Ascorbic acid (AA) is a water-soluble vitamin that is well known for its antioxidant and immune-boosting properties. Owing to the wide-range application of AA in the treatment of numerous ailments and its sweet taste, it is usually abused i.e. overused. However, the effect of the abuse has rarely received attention. Therefore, this study was designed to assess the effect of oral administration of high-dose ascorbic acid on biochemical and haematological parameters as well as the effects on the kidney, liver and lungs.adult guinea pigs were divided into four (4) groups where group 1 served as the untreated control group and groups 2-4 were dosed with 29 mg, 662 mg and 1258 mg of ascorbic acid per day, respectively for 28 days.6/µL to AA1258- 5.75 ± 0.11×106/µL ), haematocrit count (NC 39.90 ± 0.52% to AA-29-42.08 ± 0.24 and AA1258-46.13 ± 0.86%), white blood count (NC 10.15 ± 1.01 ×103/µL to AA1258- 15.18 ± 1.65×103/µL ), total lymphocytes (NC 3.5 ± 0.51×103/µL to AA29-5.28 ±0.43×103/µL), monocytes (NC 0.45 ± 0.07×103/µL to AA1258 0.80 ± 0.07×103/µL), eosinophils (NC 0.23 ± 0.03×103/µL to AA12580.40 ± 0.03×103/µL), basophils (NC0.68 ± 0.10×103/µL to AA12581.20 ± 0.10×103/µL) and neutrophil count (NC 4.73 ± 0.68×103/µL to AA1258 8.36 ± 0.71×103/µL). The histopathological indices indicate cellular necrosis in the AA662 and AA1258 treatment groups of the kidney and liver respectively compared to the normal control which has normal cells.the result revealed that administration of high dose ascorbic acid significantly (P<0.05) increased serum creatinine from 50.0 ± 7.09 (NC) to AA29- 73.8 ± 4.5, AA-662-89.7 ± 3.3 and AA1258- 79.9 ± 5.7mmol/L and urea levels in the treatment group AA-1258 -18.3 ± 0.5 µmol/L compared to the normal group (NC-2.15 ± 0.6 µmol/L). Disturbance in electrolyte balance was observed with a significant (P<0.05) increase in Na+ from NC- 131.3 ± 3.5 mmol/L to 135.7 ± 3.6 mmol/L in the AA-1258 treatment group, Cl- , and Ca2+ (NC- 2.66 ± 0.03 to AA1258- 3.36 ± 0.03 mmol/L) and a significant (P<0.05) decrease in serum K+ in the AA29-5.0 ± 0.2, AA662-5.2 ± 0.3 and AA1258-5.6 ± 0.3 mmol/L treatment groups compared to the normal group 6.6 ± 0.3 mmol/L. There was also a significant (P<0.05) increase in the differential blood count in the animals with a significant (P<0.05) increase in red blood count ( NC-5.11 ± 0.13 ×10high dose of ascorbic acid can therefore be suggested to cause damage to the cells by causing cellular necrosis as observed in the histopathology results and has effect on the blood cells as observed in the increase compared to the normal control, and the consequences are possibly triggered through inflammatory responses. Ascorbic acid (AA) is a water-soluble vitamin that is often consumed by humans as a supplement. It is known for its strong reducing property and free radical scavenging activity ,2. FurthAscorbic acid is synthesized from D-glucoronate in both plants and animals through a series of reactions with L-gulonolactone oxidase catalyzing the final step in the biosynthetic pathway. It is synthesized in the kidneys of fish, amphibians, and reptiles, and the liver of some mammals . This vi2 and L-erythrulose [Upon ingestion, ascorbic acid is partially converted into oxalate and excreted in the urine thus, increasing the potential risk of calcium oxalate formation when taken in excess due to the composition of the end products of ascorbic acid degradation . This dethrulose . The defthrulose . Adversethrulose . Sustainthrulose . As a rethrulose . TherefoStudy design and setting: the study was a randomized control trial comparing between 3 intervention groups  and control group. Like humans, guinea pigs are incapable of AA synthesis hence the choice of guinea pigs as model animals for this study. The animals were maintained under standard environmental conditions  in the animal house belonging to the Department of Veterinary Pathology, Ahmadu Bello University, Zaria- Nigeria, and were given access to a standard pellet diet  and water ad libitum. The study was conducted in Kaduna State, Nigeria between June - August 2020.Participants: twenty-four (24) healthy adult guinea pigs weighing between 250 - 500 g were obtained from the Faculty of Veterinary Medicine animal house in Ahmadu Bello University, Kaduna State, Nigeria. The inclusion criterion was the state of health after the period of acclimatization, which was two weeks.Experimental study groups and treatment: animals were further divided randomly into four treatment groups of six animals each. Each treatment group comprised six (6) animals, which were treated in the following manner: Group I- animals were given only 1 mL distilled water -Negative Control (NC). Group II- animals treated with ascorbic acid (29 mg/day) (AA29). Group III- animals treated with ascorbic acid (662 mg/day) (AA662). Group IV- animals treated with ascorbic acid (1258 mg/day) (AA1258). L-ascorbic acid was purchased from Sigma-Aldrich® scientific company . Administration of the supplements was via the oral intubation for 28 days. On the last day, we sacrificed the animals under chloroform anaesthesia and a part of the blood collected in 5 mL EDTA bottles, while another part collected in plain bottles for further analysis including serum urea, creatinine and electrolytes. We collected blood in plain bottles and allowed it to clot for 2 h at room temperature (25-26°C) before centrifuging using a KJLC-1 centrifuge  for 15 minutes at 1000 × g. The top layer was collected as a serum for assay and stored at -20°C.Haematological assays: assays for packed cell volume (PCV), haemoglobin concentration (Hb), red blood cell (RBC), leucocytes and platelet counts, and mean corpuscular volume (MCV) were carried out using the automated analyser , which operated on the principle of the cyanomethaemoglobin and light intensity for the differential leucocytes count [es count .Creatinine: Jaffe's picric acid method was used for the determination of serum creatinine as described by [ribed by . The metribed by . BrieflyUrea: the serum urea was determined using the Urease-Berthelot method as described by Doumas et al. [s et al. . This waDetermination of serum electrolytes: the method used for the determination of serum electrolytes involved the use of the ion-selective electrodes as described by Bakker and Qin [ and Qin . The eleHistopathological examination: the histo pathological examination was carried out at the histology laboratory of the Department of Human Anatomy, Faculty of Medical Sciences, Ahmadu Bello University, Nigeria. The examination was carried out by the method of Culling [ Culling . BrieflyEthical statement: ethical approval was obtained from Ahmadu Bello University, Zaria, Ethical Committee on Animal Use and Care (ABUCAUC). All animals used in this study were handled based on ethical guidelines on the use of animals for research purposes as stipulated by ABUCAUC.Statistical analyses: results in this study were expressed as mean ± standard deviation (mean ± SD) using the Statistical Package for Social Sciences (SPSS) version 20, Chicago, Illinois, USA. One-way analysis of variance (ANOVA) was used to analyze the data obtained with differences in means compared using Duncan Multiple Range posthoc test. Values of P < 0.05 were considered significant.Creatinine: serum creatinine analyses indicate that after 2 weeks of AA administration, there were significant (P< 0.05) increases in serum creatinine. The creatinine level in the AA1258 treatment group was 71.3 ± 8.9 mmol/L while it was just 29.8 ± 3.8 mmol/L in the control group, as shown in Urea: level of serum urea followed the same pattern as creatinine. Serum urea level increased significantly after administration of different doses of AA  increased from 129.1 ± 0.8 mmol/L in the NC group to 152.5 ± 3.2 mmol/L and 138.1 ± 1.3 mmol/L in the AA29 and AA1258 treatment groups respectively  in the AA29, AA662 and AA1258 treatment groups with values 124.9 ± 2.4 mmol/L, 99.7 ± 1.8 mmol/L and 10reatment . After freatment . HoweverHaematology assays: there are significant (P < 0.05) increases in erythrocyte indices in the AA-administered animals (6/µL ) whereas it was 5.75 ± 0.11(106/µL ) for the AA-1258 group  respectively compared to the control with 3.50 ± 0.51(103/µL. There was a significant (P < 0.05) increase in the AA1258 group for the neutrophil, Basophil, eosinophil and monocyte counts which were 8.36 ± 0.71, 1.20 ± 0.10, 0.40 ± 0.03 and 0.80 ± 0.07 (103/µL respectively compared to the control group. Neutrophil lymphocyte ratio (NLR) indicated significant (P < 0.05) increase in the AA1258 group with ratio (3.13 ± 0.33) when compared to the control group and AA29 groups (1.43 ± 0.27 and 1.41 ± 0.21 respectively).The white blood cell count showed a significant (P < 0.05) increase in the AA1258 treatment group with 15.18 ± 1.65  on guinea pigs, particularly focusing on the changes in their biochemical, hematological, and histopathological markers. Our study has discovered a significant shift in the renal tissue histology, along with an increase in serum creatinine and urea levels that suggest a serious renal failure. As the research continued, we also noticed a shift in the concentration of serum electrolytes: the levels of sodium, chloride, and calcium were elevated while the concentration of potassium ions decreased. The high doses of ascorbic acid administered throughout the study caused a noticeable increase in the neutrophil-lymphocyte ratio and levels of red blood cells (RBCs), hemoglobin (Hb), hematocrit (HCT), white blood cells (WBCs), neutrophils, basophils, and eosinophils.et al. [et al. [The gradual rise in the serum creatinine and urea levels during the course of our study is likely related to the enhancement of kidney malfunction or kidney disease, as exemplified in acute kidney injury (AKI) in a study by Kellum et al. . When co [et al.  who expl [et al. . Urea, aet al. [Our findings of increased sodium, chloride, and calcium levels coincide with the research outcomes of Meng  and Biobet al. , in whicet al.  were alset al. .et al.'s research [Hematology, the study of blood and blood-forming tissues' morphology, aids in assessing the health status of animals exposed to toxins and stressors, thereby assisting in diagnosing and prognosticating health conditions . Our finresearch  that obsresearch . This coresearch . A boostresearch . An incrresearch . The AA1research . The incresearch .et al. [The kidney's normal histology displays tubules and glomerulus, part of epithelial tissues containing cells responsible for reabsorbing useful materials into the blood . Tubularet al. , is a beet al. .et al. [Alveoli, the functional units of the lungs, are tiny air sacs responsible for oxygen uptake. Alveoli congestion, characterized by the alveoli filling up with excess fluid, impedes gaseous exchange, leading to erythrocyte egression in the alveolar spaces . Hypoxicet al.  using hiThis study was performed during the hot-wet season and the animal selection was randomized to eliminate bias. Analyses were conducted using one-way analysis of variance and Duncan's multiple range test was used as the post-hoc test, meaning exact p-values were not obtained as would have been the case with a T-test.From the results obtained from this study, prolonged administration of high-dose ascorbic acid causes changes in serum biomarkers of kidney function such as serum creatinine, urea and electrolytes balance which are indicators of kidney injury. The increase in RBC, WBC, neutrophils and lymphocytes is indicative of tissue injury due to the migration of neutrophils and lymphocytes to the site of damage as observed in the renal and pulmonary tissue with necrosis of cells observed. High-dose ascorbic acid thus could be said to affect electrolyte concentration and body fluid balance thus affecting equilibrium of major fluids, electrolytes.Ascorbic acid is an organic compound that occurs readily in nature;It serves as a potent antioxidant;High doses of ascorbic acid has been implicated in the treatment of cancers, common colds and burns.Evidence of pro oxidant role of ascorbic acid has been indicated by damage to renal and pulmonary cells when used in high-dose;High-dose ascorbic acid could result increase in haematological parameters such as RBC, neutrophils and lymphocytes;High-dose ascorbic acid could result in increased renal injury biomarkers like serum creatinine, urea and electrolytes.


The most frequent neurodegenerative illness among senior people and the main cause of dementia is Alzheimer’s disease. The present dementia medications available only help with the symptoms of cognitive deficits and have several negative effects. The current study’s goal is to assess the effects of curcumin and coenzyme Q10, two herbal medicines, both separately and in combination, on learning and memory before comparing them to the industry standard drug. A total of 42 adult healthy Wistar rats were used in our study. In this experiment, rats were given daily doses of 2.5 mg/kg of body weight of scopolamine hydrobromide for 7 days to induce Alzheimer’s disease. On the eighth day, behavioural testing was conducted. Following testing, scopolamine and the test medications were given daily for the following 21 days. On days 29 and 30, behavioural testing was conducted once more, and then animals were slaughtered. Brain homogenate was produced for the estimation of molecular and biochemical markers. Curcumin has demonstrated a dose–response relationship, with a higher dose (200 mg/kg b.w. p.o.) being more effective than a lower dose (100 mg/kg b.w. p.o.). Similar to the greater dose of curcumin, coenzyme Q10 (200 mg/kg b.w. p.o.) has also been found to improve memory and learning. Higher doses of curcumin and coenzyme Q10 had more pronounced and meaningful effects. Acetylcholinesterase and TNF levels increased in scopolamine-induced memory impairment, but these effects were restored by the test medications, and improved by the combined therapy. These outcomes are comparable to those of the common medication memantine. As a result, we may infer from our results that curcumin at higher doses and its combination with coenzyme Q10 (200 mg/kg b.w. p.o.) have a significant impact on cognitive impairment in animal models of Alzheimer’s disease and can be utilised alone or as an add-on therapy for the condition. A degenerative neurologic ailment called Alzheimer’s disease (AD), resulting in the death of brain cells and brain shrinkage, is characterized by a steady decline in daily living skills and cognitive function, which might result in behavioural changes. The most frequent neurodegenerative disease among senior people and the main cause of dementia is Alzheimer’s disease. Memory, thinking, orientation, comprehension, calculation, learning capacity, language, and judgement are among the multiple higher cortical functions that are disrupted by dementia, a syndrome brought on by brain diseases that are typically chronic or progressive and that might cause disability in later life . AccordiThe body’s increased susceptibility to free radical damage is a major aspect of the cognitive aging process . Free raDementia is characterised as a significant decline in cognitive performance that is severe enough to affect social interaction. There are numerous varieties of dementia, each with a unique aetiology and set of signs and symptoms. The most frequent cause of dementia is Alzheimer’s disease (AD), where 60–70% of patients exhibit dementia brought on by the accumulation of beta-amyloid plaques in the brain. Learning is the primary process by which we develop our attitudes, emotional expression, adaptive, cognitive, and affective behaviours, as well as other skills .The process through which sensory information is altered, condensed, elaborated, recovered, and put to use is referred to as cognition. In today’s demanding world, memory difficulties, such as poor recall, low retention, and slowing recall, are frequent . The exiThe first-line drugs for this are (1) Cholinergic activators/Cholinesterase inhibitors, such as rivastigmine, galantamine, and donepezil, and (2) Memantine, a glutamate (NMDA) antagonist. However, they also come with a number of undesirable side effects, such as nausea, diarrhoea, and appetite loss. From this point forward, research is concentrated on a variety of therapeutic plants that are utilized in conventional treatment and have few negative effects . The neeCurcuma longa, generally known as turmeric, is a member of the zingiberaceae family of plants. Curcumin is an active hydrophobic polyphenol that is isolated from the rhizomes of this plant. In India and China, curcumin has historically been utilised as a treatment for a variety of illnesses [The herb llnesses . Curcumillnesses .When compared to AD patients whose macrophages were not treated with curcumin, those whose macrophages were treated demonstrated improved absorption and swallowing of the plaques. Curcumin may therefore aid the immune system in eliminating the amyloid protein .In THP-1 monocytic cells, curcumin prevents the production of the Egr-1 protein and Egr-1 DNA-binding activity. Studies have demonstrated Egr-1’s contribution to the amyloid peptide-induced induction of the cytochemokine gene in monocytes. Curcumin lowers the inflammation via inhibiting Egr-1’s DNA-binding function. Curcumin can reduce the chemotaxis of monocytes, which can happen in response to chemokines from active microglia and astrocytes in the brain .Curcumin has poor bioavailability. Because curcumin is readily conjugated in the intestine and liver to form curcumin glucuronides , absorptNSAIDs, reserpine, and blood thinners are a few of the medicines that are said to interact with curcumin. The bioavailability of curcumin was significantly increased by 2000% when 20 mg of piperine, which is extracted from black pepper, was also taken as a supplement .Alzheimer’s disease, Parkinson’s disease, and other neurodegenerative disorders were found to have defects in the inner mitochondrial membrane and oxidative phosphorylation.Coenzyme Q10 is an endogenous proenzyme found in the inner mitochondrial membrane of human cells; therefore, it may also be a promising neuroprotectant. Coenzyme Q10 functions as an antioxidant, halting the oxidation of lipids within the mitochondrial membrane and other damage brought on by free radicals. CoQ10 is an essential cofactor of the electron transport chain, and an important antioxidant at both mitochondrial and lipid membranes .According to research conducted on animals, coenzyme Q10 supplementation has positive anti-oxidative effects, enhances mitochondrial activity, and guards against ATP depletion . Accordin = 6 per group) were used in this study. The animals were housed in a CPCSEA-certified institutional animal facility at King George’s Medical University in Lucknow, where they were allowed access to food and drink at will and kept in a temperature-controlled environment (25 °C). Before the trial began, a minimum of 7 days of acclimatization time was permitted, and a veterinarian routinely checked on the animals’ health. The Institutional Animal Ethics Committee (IAEC) of King George’s Medical University (K.G.M.U.), Lucknow, gave its clearance before the study could begin (Ref. No 132/IAEC/2020). All experiments in the study were conducted as per the guidelines laid down by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). Animals were obtained from IITR, Lucknow.In total, 42 mature female Wistar rats in good health weighing between 160 and 200 g (n = 6 each group) of either sex. Animals were acclimated to the environment for seven days before to the trial, during which time they received a regular pellet feed, unlimited access to water, and 12 h cycles of light and darkness.The study included a total of 42 adult, healthy Wistar rats , and pyrogallol were purchased from Sigma chemical company, USA. Memantine and all other used chemicals were purchased from TCI, Japan. ELISA kits for TNF-α and Aβ42 estimation was purchased from Real Gene, USA.A total of 42 rats were divided into 7 groups . Each grGroup 01: Control (Vehicle)Group 02: Rats were administered Scopolamine hydrochloride (2.5 mg/kg b.w. i.p.) [w. i.p.) Group 03: Rats were administered Scopolamine (2.5 mg/kg b.w. i.p.) and Curcumin (100 mg/kg b.w. p.o.) [w. p.o.) Group 04: Rats were administered Scopolamine (2.5 mg/kg b.w. i.p.) and Curcumin (200 mg/kg b.w. p.o.) [w. p.o.) Group 05: Rats were administered Scopolamine (2.5 mg/kg b.w. i.p.) Additionally, CoQ10 (200 mg/kg b.w. p.o.) [w. p.o.) Group 06: Rats were administered Scopolamine (2.5 mg/kg b.w. i.p.), Curcumin (200 mg/kg b.w. p.o.) and CoQ10 (200 mg/kg b.w. p.o.)Group 07: Rats were administered Scopolamine (2.5 mg/kg b.w. i.p.) and standard drug Memantine (10 mg/kg b.w. i.p.) [w. i.p.) Behavioural tests were performed on day 8 and then day 29 and day 30. To avoid stress to the animal, they were maintained in a quiet room for 1 h before the tests.Spontaneous alternation performance was tested as described previously , with the platform submerged in quadrant 2. The time limit was 120s/trial, with a 20 min intertrial interval. Time taken to locate the platform in the acquisition phase was analysed .The rats were each placed at the end of an open arm, away from the platform, and the time it took for them to move into either of the enclosed arms was timed . TL was the amount of time that elapsed from the moment the animal was positioned in the open arm until all of its legs had crossed the fine white line in the centre of the enclosed arm .Whole-brain samples were rinsed with ice-cold saline (0.9% NaCl) and homogenized in chilled tissue lysis buffer (RIPA + PI cocktail). The homogenates were centrifuged at 800 g for 5 min at 4 °C to separate the nuclear debris. Afterward, the lysate was centrifuged at 14,000 g for 30 min at 4 °C to obtain supernatant, which was further used for the estimation of biochemical, inflammatory, and molecular parameters.A photometric method for determining acetylcholinesterase activity of brain homogenate has been described. The enzyme activity is measured by following the increase in yellow colour produced from thiocholine when it reacts with the dithiobisnitrobenzoate ion .SOD activity in brain tissue homogenate was analysed by using the method of Marklund and Marklund and expressed in U/mg of protein. In this method, superoxide dismutase inhibits pyrogallol autoxidation. It was investigated in the presence of EDTA in the pH range 7.9–10.6. The activity was measured at 420 nm using a spectrophotometer. Protein estimation was performed by Lowry’s method.The tumour necrosis factor-alpha (TNF-α) and Aβ42 levels estimation was performed as per the instructions provided in the enzyme-linked immunosorbent assay (ELISA) kit in brain homogenate. The type of ELISA used was sandwich ELISA. The assay was performed at room temperature.Seven wells were taken for standard and one for blank. An amount of 100 μL each of the dilutions of standard, blank, and samples was added into the appropriate wells. Incubation was carried out for 2 h at 37 °C. After that, washing was performed and then 100 μL of biotinylated antibody was added and incubation was carried out for 1 h at 37 °C. Washing was performed again and then 100 μL of streptavidin-HRP was added and incubation was carried out for 1 h at 37 °C. Washing was repeated and then 90 μL of TMB substrate was added, the solution turned blue and incubation was carried out for 20 min at 37 °C. Finally, stop reagent 50 μL was added, the solution turned yellow, and the reading  was taken by microplate reader set to 450 nm.p < 0.05 in all statistical evaluations.The data obtained are expressed as mean ± SD and analysed using single-factor ANOVA. Further statistical analysis for individual groups was carried out by Tukey HSD post hoc test. The criterion for statistical significance is taken as p < 0.001) increase in transfer latency as compared to the control group (12.61 ± 1.49), which indicated a loss of memory in the scopolamine-treated groups.On Day 29, there was no significant difference found in the transfer latency amongst the groups, whereas on day 30 the scopolamine-only group (27.91 ± 1.91) showed a significant (p < 0.001) decrease in transfer latency as compared to the scopolamine group (27.91 ± 1.91). SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (15.25 ± 1.22) showed the highest decrease in transfer latency as compared to the scopolamine-only group (27.91 ± 1.91) and no statistically significant difference in transfer latency existed between the standard (14.40 ± 2.42) and SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (15.25 ± 1.22), indicating that the combination of SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (15.25 ± 1.22) showed the highest results in improving memory in elevated plus maze.The SCO + memantine group (14.40 ± 2.42) showed a statistically significant (p < 0.001) and SCO + CUR (200 mg/kg) (17.8 ± 2.55) was less significant (p < 0.050 when compared to SCO + Memantine group (14.40 ± 2.40), indicating relatively lesser improvement in memory in elevated plus maze (Increase in transfer latency in SCO + CUR (100 mg/kg) (22.91 ± 1.29) and SCO + CoQ10 (200 mg/kg) (19.98 ± 2.96) was highly significant (lus maze .10 (200 mg/kg) (15.25 ± 1.22), there was decrease (p < 0.001) in transfer latency as compared to SCO + CoQ10 (200 mg/kg) (19.98 ± 2.96) indicating strong correlating effect of combination of SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg).In SCO +CUR (200 mg/kg) +CoQp < 0.001) increase in the time taken to reach the platform as compared to the control group (50.16 ± 2.09), which indicated a loss of memory in the scopolamine-treated groups.On Day 29, the scopolamine-only group (70.55 ± 2.2) showed a significant (p < 0.001) decrease in transfer latency as compared to the scopolamine group (70.55 ± 5.69). SCO + CUR (200 mg/kg) +CoQ10 (200 mg/kg) (58.01 ± 3.36) showed the highest decrease in the time taken to reach the platform as compared to the scopolamine-only group (54.28 ± 4.95) and no statistically significant difference in the time taken to reach the platform existed between the standard (54.28 ± 4.95) and SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (58.01 ± 3.36) groups, indicating that the combination of SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (58.01 ± 3.36) showed great results in improving memory in the Morris water maze.The SCO + memantine group (54.28 ± 4.95) showed a statistically significant (p < 0.001) in the time taken to reach the platform as compared to SCO + CUR (200 mg/kg) (62.96 ± 6.94) as well as SCO + CoQ10200 mg/kg (63.4 ± 4.58), indicating a strong additive effect of the combination of SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (58.01 ± 3.36).In SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (58.01 ± 3.36), there was decrease (p < 0.001) increase in the time taken to reach the platform as compared to the control group (34.65 ± 3.97), which indicated a loss of memory in the scopolamine-treated groups.On Day 30, the scopolamine-only group (56.26 ± 2.2) showed a significant (p < 0.001) decrease in transfer latency as compared to the scopolamine group (34.65 ± 3.97). SCO + CUR (200 mg/kg) +CoQ10 (200 mg/kg) (40.75 ± 3.91) showed the highest decrease in the time taken to reach the platform as compared to the scopolamine-only group (56.26 ± 2.2) and no statistically significant difference in the time taken to reach the platform existed between the standard (38.43 ± 3.09) and SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (40.75 ± 3.91), indicating that the combination of SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (40.75 ± 3.91) showed positive results in improving memory in the Morris water maze (The SCO + memantine group (38.43 ± 3.09) showed a statistically significant (ter maze .p < 0.001) in the time taken to reach the platform as compared to SCO + CUR 200 mg/kg (44.65 ± 5.97) as well as SCO +CoQ10 (200 mg/kg) (47.7 ± 7.95), indicating a strong additive effect of the combination of SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (40.75 ± 3.91).In SCO + CUR (200 mg/kg) + CoQ10 (200 mg/kg) (40.75 ± 3.91), there was a decrease (p < 0.001) decrease in percentage alternations as compared to the control group (62.94 ± 3.84), which indicated a loss of memory in the scopolamine-treated groups.The scopolamine-only group (48.19 ± 2.55) showed a significant (p < 0.001) increase in percentage alternations as compared to the scopolamine group (48.19 ± 2.55). SCO + CUR 200 mg/kg + CoQ10 200 mg/kg (59.45 ± 2.88) showed the highest increase in percentage alternations as compared to the scopolamine-only group (48.19 ± 2.55), and no statistically significant difference in percentage alternations existed between the standard (60.48 ± 3.49) and SCO + CUR 200 mg/kg + CoQ10200 mg/kg (59.45 ± 2.88) groups, indicating that the combination of SCO + CUR 200 mg/kg + CoQ10200 mg/kg (59.45 ± 2.88) showed great results in improving memory in the Y maze (The SCO + memantine group (60.48 ± 3.49) showed a statistically significant (e Y maze .p < 0.05) in percentage alternations as compared to SCO + CoQ10 200 mg/kg (54.98 ± 3.61) as well as SCO +CoQ10 200 mg/kg (54.98 ± 3.61), indicating a strong additive effect of the combination of SCO + CUR 200 mg/kg + CoQ10 200 mg/kg (59.45 ± 2.88) on memory.In SCO + CUR 200 mg/kg + CoQ10200 mg/kg (59.45 ± 2.88), there was an increase (p < 0.001) Higher Total AChE Level as Compared to the Control Group (474.4 ± 13.70), which Indicated less Acetylcholine-Like Activity in the Scopolamine-Only Group.The Scopolamine-Only Group (922.45 ± 26.99) had a Significantly (p < 0.001) decrease in AChE level as compared to the scopolamine group (922.45 ± 26.99). SCO + CUR 200 mg/kg + CoQ10200 mg/kg (738.94 ± 24.86) showed the highest decrease in AChE level as compared to the scopolamine-only group (922.45 ± 26.99), along with a highly statistically significant (p < 0.001) difference in AChE level existing between the standard (870.58 ± 34.95) and SCO + CUR 200 mg/kg + CoQ10200 mg/kg (738.94 ± 24.86) groups, indicating that the combination of SCO + CUR 200 mg/kg + CoQ10 200 mg/kg was most effective amongst these groups in decreasing AChE level in brain homogenates (The SCO + memantine group (870.58 ± 34.95) showed a statistically significant (ogenates .p < 0.001) difference between the SCO + CUR (200 mg/kg) (779.85 ± 33.16) and SCO + CUR 200 mg/kg + CoQ10 200 mg/kg (738.94 ± 24.86) groups and SCO + CoQ10 (200 mg/kg) (789.2 ± 27.9) and SCO + CUR 200 mg/kg + CoQ10200 mg/kg (738.94 ± 24.86), with SCO + CUR 200 mg/kg + CoQ10200 mg/kg (738.94 ± 24.86) showing the highest decrease in AChE level.There was a highly significant (p < 0.001) lower total SOD level as compared to the control group (26.1 ± 1.21), which indicated more oxidative stress in the scopolamine-only group.The scopolamine-only group (11.58 ± 0.56) had a significantly (p < 0.001) increase in SOD level as compared to the scopolamine group (11.58 ± 0.56). SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (28.22 ± 0.57) showed the highest increase in SOD level as compared to the scopolamine-only group (11.58 ± 0.56) and even surpassing the standard (23.94 ± 0.45) group with a statistically significant (p < 0.001) increase in SOD level, indicating that the combination of SCO + CUR 200 mg/kg + CoQ10 200 mg/kg (28.22 ± 0.57) was most effective amongst these groups in improving SOD level in brain homogenates and acting as a powerful antioxidant (The SCO + memantine group (23.94 ± 0.45) showed a statistically significant (ioxidant .p < 0.001) difference between SCO + CUR 200 mg/kg (23.58 ± 2.14) and SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (28.22 ± 0.57) as well as SCO + CoQ10200 mg/kg (22.23 ± 1.11) and SCO + CUR 200 mg/kg +CoQ10200 mg/kg (28.22 ± 0.57), with the SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (28.22 ± 0.57) group showing a higher increase in SOD level indicating the role of SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (28.22 ± 0.57) in reducing oxidative stress.There was a highly significant (p < 0.001) Higher Total TNFα Level as Compared to the Control Group (35.94 ± 4.51), which Indicated More Inflammation in the Scopolamine-Only GroupThe Scopolamine-Only Group (279.84 ± 11.57) had a Significantly (p < 0.001) decrease in TNFα level as compared to the scopolamine group (279.84 ± 11.57). SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (144.87 ± 6.84) showed the highest decrease in TNFα level as compared to the scopolamine-only group (279.84 ± 11.57), along with a highly statistically significant (p < 0.001) difference in TNFα level existing between the standard (124.11 ± 8.61) and SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (144.87 ± 6.84) groups, indicating that the combination of SCO + CUR 200 mg/kg + CoQ10 200 mg/kg (144.87 ± 6.84) was most effective amongst these groups in decreasing TNFα level in brain homogenates (The SCO + memantine group (124.11 ± 8.61) showed a statistically significant (ogenates .p < 0.05) difference between SCO + CUR 200 mg/kg (169.80 ± 9.87) and SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (144.87 ± 6.84) and between SCO + CoQ10 200 mg/kg (169.80 ± 9.87) and SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (144.87 ± 6.84) with SCO + CUR 200 mg/kg +CoQ10 200 mg/kg (144.87 ± 6.84) showing a higher decrease in TNFα level.There was a significant  effect of curcumin and coenzyme Q10 alone and in combination. These effects were compared with the standard drug Memantine for learning and memory.Curcuma longa. We chose the oral route for giving the test drug, as it is a usual and convenient method of taking a drug and ensures better compliance.We used a purified form of curcumin (powder), which is an active compound of As mentioned in Materials and Methods, there were a total of seven groups of Wistar rats, and Alzheimer’s disease was induced in six groups out of them. The disease was induced by giving scopolamine injection intraperitoneally in all groups except the control group.As in other research, amnesia is induced using the scopolamine model (2.5 mg/kg) . It has Three behavioural tests were used in this work to examine the behavioural changes in experimental rats following scopolamine administration: the Y maze, the Morris water maze (MWM), and the elevated plus maze (EPM). To assess the impact of curcumin and coenzyme Q10, the following parameters were observed: percentage alternation in the Y maze, time to reach the platform in MWM, and transfer delay in EPM. In order to analyse them as biomarkers for learning and memory assessment, we also assessed the levels of acetylcholinesterase, TNF, Superoxide dismutase (SOD), and Amyloid-42 using rat brain homogenates .The changes observed in behaviour during the course of the experimental period can be attributed to the impact of scopolamine on the brains of Wistar rats. Treatment of rats with curcumin  and in combination with coenzyme Q10 improved the behaviour  and also decreased anxiety associated with scopolamine (2.5 mg/kg). The result of the combination (Curcumin—200 mg/kg + CoQ10200 mg/kg) treatment was found to be much better.In this study, in order to investigate oxidative stress in experimental rats after giving scopolamine, the biochemical test of SOD level was observed. Oxidative stress marker level (SOD) was improved in the group receiving curcumin (200 mg/kg) alone and in the group receiving the combination of curcumin (200 mg/kg) and coenzyme Q10 (200 mg/kg). The results of the combination (Curcumin—200 mg/kg + CoQ10200 mg/kg) treatment were found to be much better. This further strengthens the possibility of an antioxidant role of curcumin and coenzyme Q10. Acetylcholine (Ach) has a crucial role in the peripheral and central nervous systems. Cholinergic neurons located in the basal forebrain, including the neurons that form the nucleus basalis of Meynert, are severely lost in Alzheimer’s disease (AD).In this study, to investigate the level of acetylcholine in experimental rats after giving scopolamine, an AchE level was observed, which showed the maximum improvement in the group receiving the combination of curcumin (200 mg/kg) and coenzyme Q10 (200 mg/kg).In this study, to investigate inflammation in experimental rats after giving scopolamine, TNFα level was observed and Aβ42 levels were also observed with TNFα levels being decreased in the group receiving a combination of curcumin (200 mg/kg) and coenzyme Q10 (200 mg/kg), as well as in the group receiving Curcumin 200 mg/kg and CoQ10 200 mg/kg alone. The result of the combination (Curcumin—200 mg/kg + CoQ10—200 mg/kg) treatment is much better and was comparable to standard drug treatment.This study demonstrated that curcumin is capable on its own to improve the behavioural, biochemical, and inflammatory parameters of the scopolamine-induced AD model. However, when combined with coenzyme Q10 (200 mg/kg), the results are accentuated even further in the positive direction as was evident with a decrease in transfer latency in the elevated plus maze, a decrease in the time taken to reach the platform in the Morris water maze, a decrease in AchE and TNFα, and an increase in SOD levels. Based on different experimental results, the present investigation suggested that the curcumin (200 mg/kg) and coenzyme Q10 (200 mg/kg) combination displayed significantly better results in opposing the cognitive, biochemical, and inflammatory defects created by the scopolamine-induced Alzheimer’s disease model of Wistar rats.The preventive and therapeutic effects of 200 mg of curcumin on SDAT (Senile Dementia of Alzheimer’s Type) were examined by Agrawal et al. in 2010 . Oral doThe comparative protective effects of curcumin, memantine, and diclofenac against scopolamine-induced memory impairment were investigated by Elham H.A. Ali et al. (2011)  in a groZhang et al. (2015)  assessedThe expression of nuclear factor-B (NF-B), tumour necrosis factor (TNF), and interleukin-6 (IL-6), which is increased by the medication 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, has been demonstrated to be inhibited by coenzyme Q10. This has been linked to pleiotropic and anti-inflammatory effects (MPTP). According to studies on coenzyme Q10′s role, improved mitochondrial activity lowers oxidative damage, which lowers age-related cognitive decline ,38. Age-As of date, none of the studies have observed the effect of curcumin and coenzyme Q10 in combination for learning and memory enhancement (cognition) in an animal model of Alzheimer’s disease; hence, this is a combination that should be explored further.Curcumin’s antioxidant and anti-inflammatory properties account for most of its beneficial benefits on the variety of illnesses described in Jurenka JS’ review. (2009) .Curcumin has been shown to ameliorate systemic signs of oxidative stress. There is evidence that it can increase the serum activity of antioxidants such as superoxide dismutase (SOD) . Free raA few reactive oxygen/nitrogen species can also set off a chain of intracellular signals that encourage the expression of genes that cause inflammation. Inflammation has been related to several chronic illnesses and ailments in their early stages of development . One of Tumour necrosis factor (TNF-), a key mediator of inflammation in the majority of diseases, activates nuclear factor (NF)-B, a transcription factor. NF-B also regulates the expression of TNF-, despite the fact that TNF- is regarded to be the most potent NF-B activator. As was also seen in the current study, curcumin has been shown to suppress NF-B activation, which is brought on by a number of inflammatory stimuli ,46,47.Coenzyme Q10 (CoQ10) can decrease oxidative stress, hyperglycemia, and inflammatory markers, and improve vascular function. Coenzyme Q10 supplementation influences the cholinergic system and protects cholinergic neurons in patients with Alzheimer’s disease .The results of the current investigation could shed light on coenzyme Q10’s efficacy in enhancing cognitive function in scopolamine-induced amnesic rats. In various animal experiments investigating the function of oxidative stress in neurodegenerative disorders, oxidative stress indicators and antioxidant concentrations were assessed. (ND) .The use of herbal medicines as complementary and alternative medicines (CAM), particularly for neurological and mental illnesses as well as for physical wellbeing, is on the rise today all over the world. A protracted course of conventional standard treatment may be harmful and cause major adverse medication reactions. Traditional treatments, nutritional therapy, and herbal products are safer and more dependable. We attempted to combine curcumin and coenzyme Q10 in our study.Given the results that were obtained from the study of pharmacological and biochemical/molecular parameters in the present study, the following conclusion may be drawn regarding the potential effectiveness of test drugs. Curcumin has a learning and memory enhancement effect and a higher dose (200 mg/kg b.w.s p.o.) is comparatively more effective than a lower dose (100 mg/kg b.w. p.o.) and it has shown a dose–response effect. Coenzyme Q10 (200 mg/kg b.w. p.o.) also has learning and memory enhancement effects which were comparable to the effect of a higher dose of curcumin The effects of the combination of curcumin and coenzyme Q10 on learning and memory were significant and greater than when both drugs were given alone and were comparable to Memantine.The results of the present study are encouraging and may reveal the importance of curcumin and coenzyme Q10 herbal drugs and nutrients in impaired cognition states. As a result, curcumin, coenzyme Q10, and their combination, which has demonstrated the greatest benefit, may be effective in the management of learning- and memory-impaired states as an alternative, supplemental, or even preventive medication. These medications, alone or in combination, can be taken by patients of any age for a longer period of time without experiencing any negative side effects. To support our encouraging results, additional animal and clinical investigations are needed.


Penicillium chrysogenum during the period 2013–2023, together with their reported pharmacological activities. From literature surveys, 277 compounds have been identified from P. chrysogenum, which has been isolated as an endosymbiotic fungus from different host organisms, with specific attention paid to those showing marked biological activities that could be useful in the pharmaceutical industry in the future. This review represents documentation for a valuable reference for promising pharmaceutical applications or further needed studies on P. chrysogenum.Recently, it has been shown that metabolites derived from endosymbiotic fungi attracted high attention, since plenty of them have promising pharmaceutical applications. The variation of metabolic pathways in fungi is considered an optimistic source for lead compounds. Among these classes are terpenoids, alkaloids, polyketides, and steroids, which have proved several pharmacological activities, including antitumor, antimicrobial, anti-inflammatory, and antiviral actions. This review concludes the major isolated compounds from different strains of  Penicillium includes four subgenera: Penicillium, Furcatum, Biverticillium, and Aspergilloides. Penicillium is the subgenus that has been examined the most from the genus of Penicillium fungi  named PAF and PAFB were secreted from the filamentous fungus P. chrysogenum Q176 antifungal protein C (PAFC) was also characterized, and was different phylogenetically from the two Penicillium AMPs, PAF and PAFB  (9000 U/mL)  was cloned from P. chrysogenum 31B. The enzyme precisely acts on rhamnose (Rha) at the non-reducing end of RG oligosaccharides, but had not shown any action on flavonoid glycosides  was extracted from broth culture of P. chrysogenum. But further proof points to the possibility of sexual reproduction with an unidentified sexual stage. P. chrysogenum has recently been found to contain the mating-type (MAT) and pheromone signaling genes, which are associated with mating in other sexual fungus. In heterothallic ascomycete fungi, complementing MAT1-1 and MAT1-2 isolates are required for sex to occur. In contrast to the original Fleming strain, which has the opposite MAT1-2 locus, NRRL1951 (P. chrysogenum derivatives) has a MAT1-1 locus with a MAT1-1–1 gene encoding a putative alpha-box transcription factor. Sexual crosses can be used to develop new strains with improved industrial characteristics  revealed the same potency towards Fusarium oxysporum and Cryptococcus neoformans with MIC 39.06 ± 0.85 and 39.06 ± 0.98 µg/mL, respectively.According to Al-Saleem et al. , P. chryP. chrysogenum Q176 was produced together with PAF and PAFB into the culture broth. Recombinant PAFC's functional characterization revealed a promising novel molecule for anti-Candida therapy. In pre-established biofilms of two strains of C. albicans, the planktonic cells were killed by the thermotolerant PAFC while the sessile cells’ metabolic activity decreased. One of the strains was a fluconazole-resistant that displayed greater PAFC sensitivity than the fluconazole-sensitive one. The absence of hemolytic activity supports the further use of PAFC in clinical therapy.Holzknecht et al.  reportedP. chrysogenum Q176, are highly stable due to a compact disulfide-bond, β-fold structure. In micromolar doses, these two AMPs effectively prevented the growth of several fungi including: Aspergillus fumigatus, Trichophyton spp., Aspergillus niger, and Candida spp., along with the Neurospora crassa and Saccharomyces cerevisiae., which were vulnerable to both proteins since their growth diminished at 0.25–4 μM PAF or PAFB doses, respectively.Huber et al.  found thP. chrysogenum XNM-12, the marine algal-derived fungus, had a moderate antifungal potential towards the plant pathogenic fungus Alternaria alternata with MIC 8 μg/mL.Xu et al.  reportedP. chrysogenum. Botrytis cinerea and Aspergillus fumigatus, two opportunistic human and plant diseases, were discovered to be particularly inhibited, even though in vitro and in vivo tests had shown that they were inert against mammalian cells.As elaborated by Sonderegger et al. , PAF, a P. chrysogenum V11 culture. In fact, it exhibited serious inhibitory actions towards Colletotrichum gloeosporioides and Rhizoctonia solani , which were notably higher than carbendazim.Zhu et al.  have ideP. chrysogenum V11. They remarkably inhibited the plant pathogenic Rhizoctonia solani fungus ((MICs) = 11.79–23.66 μM), while chaetoglobosin A (84), chaetoglobosin E (87) and penochalasin J (88), significantly inhibited Colletotrichum gloeosporioides , revealing a higher activity than carbendazim. The findings revealed that the corresponding isolates could be greatly employed as fungicides or as primes of newly fungicidal agents against the mentioned pathogenic fungi.Previous study by Huang et al.  showed tP. chrysogenum IFL1 produced bioactive compounds that spread on agro-industrial residues, cheese whey, and grape trash. The cheese whey culture filtrate hindered the development of the fungus Fusarium oxysporum as well as the amoeba Acanthamoeba polyphaga.According to Lopes et al. , P. chryP. chrysogenum Q176, displayed that they had antiviral activity without triggering any cytotoxic effects or hemolytic activity on mammalian cells. Experiments in human cervix cancer cells showed that they both reduced Human Coronavirus cytopathogenic effects. Apparently, it was the very first study on the antiviral ability of small, cysteine-rich and cationic proteins derived from fungi.In vitro as well as in vivo studies by Huber et al.  on PAF aP. chrysogenum in a biomaltpeptone medium. This step was proceeded by cytotoxicity-guided fractionation. It showed low HCV protease potential with an IC50 value of 76.3 μM.Hawas and Abou El-Kassem  used a sP. chrysogenum strain PJX-17’s culture. Results revealed that both displayed activities against influenza virus A (H1N1), with IC50 at 85 and 113 μM, respectively.Study by Peng et al.  isolatedP. chrysogenum, separated from Eucommia ulmoides Oliver bark. The anti-inflammatory activity for all the obtained compounds were evaluated using HEK293 cells reporting that compounds (175), (152), (153) and (182) had powerful inhibitory actions on TNF-α-stimulated NF-κB activation.Liu et al.  have isoP. chrysogenum and was found to be an analogue of aspirin by structure.According to Zhang et al. , HPABA ..In a previous study for Qi et al. , Compoun, a new benzoic acid derivative, HPABA (265) from the fermented broth of P. chrysogenum., where it presented significant anti-inflammatory with pain killer activities when given at 100 mg/kg, while it showed no ulcerogenic actions.Wang et al.  isolate,50 values of 28.9 and 35.6 μM, respectively.Huang et al.  work sho50 value of 9.0 μM.Chen et al.  exposed P. chrysogenum and Kojic acid (156) exhibited strong activity with IC50 22.6 ± 0.8 and 23.4 ± 1.4 µg/mL, respectively, with respect to HEP-2 larynx carcinoma, the total extract and Kojic acid (156) had effective comparable cytotoxic profile against HEP-2 cell lines with same IC50 30.8 ± 1.3 and 30.8 ± 1.2 µg/mL, respectively. These substantial antimicrobial and cytotoxic activities may be due to the presence of penicillin G (212), Kojic acid (156), sohirnone B (217), and camptothecin (56) as major constituents in the ethyl acetate extract of P. chrysogenum.According to Al-Saleem et al. , cytotoxP. chrysogenum is an effective taxol (3) producer. Antiproliferative behavior of taxol was tested towards several cell lines like liver cancer cells (HEPG2) and breast adenocarcinoma (MCF7) cell viability. A considerable impact was revealed , since both MCF7 and HEPG2 cell viability were declined particularly by increasing taxol concentrations. The isolated taxol (3) IC50 value was indicated around 3.3 and 3.7 µM against cell lines MCF7 and HEPG2, individually.Regarding the quantitative methodology by TLC and HPLC, El-Sayed et al.  revealedP. chrysogenum strain LD-201810 fungus culture against six human cancer cell lines including A549, HeLa, THP-1, MCF-7, HepG2, and BT-549. (2’R)-westerdijkin A (224) demonstrated anticancer activity against the HepG2 cell line with an IC50 22.0 μM. Furthermore, (S)-( +)-11-dehydrosydonic acid (33) showed significant activity towards THP-1 and A549 with an estimated IC50 values of 18.2 and 21.2 μM, respectively. All results suggested that (2’R)-westerdijkin A (224) and (S)-( +)-11-dehydrosydonic acid (33) had apoptosis-triggering action towards the HepG2, A549, and THP-1 cell lines, respectively.Jiang et al.  investigP. chrysogenum S-3–25 on the human cancer cell lines, HeLa K562, BGC-823, HL-60, and A549. All the tested compounds revealed poor inhibitory action on the assayed cell lines, except chrysomutanin (4), 3-acetyl chrodrimanin F (5) and chrodrimanin F (7) which revealed higher inhibitory activities on HL-60 cells with an IC50 values 4.8, 8.1 and 8.7 μM, respectively.Using MTT method, Qiao et al.  evaluated values of 0.118 μM, 0.0746 μM for eEF2K, respectively.Cao et al.  used theP. chrysogenum S003 ethyl acetate extract yielded metabolites which were evaluated towards five human cancer cell lines by SRB assay. Ergosterol (187) and Epidioxyergosterol (194) showed promising cytotoxic activities against prostate (DU-145), hepatocellular (HepG2) cell lines, lung (A-549), and breast adenocarcinoma (MCF-7), with IC50 values of 1.50, 6.10; 2.89, 3.07 21.26, 19.3; and 16.95, 13.6; µM, respectively. However, LAMA (193) and Kojic acid (156) exhibited poor cytotoxic actions against all the corresponding cell lines.In a previous study by Alshehri et al.  stated tP. chrysogenum MCCC 3A00292 the deep-sea-derived fungus solid cultures were tested for five human cancer cell lines: BEL-7402, BIU-87, ECA109, Hela-S3 and PANC-1. Peniciversiol A (113) showed a marked inhibitory potentials towards the BIU-87 cells , while metabolites Penicilactones A-B (116–117) and Decumbenone A-B (11–119) as well Aspermutarubrol (122), 3-hydroxy-5-(3-hydroxy-5-methylphenoxy)benzoic acid (123), Cyclopenol (124), Violaceol-II (125) had inhibitory effects towards the BEL-7402, BIU-87, and ECA109 cancer cell lines with an estimated IC50 values range from 7.70 to > 20 μM.According to Niu et al. , the cytP. chrysogenum in biomaltpeptone media. This step was proceeded by cytotoxicity-guided fractionation within a group of up to 12 cancer cell lines. Results revealed substantial cytotoxicity of the metabolite, with definite selectivity for colon-38 carcinoma cells compared to human normal cells.Hawas and Abou El-Kassem  used a sP. chrysogenum MMS5 extract, shows strong antiproliferative action on breast cancer cells (MCF-7 cell line) in a real-case investigation. Its antiproliferative activity was validated with an IC50 of 0.10 μM on MCF-7 cells mainly due to the presence of high amounts of ergosterol.Ory et al.  stated tP. chrysogenum strain HLS111. In vitro cytotoxic activity against different human cell lines were estimated for these metabolites against renal carcinoma (A498), multiform glioblastoma (U87 MG), leukemia (HL60), non-small cell lung tumor (NCI-H1650), and colonic carcinoma (HT29), by the MTT method. The anticancer activities of compounds (161–163) diminished significantly compared to secalonic acid D which was previously isolated. The cytotoxic effect was reduced due to the presence of β-methyl-γ-lactone ring, meaning that the tricyclic nucleus was important to achieve bioactivity.A study by Zhen et al.  showed tP. chrysogenum AD-1540, a marine algal-derived endophytic fungus which was separated from the red alga Grateloupia turuturu ‘s inner tissue, afforded chryxanthones A and B (161–162), two novel benzophenone derivatives. Their cytotoxic profile was evaluated towards six human cancer cell lines: MCF-7, A549, HeLa, BT-549, HepG2 and THP-1. Chryxanthone A (161) exhibited moderate activity towards BT-549 and HeLa cancer cell lines, with IC50 values of 20.4 and 23.5 µM, respectively, whereas chryxanthone B (162) inhibited the growth of A549 cell line selectively with an IC50 value of 20.4 µM.Chromatographic analysis performed by Zhao et al.  on extraP. chrysogenum-1 hPc-1 isolated from Tuz Lake Turkey, were assessed by Dikmen et al. , gastric cancer cell line (SGC-7901), and a breast cancer cell line (MDA-MB-435). Penochalasin K (85) showed significant broad-spectrum inhibitory actions towards all the assayed cell lines (IC50 < 10 μM). Also, chaetoglobosin A (84), chaetoglobosin C (81) and penochalasin I (83) exhibited mild to noticeable inhibitory activities on the mentioned cell lines with IC50 values range of 6.56–37.56 μM.According to Zhu et al. , MTT tecP. chrysogenum SCSIO 41,001, displayed cytotoxic effects towards Huh-7, K562, and A549 cancer cell lines significantly with IC50 values at 2.59, 6.78, and 6.94 μM, respectively, while penicitrinone F (144) displayed a modest inhibitory effect against EV71 with IC50 14.50 μM.Chen et al.  revealedP. chrysogenum V11 for their anticancer activity. Penochalasin I (83) showed significant activity against SGC-7901 and MDA-MB-435 cells (IC50 < 10 μM), while cytoglobosin C (90) had high activity against A549 and SGC-7901 cells (IC50 < 10 μM).Huang et al.  tested cP. chrysogenum HGQ6 fermentation broth had activity towards BGC823 cell with the IC50 value of 0.19 mg/mL.Guo et al.  stated tP. chrysogenum, grown from a Gorgonian Carijoa sp. found in the South China Sea, yielded a novel flavone penimethavone A (154), with a unique methyl group at ring-B which is rarely found. It was tested in vitro for cytotoxicity against cervical cancer (HeLa), rhabdomyosarcoma, non-small cell lung cancer (A549) and human laryngeal epithelial (Hep-2) cell lines. Results indicated that it had moderate selective cytotoxic activity against (HeLa) and rhabdomyosarcoma cell lines, with IC50 values of 8.41 and 8.18 µΜ, respectively.Previous studies by Hou et al.  on P. chP. chrysogenum mycelia which could inhibit the development of human breast cancer cell lines, MDA-468, MDA-MB-231, SK BR-3, BT-474, MCF7, and MCF7-dox, while comparable therapeutic doses revealed no impact on the growth of the non-carcinogenic human mammary epithelial cells MCF10A and viability. Additionally, its therapy inhibited the HGF-induced cell migration and invasion in breast cancer cell lines in a dose-dependent behavior.Mady et al.  yielded P. chrysogenum EN118, separated from the marine brown alga Sargassum palladium, showed a weak cytotoxic activity, while 2-(4-hydroxybenzyl)quinazolin-4(3H)-one (98), N-[2-(4-hydroxyphenyl)acetyl]formamide (100) and N-[(2E)-(4-hydroxyphenyl)ethenyl]formamide (96) showed moderate activities against Du145, HeLa and A-549 cell lines with the IC50 values of 8, 20, and 20 mg/mL, respectively.Preliminary assay by An et al.  applied 50 values of 9.2 and 39.7 µM, respectively, while chloctanspirone B (151), showed no activity against the same cell lines.Bladt et al.  stated tP. chrysogenum ZZ1151. The new peniprenylphenol A (200) was found to possess promising antimicrobial activity towards the human pathogens MRSA, E. coli and C. albicans with MIC values of 6, 13, 13 mg/mL, respectively. In addition, the other known isolated compounds, preparaherquamide (203), uridine (205) and 4-hydroxybenzeneacetic acid methyl ester (207) revealed antimicrobial activity with MIC values in a range from 3 to 25 mg/mL towards the three pathogens. Meanwhile thymine (204) and clavatol (206) demonstrated antibacterial activity against MRSA and E. coli only with MIC values of 13–25 mg/mL and 2-hydroxyphenylacetic acid methyl ester (209) showed activity against both MRSA and C. albicans with MIC values of 13 and 7 mg/mL, respectively. Also, penicimumide (201) showed antibacterial activity against E. coli (13 mg/mL), communol G (102) and 4-hydroxyphenylethanone (210) had activity against MRSA (MIC: 25 mg/mL) and 2,5-dihydroxyphenylacetic acid methyl ester (208) exhibited antifungal activity against C. albicans (MIC = 25 mg/mL).According to Newaz et al. , severalP. chrysogenum for their antimicrobial activity with comparison with five types of pathogenic bacteria Staphylococcus aureus, Bacillus licheniformis, Escherichia fergusonii, Enterobacter xiangfangensis, and Ps. aeruginosa. All the samples except 6-hydroxymellein (179) revealed selective activities towards Gram-positive bacteria Staph. aureus and B. licheniformis with MIC values range 0.8 to 21.6 μg/mL. However, 4-chloro-6-hydroxymellein (180) displayed highly potent effect towards Gram-positive bacteria, with MIC 1.00 and 0.8 µg/mL−1 for Staph. aureus and B. licheniformis, respectively.Orfali et al.  investigP. chrysogenum TJ403-CA4 extract obtained from the therapeutically valuable arthropod Cryptotympana atrata. It was found that they were active against MRSA with MIC values of 4.0 and 2.0 μg/ mL, respectively. Additionally, they revealed good activity towards ESBL-producing E. coli and E. faecalis with MIC values of 32 μg/mL.Qiao et al.  isolatedP. chrysogenum strain XNM-12. It was found that oxalicine C (63) and penicierythritol A (221) revealed moderate antibacterial activity towards the plant pathogen Ralstonia solanacearum with MIC values of 8 and 4 μg/mL, respectively.Xu et al.  evaluateP. chrysogenum DXY-1, obtained from deep-sea sediments nearby the East Sea, found that tryosol had an anti-quorum sensing (anti-QS) activity. All studies implied that tyrosol (242) may act as a possible inhibitor for the QS systems to resolve the frightening crisis of bacterial resistance. It may be used as a QS inhibitor against C. violaceum and Ps. aeruginosa. The docking outcomes showed that it inhibited the QS system of CviR in C. violaceum through binding to the DNA-binding domain and blocking pathogenic gene expression.A study by Chang et al.  on tyrosP. chrysogenum HLS111 strain with the histone-deacetylase inhibitor VPA. They were examined against Staph. epidermidis , B. subtilis , Staph. aureus , Enterococcus faecalis , and E. coli . They showed the maximum antibacterial effects against B. subtilis with a MIC of 5–10 µg/mL, while they exhibited modest activities towards Staph. epidermidis and Staph. aureus with MICs of 10–80 µg/mL.Zhen et al.  treated P. chrysogenum IFL1antimicrobial activity showed that the cheese whey culture filtrate inhibited the growth of the Staph. aureus, Ps. aeruginosa and B. cereus.Lopes et al.  reportedP. chrysogenum EN118, an endophytic fungus culture extract isolated from the marine brown alga Sargassum pallidium. Its antibacterial activity towards two bacteria, E. coli and Staph. aureus was assayed, however, none of them showed any inhibitory activity.An et al.  isolated50 33.7 ± 0.8 µg/mL compared to the P. chrysogenum extract, which was nearly inactive as revealed by the DPPH free-radical-scavenging technique.According to a study by Al-Saleem et al. , Kojic aP. chrysogenum ethanolic extract which was isolated from wastewater, was found to contain higher total phenolic content and better total antioxidant capacity along with ferrous ion chelating ability.Various antioxidant activity techniques were utilized by Jakovljevic et al. , includiP. chrysogenum EN118, an endophytic fungus culture extract isolated from the marine brown alga Sargassum pallidium. Its radical-scavenging activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay but did not show any activity.An et al.  isolatedResults of Li et al.  demonstrP. chrysogenum SCSIO 07,007, were assessed for their enzyme inhibitory activities against (PTP1B). Also, the new compounds chrysopyrones A and B (158 and 159) showed noticeable inhibitory activities towards PTP1B with IC50 values of 9.32 and 27.8 μg/mL, respectively.According to Han et al. , the proP. chrysogenum MT-12, an endophytic fungus separated from the medicinal plant of Huperzia serrata. The antiplatelet activities were evaluated by applying D. S. Kim’s technique. Penicichrysogene A (16) exhibited antiplatelet aggregation activity with IC50 value of 42.7 ± 3.5 μM.Qi et al.  reportedP. chrysogenum strain (CHNSCLM-0003) yielded chrysogeamides A and B (232–233) which obviously supported angiogenesis in zebrafish at concentration 1.0 μg/mL with nontoxic effect to embryonic zebrafish at concentration 100 μg/mL.According to Hou et al.  study, tP. chrysogenum SCSIO 41,001, which were tested for their α-glucosidase inhibitory activity by PNPG method. Nine compounds  demonstrated inhibitory action against α-glucosidase with IC50 values of 0.35, 0.20, 0.04, 0.16, 0.15, 0.09, 0.14, 0.14, and 0.12 μM, respectively (IC50 0.28 μM for the standard acarbose).Wang et al.  isolatedPenicillium chrysogenum endosymbiotic fungus. The chemical structures were classified mainly as terpenoids (majority), alkaloids, polyketides, steroids, flavones (minority), and miscellaneous compounds. Penicillium chrysogenum secondary metabolites possess valuable and interesting pharmacological activities, such as antimicrobial, antifungal, cytotoxic, and miscellaneous. This review may be considered a valuable reference for promising pharmaceutical applications or further needed studies on P. chrysogenum. Reviewing such an endophyte metabolic pathway on P. chrysogenum would help to broaden the future search and discovery of several hundred novel bioactive endosymbiotic compounds with the potential for use as therapeutics. From a global perspective, the endosymbiotic metabolites remain very active and now seem to have the necessary momentum to provide additional antimicrobial, anticancer, and antifungal compounds to the marketplace soon.By revising the existing literature, a massive library of secondary metabolites was isolated and identified, and they also possessed a unique structure. Up to 277 compounds with a variety of structures that belong to various chemical classes were reported from the


Morus alba L.) is widely used as a medicinal plant in Asia. In this study, the bioactive compounds of ethanolic extracts of white mulberry leaves from the Sakon Nakhon and Buriram cultivars were evaluated. The ethanolic extracts of mulberry leaves from the Sakon Nakhon cultivar showed the highest total phenolic content of 49.68 mg GAE/g extract and antioxidant activities of 4.38 mg GAE/g extract, 4.53 mg TEAC/g extract, and 92.78 mg FeSO4/g extract using 2,2 diphenyl-1-picrylhydrazyl (DPPH), 2,20-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays, respectively. The resveratrol and oxyresveratrol compounds in mulberry leaves were also investigated by high-performance liquid chromatography (HPLC). The mulberry leaf extracts from the Sakon Nakhon and Buriram cultivars showed oxyresveratrol contents of 1.20 ± 0.04 mg/g extract and 0.39 ± 0.02 mg/g extract, respectively, whereas resveratrol was not detected. It was also found that the potent anti-inflammatory properties of mulberry leaf extracts and its compounds, resveratrol and oxyresveratrol, suppressed the LPS-stimulated inflammatory responses in RAW 264.7 macrophage cells by significantly reducing nitric oxide production in a concentration-dependent manner. These compounds further inhibited interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production and suppressed the mRNA and protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophage cells. Therefore, it is established that mulberry leaf extract and its bioactive compounds contribute to its anti-inflammatory activity.The white mulberry ( The rnd NADPH . Inflammnd NADPH .Morus alba L., or white mulberry, belongs to the genus Morus in the Moraceae family. Mulberry is a medicinal plant in Asia and is economically important to the silk industry as it serves as food for the silkworm (Bombyx mori) [κB) [2) production and inhibits iNOS and COX-2 gene expression through the suppression of NF-κB binding activity [Over the last few years, there has been increasing attention on the anti-inflammatory effects of phytochemical compounds in plant extracts that are used in traditional medicine, since inflammation is recognized as playing an important role in several diseases. yx mori) . Mulberryx mori) . Their lyx mori) ,12. Resvyx mori) . These tyx mori) , antidiayx mori)  which aryx mori) . Resverayx mori) . Resverari) [κB) ,19 and tri) [κB) ,21. Concactivity .The white mulberry in Thailand is primarily grown for its leaves, which are the preferred food of silkworms. Some people consume mulberry leaf tea as a healthy beverage that is a rich source of antioxidants. Mulberry leaves contain high levels of phenolics, flavonoids, and 1-deoxynojirimycin (DNJ) which possess beneficial effects in protecting against cardiometabolic risks, including obesity, hyperglycaemia, hypertension, hyperlipidaemia, inflammation, and oxidants, and have cardioprotective effects . Mulberr50, which is the concentration of the extracts that inhibit the initial free radical by 50%. The lower IC50 value indicated a greater potential of the substances for scavenging DPPH and ABTS, implying a stronger antioxidant activity. The IC50 value of both the DPPH and ABTS activity was lower when taken from mulberry leaf extract from the Sakon Nakhon cultivar, showing 0.78 mg/mL by the DPPH assay and 4.89 mg/mL by the ABTS assay, indicating a higher antioxidant activity. Meanwhile, the IC50 value of mulberry leaf extract from the Buriram cultivar showed 1.67 mg/mL by the DPPH assay and 15.08 mg/mL by the ABTS assay. Moreover, the antioxidant activities were also compared to the standard gallic acid in the DPPH, Trolox in the ABTS, and ferrous sulphate (FeSO4) in the FRAP assays. Mulberry leaf extract from the Sakon Nakhon cultivar exhibited higher antioxidant activity than the Buriram cultivar, with an activity of 4.38 mg GAE/g extract, 4.53 milligrams of Trolox equivalent antioxidant capacity (TEAC) per gram of extract (mg TEAC/g extract), and 92.78 milligrams of ferrous sulfate per gram of extract (mg FeSO4/g extract)  . This fiAlthough numerous studies have shown that the main bioactive components of mulberries are phenolics and flavonoids , these pThe effect of white mulberry leaf extracts on cell viability was determined after incubating cells with ethanolic mulberry leaf extract at concentrations of 312, 625, 1250, 2500 and 5000 µg/mL for 24 h. The results of the MTT assay indicated that mulberry leaf extract at a concentration of up to 1250 µg/mL had no significant toxicity effects on the viability of RAW 264.7 cells. The effect of resveratrol and oxyresveratrol on cell viability was determined after incubating cells at concentrations of 3.12, 6.25, 12.5, 25 and 50 µg/mL. The results showed that resveratrol and oxyresveratrol were also nontoxic on RAW 264.7 cells at concentrations of up to 12.5 and 25 µg/mL, respectively. Therefore, these concentrations of mulberry leaf extract as well as resveratrol and oxyresveratrol were chosen for further experiments .The effects of mulberry leaf extract, resveratrol, and oxyresveratrol on LPS-stimulated nitric oxide production on RAW 264.7 cells are shown in Similarly, the results showed that resveratrol and oxyresveratrol were able to inhibit nitric oxide production in a concentration-dependent manner and the nitrite levels were found to be between 9.54 and 50.42 µM . AdditioiNOS and COX-2 in LPS-stimulated RAW 264.7 cells were evaluated using real-time quantitative PCR. The results revealed that mulberry leaf extract inhibited the mRNA expression of iNOS and COX-2 in a concentration-dependent manner . Moreover, the highest concentration of mulberry leaf extract at 1250 µg/mL had the greatest inhibitory effect on the mRNA expression of iNOS and COX-2. Regarding resveratrol and oxyresveratrol, these compounds were able to inhibit the mRNA expression of iNOS in a concentration-dependent manner. Resveratrol at a concentration of 6.25–12.5 µg/mL and oxyresveratrol at a concentration of 12.5–25 µg/mL could suppress the mRNA expression of iNOS by more than 50% and inhibited COX-2 when treated with resveratrol and oxyresveratrol at concentrations of 12.5 and 25 µg/mL, respectively.The effects of mulberry leaf extract, resveratrol, and oxyresveratrol on the mRNA expression of t manner . In the TNF-α and IL-6 using real-time quantitative PCR. As shown in TNF-α and inhibited the expression levels of IL-6 in a concentration-dependent manner. The mulberry leaf extract from the Buriram cultivar at a concentration of 625–1250 µg/mL, the resveratrol at a concentration of 12.5 µg/mL, and the oxyresveratrol at a concentration of 6.25–25 µg/mL could suppress the mRNA expression of IL-6 by more than 50%, compared to the cells treated with LPS alone (p < 0.05). However, the mulberry leaf extract from the Sakon Nakhon cultivar did not downregulate the mRNA expression levels of IL-6 (p < 0.05) in the same conditions. The expression of the housekeeping gene β-actin was not induced by the treatment.The inhibitory effects of mulberry leaf extract, resveratrol, and oxyresveratrol on the production of pro-inflammatory cytokines were further investigated with consideration of the transcription of p < 0.05) (p < 0.05) (p < 0.05) and also downregulated the COX-2 expression by more than 50% at concentrations of 6.25–12.5 and 12.5–25 µg/mL of resveratrol and oxyresveratrol, respectively. Furthermore, the inhibition of the protein expression of iNOS and COX-2 by the mulberry leaf extract as well as oxyresveratrol and resveratrol correlated well with the iNOS and COX-2 mRNA expression.The effects of mulberry leaf extract, resveratrol, and oxyresveratrol on the protein expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells were determined by a Western blot analysis. The mulberry leaf extracts from both the Sakon Nakhon and Buriram cultivars reduced the protein expression of iNOS and COX-2 ( < 0.05) A. The mu < 0.05) B. The reNumerous studies have confirmed that the phenolics and flavonoids found throughout medicinal plants  are majoPhenolic and flavonoid compounds are confirmed to exhibit antioxidant , anti-in2−). Therefore, it is defined by assessing the level of nitrite, commonly used as a surrogate marker of nitric oxide generation [Lipopolysaccharide (LPS) was required for the full activation of RAW 264.7 macrophage cells . LPS canneration . In thisneration . TherefoiNOS mRNA expression level. These findings showed that nitric oxide inhibition may be due to the inhibition of iNOS during LPS-stimulation on macrophages [iNOS and have no effect on COX-2 mRNA expression. However, in a concentration-dependent manner, resveratrol and oxyresveratrol significantly suppressed the protein expression levels of both iNOS and COX-2. Thus, it is considered that resveratrol and oxyresveratrol might influence the process of iNOS and COX-2 translation from gene to protein. Additionally, these active compounds might inhibit the formation of prostaglandin E2 (PGE2) induced by LPS by reducing the expression level of the COX-2 gene [Moreover, the inhibitory effect on nitric oxide production in white mulberry leaf extracts from both the Sakon Nakhon and Buriram cultivars was higher than resveratrol and oxyresveratrol, since the extracts contain phenolic and flavonoid polyphenols. Apart from these polyphenols, oxyresveratrol possibly accounts for the high anti-inflammatory properties of mulberry leaf extract. White mulberry leaf extract, resveratrol, and oxyresveratrol were also found to suppress the rophages . The oxirophages . Thereforophages . In thisX-2 gene .TNF-α, but did not affect the expression of IL-6. A prior study found that NF-κB, a crucial transcriptional regulator, regulates the production of pro-inflammatory cytokines, iNOS and COX-2, at the transcriptional level [The inhibition of pro-inflammatory cytokines by mulberry leaf extract was further examined for anti-inflammatory activity as well as its bioactive substances. The inflammatory response can be orchestrated by pro-inflammatory cytokines, such as TNF-α and IL-6, and it can be mediated through NF-κB transcriptional factor . Nitric al level , and is al level . This fial level .Among the phytochemical compounds in mulberry leaf extracts, resveratrol and oxyresveratrol have sparked a lot of curiosity based on their abilities for antioxidant, antibacterial, and anti-inflammatory activity. Our research indicated that these bioactive compounds appeared to decrease the expression of iNOS and COX-2 proteins better than crude mulberry leaf extract. This might be due to the amount of resveratrol and oxyresveratrol in the test being greater than the compound content that found in the mulberry leaf extract. Although, in this study, only oxyresveratrol was detected in the mulberry leaf extract, the inhibitory ability of these compounds has also been supported in other studies . ResveraApart from resveratrol and oxyresveratrol, numerous natural antioxidant substances have been found to inhibit NF-κB-dependent cytokines as well as iNOS and COX-2 levels. The suppressive influence on the generation of inflammatory mediators is linked to their antioxidant activity . Among tEscherichia coli O111:B4 were obtained from Sigma-Aldrich . RAW 264.7 cell culture media and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco . 3--2,5-diphenyl tetrazolium bromide (MTT) was obtained from Bio Basic . Primary antibodies, anti-beta-actin monoclonal antibody, anti-iNOS/NOS II polyclonal antibody, and anti-COX-2 polyclonal antibody, as well as the peroxidase-conjugated secondary antibody were purchased from EMD Millipore corporation .Folin-Ciocalteu reagent, TPTZ -s-triazine), and quercetin dehydrate were purchased from Merck . 2,2-diphenyl-1-picrylhydrazil (DPPH), 2,2′-azinobis-(3-ethylbenzothiazolin-6 sulfonic acid) (ABTS), 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox), gallic acid monohydrate, resveratrol, oxyresveratrol, and lipopolysaccharide (LPS) from M. alba L.) from Sakon Nakhon and Buriram cultivars were collected during May–June in Chiang Mai province  under the supervision of the Queen Sirikit Department of Sericulture. The leaves were dried in an oven at 60 °C for 48–72 h and blended into powder with a blender. The samples were kept in airtight plastic bags for around 1–2 days prior to extraction.White mulberry leaves (Mus musculus) and was purchased from ATCC with the accession number ATCC-TIB-71.RAW 264.7 macrophage cell line originated from mice (w/v) and was shaken at 150 rpm  for 72 h at room temperature. The extracts were then filtered through a Whatman No.1 filter paper and vaporized by a rotary evaporator  under a vacuum at 45 °C. Afterward, the extracts were freeze-dried by lyophilization  and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL until they were used for experimentation. Mulberry leaf powder was extracted with 95% ethanol in a ratio of 1 to 10 (w/v sodium carbonate solution (250 µL). After incubation at room temperature for 1 h, the absorbance was measured at 725 nm using a spectrophotometer . The total phenolic content was expressed as milligrams of gallic acid equivalents (GAE) per 1 g of extract (mg GAE/g extract).Total phenolic content was investigated by the Folin–Ciocalteu method . BrieflyTotal flavonoid content was determined by the aluminum chloride colorimetric method . Briefly50. Gallic acid was used as a standard and the antioxidant activity is presented as milligrams of gallic acid equivalent per 1 g of extract (mg GAE/g extract).The radical scavenging activity was evaluated using 2,2-diphenyl-1-picrylhydrazil (DPPH) assay  with min+) assay [+ solution containing an absorbance of 0.700 ± 0.020 at 734 nm. After incubation for 10 min, the absorbance was measured at 734 nm using a microplate reader . The radical cation decolorization activity of the mulberry leaf extracts is presented as IC50. Trolox was used as a standard, and the antioxidant activity is presented as milligrams of Trolox equivalent antioxidant capacity (TEAC) per 1 g of extract (mg TEAC/g extract).The radical cation decolorization activity was determined using 2,2′-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS.+) assay  with som4) per 1 g of extract (mg FeSO4/g extract).The ferric reducing antioxidant power was estimated using reductants in a redox-linked colorimetric method . The mulv/v) and B (acetonitrile) were used with the gradient program set as follows: 0–30 min, linear change from A-B  to A-B . The peak area of resveratrol and oxyresveratrol was calculated by the Agilent ChemStation level-5 program, and the content of each compound in the mulberry leaf extracts was calculated from the standard graph within a range of 7.8 to 125 µM  of resveratrol and a range of 7.8 to 125 µM  of oxyresveratrol.The resveratrol and oxyresveratrol found in mulberry leaf extracts were determined using high-performance liquid chromatography (HPLC) with some modifications . These c2 incubator.RAW 264.7 macrophage cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin) and maintained at 37 °C in a 5% CO5 cells/well and incubated at 37 °C in a 5% CO2 incubator for 24 h. The media were removed and the cells were then treated with 100 µL of the control medium or the medium supplemented with various concentrations of mulberry leaf extract (312–5000 µg/mL), resveratrol (3.12–50 µg/mL), or oxyresveratrol (3.12–50 µg/mL) and incubated at 37° for 24 h. MTT solution  was added to each well and the plates were incubated at 37 °C for 4 h.Cell viability was determined by 3--2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, the cells were grown in 96-well plates at a density of 2 × 10Next, the medium from the wells was removed, and 200 µL of DMSO was added to the wells to solubilize the formed formazan crystals. The absorbance was measured on a microplate reader  using a test wavelength of 540 nm and a reference wavelength of 630 nm [Treatment before stimulation with LPS5 cells/well in 96-well plates and incubated at 37 °C in a 5% CO2 incubator for 24 h. The media were then removed and the cells were treated with 100 µL of non-toxic concentration of mulberry leaf extract (312–1250 µg/mL), resveratrol (3.12–12.5 µg/mL), and oxyresveratrol (6.25–25 µg/mL) for 1 h, then LPS at 1 µg/mL (30 µL) was added to stimulate inflammation. After incubation for 24 h, the nitric oxide level in the cell culture medium was measured using Griess reaction assay [w/v) sulfanilamide in 5% (v/v) phosphoric acid and 0.1% (w/v) naphthyl ethylenediamine dihydrochloride), and incubated at room temperature for 15 min. Then the absorbance was measured at 540 nm by a microplate reader . The nitrite concentration was calculated using sodium nitrite as a standard within a range of 1 to 100 µM .The cells were plated at a density of 2 × 10on assay . BrieflyTreatment after stimulation with LPS5 cells/well in 96-well plates and incubated at 37 °C in a 5% CO2 incubator for 24 h. LPS stimulation was performed using LPS at 1 µg/mL (30 µL) for 10 min, then LPS was removed, followed by incubation with 100 µL of non-toxic concentration of mulberry leaf extract (312–1250 µg/mL), resveratrol (3.12–12.5 µg/mL), and oxyresveratrol (6.25–25 µg/mL) for 24 h. Nitric oxide levels in cell culture medium were measured using Griess reaction assay [The cells were plated at a density of 2 × 10on assay .6 cells/well in 24-well plates and stimulated with LPS at 1 µg/mL for 30 min prior to incubation with a non-toxic concentration of mulberry leaf extract (312–1250 µg/mL), resveratrol (3.12–12.5 µg/mL), and oxyresveratrol (6.25–25 µg/mL). Total RNA was isolated from the cells using Trizol® reagent . The total RNA was converted into cDNA with a reverse transcription system containing 2 µL of 5× RT Master MIX , 2 µg total RNA, and 6 µL RNase-free water. The reaction was performed at 37 °C for 15 min, followed by incubation at 50 °C for 5 min and heating at 98 °C for 5 min, respectively.The cells were plated at a density of 1.0 × 10® No-ROX Kit  for analysis of iNOS, COX-2, TNF-α, and IL-6. The qPCR reaction system contained 10 µL SensiFAST SYBR® (2×), 0.8 µL forward primer (10 µM), 0.8 µL reverse primer (10 µM), and 8.4 µL cDNA under the following reaction conditions: 95.0 °C for 2 min, followed by 40 cycles at 95.0 °C for 5 s and 65.0 °C for 30 s. The amplification results were expressed as the threshold cycle (Ct) value, which represented the number of cycles needed to generate a fluorescent signal greater than a predefined threshold. The expression levels of iNOS, COX-2, TNF-α, and IL-6 were normalized using β-actin as an internal control. The primers for iNOS, COX-2, TNF-α, IL-6, and β-actin genes were used as shown in The cDNA was used for real-time qPCR amplification by SensiFAST™ SYBR6 cells/well in 24-well plates and incubated at 37 °C in a 5% CO2 incubator for 24 h. LPS stimulation was performed at 1 µg/mL for 30 min prior to incubation with a non-toxic concentration of mulberry leaf extract (312–1250 µg/mL), resveratrol (3.12–12.5 µg/mL), and oxyresveratrol (6.25–25 µg/mL) for 12 h. Afterwards, cell suspension was centrifuged at 1200 rpm at 4 °C for 10 min. The cell pellet was washed with phosphate buffer saline (PBS) (pH of 7.4) twice by centrifugation at 1200 rpm for 5 min. After that, cell extraction was performed by adding 60 µL of RIPA lysis buffer  containing protease inhibitor cocktails with a ratio of 1:1000. The lysates were clarified by centrifuge at 3300 rpm at 4 °C for 15 min. The protein concentration was determined using BCA protein assay reagent . The protein equivalents of samples (30 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (15% SDS-PAGE) using Bio-Rad Mini-PROTEAN Tetra Handset . SDS-PAGE gel electrophoresis was performed at 60 volts for 3 h 30 min to determine proteins and transferred to polyvinylidene fluoride (PVDF) membranes at 20 volts and 200 milliampere (mA) for 45 min using the semi-dry blotting method. The membranes were blocked with 5% skim milk for 1 h and incubated overnight at 4 °C with the primary antibodies, including iNOS (1:2000), COX-2 (1:1000), and β-actin (1:2000) by dilution in 5% skim milk. Then, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:3000) for 1 h at room temperature. Antibody-specific proteins were visualized using enhanced chemiluminescence reagent . Visualization of target proteins was detected through chemiluminescence detection .The cells were plated at a density of 1 × 10p < 0.05 were considered statistically significant.Statistical analysis of data was performed using SPSS 17.0 statistical analysis software . All quantitative data were verified in triplicate and presented as mean ± standard deviations. A one-way analysis of variance (ANOVA) was performed and the differences at As a whole, white mulberry leaf extract appears to be a good source of phenolics and flavonoids that influence antioxidant activity, as determined by DPPH, ABTS, and FRAP assays. Although only oxyresveratrol was detected in this study, the crude mulberry leaf extract and its bioactive compounds demonstrated anti-inflammatory activity by suppressing nitric oxide generation in LPS-stimulated RAW 264.7 macrophage cells and substantially decreasing the protein expression of iNOS and COX-2 in a concentration-dependent manner. Additionally, pro-inflammatory cytokines, e.g., TNF-α and IL-6, were also downregulated. This study applied an in vitro approach to white mulberry leaf extract, as well as resveratrol and oxyresveratrol, regardless of its in vivo actions that may affect their potential anti-inflammatory activities. Moreover, the analysis of physiologically relevant reactive molecules, e.g., superoxide anions, hydrogen peroxide, and hydroxyl radicals, should be considered. As a result, animal models may help to investigate these issues and ultimately assess the benefits for human health.