effective alternative for preventing kidney stone formation. ### Research Question This study will determine the synergistic effect of *Citrus limon* (lemon) juice and Cucumis sativus (cucumber) juice on inhibition of calcium oxalate crystal formation using an in vitro model. It will answer the following questions: 1. Can the synergistic effect of Citrus limon (lemon) juice and Cucumis sativus (cucumber) juice inhibit calcium oxalate crystal formation using an in vitro model at varying concentrations? 2. Is the inhibitory effect of synergistic *Citrus limon* (lemon) juice and *Cucumis sativus* (cucumber) juice on calcium oxalate crystals more potent than the inhibitory effect of *Citrus limon* (lemon juice) on calcium oxalate crystals? 3. Is the inhibitory effect of synergistic *Citrus limon* (lemon) juice and *Cucumis sativus* (cucumber) juice on calcium oxalate crystals more potent than the inhibitory effect of *Cucumis sativus* (cucumber) on calcium oxalate crystals? ### Specific Objectives 1. To evaluate the percentage inhibition of calcium oxalate crystal formation using the synergistic effect of Citrus limon (lemon) juice and Cucumis sativus (cucumber) juice at varying concentrations: a. 10% concentration b. 25% concentration c. 50% concentration 2. To compare the inhibitory effect of the synergistic *Citrus limon* and *Cucumis sativus* juices with the inhibitory effect of *Citrus limon* juice alone on calcium oxalate crystal formation. 3. To compare the inhibitory effect of the synergistic *Citrus limon* and *Cucumis sativus* juices with the inhibitory effect of *Cucumis sativus* juice alone on calcium oxalate crystal formation. ### Research Hypothesis The null and alternative hypotheses have been established for each research question to facilitate accurate statistical inference, thereby providing a robust foundation for research-based conclusions. 1. Can the synergistic effect of *Citrus limon* (lemon) juice and *Cucumis sativus* (cucumber) juice inhibit calcium oxalate crystal formation using an in vitro model? - **Null Hypothesis (H₀₁):** The synergistic effect of Citrus limon (lemon) juice and Cucumis sativus (cucumber) juice does not significantly oxalate crystal formation at varying concentrations in an in vitro model. - **Alternative Hypothesis (H₁₁):** The synergistic effect of Citrus limon (lemon) juice and Cucumis sativus (cucumber) juice significantly inhibits calcium oxalate crystal formation at varying concentrations in an in vitro model. 2. Is the inhibitory effect of synergistic *Citrus limon* (lemon) juice and *Cucumis sativus* (cucumber) juice on calcium oxalate crystals more potent than the inhibitory effect of *Citrus limon* (lemon juice) on calcium oxalate crystals? - **Null Hypothesis (H₀₂):** The inhibitory effect of the synergistic Citrus limon (lemon) juice and Cucumis sativus (cucumber) juice on calcium oxalate crystals is not significantly more potent than the inhibitory effect of Citrus limon (lemon) juice alone. - **Alternative Hypothesis (H₁₂):** The inhibitory effect of the synergistic Citrus limon (lemon) juice and Cucumis sativus (cucumber) juice on calcium oxalate crystals is significantly more potent than the inhibitory effect of Citrus limon (lemon) juice alone. 3. Is the inhibitory effect of synergistic *Citrus limon* (lemon) juice and *Cucumis sativus* (cucumber) juice on calcium oxalate crystals more potent than the inhibitory effect of *Cucumis sativus* (cucumber) on calcium oxalate crystals? - **Null Hypothesis (H₀₃):** The inhibitory effect of the synergistic Citrus limon (lemon) juice and Cucumis sativus (cucumber) juice on calcium oxalate crystals is not significantly more potent than the inhibitory effect of Cucumis sativus (cucumber) juice alone. - **Alternative Hypothesis (H₁₃):** The inhibitory effect of the synergistic Citrus limon (lemon) juice and Cucumis sativus (cucumber) juice on calcium oxalate crystals is significantly more potent than the inhibitory effect of Cucumis sativus (cucumber) juice alone. ### Conceptual Framework The study will investigate the potential of Citrus limon (lemon) and Cucumis sativus (cucumber) juices as inhibitors of calcium oxalate crystal formation. The researchers will employ nucleation and aggregation assays, with Cystones as the positive control and distilled water as the negative control. Variations in the concentrations of lemon and cucumber juices will also be tested. The experiment will be observed under a microscope to evaluate the initiation, growth, aggregation, and inhibition of calcium oxalate crystallization. The researchers hypothesize that the combined juices will exhibit a synergistic effect, potentially offering a more potent inhibition of crystal formation compared to individual juices. ### Scope and Limitation This study focuses on the potential synergistic effect of *Citrus limon* (lemon) and *Cucumis sativus* (cucumber) juices in inhibiting calcium oxalate crystal formation in vitro. The study will evaluate both the individual and combined effects of these natural substances at varying concentrations (10%, 25%, and 50%) in inhibiting crystal nucleation and aggregation. The inhibitory potential will be assessed through nucleation and aggregation assays, with the results measured using spectrophotometry at 620 nm. A positive control (Cystone filtrate) will be included to ensure accuracy. The study is limited to in vitro conditions, which may not fully replicate the exact physiological environment of the human body. This model also does not take into account factors such as pH changes, metabolism, absorption, or interactions with other foods. Additionally, the study does not explore long-term effects, potential side effects, or the optimal dosage of the natural compounds. ### Significance of the Study This study makes some important contributions to preventing and treating the formation of kidney stones and may prove to be useful to the following groups: The ***academe***. The research could improve knowledge and promote awareness about prevention of kidney stones. Furthermore, it may provide information regarding possible natural remedies for kidney stone inhibition. ***Patients.*** The findings of this study suggest the synergistic effect of *Citrus limon* (Lemon) and *Cucumis sativus* (Cucumber) juices could aid in the development of inexpensive alternative treatments for kidney stones by naturally inhibiting calcium oxalate crystal formation, reducing the patient\'s dependence on synthetic medications. This study analyzes a natural, plant-based treatment that may offer the possibility of helping patients who experience kidney stone formation. ***Healthcare system.*** The study aims to educate patients on the risks and treatments for kidney stones and provides an alternate, inexpensive method for anti-urolithiasis. It highlights the need of researching alternative treatments for diseases. The likelihood that lemon and cucumber juice can be utilized to prevent kidney stones could lead to additional research collaboration for the product\'s potential use in healthcare. ***Future researchers.*** The study provides future researchers a foundation of how bioactive chemicals in select natural products (Lemon and Cucumber) interact to prevent crystal formation. Using an in vitro model creates a controlled experimental framework that may be replicated or altered for the purpose of future research. ### Definition of Terms - ***Agglomeration.*** It is a process in which fine particles stick together to form larger and denser particles. - ***Anti-inflammatory.*** It is an action of a substance or process in which it inhibits the inflammatory effect on the human body. - ***Antimicrobial.*** It is an action of a substance where it inhibits the presence of any microbes including bacteria, fungi, virus, parasites, and prions. - ***Antioxidant.*** It is a property of a substance that prevents cells from being damaged by free radicals, which are unstable molecules created by oxidation during metabolism. - ***Calcium Oxalate.*** A chemical compound that is a major component of kidney stones when formed into crystals. - ***Chelating Agent.*** A chemical compound that binds to metal ions to form a ring-like structure called a chelate. A characteristic of citric acid that reacts with calcium to form calcium citrate which can be easily removed from the body. - ***Citric Acid.*** Components of citrus fruits, including the Citrus limon in which it has an ability to inhibit the formation of calcium oxalate crystals. - ***Citrus limon.*** It is the scientific term for lemon, a citrus fruit with pale yellow skin and a sour juice which has substances such as citric acid and polyphenols that could inhibit the calcium oxalate crystal formation. - ***Cucumis sativus.*** It is the scientific term for cucumber, an annual, tendril-bearing vines of the gourd family that has a diuretic properties which can potentially inhibit the calcium oxalate crystal formation. - ***Flavonoid.*** Typically biologically active-water soluble compounds (such as anthocyanin and flavones) which compose several fruits such as citrus fruits and have antioxidative properties. - ***High Performance Liquid Chromatography.*** A technique that separates and analyzes specific compounds present in a liquid solution or sample. - ***Hypercalciuria.*** It is a condition when there is a presence of excess calcium in the urine. - ***Hyperoxaluria.*** A condition when there is an oxalate excess in the urine. - ***Hyperuricemia.*** A condition that occurs when there is an excessive amount of uric acid in the urine. - ***Hypocitraturia.*** A condition where there is relatively low amount of citrate in the urine, which can increase the risk of forming kidney stones or having nephrolithiasis. - ***Hypomagnesuria.*** A condition where the amount of magnesium excreted in the urine is less than 50 mg per day. This is a common symptom of patients associated with nephrolithiasis. - ***In vitro.*** A laboratory procedure that is performed outside of a living organism. It is usually done using test tubes, culture dish, and any artificial conditions - ***In vivo.*** A laboratory procedure that is performed within the living organisms. It is usually done in clinical trials of safety and efficacy of certain drugs in humans or animals. - ***Nephrolithiasis.*** A condition where mineral deposits form nephroliths or renal calculi in the kidney, ureters, or bladder that hinders the excretion and urination of a person. - ***Nucleation.*** A process in which monomers, like atoms,ions, or molecules, form a new structure or configuration at the atomic or molecular level. - ***Polyphenols.*** A compound found in plant foods like fruits and vegetables that contains more than one phenolic hydroxyl group. - ***Synergistic.*** Interaction of two or more substances to produce a combined effect greater than the sum of their separate effects. - ***Urolithiasis.*** A complex urological disorder characterized by the production of calculi in the kidneys, bladder, and urethra. ### Acronyms - ***CaOx*** -- Calcium Oxalate - ***CUPRAC*** -- Cupric Reducing Antioxidant Capacity - ***DPPH*** -- 2,2-diphenyl-1-picrylhydrazyl - ***HPLC*** -- High Performance Liquid Chromatography - ***TFC*** -- Total Flavonoid Content - ***TPC*** -- Total Polyphenol Content CHAPTER 2 ========= REVIEW OF RELATED LITERATURE ---------------------------- ### Calcium Oxalate Crystal Formation and Pathophysiology Calcium oxalate (CaOX) crystal formation is a primary contributor to nephrolithiasis, commonly known as kidney stone disease. Kidney stones are mineral deposits found in the calyces or pelvis, either free-floating or attached to the renal papillae. They consist of crystals and organic materials, forming when urine becomes supersaturated with minerals (Tamborino et al., 2024). According to recent studies, nephrolithiasis affects 2--15% of the global population and varies regionally, with rates of 13% in North America, 5--9% in Europe, and 1--5% in Asia (Sui et al., 2020; Kim et al., 2022). Tamborino et al. (2024) emphasize that understanding the formation and pathophysiology of these crystals is critical for developing effective prevention and treatment strategies. CaOX stones primarily occur in two forms: monohydrate and dihydrate. The monohydrate form is more thermodynamically stable, while the dihydrate form exhibits higher solubility (Tamborino et al., 2024). According to the study Allam (2024), CaOX crystal formation initiates when urine becomes supersaturated with calcium and oxalate ions, leading to nucleation---the step where these ions combine to form microscopic crystals. Subsequent crystal growth and aggregation result in larger stones capable of adhering to renal epithelium, potentially causing urinary obstruction and renal injury. The attachment of crystals to renal epithelial cells triggers inflammation, oxidative stress, and cellular injury, promoting immune cell recruitment and mediator release. This cycle intensifies stone growth, aggregation, and urinary cast formation, obstructing urine flow and advancing kidney stone disease. Factors such as the degree of urinary supersaturation, urinary pH, and the presence of specific inhibitors and promoters significantly influence this process. The multifaceted nature of CaOX stone formation extends beyond these biochemical interactions, encompassing genetic, metabolic, and environmental components (Shastri et al., 2023). Recent studies have highlighted the contribution of metabolic disorders, such as hypercalciuria, hyperoxaluria, hypocitraturia, hypomagnesuria, and hyperuricemia, to an increased risk of stone development (Geraghty et al., 2020; Tamborino et al., 2024). Furthermore, kidney stones are increasingly recognized as a risk factor for various systemic diseases, including diabetes, cardiovascular disease, bone fractures, and chronic kidney disease. Conversely, these systemic conditions also play a role in the development of kidney stones. The authors suggest that shared risk factors play a significant role in both stone formation and the progression of these systemic health issues (Stamatelou & Goldfarb, 2023). Additionally, the balance between promoters, such as calcium and oxalate, and inhibitors, such as citrate and magnesium, plays a critical role in determining crystal growth, aggregation, and attachment. According to Wang et al. (2021), calcium, oxalate, urate, and phosphate ions are key promoters of crystal formation, facilitating the crystallization of stone components or their aggregation by triggering various mechanisms. Conversely, inhibitors like citrate and magnesium play a protective role by efficiently hindering crystal growth and aggregation, with citrate showing effectiveness at concentrations above 0.1 mM and magnesium enhancing its action synergistically in acidic environments. These interactions highlight the complexity of calcium oxalate stone formation, emphasizing the need for comprehensive strategies to prevent and manage this condition effectively. Stamatelou and Goldfarb (2023) highlight that calcium oxalate remains the predominant component of kidney stones worldwide. To prevent recurrent kidney stone disease, Barghouthy and Somani (2021) emphasize the importance of a combination of dietary and lifestyle modifications, along with medical management in some cases. Key strategies for prevention include adequate hydration, minimizing urinary oxalate and uric acid overload, regulating urinary pH to prevent acidic urine, and avoiding low urinary citrate levels. These approaches align with the need for comprehensive strategies to manage and prevent kidney stones, with research into inhibitors of calcium oxalate crystal formation offering promising avenues for therapeutic interventions (Allam, 2024). ### Role of Natural Products in Stone Inhibition The study of natural products with regards to inhibition of stones has gained pace over these years due to the potential therapeutic action against urolithiasis, in particular those from the calcium oxalate stones, the most common urinary calculi. Indeed, some of these phytochemicals demonstrated inhibitory actions towards the nucleation and growth of those stones. Among these, Cystone is a polyherbal formulation containing herbs like *Didymocarpus pedicellata*, *Saxifraga ligulata*, and Gokshura, which are well studied against renal calculi. This has emerged as a standard reference in various studies that have evaluated anti-urolithiatic properties in other herbal extracts, particularly in the context of inhibition of calcium oxalate crystals (Sewwandi, 2023; Al‐Mamoori & Aburjai, 2023). In comparative studies, Cystone has evidenced marked inhibition of calcium oxalate crystallization and thus established its value as a control in experimental setups that assess the efficacy of other phytochemicals. For instance, it was reported that Cystone can inhibit nucleation by 17.4% and aggregation by 44.27% at a concentration of 2000 µg/mL, comparable to various tested plant extracts (Sayed et al., 2023). Further, Cystone was noted to influence the urinary composition by reducing the excretion of calcium ions and increasing the level of citrate, adding to its anti-urolithiatic action. Citrate is considered an important inhibitor of the formation of calcium oxalate stones in the urine, hence making dietary factors an important aspect in the management of urolithiasis (Oumari et al., 2022). Various studies have thrown light on the role of phytochemicals in Cystone, which act through antioxidant and anti-inflammatory actions. The bioactive phytoconstituents, including flavonoids and polyphenols from the herbal extracts, are responsible for the synergistic contribution to the antilithiatic effects seen in Cystone (Menyiy et al., 2021). Clinical evidence supports the efficacy of the formulation by showing that the intervention improves urinary composition and decreases stone formation even over prolonged periods (Agdamag et al., 2020). This coincides with reports that have collated the efficacy of various phytomolecules to prevent urinary stone formation, among which are the phytomolecules quercetin and rutin (Chinnappan, 2023). Further, the section deals with protective urinary proteins that usually prevent stone formation, one of which is Tamm-Horsfall protein. Prevention of crystal aggregation and adhesion bacteria by THP has been believed to reduce the risk of urolithiasis. Recent studies indicate that urinary THP may predict the recurrence of the stones, thus underlining its importance in the development of urinary calculi (Djafari, 2024). The interaction of dietary factors, natural products, and urinary proteins shows the multifaceted approach required for the effective management of kidney stones. This agrees with the therapeutic potential of natural products such as Cystone in addressing urolithiasis (Al‐Mamoori & Aburjai, 2023). In conclusion, the integration of natural products, like Cystone, into the management of urolithiasis offers hope for newer dimensions of therapeutic development. Evidence of the efficacy of phytochemicals and urinary problems inhibiting stone formation warrants further study to explore these natural alternatives in conjunction with conventional treatments. ### Citrus limon (Lemon) *Citrus limon*, also known as lemon, is species under the Rustaceae family and is shown to have many beneficial effects in the human body. Klimek-Szczykutowicz et. al (2020) conducted a review study on the chemistry, pharmacological properties, as well as the application of *C. limon* (Lemon) in modern pharmaceutical, food, cosmetics and biotechnological industries. It highlights the biological activities that *C. limon* (lemon) are capable of due to its abundance in compounds such as flavonoids, phenolic acids, and essential oils (D-limonene). Several studies also showed the potential therapeutic properties of *C. limon* including anti-inflammatory, antimicrobial, anticancer, and antioxidant effects. One of the chemical properties of *C. limon* that could inhibit calcium oxalate crystal formation is citric acid. The study of Li et. al (2020) conducted a study to evaluate the role of citric acid in calcium oxalate crystallization. It stated that from previous studies, it have been found that citric acid inhibits crystallization of calcium oxalate through inhibiting the crystal nucleation and aggregation. In addition, the study explore other mechanism that the citric acid influences the crystallization of calcium oxalate, which is the (1) modulation of water content which is greatly affected by the citric acid leading to precipitation of amorphous calcium oxalates; (2) pathway shifts which occurs when there is presence of citric acid that leads to formation of various calcium oxalate hydrates; and (3) structural similarities which suggests a direct link between the early stages of precipitation and the crystal structure. This findings provide insight for the development of more effective strategies for the prevention and treatment of kidney stones. Other than citric acid, *C. limon* is also rich in flavonoids and polyphenols which have diverse pharmacological properties. The study of Rizaldy et. al (2022) assessed the antioxidative activity and marker compound of lemon peel and flesh and its relationship to total phenolic content (TPC) and total flavonoid content (TFC). TPC and TFC are evaluated using the colorimetric method, Folin-Ciocalteu and Chang respectively; while the antioxidative activities are measured using DPPH and CUPRAC; and HPLC are used to analyze the marker compound. The study showed a strong correlation between TPC and antioxidant activity in lemon peel extract that is measured by DPPH. However, it did not find a significant correlation between the TFC and antioxidant activity using CUPRAC. Despite this, it is important to take note that flavonoids are a part of phenolic compounds, hence TPC reflects all combined contributions of various phenolic compounds, including flavonoids. Moreover, there are different flavonoids that exhibit different antioxidant properties. In summary, this study highlighted the importance of phenolic compounds, including the flavonoids in the antioxidant activity of lemon peel and flesh. It contributes to a deeper understanding of the relationship between antioxidant properties of *C. limon* but further research is needed to know specific roles of different flavonoids and other phenolic compounds. The findings of this study are further supported by the study Haida et. al (2021) on which evaluates the different factors that affect the recovery of total polyphenols, phenolic acids, and flavonoids content from *C. limon* peel. It assesses the effect of five independent factors which includes drying temperature, methanol concentration, extraction temperature, extraction time, and storage duration. Among these factors, the most significant and contributing factors that affect the extraction of total polyphenols, flavonoids, and phenolic acids are: (1) drying temperature; (2) storage duration; and (3) extraction time. *C. limon* are rich in vitamin C or ascorbic acid, however different from citric acid and polyphenol, this property of *C. limon* can promote the formation of calcium oxalate crystals. Vitamin C overdose, along with enteric hyperoxaluria, and ingested ethylene glycol may cause calcium oxalate deposition in kidneys (Geraghty, 2020). It is contradictory since vitamin C are considered to be one of the factors that contributes to the increase in formation of calcium oxalate crystals. In the study of Karam et. al (2022), where they assess the prevalence of urolithiasis due to vitamin C and D supplementation during COVD-19 pandemic, it explains how excess vitamin C intake can lead to the calcium oxalate crystals formation. It stated that while vitamin C is essential for its antioxidant properties and immune function, it undergoes a metabolic process that leads to the production of oxalate. Increased intake of vitamin C could lead to increased production of oxalate production in the body. Elevated levels of urinary oxalate are a major risk factor for the formation of calcium oxalate crystals. Furthermore, Bouha et. al (2024), evaluates the lemon juice as a chelating agent for calcium oxalate crystal prevention using electrochemical techniques. The findings obtained using the calcium carbonate simplified model showed the ability of lemon juice to reduce the availability of complex calcium ions for crystal formation. Thus, it has a great potential in preventing the calcium oxalate crystal formation. However, as promising as it can be, further research is advised to evaluate its in vivo and clinical efficacy. In relation to its vitamin C content, although it is a factor which increases the calcium oxalate crystal formation, *C. limon* has the inhibitory effect primarily because of its other chemical properties such as citric acid and polyphenols. Moreover, studies show that only supplements that have higher concentrations of vitamin C can increase the calcium oxalate crystal formation. C. limon, which is relatively rich in vitamin C as it is one of the citrus fruits, can increase the formation of calcium oxalate crystals but it can also be an inhibitory substance due to its citric acid and polyphenols (flavonoids) properties. ### Cucumis sativus (Cucumber) ***C***ucumber (*Cucumis sativus*) is a plant that belongs to the *Cucurbitaceae* family together with pumpkin, melon, and squash. It is one of the popular vegetable crops known for its high water content of 96% (Chakraborty & Sadhana Rayalu, 2021). As described by Pal (2020), Cucumber contains numerous amount of nutrients despite the fruit being low in calories, It contains significant amounts of Vitamin K (19%), Molybdenum (12%), Pantothenic acid (5%), Copper (4%), Phosphorus (4%), Vitamin C (4%), Biotin (4%), Vitamin B1 (3%), Potassium (3%), Magnesium (3%), and Manganese (3%). These nutrients make cucumber a nutritious and valuable addition to a healthy, balanced diet. Cucumbers contain a significant amount of dietary fiber, potassium, and magnesium. Mallick (2022) has found that cucumbers are vital for maintaining cardiovascular health due to their ability to lower blood pressure. Furthermore, with the vitamin K content of the fruit, cucumber has the potential to regulate the clotting tendencies of the body and maintenance of the structure of bones. Lignans, one of the contents of cucumber, are an antioxidant that inhibits the high reserve of free radicals in the body, which if stored in high amounts, can result in cell damage and various diseases. The body's hydration is imperative in preventing the formation of calcium oxalate crystals. As stated by Parveen Akhtar et al. (2020), regular consumption of *Cucumus sativus* (cucumber) will aid in the dissolution of kidney stones as the content of such is mainly water and essential electrolytes, therefore dehydration would be prevented. Moreover, the water content of such fruit can serve as a diuretic, enabling the body to remove as much as metabolic waste in urination. Supersaturation of calcium oxalate is one of the vital risks of the formation of its crystals, as the formation of such originates from supersaturation (Susilo et al., 2021). In a research conducted by (Muhamad Insanu et al., 2022), results indicate the flavonoid content of cucumber. Cucumber has 0.00114% quercetin content in ethanol cucumber pulp extract, which concluded that the pulp and leaves of cucumber are good sources of natural antioxidants. Quercetin, a flavonoid, is discovered to be inhibitory to the growth of calcium oxalate (Sakdithep Chaiyarit et al., 2023). ### Synergistic Effects of Combined Extracts Although no direct studies have been conducted on the combined effects of *Citrus limon* (lemon) and *Cucumis sativus* (cucumber) in inhibiting calcium oxalate crystallization, it is hypothesized that these two fruits may exhibit synergistic properties. Lemon juice, characterized by its high citric acid content, has the capacity to chelate calcium ions and reduce supersaturation, thereby inhibiting crystal formation. On the other hand, cucumber juice, known for its potent antioxidant properties, may mitigate oxidative stress and impede crystal aggregation. Synergy, a phenomenon where the combined effect of two or more components exceeds the sum of their individual effects, is frequently observed in plant-based research. According to Pezzani et al. (2019), such synergistic interactions enhance antioxidant activity, improve bioavailability, and amplify both therapeutic and preventive outcomes, underscoring the potential efficacy of plant-derived compounds in addressing medical and nutritional challenges. Dietary interventions using citrate-rich juices have demonstrated significant effects in modulating urinary parameters associated with calcium oxalate stone formation. A study conducted by Gopala et al. (2023) explored the effects of lemon--tomato juice in a randomized crossover clinical trial involving 22 patients with calcium oxalate stones. The findings revealed that consumption of lemon--tomato juice led to a significant reduction in urinary optical density (mean = 0.131 for milk only vs. 0.053 for milk and lemon--tomato juice, p \< 0.001), a marker for calcium oxalate supersaturation. Additionally, the calcium--creatinine ratio in the urine decreased notably (mean = 0.141 for milk only vs. 0.076 for milk and lemon--tomato juice, p = 0.019), indicating reduced risk factors for stone formation. Numerous studies have highlighted the enhanced health benefits arising from synergistic interactions between bioactive compounds found in fruits and vegetables. Such interactions often improve bioavailability and therapeutic efficacy. For example, research published in the *Proceedings of the Nutrition Society* has demonstrated that combinations of fruits and vegetables, such as citrus fruits and other botanicals, can yield amplified preventive and therapeutic outcomes (Eivers et al., 2018). These synergistic effects are particularly evident in the context of urinary health, where combined bioactive components have been shown to reduce oxidative stress and inhibit factors that promote calcium oxalate crystallization. ### Gaps in Current Literature Despite extensive research into natural inhibitors of calcium oxalate crystallization, several gaps remain in the current literature, particularly concerning the combined effects of *Citrus limon* (lemon) and *Cucumis sativus* (cucumber) juices. While individual studies have demonstrated the inhibitory effects of lemon juice on calcium oxalate crystallization (Barghouthy & Somani, 2021), there is a lack of research exploring the potential synergistic effects when combined with cucumber juice. Most existing studies have focused on the effects of these substances separately, leaving the combined impact unexplored. For instance, Gopala et al. (2023) investigated the effects of lemon-tomato juice consumption on risk factors for calcium oxalate stone formation in patients. The results showed that the optical density of urine samples was significantly lower after consuming milk and lemon-tomato juice compared to milk only (mean = 0.053 vs. 0.131, p \< 0.001). Additionally, the urine calcium-creatinine ratio was also lower in the lemon-tomato juice group (mean = 0.076 vs. 0.141, p = 0.019). These findings suggest that lemon juice, particularly when combined with other substances like tomato, can help reduce risk factors for calcium oxalate stone formation. However, research specifically investigating the combined impact of lemon and cucumber juices on calcium oxalate crystal formation is lacking. Moreover, there is a notable gap in research regarding the role of cucumber (*Cucumis sativus*) in inhibiting calcium oxalate formation. While cucumber has been recognized for its hydrating properties and high water content, few studies have explored its potential as a natural inhibitor of calcium oxalate crystallization (Sariyanto, 2020). Most existing research has concentrated on substances like lemon juice, which has demonstrated promising results in reducing supersaturation, crystal nucleation, and aggregation in vitro. However, the absence of studies exploring cucumber\'s individual effects on calcium oxalate crystals presents a significant gap in understanding its potential as a complementary treatment for kidney stone prevention. The combined effects of cucumber and lemon juices on calcium oxalate crystallization remain to be investigated, despite the potential for synergistic interactions between these two natural substances. Addressing this gap could yield valuable insights and contribute to the development of more effective dietary strategies for the prevention of kidney stone formation. Further research in this area has the potential to advance the understanding and facilitate the implementation of accessible, natural interventions for kidney stone prevention. CHAPTER 3 ========= METHODOLOGY ----------- ### Study Design The study will employ an experimental research design to investigate \"The Synergistic Effect of *Citrus limon* (Lemon) and *Cucumis sativus* (Cucumber) Juices on Calcium Oxalate Crystal Inhibition in an In Vitro Model.\" The inhibition activity will be evaluated by testing the combined juices against calcium oxalate crystals to determine the relationship between the independent variables (*Citrus limon* and *Cucumis sativus* juices) and the dependent variable (degree of crystal inhibition). This design will allow for controlled manipulation of variables under standardized laboratory conditions. ### Study Setting The researchers will conduct the experimentation at the National University---Mall of Asia. The study will take place in the university's laboratory facilities, ensuring the availability of the necessary equipment and resources for precise data collection and analysis. All experiments will be carried out in compliance with institutional protocols and safety guidelines to maintain the integrity of the research process. ### Purchasing and Certification of Plant Material The researchers will develop a plan for the experiment that includes technical frameworks, task responsibilities, resource management, communication, financial planning, and a timeline. The researchers will also identify potential obstacles in data collection and create backup plans to address these challenges. The Cystone, available at a cost of PHP 320, will be procured from the Himalaya Drug Company. Key chemicals required for the experiment include calcium chloride dihydrate (PHP 290 for 50g), sodium oxalate (PHP 490 for 50g), Tris buffer (PHP 780 for 20g), and sodium metabisulfite (PHP 560 for 100g), all of which will be sourced from DKL Laboratory Supplies. Certified specimens of *Cucumis sativus* (cucumber) and *Citrus* *limon* (lemon) will be sourced from reputable local suppliers. Certification will be obtained from the Bureau of Plant Industry to ensure their quality and compliance with the study\'s requirements. The researchers will carefully select all materials to maintain the study\'s integrity. ### Extraction of Citrus limon (Lemon) Material The fresh C. limon (lemon) will be cut open with a sterilized knife, cleaned in lab with running tap water, surface sterilized with 70% alcohol, and their juices will be squeezed out into a sterile universal container before being filtered into another sterile the seeds and other tissues from the fruit (Mahjaf et al., 2024). The lemon juices will be put into three containers with varying concentrations of 10%, 25%, and 50%. ### Extraction of Cucumis sativus (cucumber) The fresh cucumber will be cleaned and dried. It will then peel, its seed will be cleaned and peeled fruit will be cut into pieces. Using a laboratory mortar and pestle it will be then crushed in order to extract all the juices (Amani et al., 2024). It will then be filtered by muslin cloth and a Whatman filter paper in order to remove other impurities. The extracted juices will then transferred in to the container with varying concentration of 10%, 15%, 50%. ### Preparation of Calcium Oxalate Crystals Calcium oxalate crystals will be synthesized following the procedure described by Hennequin et al., with slight modifications. Equal volumes of 0.735 g calcium chloride (CaCl₂) and 0.67 mg sodium oxalate (Na₂C₂O₄) will be prepared in a buffer containing 10 mM Tris-HCl and 90 mM NaCl at pH 6.5. The solutions will be mixed at 37°C, resulting in a white turbid suspension, and stirred at 400 rpm for 24 hours. After stirring, the mixture will be left undisturbed to allow the crystals to settle. The supernatant will be discarded, and the crystals will be washed twice with ethanol and once with distilled water before being subjected to lyophilization (Vaitheeswari et al., 2015). The inhibitory potential of Citrus limon and Cucumis sativus juices will be evaluated by incorporating the juices into the synthesis procedure at various concentrations, and the effects on crystal formation will be analyzed. ### Nucleation Assay The determination of effectivity of the juices will be measured using nucleation and aggregation assay. Crystallization will be done by mixture of 4 mmol/L of calcium chloride and 7.5 mmol/L of sodium oxalate and it will be prepared at 6.5 pH and at temperature of 37 degrees Celsius in a Tris buffer which contains 0.05 mol/L of Tris and 0.15 mol/L of NaCL as the synthetic urine (Zarin, et.al, 2020). A varying concentration of the combined *Citrus limon* and *Cucumis sativus* juices (10%, 25%, and 50%) will be added to the solution to assess its inhibitory effect in the crystal formation of calcium oxalate after the incubation of 6 hours at 37 degrees Celsius. Cystone filtrate will be used as a positive control to assure the accuracy and precision of the results. The inhibition reaction will be measured by reading the absorbance of the solution using a spectrophotometer at 620 nm for each concentration (Zarin, et. al, 2020). Percentage of inhibition will be calculated using the formula: The same procedure will be done using *Citrus limon* and *Cucumis sativus* with the same concentration, individually and the percentage inhibition will be compared with the combined concentration of the two juices. ### Aggregation Assay In accordance with Zarin et al. (2020), the preparation of calcium oxalate crystals includes mixing 50 mmol/L of calcium chloride and sodium oxalate. The solutions will be submerged in a water bath at 60°C for 60 minutes, which will then be cooled to 37°C before evaporation of the solution. Tris buffer which is prepared by combining Tris (0.05 mol/L) and NaCl (0.15 moL/L) will be added to the crystals, resulting for the calcium oxalate crystals to have a final concentration of 0.8 mg/mL. The fruit extract of Cucumus Sativus (cucumber), Citrus limon (lemon), and the combination of the two in different concentrations (10%, 30%, 50%) will then be tested with the utilization of spectrophotometry in wavelength of 620 nm. The results will then be calculated using the formula below:  The aggregation, growth, and inhibition rate of calcium oxalate crystals will be observed and analyzed using a brightfield microscope. The ability of the calcium crystals to aggregate will be graded as follows ### Statistical Analysis The following statistical tools will be utilized when processing the data: ***Research Flowchart*** Agdamag, A., Aggabao, L., Agudo, M., Alcachupas, F., Alejo, J., Altamera, S.,... & Rosa, T. (2020). Anti-urolithiatic activity of sambong (blumea balsamifera) extract in ethylene glycol-induced urolithiatic wistar rats (rattus norvegicus). Acta Medica Philippina, Alomair, M., Alobaid, A., Almajed, M., Alabduladheem, L., Alkhalifah, E., Mohamed, M.,... & Younis, N. 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International Journal of Molecular Medicine, 48(2). ***APPENDICES*** **Appendix A: Gantt Chart** GANTT CHART =========== ACTIVITIES NOV 29 DEC 6 – 2024 DEC 6-13 2024 DEC 14-23 2024 JAN 6-10 2025 JAN 11-16 2025 JAN 17 2025 JAN 18-31 2025 FEB 1-28 MAR 5 2025 MAR 17-31 2025 APR 1-30 2025 MAY 1-31 2025 JUNE 1-30 Initial Title (Form 1) Refined Research Title (Form 2-3) Title Deliberation and Submission of Title Proposal (General Format Form) Chapter 1 Chapter 2 Chapter 3 Proposal Defense Submission of Manuscript Initial Approval of Panel on Chapters 1-3 Authentication of Plant Material Purchasing of Plant and Reagents Research Experiment Chapter 4 Chapter 5 Final defense Submission of Final Hardbound Manuscript **Appendix B: Budget proposal** **Expenses** **Quantity** **Cost (PHP)** ---------------------------- -------------- ---------------- **Reagents** Cystone 60 tablets 320 Calcium Chloride Dihydrate 50g 290 Sodium Oxalate 50g 490 Sodium Chloride 50g 290 Tris Buffer 20g 780 Tri-HCl 10g 390 Distilled Water 1 liter 75 Ethanol 100ml 280 **Plant material** Cucumber 400-500g 105 Lemon 1 pack 137 **Other materials** Whatman no.1 filter paper 1 box 1,896 Muslin cloth 60 **Total** **5,113**