The effects of a green rooibos extract on the reproductive function of obesity-induced insulin resistant or hypertensive male wistar rats

Manirafasha, Claudine (2019-12)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Diet-induced obesity (DIO) due to a high caloric diet (HCD) predisposes an individual to the development of diabetes and cardiovascular diseases, with high prevalence in young populations. Existing evidence supports the sentiment that insulin resistance and hypertension (HT) affect male reproduction. A greater understanding of the influence of insulin resistance and/or HT on male reproduction is required in order to prevent or treat male infertility. Due to the limitations of orthodox drugs, there is currently a strong movement towards and support for studies on phytomedicine. Rooibos (Aspalathus linearis) has been used in several studies and is known to have natural antioxidant effects and anti-obesogenic, antidiabetic, anti-hypertensive and anti-infertility activities. Currently, the company Afriplex (Pty) Ltd is producing an aspalathin-rich laboratory standardized extract prepared from green rooibos called Afriplex GRTTM (GRT). However, there is very little knowledge regarding the use of GRT in obesity-related insulin resistance and/or HT, and specifically, its effects on male reproductive health. Aim This study aimed to explore the effect of GRT on the reproductive function in obesity-induced insulin resistant and hypertensive male Wistar rats. Methods A prospective randomized control and experimental animal study design was used. Two different diets were used to induce obesity-related insulin resistance with or without HT in male Wistar rats. Subsequently, the possible protective properties of GRT on the male reproductive system were evaluated. Animals (weighing 120 ±10 g, approximately 7 weeks old) were randomly assigned to seven groups with seven rats each. All rats had unrestricted access to their respective diets and water for 16 weeks. At baseline (week 0–10), we had three groups: 1) lean control (LC) – animals that received standard rat chow; 2) obese (OB) – animals that received a diet to induce obesity associated with insulin resistance; and 3) obese with hypertension (OBHT) – animals were placed on a slightly modified DIO and additionally developed HT. From weeks 11 to 16, one LC, OB and OBHT group were each treated with GRT (prepared and supplied by Afriplex (Pty) Ltd) at 60 mg/kg/day as a dietary supplement in the form of jelly blocks. An additional group of OBHT animals was treated during the same period for 6 weeks with Captopril, an angiotensin-converting-enzyme (ACE) inhibitor (positive control for HT) at 60 mg/kg per day. Food and water intake were monitored on a daily basis. An oral glucose tolerance test was performed during the 10th week after the onset of the respective diets and during the 16th week, after which the animals were sacrificed. Blood pressure measurements were taken once per week throughout the experimental period. After the 16-week period, animals were killed and blood, testis and epididymal tissue were harvested for further analysis. Body weight, intra-peritoneal fat, non-fasting glucose levels, IL-1β, IL-6, IL-12, IL-18 and TNFα, oxidative stress (OS) markers (superoxide dismutase and catalase activity, malondialdehyde), testosterone and estradiol, sperm concentration, viability, morphology, total motility, progressive motility and various velocity parameters were measured. Results and conclusion Both diets successfully induced insulin resistance with or without hypertension and demonstrated detrimental effects on male reproductive function as evidenced by OS and hormone dysregulation. Treatment with GRT reversed OS and balanced the androgens. This study provided insight into the pharmacological effects of GRT in the treatment of pathophysiological changes that occur in DIO associated with insulin resistance or HT. These findings will hopefully inspire further research into the clinical setting related to the GRT and could possibly lead to the development of new drugs from this compound.

AFRIKAANSE OPSOMMING: Dieet-geïnduseerde vetsug (DGV) as gevolg van ʼn hoë kalorie-dieet (HKD) maak individue vatbaar vir die ontwikkeling van diabetes en kardiovaskulêre siektes, met hoë voorkoms in jong populasies. Bestaande bewyse ondersteun die sentiment dat insulienweerstand en hipertensie (HT) manlike voortplanting beïnvloed. ’n Beter begrip van invloed van insulienweerstand en/of HT op manlike voortplanting is nodig om manlike onvrugbaarheid te voorkom of te behandel. As gevolg van die beperkings van ortodokse medisyne is daar tans ʼn sterk beweging na en ondersteuning van die studie van fitomedisyne. Rooibos (Aspalathus linearis) is in verskillende studies gebruik en is bekend vir sy natuurlike antioksidant-effek en anti-obesogeniese, anti-diabetiese, anti-hipertensiewe en anti-onvrugbaarheidsaktiwiteite. Tans vervaardig die maatskappy Afriplex (Edms.) Bpk. ʼn aspalatienryke laboratorium gestandaardiseerde ekstrak, wat uit groen rooibos ekstrak voorberei is, genaamd Afriplex GRTTM (GRT) ekstrak. Daar is egter baie min kennis aangaande die gebruik van GRT-ekstrak in obesiteitverwante insulienweerstand en/of hipertensie en spesifiek wat die effek daarvan op manlike voorplantingsgesondheid is. Doelstelling Die doel van hierdie studie is om die effek van GRT-ekstrak op die voortplantingsfunksie van obesiteit-geïnduseerde insulienweerstandige en hipertensiewe manlike Wistar-rotte te ondersoek. Metodes Hierdie studie het ʼn prospektiewe ewekansige kontrole en eksperimentele ontwerp. Twee verskillende diëte is gebruik om obesiteit-verwante insulienweerstandigheid en/of hipertensie in manlike Wistar-rotte te bewerkstellig. Vervolgens is die moontlike beskermende eienskappe van GRT-ekstrak op die manlike voortplantingstelsel geëvalueer. Diere (120 ± 10g gewig, ~ 7 weke oud) is lukraak toegewys aan sewe groepe en elke groep het sewe rotte bevat. Alle rotte het vir 16 weke onbeperkte toegang tot hul onderskeie diëte en water gehad. By aanvang (week 0–10) was daar drie groepe: 1) maer kontrole (LK) diere wat standaard rotvoer ontvang het; 2) obees (OB) rotte wat ʼn dieet ontvang om vetsug te veroorsaak wat verband hou met insulienweerstandigheid; en 3) obees met hipertensie (OBHT) was diere wat op ʼn aangepaste dieet geplaas is om sodoende ook HT te veroorsaak Van weke 11 tot 16 is een LK, OB- en OBHT-groep elk met 60 mg/kg/dag GRT-ekstrak (vervaardig en verskaf deur Afriplex (Edms.) Bpk.) as ʼn dieetaanvulling in die vorm van jellie-GRT-blokke behandel. ʼn Verdere groep OBHT-diere is gedurende dieselfde tydperk vir 6 weke daagliks behandel met 50 mg/kg Captopril, ʼn ACE-inhibeerder (positiewe kontrole vir HT) (Huisamen et al., 2013). Voedsel- en waterinname is daagliks gemonitor. ʼn Orale glukose-toleransietoets is gedurende die 10de week na die aanvang van die onderskeie diëte en gedurende die 16de week uitgevoer, waarna die diere doodgemaak is. Bloeddrukmetings is een keer per week tydens die eksperimentele periode geneem. Na die 16-week periode is die diere doodgemaak en is bloed, testis en epididimale weefsel geoes vir verdere analise. Liggaamsgewig, intraperitoneale vet, nie-vastende glukosevlakke, IL-1β, IL-6, IL-12, IL-18 en TNFα, superoksied dismutase en katalase aktiwiteit, malondialdehied, testosteroon en estradiol, spermkonsentrasie, lewensvatbaarheid, morfologie, totale motiliteit, progressiewe motiliteit en verskeie snelheidsparameters is gemeet. Resultate en gevolgtrekking Albei diëte was suksesvol in die bewerkstelling van insulienweerstandigheid met of sonder hipertensie en het nadelige uitwerkings op manlike voortplantingsfunksie, soos gesien in OS en hormoondisregulasie, veroorsaak. Behandeling met GRT-ekstrak het OS omgekeer en het androgeenvlakke gebalanseer. Hierdie studie het insig gebied in die farmakologiese effekte van GRT in die behandeling van patofisiologiese veranderinge wat voorkom gedurende DGV wat verband hou met insulienweerstandigheid of hipertensie. Die resultate sal hopelik verdere kliniese studies oor GRT-ekstrak inspireer en kan moontlik lei die ontwikkeling van nuwe medisyne gemaak van hierdie samestelling.

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