CHARACTERIZATION OF EXTRACTABLE HYPOGLYCAEMIC PRINCIPLES FROM SECURIDACA LONGEPEDUNCULATA LEAVES

ABSTRACT

This work was undertaken to characterize the extractable hypoglycaemic principles from Securidaca longepedunculata leaves that have improved pharmacological agent(s) that could be used in managing diabetes mellitus with relatively lesser side effects. Six groups of five Wistar rats each were used. Group one received 3 ml/kg bw of normal saline, group two received no treatment after induction, group three received 5 mg/kg bw glibenclamide; groups four, five and six received 100, 200, 400 mg/kg bw respectively. Groups two to six were induced with diabetes mellitus by treatment with 100 mg/kg bw alloxan. The methanol extract of S.   longepedunculata (MESL) contained alkaloids, flavonoids, terpenoids, steroids, saponin, phenolics, tannins and reducing sugar. Diabetic but treated groups four, five and six significantly (p < 0.05) lowered the blood glucose of the Wistar rats compared to the diabetic control.  Diabetic but treated groups four to six showed significant (p < 0.05) decrease in the liver functions as shown by serum, AST, and ALP activities (100 mg/kg bw), with the exception of ALT activity at 200 and 400 mg/kg bw, total, conjugated and unconjugated bilirubin levels compared to the diabetic control. Diabetic but treated groups four to six had varied effects on the kidney functions as shown by serum concentrations of Na+, K+, Cl-  and HCO3-  concentrations compared to the diabetic control. Diabetic but treated groups four to six showed significant (p < 0.05) decrease in urea and creatinine levels compared to the diabetic control. The histology of the pancreas showed  regeneration  of  beta-cells  of  islets  of  Langerhans  in  the  diabetic  but  treated  group  six compared to the diabetic control. The diabetic but treated groups four to six caused a significant (p <

0.05) increase in serum antioxidant activities as shown by catalase and glutathione peroxidase activities, with the exception of superoxide dismutase  activity and also a significant (p < 0.05) decrease in malondialdehyde concentrations compared to the diabetic control. Liquid-liquid fractionation yielded four fractions: n-hexane, dichloromethane, ethylacetate and aqueous-methanol fractions. Oral glucose tolerance test of the crude and aqueous-methanol fractions showed significant reductions in blood sugar. The 0.2% crude extract and the four fractions of MESL at 10, 20, 30 and 40 µl significantly (p < 0.05) inhibited the activity of disaccharidase A2. However, aqueous-methanol fraction showed the highest inhibitory activity of disaccharidase A2. The transports of ion across intestine showed that sodium ion was inhibited by MESL at 5 and 7.5 mg/ml, while potassium ion was inhibited at 5 mg/ml only. The UV-Visible scan of chloroform: acetic acid: methanol (18:1:1) column chromatography eluent showed a lamda max at 275 nm. The FTIR spectroscopy of the eluent showed molecular vibrations at 3407.17 cm-1 (O-H and N- stretching), 3199.80 and  3011.496 cm-1 (- C=C-H,  C=CH,  Ar-H,  C-H stretch) 2863.64  and  2711.151 cm-1      (CH3-, CH2   and  C=O stretch)

1643.91 (C=N stretching), 1377, 1219.24 and 1055.29, cm-1  (C-H bending), 833.81 cm-1  (Ar finger

print). The GC-MS of the eluent identified five groups of compounds namely 2,4-bis (1,1-dimethyl) phenol, cyclic octaatomic sulphur, 5-methyl-3-(1-piperidylmethyl) -1,2,4-oxadiazole, oils (n- hexadecanoic acid, octadecanoic acid and oleic acid- an essential oil). The GC-MS of Sephadex G75 column chromatography eluent yielded a pure compound di-n- octylphthalate. The plant’s active principles from the study have anti-diabetic and anti-hyperglycaemic potencies. However it was found to have toxic principle with increase in dosage.

ABSTRACT

This work was undertaken to characterize the extractable hypoglycaemic principles from Securidaca longepedunculata leaves that have improved pharmacological agent(s) that could be used in managing diabetes mellitus with relatively lesser side effects. Six groups of five Wistar rats each were used. Group one received 3 ml/kg bw of normal saline, group two received no treatment after induction, group three received 5 mg/kg bw glibenclamide; groups four, five and six received 100, 200, 400 mg/kg bw respectively. Groups two to six were induced with diabetes mellitus by treatment with 100 mg/kg bw alloxan. The methanol extract of S.   longepedunculata (MESL) contained alkaloids, flavonoids, terpenoids, steroids, saponin, phenolics, tannins and reducing sugar. Diabetic but treated groups four, five and six significantly (p < 0.05) lowered the blood glucose of the Wistar rats compared to the diabetic control.  Diabetic but treated groups four to six showed significant (p < 0.05) decrease in the liver functions as shown by serum, AST, and ALP activities (100 mg/kg bw), with the exception of ALT activity at 200 and 400 mg/kg bw, total, conjugated and unconjugated bilirubin levels compared to the diabetic control. Diabetic but treated groups four to six had varied effects on the kidney functions as shown by serum concentrations of Na+, K+, Cl-  and HCO3-  concentrations compared to the diabetic control. Diabetic but treated groups four to six showed significant (p < 0.05) decrease in urea and creatinine levels compared to the diabetic control. The histology of the pancreas showed  regeneration  of  beta-cells  of  islets  of  Langerhans  in  the  diabetic  but  treated  group  six compared to the diabetic control. The diabetic but treated groups four to six caused a significant (p < 0.05) increase in serum antioxidant activities as shown by catalase and glutathione peroxidase activities, with the exception of superoxide dismutase  activity and also a significant (p < 0.05) decrease in malondialdehyde concentrations compared to the diabetic control. Liquid-liquid fractionation yielded four fractions: n-hexane, dichloromethane, ethylacetate and aqueous-methanol fractions. Oral glucose tolerance test of the crude and aqueous-methanol fractions showed significant reductions in blood sugar. The 0.2% crude extract and the four fractions of MESL at 10, 20, 30 and 40 µl significantly (p < 0.05) inhibited the activity of disaccharidase A2. However, aqueous-methanol fraction showed the highest inhibitory activity of disaccharidase A2. The transports of ion across intestine showed that sodium ion was inhibited by MESL at 5 and 7.5 mg/ml, while potassium ion was inhibited at 5 mg/ml only. The UV-Visible scan of chloroform: acetic acid: methanol (18:1:1) column chromatography eluent showed a lamda max at 275 nm. The FTIR spectroscopy of the eluent showed molecular vibrations at 3407.17 cm-1 (O-H and N- stretching), 3199.80 and  3011.496 cm-1 (- C=C-H,  C=CH,  Ar-H,  C-H stretch) 2863.64  and  2711.151 cm-1      (CH3-, CH2   and  C=O stretch) 1643.91 (C=N stretching), 1377, 1219.24 and 1055.29, cm-1  (C-H bending), 833.81 cm-1  (Ar finger print). The GC-MS of the eluent identified five groups of compounds namely 2,4-bis (1,1-dimethyl) phenol, cyclic octaatomic sulphur, 5-methyl-3-(1-piperidylmethyl) -1,2,4-oxadiazole, oils (n- hexadecanoic acid, octadecanoic acid and oleic acid- an essential oil). The GC-MS of Sephadex G75 column chromatography eluent yielded a pure compound di-n- octylphthalate. The plant’s active principles from the study have anti-diabetic and anti-hyperglycaemic potencies. However it was found to have toxic principle with increase in dosage.

CHAPTER ONE

INTRODUCTION

Diabetes mellitus is an endocrine-disorder that has been ranked among the first ten causes of mortality worldwide (Chakraborty and Das, 2010; Shaw et al., 2010 and IDF, 2013). Two major types of diabetes mellitus are most prevalent. They are type I and type II otherwise known as insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM) respectively (Nelson and Cox, 2005). While insulin therapy is ideal for type I, oral hypoglycaemic principles are ideal for type II. The oral hypoglycaemic principles available, currently are sulfonylureas like chlorpropamide, tolbutamide, glibenclamide, glipizide, among others; biguanides like phenformin and metformin; α- glucosidase inhibitors like acarbose and meglitol; sodium-glucose co-transporter 2 (SGLT 2) inhibitors, like canagliflozin (Katzung et al., 2009). These hypoglycaemic principles are characterized with short-comings ranging from beta-cells depletion, hypoglycaemia and monotherapy- inadequacy (Ukwe, 2005; Wadkar et al., 2008; Shammi et al., 2010). In addition oral hypoglycaemic principles cause serious side effects which included weight gain, gastrointestinal discomfort, nausea, liver and heart failure and diarrhoea (Kane et al., 2005). These  challenges  have  forced  the  rural,  urban  communities  and  the  World  Health Organization (WHO) to source for alternative medicines in the management of diabetes mellitus (WHO, 1980; Njike et al., 2005; Ezeigbo and Asuzu 2012). Different plants have been studied for their abilities to reduce high blood glucose which is the principal feature in diabetes mellitus (Arumugam et al., 2013). Securidaca longepedunculata which belongs to the family of polygalacae has been credited for ameliorating various illnesses (Wannag,

2005; Mohammed et al., 2012). A couple of researchers have reported its hypoglycaemic potency (Gbile and Adesina, 1987; Ojewole, 2008). However, from literature, there are no reports on the active agents that are responsible for the reported hypoglycaemic potency. Hence this work was undertaken to characterize the extractable hypoglycaemic principles from the plant with the view to finding a hypoglycaemic principle with comparative advantages over the conventional hypoglycaemic agents.

1.1 Securidaca longepedunculata

1.1.1 Taxonomy and distribution of Securidaca longepedunculata

Securidaca longepedunculata as shown in Figure 1 belongs to the family polygalaceae, and is a spinny semi-deciduous shrub that grows to about 12 m tall, (Owoyele et al., 2006). It is widely distributed in West and South African regions (Abdullahi and Lawal, 2010). It is a tropical plant (Mohammed et al., 2012). The plant is also known as Rhodes’ violet wild wisteria (English), uuwar maagunguna (Hausa), enu-opiri (Yoruba), ezeogwu or atumaka (Ibo) and aalali (Fulani). In Nigeria it is used as vegetable. It is used in traditional mid-wifery and to induce abortion. In Nigeria ethnopharmacology the natives dry the leaves and subsequently inhale the smoke for the amelioration or cure for head ache (Alqasim, 2013). It is among Nigeria ethno-pharmacological agents used in tackling diabetes mellitus (Gbile and Adesina, 1987). It is distributed across Nigeria. It is common in North Central and South Eastern Nigeria.

1.1.2 Traditional and Pharmacological Uses of Securidaca longepedunculata

Despite immense technological advancements in modern medicine, many people in the developing countries still rely on the healing practices based on medicinal plants for their daily health care needs (Ojewole, 2004). The young leaves of Securidaca longepedunculata are cooked and eaten as vegetable or in sauces. The cooked leaves are said to be effective in the treatment of snake bites. Leaves and roots are used to induce abortion used in boosting libido/genital stimulant (Alqasim, 2013). Erectile dysfunction is a common problem for men who have diabetes. It (erectile dysfunction) can stem from problems caused by poor long term blood sugar control which damages nerves and blood vessels. This condition is also seen in diabetic men with high blood pressure and coronary artery disease. Securidaca longepedunculata is reported to have analgesic, anti-inflammatory and hypoglycemic potentials (Ajiboye et al., 2010). There are also reports on its use in treating malaria and rheumatism (Gill, 1992). It is also used in bacterial chemotherapy and in the treatment of cough, headache, constipation, wound, sore throat and gout (Akinniyi et al., 1996; Ojewole,

2008; Mohammed et al., 2012). The anti-snake venom potential has  also been reported (Wanag et al., 2005). Indole and ergot alkaloids, flavonoids and methyl salicylate are some secondary   metabolites   that   have   been   reported   as   being   present   in   Securidaca

longepedunculata (Iddagoda and Thamara, 2003; Abdullahi and Lawal, 2010). Methyl salicylate is toxic at certain concentration. In ethnopharmacology the natives dry the leaves and subsequently inhale the smoke for the amelioration or cure for head ache (Alqasim,

2013). Alqasim (2013) also reported that the leaves were the least used (23.8%) while the barks were the second most used parts (28.6%) whereas the roots were the highest used (47.6%). Methanol extract of Securidaca longepedunculata was reported to cause non- significant alterations in the haematological parameters and serum electrolytes at long term administration to rats (Owoyele et al., 2006).

The antibacterial activity of chloroform, methanol and aqueous extracts of the roots and leaves of Securidaca longepedunculata against some selected microorganisms demonstrated inhibitory  activity  against  the  tested  organisms.  The  chromatography  of  methanol  and aqueous extracts of the leaves revealed two major spots. The phytochemical screening of the extracts (roots and leaves) revealed the presence of alkaloids, flavonoids, saponins, tannins, cardiac glycosides, anthraquinones, steroids, balsams and reducing sugars (Mohammed et al.,2012).

Abdullahi  and  Lawal,  (2010)  reported  that  the  result  of  phytochemical  screening  of Securidaca longepedunculata demonstrated the presence of steroids, flavonoids, cyanogenic glycosides, anthraquinones and flavotannin in the solvents used. However alkaloids were only present in the ethanol extract, while saponin was found in the water extract only. The thin layer chromatography, on its own, showed four and two spots for ethanol and aqueous extracts respectively, though aqueous extract yielded more extract than the ethanol extract.

The proximate composition carried on this plant showed that dry matter represented 94.5 % while moisture represented 5.5%. Carbohydrate was found to be 89.28 %, nitrogen free extract 64.74 %, ash 2.0 % and protein was found to be 1.22 % (Mustapha et al., 2012). The levels of twelve elements, namely sodium, potassium, magnesium, calcium, cadmium, chromium, iron, nickel, zinc, cobolt, manganese and lead were found to be within the permissible limit set by the World Health Organisation (Mustapha et al., 2012).

Fig.1 Securidaca longepedunculata leaves

1.2.1    Extraction of Active Principles from Plants

Extraction is the crucial first step in the analysis of medicinal plants. The basic operation include steps, such as pre-washing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Proper actions must be taken to ensure that potential active constituents are not lost, distorted or destroyed during the preparation of the extract from the plant samples (Sasidharan et al., 2011). If the plant was selected on the basis of traditional uses then there will be need to prepare the extract as described by the traditional healer in order to mimic as closely as possible the ‘herbal’ drug (Fabricant and Farnsworth, 2001). The study of medicinal plants starts with the pre-extraction and the extraction procedures, which is an important step in the processing of the bioactive constituents from plant materials. Traditional methods such as maceration and  extraction are commonly  used  for  prototype  standard.  Significant  advances  have  been  made  in  the processing of medicinal plants such as the modern extraction methods; microwave-assisted (MAE), ultrasound-assisted (UAE) and supercritical fluid extractions (SFE), in which these advances are aimed to increase yield at lower cost. Moreover, modifications on the methods are continuously developed. With such varieties of methods present, selection of proper extraction method needs meticulous evaluation (Azwadina, 2015). All the methods that employ solvents in the procedures maceration, microwave-assisted, ultrasound-assisted and supercritical  fluid  extractions  are  critically influenced  by the  solvents  types  (Azwadina,

2015). However, no significant effect was caused by the solvent volume using three methods (maceration, MAE and UAE) on the biologically active compounds at ratio (1:10 w/v). All stages of extractions, from the pre-extraction to extraction proper are equally important in the study of medicinal plants. The sample preparation such as grinding and drying do affect the efficiency and phytochemical constituents of the final extractions; that eventually have an effect on the final extracts. Previously optimized methods can be used in the selection of suitable methods however; evaluation and selection of pre-extraction preparation and extraction methods depend on the objectives, samples, and target compounds (Azwadina,2015).

1.2.2 Evaluation of Active Principles of Compounds from Plants

Natural products, such as plants extract, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity (Cosa et al., 2006). Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases  (Duraipandiyan  et  al.,  2006).  Due  to  the  development  of  adverse  effects  and microbial  resistance  to  the  chemically  synthesized  drugs,  men  turned  to ethnopharmacognosy. They found literally thousands of phytochemicals from plants as safe and broadly effective alternatives with less adverse effect. Many beneficial biological activity such as anticancer, antimicrobial, antioxidant, antidiarrhoeal, analgesic and wound healing activities have been reported (Sasidharan et al., 2011). In many cases the people claim the good benefit of certain natural or herbal products (Sasidharan et al., 2011). However, clinical trials are necessary to demonstrate the effectiveness of a bioactive compound to verify this traditional claim. Clinical trials directed towards understanding the pharmacokinetics, bioavailability,   efficacy,   safety  and   drug  interactions   of  newly  developed  bioactive compounds and their formulations (extracts) require a careful evaluation. The premier steps to utilizing the biologically active compound from plant resources are extraction, pharmacological screening, isolation and characterization of bioactive compound, toxicological evaluation and clinical evaluation. These protocols constitute the standardizing procedures   required   to   place   the   active   principles   of   plant   sources   to   the   right pharmacological use.

1.2.3    Importance of Identifying Active Principles from Natural Sources

Natural products are expected to play an important role as one of the major sources of new drugs in the years to come because of (i) their incomparable structural diversity, (ii) the relatively small dimensions of many of them (<2000 Da), and (iii) their ‘‘drug like’’ properties, that is their ability to be absorbed and metabolized (Otto, 2008). Isolation of natural products from higher plants, marine organisms and microorganisms require methodologies for separation and isolation procedures. Isolation of natural products generally combines  various  separation  techniques,  which  depend  on  the  solubility,  volatility  and stability of the compounds to be separated. The choice of different separation steps is of great importance and an analytical-scale optimization of the separation parameters is worthwhile

1.2.4 Identification and Characterization of Active Principles from Plants

Due to the fact that plant extracts usually occur as a combination of various types of bioactive compounds or phytochemicals with different polarities, their separation still remains a big challenge for the process of identification and characterization. It is a common practice in isolation of these bioactive compounds that a number of different separation techniques such as TLC, column chromatography, flash chromatography, Sephadex chromatography and high performance liquid chromatography (HPLC), should be used to obtain pure compounds. The pure compounds are then used for the determination of the structure and biological activity. Beside that, non-chromatographic techniques such as immunoassay, which uses monoclonal antibodies (MAbs), phytochemical screening assay, Fourier-transform infrared spectroscopy (FTIR), can also be used to obtain and facilitate the identification of the bioactive compounds (Sasidharan et al., 2011).

1.2.5 Role of Polarity Index in Isolating Phytoconstituents

The differences in polarity of solvents determine differences in types and compositions of phytochemicals extracted by the solvents (Dehkharghanian et al., 2010). The selection of solvent system largely depends on the specific nature of the bioactive compound being targeted. Different solvent systems are available to extract the bioactive compound from natural products. The extraction of hydrophilic compounds uses polar solvents such as methanol, ethanol or ethyl-acetate (Cosa et al., 2006) while the extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/methanol in ratio of 1:1 v/v are used. In some instances, extraction with hexane is used to remove chlorophyll (Cosa et al., 2006). Widyawati et al. (2014) reported that methanol was the most effective solvent to extract phytochemical compounds compared to aquadest, ethanol, ethylacetate and hexanes. They identified compounds like sterol, flavonoid, saponin, tannin, phenolic, alkaloid and glycosides. Previous research showed that methanol and ethanol can dissolve polar compounds, such as sugar, amino acid, glycoside compounds, phenolic compounds with low and medium molecular weights and medium polarity aglycone flavonoid (Dehkharghanian et

al., 2010), anthocyanin, terpenoid, saponin, tannin, xantoxilin, totarol, quacinoid, lactone, flavone, phenone, and polyphenol.

1.3.0 Separation Techniques for Studying Active Principles from Plants

1.3.1 Thin Layer Chromatography (TLC) in Characterizations of Samples

Thin layer chromatography (TLC) and column chromatography are classified as liquid chromatography. They are used in carbohydrate and protein separations. The principle of separation of thin layer chromatography is either partition or adsorption. The constituent of a mixture which is having more affinity for mobile phase moves with it, while the constituent which is having more affinity for stationary phase gets adsorbed on it. This way various compounds appear as a band on the TLC plate, having different rate of flows (Rf) values (Balaji, and Kilimozhi, 2014).

1.3.2 Gas Chromatography

Gas chromatography can also be termed gas-liquid chromatography. It separates molecules principled upon the partition chromatographic separation of components of a mixture through the use of a liquid phase held in place on a support and a gas phase flowing over the liquid phase in a controlled fashion. In gas chromatography, gas-liquid system is involved (Horning and VandenHeuvel, 1963).

Gas-chromatography is used to analyze compounds that can be vaporized without decomposition. The use of gas chromatography encompass testing the purity of a substance, or separating the different components of a complex mixture. When employed to prepare pure compounds from a mixture, GC separates volatile components of mixtures by a differential migration through a column containing a liquid or stationary phase (Wu et al.,

1.3.3 Gel filtration

More discriminating separations on the basis of size can be effected using gel-filtration chromatography. Sephadex, sepharose, and biogel are examples of gels used in gel filtration. These beads have diameter of about 100 µm. Small molecules can enter these beads, but large ones cannot. This leads to the distribution of the small molecules in the solution both inside the beads and between them, while large molecules are located only in the solution between

the beads. The large molecules flow more rapidly through the column and emerge first as a result of the fact that the smaller volume is accessible to them. While the molecules that are sizeable enough to occasionally enter a bead will flow from the column at an intermediate position, smaller molecules which take a longer path will leave last from the column (Berg, et al., 2002).

Sephadex is also known as dextran while agarose is called sepharose, though both are carbohydrates Gel chromatography involves size exclusion. Here molecules are separated on the basis of their size and shape (Hamesa and Hooper, 2002). Gel filtration can be used to purify biological molecules like protein and plant extract.

1.4.1 Ultraviolet and Visible Spectroscopy

This absorption spectroscopy uses electromagnetic radiations between 190 to 800 nm and is divided into the ultraviolet and visible regions. Since the absorption of ultraviolet or visible radiation by a molecule leads to transition among electronic energy levels of the molecule, it is also often called electronic spectroscopy. The information provided by this spectroscopy when combined with the information provided by NMR and IR spectral data leads to valuable structural proposals (Akpanisi, 2004). Energy absorbed in the UV region produces changes in the electronic energy of the molecule. As a molecule absorbs energy, an electron is promoted from an occupied molecular orbital to an unoccupied molecular orbital

For  presenting  the  absorption  characteristics  of  a  spectrum,  the  positions  of  peaks  are

reported as λmax (in nm) values and the absorptivity is expressed in parenthesis (Kumar,

2006). Absorbance, therefore gives a measure of the photons of light absorbed by a given chemical species. The concentration and nature of the species determine how much light that would be absorbed. Therefore this spectroscopy enables the researcher to identify the unique molecules that are absorbing the light.

1.4.2 Fourier Transform Infra-Red Spectroscopy (FTIR)

Fourier transform infrared spectroscopy (FTIR) is an excellent analysis medium for characterizing and identifying organic molecules. It is a spectroscopic technique that is most widely used for determining the characteristics of new membranes.

Fourier  transform  infrared  spectroscopy  identifies  chemical  bonds  in  a  molecule  by producing an infrared absorption spectrum. The spectra produce a profile of the sample, a distinctive molecular finger. Fourier transform  infra red spectroscopy is most useful for identifying chemicals. The term Fourier transform infrared spectroscopy refers to a development in the manner in which the data is collected and converted from an interference pattern to a spectrum. Ultra-violet region spans 200-400nm while visible region ranges between 400 to 750nm, though 800nm could be considered. A few functional groups may be detected, but unsaturation in compounds; especially conjugated systems are chiefly tracked by UV-Vis. Infra-red spectroscopy ranges between 4000-650 cm-1, though 600 cm-1  can be included. It leads to a great deal of information, for example, the presence of various functional groups, hydrogen bonding (intra-molecular and intermolecular), the identification of cis- and trans- isomers, conformational orientation and orientation in aromatic compounds (Finar, 1973). The essential requirement for a substance to absorb in this region is that vibrations in the molecule must give rise to an unsymmetrical charge distribution. Just as electronic transitions are in discreet form, so are rotational and vibrational energy levels also in discreet form. Absorption in the near infra-red is due to changes in vibrational energy levels. A vibration that occurs along the bond causes stretching while the one that results in deformation is called bending (Finar, 1973).

1.4.3 Mass Spectrometry (MS)

Mass spectrometers utilize the discrepancy in mass to charge ratio (m/z) of ionized atoms or molecules spectroscopy quantify atoms or molecules and provide structural information by the identification of distinctive fragmentation patterns. Generally it operates by creating gas- phase ions, separating the ions created in space or time based on their mass-to-charge ratio and measuring the quantity of ions of each mass-to-charge ratio (Uggerud et al., 2003).

The use of mass-spectrometry to identify compounds is possible because when electrons interact with a given molecule, an excess of energy results in the formation of a wide range of positive ions. The resulting mass distribution is characteristic (the fingerprint) for that given molecule. There are some similarities with infra-red and nuclear magnetic resonance (Vangalen and Feiters, 2016).

1.5.0 Phytochemistry

The use of herbs has been a matter of antiquity; however, it was around the 19th century that concerted efforts were made to identify the active principles that are responsible for one activity or the other (Ameyaw and Duker-Eshun, 2009). The significance of medicinal plants is  directly linked  to  the  wide  range  of  chemical  compounds  synthesized  in  the various biochemical pathways. These compounds are classified as secondary metabolites (Ameyaw and Duker-Eshun, 2009). Secondary metabolites, are small molecules (mol wt C 1500 amu approx) produced by an organism but that are not strictly necessary for the survival of the organism, unlike the more prevalent macromolecules such as proteins, nucleic acids, and polysaccharides that make up the basic machinery for the more fundamental processes of life. The importance of natural molecules in medicine lies not only in their pharmaceutical or chemotherapeutic effects but also in their role as template molecules for the production of synthetic drugs. Most anti-malarial drugs currently in use are quinoline derivatives modeled on the quinine molecule (Ameyaw and Duker-Eshun, 2009). There are three large classes of secondary metabolites in plants: Nitrogen-containing compounds, terpenoids and phenolics (Harborne, 1978). Some of these phytochemicals are alkaloids, flavonoids, saponin, resins, and tannins.

1.5.1 Alkaloids

Alkaloids are defined as a group of naturally occurring chemical compounds that contain mostly basic nitrogen atom, though some related compounds with neutral and even weakly acidic  property  are  included  (Manske,  1965).  Besides  carbon,  hydrogen  and  nitrogen, alkaloids may also contain oxygen, sulfur, chlorine, bromine and phosphorus (Robert, 1998). Many alkaloids are purified from crude extracts by acid-base extraction and are toxic to other organisms.  They  have  medical  significance  and  are  used  as  anesthetic  and  stimulant. Examples of alkaloids that are used as stimulant are cocaine, caffeine and nicotine. Morphine serves as an analgesic while berberine has antibacterial potency. Vincristine and reserpine act as anticancer and antihypertension agents respectively. Others are the cholinomimetic principle  known  as  galantamine,  the  spasmolysis  agent  called  atropine,  the  vasodilator referred   to   as   vincamine,   the   antiarhythmia   compound   known   as   quinidine,   the hypoglycaemic principles referred to as guanidine, piperidine alkaloids, the anti-asthma therapeutic ephedrine and the popular anti-malarial drug known as quinine. Alkaloids have characteristically bitter taste (Rhoades, 1979). Some authorities argue that amino acids, peptides, proteins, nucleotides, nucleic acid –though nitrogen containing compounds are not alkaloids,  but  some  researchers  refer  to  alkaloids  as  special  amines  (McNaught  and. Alkaloids have pharmacological effects and biological activities such as anti-malarial, anti- microbial, anti-hyperglycemic and anti-inflammatory effect and are used as medications, recreational drugs,  or in etheogenic rituals (Tackie and Schiff, 1993)  besides the above mentioned potencies. Tiong et al. (2013) reported that alkaloids showed improved uptake of glucose and inhibited tyrosine phosphatase activity implying good potency against type 2 diabetes mellitus. There is, also the non-basic forms such as quaternary compounds and N- oxides (Ameyaw and Duker-Eshun, 2009). Many alkaloids are poisonous to other organisms.

1.5.2 Flavonoids

Flavonoids are defined as plants’ pigments that have a structure based on or similar to that of flavones. They are among the antioxidants produced by plants for their own sustenance (Ali et al., 2010). Various solvents of varying polarities are employed in the extraction of flavonoids. Dichloromethane, chloroform, diethylether, or ethyl acetate could be used for less polar flavonoids like isoflavones, flavanones, methylated flavones, and flavonols, while more polar flavonoids are extracted using alkanol or alkanol-water mixture (Markston and Hostettmann, 2006). Flavonoids have hydroxyl group (OH). The effect of hydroxyl moiety of flavonoids on protein targets varies depending on position and number of the moiety on the flavonoid skeleton (Hyunchu et al., 2010). Flavonoids are a class of phytochemicals that have various biological activities. They are classified into (a) flavones from 2-phenyl chromen-4- one (2-phenyl-1-4-benzophyrore) structure (examples:  quercetin,  rutin)  (b) isoflavoriouds from 3-phenyl chromen-4-one (3-phenyl-1, 4-benzopyrone) structure (c) neoflavonods from

4-phenyl coumarine (4-phenyl-1, 2-bioflavonoid (McNaught and Wlkinson, 1997).

The bioavailability, metabolism and biological activity of flavonoids depend on the configuration, total number of hydroxyl groups and substitution of functional groups about their nuclear structure. Fruits and vegetables are the main dietary sources of flavonoids for humans,  along with  tea  and  wine.  Flavonoids  have been  attributed  to  have antioxidant, antibacterial,  antiviral and anticancer among others. They can  prevent fat oxidation and protect vitamins and enzymes. They can lower blood low density lipoprotein cholesterol  and blood  pressure,  thereby  protecting  the  body  against  cardiovascular  disease  (Agrawal,

2011;Mishra et al., 2013). Flavonoids have anti-inflammatory, antithrombogenic, antiosteoporetic, antiviral, antibacterial and antifungal effects. They are known to inhibit

aldose reductase, xanthine oxidase, phosphodiesterase, Ca2+ ATPase, lipoxygenase, cyclooxygenase and adenosine deaminase (Agrawal, 2011). Salib et al. (2013) reported antidiabetic property of flavonoids.

1.5.3 Tannins

Tannins are naturally occurring plant polyphenols. They bind and precipitate proteins. They form complexes, also with carbohydrates, bacterial cell membranes and enzymes involved in protein and carbohydrate digestion (Reed, 1995). The tannin phenolic group is an excellent hydrogen donor that forms strong hydrogen bonds with the protein’s carboxyl group (Reed,

1995). The anti carcinogenic and anti mutagenic potentials of tannins may be related to their antioxidant property (Chung et al., 1998). The anti-microbial properties of tannins are associated with the hydrolysis of ester linkage between gallic acid and polyols hydrolyzed after ripening of many edible fruits (Chung et al., 1998). Tannins impact immensely on animal  nutrition  in  that  they  complex  with  carbohydrates,  proteins,  polysaccharides), bacterial cell membranes and enzymes involved in protein and carbohydrates digestion. Tannins are giant molecules and therefore form good binding with proteins (which are rich in protein. These bonds between tannins and proteins are hydrophobic and hydrogen bonds (Reed, 1995).

1.5.4 Total Phenolics

Phenols are defined as phytochemicals that have a benzene ring and atleast one hydroxyl group. Examples are monophenolics, diphenolics and polypenolics. Most tannins and flavonoids are phenolics but some phenols are neither tannins nor flavonoids for example xanthone (Harbone, 1978). Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids (Kahkonen et al., 1999). Antioxidant activity of plant extracts is not limited to phenolics. Crude extracts of fruits, herbs, vegetables, cereals and other plant materials rich in phenolics are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional  value  of  food  (Shahidi  et  al.,  1992).  Phenolics  are  the  main  antioxidant components of food. While in plants, oils are basically monophenolics such as tocopherols, water-soluble polyphenols are more typical in water-soluble products like fruits, vegetables, tea, coffee, wine, among others (Roginsky and Lissi, 2004). Polyphenolic compounds are known to have antioxidant activity. This activity is believed to be mainly due to their redox

properties which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Ali et al., 2010).

1.5.5 Saponin

Saponins are defined as glucosides that occur in plants and are characterized by producing a soapy lather. They are classified into two: first on the basis of the soap-like foaming they produce when shaken in aqueous solutions and second on the basis of structure (by their composition of one or more hydrophilic glycoside moieties in combination with lipophilic triterpene derivatives (Hostettman and Marstton, 1995). The aglycone parts of saponins are called sapogenines (Hostettmann, and Marston, 1995). These classes of phytochemicals can cause haemolysis, serve as surfactants and adjuvant for vaccines. While some are poisonous, some increase and accelerate calcium and silicon absorptions (Cornell University, 2013).

Saponins have been reported to bring about the secretion of insulin from beta-cells, ameliorating oxidative stress and  reduction of advanced  glycation end  products, thereby playing a role in antidiabetic potency (Elekofehinti, 2015).

1.5.6 Terpenoids

Terpenoids are defined as any of the class of hydrocarbons that consist of terpenes attached to an oxygen-containing group. They are widely found in plants and can form cyclic structures such  as  sterols.  Plant  terpenoids  play  some  important  roles  in  ethnopharmacological practices. Their antibacterial, antineoplastics and other therapeutic benefits have been investigated (Nita et al., 2014).

Terpenoids   are   classified   as   monoterpenoids   (C10H16),   sesquiterpenoids   (C15H24), diterpenoids (C20H32), sesterpenoids (C25H40), troterpenoids (C30H48), tetraterpenoids (C40H64) and polyterpenoids (C5H8)n (Nita et al., 2-014). Croteau et al. (2000) reported that terpenes form 55% of the three major groups of plant secondary metabolites. Based on their numbers and diversity, terpenes have much potential in industry and medicine. Pharmaceutical and food industries  have  exploited terpenes  for their  effectiveness  as  medicines  and  flavour enhancers (Zwenger and Basu, 2008). Shori, (2015) has reported that terpenoids have antidiabetic property.

1.5.7 Steroids

Steroids have been defined as compounds possessing the skeleton of cyclopenta [α] phenanthrene  or  a  skeleton  derived  there  from  by  one  or  more  bond  scissors  or  ring expansions or contractions. Methyl groups are normally present at C-10 and C-13 and most of the skeleton of chostane (Moss, 1989). Examples of steroids and sterols are cholesterol, ergosterol,  found  in  yeast  and  many  fungi  and  fucosterol  found  in  coconut.  Three phytosterols  are sitosterol,  stigmasterol  and  campesterol  (Harborne,  1978). Some steroid derivatives may induce changes on glucose levels (Figueroa-Valverde et al., 2014).

Steroids are known for tackling inflammation and are immune-modulating agents (Ericson- Neilsen  and  Kaye,  2014).  Some  side  effects  of  the  use  of  steroids  are  acne,  Cushing syndrome that can result in diabetes mellitus and potentially life threatening heart disease if untreated (Ericson-Neilsen and Kaye, 2014).

1.5.8 Essential Oils

Essential oils, have been known to man for several centuries. Different uses have been employed ranging from use in perfumery to flavoruing, to food and beverages or therapeutics (Baris et al., 2006).

Essential oils have excellent bioavalaibility. Their oral administration does not inhibit their biological action. They are absorbed and even cross blood –brain-barrier. Subsequently they effect  their  different  pontencies  such  as  relaxation  digestion,  sleep  among  others.  They mostly undergo conjugation before elimination via the lungs or kidneys and thus pose less risk of accumulation (Kohlert et al., 2000).

Essential oils have been shown to posses various pharmacological potencies such as antimicrobial, antioxidant and antidiabetic, among others (Yen et al., 2015, Bakkali, et al., 2008, Hsieh et al., 2011).

1.6.0 Diabetes

Diabetes mellitus is a chronic disease caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. Such a deficiency results in increased concentrations of glucose in the blood, which in turn damage many of the body’s systems, in particular the blood vessels and nerves (WHO, 2018).

Diabetes  is  one  of  the  common  endocrine  metabolic  disorders.  There  are  two  types  of diabetes mellitus. Type 1 called insulin dependent diabetes mellitus (IDDM) while type 2 is known as non-insulin dependent diabetes meliitus (NIDDM).   Types I and II diabetes are characterized by hyperglycacmia, abnormal lipid and protein metabolism (Nathan, 1993). Insulin-dependent diabetes mellitus is an autoimmune disease. Insulin is absent in this type of diabetes. This type peaks at about 12 years of age (Nester et al., 2004). This informs the term

‘juvenile-onset’ diabetes. These patients therefore need daily insulin injections. In this condition,  although  auto-antibodies  are  present,  the  major  damage  is  destruction  of  the insulin-producing cells of the pancreas by infiltrating cytotoxic T-cells (Nester et al., 2004). Oral hypoglycaemic agents are prescribed for type II. This is so because insulin resistance is prominent here. Insulin may be available in enough quantity but its secretion, receptance, and utilization might have been totally or partially compromised (Nester et al., 2004). The oral hypoglycaemic medications available, currently are sulfonylureas like chlorpropamide, tolbutamide, glibenclamide, glipizide, gimepiride, among others; biguanides such as phenformin and metformin; α- glucosidase inhibitors such as acarbose and meglitol; sodium- glucose co-transporter 2 (SGLT 2) inhibitors, like canagliflozin, among others (Ukwe, 2005; Wadkar et al., 2008 and Katzung et al., 2009). The findings of Yamamoto et al. (2018) indicated that modification of diets has been reported to decrease post-prandial glucose level and reductions in medications among diabetics. Furthermore, they reported that modified dietary interventions favourably influenced outcomes related to maternal glycaemia and birth weight. This is so since diabetes mellitus tend to modulate the weights of neonatals.

1.6.1 Insulin

Insulin, an endocrine hormone is produced in the beta-cells of the islets of Langerhans in the pancreas. It is composed of two peptide chains (alpha & beta) connected by two disulfide bridges, its amino acid sequence was determined by Sanger and his co-workers in 1955 (Nelson and Cox, 2005; Ukwe, 2005). The alpha chain has 21 and the beta chain has 30 amino acid residues. Granules within the β-cells store the insulin in the form of crystal consisting of two atoms of zinc and six molecules of insulin. In response to a variety of stimuli, especially glucose, the granules adjacent to the beta-cells membrane fuse with it and empty their contents into the pericapillary space. Insulin is then liberated and enters the capillary circulation in solution. Nelson and Cox, (2005) stated that insulin receptor consists of two α-chains on the outer face of the plasma membrane and two β-chains that transervese the membrane and protrude from the cytoplasmic face.

1.6.2 Induction of Diabetes

Some substances like streptozotocin and alloxan are used to induce diabetes mellitus in albino wistar rats. The potency of alloxan to induce diabetes in laboratory animals lies in its ability to selectively kill insulin-producing beta cells found in the pancreas (Loreto and Elina,

2009; Danilova et al., 2014). This occurs most likely because of selective uptake of the compound due to its structural similarity to glucose as well as the beta-cells’ highly efficient uptake mechanism (GLUT 2). The three major stimuli for β-cells’ apoptosis as adduced by Hui et al, (2004) are cytokines, lipotoxicity and gluco-toxicity. Cytokines are known to be inflammatory mediators. They give signals to T-cells (T-lymphocytes) to come for an attack. The T-lymphocytes then attack the β-cells since β-cells are seen as antigen and would be attacked leading to apoptosis. In addition, alloxan has a high affinity to SH- containing cellular compounds and, as a result, reduces glutathione content (Szkudelski, 2001). Furthermore, alloxan inhibits glucokinase an SH-containing protein essential for insulin secretion induced by glucose (Szkudelski, 2001). Alloxan therefore disrupts the required processes for insulin metabolism. It is an endocrine disruptor. There is accumulating evidence suggesting that the increased presence of endocrine-disrupting chemicals in the environment may, also explain an essential component in the incidence of metabolic diseases such as obesity and type-2 diabetes mellitus.

1.6.3 Glycations in Diabetes

Hyperglycaemia is also observed to be pathogenetic. This condition could accelerate atherogenesis and increase chronic heart disease via these mechanisms: glycation of collagen, other vessel-wall proteins and lipoproteins; accelerated generation of reactive oxygen species and hightened oxidative stress on glycated end products, LDL-cholesterol and vascular endothelial cells; alteration in haemorrheological characteristics or changes in vascular reactivity (Brownlee, et al., 1988; Guiliano et al., 1996). Irregularities in different cellular processes have been implicated in diabetic patients. Direct effects of hyperglycaemia, glycation processes formation of advanced glycation products and increased modification through the polyol and hexosamine pathways have all been described in the pathogenesis of diabetic nephropathy. Oxidative stress has been shown to be the linking factor among these different factors. Oxidative stress represents an abnormality in the mitochondrial electron transport chain resulting in increased production of reactive oxidative stress molecules that modulate each of the above pathways (Brownlee, 2001, Sahib, 2009). The three major stimuli

for β-cells’ apoptosis as adduced by Hui et al, (2004) are cytokines, lipotoxicity and gluco- toxicity; cytokines are known to be inflammatory mediators.

1.6.4 Epidemiology of Diabetes Mellitus

Diabetes mellitus is a common major degenerative and very prevalent disease affecting and afflicting the citizens of both developed and developing countries today). It is estimated that

25% of the world population is affected by this disease (Mbaka et al, 2012 and Arumugam et al.,  2013). Diabetes  is  a  deadly  disease  that  affected  an  estimated  285  million  people worldwide in 2010; the number is increasing in rural and poor populations throughout the world and is projected to become one of the world main disablers and killers within the next

25years (Shaw, et al., 2010). Environmental and lifestyle changes resulting from industrialization and migration to urban environment from rural settings are speculated to be possible reasons for the observed rise from 409 million in the year 2000 to estimated 69.9 million by 2025 in Asian pacific (India) region (Sicree et al., 2006; Mohan and Pradeepa

2009). Though diabetes is a non-communicable disease, it is considered to be one of the five leading causes of death in the world (Chakraborty and Das, 2010). Recent data indicate that more than 371 million people have diabetes mellitus and 4.8 million people died, due to diabetes mellitus and these figures are likely to double by 2035 (IDF, 2013).

Large countries such as India, China and the United States of America, are presently the countries  with  the  leading  number  of  diabetics.  United  States  has  seven  percent  of  its residents that are diabetics (Wei et al., 2015). Globally, an estimated 422 million adults are living with diabetes mellitus according to the latest 2016 data from the World Health Organisation (WHO). The latest estimates are 451 and 500 million for 2017 and 2018 respectively. Previous epidemiological survey presented 381 million in 2013. Types 2 was said to account for 85-90% of all cases. The disease was reported to be more common in the more developed countries. The same World Health Organization alleged that 1.5 million death cases occurred in 2012 thereby making diabetes the 8th  leading cause of death. They estimated 1.707 million cases for Nigeria in the year 2000 and projected an astronomical increase to 4.835 million in 2030, while African continent had 7.02 million in the same year 2000 and a projection of 18.234 million in 2030.

1.6.5 Complications in Diabetes

The long term effects of diabetes mellitus include progressive development on the specific complications of retinopathy, nephropathly, and/or neuropathy (Nathan, 1993). The major goals in the treatment of diabetes has been to keep both short-term and long-term glucose levels within acceptable limits, thereby reducing the risk of long term complications (Park et al., 2009). This could be achieved by optimizing both fasting blood glucose and postprandial glucose levels which have been found to be very important in achieving near normal glucose levels (Emordi et al., 2015). Postprandial glucose levels have been reported to serve as a better marker of glycaemic control than fasting blood sugar levels (Park et al., 2009). The disease is a complex metabolic disorder of the endocrine system with dynamic expression of pathological disequilibria, resulting in various micro and macro vascular complications (Wei et al., 2015).

1.6.6 Risk Factors of Diabetes Mellitus

Different factors predispose to diabetes mellitus. Excessive food consumption regarding to high calorie, obesity, cardiovascular disease, stress, and lack of exercise are risk factors for diabetes mellitus (Porika, and Estari, 2015). Obesity as one of the predisposing factors in this case, is because lipid cells or adiposities tend to resist smooth glucose metabolism in obessed individuals than non-obesed individuals. Murray et al, (2003) had reported that diabetes has a genetic component. They suggested that distortions to cellular functions that lead to diabetes were caused by genetic mutations. Other predisposing factors include sedentary lifestyle and tobacco smoking-which may be as a result of the oxidative effects of tobacco smoke. Nelson and Cox (2005) had also implicated mutation in the mitochondrion. The mutation in the mitochondrial lysyl-tRNA gene has also been implicated as a possible cause of adult onset (type 11) diabetes mellitus.

1.6.7 Plants with Antidiabetic Properties

Different plants of different species have been mentioned to have anti-diabetic, hypoglycaemic, anti-hyperglycaemic or anti-hyperlipidemic potency. They are Albizia odoratissima, Alangium lamerckii, Axonopus compressus, Berberis vulgaris, Caesalpnia digyna, Catharanthus roseus, Brassssica jincea, Centaurium erythrea, Chaenomeles sinensis, Cocos nicifera,  Costus speciosus,  Cyclocarya paliunus.   The report of Arumugam et al.

(2013) seemed to suggest that among the different solvents used in extracting the active principles the most of the active anti-diabetic phytochemicals were extracted using polar solvents like methanol, aqueous, ethylacetate, hydro-methanol, hexane, pet ether, n-butanol, ethanol  extracts  among  others.    However,  the  featuring  of  n-hexane  and  ethylacetate suggested diversity in polarity and nature of herbal anti-diabetics.

Table 1: Some plants with antidiabetic/hypoglycaemic potencies

Plant name	Family	Parts used	Type of extract	Activity
Alangium lamerckii	Alangiaceae	Leaves	Alcoholic	Antidiabetic
Albizia odoratissima	Mimosaceae	Bark	Methanol	Antidiabetic
Axonopus compressus	Poaceae	Leaves	Methanol	Antidiabetic
Berberis vulgaris	Berberidaceae	Root	Aqueous	Hypoglycaemic
Brassssica jincea	Cruciferae	Seed	Aqueous	Hypoglycaemic
Caesalpnia digyna	Fabaceae	Root	Methanol	Antidiabetic
Catharanthus roseus	Apocynaceae	Leaf	Methanol	Antidiabetic
Centaurium erythrea	Gentianaceae	Leaf	Aqueous	Antidiabetic
Chaenomeles sinensis	Rosaceae	Fruits	Ethylacetate	Antidiabetic
Cocos nicifera	Arecaceae	Leaf	Hyoro-methanol	Antihyperglycemic
Costus speciosus	Costaceae	Rhizome	Hexane	Antidiabetic
Cyclocarya paliunus	Cyclocaryaceae	Bark	Aqueous, pet ether, ethylacetate, butanol	Hypoglycemic
Dillenia indica	Dilleniaceae	Leaves	Methanolic	Antidiabetic
Embelia ribes	Myrsinaceae	Berries	Hexane	Antidiabetic
Hybanthus enneaspermus	Violaceae	Whole plant	Alcoholic	Antidiabetic
Lippa nodiflora	Vernenaceae	Whole plant	Methanol	Antidiabetic   hypolipidemic
Lithocarpus polystachyus	Fagaceae	Leaves	Ethanol             and   Aqueous methanol	Hypoglycemic
Marrubuim vulgare	Lamiaceae	Arial part	Methanol	Hyperglycemia   dispidemia
Cassia auriculata	Caesalpiniaceae	Leaves	Aqueous	Antihyperglycemic
Vitex nejundo	Lamiaceae	Leaves	Methanol	Antihyperglycemic
Solanum xauthocarpum	Solanaceae	leaves	Aqueous            and methanol	Antihyperglycemic

Source: Arumugam et al., (2013)

1.7.0 Herbal Medicine

Herbal medicines may be safer than synthetic medicines because of their phytochemicals that target the biochemical pathway (Kirtikar et al., 1975). Medicinal plants have been used all over the world for the treatment and prevention of various ailments, particularly in developing countries where infectious diseases are endemic and modern health facilities and services are inadequate (Kirtikar et al., 1975; Leung, 1980).

1.7.1 Herbal Medicine in Treating Diabetes Mellitus

Plants have played a significant role in the introduction of new therapeutic agents. Metformin was discovered from Galega officinalis, a medicinal plant (Ezeigbo and Asuzu, 2012). In Nigeria traditional medicine occupies a unique position in the health care delivery especially among the rural populace. However, the activities of herbalists are surrounded with a lot of secrecy and lack of scientific procedure, hence the need to standardize the practice of traditional medicine (Ezigbo and Asuzu, 2012).

In Africa, hundreds of plants are used traditionally for the management of diabetes mellitus. Up to date, however, only a few of these African medicinal plants have received scientific scrutiny, (Njike et al., 2005) despite the fact that the World Health Organization has recommended that medical and scientific examinations of such plants should be undertaken (WHO, 1980 Njike et al., 2005). The use of herbal medicines for the treatment of diabetes mellitus has gained importance throughout the world and there is an increased demand to use natural products with antidiabetic activity due to the side effects associated with the use of insulin and oral hypoglycemic agents (Porika and Estari 2015). In Nigeria it seemed that some bitter herbs are employed  as  sauce  for  diabetics  giving  more  credence  to  the  use  of  alternative  medicine. Diabetes mellitus has recently been identified by Indian Council of Medical Research (ICMR) as one of the refractory diseases for which satisfactory treatment is not available in modern allopathic system of medicine and suitable herbal preparations are to be investigated.

1.7.2 Mechanisms of Actions of Orthodox/Herbal Antidiabetics: Overview

Some of the modes of reduction of hyperglycaemia effects  are insulinomimetic and insulin secretagogues activity (Patel, et al., 2012). The mechanisms of action of herbal antidiabetics include adrenomimeticism, pancreatic β-cell potassium channel blocking, cAMP (second messenger) stimulation. This is obvious since cAMP amplifies signals that are received at the cell membrane. Other modes of actions are inhibition in renal glucose reabsorption, stimulation of insulin secretion from β-cells of islets of Langerhan and inhibition of insulin degradative processes. Noteworthy is the fact that reduction in insulin resistance, providing certain necessary elements like Ca, Zn, Mg, Mn and Cu for the β-cell, regenerating and/or repairing pancreatic β- cell, are also other mechanism of actions of anti-diabetic agents. Increasing the size and number of cells in the islets of langerhans, stimulation of glycogenesis and hepatic glycolsis, protective effect on the destruction of the β-cell, improvement in digestion along with reduction in blood sugar and urea also represent other modes of actions of anti-diabetics. Prevention of pathological conversion of starch to glucose inhibition of β-galactosidase and α-glucosidase, cortisol lowering activities, inhibition of α-amylase, and preventing oxidative stress are employed by anti-diabetic herbs in ameliorating hyperglycaemia (Creutzfeldt and Soling, 1961; Katzung et al., 2009).

1.8.0 Free Radicals in the Pathology of Diabetes

In diabetes, oxidative stress has been found mainly due to an increased production of oxygen free radicals and a sharp reduction of antioxidant defenses (Oberly, 1988). Reactive oxygen species (ROS), in particularly free radical induced lipid peroxidation, cause tissue damage that has been implicated in the pathogenesis of various diseases including diabetes (Templar et al., 1994; Ozben and Nacitarhan 1995). These radicals further damage other important biomolecules including carbohydrates, proteins and deoxyribonucleic acid (DNA) (Baynes, 1991). Increased blood glucose levels in diabetes produce superoxide anions, which generate hydroxyl radicals via Haber-Weiss reaction, resulting in peroxidation of membrane lipids and protein glycation. This leads to oxidative damage of cell membranes. Subsequently, free radicals change lipid/protein ratio of membranes by affecting polyunsaturated fatty acids and lipid peroxidation and cause functional irregularities of several cellular organelles (Yagi, 1984; Cheesman and Slater 1993). Lipid peroxides are disintegrated quickly and form reactive carbon compounds, among these, malondialdehyde (MDA) is an important reactive carbon compound which is used commonly as

an indicator of lipid peroxidation, and has become one of the widely reported analytes for the purpose of estimating oxidative stress effects on lipids (Gallou et al., 1993; Jacob and Burri

1996). Since free radical production is increased whereas capacity of antioxidant system is reduced in diabetes, it has been proposed that diabetic patients may require more antioxidants compared to healthy individuals (Langenstroer and Pieper 1992; Sundaram et al., 1996). Since the effect of free radicals in diabetes is now documented, it has been proposed to use antioxidant vitamins to block formation of free radicals and hence prevent development of diabetes complications (Ceriello et al., 1997).

1.8.1 Glutathione Peroxidase and Superoxide Dismutase in Diabetes Mellitus

Antioxidants are agents that protect, prevent, or reduce the extent of oxidative damage to biomolecules.  These  agents  may  be  enzymatic,  non-enzymatic,  or  metal  chelators.  The enzymatic   antioxidants   include   catalase,   superoxide   dismutase   (SOD),   and   glutathione peroxidase (GPx). Superoxide dismutase, a copper, zinc and manganese-containing enzyme, reacts with superoxide radical to form hydrogen peroxide, which is then converted to water by GPx (a glutathione-dependent selenoprotein), or catalase, a heme enzyme. Decreased activity of these antioxidant enzymes may increase the susceptibility of diabetic patients to oxidative injury (Hisalkar et al., 2012).

Increased levels of the products of oxidative damage to lipids and protein have been detected in the  serum  of  diabetic  patients  and  their  presence  correlates  with  the  development  of complications (Brownlee, 2001). Different studies have provided evidence of increased oxidative stress with depleted antioxidant enzymes and vitamins in both type 1 and 2 diabetes (Lapolla et al., 2007;  Lodovici et  al., 2008;  Likidlilid et al., 2010; Al-Rawi, 2011). Hyperglycemia, a hallmark of diabetic condition, depletes natural antioxidants and facilitates the production of ROS, which has the ability to react with all biological molecules like lipids, proteins, carbohydrates, DNA and exert cytotoxic effects on cellular components (Dincer et al., 2002). Thus, increased ROS and impaired antioxidant defense contribute to initiation and progression of micro- and macrovascular complications in diabetics (Ceriello et al., 1998; Ceriello and Motz,2004).

Several studies have reported lower concentrations of non-enzymatic antioxidants as well as enzymatic antioxidants in type 2 diabetes (Lodovici et al., 2009; Likidlilid et al., 2010; Bigagli et al., 2012). Superoxide dismutase is a natural occurring enzyme that protects the body against active oxygen free radicals by scavenging excess superoxide. Lower undetectable levels of superoxide dismutase allow oxygen radicals to form in anaerobic bacteria and to inactivate other bacterial enzyme systems. Superoxide dismutase has particular value as an antioxidant that can help to protect against cell destruction (Math et al., 2004). The glutathione peroxidase enzyme takes part in a system that converts intracellular free radicals into less reactive or neutral components (Mahendra et al., 2014).

Murray et al, (2006) further stated that the transfer of a single electron from substrate to O2 generates the potentially damaging superoxide anion free radical (O2-.) via electron leakage. Electron leakage also occurs when NADPH-cytochrome P450  reductase reduces oxidized FAD+ to FADH2, the destructive effects of which are amplified by its giving rise to free radical chain reactions. This is shown in Figure 2. The ease with which superoxide can be formed from oxygen in tissues and the occurrence of superoxide dismutase, the enzyme responsible for its removal indicate that the potential toxicity of oxygen is due to its conversion to superoxide.

1.8.2 Catalase

Catalase is  a common  enzyme found in  nearly all  living organisms  exposed  to  oxygen.  It catalyzes the decomposition of hydrogen peroxide to water and oxygen (Chelikani et al., 2004). It is very much needed in the protection of cells from oxidative damage by reactive oxygen species (ROS). It also has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each second. Catalase is a tetramer of four polypeptide chains, each over 500 amino acids long (Boon et al., 2007). It contains four porphyrin heme (iron) groups that allow the enzyme to react with H2O2. Human catalase is reported to have optimum PH of 7 (Maehly and Chance, 1954) and has a fairly broad maximum between ρH 6.8 and 7.5 (Aebi, 1984). The reactivity of the iron centre may be improved by the presence of the phenolate ligand of Tyr 357 in the fifth iron ligand which facilitates the reduction of hydrogen peroxide (Boon et al., 2007). A catalase deficiency may increase the likelihood of developing type 2 diabetes mellitus.

1.8.3 Lipid Peroxidation in Diabetes Mellitus

Oxidative stress, through the production of reactive oxygen species (ROS), has been proposed to be the unifying link between the various molecular disorders underlying the development of insulin resistance, β-cell dysfunction and impaired glucose tolerance leading to the development of type 2 diabetes mellitus (Santini et al., 1997; Ceriello and Motz, 2004). Oxidative stress, secondary to persistent hyperglycaemia and dyslipidemia, play key role in the pathogenesis of type 2 diabetes mellitus (T2DM). They also play a major role in the complications of T2DM by excess ROS generation, auto-oxidation of glucose, non enzymatic protein glycosylation, lipid peroxides formation, impaired glutathione metabolism, impaired activities of antioxidant defence enzymes and decreased concentrations of low molecular weight antioxidants. Malondialdehyde (MDA), as TBARS is frequently used to determine the prooxidant/antioxidant balance in type 2 diabetic patients as they are stable and easily measurable lipid peroxidation products (Kumari and Sankaranarayana, 2014). Type 2 diabetes mellitus is a chronic progressive disease, characterised by hyperglycaemia and dyslipidemia, causing an increased susceptibility of cells to lipid peroxidation and inflammation due to oxidative stress which plays a major role in the pathogenesis of diabetes and its complications.  Poor metabolic control and dyslipidemia in

1.9 Role of the Liver in the Metabolism of Drugs

Liver is a very vascular glandular organ of vertebrates that secretes bile and causes important changes in many of the substances contained in the blood. It plays very important roles in metabolism such as gluconeogenesis, glycogenesis, storage of glycogen, storage of blood and metabolism of xenobiotics (Nelson and Cox, 2005). Alanine aminotransferase (ALT) is known as L-alanine 2-oxoglutarate aminotransferase (EC 2.6.1.2). The enzyme is also called pyruvate aminotransferase or glutamate pyruvate aminotransferase. It was formally known as alanine transaminase. It is an intracellular cytoplasmic enzyme. It is widely distributed throughout the body’s tissues with the greatest amounts in the liver and the kidneys. Its plasma half-life is approximately 48 hours. It catalyzes the transfer of amino group from alanine to 2-oxo-glutarate forming glutamate. It is a key enzyme in gluconeogenesis. Increased quantity in the plasma is due to tissue damage particularly hepatic damage. Its quantities are measured by the activity of the enzyme (William, 2013).

Aspartate aminotransferase (AST, EC 2.6.1.1) is an enzyme found mainly in the liver, it is also found in red blood cells, heart, muscle tissue and other organs, such as the pancreas and kidneys (Xing-Jiu, 2006). It is found alongside ALT and both are important in the diagnosis of liver disease and integrity. The normal concentrations in the blood are from 5 to 40 U/L for AST and from 5 to 35 U/L for ALT. However, when body tissue or an organ such as the liver or heart is diseased or damaged, additional AST and ALT are released into the blood stream, causing levels of the enzymes to rise. This implies that the level of ALT or AST in the blood is a direct function of the degree of the liver or tissue damage (Xing-Jiu, 2006).

Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters in an alkaline environment resulting in the formation of an organic radical and inorganic phosphate. In mammals, this enzyme is found mainly in the liver and bones. Marked increase in serum ALP has been associated with malignant biliary obstruction, primary biliary cirrhosis, hepatic lymphoma and sarcoidosis (Wan, 2007).

Ajiboye et al. (2010) observed a significant serum increase in ALP after 28 days administration of aqueous root extract of Securidaca longepedunculata. Dapar et al. (2007) reported elevated levels in alanine aminotransferase and aspartate aminotransferase on the serum of animals treated with Securidaca longepedunculata. Ajiboye et al. (2010) further stated that aqueous root extract of Securidaca longepedunculata caused decreased activities of superoxide dismutage, catalase and glutathione peroxidase in the liver and kidney of rats. They also noticed increase in the concentration of malondialdehyde.

1.10 Role of the Kidneys in the Diabetes Conditions

The kidneys are a pair of small bean-shaped organs in the body that take away waste substances from the blood. Urea cycle is partly completed here. Reabsortion of water, glucose and minerals occurs here (Nelson and Cox, 2005).Yakubu, et al. (2010) reported elevated creatinine and urea levels in diabetic, but untreated animals (rats) and a reversal of elevated creatinine and urea to normal levels in glibenclamide and Cochlospermum planchonii aqueous root extract. Kiberd, (2006) found out that diabetes is the leading cause of established renal failure in the western world. One of the most important determinants of kidney function is level of cretinine. The value is estimated by the rate of production of creatinne which is a function of muscle mass (Thomas and Gallagher, 2007). It is recommended that people with diabetes are at a risk of renal disease. This can slow down the progression of chronic kidney disease; by strict blood pressure and blood sugar control have prescribed medicines that modify the reninangiotensin system and lifestyle changes (Thomas, 2009).

1.11 Disaccharidase A2 Activity

Disaccharides are carbohydrates that are made up of two monosaccharide units. Examples are sucrose, maltose and lactose. Disaccharidases are the enzymes that convert these disaccharides to monosaccharides. Competitive inhibitors of the intestinal α-glucosidase: sucrase, maltase, glucoamylase, dextranase and isomaltase function by delaying the digestion and absorption of starch  and  disaccharides.  Only  monosaccharides,  such  as  glucose  and  fructose,  can  be transported out of the intestinal lumen and into the bloodstream. Complex starches, oligosaccharides,  and  disaccharides  must  be  broken  down  into  individual  monosaccharides before being absorbed in the duodenum and upper jejunum. This digestion is facilitated by

enteric enzymes, including pancreatic α-amylase and α-glucosidases that are attached to the brush border of the intestinal cells Katzung et al, (2009). This enzyme inhibition is to minimize upper intestinal digestion and slow digestion (and thus absorption) of the ingested starch and disaccharides to the distal small intestine, thereby lowering post-meal glucose as much as 45-60 mg/dl and creating an insulin-sparing effect (Katzung et al., 2009).

1.12 Transports across Intestine

The lipid bilayer of biological membranes is intrinsically impermeable to ions and polar molecules. Permeability is conferred by two classes of membrane proteins- pumps and channels. Pump action is an example of active transport. Channel action illustrates passive transport, or facilitated diffusion (Berg et al., 2002). In the small intestine potassium can be absorbed via active transport. Monovalent ions are absorbed with ease and in great quantities. However, sodium ion is a counter ion to potassium. Sodium is an extracellular fluid ion and the ions are more abundant outside while potassium ions are more inside the cell. Three moles of sodium are pumped inside in exchange of two moles of potassium ion that are pumped outside using the Na+-K+  ATPase. Glucose is transported by a sodium-glucose co-transport mechanism. In the absence of sodium transport through the intestinal membrane, virtually no glucose can be absorbed.  They  need  to  go  in  tandem.  Sodium  ion  can  be  transported  into  the  cell  from endothelial epithelial cells by either active transport or facilitated diffusion. Intestinal glucose combines simultaneously with the same transport protein that sodium uses and then both the sodium ion and glucose molecule are transported together to the interior of the cell. Thus, the low concentration of sodium inside the cell drags to the interior of the cell and along with it the glucose at the same time and from there to the blood (Guyton and Hall, 2006). In the small intestine, potassium can be absorbed via active transport. Monvalent ions are easily absorbed in great amount. Sodium, however, is a counter ion to potassium. Sodium is extracellular fluid ion, while potassium is more abundant inside the cell. Three moles of sodium ions are pumped in while two moles of potassium ions are pumped outside using Na+- K+ ATPase (Guyton and Hall, 2006).

The Na+-K+ ATPase converts the free energy of phosphoryl transfer into the free energy of a Na ion gradient. The ion gradient can then be used to pump materials into the cell, through the action of a secondary transporter such as the Na+-glucose symporter (Berg et al., 2002). Glucose is mainly transported alongside sodium ion. They have a symporter. This is illustrated in Figure

Table 2: Glucose transporters in human genome

Transporter	Tissue (s) where expressed	Gene	Role
GLUT1     GLUT2	Ubiquitous     Liver, pancreatic islets, intestine	SLC2A1     SLC2A2	Basal glucose uptake     In liver, removal of excess glucose from blood; in pancreas, regulation
GLUT3	Brain (neuronal)	SLC2A3	of insulin release     Basal glucose uptake
GLUT4     GLUT5     GLUT6           GLUT7     GLUT8     GLUT9     GLUT10     GLUT11	Muscle, fat, heart     Intestine, testis, kidney, sperm     Spleen, leukocytes, brain           Liver microsomes Testis, blastocyst, brain Liver, kidney Liver, pancreas     Heart, skeletal muscle	SLC2A4     SLC2A5     SLC2A6           SLC2A7     SLC2A8     SLC2A9     SLC2A10     SLC2A11	Activity increased by insulin Primarily fructose transport Possibly no transporter function –     –     –     –     –
GLUT12	Skeletal muscle, adipose, small     Intestine	SLC2A12	–

Source: Nelson and Cox, (2005)

1.13 Rationale of the Study

Diabetes mellitus is a non-communicable disease that is increasing in different populations. The current orthodox anti-diabetic drugs have their adverse effects, ranging from stomach upset, to excessive hypoglycaemic states. Plant materials are alternatives; however, the bio-principles identification, the mechanisms of action and toxicity profile remain a huge challenge. This study aims at investigating the possible mechanism(s) of action and toxicity profile of the active hypoglycaemic principle extractable from Securidaca longepedunculata leaves.

1.14.0 Aim and Specific Objectives

1.14.1 Aim: The study was designed to characterize the extractable hypoglycaemic principles from  Securidaca  longepedunculata  leaves  and  evaluate  their  mechanism  of  hypoglycaemic action and toxicity profile.

1.14.2 Specific Objectives: The study was designed to achieve the following objectives:

I.         To extract the phytochemical constituents of Securidaca longepedunculata leaves.

II.        To establish the hypoglycaemic potency of the Securidaca longepedunculata leaf- extact.

III.       To determine the possible mechanism(s) of action of the hypoglycaemic principles extractable from Securidaca longepedunculata leaves.

IV.      To determine the toxicity profile of the extractable hypoglycaemic principles from

Securidaca longepedunculata (MESL) leaves.

V.        To fractionate the crude 80% aqueous-methanol extract. VI.      To further purify the most active fraction. VII.     To   chemically   identify   the   compounds   in   the   active   fraction   using   gas chromatography- mass spectrophotometry (GC-MS).

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