INTRODUCTION:

Present verification signify that a developed up human intake diet flush in flavonoids remind a moderation of combination cholesterol, low-thickness lipoproteins, and triglycerides in plasma, and diminish incident of cardiovascular diseases and osteoporosis. Often flavonoids, naringenin and hespered in are very common in some eadable of the soil as aglycons and glycosides^1,2. Naringin is rich in grapefruit and it accounted for the hypocholesterolemic, antiestrogenic, hypolipidemic, antihypertensive drug, and anti-inflammatory activities, antioxidant activity, and free radical scavenging properties and some defense against lipid peroxidation have been recorded^3,4. Human body do not produce any flavonoids but are taken through daily. The result proved that flavonoids inhibit a vital biological role, in addition to the function of scavenging the reactive oxygen species^5,6.

Lupeol is pharmacologically active triterpenoid found in dietary fruits and vegetables such as mango, strawberry, grapes, tomato and cabbage nuts and medicinal plants including gingseng and aloe vera. Lupeol has been reported for medicinal effectiveness against disorders involving multiple diseases such as cardiovascular disease, kidney disease, wound healing, arthritis and diabetes^7,8.

MATERIALS AND METHODS:

Chemicals:

In this study all of the chemicals and reagents were used of very high analytical grade and were purchased from companies such as Sigma-Aldrich, Merck, and others.

Animals:

Sprague Dawley rats weighing 160-180gm were collected from the animal centre house of the Central Drug Research Institute, (CDRI), Lucknow (U.P.). The animals were housed in polypropylene cages at temperatures of 22±2°C, relative humidity (55±10%) and 12 hours of light/dark cycles and supplemented with standard laboratory animal diet and water ad libitum. The animals were acclimated to the laboratory environment prior to procedure. The animals were housed in sterile polypropylene cages for 5 days before to being labeled for individual marking to the establish the experimental work.

Approval from animal’s ethical committee:

The study was carried out after the permission was received from the Institutional Animal Ethical committee, (IAEC), (Faculty of Pharmacy), Integral University, Lucknow–226026 (U.P) Approval No./Proposal No. IU/IAEC/18/35., under strict guideline of CPCSEA.

Toxicity studies of Lupeol+Naringin combination:

The oral acute toxicity analysis of Lupeol+Naringin combination was assessed in compliance with guideline OECD NO.423TG⁹.

Acute toxicity assay:

A healthy female young and nulliporous, non-pregnant Sprague Dawley (S.D) rats aged between 160-180gm and 8-12 weeks were picked and housed under normal conditions for five days; then rats were left without food for 3-4hours with access to water ad libitum before receiving a single dose of 2000mg/kg orally, limit test has been performed.

The dosage was administered according to body weight for each rat, and then closely observed followed for the first 30 minutes, and for the next 4 hours. After 1-2 hours of dosing, the standard pellet diet was given. The vehicle-treated group of five rats performed the same treatment of 1% carboxymethyl cellulose (CMC) gel was given in the equivalent amount as the treated group. Any form of toxic effect was detected within 6 hours of both groups and continuously for a total duration of 14 days. Rats were weighted at the ending of the research study period and blood samples were obtained via direct cardiac puncture method by given under isoflurane anesthesia. Obtained Serum was isolated for the assessment of biochemical’s and haematological parameters. Rats were sacrifice by cervical dislocation, weight of vital organ (liver, heart, and kidney) was noticed and it was preserved in a 10% formalin solution for the histopathological estimation.

Biochemical analysis:

Serum were collected to determine liver and renal toxicity marker enzyme like, Alkaline phosphate (ALP), Alanine amino transferase (ALT), Aspartate amino transferase (AST),glucose, Total protein (TPRO), triglycerides (TG), cholesterol (CHO), bilirubin, urea, creatinine (CK) using a Span diagnostic assay kit and absorbance was taken by the help of UV Spectrophotometer.

Haematological analysis:

The blood sample was obtained from animal both control and tested group for hematological analysis in EDTA containing tubes., hemoglobin (Hb), packed cell volume (PCV) White blood cells count (WBC), Red blood cells count (RBC) Mean corpuscular hemoglobin concentration (MCHC), Mean corpuscular volume (MCV), and eosinophils (E), lymphocyte (L), monocyte (M), neutrophils (N), platelet count (PLT). The estimation were made with auto analyzer.

Histopathological study:

The examination of histopathology of all vital organs like, liver, heart, and kidney were collected from animal. The organ was weighed and preserved in 10% formalin solution. The small tissue were trimmed and processed, after processing was done the tissue section was trimmed to a thickness of around 5µmM using a rotatory microtome, and the tissue section was staining with hemotoxylin and eosin dye (H&E) for microscopic examination.

Statistical analysis:

All important data is expressed as mean±SD and analysed using one way analysis of variance ANOVA with Student-Newman-Keuls test, with a significance level of (P ≤ 0.05).

RESULTS:

Acute toxicity study:

A single dose of Lupeol+Naringin combination (2000 mg/kg+2000mg/kg body weight) in rats did not show any mortality or signs of toxicity. As results the LD50 value may be higher than the 2000mg/kg.

Body weight:

The body weights in regulated and Lupeol+Naringin combination test animals were steadily increased, Table 1.

Measurement of relative organ weight:

No significant variation was observed in weight of the heart, liver, kidney, treated and control groups (P≤ 0.05), the relative organ weight was insignificant when compared to test groups.

Behavioral observation:

Lupeol+Naringin combination and control group were regularly evaluated for the 14 days, all monitoring was closely reported and separated notes were written for each animal. Observation to distinguish such differences in pattern of the skin, hair, eye, mucus membrane, and usual behaviors. Tremor, salivation, convulsion, diarrhoea, sleep, lethargy, coma and mortality was monitored. Behavioral and clinical observation is summarized in Table 2.

Feed and water intake:

The feed and water intake of both control and Lupeol+Naringin combination treated animals was gradually enhanced over the study period duration as shown in Table 3.

Biochemical analysis:

The analysis of liver function markers enzyme like, Alkaline phosphate (ALP), Aspartate amino transferase (AST)) Alanine amino transferase (ALT), used in the acute toxicity analysis indicated no significance difference (p≤ 0.05), involving the control and the test drug administered orally to rats. In Table 4, displays the findings of the renal function investigation used to determine the status of kidney function in the acute toxicity. Even the Lupeol+Naringin combination (2000 mg/kg+2000mg/kg body weight) treated rats displayed no significance difference in the levels of renal toxicity indicators such as, creatinine, urea and content of total protein relative to control rats.

Table1: Effect of Lupeol+Naringin combination on body weight in rats.

Treatment	Body weight (g)
Treatment	0 day	1st week	2nd week
Control (CMC1% gel)	128.1±1.18	138.2±2.34	151.7±2.53
Lupeol+Naringin combination	126.9±1.34	139.6±2.24	154.9±3.62

Values are mean ± SD, N=5

There is no toxicologically significant difference in body weight between the control and treated groups.

Hematological analysis:

It can be shown hematological parameter that there are no major alteration in hemoglobin (Hb), White blood cells count (WBC), Red blood cells count (RBC). However, there was no significance difference (P<0.05) MCHC, packed cell volume (PCV) platelet count, and lymphocyte (L), eosinophils (E), monocyte (M), neutrophils (N), compare with normal control group. (Table-2).

Histopathological study

The acute toxicity study histopathology finding of vital organ like liver, heart kidney of Lupeol+Naringin combination treated animals showed in the Fig.1 no any pathological changes in test group and compared to control group animals.

Heart: The cardiac tissue portion has been arranged in a thick and compact way with no evidence of toxicity, normal tecture of heart muscle fibre.

Liver: No lesions from hepatic parenchyma, normal lumen vein tecture, intact nucleal and cellular lumen vein has been arranged in the hepatic tissue portion.

Kidney: The kidney tissue section show normal glomeruli, Bowmans space, renal tubules in cortex and medulla.

Table:2 Behavioral parameter of Lupeol+Naringin combination treated groups

Observations of vehicle control and Lupeol+Naringin combination (2000mg/kg/p.o.) treated groups

Parameters

30 MINUTES

4 HOURS

24 HOURS

48 HOURS

7 DAYS

14 DAYS

C.G.

T.G.

C.G.

T.G.

C.G.

T.G.

C.G.

T.G.

C.G.

T.G.

C.G.

T.G.

Skin and Fur

Mucous membrane

Eyes

Salivation

Sleep

Lethargy

Convulsion

Coma

Tremors

Diarrhea

Morbidity

Mortality

Key: CG =Vehicle Control group (CMC1% gel),TG =Lupeol+Naringin combination treated groups. NR= Normal, NF = Not Found

Table 3: Effect of Lupeol+Naringin combination on feed and water intake in rats.

Feed intake (g)
Treatment	1st week	2nd week
Control (CMC1% gel)	80.71±2.73	107.4±11.85
Lupeol+Naringin combination	93.18±4.19	156.2±12.59
Water intake (ml)
Treatment	1st week	2nd week
Control (CMC1% gel)	161±17.43	198.1±18.49
Lupeol+Naringin combination	176.7±18.90	211.6±20.01

Value are mean ± SD (n=5) Regular variation showed in food and water intake compare with control groups.

Table 4: Effect Lupeol+Naringin combination on relative organ weight

Groups	Relative weight of organ
Groups	Liver	Kidney	Heart
Control (CMC1% gel)	3.85±0.03	0.95±0.03	0.67±0.03
Lupeol+Naringin combination treated groups	3.82±0.02	0.93±0.03	0.65±0.02

Value are mean ± SD (n=5), control groups compare to treated groups. There were no any significant changes

Biochemical parameters:

Table 5: Effect Lupeol+Naringin combination on biochemical parameters in rats

Parameters	Control (CMC1% gel)	Test
Glucose(mg/dl)	109.2±7.92	103.2±10.94
ALT(IU/L)	45.18±2.13	57.30±4.92
AST(IU/L)	130.9±11.1	110.2±9.28*
ALP (IU/L)	310.6±82.65	210.7±80.03*
Total Protein (g/dl)	5.80±0.32	5.20±0.12
Triglycerides(mg/dl)	102.9±12.4	84.38±10.70*
Cholesterol(mg/dl)	94.80±5.40	91.08±4.14
Bilirubin (mg/dl)	0.270±0.012	0.21±0.023
Urea(mg/dl)	34.21±2.08	37.91±2.24
Creatinine(mg/dl)	0.50±0.021	0.52±0.01

Value are mean±SD (n=5).p˂0.05; Student-Newman–Keul Test; control groups compare to treated groups. There was no any significant change in the biochemical parameters of drug treated groups as compare with the control groups.

Hematological parameters:

Table 6: Effect of Lupeol+Naringin combination on hematological parameters in rats.

Parameters	Control(CMC1% gel)	Treated
WBC(103/uL)	15.38±1.80	14.43±1.70
RBC(106/uL)	6.50±0.22	7.022±0.036
Hb.(g/dL)	13.38±0.63	13.10±0.13
HCT(%)	41.64±1.47	40.3±0.61
MCV(fL)	52.05±0.82	56.34±1.21
MCH(pq)	18.72±0.39	19.70±0.26
MCHC(g/dL)	31.9±0.56	31.77±0.28
PLT(103/uL)	810±77.53	1001±43.02
N (%)	2.17±0.22	1.66±0.114
L(%)	10.51±1.70	12.91±1.74
M(%)	0.52±0.80	0.55±0.37
E(%)	0.24±0.21	0.26±0.050
B(%)	0.015±0.03	0.01±0.002

Value are mean±SD (n=5).p˂0.05; Student-Newman–Keul Test; control groups compare to treated groups.

There was no any significant change in the hematological parameters of drug treated groups as compare with the control groups.

Mean corpuscular hemoglobin concentration (MCHC), packed cell volume (PCV) White blood cell count (WBC), Red blood cell count (RBC), Mean corpuscular volume (MCV), hemoglobin (Hb), and eosinophils (E), lymphocyte (L), monocyte (M), neutrophils (N), platelet count (PLT).

In all of the hematologic parameters was observed in control groups compare with treated groups of rats no toxicologically relevant results were found, except that in treatment groups the platelet counts were elevated compared with the control group.

Figure 1: Histopathological results of kidney (P, Q), liver(R, S) and heart (T, U) of control and treated groups.

P, R, T= Control groups (CMC1% gel) Q, S, U =Lupeol+Naringin combination treated groups

DISCUSSION:

Natural herbal drugs are also commonly used in primary care in developed countries. The World Health Organization estimates that 82% of the people of distant areas depend on herbal drug there is a tradition of about 50,000 years of human used as medicine, herbal plants¹⁰.The most common naturally bioactive derivative metabolites in plants are flavonoids and fagarsterol. Their pharmacological properties include hepatoprotective, anti-cancerous, immunomodulater and cardiovascular protection, both of which are used in the management of various diseases. In acute toxicity study, according to OECD Guidelines423TG, the maximal dosage of 2000 mg/kg body weight of the Lupeol+Naringin mixture has been given no any signs of toxicity or mortality was observed over 14 days. The toxic effect of chemical and drugs decrease or increase the body weight. In both the controls and treated drug groups, the relative weight of essential organs like, heart, liver and kidney is considered normal, and statistically significant variation (P˂0.05) was found to have no toxic effect. Lupeol+Naringin combination hematologic parameters were within the reference range and were comparable to the control rat group⁹.The potential of Lupeol+Naringin combination hepatotoxicity treated without any major alterations for ALT, AST or ALP, indicating that Lupeol+Naringin treated combination does not cause any inflammatory or necrotic damage to liver. Histopathologic findings of section of the kidney, liver and heart of the rat treated with Lupeol+Naringin are evidence that the normal showing integrity of the hepatic cell does not cause any damage to the liver.

CONCLUSION:

The oral doses of Lupeol+Naringin were found to be safe and did not cause any adverse effects in rats in acute toxicity studies. This research provides useful acute toxicity evidence of the combination of Lupeol+Naringin. Since no mortality or signs of toxicity in treated rats have been shown, it is possible to say that the research feasible to propose the LD50 are orally controlled doses of more than 2000 mg/kg. Monitoring made during the acute toxicity analysis suggests, in compare to control group rats, that the dosage levels examined do not produce toxic effects in treated rats. There is also a wide safety margin and ability to improve the new therapeutic agent of Lupeol + Naringin combination.

ACKNOWLEDGEMENT:

All authors contributed equally to this work. The authors are thankful to the Integral University for providing facilities, critical suggestion regarding the improvement of the manuscript and special thanks to research community for providing manuscript communication number for further communication–IU/R&D/2021-MCN0001122

CONFLICTS OF INTEREST:

The authors declare no conflict of interest.

REFERENCE:

1. Mukherjee PK. Quality control of herbal drugs. Business Horizons, New Delhi, India. 2002.

2. Suseem SR, Joseph D. The Myth and The fact on Naringin -A Review. Research Journal of Pharmacy and Technology. 2019; 12(1): 367-74. doi: 10.5958/0974-360X.2019.00067.2

3. Wongmekiat O, Leelarugrayub N, Thamprasert K. Beneficial effect of shallot (Allium ascalonicum L) extract on cyclosporine nephrotoxicity in rats. Food and Chemical Toxicology.2008 May;46(5):1844-50. doi: 10.1016/j.fct.2008.01.029

4. Santos FV, Colus IMS, Silva MA, Vilegas W, Varanda. EA. Assessment of DNA damage by extracts and fractions of Strychnospseudoquina, A Brazilian medicinal plant with anti-ulcerogenic activity. Food and Chemical Toxicology.2006 Sep;44(9):1585-9. doi: 10.1016/j.fct.2006.03.012

5. Khan N, Sultana S. Chemomodulatory effect of Ficusr acemosa extract against chemically induced renal carcinogenesis and oxidative damage response in Wistar rats. Life Sciences. 2005 Jul 29;77(11):1194-210. doi: 10.1016/j.lfs.2004.12.041

6. Keles H, Fidan AF, Cigerci IH, Kucukkurt I, Karadas E, Dundar Y. Increased DNA damage and oxidative stress in chickens with natural Marek's disease. Veterinary Immunology and Immunopathology. 2010 Jan 15;133(1):51-8. doi: 10.1016/j.vetimm.2009.07.003

7. Moreau RA, Whitaker BD, Hicks KB. Phytosterols, phytostanols, and their conjugates in foods: structural diversity, quantitative analysis, and health-promoting uses. Progress in lipid research. 2002 Nov;41(6):457-500. doi: 10.1016/s0163-7827(02)00006-1

8. Fernández, M. A., de lasHeras, B., García, M. D., Sáenz, M. T., &Villar, A. (2001). New insights into the mechanism of action of the anti-inflammatory triterpenelupeol. The Journal of pharmacy and Pharmacology. 53(11), 1533-9. https://doi.org/10.1211/0022357011777909

9. OECD. OECD guidelines for the testing of chemicals, section 4, test No. 423: acute oral toxicity - up-and-down procedure. OECD Publishing. 2001.

10. Fadholly A, Ansori AN, Sucipto TH. An Overview of Naringin: Potential Anticancer compound of Citrus Fruits. Research Journal of Pharmacy and Technology. 2020; 13(11):5613-9. doi: 10.5958/0974-360X.2020.00979.8

Received on 05.06.2021 Modified on 23.07.2021

Research J. Pharm. and Tech. 2022; 15(8):3447-3451.

DOI: 10.52711/0974-360X.2022.00577

Relationship between the Dose administered and Toxicity level after Acute Oral Exposure to Lupeol and Naringin combination in rats

The oral acute toxicity analysis of Lupeol+Naringin combination was assessed in compliance with guideline OECD NO.423TG9.

Acute toxicity assay:

The oral acute toxicity analysis of Lupeol+Naringin combination was assessed in compliance with guideline OECD NO.423TG⁹.