Use of actinidia deliciosa and olea europea plants bioengineered to over-express the osmotin plant protein and their derivates effectively lowers plasma glucose levels in type 2 diabetes mellitus patients and activates glycosuria

Abstract

The present invention is related to powder, extracts, capsules, other formulations and foods derived or developed from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant gene as having anti-diabetes effects and more particularly, glycosuria activating effects in Type 2 Diabetes Mellitus (T2DM) patients with no negative side effects. The present invention can be used to ameliorate glucose metabolism, lower blood sugar in adults with T2DM by causing the kidneys to remove sugar from the body through the urine, and treat T2DM. Thus, this invention represents a novel T2DM therapy and has the potential to be an efficacious treatment for managing T2DM associated hyperglycaemia.

Description

[0001] Use of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant protein and their derivates effectively lowers plasma glucose levels in Type 2 Diabetes Mellitus patients and activates glycosuria.

[0002] Inventor: Dr. Bruno Ruggiero, Ph.D.

[0003] ABSTRACT

[0004] The present invention is related to powder, extracts, capsules, other formulations and foods derived or developed from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant gene as having anti-diabetes effects and more particularly, glycosuria activating effects in Type 2 Diabetes Mellitus (T2DM) patients with no negative side effects. The present invention can be used to ameliorate glucose metabolism, lower blood sugar in adults with T2DM by causing the kidneys to remove sugar from the body through the urine, and treat T2DM. Thus, this invention represents a novel T2DM therapy and has the potential to be an efficacious treatment for managing T2DM associated hyperglycaemia.

[0005] DESCRIPTION

[0006] Use of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant protein and their derivates effectively lowers plasma glucose levels in Type 2 Diabetes Mellitus patients and activates glycosuria.

[0007] FIELD OF INVENTION The present invention is related to powder, extracts, capsules, other formulations and foods derived or developed from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant gene with antidiabetes effects and more particularly, glycosuria activating effects in T2DM patients with no negative side effects.

[0008] RELATED ART

[0009] T2DM is a degenerative disease that occurs either when the pancreas does not produce enough insulin or when the body cannot effectively use the insulin it produces. Glucose levels in the blood are controlled by the insulin hormone. According to Y Wu et al. T2DM patients represent 90% of all diabetes cases [1], The World Health Organisation (WHO) reports that in 2014 diabetes afflicted 8.5% of all adults aged 18 years and older while in 2019, 1.5 million deaths were directly caused by diabetes and nearly half of all diabetes deaths occurred in patients younger than 70 years of age. The WHO also estimates that by 2030 the number of diabetes patients is expected to reach 552 million. Diabetes has caused an additional 460 000 kidney disease related deaths, and approximately 20% of cardiovascular deaths are caused by increased blood glucose [2], Hyperglycaemia is a common effect of uncontrolled T2DM, and it manifests as a increase in blood glucose levels disrupting many of the body’s organ systems, especially the nerves and blood vessels.

[0010] The kidneys play an important role in glucose homeostasis via glucose utilisation and reabsoprtion which have been reported to take place via the sodium glucose cotransporters (SGLTs) and glucose transporters. [3] Glycosuria is the process whereby excess glucose is excreted by the kidneys through the urine. In normal conditions, only minimal traces of glucose can be found in the urine. In patients with T2DM, the renal threshold for glucose excretion (RTG) is increased, which is thought to take place due to the upregulation of SGLT2 and SGLT1 expression. Such up regulation could be one of the causes contributing to hyperglycaemia in patients with T2DM. [3] Glycosuria activation in T2DM patients is, therefore, a valid strategy to control glycemic levels in T2DM patients.

[0011] Glycosuria is activated via increasing plasma adiponectin levels through stimulating PPARd in adipose tissue. Enhanced adiponectin downregulates renal SGLT2, which in turn reduces reabsorption of sodium and glucose. This mechanism is dampened by hyperglycaemia in T2DM [4],

[0012] SGLT2 inhibitor class of drugs represent an important step forward in the treatment of T2DM. Drugs of this class have shown an ability to ameliorate glycemic control with weight loss by inducing glycosuria with calorie loss. However, chemical inhibitors of SGLT2 carry considerable side-effects and the currently available inhibitors of this class may increase the risk of genital and urinary tract infections and dehydration [5].

[0013] Adiponectin is an important insulin-sensitizing hormone and adipokine protein that affects several metabolic processes and is involved in the regulation of glucose levels. Adiponectin has direct anti-diabetic, anti -atherogenic and anti-inflammatory activities, enhancing glucose utilisation. Higher Adiponectin levels are consistently associated with a lower risk of T2DM across diverse populations, consistent with a doseresponse relationship [6], It is known, in fact, that osmotin belongs to the PR (Pathogen Related) protein family, of the 5th type [7] and, in nature, it is only produced by plants in response to pathogen attacks or abiotic stresses of various kinds. Plants over-expressing osmotin, in addition to representing an opportunity for sustainable agriculture, through the manifestation of greater resistance in terms of biotic and abiotic stresses, could also have important implications for the protection of human health [8], In terms of human health, of particular importance is the role that osmotin plays in the veterinary and human held of applications, inasmuch as this plant protein is capable of mimicking the activity of the human hormone Adiponectin [9], For these reasons, today there is a growing interest in the research on osmotin, seen as a protein with a therapeutic potential targeted to humans. Recent publications have demonstrated that osmotin is capable of recovering damages caused by senile dementia, Alzheimer’s,

[0010] and T2DM [9], thanks to its capacity to activate the Adiporl / Sirtl receptors

[0010] , activating the cellular enzymatic cascade improving the functionality of mitochondria and, thus, potentiating the cell’s vital activity, including the control of neoplastic growth.

[0014] Kiwi plants (Actinidia deliciosa) and olive plants {Olea europea) bioengineered to over-express the osmotin gene were developed and tested as described by Ruggini et al. these plants, express the recombinant gene for osmotin from tobacco Wisonsin 38 with a 35S viral promoter. These genotypes are, de facto, perfect bioreactors capable of supplying osmotin, of great therapeutic worth, in good quantities and in a context of being ready for human and animal consumption, making the commercialisation of this substance economically advantageous for the pharmaceutical industry and plant suppliers alike. [8] Furthermore, plant cells are now approved by the FDA for cost-effective production of protein drugs (PDs). PDs are stable and maintain their folding and efficacy at ambient temperature for several years when bioencapsulated in lyophilised plant cells. In oral delivery, PDs bioencapsulated in plant cells are protected in the stomach from acids and enzymes but are subsequently released into the gut lumen by microbes that digest the plant cell wall. The large mucosal area of the human intestine offers an ideal system for oral drug delivery

[0011] ,

[0015] Oral PDs administration via bioencapsulation in plants is non invasive and effective. Moreover, plants do not host human pathogens. Human gastric enzymes are incapable of digesting plant cell walls; therefore, when bioencapsulated in plant cells PDs are protected until they reach the intestine where the cell walls are digested by the gut microbiome and the PDs are released into the gut lumen

[0011] , In addition, when bioencapsulated in lyophilised plant cells, PDs can be stored at room temperature indefinitely without compromising their efficacy, rendering this method ideal under all aspects for mass production, distribution, and administration worldwide.

[0016] SUMMARY

[0017] It is an object of the present invention to provide methods for the treatment of T2DM via the activation of glycosuria by oral administration to patients of powder, extracts, capsules, other formulations and foods derived or developed from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to overexpress the osmotin plant gene.

[0018] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention is explained in detail by the following examples. However, the examples are provided for illustration of the present invention not for limitation thereof.

[0019] Example 1

[0020] In one embodiment kiwi plants (Aciinidia deliciosa) and olive plants {Olea europea) were bioengineered to over-express the osmotin gene and tested as described by Ruggini et al. [8] The leaves of the plants were collected, washed, dried at 40C° for 24 hours and incapsulated in size 00 gelatine capsules of 0.5 gram each; 5 capsules per day were administered orally to T2DM patients for a period of 30 days. Patients were asked to perform blood and urine tests before and after treatment. The 30 day treatment with capsules containing 0.5 gram each of lyophilised bioengineered leaves over-expressing the osmotin gene activated glycosuria from 0 mg / dl to 250 mg / dl in the urine tests (Table 1), while the blood tests showed that glucose plasma levels were lowered from 126 mg / dl to 123 mg / dl and HDL plasma levels were increased (Table 2).

[0021] Example 2

[0022] In a second embodiment kiwi plants {Aciinidia deliciosa) olive plants {Olea europea) bioengineered to over-express the osmotin gene were developed and tested as described by Ruggini et al. [8]. The juice from the plants leaves is collected and diluted in water at a concentration of 10% water volume. The solution is administered orally to T2DM patients for a period of 30 days at a dose of 200 ml per day.

[0023] Example 3 In a third embodiment the osmotin protein is extracted from kiwi plants (Aciinidia deliciosa) and olive plants (Olea europea) bioengineered to over-express the osmotin gene and is administered orally at a dose of 10 microgram per kilogram of body weight daily for a period of 30 days

[0024] Table 1 I

[0025] Table 2

[0026] DETAILED DESCRIPTION

[0027] It is an object of the present invention to provide methods for the treatment of T2DM via the activation of glycosuria by oral administration to patients of powder, extracts, capsules, other formulations and foods derived or developed from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to overexpress the osmotin plant gene.

[0028] The kiwi and olive leaves from plants bioengineered to over-express the osmotin gene were administered as dried powder incapsulated in 0.5 gram gelatine capsules at a dose of 5 capsules per day for 30 days. Prior to starting treatment, the patient performed urine tests, as shown in Table 1 , and a blood test as shown in Table 2. The urine glucose level at the beginning of the test was 0 (not detectable). After 30 days treatment the glucose level in the urine was 250 mg / dl. These results show, for the first time, that plants over-expressing the osmotin gene can induce glycosuria in T2DM patients, while maintaining the rest of the values in the normal range. Therefore, these plants and their derivates represent a valid treatment to manage T2DM related hyperglycaemia. It is important to note that in absence of hyperglycaemia the administration of the plant powder does not cause glycosuria. This implies a selective inhibition of glucose retention by the osmotin-rich plant powder only in T2DM patients. Of further note, compared to generally healthy individuals glycosuria is dampened in T2DM patients; therefore, its reactivation only in T2DM patients shows that the osmotin-rich plant powder works by blocking the diabetes- related inhibition of glycosuria rather than by selectively blocking SGLT2. Table 2 also shows that administration of the the osmotin-rich powder lowered plasma glucose levels from 126 mg / dl pre -treatment to 123 mg / dl post treatment. Although these changes do not appear substantial, it is important to note that a glucose plasma level of 126 is the threshold at which a diagnosis of T2DM can be made. A lower level indicates a pre-diabetic condition. In addition this data is coherent with the theory that glycosuria lowers plasma glucose levels, and here we show that the invention is capable of lowering such plasma levels, making it a perfect candidate for the treatment of T2DM induced hyperglycaemia. In addition, these plants are considered GRAS (Generally Recognised As Safe.) In fact, osmotin has show no toxicity and as a thaumatin- group protein, osmotin is already approved as a sweetener for the marked in the USA and the European Union. This is the first time that the ability of osmotin and plants over-expressing osmotin to induce glycosuria is described and represents a total novelty. It had been shown that osmotin activates the AdipoRl and Adipor2 receptors and ameliorates glucose consumption by the cell, but osmotin’s ability to induce glycosuria has never been described prior to this invention. This new function of osmotin and plants over-expressing osmotin to induce glycosuria in T2DM patients thus renders the bioengineered osmotin-rich plants and their derivates a valid method to treat T2DM related hyperglycaemia. The same results can be obtain by the administration of the juice from plants bioengineered to over-express the osmotin gene in a different embodiment.

[0029] Additionally, in a different embodiment, the same results can be obtained by administering the pure osmotin protein extracted from transgenic kiwi and olive plants over expressing the osmotin gene.

[0030] REFERENCES Wu Y, Ding Y, Tanaka Y, Zhang W. Risk factors contributing to type 2 diabetes and recent advances in the treatment and prevention. Int J Med Sci. 2014 Sep 6; 11(11): 1185-200. doi: 10.7150 / ijms.10001. PMID: 25249787; PMCID: PMC4166864. World Health Organisation. Global Report on Diabetes. 2022 Sep 16. John PH. Wilding, The role of the kidneys in glucose homeostasis in type 2 diabetes: Clinical implications and therapeutic significance through sodium glucose cotransporter 2 inhibitors, Metabolism, Volume 63, Issue 10, 2014, Pages 1228-1237, ISSN 0026-0495, https: / / doi.org / 10.1016Zj.metabol.2014.06.018. Zhao Y, Gao P, Sun F, Li Q, Chen J, Yu H, Li L, Wei X, He H, Lu Z, Wei X, Wang B, Cui Y, Xiong S, Shang Q, Xu A, Huang Y, Liu D, Zhu Z. Sodium Intake Regulates Glucose Homeostasis through the PPARb / Adiponcclin-Mcdialcd SGLT2 Pathway. Cell Metab. 2016 Apr 12;23(4):699-711. doi: 10.1016 / j.cmet.2016.02.019. Epub 2016 Mar 24. PMID: 27053360. Liman MNP, Jialal I. Physiology, Glycosuria. [Updated 2022 Mar 18], In: StatPearls

[0031] [Internet], Treasure Island (FL): StatPearls Publishing; 2022 Jan-. Available from: https : / / w w w. ncbi . nlm . nih. gov / books / NB K557441 / Li S, Shin HJ, Ding EL, van Dam RM. Adiponectin levels and risk of type 2 diabetes: a systematic review and meta-analysis. JAMA. 2009 Jul 8;302(2): 179-88. doi:

[0032] 10.1001 / jama.2009.976. PMID: 19584347. Anil Kumar S., Hima Kumari P, Shravan Kumar G., Mohanalatha C., Kavi Kishor P.

[0033] B. Osmotin: a plant sentinel and a possible agonist of mammalian adiponectin; Frontiers in Plant Science ; Volume 6; 2015 DOI 10.3389 / fpls.2015.00163 ; ISSN=1664-462X Eddo Rugini, Muhammad Ajmal Bashir, Stefania Astolfi, Valerio Cristofori, Bruno Ruggiero, Cristian Silvestri. Transformation of kiwi (Actinidia deliciosa A. Chev.) and olive {Olea europea L.) with tobacco osmotin gene confers drought-stress tolerance and provides a health-promoting protein source. Proceedings of The 2nd International Conference of Biotechnology and Environment (ICBE 2018) 28-30 March 2018, Alexandria - Egypt. Kadowaki T, Yamauchi T. Adiponectin and adiponectin receptors. Endocr Rev. 2005 May;26(3):439-51. doi: 10.1210 / er.2005-0005. PMID: 15897298. . Shah SA, Yoon GH, Chung SS, Abid MN, Kim TH, Lee HY, Kim MO. Novel osmotin inhibits SREBP2 via the AdipoRl / AMPK / SIRTl pathway to improve Alzheimer's disease neuropathological deficits. Mol Psychiatry. 2017 Mar;22(3): 407-416. doi: 10.1038 / mp.2016.23. Epub 2016 Mar 22. PMID: 27001618; PMCID: PMC5322276. . Kwang-Chui Kwon, Henry Daniell. Oral Delivery of Protein Drugs Bioencapsulated in Plant Cells. Mol Then 2016 Aug; 24(8): 1342-1350; doi: 10.1038 / mt2016.115 PMID: 27378236

Claims

CLAIMS1. Oral administration of powder, extracts, capsules, other formulations and foods derived or developed from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant gene is a novel and valid method to induce glycosuria in T2DM patients.

2. Oral administration of powder, extracts, capsules, other formulations and foods derived or developed from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant gene lowers plasma glucose levels in T2DM patients.

3. Powder, extracts, capsules, other formulations and foods derived or developed from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant gene can be used to ameliorate glucose metabolism, lower blood sugar in adults with T2DM by causing the kidneys to remove sugar from the body through the urine, and represents a novel treatment to ameliorate T2DM-related hyperglycaemia.

4. Osmotin protein extracted and purified from the leaves, fruits, and biomass of Actinidia deliciosa and Olea europea plants bioengineered to over-express the osmotin plant gene can be used to ameliorate glucose metabolism, lower blood sugar in adultswith T2DM by causing the kidneys to remove sugar from the body through the urine, and represents treatment to ameliorate T2DM.

Images