VALIDATION OF PHARMACOLOGICAL AND PHYTOSYNERGIC ASPECTS OF POLYHERBAL ANTIDIABETIC AFRICAN TRADITIONAL MEDICINES

VALIDATION OF PHARMACOLOGICAL AND PHYTOSYNERGIC ASPECTS OF POLYHERBAL ANTIDIABETIC AFRICAN TRADITIONAL MEDICINES (ATMs) USED IN RIFT VALLEY
SALINA RUTTO
SC/PGC/037/13
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN ORGANIC CHEMISTRY
UNIVERSITY OF ELDORET
FEB 2018
CHAPTER ONE
BACKGROUND INFORMATION
Most communities depend on plants for medicine. Over 80% of the population in Africa depend largely on traditional medicine to meet their basic health care needs. (WHO,2008) Globally, nearly three quarters of drugs are derived from plants.(Mustafa et al,2010)
According to the World Health Organization, Phytomedicine is defined as herbal preparations produced by subjecting plant materials to extraction, fractionation, purification, concentration or other physical or biological processes. There preparations may be produced for immediate consumption or on the basis for other herbal products. Such plant products may contain recipient or inert ingredients, in addition to the active secondary metabolites (WHO, 2001).
In the past few years there has been tremendous growth in study in the field of herbal medicine and its products and is gaining popularity in developing and developed countries. The herbal medicine are extracted from different parts of the plant, such as leaves, barks, seeds and flowers and prepared from a single plant or a combination of different plant species(Savithramma et al,2011).This bioactive compounds has the ability to heal. These include saponins, tannins, essential oils, flavonoids, alkaloids and other chemical compounds found as secondary metabolites in plants(Pavirini et al,2012).Secondary metabolites are potential source of novel antibiotics, insecticides and herbicides. This is because they possess antioxidant, anti-aging, anti-atherosclerotic, antimicrobial and anti-inflammatory activities (Mulaudzi et al,2012) The mixture could be simple, that treats minor illnesses such as colds, stomach, headaches and menstrual pains or complex preparations that are used for chronic diseases like diabetes, tuberculosis, epilepsy and Cancer (Ndhalal et al,2011)
It is reported that about 74% of plant-derived drugs that are currently in the market were actually derived from the knowledge of traditional herbalist on ethno medicines (Farnsworth et al.,1985).For example, the anti malarial artemisinin originally isolated from the Chinese plant Artermisia annua L. enabled the synthesis of more potent and water soluble artemeter,arteether and sodium Artesunate (Rashmi et al., 2014)
ARTEMISIN

Artemeter

Arteether

Sodium Artesunate

Quinine was first isolated in 1820 from cinchona officinalis L bark by polletier and convention and large numbers of derivatives was synthesized to more potent drugs such as chloroquine, Mefloquine,quinidine,cincholine and cinchonidine (Cechinel-Filho,2012).

Quinine

Chloroquine

Melfoquine

Cinchonine

Cinchonidine

Diabetes
Diabetes is a metabolic disorder, a life-long disease that affects the way body handles glucose, a kind of sugar in the blood of which in this case is high in blood. over a prolonged period (March,2014). It takes long time for one to realize that he/she has this chronic disease which is a threat to mankind globally. It is predicted that the number of diabetes person in the world could reach upto 366 million by the year 2030 (International Diabetes Federation,2016)There are different types of diabetes mellitus namely;
Type 1 or Diabetic Insipidus. This type of diabetes starts in childhood .In this condition pancreas do not produce enough insulin or failure of insulin to work efficiently (WHO,2013)
Type 2 or Diabetes mellitus. This type of diabetes, the pancreas makes insulin that gets glucose into the cells to give energy. But eventually it cannot keep up and the sugar build up in the blood instead causing resistance(Jaypee et al,2012)
Gestational diabetes mellitus occurs when pregnant women have high blood glucose levels. This is due to hormones produced in pregnancy. Sometimes it disappears after delivery or develops prediabetic condition, or eventually to type 2 (Cash Jill,2014)
Rare types of diabetes mellitus such as MODY, LADA and secondary diabetes mellitus caused by pancreatic disease, drug side effects or endocrine disorders( March,2017)
Brittle diabetes. is a sub-type of type 1 diabetes. It is very rare (Monogenic Forms of Diabetes (March,2017)
Pre diabetes (march,2017)
CONVECTIONAL DRUGS
Change of lifestyle and exercise should be adhered to. Blood pressure should be controlled and proper foot care should be maintained. Hypoglycaemic treatments include drugs that (1) increase the amount of insulin secreted by the pancreas, (2) increase the sensitivity of target organs to insulin, and (3) decrease the rate at which glucose is absorbed from the gastrointestinal tract (WHO,2013).

Classes of hypoglycaemic agents
Insulin
Sensitizers
Biguanides
Thiazolidinediones
Lyn kinase Activators
Secretogogues
Sulfonylureas
Nonsulfonylurea secretogogues
Alpha-glucosidase inhibitors
Peptide analogs
Injectable incretin mimetics
Injectable Amylin analogues
Glycosurics
INSULIN
Is given as an injection subcutaneously or by an insulin pump and sometimes intravenously. It is safe to be used in pregnancy. There are three types of insulin depending on absorption rate in the body (Agabegi et al.,2008)These are;
Rapid acting insulin,
Regular insulin (Humulin R, Novolin R)
Insulin lispro (Humalog)
Insulin aspart (Novolog)
Insulin glulisine (Apidra)
Prompt insulin zinc (Semilente, Slightly slower acting)
Intermediate acting insulin include
Isophane insulin, neutral protamine Hagedorn (NPH) (Humulin N, Novolin N)
Insulin zinc (Lente)
Long acting insulin include
Extended insulin zinc insulin (Ultralente)
Insulin glargine (Lantus)
Insulin detemir (Levemir)(Agabegi et al.,2008)
BIGUANIDES
Biguanides reduces hepatic glucose output and increase peripheral glucose uptake which includes skeletal muscle.(Eurich et al,.2007)
METFORMIN

Metformin decreases hepatic glucose production, intestinal absorption of glucose, and improves insulin sensitivity by increasing peripheral glucose uptake and utilization. It decreases glycated hemoglobin (Hb1Ac) values by 1.5-2.0% (Fimogreni et al,.2006)
THIAZOLIDINEDIONES
Thiazolidinediones also called glitazones binds to PPAR that is involved in transcription of genes regulating glucose and fat metabolism. It acts on peroxysome proliferator responsive elements (PPRE)This PPREs influence insulin-sensitive genes which enhance production of mRNAs of insulin-dependent enzymes. This result to better uptake of glucose by the cells. This causes a decrease of glycated hemoglobin values by 1.5-2.0%(Grant et al.,2008)
PIOGLITAZONE

Pioglitazone acts by improving insulin sensitivity through its action at PPAR gamma 1 and PPAR gamma 2, and affects lipid metabolism through action at PPAR alpha. The results cause increase in glucose transporters 1 and 4, lowered free fatty acids, enhanced insulin signaling, reduced tumor necrosis factor alpha (TNF alpha) and remodeling of adipose tissue.. Pioglitazone has been discontinued because it causes bladder tumors (Dormandy et al,.2007)
SULFONYLUREAS SECRETAGOGUES
Sulfonylureas binds to plasma proteins and stimulates endogenous release of insulin
It reduces glycated hemoglobin (A1C) values by 1.0–2.0% (Seino S, 2012).

First-generation agents
tolbutamideacetohexamidetolazamidechlorpropamideCHLORPROPAMIDE

Second-generation agents
glipizideglyburide or glibenclamideglimepiridegliclazideglyclopyramidegliquidoneGLIPIZIDE

Glipizide acts by blocking potassium channels among beta cells of pancreatic islets of langerhans.The cell depolarizes which results in the opening of voltage-gated calcium channels. The resulting calcium influx encourages insulin release from beta cells (Harrower A,.2000).

NON SULFONYLUREUS SECRETAGOGUES
Meglitinides is one of the Non sulfonylurea secretogogues, helps the pancreas produce insulin by closing the potassium channels of the pancreatic beta cells thus opening the calcium channels thereby enhancing insulin secretion. They are taken with or before meals to boost the insulin response to each meal. If a meal is skipped the drug is also skipped. It reduces glycated hemoglobin (1Ac) values by 0.5–1.0%. (Blicklé JF . 2006). 
repaglinidenateglinideSTRUCTURE OF REPAGLINIDES

ALPHA GLUCOSIDASE INHIBITORS
These hypoglycaemic agents slow the digestion of starch in the small intestine so that glucose from the starch of a meal enters the bloodstream slowly and be matched effectively by an impaired insulin response or sensitivity. This reduces glycated hemoglobin (A1C) values by 0.5–1.0%.(Benalla et al,.2010).

miglitolacarbosevogliboseSTRUCTURE OF ACARBOSE

Injectable Incretin mimetics
Incretins are secretogogues. Glucagon-like peptide-1(GLP-1) and gastric inhibitory peptide (GIP) .This is inactivated by the enzyme dipeptidyl peptidase-4(DPP-4)(Inzucchi SE, et al. 2012).

Injectable Glucagon-like peptide analogs and agonists
Glucagon-like peptide (GLP) agonist binds to a membrane GLP receptor causing increase in release of insulin from the pancreatic beta cells(Bajaj, M ,2006). Exenatide; is a GLP agonist and reduces glycated hemoglobin by 0.5-1.0% (Gallwitz (2006).

Liraglutide,
Taspoglutide 
Gastric inhibitory peptide analogs
Sitagliptin is a gastric inhibitory peptide analogs, it acts by blocking the enzyme dipeptidyl peptidase 4 (DPP-4) through incretins GLP-1 and GIP inactivation. Hence escalating the secretion of insulin and suppressing the release of glucagon by the alpha cells of the pancreas.

It causes reduction of HbA1c level by about 0.7% (Mentlein R,2005).

sitagliptin phosphate
Injectable Amylin analogues
Amylin agonist analogues slows gastric emptying and suppresses glucagon. Pramlintide is the only amylin analogue available and is administered subcutaneously. It reduces glycated hemoglobin values by 0.5-1.0% (Ludyik et al,.2003).

Glycosurics
Glycosurics acts by blocking the re-uptake of glucose in the renal tubules, hence promoting loss of glucose in the urine (Dietrich et al,.2013)
Examples include:
DapagliflozinCanagliflozinEmpagliflozinSTRUCTURE OF DAPAGLIFOZIN
NATURAL REMEDIES
The medicinal components in plants are due to chemical compounds secondary metabolites that can be synthesized and are responsible for their diverse biological activities (Khan et al.,2011)Also in pharmaceutical industries, this plants are regarded as potential sources of lead compounds of drug development (Severino et al.,2011)
The mechanisms of action of hypoglycaemic herbs are multiple such as increasing insulin secretion for example Ginseng, Bitter lemon. Enhancing glucose uptake by adipose and muscle tissues forexample cinnamon. Inhibiting glucose absorption from intestine for example myrcia and inhibiting glucose production from heptocytes for example Berberine leaves(Manisha et al,.2007).

Ginseng
The therapeutic potency of ginseng relies on its geographical locality, dosage, processing and types of diabetes. Panax ginseng has the highest therapeutic potency. Its anti-diabetic effects have been investigated with aqueous or ethanol ginseng extracts. A proposed action mechanism has been tested on various animal models Korean red ginseng (0.1–1.0 g/ml) significantly stimulated insulin release from isolated rat pancreatic islets at 3.3 mM glucose concentration (Kim et al.,2015)
Ginsenosides
Momordica charantia (bitter melon)
Its hypoglycemic effects were demonstrated in cell culture, animal and human studies This components include charantin, vicine, polypeptide-p, alkaloids and other non-specific bioactive components such as anti-oxidants. The major compounds in bitter melon methanol extract, including 5-?, 19-epoxy-3-?, 25-dihydroxycucurbita-6,23(E)-diene (4) and 3-?,7-?,25-trihydroxycucurbita-5,23(E)-dien-19-al (5) showed hypoglycemic effects in the diabetic male ddYmice at 400 mg/kg .Oleanolic acid glycosides, improved glucose tolerance in Type II diabetics by preventing sugar from being absorbed into intestines. Saponin fraction (SF) extracted showed hypoglycemic activity in alloxan-induced hyperglycemic mice . Bitter melon increased the mass of ? cells in the pancreas and insulin production ( Hazarika et al,.2012)
3-b-trihydroxycucurbita

ADHF (anti-diabetes herbal formulation)
Studies was done in Anti-diabetic herbal formulation in diet-induced type II diabetic animals . These showed that there was a markedly reduction of blood glucose level in the mice fed with a diet containing 4% or 8% ADHF. Insulin insensitivity, histopathological changes in the pancreas and liver were improved significantly in the mice fed with ADHF(Rajesham et al,.2012).
Standardization approaches for herbal drugs
This is critical analysis of each step during the establishment of samples which is essential in order to ensure quality, safety, and reproducibility. With this objective, Chawla et al. proposed a copy for developing these challenges in clinical studies.He further suggested studies to guarantee effective herbal drug standardization methodology, as well as a regulatory standard guide for future research. This is by utilizing holistic approach of herbal drugs, only if these drugs undergo a critical standardization process (Folashade,et al,2012).

STATEMENT OF THE PROBLEM
Diabetes mellitus is an endocrine metabolic disorder that is currently managed by different types of synthetic oral hypoglycemic agents and insulin. Most of the current antidiabetic agents have a major shortcoming in that they are only designed to alleviate T2DM and not to cure it fully. It is evidently that targeting one or two metabolic pathways is not sufficient to cure T2DM but those with more metabolic pathways show more encouraging results than those that target a single pathway (Habeck, M, 2003).Some of this drugs cause serious side effects that through research some have been discontinued though been used over sometime. For example Pioglitazone was discontinued being associated with bladder tumours (Dormandy et al,.2007).

This convectional drugs are not readily available and if available is very expensive that not everyone can afford. This are used for a life time and prolong use of this medicament sometimes damage body organ like kidneys and liver(Upendra et al,.2010).

Medicinal plants have been used for a long time in developed and developing countries. They are used to alleviate illness and some are used as food. Their side effects are minimal as compared to conventional medicine. However, continued use of extracts without scientific evaluation may lead to serious complications in relation to overdose or under dose such as kidney damage and lack of awareness about the presence of toxic substances that would poison the body cells (Pritesh et al,.2012)
It is necessary to establish a proper scientific procedure for preparation and use in herbal medicines through toxicological, pharmacological and chemical studies (Nkunya et al, 1990). This attempts to isolate and identify those active compounds, their structural modification be performed so as to synthesize more derivatives. This bioactive compounds can also be used as templates for synthesizing drugs for clinical use (Murray, 1995).
To ensure patients’ safety, there is still an obvious need to develop antidiabetic medicines with satisfactory efficacy and no severe adverse effects (Zimmet,et al.2001).It is in this light that this study aimed at conducting phytochemical and structural investigation on the leaves of pavonia urens that is readily available and is used by communities in washing utensils. It is claimed by the traditional medicine men that it reduces high blood sugar levels.

JUSTIFICATION OF THE STUDY
Many researchers have shown that medicinal plants have been the most promising source of lead structure with the development of new drugs (Allison, 2006).The rise in the number of reviews and research publications clearly confirms that medicinal plants are of great use in human health (Gantait et al.,2014). For example, Anti-malarial drugs such as Artemisinin was isolated from Artemisia annual as other drugs were synthesized (Rashmi et al.,2014)
Plant species belonging to the species pavonia urens have been widely used by the people of North Rift Valley not only as medicine also in washing utensils. This herb is user-friendly. They use this species as hypoglycaemic and the properties has been associated with the presence of bioactive compounds. Therefore, there is need to study this species in order to understand its efficiency, safety and activity.

Research questions
1.Medicinal plants in the same genus have similar antihyperglycemic properties and compounds.

2. The bioactive compounds are stable enough to be extracted, isolated and identified.

3.The biological activity of medicinal plants is due to the secondary metabolites which remain bioactive after isolation from the plants.

4.This medicinal plants do not contain heavy metals like lead, mercury.

6.The bioactive components are not toxic to body cells when consumed.

Objectives
General ObjectiveTo scientifically validate FIVE Polyherbal formulations used in the treatment of diabetes.

Table 1.

FORMULATION
1 FORMULATION 2 FORMULATION 3 FORMULATION 4 FORMULATION 5
Kabukel
Kimolwo
Kiptebes
Korwol
Kiplak
Chepili
Tengeretwo
Torwet
Sigowet
Soget Singorwet
Morkwo
Rekerio
Cheseria
Kuryot
Losin
Tabirirwo
Maririch
Kiter
Marsitet Chebisayat
Motosiet
Kurbanyat Akelkeltet
Siryat
Bamustanyit
Lelek
Sasayat
Abaibatet
Kuryot Plisli
Chemosong
Rekerio
Specific objectives
1.To standardize five antidiabetic Polyherbal formulations;
(i)To collect plant materials comprising the five antidiabetic formulations.

(ii)To taxonomically identify plants comprising the antidiabetic formulations.

(iii)To prepare five herbal drugs under the direction of traditional medicine men
2.To extract, isolate and identify the bioactive compounds with antidiabetic properties from the leaves of pavonia urens.

(i)To carry out sequential extraction of the leaves of pavonia urens using solvents extraction of increasing polarity (hexane, DCM and EtOACs).

(ii)To isolate compounds from the crude extracts of pavonia urens using chromatographic Techniques (TLC,CC (silica gel),preparative TLC and sephadex LH-20)
3.To carry out qualitative pharmacological evaluation of the pavonia urens leaves
(i) To test for the presence of alkaloids
(ii) To test for the presence of antioxidant activity.

(iii) To test for the presence of flavonoids.

5. To chromatographically and spectroscopically examine compounds present in the leaves of pavonia urens
(i)To determine the structures of the isolated compounds using spectroscopic techniques (IR,UV,MS,1H -NMR and 13 C-NMR and 2D NMR).

Overall significance of the study
This study might find natural plants as an effective nutraceuticals which will offer an affordable alternative for synthetic oral hypoglycemic agents including insulin which have been associated with unwanted side effects and several complications. Be able to come up with a drug of good standard in order to ensure quality, safety, and reproducibility. This will reduce global burden and the number of new cases will be minimal, including full treatment and eradication of disease.

CHAPTER TWO
LITERATURE R
First medicinal plant
The first medicinal plant discovered with a clear antidiabetic effect was Galega officinalis L. (Fabaceae), has been in use since the Middle Age to treat diabetes mellitus. From this plant, other name goat?s rue, French lilac, a guanidine derivative, galegine,(Fig.8) was isolated. This compound, its chemical structure is the same as hypoglycaemic agent metformin,(Fig.9) responsible for the lowering of blood glucose extracted from plant.(Perla,et al,2013).

FIG.8
FIG.9.HCL
When analyzed, it turned out to contain compounds related to guanidine, a substance that decreases blood sugar by causing a decrease in insulin resistance but found that it was too toxic for human use (Bailey et al,2007).

2.3.Mechanism of Action of Herbal Antidiabetics
The mechanism of action of herbal anti-diabetic could be grouped as-
Adrenomimeticism, pancreatic beta cell potassium channel blocking.

Reduction of insulin resistance and renal glucose reabsorption
Stimulation of insulin secretion from beta cells of islets as well as inhibition of insulin degradative processes
Providing elements like calcium, zinc, magnesium, manganese and copper for the beta-cells
Regenerating and repairing pancreatic beta cells
Growing the size and number of cells in the islets of Langerhans
Stimulation of glycogenesis and hepatic glycolysis
Shielding effect on the destruction of the beta cells
Enhancement in digestion along with reduction in blood sugar and urea
Prevention of pathological change of starch to glucose
Inhibition of ? -galactocidase and ?–glucocidase
Cortisol lowering activities
Inhibition of alpha-amylase6 lation of insulin secretion (Manisha et al,2007)
2.4.Global medicinal plants
In India, many herbal drugs in use are derived from medicinal plants, minerals and organic matter (Glover et al, 2002).Is the main producer of medicinal herbs hence the name botanical garden of the world (Seth,et al,2004).Tinospora cordifolia,commonly known as Guduchi is widely used in Indian ayurvedic medicine for treating diabetes mellitus(Stanley et al,2001).It belongs to the family Menispermaceae .The crude extract of Tinospora cordifolia roots given orally for 6 weeks resulted in a reduction of sugar in urine, blood , lipids in serum and tissues in alloxan diabetic rats. It also causes reduction in body weight(Stanley,et al,2003). Tinospora cordifolia contains bioactive constituents such as glycosides, diterpenoid,lactones, alkaloids, phenolics, sesquiterpenoid,steroids, aliphatic compounds and polysaccharides (Gaur et al,.2014)
BERBERINE
PALMATINE
TEMBETARINE
ISOCOLUMBIN
MAGNOFLORINE
CHOLINE
TETRAHYDROPALMATINE
SYRINGIN

TINOCORDISIDE
CORDIFOLIOSIDE A
STEROIDS
SITOSTEROL
ECDYSTERONE
ALIPHATIC COMPOUNDS
OCTASONALON

HEPTACOSANOL

JATRORRHIZINE
Elsewhere in India,Raju et al, evaluated hypoglycaemic activity of polyherbal extracts of Benincasa hispid fruit, Acacia arabica bark Tinispora cordifolia stem,(Patel et al,2009) Ocimum sanctum areal parts and Jatropha curcus leaves. The extracts were orally administered at different strengths of 250 and 500mg/kg body weight in Alloxan induced rats for two weeks in different groups where as tolbutamide 80 mg/kg body weight was used as reference standard . Treatment with this Polyherbal formulation and tolbutamide resulted in a significant reduction of blood glucose and increase in plasma insulin reducing elevated level of serum glucose and reverse the cholesterol, triglyceride, HDL and LDL values when compared to untreated diabetic rats (Nanjwade B.K,.2010).
Studies have been done on the action of cinnamon on diabetes and metabolic syndrome(Qin,et al,2010).It was shown to diminish blood glucose through fall of insulin resistance and boost of hepatic glycogenesis(Couturier,et al,2011). Cinnamon (Fig.10) an active metabolite, phenolics was proposed to be the active compounds in modulation of insulin signaling(Qin,etal,2012).In addition, cinnamaldehyde from cinnamon extract has hypoglycaemic and antihyperlipidemic effects on animal models of diabetes rat (Li et al,2012) and is thought of as a potential antidiabetic agent (Subash ,et al,2007).But its molecular target of cinnamon and cinnamaldehyde remains unclear.
FIG.10
Safflower Carthamus tinctorius seeds are used as a herbal medicine for menstrual pain, trauma, constipation, and diaphoresis in Asian countries(Takahashi,etal,2012). Hydroalcoholic extract of safflower exhibit antihyperglycemic properties through enhancing insulin secretion in alloxan-induced diabetic rats (Asgary,et al,2012).Two derivatives of serotonin isolated from safflower seed were shown to suppress ? -glucosidase activity to a greater degree than the positive control acarbose (Takahashi,et al,2012).

Weisberg and colleagues, agreed that curcumin reverses various inflammatory and metabolic derangements connected with obesity and improves glycemic control in animal models of type 2 diabetes (Weisberg,et al,2008).

Curcumin (Fig.11) is a major constituent of the rhizomatous powder of turmeric Curcuma longa commonly used as food and medicine in southern Asia. Curcumin and turmeric rhizomes demonstrate bioactivities such as antioxidant, anti-inflammatory, antidiabetic, and immunomodulatory (Meng,et al,2013) and has been used to treat T2DM (Chuengsamarn,et al,2012).
FIG.11
Turmeric (Fig.12) shows hypoglycemic and hypolipidemic effects on diabetic mice(Madkor,et al,2011).It also increased postprandial serum insulin levels to maintain blood glucose levels (Wickenberg,et al,2010
FIG.12
Soy protein and isoflavonoids in soybeans progress insulin resistance and enhancement of insulin release (Kwon,et al,2007).In recent times, Genistein(Fig.13) a key isoflavone present in soybean (Jang,et al,2007) has been reported to treat obesity and diabetes(Behloul,et al,2013).
FIG.13
Genistein preserved islet mass by increasing ?-cell count, proliferation, and survival in the pancreas(Zhang,et al,2010).
The data demonstrated it could prevent T2DM via a direct protective action on cells without alteration of periphery insulin sensitivity(Fu,et al,2012).

In addition, its antidiabetic mechanism involves activation of protein kinase A (PKA) and extracellular-signal-regulated kinases (ERK)1/2. Nevertheless, another review stated that genistein could activate AMPK and, in turn, led to a decrease in insulin sensitivity(Leiherer,et al,2013). Genistein enhanced diabetic complications such as vascular dysfunction and wound healing(Tie,et al,2013). In a clinical trial, genistein and/or soybean extract reduced the risk of T2DM in overweight women(Nanri,et al,2010).

Quercetin(Fig.14) is a flavonoid found in plants and their products .It is used to treat cancers, viral infections, inflammation and metabolic syndrome. In recent time, studies indicated that it can treat T2DM in Streptozocin and alloxan-treated mouse models (Bansal,et al,2012)and was shown to lower fasting and postprandial blood glucose levels in diabetic mice without any variation in serum insulin level (Kim,et al,2011). Furthermore, like acarbose, quercetin inhibited glucosidase activity (Li,et al,2009). On the other hand, quercetin also suppressed DPP-4 activity in vitro(Bansal, et al,2012). Nevertheless, it remains unclear whether this compound can stimulate GLP-1 production in vivo.

FIG.14
FAMILY OF MALVACEAE
Malvaceae is a widespread family with about 243 genera and 4225 species, it is distributed in tropical areas.(Costa et al,2007)Natural products extracted from plants that belong to the Malvaceae family are used in the treatment of many diseases worldwide.(Maganha et al,2009) Some of these include snakebites, asthma, diuretic, treatment of rheumatism and gastrointestinal disorders, among others (Darwish et al.,2005)
Sidastrum micranthum belongs to the family of malvaceae.Natural compounds were isolated from this plant and evaluated as modulators of antibiotic resistance.Flavonoids acacetic and 7,4′-Di-o-methyllsoscutellarein were evaluated as modulators of antibiotic resistance using efflux strains of staphylococcus aureus
Compound 1 Vanillin

Compound 2 Salicylic acid

Compound 3 Quercetin (3,7,3′,4′-tetrahydroxy-flavone)

Compound 4 Acacetic (5,7-dihydroxy-4′-methoxyflavone)

Compound 5. 7,4′-Di-o-methylisoscutellarein (5,8-dihydroxy-7.4′-dimethoxyl-flavone)

Compound 6

Compound 7

All these compounds were based on the spectral analysis and were good in agreement with those reported in literature. This was the first study report of the compounds from the malvaceae family.

The Infra Red spectrum of 7,4′-Di-o-methylisoscutellarein (5,8-dihydroxy-7.4′-dimethoxyl-flavone) showed a hydroxyl signal at 3458 cm-1, a conjugated carbonyl hydroxyl absorbance at 1665 cm-1 and aromatic C=C stretching absorptions at 1609 and 1510 cm-1.
The 1H Nuclei Magnetic Resonance spectra showed a pair of double doublets at ?H 8.09 (J = 7.0 Hz and J = 2.0 Hz) and ?H 7.12 (J = 7.0 Hz and J = 2.0 Hz), which suggested an AA’BB’-type system of hydrogens bonded to aromatic carbon. There is a methoxyl group in one of the aromatic nuclei that was inferred by the signal at ?H 3.88.

The 13C Nuclei Magnetic Resonance spectral data strengthened the information provided by the 1H Nuclei Magnetic Resonance spectra. This emphasized the presence of the AA’BB’ system due to signal at ?C 128.5 and ?C 114.5.

The methoxyl groups were established by HMBC spectrum, which showed interactions between the absorptions at ?H 3.85 (s, OCH3-4´) and ?C 162.4 (C-4´) adding to interactions between the absorptions at ?H 3.88 (s, OCH3-7) ?C 154.3 (C-7). The analysis of NOESY spectrum was possible to strengthen the positions of methoxyl groups by correlations between the absorptions ?H 3.88 (s, OCH3-7) and ?H 6.53 (s, H-6) adding to the interactions between absorptions at ?H 3.85 (s, OCH3-4´) and ?H 7.12 (d, J = 9.0 Hz, H-3´/5´).
Two-dimensional spectra (HMBC and NOESY) are shown below
 

Some correlations observed at the HMBC (Jn) and NOESY (NOE) spectra of 5
The combination of spectral analysis of Infra Red spectroscopy, 1H and 13C nuclear magnetic resonance, with aid of two dimensional techniques, and by. comparisons with literature data, confirmed that the compound 5 as being 7,4?-di-O-methylisoscutellarein, flavonoid unpublished in the Malvaceae family.

Modulation of antibiotic activity
Acacetin and 7,4?-Di-O-methylisoscutellarein showed no antibacterial activity at 256 µg/mL against any strains of S. aureus used. When the compounds were incorporated into the growing medium at 64 µg/mL (¼ MIC), a two-fold reduction in the MIC was observed for norfloxacin and ethidium bromide. Regarding tetracycline and erythromycin a two-fold reduction in the MIC was observed only with 7,4?-Di-O-methylisoscutellarein.
 The flavonoids modulated activities of the antibiotics by reducing the concentration of antibiotics needed to inhibit the growth of the drug resistant efflluxing bacteria. At least regarding tetracycline and erythromycin, 7,4?-Di-O-methylisoscutellarein can be favorably compared with acacetin, which may be related to its higher lipophilicity due to an additional methoxyl group at C-7, since lipophilicity is a common feature of several EPIs and may be a key factor for the inhibition in Gram-positive bacteria. Ethidium bromide is a well known substrate for NorA efflux protein, and active efflux is the only known mechanism of resistance to this DNA-intercalating dyes. Therefore, the use of ethidium bromide against the strain SA-1199B is of worth to demonstrate that the flavonoids evaluated in this case modulated the resistance to norfloxacin by inhibition of efflux pump (Stavre et al,.2007) These findings indicates that plants of the Malvaceae family could be useful as a source of potential adjuvant of antibiotics.

GENUS PAVONIA
Pavonia is a genus of flowering plants in the mallow family malvaceae. It is a large genus of herbaceous perennials and shrubs native to Africa, South America and North America. The plant includes 494 scientific plant names of species rank for the genus pavonia of these 164 are accepted species names (United States Department of Agriculture. 2007)
ESSENTIAL BIOACTIVE COMPOUNDS IN PAVONIA GENUS
Pavonia genus have been reported to possess terpenoids mainly the triterpenoids and sesquiterpenes (The wealth of India,1992). Sesquiterpenes are class of compounds that are made up of fifteen carbons atoms and are assembled from three isoprenoid also called terpenoids units, while triterpenoids contain thirty carbon atoms. They are naturally occurring organic chemicals. (Richard 2010). Plant terpenoids are used widely for their aromatic qualities and play a great role in traditional herbal medicine. Well known terpenoids include citral,menthol,camphor,salvinorin-A,the cannabinoids.ginkgolide and bilobalide,curcuminoids found in turmeric and mustard seed (Richard 2010).

Chemical structure of terpenoids
BIOSYNTHESIS OF TERPENOIDS
Two Metabolic pathways
1. Mevalonic acid pathways
Organisms manufacture terpenoids through HMG-CoA reductase pathway which also produces cholesterol. The reactions takes place in the cytosol. This pathway begins the acetyl-CoA and ends with the production of IPP and DMAPP. Best known as the target of statins, a class of cholesterol lowering drugs (Serrantona,2005).

2. Non-mevalonate Pathway (MEP)
This starts with the condensation of pyruvate and D-glyceraldehyde-3-phosphate to 1-deoxy-D-xylulose-5-phosphate(DMAPP) which is subsequently formed via a series of enzymatic steps starting with the conversion of DXP to 2C-methyl-D-erythritol-4-phosphate(MEP). Enzymes of this non-mevalonate pathway are attractive targets for the development of drugs targeting infectious diseases such as tuberculosis and malaria, because this pathway occurs in pathogenic prokaryotes but is absent in human metabolic pathways (Berg et al 2012).

1.Acetyl-Coenzyme A
2. Acetyl-CoA, HS-CoA
3.acetyl-coenzyme A
4.Acetyl-CoA, HS-CoA
5.3-Hydroxy-3-methylglutaryl-Coenzyme A
6. 2 NADP+, 2NADPH
7. Mevalonate
8. ATP,ADP
9. Mevalonate-5-phosphate
10. ATP, ADP
11. Mevalonate-5-pyophosphate
12. ATP, ADP, CO2
13. Isopentenyl-pyrophosphate
14. 2C-Methyl-D-erythritol-2,4-cyclophosphate
15.2C-Methyl-D-erythritol-4-phosphate
16. NADP+, NADPH
17. 1-Deoxy-D-xylulose-5-phosphate
18. CO2
19. Pyruvate
20. D-Glyceraldehyde-3-phosphate
The ethanolic extract, from the leaves of the P. multiflora, was partitioned to the partitions of hexane and of ethyl acetate. This partitions were subjected to various chromatographic techniques, including thin layer chromatography, column liquid chromatography, preparative thin layer chromatography, vacuum liquid chromatography, and a high-performance liquid chromatography using different stationary phases and eluents, to isolate the chemical components. Using spectroscopic and spectrometric methodologies, ten substances, including four phenolic compounds (p-hydroxybenzoic acid, p-coumaric acid, vanillic acid and ferulic acid), five terpene derivatives (loliolide, vomifoliol, 4,5 dihydroblumenol A, 3-oxo-?-ionol and blumenol C), and an esterified derivative of triterpenoid taraxerol, taraxerol p-methoxybenzoate (novel compound)were extracted  “Pavonia multiflora”. Tropicos. Missouri Botanical Garden. Retrieved 15 April 2017.

P-COUMARIC ACID STRUCTURE

p-hydroxybenzoic acid

Vanillic acid

taraxerol structure

Pavonia odorata (Maivaceae) is an indigenous medicinal plant from India, mainly used in Ayuverdic system of medicine on a diuretic diaphorelte and demulant. When combined with other drugs, it is used as an astringent and tonic for fever, inflammation and hemorrhage. The aromatic roots contain refrigerant,antipyretic,stomachic and astrigent properties,also controls diabetes, tumor and blood pressure.(Kashuma et al 2014).It was also reported to contain sesquiterpenes alcohol known as pavonenol(C15H24O)(The wealth of India,1992).

In an earlier study, the alcoholic fraction of P.odorata showed in vitro Cytotoxicity against Er lich’s ascites carcinoma cells (EAC) (Selvan et al 2007)
In another study, it was found that the extract of P.odorata, was effective in inducing Cytotoxic effect upon Human Breast cancer, Prostate cancer and lung cancer cell lines (Girish et al.,2016)
Preliminary phytochemical examination of ethanolic fraction of P.odorata has been reported to contain flavonoids which are known to process remarkably anti-tumor activities (Carlos et al 1999)
Ageratochromene
  Aromadendrene
Alpha-terpinene
Azulene

Methyl heptenone
Alpha-pinene
Beta-caryophyllene
A study was done on the antioxidant activity of Pavonia sepioides. The results showed that different phenolic compounds of low molecular weight were identified by Gc/ms those are salicylic acid, cinnamic acids, P-hydroxybenzoic acid, P-hydroxyphenylacetic acid, P-hydrocinnamic acid, valinnic acid, gentisic acid, P-cinnamic acid, protocatechilic acid, syringic acid, ferulic acid and caffeic acid (Christian et al.,2013).

Pavonia zeylanica is a plant that has shown potential as a source of Chemotherapeutic compounds. Phytochemical studies revealed that the plant is rich in flavonoids and other water soluble polyphenolic compounds (Surabhil et al,.2011).

Pavonia urens is widely used in African traditional medicine. In Kenya the bark is used for making baskets. The wood is used for roof rods and as fuel wood. The leaves are browsed by sheep and goats, and are fed to calves. Also used for cleaning the hands and utensils, and as toilet paper for infants. The flowers may be cooked as a vegetable. The plant has ornamental value (Njuguna,P.K.1995).

In DR Congo the scraped and softened root is applied on wounds, and the leaves are boiled in water after which the warm leaf is used to pat the eyes in case of weak eyesight. In Burundi the pounded roots and leaves are applied as dressings on fractures and dislocations, a decoction of the roots and leaves is given for the treatment of diarrhoea in babies, a leaf decoction is taken against cough and insanity, a leaf infusion is drunk against nausea and abdominal pain during pregnancy and leaf powder is taken as an oxytocic(de Boer,et al,.2005)
In Ethiopia root powder is taken orally against impotence, and leaf preparations are applied on ulcers. In Tanzania root preparations are taken for the treatment of stomach problems and pneumonia; the crushed leaf is also taken against stomach problems (Latham,P.,2007). In Madagascar the root is used against stomach-ache and a decoction of the aerial parts is inhaled in case of fever (Vollesen,K.1995).

   
CHAPTER THREE METHODOLOGY
3.0 PLANT MATERIALS
3.1 PLANT COLLECTION AND IDENTIFICATION
3.2 INTRODUCTION
Information was gathered through the help of traditional health practioners and herbarium personnel who assisted in plant collection. The traditional health practioners assisted in providing the local plant names, parts of the plant used, mode of preparation, administration and its medicinal use.

3.3 MATERIALS
1.Field note book and pencil
2. Small shovel, sharp knife for digging roots
3.Clippers or pruning shears for cutting branches
4.Plastic bags to hold the plants until they are ready to press
5.Plant press
6.Camera for recording appearance of the live plant and habitat.

3.4 PLANT COLLECTION
Permit was secured from National Forest Office for the collection of plant specimens. The traditional health practioner,researcher and herbarium personnel accompanied to assist in collection.
3.5 GENERAL EXTRACTION
The leaves of pavonia urens were dried in an aerated room at temperatures between 15-20oC. Five kilograms (5 kg) of plant material was then ground into fine powder using laboratory electric mill and then extracted thrice in 15 litres each of n-hexane, Acetone and Methanol starting from the least polar (hexane to pure Methanol) for 48 hours with occasional swirling to ensure thorough extraction. The filtrates were concentrated and the solvents recovered by distillation using a rotary evaporator (Buchi Rotavapor R-205, England) under vacuum and temperature of about 45oC (Harborne, 1998). The concentrates were transferred to sample bottles, dried under vacuum and stored at – 20oC until required for isolation.

3.6 PHYTOCHEMICAL SCREENING
3.6.1 p-Anisaldehyde (for steroids, terpenes, flavonoids and sugars)
This was prepared by mixing 0.5 ml 4-methoxybenzaldehyde (p-anisaldehyde), 90 ml distilled ethanol (chilled for 3 hours), 5 ml (98%) sulphuric acid and 1 ml ethanolic acid.
3.6.2 Dragendoff’s reagent (for alkaloids)
(a) Solution A
This was prepared by mixing 0.85 g bismuth (III) nitrate, 10 ml glacial acetic acid and 40 ml distilled water.
(b) Solution B
This was prepared by mixing 8 g potassium iodide and 20 ml distilled water.
Stock solution: This was prepared by mixing equal parts of solution A and solution B.
Spray solution: It was prepared by mixing 1 ml stock solution with 2 ml glacial acetic acid and 10 ml distilled water.
Chromatographic materials
Percolated thin layer chromatography (TLC) plates with fluorescent material were used (AlugramR Sil G/Uv 254, Macherey-Nagel – Germany). Silica gel 60 (0.063-0.2 mm, Merch Chemicals Ltd – South Africa) was used for vacuum liquid chromatography (VLC) while silica gel 60 (70-230 mm, Merch Chemicals Ltd- South Africa) was used for column chromatography (CC). Filter gel (Sephadex LH-20) was used for the sephadex column
Instrumentation
The developed TLC plates were viewed using UV lamp at 254 and 366 nm. Melting points were determined on a Gallenkamp-Sanyo (UK) machine. UV spectra were recorded on a Cecil CE 2041–2000 series spectrometer while IR spectra were recorded on a FTIR-8400 spectrometer, Shimadzu-Japan. 1H-NMR and 13C-NMR spectra were measured on an Oxford Varian Mercury YH-400 MHz and Jeol JNM-400 MHz spectrometers using deuterated solvents chloroform and methanol as a solvents and tetramethylsilane (TMS) as an internal reference.
3.7 Isolation of the compounds
3.7.1 Pavonia urens hexane and Acetone extracts
The hexane and Acetone leaves of extract of pavonia urens were combined based on the TLC profiles (hexane: Acetone = 3:2) giving 61 g (scheme 3.8.1). 2 g of this extract was put in a vial and kept in a deep freezer for isolation. The rest (59 g) was adsorbed on silica gel and subjected to column chromatography (diameter = 4 cm, length = 45 cm) over a column of silica gel 60 (Merch brand,70-230 mesh ASTM, 70g, Merch Chemicals Ltd. South Africa) using solvents
of increasing polarity from hexane through Methanol (hexane: Acetone; 100:0 to Acetone: Methanol; 0:100) to give 85 fractions (200 ml).

CHAPTER FOUR
4.0 RESULTS AND DISCUSSION
4.1 PLANT COLLECTION
4.2.Plant materials
The leaves parts of pavonia urens were collected in January, 2018 from near Eldoret town, Uasin-Gishu district of Rift-Valley province. The plants was identified by the herbalist who dispenses and taxonomist and voucher specimens were deposited at the University of Eldoret Herbarium for future references.

Cutting knife and shovel was used. The fibrous roots were trimmed and soil washed from the specimens. Plant specimen were placed in labeled plastic bag and a sprinkle of water was added.

4.3 PRESSING AND DRYING PLANT SPECIMENS
Specimens were pressed in a plant press which consists of a wooden frame, blotter paper and a folded newspaper. The plant press was tightened using straps with nuts. Most of the specimen consisted of a stem with attached leaves while others with a flower or a fruit. The roots of herbaceous plants was also included. Each collection was assigned a collection number and data for each collection entered in a field notebook.

Pressing of material was done immediately upon collection. The plant press was kept tight and thoroughly dried prior to storage and mounting.

PHOTOS for every plant pressed
4.4 MOUNTING HERBARIUM SPECIMENS
Dried pressed plant specimen and its label was mounted to a sheet of heavy paper. Glue was used to attach the larger portions of the plant such as stems, large leaves and fruits.

photos filing
4.5 IDENTIFICATION OF PLANT SPECIMENS
A thorough literature review and consultation with herbarium personnel was done. The identification of unknown plant material was accomplished with the use of published plant description and comparing with properly identified herbarium specimens. It was roughly grouped by Family, botanical name and the medicinal plant part used. Information of the plant’s growth habit; size and the habitat where it is found was captured. The label data was attached.

FORMULATION 1 FORMULATION 2 FORMULATION 3 FORMULATION 4 FORMULATION 5
Kabukel
Kimolwo
Kiptebes
Korwol
Kiplak
Chepili
Tengeretwo
Torwet
Sigowet
Soget Singorwet
Morkwo
Rekerio
Cheseria
Kuryot
Losin
Tabirirwo
Maririch
Kiter
Marsitet Chebisayat
Motosiet
Kurbanyat Akelkeltet
Siryat
Bamustanyit
Lelek
Sasayat
Abaibatet
Kuryot Plisli
Chemosong
Rekerio
FORMULATION 1( JULIUS KIPRONO)
KALENJIN NAME FAMILY BOTANICAL NAME COLLECTED FROM MEDICINAL PART
Kabukel LAMIACEAE Ocimum suave Uasin Gishu Root
Kimolwo RUBIACEAE
vanguera madagascariensis Nandi Root, bark
Kiptebes MIMOSACEAE Albizia gummifera Nandi Root
Korwol OLEACEAE Olea welwitschii Uasin Gishu Root
Kiplak NO NAME Not identified Uasin Gishu Leaves
Chepili BIGNONIACEAE Mankhamia lutea Nandi Root
Tengeretwo MORACEAE Combretum molle Nandi Root, Bark
Torwet VITACEAE Rhoicissus tridentate Uasin Gishu Bark
Sigowet SOLANACEAE solanum aculeastrum
Uasin Gishu Root
Soget CANELLACEAE
Warburgia ugandensis
Uasin Gishu Leaves, Bark
(i) Torwet Rhoicissus tridentate
Rhoicissus tridentate is a deciduous shrubby creeper in the family Vitaceae, common name is wild grape. The leaves are trifoliate with wedge-shaped leaflets. Decoctions and infusions of R. tridentate roots or tubers are taken orally in many South African pregnancy related traditional herbal remedies(Katsoullo et al,2000).
Also used to, ease indigestion through chewing, for abdominal pain during menstruation, treatment of swollen glands by warming the roots in fire and then pressing against the glands, its sap is reported to have healing and anesthetic properties. (kolwarojo,1976 R. tridentata is used in South Africa for gynaecological purposes and diarrhea. In other studies, R. tridentata is reported to have direct uterotonic activity.(Samie, et al,2005).

The collected plant materials were chopped into small pieces, dried under shade at room temperature for four weeks. The grounded powders were packed in clean dry plastic air tight bags.
The table below, shows qualitative screening of aqueous extracts of R. tridentata indicated the presence of phenols, alkaloids, flavonoids, tannins, saponins, and absence of phylobatannins and cardiac glycosides.
Phytochemical composition of the aqueous leaf extracts of R.tridentate
PHYTOCHEMICALS PRESENT QUANTITY
Phenols (mg/g) 1.33
Alkaloids (mg/g) 2.90
Flavonoids (mg/g) 0.14
Tannins (mg/g) 0.97
Saponins (mg/g) 8.53
Cardiac glycosides (mg/g) Not detected
Phylobatannins (mg/g) Not detected
(ii) Komolwet/vanguera madagascariensis
Spanish tamarind is a branched evergreen shrub, growing from 2 – 15 metre tall. The plant is cultivated for its fruits in several places in tropical Africa, as well as in India, China Singapore, N Australia and Trinidad (worldagroforesty website).

Medicinal
Roots and bark are used in traditional medicine; in Tanzania roots extract is used to treat intestinal worm infections and bark infusion is used in treating malaria
(iii) Soget/Warburgia ugandensis
Warburgia ugandensis Sprague, belongs to a member of Canellaceae family, is a small evergreen tree distributed in eastern and southern Africa. Warburgia bark has been used as traditional medicines for the treatment of gastro-intestinal disorders, cold, cough and sore throat, fever, malaria, respiratory and odontological problems in African countries (Maroyi,A,2014).

Phytochemical Analysis
The screening for phytochemicals was carried out qualitatively on the aqueous and methanolic extracts using standard established procedures for identifying plant constituents as described by Sofowora (1982) and Harbone (1973). An aliquot of every plant extract was analysed for the presence of saponins, alkaloids, terpenoids, steroids, cardiac glycosides, anthraquinones, tannins and reducing sugars.
The phytochemical tests showed presence ofsaponins,terpenoids,steroids,anthraquinones,tannins but absence of alkalloids.

(iv) SIGOWET/solanum aculeastrum
Solanum aculeastrum is used in traditional medicine to treat various human and animal diseases, especially stomach disorders and various cancers in Eastern Cape Province of south Africa. Biological active compound present in the medicinal plant have always been of great importance to scientist. The extract from Solanum aculeastrum is a rich source of saponins, glycoalkaloids and steroidal alcohols which are active against microorganisms(esthermwanzia,2014)
FORMULATION 2 ( Ruth Toroitich)
KALENJIN NAME FAMILY BOTANICAL NAME COLLECTED FROM MEDICINAL PART
Rekerio LAMIACEAE Acyrospermum schinipea Elgeyo Marakwet leaves
Singorwet BORAGINACEAE Cardia africana Elgeyo Marakwet Leaves
Tabirirwo MORACEAE Combretum molle Elgeyo Marakwet Bark
Maririch NO NAME Cardia africana Elgeyo Marakwet Leaves
Marsitet TILIACEAE Grewia similis Elgeyo Marakwet Bark
Kuryot RUTACEAE Teclea nobilis Elgeyo Marakwet Leaves
Morkwo MORACEAE Syzgium gumeene Elgeyo Marakwet Leaves
Losin SAPINDACEAE Allophylus abyssicus Elgeyo Marakwet Leaves
Cheseria CUCUMBABATACEAE
Momordica foetida Elgeyo Marakwet Leaves
Kipter EUPHOBIACEAE
Croton megaluspus
Elgeyo Marakwet Bark
LOSIN (Allophylus abyssicus)
The leaves of Allophylus abyssinica (Sapindaceae) are used fot the treatment of wounds, burns, skin diseases and to arrest bleeding in the Ethiopian folk medicine. The active ingredient appear to reside mainly in the methanolic fraction suggestion that they are polar.(Azeez et al,2007)
CHESERIA (Momordica foetida)
Previous phytochemical studies resulted in the isolation of cucurbitane triterpenoids from a leaf extract (Mulholland, 1997), alkaloids and glycosides from the complete plant (Olaniyi; Marquis, 1975; Olaniyi, 1975) and the identification of sitosteryl glycoside, 5,25-stigmastadien-3b-yl-glucoside and 1b-hydroxyfriedel-6-en-3-one (Olaniyi, 1980). In vivo studies with water extracts showed that M. foetida orally given in different doses prolonged the survival of Plasmodium berghei infected mice (Waako et al., 2005)
Spectrometric methods
1H NMR spectra were recorded on a Bruker AVANCE DPX 400-spectrometer (400 MHz) using TMS as internal standard. Samples were dissolved in acetone-d6, chloroform-d1 or methanol-d4. FAB-MS were obtained on a Varian MAT CH5DF instrument with DMSO/Glycerin, Xenon; CHCl3/slm-Nitrobenzylalkohol, Xenon or MeOH/Glycerin, Xenon as solvents. EI-MS were recorded on a Varian MAT CH7A, (70 eV).

KURYOT(Teclea nobilis)
The leaves and bark of Teclea nobilis(Rutaceae) locally called Al-Thureim are known in ethno-medicine having properties to reduce pain and fever (Inga and Sue 1989).

phytochemistry of teclea nobilis
Analysis of the Essential oil
Fresh leaves were used for the grounding of essential oil by hydro distillation. Analysis of the oil was performed by Gas Chromatography/Mass spectrometer. Oil components were identified by comparing their retention times and mass spectral data. The results showed that sesquiterpene and monoterpene hydrocarbons were the major metabolites in the oil.(Kuete et al.,2008).

The essential oils of the leaves of Teclea nobilis showed significant analgesic and antipyretic activity in mice (Al-Rehaily, 2001). The crude extracts and lupeol isolated from Teclea nobilis also showed anti-inflammatory activity on rats without causing apparent deleterious effects (Al-Rehaily et al., 2001; Mascolo et al., 1988; Adnan et al., 2001).

FORMULATION 3 (Elizabeth Chirchir)
KALENJIN NAME FAMILY BOTANICAL NAME COLLECTED FROM MEDICINAL PART
Chebisayat ASPARAGACEAE
Asparagus racemosus Timboroa Leaves
Motosiet MALVACEAE Pavonia urens
Timboroa Leaves
Kurbanyat EUPHOBIACEAE
Clutia kilimandscharica Eng. Timboroa Root, Bark
CHEBISAYAT (Asparagus racemosus)
The ethanol extract of Asparagus racemosus Willd was examined for antioxidant properties. Phytochemical investigation was also done to identify the presence of phytochemical compounds. Antioxidant activity of the ethanol extract was determined according to their scavenging activity of the stable DPPH (1,1-diphenyl-2-picryl hydrazyl) free radical and 10% H2SO4. In the qualitative antioxidant assay, the extract showed free radical scavenging properties. Preliminary phytochemical analysis of the plant extract showed the presence of alkaloids, tannins, saponins, glycosides, flavonoids and carbohydrate which could be responsible for antioxidant properties justifying the ethno medicinal applications of Asparagus racemosus. Thus, further advanced research is necessary to isolate and characterize the chemical compounds responsible for the therapeutic activities of the plant.

MOTOSIET(Pavonia urens)
No phytochemistry study documented.

KURBANYAT (Clutia kilimandscharica Eng)
Also called Kirbanyat by Ogiek community. Used as aphrodisiac tonic. About 100gm of root bark is pounded, cooked in 500gm of meat into a soup taken as an aphrodisiac. No phytochemistry study done.

FORMULATION 4 (Talai Kosgei)
KALENJIN NAME FAMILY BOTANICAL NAME COLLECTED FROM MEDICINAL PART
Lelek OLEACEAE
Olea africana West Pokot Leaves
Siryat TILIACEAE
Grewia similis Timboroa Root
Sasayat VERBANACEAE
Lantana camara Uasin Gishu Root
Bamustanyit BORAGINACEAE
Cardia abyssinica
Nandi Bark
Abaibatet OLEACEAE
Olea welwitschii
Uasin Gishu Root
Kuryot RUTACEAE
Teclea nobilis Elgeyo Marakwet Root
Akelkeltet RUTACEAE
Teclea nobilis Elgeyo Marakwet Root
(i) SASAYAT(Lantana camara)
The preliminary phytochemical studies were carried out by the methods described by Harborne and Kokate et al. The results of preliminary phytochemical screening of methanol, ethyl acetate and aqueous leaf extracts of Lantana indica was done The methanol and aqueous extracts showed the presence of flavonoidal glycosides, carbohydrates, proteins, triterpenoids and tannins.(Singh et al,1990)
(ii) SIRYAT(Grewia similis)
Phytochemical characterization of the compounds isolated from the hexane/DCM root extract of G. similis showed presence of bioactive compounds.(James,2005)
(iii) LELEK (Olea africana)
 The leaf extracts of O. africana contain compounds has antioxidant activity, antibacterial and antifungal activity. (Masoko et al,2015)
(iv) BAMUSTANYIT. No documented study
(v) ABAIBATET. No documented study
(vi) AKELKELTET. No documented study
FORMULATION 5 (Marina Kibor)
KALENJIN NAME FAMILY BOTANICAL NAME COLLECTED FROM MEDICINAL PART
Rekerio LAMIACEAE
Acyrospermum schinipea
Elgeyo Marakwet Leaves
Plisli (Tabirirwo) RUTACEAE
Teclea nobilis Nandi Root, Bark
Chemosong VERBENACEAE
Lippia japonicum/lantana camara Nandi Root, Bark
(i) Chemosong
Phytochemical evaluation indicates the presence of saponins, tannins and terpenoids in Ethanolic fraction and terpenoids in Essential oil. These compounds are believed to be responsible for the broad spectrum activity of the plant extracts. (Wadhai, V.S. 2012
(ii) Rekerio. No documented study
(iii) Plisli. No documented study
Most of the plants in the five formulations have been studied and this confirms that their phytochemistry showed presence of bioactive secondary metabolites.

This study focuses on Formulation three (3) for the herbalist Elizabeth Chirchir and the plant of study is Motosiet (Pavonia urens).This draws attention because this plant is very available non poisonous as the local community uses for domestic, like washing utensils and as tissue paper. Besides this, no study has been done to ascertain the active metabolites that the herbalist claims it causes hypoglycemia.

4.6.PLANT EXTRACTION
4.6.1 Yields of the extracts
The pavonia urens afforded 107 g (2.1%), 60 g (1.2%) and 68 g (1.4%) for hexane, Acetone and Methanol extracts, respectively.
4.6.2 Structural elucidation of isolated compounds
4.7 PHYTOCHEMICAL SCREENING
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