Phytochemical and biological investigation of Lathyrus linofilius as a possible treatment in diabesity

Previously held under moratorium from 10 February 2020 until 10 February 2022Diabetes is a world-wide issue which can affect people at any age. It is ranked as one of the top 10 diseases responsible for death worldwide. According to the International Diabetes Federation, 424.9 million people worldwide were estimated to have diabetes in 2017 (90% with type 2 diabetes) and this number is projected to reach 629 million patients by 2045. The current treatments for managing diabetes are linked with a number of side effects including severe hypoglycaemia, permanent neurological deficit, stomach-ache, headache, lactic acidosis, liver damage, dizziness and death in some cases. Therefore, there is a need for improved anti-diabetic drugs with fewer side effects to enhance patient compliance and to control blood glucose levels more tightly. Natural products and plants in particular, offer an alternative to synthetic drugs. Lathyrus linifolius is a plant whose tubers have historically been used as an appetite suppressant during medieval times in times when food was scarce. Due to the close link between appetite suppressants and the treatment of diabetes/obesity, the plant’s leaves and tubers were examined for their potential anti-diabetic and antiobesity activity. This project aimed to: 1) produce Lathyrus linifolius extracts using Soxhlet apparatus extraction from which bioactive compounds could be isolated by column chromatography and characterised by nuclear magnetic resonance (NMR); 2) assess the anti-diabetic and anti-obesity activity of the extracts and compounds; 3) investigate the anti-inflammatory and anti-oxidant activity of the extracts and compounds in in vitro assays; 4) identify the effects of the tuber treatment on the gene expression of appropriate diabesity components in normal rat pancreatic tissues obtained from a previous study in which rats were fed with tubers; and 5) re-assess Lathyrus linifolius effects on obese Zucker rats and monitor the effects on body weight, food intake, water intake and blood glucose levels. 1) Four compounds were isolated for the first time from the ethyl acetate extract of the tubers; these were betulinic acid, lupeol, stigmasterol and β-sitosterol. The isolated compounds are known for their beneficial effects on hyperglycaemia and obesity to some extent. Betulinic acid was the major component in the tubers and leaves. 2) In biological assays, the ethyl acetate extract and betulinic acid from the tubers were potent α-glucosidase inhibitors (P<0.05) with IC50 of 9.5 µg/ml and 5.5 µM, respectively. Moreover, the leaf ethyl acetate and methanol extracts were also strong inhibitors of α-glucosidase (P<0.05) with IC50 of 0.58 µg/ml and 4.3 µg/ml, respectively. In an anti-obesity assay, based on pancreatic lipase, the ethyl acetate tuber extract at 30 µg/ml and betulinic acid at 30 µM showed 50% enzyme inhibition (P<0.05); and the hexane extract from the leaves showed 30% inhibition. In HepG2 cells, leaf hexane extract showed a significant increase in glucose uptake which was comparable to that of insulin. These findings showed promising results for the plant to be used as an anti-diabetic and anti-obesity agent. Inflammation and oxidative stress are implicated in diabesity and determined the subsequent investigation. 3) Moreover, all the plant extracts and isolated compounds at 30 µg/ml or 30 µM showed a greater than 70% protection (P<0.05) in L929 cells (mouse fibroblasts commonly used for anti-inflammatory studies) from the cytotoxic effects of tumour necrosis factor alpha (10 µM). This was followed by investigating the ability of Lathyrus linifolius to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ƙB) in the NCTC cell line (human skin cells transfected with a NF-ƙB luciferase reporter vector, used in anti-inflammatory studies). Betulinic acid, ethyl acetate extract of the tubers, hexane and ethyl acetate extracts of the leaves inhibited NF-ƙB which gave a NF-ƙB % inhibition of 70, 40, 30 and 30, respectively. A betulinic acid inhibition curve was performed and found to inhibit NF-ƙB expression with an IC50 of 22.8 µM. Oxidative stress was then investigated following the positive findings in the anti-inflammatory assays. Lathyrus linifolius extracts and isolated compounds were not reactive oxygen species-generators and significantly (P<0.05) protected cells from reactive oxygen species generated by tert-butyl hydroperoxide in SH-SY5Y (neuronal), HepG2 (hepatic) and Panc1 (pancreatic) cells, except lupeol in HepG2 cells. In HepG2 cells, glutathione levels were significantly (P<0.05) increased by ethyl acetate extract of the tubers which confirmed the potential anti-oxidant activity of Lathyrus linifolius. 4) It was considered pertinent to carry out a retrospective investigation into any gene changes using RNA-Sequencing and real-time quantitative polymerase chain reaction (RT-qPCR) on RNA isolated from rat pancreatic tissues in a feeding study with Lathyrus linifolius tubers carried out by Woods (2017). It was found that the adiponectin gene (Adipoq) was highly up-regulated (P<0.05) whereas genes that contribute to pancreatic β-cells damage for example Il1β and Tnfrsf19 were downregulated (P<0.05). 5) Then, the biological effects of Lathyrus linifolius on an animal model for diabesity were followed up. Dried, powdered Lathyrus linifolius tubers (100-200 mg/kg BW) were dissolved in deionised water, mixed with Farley’s rusks and given to obese Zucker rats daily for 16 days. However, the results showed that there were no effects on the body weight, food intake, water intake or blood glucose of Zucker rats and this may have occurred due to the low Lathyrus linifolius dosage used and the short duration of the study. It was decided not to carry out any further investigation (such as RNA- Sequencing) after this as there was no significant difference in results between the obese Zucker rats and normal rats as observed in the previous feed study. These findings suggest that compounds from Lathyrus linifolius could possibly be used as anti-diabetic and anti-obesity agents in the future, based on the positive results obtained from the in vitro and ex vivo assays in this study. This study should be followed by assessing the effects of Lathyrus linifolius on body weight, food intake, water intake and blood glucose in humans.Diabetes is a world-wide issue which can affect people at any age. It is ranked as one of the top 10 diseases responsible for death worldwide. According to the International Diabetes Federation, 424.9 million people worldwide were estimated to have diabetes in 2017 (90% with type 2 diabetes) and this number is projected to reach 629 million patients by 2045. The current treatments for managing diabetes are linked with a number of side effects including severe hypoglycaemia, permanent neurological deficit, stomach-ache, headache, lactic acidosis, liver damage, dizziness and death in some cases. Therefore, there is a need for improved anti-diabetic drugs with fewer side effects to enhance patient compliance and to control blood glucose levels more tightly. Natural products and plants in particular, offer an alternative to synthetic drugs. Lathyrus linifolius is a plant whose tubers have historically been used as an appetite suppressant during medieval times in times when food was scarce. Due to the close link between appetite suppressants and the treatment of diabetes/obesity, the plant’s leaves and tubers were examined for their potential anti-diabetic and antiobesity activity. This project aimed to: 1) produce Lathyrus linifolius extracts using Soxhlet apparatus extraction from which bioactive compounds could be isolated by column chromatography and characterised by nuclear magnetic resonance (NMR); 2) assess the anti-diabetic and anti-obesity activity of the extracts and compounds; 3) investigate the anti-inflammatory and anti-oxidant activity of the extracts and compounds in in vitro assays; 4) identify the effects of the tuber treatment on the gene expression of appropriate diabesity components in normal rat pancreatic tissues obtained from a previous study in which rats were fed with tubers; and 5) re-assess Lathyrus linifolius effects on obese Zucker rats and monitor the effects on body weight, food intake, water intake and blood glucose levels. 1) Four compounds were isolated for the first time from the ethyl acetate extract of the tubers; these were betulinic acid, lupeol, stigmasterol and β-sitosterol. The isolated compounds are known for their beneficial effects on hyperglycaemia and obesity to some extent. Betulinic acid was the major component in the tubers and leaves. 2) In biological assays, the ethyl acetate extract and betulinic acid from the tubers were potent α-glucosidase inhibitors (P<0.05) with IC50 of 9.5 µg/ml and 5.5 µM, respectively. Moreover, the leaf ethyl acetate and methanol extracts were also strong inhibitors of α-glucosidase (P<0.05) with IC50 of 0.58 µg/ml and 4.3 µg/ml, respectively. In an anti-obesity assay, based on pancreatic lipase, the ethyl acetate tuber extract at 30 µg/ml and betulinic acid at 30 µM showed 50% enzyme inhibition (P<0.05); and the hexane extract from the leaves showed 30% inhibition. In HepG2 cells, leaf hexane extract showed a significant increase in glucose uptake which was comparable to that of insulin. These findings showed promising results for the plant to be used as an anti-diabetic and anti-obesity agent. Inflammation and oxidative stress are implicated in diabesity and determined the subsequent investigation. 3) Moreover, all the plant extracts and isolated compounds at 30 µg/ml or 30 µM showed a greater than 70% protection (P<0.05) in L929 cells (mouse fibroblasts commonly used for anti-inflammatory studies) from the cytotoxic effects of tumour necrosis factor alpha (10 µM). This was followed by investigating the ability of Lathyrus linifolius to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ƙB) in the NCTC cell line (human skin cells transfected with a NF-ƙB luciferase reporter vector, used in anti-inflammatory studies). Betulinic acid, ethyl acetate extract of the tubers, hexane and ethyl acetate extracts of the leaves inhibited NF-ƙB which gave a NF-ƙB % inhibition of 70, 40, 30 and 30, respectively. A betulinic acid inhibition curve was performed and found to inhibit NF-ƙB expression with an IC50 of 22.8 µM. Oxidative stress was then investigated following the positive findings in the anti-inflammatory assays. Lathyrus linifolius extracts and isolated compounds were not reactive oxygen species-generators and significantly (P<0.05) protected cells from reactive oxygen species generated by tert-butyl hydroperoxide in SH-SY5Y (neuronal), HepG2 (hepatic) and Panc1 (pancreatic) cells, except lupeol in HepG2 cells. In HepG2 cells, glutathione levels were significantly (P<0.05) increased by ethyl acetate extract of the tubers which confirmed the potential anti-oxidant activity of Lathyrus linifolius. 4) It was considered pertinent to carry out a retrospective investigation into any gene changes using RNA-Sequencing and real-time quantitative polymerase chain reaction (RT-qPCR) on RNA isolated from rat pancreatic tissues in a feeding study with Lathyrus linifolius tubers carried out by Woods (2017). It was found that the adiponectin gene (Adipoq) was highly up-regulated (P<0.05) whereas genes that contribute to pancreatic β-cells damage for example Il1β and Tnfrsf19 were downregulated (P