Abstract: Background and Objectives: Pumpkin leaves (Telfairia occidentalis) are extensively used in homes in Nigeria and some West African countries for soup making and sometimes extracted in water for drinking as medicament for stimulating erythropoiesis. The present study was aimed at evaluating the in vitro effect of pumpkin leaves extract on human erythrocytes with an interest in obtaining base data on the phyto-toxicity. Materials and Methods: Pumpkin leaves weighing 250 g was extracted in 250 mL cold distilled water to obtain a stock solution of 1 g mL–1 pumpkin leaves extract. The stock solution was further diluted in sterile test tubes to obtain a concentration range of 10, 20, 40 and 640 μg m–1 by using cold sterile distilled water. The test required the addition of 0.5 mL of each pumpkin leaves extracted concentration to a sterile test tube containing 2.5 mL of 5% human erythrocyte in sterile physiological saline and allowed to remain at room temperature (27+1°C) for 30 min. The hemolysis occurring in the tubes was read spectrophotometrically (Cornings) at 540 nm wavelength along with the neat and saline control. Results: The absorbance reading reflected corresponding increase in the concentration of pumpkin leaves extract by using phosphate. This showed a significant increase in toxicity in vitro as the concentration gradient was increased, the least concentration with stability of the erythrocyte in vitro as compared with the phosphate buffer is termed an equivalent for the determination of toxicity in erythrocyte in vitro-in vivo by other phyto products. Conclusion: The data obtained from this study has value in determining phytotoxicity with pumpkin leaves and any other phyto-products on human erythrocytes with all its determining variables in consideration.
INTRODUCTION
Biomaterials are either derived from nature or synthesized by using polymers, ceramics, metals and composite materials. Specifically, biomaterials have been extensively applied to controlled release systems since 1976 that continually released macro-molecules to inhibit angiogenesis1. There have been many studies on drug delivery1, but in vitro toxicity assessments a carrier must be developed. Most reports have assessed cytotoxicity by using only target cells or non-specific cells from animals. This approach cannot represent the overall toxicity for humans because of the differences between many of the cells used in these studies and human cells2.
Charles Telfair (1778-1835), an Irish botanist who lived in Mauritius, sent an African genus of the cucumber family (Cucurbitaceae) from Mauritius to Sir Williams Jackson Hooker (1785-1865) for identification. In honor of Dr. Telfair, the plant Telfairia occidentalis was named after him2. However, the earliest reference to Telfairia was made by Oliver in 1871 and it recorded its presence in Upper Guinea areas of Sierra Leone, Fernando Po and Abeokuta (Nigeria)3-5.
Telferia occidentalis which belongs to the family Cucurbitaceae is one of the leafy vegetables widely consumed in Nigeria for its nutritional and therapeutic benefits. It is widely cultivated in the south-eastern, south-western and south-southern parts of Nigeria and utilized in the preparation of soups and medicines. Dietary intake of Telferia occidentalis could reduce garlic induced hemolysis in rats3,4. The aqueous extracts of Telferia occidentalis has been found to reduce blood glucose levels and also have anti-diabetic effects in glucose induced hyperglycemic and streptozotocin induced diabetic mice5,6. Also, aqueous extracts of Telferia occidentalis has been reported to assist in the purging of the gastro-intestinal tract as revealed by the purgative effects of the aqueous extracts of Telferia occidentalis leaf on isolated Guinea pig ileum7-10.
The leaves are rich in vitamins and minerals such as; Ca, P and Fe etc. The seed is also eaten as food. The oil obtained from the seed is used in cooking4,5,10. Telfairia occidentalis has been reported to possess antioxidant property. The aqueous extract had a high total phenol, reducing power and free radical scavenging ability (12.2%, 1.9 OD700 and 92%, respectively) than the ethanolic extract which had total phenol, reducing power and free radical scavenging ability of 5.5%, 1.5 OD700 and 25%, respectively9,10. The free soluble polyphenols content in the leaf of the plant which was higher than the bound polyphenols had higher antioxidant activity as typified by their higher reducing power and free radical scavenging ability than the bound polyphenols6,7. Telfairia occidentalis leaf contained a significantly high amount of vitamin C, total flavonoids and phenolics than Psidium guajava stem bark. The leaf inhibited more free radicals than Psidium guajava stem bark10,11. The n-hexane fraction had the highest flavonoid content and free radical scavenging activity comparable to that of the commercial antioxidant12,13. The ability of the leaf of Telfairia occidentalis to reduce iron (III) to iron (II) was also reported13. The antioxidant property of Telfairia occidentalis is attributable to the high content of polyphenols, especially flavonoids. In the search for methods to evaluate the toxicological risk of chemicals without employing animal experimentation, much emphasis has been put on the replacement of acute toxicity (LD50) determinations. One important assumption was that acute toxicity is related to a compound’s basal cytotoxicity13. The erythrocytes of humans and mammals represent a good model to evaluate the cytotoxicity of molecules, organic and inorganic, natural or synthetic by cellular damage measure14-16. Indeed, before any investigation on the mechanism of action of different molecules, it is important to perform a cytotoxicity assay7. Among the different cytotoxicity assays that assess a possible toxicity in the red blood cells is the rate of hemolysis7. This essay is based on the evaluation of the alterations of red cell membranes in the presence of an eventual xenobiotic17,18. Red blood cells are the main cells in circulation and they are responsible for transporting oxygen; in fact, any alterations of this process could be lethal. The present study was aimed at evaluating the in vitro effect of pumpkin leaves extract on human erythrocytes with an interest in obtaining base data on the phyto-toxicity.
MATERIALS AND METHODS
Study area: This study was carried out between the month of March and September, 2018 in the University of Benin, Health Services Department, Laboratory Unit, University of Benin and the Laboratory in the Department of Medical Laboratory Sciences, University of Benin, Nigeria.
Inclusion criteria: Red blood cell with the ABO blood group types from healthy patients to determine the in vitro phyto-toxicity of pumpkin leaves.
Exclusion criteria: Blood samples from patients who are on any form of medication which could interfere with the toxicity studies.
Ethical approval: Permission shall be sought and obtained from the Ministry of Health, Benin City, Edo state, Nigeria.
Sample collection
Blood sample: Arterial/Venous blood is collected aseptically from the cubital fossa and dispensed into a EDTA coagulated specimen bottles (5 mL) was deposited.
Methodology
Phyto-material extraction: The leaves of the plant were collected, dried in the shade and powdered using mortar and pestle. The powdered processed leaves were stored in airtight containers and labeled properly. Each of the dried grounded material weighed 500 g. Extraction of the phyto-product was carried out by using 2 L of methanol and normal saline by cold maceration for 7 days in large amber bottles with intermittent shaking. Filtration by using the Whatman filter paper (No. 42) was used to separate the artifacts and macro-substance.
Collection of blood and preparation for analysis: Using a sterile 5 mL syringe, 5 mL of blood was collected by veni-puncture from the cubital fossa of healthy patients without gender discrimination. The blood was dispensed into sodium EDTA specimen bottles (green cap), it was mixed gently and thoroughly rolling the bottle. Centrifugation was carried out to separate plasma from the packed erythrocytes. The separated packed erythrocytes were washed 3 times with phosphate buffered normal saline and the supernatant decanted. The time of centrifugation was 5 min at a speed of 626 (xg)8,9. The washed packed cells were used for the toxicity test by in vitro red cell hemolysis.
Haemolysis study: Hemolytic toxicity of pumpkin equivalent was checked by incubating the formulations with red blood cells separated from human blood by centrifugation at low speed and analyzing the samples for hemoglobin release at 541 nm. Hemolysis with different formulations were compared with that obtained with Triton-X100 as a positive15.
Cell viability test: Hemolysis potentials of the pumpkin leave extract equivalent were added to the RBC concentrate and gently mixed. The concentrate was then incubated at 37°C for 30 min in incubator.
After incubation, it is again at 3000 rpm for 5 min to separate the pellet. The supernatant was analyzed for absorbance at 540 nm in UV spectrophotometer against normal saline as blank.
Percentage of hemolysis was determined for different samples considering the absorbance value of sample treated with 0.5% Triton-X100 to represent 100% hemolysis and normal saline treated samples to serve as negative control. Relative hemolysis (%) was determined by following expression18:
About Table 1, a varied concentration the lethal dosage of pumpkin leaves extract was determined with doubling dilutions of 101-109 by using the heamolytic assay to determine cytotoxicity on human red blood cells. The absorbance values at 540 nm as measure of its toxicity.
At 125, 250, 500 and 1000 mg doses, respectively, the concentration gradient from 101-109, the effect of pumpkin leave extract determine its equivalent (X). A steady increase in absorbance values as the concentration of the pumpkin leave extract continued to increase exponential.
About Table 2, a varied concentration the lethal dosage of pumpkin leaves extract was determined with doubling dilutions of 101-109 by using the heamolytic assay to determine cytotoxicity on human red blood cells. The percentage haemolysis was determined by the formula:
Comparatively the percentage haemolysis at 125, 250, 500 and 1000 mg doses, respectively, is determined by comparing the positive and negative controls (Triton-X and PBS) against the concentration gradient from 101-109, of pumpkin leave extract and human red blood cells. Its equivalent (X) showed a steady increase in hemolysis values as the concentration of the pumpkin leave extract continued to increase exponentially.
Statistical analysis: Statistical analysis including descriptive statistics carried out by using the Statistical Package (Graph Pad Prism). All values will be expressed as Mean±SE (Mean standard error of mean). The Analysis of Variance (ANOVA) used to determine significant difference in test and control groups (p<0.05) at confidence limit will be set at 95%.
| Table 1: | Determination of lethal toxicity dosage of pumpkin extract on human red blood cells in vitro using a doubling dilution concentration vs. the absorbance value (Mean±SEM) | |||||||||
| Dose(μg) | 101 |
102 |
103 |
104 |
105 |
106 |
107 |
108 |
109 |
p-value |
| 125 | 0.3306±0.001 |
0.3304±0.002 |
0.3375±0.002 |
0.3394±0.002 |
0.3716±0.004 |
0.4034±0.002 |
0.4078±0.002 |
0.4955±0.002 |
0.5567±0.009 |
0.0001 |
| 250 | 0.3067±0.003 |
0.3124±0.002 |
0.3206±0.004 |
0.3394±0.003 |
0.3598±0.015 |
0.3953±0.005 |
0.4037±0.002 |
0.4184±0.002 |
0.4470±0.008 |
0.0001 |
| 500 | 0.4955±0.002 |
0.4975±0.001 |
0.5001±0.000 |
0.5019±0.001 |
0.6800±0.006 |
0.7600±0.005 |
0.8710±0.008 |
1.033±0.020 |
1.0780±0.0233 |
0.0001 |
| 1000 | 0.8599±0.005 |
0.8757±0.006 |
0.9731±0.001 |
1.033±0.020 |
1.048±0.021 |
1.078±0.023 |
1.133±0.003 |
1.773±0.064 |
2.0190±0.038 |
0.0001 |
| Table 2: Comparing the mean absorbance and percentage hemolysis of Telfaira occidentalis leaves extract to determine its equivalent (X) | ||
| Dose | Absorbance (540 nm) |
Hemolysis (%) |
| 125 μg | 0.3969 |
3.40 |
| 250 μg | 0.3670 |
2.30 |
| 500 μg | 0.7130 |
0.15 |
| 1000 μg | 1.1991 |
32.90 |
| PBS | 0.3023 |
0.00 |
| Triton-X | 3.0200 |
100.00 |
RESULTS
The phytotoxic effect of pumpkin leaves equivalent was determined by carrying out a hemolytic assay on human red blood cells. About 5 mL of the various ABO blood group was collected from 292 healthy human subjects and dispensed into the EDTA bottle this selection was void of gender bias. Telfaira occidentalis were extracted at varied concentration, 125, 250, 500 and 1000 μg by using water as the solvent of extraction.
The pumpkin equivalent was determined comparing the least and the highest dose concentration gradient (125, 250, 500 and 1000 μg), there is a significant difference due to dose and concentration gradient, with 250 μg showed the closest when compared with phosphate buffered saline (2.3% hemolysis) (Table 1 and 2).
From the observations of this study, Telfaira occidentalis extract showed a dose dependent increase in heamolytic activity, there was low to mild heamolytic effect towards human erythrocyte, the dose of 125 μg showed an absorbance value of 0.3969 (3.4%) on the erythrocyte which was close to the absorbance value of phosphate buffered saline erythrocyte, 0.3023 (0%).
The pumpkin equivalent was determined to comparing the equivalent with phosphate buffered saline (0%) hemolysis and Trition-X (100%) hemolysis. The pumpkin equivalent closet to the phosphate buffered saline was at 250 μg with no significant difference across the ABO blood groups (p>0.005).
DISCUSSION
The in vitro effect of pumpkin leaves extract on human erythrocyte was determined comparing its hemolytic effects on human erythrocyte as a measure of its toxicity this led to the discovery of an equivalent lethal toxic concentration12,13.
Telfaira occidentalis leaves extract showed significant less toxic effect in vitro at room temperature when compared with Ocimium gratissimum and Vernonia amygdalina (p<0.005) there was similarity when compared with works carried out on the stabilizing effect of pumkin leave extract on sickle cell erythrocyte17.
All of the concentration of Telfaira occidentalis leave extract showed minimal heamolytic effect on human erythrocyte, 3.4, 2.3, 15 and 32.9%, respectively as compared with Triton-X and PBS solution, this coincides with previous studies which showed the stabilizing effect of Telfaira Occidentalis leave extract on human erythrocyte in vitro10-12.
This study showed an elevation in the hemolytic effect on the human erythrocyte as the concentration increased which showed that at a higher concentration Telfaira occidentalis showed some level of toxicity on human erythrocyte conferring with the dose dependent toxicity this is synonymous with studies carried out by Foller et al.13 who discovered a lethal concentration of Telfaira occidentalis at high concentration.
CONCLUSION
From the results obtained in this study Telfiara occidentalis has a potential as a tool for determining phyto-toxicity, this is due to its stabilizing effect on the erythrocyte at a lower concentration. The pumpkin leave equivalent will provide an alternative from by using laboratory animals for toxicity studies, but to replace them with erythrocyte in vitro as a measure in vivo for determine toxic concentration across a concentration gradient in any phyto-product. However, there is still need for further studies to evaluate its sensitivity, specificity and accuracy.
SIGNIFICANCE STATEMENT
This study has discovered a significant similarity with phytotoxicity studies done in vitro comparing with in vivo by using human erythrocyte as alternative instead of laboratory animals and bring to the fore front the use of cells as an alternative for laboratory animals in both one phase and multi-phase toxicity studies considering sequestration points and active sites.
ACKNOWLEDGMENT
Many thanks to the National Academy for The Advancement of Science for grants given by providing a working environment.