Analysis of Biochemical Compounds in ‘gynura Procumbens’

Extraction of Biochemical Compounds in ‘gynura Procumbens’ Using 3 Mixture Design and Gcms
Name : Syazliana Syamira Binti Hashim
NOVEMBER 2014
Gynura procumbens or known locally in Malaysia as ‘Sambung Nyawa’ or in chinese, it is called ‘Nan Hui Ye’ is a plant that was found in South East Asia usually inThailand, Indonesia and Malaysia.This plant belongs to the Asteraceae Family.It is a famous traditional herb in South East Asia for the treatment of diabetic, high cholesterol level, cancer, high blood pressure, kidney discomfort and inflammation.The present study aims to study the antidiabetic activity of Gynura procumbens leaves to manage diabetes and to study the potential toxicity for many of the biochemical compounds in this plant leaves by using the three mixture design and analysis by CGMS technique.
In a mixture experiment, the independent factors are proportions of different components of a blend. The fact that the properties of the different factors must sum to 100% complicates the design as well as the analysis of mixture experiments.In this work,a mixture design will be applied to optimize solvent mixtures for selective extraction of biochemical compounds from Gynura procumbens leaves.Pressurized solvent extractions(PSE) will be performed at 313K and 20MPa,using homogeneous,ethanol(EtOH) and O solvent mixtures.The operating conditions to extract biochemical compounds from Gynura procumbens leaves will be optimized.The ranges of extraction temperatures and pressures are 25 – 60 and 8 – 25MPa, respectively.GC coupled with MS detection will be used to characterize the extracts.
Introduction
The modern lifestyle has become a major threat to the health of people around the world.There are many cases that involve arteriosclerosis, obesity, diabetes mellitus and cancers and these diseases remind us about our unhealthy lifestyles and thus give rise in circulatory system diseases such as hypertension, pre-hypertension and heart disease in developed countries (Johnson and Turner, 2005; Kearney et al., 2005).
From the previous time,plants have often been used to cure human diseases (Stepp and Moerman,2001; Yesilada, 2005).Today,with the arising threat of intractable diseases,a research is to focus on the finding bioactive molecules from plants(Amos et al.,2003; Fatehi et al.,2005).The one such plant is ‘Sambung Nyawa’ or its scientific name is Gynura procumbens is a member of the Asteraceae family(Wiart,2002).
Gynura procumbens is commonly used as a traditional medicinal plant in Malaysia to treat many diseases. Recently, the use of herbal medicines for complementary treatments of some diseases has been popular and researchers have shown that these herbs are commonly used by cancer patients to manage their diseases (Riboli and Norat, 2003; Van Duijnhoven et al., 2009).
Pharmacologic studies also have proved that Gynura procumbens possesses anti-herpes simplex virus, (Nawawi et al.,1999), anti-ulcerogenic activities (Mahmood et al., 2010) anti-inflammatory (Iskander et al.,2002), and anticancer properties(Agustina et al.,2006).
Problem Statement :
Gynura procumbens had been given attention in the pharmacology of antidiabetic medicinal plants probably because of its solid evidences and efficiency in the traditional management of diabetes mellitus.But in contrast,the scientific reports on the antidiabetic activity of this plant had their own conflicts and inconsistent.For example,Zhang and Tan had reported that 95% ethanol extract improved glucose tolerance in STZ-induced diabetic rats, but not in normal rats(Zhang and Tan).These authors also reported that its aqueous extract to exert significant anti hyperglycemic action in STZ-induced diabetic rats.But after that,Akowuah et al., on the contrary indicated its glucose lowering effect in normal rats(Akowuah et al.,2001).In the most recent study, the extract of Gynura procumbens was reported to generate significant elevation in the fasting blood glucose(FBG) levels of normal rats but a decrease in diabetic rats(Hassan et al.,2010).

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Eventhough Gynura procumbens is useful in the treatment of many ailments,however there is a little toxicological information that is available regarding to the safety due to the repeated exposure.Thus,a study need to be conducted to improve these reports and give more useful informations about its widespread uses.Therefore,the present research is to study the antidiabetic activity of Gynura procumbens leaves to manage diabetes and also to study the potential toxicity for many of the biochemical compounds in Gynura procumbens leaves.
Objectives :

To study the antidiabetic activity of Gynura procumbens leaves to manage diabetes.
To study the potential toxicity for many of the biochemical compounds in Gynura procumbens leaves.

2.0 Literature Review
2.1 General Overview of Gynura procumbens
Gynura procumbens that is well-known in South East Asia has traditionally been used to cure rash, eruptive fevers, kidney disease, constipation, migraines, diabetes mellitus, hypertension and cancer(Perry,1980).Recently, pharmacological studies have state that this plant has anti-hyperglycaemic(Akowuah et al.,2001; Akowuah et al.,2002),anti-hyperlipidaemic(Zhang and Tan,2000),anti-inflammatory(Iskander et al.,2002) and blood hypertension reduction capabilities(Lam et al.,1998; Kim et al.,2006).
The advantages of the traditional use of Gynura procumbens leaves have been supported the isolation and identification of several possible active chemical constituents from this herb, including flavonoids, tannins, saponins and terpenoids(Akowuah et al.,2002).Gynura procumbens is usually used as a traditional medicinal plant in Malaysia to cure many ailments. It is an annual ever- green shrub with a fleshy stem and purple tint.In Malaysia,this plant is called as ‘Sambung nyawa’, and it is commonly used in South-East Asia,especially in Malaysia, Indonesia, and Thailand.
In spite of the various uses over long time periods, little toxicological information is available regarding safety following repeated exposure to Gynura procumbens. Currently, Malaysian authorities are paying more attention to the safety and potential toxicity of botanicals, including medical plants and edible materials. Therefore,the objective of the present study was to provide scientific data on the safety of Gynura procumbens, that focus on the acute and 90days (13weeks) sub-chronic toxicity of a methanol extract that was orally administered to Sprague Dawley (SD) rats.
Gynura procumbens has recently received particular attention in the pharmacology of antidiabetic medicinal plants,probably because of its admitted empirical evidence and efficiency in the traditional management of diabetes mellitus.Besides that, these study designs are not targeted at natural product discovery or production of standardized herbal forms. Satisfactory research on medicinal plants beyond screening for biological activity should be conducted with the objective to systematically standardize and possess them into natural products or dosage forms which should effectively complement or supplement existing conventional measures(Ali et al.,2012).
2.2 Current Research of Gynura procumbens
There is currently a good deal of research interest to utilize plant compounds against human diseases, that includes hypertension. The present study investigated the effects of different extracts and fractions from leaves of Gynura procumbens on rat atrial contraction in vitro. Isolated left and right atria were mounted in a 20-ml organ bath and they were allowed to equilibrate for 15 minutes before the application of the extracts or fractions. The extracts ( methanol extract (ME) and petroleum-ether extract (PE) ) and the fractions (chloroform fraction(CHL), ethyl-acetate fraction (EA), n-butanol fraction (NB) and water fraction (WA) of the methanol extract) were tested at three concentrations (0.25, 0.5 and 1.0 mg/ml), with a b-adrenergic agonist (isoprenaline) as a control.
All the data on the contraction responses were log-transformed and had been analyzed. When exposed to the different extracts, both atria tended to exhibit greater contractive responses with the NB whereas cardiac contractions had a tendency to be reduced with most other extracts. For a given extract, the contraction responses were particularly greater at 0.5 mg/ml for the right atrium and at 1 mg/ml for the left atrium. For the further analysis is to focus on the NB fraction revealed that positive inotropism was greater in left atria exposed to highly-concentrated F2 and F3 sub-fractions.
Taken together,the results suggest that NB extracts and fractions from the Gynura procumbens-leaf methanol extract have positive inotropic activities and, hence, can be considered as an alternative or traditional medicine against increased blood pressure in humans or can be used in strategies aimed at finding antihypertensive biomolecules from an accessible source.
In each of these studies, the approach was not bioassay directed, an approach basis to the discovery of novel natural products from their natural sources and or to increase their standardization preparation to formulate into dosage forms for human usage after clinical trials. In summary, there is little analysis on the importance of the different extracts and fractions of Gynura procumbens on heart tissues. Therefore, the present study examined the effects of Gynura procumbens leaves extracts and fractions of the active extract on the contraction of left and right atria (LA and RA, respectively) isolated from Sprague Dawley (SD) rats with the aim of exploring the possibilities of its standardization or industrial scaling up for natural products against hypertension.
2.3 Functions of Gynura procumbens
In South-East Asia, this plant is widely distributed and has often been used to treat diseases. In Indonesia, leaves of the Compositae family are routinely used for treatment in kidney diseases, eruptive fevers, rash, hypertension, diabetes mellitus, and hyperlipidemia(Perry,1980). In Thailand, where this plant is empirically used against topical inflammation, rheumatism, and viral ailments, Iskander and colleagues have reported the anti-inflammatory actions of its extracts(Iskander et al.,2002).
Recently, pharmacological investigations in Singapore have shown that extracts of Gynura procumbens reduced serum cholesterol and triglyceride levels in streptozotocin induced diabetic rats(Zhang and Tan,2000). In Malaysia, evidence exists that Gynura procumbens has antidiabetic properties(Akowuah et al.,2001; Bohari et al.,2006). In this country, the currency of hypertension is high, but the levels of awareness, control, and treatment are still low(Rampal et al.,2008).This species has been used as a traditional medicine to treat inflammation, cancer, rheumatism, and viral infections. Among the various ethnobotanical practices of this plant, the application as a poultice for diverse skin diseases was found to be a universal usage in many countries (Perry, 1980). The leaves of this plant are not toxic(Rosidah et al., 2009). Gynura procumbensfrom Asteraceae family has been used as a traditional remedy for various skin diseases in certain areas of Southeast Asia(Junoh et al.,2011).
2.4 Related Diseases
There were many attempts to control increased blood pressure that have been used as strategies to control arterial hypertension(Collins et al.,1990; Lang et al.,2001). Eventhough the treatment of arterial hypertension has been shown to be quite efficacious in reducing cardiovascular mortality and morbidity (Collins et al.,1990), hypertension control at the population level has been generally considered as not enough yet. In the United Kingdom, for instance, 94% of hypertensive patients still have their blood pressure higher than the normal(Colhoun et al.,1998), whereas in the United States, there is only 27% had a normal blood pressure following treatment(Burt et al.,1995). This intractability of hypertension has not changed, and recently there is a noticeable increase in hypertension cases(Kearney et al.,2004); about 26% of adults worldwide in 972 million are known to have hypertension(Kearney et al.,2005).
Current optimism based on pharmacological therapy, which consists of developing drug formulations, is severely impeded by drug interactions, dose dependence, and adverse effects such as the possibility of depression. All in all,it is clear that the development of alternative strategies complementary to existing control methods is needed.
2.5 Acute and Sub-chronic Toxicity
The Gynura procumbens methanol extract was suspended in 1% (w/v) carboxylmethylcellulose (CMC) and it was administered orally (1250, 2500, and 5000 mg/kg) in single doses to both female and males rats (n = 10; 5 males and 5 females). The rats need to fast overnight due to dosing. The general behaviour of the rat was continuously monitored for 1 hour after dosing, periodically during the first 24 hour (with special attention given during the first 4 hour), and daily thereafter for a total of 14 days. All animals were humanely killed by inhalation after 14 days.The selected vital organs were excised, weighed, and macroscopically examined(Ghosh,1984).
Healthy rats of both sexes were randomly assigned to control and treatment groups (n = 10; 5 males and 5 females). The Gynura procumbens methanol extract was suspended in 1% CMC and administered orally on a daily basis for 90 days (13 weeks) at doses of 125, 250, or 500mg/kg. Based on the report by Suharmiati (2003), traditional healers typically prescribe seven leaves each day for an adult, which is equivalent to a 125 mg/day dose. Therefore, doses ranging from of 125–500mg/(kg day) were selected as the tested doses. All rats were anesthetized under inhalation at the end of 13 weeks. Blood samples were collected via cardiac puncture into non-heparinized and EDTA containing tubes for biochemical and haematological analyses, respectively (Winarto, 2003; Petterino and Argentino-Storino, 2006). After cardiac puncture, the rats were killed by clavicle dislocation.Those vital organs were excised, weighed, and macroscopically examined and then they were fixed in 10% formalin for histopathological study.
2.6 Diabetes Mellitus
Diabetes causes oxidative stress through the autooxidation of glucose, protein glycation and lipid peroxidation; and as a consequence, structural and functional alterations occur in the lungs of diabetics, as well as other organs like the heart, brain, eyes and kidneys. There is growing, well-established evidence regarding such changes in diabetic lungs(Gulay et al.,2014). Oxidative stress plays an important role in the pathogenesis of diabetes mellitus, contributing not only to the development, but also to the progression of diabetes and its related complications. The immunosuppressive and antioxidant effects of resveratrol in attenuating the increased oxidative stress due to responses of β-cells to leukocyte activation have been implicated in the prevention of type 1 diabetes mellitus. Resveratrol affords advantageous effects by decreasing the oxidative injury and the recruitment of the nutritive precapillary arterioles in the context of disease states associated with insulin resistance, such as metabolic syndrome, pre-diabetes, and type 2 diabetes mellitus.
Globally, more than 366 million individuals currently live with diabetes mellitus, with this figure expected to rise to 552 million by 2030 (Whiting et al., 2011). The World Health Organization estimates that around 1.1 million deaths occur per year as a result of the disease (World Health Organization, 2011). There are around 2.9 million individuals in England currently have diagnosed with diabetes, a currency of 4.5% (NHS & The Information Centre, 2011). Projections for England estimate that by 2030 over 4.5 million people will be living with diabetes (APHO, 2011). The number of individuals that are living with diabetes means that if there was found to be an excess in mortality caused by the disease, this could result in a large number of premature deaths.
The relation between diabetes and increased all-cause death, particularly from cardiovascular disease (CVD) and renal disease, is well recognized. C There is a limited amount of evidence relating to diabetes and respiratory disease mortality (Dawson et al., 2008;De Marco et al., 1999andMurugan and Sharma, 2008).Current evidence suggests a biological link between inflammation, reduced levels of adiponectin within the body and the development of diabetes and respiratory disease (Sevenoaks & Stockley, 2006). De Marco et al. concluded that these may be caused by differences in the severity of diabetes or differing in national treatment cultures (De Marco et al., 1999). Therefore, research is required that further explores the relation between diabetes and mortality from cancer and respiratory disease. Current research indicates that, for mortality from a number of causes, it may be diabetes-related comorbidities that increase an individual’s risk of death rather than the diabetes itself. (Mohammadi et al., 2007) Morbidity and mortality of type 2 diabetes mellitus (DM) is closely related to development of cardiovascular disease (Haffner et al.,1998).
Although the manifestations and complications of diabetes mellitus have been extensively studied, the knowledge regarding the effects of diabetes on bone mineral metabolism is still limited and inconsistent. Since Albright and Reifenstein(Anwar et al.,2008)first reported the occurrence of osteoporosis in patients with poorly controlled diabetes in 1948, many authors have attempted to describe alterations of bone mineralization in diabetic subjects by using various techniques and biochemical markers(Asbun and Villareal,2006).
The types of diabetes caused by other conditions or found in increased frequency with other conditions (imply an etiologie relationship) be considered a third subclass of diabetes mellitus that is the diabetes that associated with certain conditions and syndromes. This subclass has been divided according to the known or suspected etiologie relationships.The class gestational diabetes has been restricted to women in whom glucose intolerance develops or is discovered during pregnancy (National Diabetes Data Group,1979).