Assessment of hepatoprotective, nephroprotective efficacy, and antioxidative potential of Moringa oleifera leaf powder and ethanolic extract against PCOS‐induced female albino mice (Mus Musculus)

Abstract

Moringa oleifera is a medicinal plant that has anti‐inflammatory, antihypertensive, antidiabetic, tissue‐protective, and antioxidant activities. Here, we evaluated the protective effect of M. oleifera leaf powder (MoLP) and 70% ethanol M. oleifera leaf extract (MoLE) on mitigating polycystic ovary syndrome (PCOS)‐induced liver and kidney dysfunction via regulating oxidative stress in female albino mice (Mus musculus). The efficacy of M. oleifera was compared with metformin (standard medicine used to treat infertility in women). PCOS was induced by intramuscular injection of testosterone enanthate at 1.0 mg/100 g BW for 35 days. PCOS‐induced mice were treated with MoLP (250 and 500 mg/Kg), MoLE (250 and 500 mg/kg), and metformin (250 mg/kg) orally for 14 days. Renal function test (RFT), liver function test (LFT), and oxidative stress biomarker malondialdehyde (MDA) were quantified in serum at 0, 7, and 14 days of intervention. Mice treated with M. oleifera and metformin showed a significant decrease (p < .001) in alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphate (ALP), total bilirubin, urea, creatinine, and a significant increase (p < .001) in total protein, albumin, globulin, and albumin/globulin (A/G) ratio. Oxidative stress decreased significantly (p = .00) with respect to treatments, exposure days, and their interaction in metformin and all M. oleifera‐treated groups. M. oleifera leaf powder and extract reduce oxidative stress and enhance nephron‐hepatic activity in PCOS‐induced female albino mice.

This study highlights the potential of both Moringa oleifera leaf powder and extract as effective therapeutic options for managing PCOS, and liver and kidney diseases. It also provides new opportunities for further research in this field.

1. INTRODUCTION

Polycystic ovary syndrome (PCOS) is a complex disorder whose etiology seems to be polygenic, multifactorial, and multisystem hormonal problems. It affects approximately 5–10% of women during puberty and is characterized by elevated androgen levels, lack of ovulation, insulin resistance (IR), and type II diabetes (Broskey et al., 2018). Women with PCOS may have hyperinsulinemia, hyperlipidemia, and a high LH/FSH ratio that affects oocyte quality (Siahaan et al., 2022). The use of androgens (testosterone, testosterone propionate, enanthate) or estrogens (estradiol valerate) is a technique to develop a type of physiopathology in animal models similar to women with PCOS (Anesetti & Chávez‐Genaro, 2016). Insulin resistance (IR) due to elevated levels of free fatty acids and hyperglycemia leads to the activation of reactive oxygen species (ROS) and enhances oxidative stress (Velaga et al., 2017).

Oxidative stress is caused by a deskbound lifestyle, obesity, and exposure to toxic compounds, pesticides, and heavy metals, which raises the risk of diabetes, metabolic disorders, inflammation, and infertility (Kostoff et al., 2020; Tsatsakis et al., ²⁰¹⁹). Oxygen free radicals are superoxides that can cause cell damage and apoptosis, leading to cancer and myocardial infarction (Padureanu et al., 2019; Tsatsakis et al., ²⁰¹⁹). Oxidative stress also results in PCOS and leads to a high level of malondialdehyde (MDA) (Kalhori et al., 2018). The presence of toxic substances in the body, such as MDA, free radicals, and other lipid peroxidation products causes oxidative stress and can contribute to insulin resistance (IR) (Murri et al., 2013).

PCOS patients are increased risk of diabetes, abdominal adiposity, and metabolic disorders like liver and kidney damage and steatosis (Vassilatou, 2014). In women with PCOS, IR, and hyperandrogenism, double the chances of fatty liver disease (steatosis), which can cause more severe and rapidly progressive liver disease at a young age (Javed et al., 2019). PCOS can cause kidney cysts that cause enlarged kidneys and loss of function. However, women with PCOS may have an increased risk of developing chronic kidney disease (CKD). CKD damages renal activity, ultimately leading to end‐stage kidney function, and is one of the leading causes of mortality (Fraser et al., 2015). Several studies have shown that decreased glomerular filtration and microalbumin in urea are vital markers of kidney dysfunction in PCOS women (Song et al., 2019). Escobar‐Morreale (2018) found more cases of age‐dependent CKD in PCOS‐induced female rats.

Metformin is an antidiabetic drug that shows redox‐modulating effects and addresses numerous ailments linked with oxidative stress, and can chelate polyvalent metal ions, reduce oxidative stress, and mitigate toxicity (Karmanova et al., 2023). Metformin, clomiphene citrate, and tamoxifen are commonly prescribed for PCOS. Studies have shown that metformin ameliorates hormonal disorders, maintains ovarian physiology, and reduces obesity in PCOS patients by inhibiting hepatic glucose uptake and promoting peripheral glucose uptake (Chukwunonso Obi et al., 2016). Long‐term use of these drugs may have limited efficacy and cause many side effects, such as abdominal disturbance, nausea, headache, and weight gain (Domecq et al., 2013; Younas et al., ²⁰²²). However, it is necessary to identify and develop alternative and safe sources to prevent and treat PCOS. This is a leading cause of infertility and oxidative stress (Jelodar & Askari, 2012).

Moringa oleifera is a food plant and is often called golden tree, magic plant, drumstick, and sohanjna (Grosshagauer et al., 2021). It is grown for its edible leaves and flowers, nutritious pods, and extensively consumed as food, medicines, cosmetic oil, or fodder for livestock (Vergara‐Jimenez et al., 2017). It contains multiple therapeutically active chemicals that make it an ideal candidate for supplementation with side effects suppression. Historically, it has been used as an antidiabetic, antioxidant, antimicrobial, and anti‐inflammatory remedy to cure more than 300 diseases (Zucca et al., 2016). It is a storehouse of nutrients, such as minerals, vitamins, flavonoids, and phenols. Flavonoids like quercetin possess strong antioxidant properties, inhibit various enzymes, quench ROS production, and chelate all the metals used in radical chain reactions (Banafsheh & Sirous, 2016; Kumar & Pandey, ²⁰¹³; Shah & Patel, ²⁰¹⁶). Polyphenols (gallic acid) have antiobesity, antidiabetic, anti‐inflammatory, and antioxidative activity (Esmaeilzadeh et al., 2020). Chlorogenic acid (CGA) elevates nonenzymatic antioxidant activity. Ferulic acid is a strong antioxidant that provides lipid peroxidation protection, scavengers free radicals, and binds to Fe and Cu (Tee‐ngam et al., 2013; Zduńska et al., ²⁰¹⁸). Sinapic acid and p‐Coumaric acid possess anti‐inflammatory, antimicrobial, antimutagenic, antioxidant, antidiabetic, and antihyperlipidemic properties (Chen, 2016).

A kidney function test (RFT), such as urea and creatinine, and a liver function test (LFT) are biochemical blood tests designed to assess the working of the kidneys and liver. Examining hepatic injury in patients often involves analyzing the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are both important biomarkers. Albumin and transferase tests are related to cellular integrity, while alkaline phosphatase is linked to the biliary tract (McClatchey, 2002). A high level of BUN (blood urea nitrogen) is caused primarily by a high‐protein diet, congestive heart failure, gastrointestinal hemorrhage, and increased catabolism (Braunwald et al., 2005).

Considering the alarming prevalence of PCOS and poor physical, mental, and social outcomes, a study was carried out to investigate the hepatoprotective and nephroprotective efficacy of M. oleifera leaf powder (MoLP) and M. oleifera leaf extract (MoLE) against PCOS‐induced albino mice. We hypothesized that MoLP and MoLE are effective therapeutic interventions to reverse PCOS. We further presume that the effects of MoLP and MoLE will be comparable to metformin. Thus, it is possible that this could reverse the kidney and liver damage caused by PCOS in humans.

2. MATERIALS AND METHODS

2.1. Plant collection and preparation of aqueous and ethanolic extracts

Moringa oleifera fresh leaves were collected from the garden of the University of Sargodha. The detailed procedure for preparing aqueous and ethanol extract was previously described in Khalid et al. (2023).

To prepare the ethanol extract (MoLE), 300 g of the powder was macerated in 70% ethanol (5000 mL) using a shaking incubator. The resulting mixture was filtered, evaporated using a rotary evaporator under reduced pressure, and stored at 4°C. Seventy‐percent ethanolic extract of M. oleifera leaves (MoLE) was used as treatment dosage.

2.2. Development of basal and focused feed

The feed was prepared following proximate and HPLC analyses of MOLP extracts. Basal feed was composed of carbohydrates (16%), proteins (18%), and fats (2%), while focused feed was prepared as per table.

2.3. Experimental animals

For the experiment, 100 female albino mice (Mus musculus) were used. They were 8–9 weeks old and weighed 26 ± 2 g. Mice were obtained from the stock of mice bred in the animal house of the University of Sargodha, Pakistan. They were kept under standard housing conditions with a temperature of 22 ± 2°C, relative humidity of 45 ± 5%, and 12 h of light/dark cycles. Before the experiment, the mice were acclimatized for 10 days. Mice with anatomical abnormalities or pregnancies were excluded from the experiment during the acclimatization days (Amelia et al., 2018). Ten albino mice were randomly selected, and their biomarker, including malondialdehyde (MDA), renal function test (RFT), and liver function test (LFT), were measured.

2.4. Induction of PCOS

After a period of acclimatization, 1.0 mg/100 g BW testosterone enanthate was dissolved in sesame oil (100:9900 μL), and injected in into thigh muscle of albino mice for 35 days to induce PCOS (Kalhori et al., 2018).

2.5. Experimental design

PCOS‐induced albino mice (n = 76) were randomly divided into six treatment groups, while non‐PCOS‐induced mice (n = 10) were kept as a negative control group. The negative control group (non‐PCOS‐induced mice) was assigned to group $T_0^{-}$. PCOS‐induced mice were randomly divided into six groups T ₀ (Placebo), $T_0^{+}$ (Positive control, Basal Feed + Metformin at 250 mg/kg BW), T ₁ (MoLP‐250, Basal Feed + MoLP at 250 mg/kg BW), T ₂ (MoLP‐500, Basal Feed + MoLP at 500 mg/kg BW), T ₃ (MoLE‐250, Basal Feed +70% MoLE at 250 mg/kg BW), and T ₄ (MoLE‐500, Basal Feed +70% MoLE at 500 mg/kg BW). The study comprised 10 days of acclimatization, 35 days of induction, and 14 days of intervention (Amelia et al., 2018).

2.6. Biochemical analysis

Various biomarkers of albino mice were studied during acclimatization, PCOS induction, intervention period, and at the completion of the study.

2.7. Interventions among infertile albino mice

The biomarkers were reexamined at the end of the experiment in all treatment groups by following treatment for 14 days in PCOS‐induced albino mice.

2.8. Lipid peroxidation

Malondialdehyde (MDA) concentration was determined using the Buege and Aust (1978) methodology based on thiobarbituric acid reactivity. A volume of 50 μL of serum was taken, and 1 mL of trichloroacetic acid TCA‐2‐thiobarbituric acid (TBA)–HCl reagent was added, and then the samples were placed in boiling water for 10 min. After cooling, the reaction mixture was centrifuged, and the supernatant was separated. The absorbance was read at 535 nm. 1.56 × 105/mol/cm as the molar absorbance coefficient was used to calculate MDA concentration (Khalofah et al., 2020).

2.9. Safety biomarkers

To ensure the safety of interventions on albino mice, blood samples were collected to investigate LFT and RFT at the University Medical and Diagnostic Center (UMDC) at 0, 7, and 14 days.

2.9.1. Liver function test (LFT)

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using the method of Reitman and Frankel (1957). ALT and AST were analyzed by monitoring the concentrations of pyruvate hydrazine and oxaloacetate hydrazine, respectively, using 2,4 dinitrophenylhydrazine. For the experiment, 0.5 mL of ALT and AST substrate phosphate buffer solutions was mixed with 0.1 mL of serum in test tubes. In blank test tubes, 0.1 mL of distilled water was poured instead of serum. These test tubes were incubated in a water bath at 37°C for 25 min. Then, 2,4 dinitrophenylhydrazine (0.5 mL) was added to all test tubes, and the blank test tubes received only a 0.1 mL of sample, which was allowed to stand at room temperature for 20 min. In the end, 5.0 mL of NaOH was pipetted into both test tubes. The absorbance of the sample was recorded at 550 nm after 5 min.

Alkaline phosphate (ALP) was detected by ELITech kits as described by Young (1997). ALP concentration was measured using 2‐amino, 2‐methyl, 1‐propanol (AMP), and p‐nitrophenyl phosphate as substrates. A volume of 200 μL of ALP substrate phosphate buffer solutions and 5 μL serum were added to sample test tubes. In blank test tubes, 200‐μL AMP substrate phosphate buffer solutions and 5‐μL distilled water were poured using a pipette. Both test tubes were incubated in a water bath at 37°C for 15 min. After incubation, the sample absorbance was recorded at 405 nm after 5 min.

Total protein, albumin, globulin, and albumin/globulin (A/G) ratio were evaluated using the method of Lowry et al. (1951). Protein was assessed by arranging different sets of test tubes in racks and filled with bovine serum albumin (BSA) with concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μL. Two milliliters of solution (48 mL of 2% Na₂CO₃ in 0.1 mL NaOH; 1 mL of 0.5% CuSO₄. 5H₂O in water) and 1 mL of 1% NAK Tartarate in water were incubated at 25°C for 10 min. In the end, 0.2 mL of diluted Folin phenol was mixed into each test tube, vortexed, incubated for 25 min, and the absorbance was recorded at 600 nm.

Total bilirubin was measured using Jendrasskik and Grof (1938). A volume of 0.2 mL of sulfanilic acid was poured into the sample test tube and the blank test tube by a pipette. A volume of 0.05 mL of sodium nitrite was added to the sample tube. Ten milliliters of caffeine and 0.2 mL of sample were added to both test tubes. They were mixed thoroughly and incubated at 25°C for 10 min. Tartrate (1.0 mL) was poured in the end and incubated at 25°C for 25 min, and the absorbance of the sample was measured at 578 nm (Nwankwo et al., 2015).

2.9.2. Renal function test (RFT)

Serum urea and creatinine were assessed using method of Tietz (1994). Serum urea was analyzed by taking 10 μL of sample in a sample test tube, 10‐μL standard solution in the standard test tube, and 10‐μL distilled water in the blank test tube. Ten‐microliter nitroprusside and urease were added to all three test tubes. Test tubes were vortexed and incubated at 37°C for 10 min. In the end, 2.0 mL of phenol was added to the test tubes, followed by 2.50 mL of sodium hypochlorite. The tubes were incubated at 37°C for 10 min. The sample absorbance was recorded at 546 nm (Nwankwo et al., 2015).

To measure creatinine concentration, 100 μL of the sample was added to the sample test tube, 100 μL of distilled water into the blank test tube, and 100 μL of the standard solution in the standard test tube. The tubes were vortexed after adding standard reagent, and the absorbance of the sample was measured at 492 nm (Nwankwo et al., 2015).

2.10. Data analysis and statistical application

All results were expressed as mean ± SEM of ten replicates. The experimental data were tested for normal distribution with Kolmogorov's test. Multifactor analysis of variance (two‐way ANOVA) with ‘treatments’ and ‘days’ as independent variables was applied to test the main effects and interactions (Benjamini & Braun, 2002; Jaccard et al., ¹⁹⁸⁴; Sawyer, ²⁰⁰⁹).

3. RESULTS AND DISCUSSION

3.1. Lipid peroxidation (nM/mL)

The oxidative stress in the current study is measured by an increase in malondialdehyde (MDA), a marker of lipid peroxidation. Serum MDA concentration was significantly increased with time in PCOS‐induced (T ₀) albino mice compared to the negative control group ($T_0^{-}$), as shown in Table 1. Increased testosterone levels caused oxidative stress in albino mice. Rojas et al. (2014) observed that oxidative stress causes infertility and heterogeneous disorders in PCOS patients. Murri et al. (2013) further elaborated that under oxidative stress, the production of highly poisonous products such as MDA, free radicals, and other lipid peroxidation products results in insulin resistance.

TABLE 1.

Mean values indicating the effect of treatments and days on MDA (nM/mL) of albino mice.

Days
Treatments	0	7	14	Means ± SEM
T 0	869.5 ± 11.54c	1142.6 ± 15.17b	1213.8 ± 26.46a	1075.3 ± 104.93A
T 0 −	258.69 ± 3.84h	261.09 ± 4.85h	264.1 ± 5.85h	261.29 ± 1.56F
$T_0^{+}$	871.08 ± 8.96c	785.72 ± 4.09d	504.01 ± 6.01f	720.27 ± 110.90B
T 1	868.39 ± 5.12c	681.8 ± 5.53e	493.09 ± 4f	681.09 ± 108.34C
T 2	870.25 ± 4.53c	489.78 ± 3.67f	391.83 ± 4.6g	583.95 ± 145.91D
T 3	878.44 ± 9.23c	479.99 ± 3.22f	383.1 ± 3.86g	580.51 ± 151.56D
T 4	871.33 ± 4.65c	383.09 ± 3.8g	298.51 ± 4.73h	517.64 ± 178.52E
Means ± SEM	783.95 ± 87.55A	603.44 ± 111.69B	506.92 ± 122.56C

Note: T ₀ = Basal Feed +5% Tween 80; $T_0^{-}$ = Basal Feed; $T_0^{+}$ = Basal Feed + Metformin at 250 mg/kg BW; T ₁ = Basal Feed + MoLP at 250 mg/kg BW; T ₂ = Basal Feed + MoLP at 500 mg/kg BW; T ₃ = Basal Feed + MoLE at 250 mg/kg BW; T ₄ = Basal Feed + MoLE at 500 mg/kg BW.

Oxidative stress decreased significantly (p = .00) with respect to treatments, exposure days, and their interaction in the metformin group ($T_0^{+}$) and all M. oleifera‐treated groups from T ₁ to T ₄ (Table S1). The lowest MDA level was recorded during T ₄ treatment (298.51 ± 4.73 nM/mL), where it was almost four times lower than T ₀ (1213.8 ± 26.46 nM/mL) after 14 days of exposure. Research by Siahaan et al. (2022) proved that M. oleifera leaves reduced IR and diabetes. Moreover, moringa extract is a powerful antioxidant that binds to reactive oxygen species (ROS) and limits oxidative stress in PCOS‐induced rats. Previous studies suggested that antioxidant treatment improves insulin function in diabetic patients by reducing oxidative stress. T ₀ subjects treated with T ₂ and T ₄ at a dose of 500 mg/kg BW showed significantly less oxidative stress, as shown in Table 1. Singh et al. (2009) claimed that flavonoids and phenols in plant leaves were antioxidants and inhibited lipid peroxidation.

In the present research, the MDA level of $T_0^{+}$ (504.01 ± 6.01 nM/mL) was approximately four times lower than T ₀ (1213.8 ± 26.46 nM/mL) on the 14th day of exposure (Table 1). Chukwunonso Obi et al. (2016) found that oxidative stress and free radicals cause diabetic complications, whereas metformin as an antioxidant helps combat these pitfalls. Several studies have shown that Moringa and metformin have a parallel relationship. However, Huang et al. (2015) reported that metformin long‐term administration causes indigestion in patients aggravating diarrhea and abdominal distress. With minimal side effects, herbal plants could be the best option. Gopalakrishnan et al. (2016) suggested that M. oleifera plant extract has the potential as antimicrobial, antioxidant, and antidiabetic.

3.2. Biosafety markers

Blood samples of mice from all treatment groups were collected and liver function test (LFT) and renal function test (RFT) were conducted at 0, 7, and 14 days.

3.2.1. Liver function test (LFT)

The end product of lipid peroxidation, malondialdehyde (MDA), is concentrated in the liver of PCOS‐induced albino mice and enhances oxidative damage to lipids. MDA increased oxidative stress and impaired liver enzyme function compared to mice treated with metformin, M. oleifera leaf powder, and extract. Patients are diagnosed with liver disease by passing through a liver function test (LFT). Sustained liver injury causes hepatocellular injury and excessive collagen damage through the activation of hepatic satellite cells and liver fibrogenesis (Ahmed et al., 2018). A liver function test (LFT) is a biochemical blood assay designed to describe the working of the liver in the patient's body (Lee, 2009). Targher et al. (2016) stated that nonalcoholic fatty liver disease (NAFLD) and polycystic ovarian syndrome (PCOS) are related to each other. Oxidative stress and insulin resistance are common pathogenetic mechanisms in both diseases.

It has been shown in our study that antioxidants can reduce liver stress and PCOS. Metformin, M. oleifera leaf powder (MoLP), and M. oleifera leaf extract (MoLE) are used to establish the fact that they serve as protective antioxidant and antidiabetic drugs, thereby shielding the vital organs of the human body, such as the pancreas, liver, heart, and kidney from oxidative stress induced during PCOS complications. According to Altaee and Fadheel (2021), M. oleifera ethanol extracts inhibited lipid peroxidation and ROS in rabbits injected with iodide. Akter et al. (2021) found that M. oleifera extract limits free radical overproduction in rats induced by tilmicosin and Hg by inhibiting MDA concentration. ANOVA tables of different LFT parameters showed highly significant (p = .00) effects of treatments, days, and interaction (Tables S3–S10).

Serum aspartate transaminase (U/L)

In albino mice, higher serum aspartate transaminase (AST) is associated with liver disease. A significant (p = .00) change in AST was observed due to treatments, days, and interactions (Table S2). There was an increase in AST levels in PCOS‐induced mice (T ₀) due to stress‐induced liver damage caused by injecting testosterone enanthate, with the highest value observed at day 14 (59.49 ± 1.49 U/L). Mice treated with M. oleifera and metformin showed a significant decrease in AST with time, which might be due to stress reduction. The lowest AST (32.89 ± 0.87 U/L) was found in T ₄ (MoLE extract 500 mg/kg BW) mice, as depicted in Figure 1a.

FIGURE 1.

Effect of treatments and days on serum AST (a), ALT (b), bilirubin total (c), ALP (d), total protein (e), serum albumin (f), serum globulin (g), and A/G ratio (h) of albino mice (n = 10 mice per week). T ₀ = Basal Feed +5% Tween 80; $T_0^{-}$ = Basal Feed; $T_0^{+}$ = Basal Feed + Metformin at 250 mg/kg BW; T ₁ = Basal Feed + MoLP at 250 mg/kg BW; T ₂ = Basal Feed + MoLP at 500 mg/kg BW; T ₃ = Basal Feed + MoLE at 250 mg/kg BW; T ₄ = Basal Feed + MoLE at 500 mg/kg BW.

Vassilatou (2014) examined the high AST level in PCOS patients with fatty liver disease. According to another study (Schwimmer et al., 2005), elevated AST was found in 12% of 70 patients examined in an infertility clinic. Del Rio et al. (2005) claimed that serum catalase activity in diabetic rats was significantly lower than in rats treated with metformin. Iweala and Okeke (2005) also supported metformin's role in reducing oxidative stress induced by damaged liver physiology.

Serum alanine transaminase (U/L)

Higher serum ALT indicates liver disease in albino mice. A highly significant (p = .00) change in serum ALT in albino mice was observed due to treatments, days, and interactions (Table S3). Serum ALT increased significantly with time in PCOS‐induced mice (T ₀). The highest rise in serum ALT (69.5 ± 0.45 U/L) was seen in T ₀ mice may be due to stress imposed on the liver by injecting testosterone enanthate, and the lowest (40.9 ± 1.09 U/L) in T ₄ (MoLE extract 500 mg/kg BW) mice on day 14 might be due to stress reduction (Figure 1b). Macfarlane et al. (2000) explored that the rise in serum ALT is reflected in situations involving the necrosis of erythrocytes, myocardial cells, hepatocytes, and skeletal muscles.

Schwimmer et al. (2005) examined 70 female patients with PCOS in an infertility clinic and noted that 30% of females had high ALT levels in their serum. He found that most PCOS and liver disease females are insulin resistant and have abnormal ALT levels. These females with increased ALT had significantly (p < .01) more waist circumference, body mass index (BMI), total cholesterol‐to‐HDL‐cholesterol ratio, serum triglycerides, and degree of IR. Erbey et al. (2000) conducted a population‐based study including 18,825 adult women. The study recorded abnormal ALT in 2.2% of all women, 5.3% of diabetics, and 7.1% of obese women. Another research performed by Ruhl and Everhart (2003) on 5724 female patients showed that ALT increased with BMI: 1.0% normal body weight, 3.3% overweight, and 6.6% more in obese women.

Serum bilirubin total (mg/dL)

Higher serum levels of bilirubin indicate liver damage in albino mice. Highly significant (p = .00) changes in bilirubin in albino mice were observed due to treatments, days, and interaction (Table S4). High bilirubin levels were recorded in T ₀ over time. Statistical analysis revealed a highly significant (p = .00) decrease in serum bilirubin with time in albino mice treated with metformin and M. oleifera compared to mice with PCOS. The elevation in bilirubin (1.68 ± 0.29 mg/dL) was seen in T ₀ albino mice, which might be due to oxidative stress and reduction (0.95 ± 0.02 mg/dL) in T ₄ mice on day 14 of treatment is due to polyphenols in MoLE (Figure 1c).

Serum bilirubin is used as a diagnostic test for hepatic disorders. Its level depicts hepatotoxicity in humans (Saravanan et al., 2006). Serum bilirubin of more than 17 μmol/L indicates liver infection, and a level above 24 μmol/L shows abnormal liver laboratory tests (Thapa & Walia, 2007; Wong et al., ²⁰⁰⁴). Adewusi and Afolayan (2010) found that bilirubin levels were elevated in diseased groups due to hepatic damage and reduced in treatment groups by decreasing liver marker enzyme activity. The decrease in serum bilirubin after treatment with Pelargonium reniforme proved the efficacy of the plant‐based drug in regulating normal liver health. Abd‐Elhakim et al. (2021) emphasized in their study of rats that ethanol extract from M. oleifera detoxified plasma by lowering bilirubin.

Thus, plant extracts are enriched with antioxidants and free radical scavenging phenols, which have a strong reducing power. The results clearly showed that the plant was curative to alcohol‐induced liver damage by restoring bilirubin, liver marker enzymes, and other LFT parameters.

Serum alkaline phosphatase (U/L)

In albino mice, higher serum ALP causes liver damage. The change in serum ALP in albino mice was highly significant (p = .00) concerning treatments, days, and interaction (Table S5). The highest serum ALP (251.2 ± 1.68 U/L) was recorded in T ₀ mice after 14 days of exposure. PCOS‐induced mice treated with metformin and M. oleifera demonstrated a significant decrease in ALP with time. The lowest value (139.2 ± 0.72 U/L) was measured in the T ₄ group on the 14th day (Figure 1d).

According to Ojan and Nihorimbere (2004), an increase in serum ALP levels may be a sign of hepatobiliary diseases. Singha et al. (2007) have identified ALP as a sensitive marker enzyme for hepatocellular and liver damage. The serum of alcohol‐induced rats contained high levels of ALP, indicating aggravated permeability, damage, and necrosis. Furthermore, moringa promotes hepato‐protective effects against hepatocellular injury by attenuating serum ALP activities.

Serum total protein (g/dL)

Total protein levels varied significantly (p = .00) in all experimental groups with reference to treatments, days, and interactions (Table S6). Albino mice with PCOS showed a significant reduction in total protein with time (T ₀). The protein level of metformin ($T_0^{+}$) and all M. oleifera‐treated groups (T ₁–T ₄) significantly increased with time. The highest total protein (7.05 ± 0.08 g/dL) was recorded in T ₄ mice, which may be that MoLE reduced liver inflammation and oxidative stress, while the lowest level of total protein (2.8 ± 0.08 g/dL) was measured in T ₀ mice, as indicated by Figure 1e.

In a study carried out by Adewusi and Afolayan (2010), there was a significant decline in serum total protein levels in ethanol‐fed rats compared to the control group. The decline in total protein indicated a high level of cellular abnormality in chronic liver infection, and the rise in total protein justified liver inflammation recovery. This improvement is due to the phenols present in plant extract having strong antioxidative activity and redox properties. These phenols absorb and neutralize free radicals, quench atomic and triatomic oxygen, and decompose peroxides.

Serum albumin (g/dL)

Highly significant (p = .00) changes in albumin in albino mice were observed with treatments, days, and interactions (Table S7). PCOS‐induced mice showed decreased albumin levels with time, while mice treated with metformin and M. oleifera showed significant increases with time. The highest albumin (3.91 ± 0.05 g/dL) was measured in T ₄ mice due to the presence of antioxidants, gallic acid, sinapic acid, ferulic acid, and chlorogenic acid in MoLE and the least (1.1 ± 0.04 g/dL) in T ₀ group at 14th day of exposure (Figure 1f).

Albumin is a serum protein considered a biomarker of the nutrition and inflammation of the body (Don & Kaysen, 2004). The rise in albumin showed liver inflammation reduction. Cadet et al. (2012) claimed that chronic diseases and oxidative stress can be managed by selecting efficient antioxidants. Antioxidants are effective in countering oxidative stress and can inhibit, manage, and treat various pathologies (Pizzino et al., 2017). Antioxidants stop radical chain reactions, mitigating oxidative stress‐related damage.

Serum globulin (g/dL)

Highly significant (p = .00) differences in serum globulin in albino mice were recorded due to treatments, days, and interactions (Table S8). Albino mice with PCOS showed a decrease in globulin with time, while mice treated with metformin and M. oleifera showed a significant increase with time. The highest globulin (2.84 ± 0.05 g/dL) was measured in T ₄ and the least (1.31 ± 0.02 g/dL) in the T ₀ group on the 14th day of exposure (Figure 1g). Schiødt (2008) discussed globulin's multifunctional nature, as a patient with chronic liver disease has a low globulin level. Its function is to scavenge free radicals and control oxidative stress in hepatic tissues. Moringa extract may reduce liver inflammation and oxidative stress due to polyphenols and flavonoids. Moringa has a plethora of health benefits and improves the liver by raising globulin level.

Globulin is a reliable biomarker, a major protein in serum that is synthesized and secreted by plasma and hepatic cells in response to infection and inflammation. Inflammatory cytokines and antibodies are its two major constituents. Meyer et al. (2016) reported that patients with infection had high levels of globulin in their serum, indicating its role in immunity.

Serum albumin/globulin ratio

A highly significant (p = .00) variation in albumin‐to‐globulin ratio (A/G) in albino mice was studied due to treatments, days, and interaction (Table S9). Albino mice with PCOS showed a decrease in serum A/G ratio with time. In contrast, mice treated with metformin and M. oleifera showed a significant increase with time. The highest A/G ratio (1.48 ± 0.02) was recorded in the T ₄ mice, while the least (1.31 ± 0.02) in T ₀ mice, possibly due to disease severity, after 14 days of exposure (Figure 1h). The rise in globulin and A/G ratio indicated liver function recovery.

Hill et al. (2016) also demonstrated that the albumin‐to‐globulin ratio in patients decreases with infection and malignancy, suggesting their vital role in inflammation and the immune system. According to Yang et al. (2022), A/G is indicative of inflammation in the body, as well as stroke, infection, cancer, and autoimmune disorders. Yoshino et al. (2019) revealed that the opposite correlation between albumin and globulin decreases the A/G ratio. The A/G ratio is a measure of infection and inflammation, which indicates a higher level of infection.

3.2.2. Renal function test (RFT)

The adverse symptoms of PCOS are hyperandrogenism, hypertension, insulin resistance, diabetes type 2, adiposity obesity, and hyperlipidemia, which results in infertility in women and irreversibly impairs renal physiology. Hyperandrogenism is correlated with kidney dysfunction and causes severe tubular epithelial cell injury in women with PCOS (Castro & Coresh, 2009; Webster et al., ²⁰¹⁷). High testosterone in serum activates the apoptotic pathway in human renal tubule cells and increases kidney inflammation (Verzola et al., 2004). Sirmans and Pate (2014) found that insulin resistance in PCOS also leads to chronic kidney damage. Patil et al. (2017) observed renal function in hyperandrogenic female rats and reported a higher risk of kidney injury.

Kidney or renal function tests (RFT), such as urea and creatinine, are common laboratory tests used to study kidney function. Urea is a waste metabolite of protein catabolism by the liver. Allen (2012) describes creatinine as a crucial biosafety marker in renal health. It is produced during muscle catabolism and removed unchanged from the kidney. Song et al. (2019) investigated the link between kidney damage and PCOS. They selected 69 healthy and 55 PCOS women and assessed their testosterone levels and renal activity. Results showed that the urinary albumin‐to‐creatinine ratio in PCOS women was significantly higher than in non‐PCOS women, leading to kidney damage. They concluded that serum testosterone and PCOS cause kidney inflammation.

The present study demonstrated that M. oleifera is a preferred herbal remedy for treating kidney disorders, which is consistent with the findings of Saleh (2019). Akpan et al. (2018) proved that M. oleifera alleviated diabetic nephropathy in alloxan‐treated rats. Kagbo and Abaekwume (2021) reported that moringa taken at 500 mg/kg cured hepato‐renal toxicity caused by acetaminophen‐induced diabetes in Rattus norvegicus. Arafat et al. (2018) supported that M. oleifera leaf extracts decreased MDA, liver and kidney damage. Kakalij et al. (2014) documented that serum urea and serum creatinine can be used to measure drug‐induced nephrotoxicity. They claimed that nephrotoxicity in kidney activity is due to increased levels of blood urea, serum urea, serum creatinine, and urine creatinine.

Analysis of the variance of serum urea and creatinine was measured. Results indicated a highly significant (p = .00) reduction in urea and creatinine (Tables S10 and S11).

Serum urea (mg/dL)

A highly significant (p = .00) decline in serum urea in albino mice due to treatments, days, and their interaction was observed (Table S10). Serum urea concentration was significantly higher in T ₀ albino mice than $T_0^{-}$ with time. The highest serum urea (58.6 ± 1.09 mg/dL) was recorded in T ₀ mice due to PCOS induction and oxidative stress, and the least (25.8 ± 0.32 mg/dL) in T ₄ mice at the 14th day of exposure, indicating that M. oleifera leaf extract effectively restored the antioxidant activity of testosterone affected kidneys as presented in Figure 2a. There was a decline in urea, which indicated better renal function.

FIGURE 2.

Effect of treatments and days on urea (a) and serum creatinine (b) of albino mice (n = 10 mice per week). T ₀ = Basal Feed +5% Tween 80; $T_0^{-}$ = Basal Feed; $T_0^{+}$ = Basal Feed + Metformin at 250 mg/kg BW; T ₁ = Basal Feed + MoLP at 250 mg/kg BW; T ₂ = Basal Feed + MoLP at 500 mg/kg BW; T ₃ = Basal Feed + MoLE at 250 mg/kg BW; T ₄ = Basal Feed + MoLE at 500 mg/kg BW.

Previous studies have found that serum urea rises in response to glomerular injury and also in response to a decrease in glomerular filtration injuries (Manikandan et al., 2011). Ahmadvand et al. (2016) demonstrated that urea was four times higher in the serum of untreated nephrotoxic rats than in healthy rats. In their study, serum urea levels were significantly reduced in carvacrol treatment groups. As an antioxidant, carvacrol reduces kidney inflammation. Similarly, Akter et al. (2021) observed that oxidative stress and inflammation associated with kidney disease can be relieved by M. oleifera, an antioxidant food plant. They further suggested that moringa‐based medication would be a protective choice against various risk factors linked to renal pathologies.

Creatinine (mg/dL)

Highly significant (p = .00) variations in creatinine in albino mice were recorded due to treatments, days, and their interactions (Table S11). M. oleifera and metformin significantly decreased creatinine levels in PCOS‐induced mice. The highest creatinine level (1.62 ± 0.04 mg/dL) was recorded in T ₀ and the least (1.1 ± 0.02 mg/dL) in T ₄ on the 14th day of exposure (Figure 2b). The rise in creatinine showed poor kidney function may be due to decreased tubular secretion capacity, as supported by Traynor et al. (2006).

In the present study, MoLP and MoLE protected kidneys from PCOS‐induced testosterone imbalance by activating endogenous antioxidant systems and enzymatic mechanisms that counteract oxidative stress. Pasaban and Niazmand (2021) observed that metformin limits intracellular ROS by activating the antioxidative system. Karthivashan et al. (2016) showed that Moringa powder and extract enriched with minerals, flavonoids, phenols, and minerals enhanced renal protection. Ahmed et al. (2020) found that moringa extract decreased serum creatinine and BUN in chronic kidney disease patients. Tang et al. (2017) also supported the role of ethanolic extract of M. oleifera in reducing low‐density lipoproteins (LDL) to inhibit oxidative stress and atherosclerosis in kidney infection. Ling and Kuo (2018) claimed that ethanolic extracts elevated creatinine clearance and limited plasma creatinine.

4. CONCLUSION

The present study shows that M. oleifera leaf powder and extract minimizes oxidative stress and reduces liver and kidney inflammation in female albino mice (Mus musculus). Furthermore, M. oleifera is nontoxic and enhances nephro‐hepatic activity in female albino mice with PCOS. The research demonstrated that administering M. oleifera at a dosage of 500 mg/kg BW in female albino mice was more effective in reducing the risk of liver and kidney diseases than metformin (250 mg/kg BW). The current study provided a direction that M. oleifera leaf powder and leaf extract have promising therapeutic potential for managing PCOS and infertility. However, the results of our research are limited to mice only, further studies with long‐term follow‐up and more diverse phenotypes are needed to endorse the potential of M. oleifera leaf powder and leaf extract to manage hepatic and renal damages caused by PCOS in human. Overall, this study highlights the potential of both M. oleifera leaf powder and extract as effective therapeutic options for managing PCOS, and liver and kidney diseases. It further promoted that M. oleifera‐based medication would be a better option to treat oxidative stress and several other risk factors linked with PCOS, and liver and kidney disorders.

AUTHOR CONTRIBUTIONS

Shakeela Khalid: Resources (equal). Muhammad Arshad: Software (equal). Shahid Mahmood: Writing – original draft (equal). Farzana Siddique: Funding acquisition (equal). Nida Aziz: Methodology (equal). Sarfraz Khan: Visualization (equal). Komal Raza: Investigation (equal). Waseem Khalid: Funding acquisition (equal). Ammar AL‐Farga: Project administration (equal). Faisal Aqlan: Writing – review and editing (equal).

FUNDING INFORMATION

This research did not receive any specific grant from funding agencies in the public, commercial, or non‐profit sectors.

CONFLICT OF INTEREST STATEMENT

The authors have no conflict of interest to declare.

ETHICS STATEMENT

All ethical principles were considered in this article.

Supporting information

Tables S1–S11

Khalid, S. , Arshad, M. , Raza, K. , Mahmood, S. , Siddique, F. , Aziz, N. , Khan, S. , Khalid, W. , AL‐Farga, A. , & Aqlan, F. (2023). Assessment of hepatoprotective, nephroprotective efficacy, and antioxidative potential of Moringa oleifera leaf powder and ethanolic extract against PCOS‐induced female albino mice (Mus Musculus). Food Science & Nutrition, 11, 7206–7217. 10.1002/fsn3.3646

DATA AVAILABILITY STATEMENT

Samples of the compounds and data used during the current study are available from the corresponding author.