University of Khartoum Graduate College Medical and Health Studies Board Activity

University of Khartoum

Graduate College Medical and Health Studies Board

Activity- guided Isolation and Structure Determination of Antioxidant and Antidiabetic Compounds from

Bauhinia rufescence L.

By: Wadah Jamal Ahmed Osman

B. Pharm. U of K. (2003) M. Pharm. King Saud University (2012)

A thesis submitted in fulfillment of the requirement for the degree of PhD in Pharmacognosy

Supervisor

Prof.Abdelkhaleig Muddathir, (B.Pharm.M.pharm., PhD) Professor of Pharmacognosy, U.of K.

2014

Co-Supervisor:

Prof. Dr. Hassan Elsubki Khalid B.Pharm., PhD Professor of Pharmacognosy, U.of K.

DEDICATION First of all I thank Almighty Alla for his mercy and wide guidance on a completion of my study.

This thesis is dedicated to my parents, who taught me the value of education, to my beloved wife and to my beautiful kids.

I express my warmest gratitude to my supervisor Professor Dr Prof. Abdelkhaleig

Muddathir and Prof. Dr. Hassan Elsubki for their support, valuable advice, excellent supervision and accurate and abundant comments on the manuscripts taught me a great deal of scientific thinking and writing.

In addition, I would like to express my appreciation to all members of the

Pharmacognosy Department for their encouragement, support and help throughout this study.

Great thanks for Professor Kamal Eldeen El Tahir (King Saud University,

Riyadh) and Prof. Sayeed Ahmed (Jamia Hamdard University, India) for their co-operation and scientific support during the laboratory work.

Wadah jamal Ahmed

July, 2018

III

Contents

1. Introduction and Literature review 1.1.Oxidative Stress and Reactive Metabolites 1 1.2. Production Of reactive metabolites 1 1.3. Biological roles of Free radicals. 2 1.4.Antioxidants 3 1.5.Diabetes: 3 1.6. Complications of Diabetes mellitus 4 1.7. Available therapy for Diabetes mellitus 4 1.8. Oxidative Stress and diabetes 5 1.9.Oxidative stress and diabetic complications 5 1.10. Medicinal plants 6 1.11. Medicinal plants, antioxidants and 7 diabetics complications 1.12. Medicinal plants with ant diabetic activity 8 1.13. Phytochemicals and Management of 15 Diabetes mellitus. 1.14. Flavonoids 15 1.15. Alkaloids 21 1.16. Terpenoids and Steroids 26 1.17. Glycosides 33 2.1.Family Fabaceae 36 2.2 Bauhinia rufescens 36 2.3.Scientific classification: 36 2.4.Description 36 2.5.Medicinal uses of B. rufescens: 37 2.6.Local uses of B. rufescens: 38 2.7.Biological Activities of the Genus Bauhinia 38 2.8. Anti-diabetic Properties 38 2.9.Antimicrobial studies 39

2.10.Analgesic Potential Studies 41 2.11.Anti-inflammatory activity 41 2.12.Pharmacological and Toxicological 43 Investigations of Genus Bauhinia 3. Materials and Methods 3.1. Plant material 47 3.2.Extraction procedure 47 3.3.In vivo anti diabetic assay of plant extracts 47 3.4. In vitro anti diabetic assay of most active 48 fraction using TLC bio autographic method for the detection Amylase enzyme inhibitors. 3.5.Antioxidant assay 49 3.6. Quantitative antioxidant assay using DPPH 49 radical scavenging assay 3.7. Qualitative antioxidant assay using 49 Bioautography method 3.8.LC-MS analysis of the active fraction 50 3.9.High performance thin layer chromatography 51 (HPTLC) analysis of active fraction 3.10. Isolation of some active compounds and 52 structures determination using Nuclear Magnetic Resonance (NMR). 3.11.In silico assessment of the compounds from 53 active fraction 4.Results and Discussion 4.1. Anti diabetic assay of plant extracts. 55 4.2.Quantitative antioxidant assay using DPPH 56 radical scavenging assay of plant extracts 4.3.Fractionation of active methanol extract 56 4.4. Quantitative antioxidant assay using DPPH 57 radical scavenging assay of methanolic fractions (ethyacetate, n-butanol, chlorororm and aqueous fractions). 4.5. In vitro anti diabetic assay and qualitative 58 antioxidant evaluation of most active fraction (ethyl acetate fraction).

4.5. Isolation and structure determination of 60 compound (1) and compound (2) from ethyl acetate fraction. 5-1 4.6.1.isolation of the active constituents 60

4.6.2 Structure elucidation of compound (1) 60 4.6.2 Structure elucidation of compound (2) 66 7 4.7.Identification of Phytoconstituents from 69 ethyl acetate fraction by LC-MS 8. 4.8.In silico assessment of the identified 77 compounds as inhibitors for α-amylase and α- glucosidase enzyme. 9. 4.9.Standardization and High performance thin 91 layer chromatography (HPTLC) fingerprint analysis References 96

List of Figures Fig. (1): Some classes of flavonoids. 16 Fig. (2) Flavonoids for diabetes management 20 Fig. (3): Glycosides for diabetes management 35 Fig. (4): Bioautographic assay showing the inhibition of α-amylase by some 59 compounds developed on a silica gel G60 F254 plate Fig. (5): Bioautographic method showing the antioxidant compounds (yellow 59 color spots) after developing on a silica gel Fig. (6): 1H-NMR-spectrum of compound -1 (Rutin), (700 MHz, DMSO). 63 Fig. (7): 13C-NMR-spectrum of compound -2 (Rutin), (700 Hz, DMSO). 64 Fig. (8): HMBC-spectrum of compound -1, showed some two and three bond 65 correlations, (700 MHz, DMSO). Fig. (9): HSQC-spectrum of compound -1 (Rutin), showed direct proton-carbon 69 correlation (700 MHz, DMSO). Fig. (10): 1H-NMR-spectrum of compound -2 (Kaempferol -3-O-rutinoside), 70 (700 MHz, DMSO).

Fig. (11): 13C-NMR-spectrum of compound -2 (Kaempferol -3-O-rutinoside), 76 (700 Hz, DMSO). Fig. (12): Total MS chart represent molecular ion peaks of identified compounds from 77

active fraction analyzed by LC/MS/MS.

Fig. (13): HPLC peaks of compounds identified by LC/MS/MS. 82

Fig. (14): In silico assessment of identified compounds against human α- 86 glucosidase enzyme Fig. (15): In silico assessment of identified compounds against human α-amylase 92 enzyme Fig. (16): HPLC analysis of ethyl acetate fraction of Bauhinia rufescense 92 Fig. (17): 3D graph of HPTLC chromatogram 93 Fig. (18 TLC chromatogram of ethyl acetate fraction as shown by TLC scanner 94 (254 and 366 nm), List of Tables Table 1: : List of plants having insulin mimetic or insulin secreatory activity 9 Table 2: List of important alkaloids used in the treatment of Diabetes mellitus 21 Table 3: List of important Terpenoids and Steroids used in the treatment of Diabetes 26 mellitus Table 4: Invivo- Anti diabetic assay of methanol, chloroform and pet.Ether extracts 55 of Bauhinia rufescens . Table 5: The results of antioxidant Activity of methanol ,chloroform and pet.ether 56 extracts Table 6: The results of antioxidant Activity of different methanolic fractions 57 Table 7: 1H- and 13C-NMR assignments of compound -2 (700 MHz, DMSO) in 62 comparison with reported data for Rutin (Quercetin-3-Orutinoside) (100 MHz, CD3OD) Table 8: 1H- and 13C-NMR assignments of compound -1 (500 MHz, DMSO) in 67 comparison with reported data for Kaempferol -3-O-rutinoside (100 MHz, CD3OD) Table 9: Identified compounds using LC-MS/MS 62 Table 10: : In silico assessment of identified compounds against human α- 78 glucosidase and α-amylase enzymes Table 11: HPTLC profile at 366 (nm) of ethyl acetate fraction 92

VII

List of Abbreviations

δ Chemical shift value

13C NMR Carbon 13-nuclear magnetic resonance

1HNMR proton nuclear magnetic resonance

DEPT Distortionless Enhancment by Polarization Transfer

Hz Hertz

MS Mass spectrum

[M]+ Molecular ion peak m/z Mass/ charge ratiomp nm Nanometer

Rf Relative to solvent front

TLC Thin layer chromatography

ROS Reactive oxygen species

HPTLC High performance Thin layer chromatography

HMQC Heteronuclear Multiple Quantum Correlation

HMBC Heteronuclear Multiple Bond Correlation

PTLC Preparative thin layer chromatography

VIII

A b s t r a c t

Background:

Diabetes mellitus is a group of disorders of multiple etiologies resulting from a defect in insulin secretion, insulin action, or both. Diabetes remain one of the world’s major health problems and many repots have been supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes.

This study aimed to evaluate the antidiabetic and antioxidant activity of Bauhinia rufescens a plant used in Sudanese folkloric medicine for treatment of diabetes. It was also aimed to identify isolates and characterize the bioactive antidiabetic and antioxidant compounds using bioactivity guided fractionation followed by High Performance Thin Layer

Chromatography (HPTLC) autobiography, Liquid-Mass spectroscopy analysis and nuclear magnetic resonance (NMR).

Method:

The powdered plant leaves were exclusively extracted using petroleum ether, chloroform and methanol, successively.

These extracts were tested in streptozotocin-induced diabetic Wistar rats for antidiabetic as well as DPPH antioxidant potential. Standard Propyl Gallate was used as (+ve) control and the difference in results was considered significant when P < 0.05. The best active extract was fractionated with chloroform, ethyl acetate and butanol along with the remaining aqueous fraction. The fractions are tested for antioxidant and antidiabetic activity using

HPTLC autobiography (α amylase and α glucosidase).

Further, bioactive compounds were isolated by Preparative Thin Layer Chromatography and identified using LC/MS and NMR spectroscopy.

Results:

The methanol extract significantly decreased the blood glucose level in normal fasted rats by

27 %; two hours following administration and decreased the blood glucose level in streptozotocin-treated rats by 48.9%; two hours following oral administration, while chloroform and pet. Ether extracts not significantly decreases the glucose level.Significantly the methanol extract also showed strong antioxidant activity (89%) compared with other extracts; thereforeit was further subjected for fractionation by using chloroform, ethyl acetate, n-butanol and water sequentially, and then tested for their antioxidant activity; where the ethyl acetate fraction was showed good activity compared with other fractions.

Bioactivity guided fractionation of Bauhinia rufescensled to isolation and characterization of two compounds with potent antidiabetic and antioxidant activity namely; Quercetin-3-O- rutinoside and Kaempferol -3-O-rutinoside.

These compounds were isolated by using preparative thin layer c chromatographic techniques (PTLC) and their structures were identified by 1D NMR spectrum (C13and 1H-

NMR), 2D NMR (COSY, HSQC, HMBC Correlation) and confirmed bycomparing with reported data.

LC/MS analysis led to identified more than thirty compounds; which were subjected to in silico assessment against human α-glucosidase and α-amylase enzymes. Insilico assessment showed that more than 14 compounds showed potent activity compared with acarbose

(standard anti-diabetic drug).

High performance thin layer chromatography (HPTLC) for the first time of the active fractionof the plant revealed the presence of different spots/peaks in different Rf values, corresponding to different compounds.

Conclusion:

This study validated the traditional uses of the evaluated plant in treating diabetes; beside that it was found a good antioxidant. The compounds which were isolated and identified may provide a new lead for more potent analogues for drug discovery. The anti-diabetic potential and antioxidant activity reported in present investigation is reported for the first time on the aerial plant. Further the isolated and characterized compounds from this study are also being reported for the first time.

Toxicological studies may further be planned to develop it in the form of new drug for management of diabetes, however its use is available as per traditional claim.

المستخــــــلص

المقدمة: دا السكري هو مجموعة من االضطرابات التي تنتج من خلل في افراز االنسولين,عمل االنسولين او االثنين معا.يعتبر داء السكري واحد من اهم المشاكل الصحية العالمية وهنالك عدة تقاريرتفيد بدور جزئيات االكسجين النشطة في مرض السكري بشقيه النوع االول والثاني.

هدفت هذه الدراسه الي تقييم علمي لنشاط المضاد للسكري والمضاد لالكسدةلنبات الكلكل المستخدم في الطب الشعبي السوداني لعالج السكري كما هدفت ايضا لفصل والتعرف علي المركبات الفعالة المضادة للسكري والمضادة لالكسدة باستخدام التجزئية الموجهة بيولوجياثم باستخدام ).LC-MS, HPTLC and NMR

الطريقة:

تم استخالص بودرة االوراق المجففة باستخداممذيبات االيثر البترولي والميثانول. هذه المستخلصات تم تقيم فعاليتها ضد مرض السكريلالكسدةباستخدامفئيرانمصابةبداءالسكربعدتناولهامادةالستربتوزوتوسين وكمضادات االكسدة باستخدام مادة الدي بي بي اتش.تم استخدام مادة البروبايل قالكيت كمنظم موجب ويعتبراالختالففيالنتائجمميزعندماتكونP < 0.5.

المستخلص الفعال بعد ذالك تمت تجزئيته بواسطة مذيبات الكلورفورم, االيثايل استيت واالبيتانول وتم

اختبار هذه االجزاء مع الجزء المائي المتبقي كمضادات اكسدة واختبارها كمضادات للسكريضدانزيمالفا- امايليزوالديبيبياتشعليالتوالي باستخدام طريقة البايوتوغرافيا..

تم بعد ذالك فصل المركبات المسئولة عن الفعالية في النبات عن طريق Preparative Thin LayerChromatographyثم التعرف علي المركبات المفصولة باستخدام

(LC/MS, HPTLC and NMR spectroscopy).

النتائج:

المستخلص الميثانولي ادي الي خفض واضح في مستوي السكر في الفئيران العادية المصومة بنسبة ) 27.%( بعد ساعتين من اعطائها المستخلص وادي ايضا لخفض السكر في الفئيران المصابة بالسكري بعد معالجتها بمادة االستربتوزوتوسين بنسبة )48 %( بينما مستخلص الكلورفورم وااليثر البترولي لم يكن لهما اثر واضح.

المستخلص الميثانولي مضاد قوي لالكسدة بنسبة تصل الي )89%( مقارنة ببقية المستخلصات. اظهرت لهذا السبب فقد تمت تجزئية المستخلص الكحولي بواسطة الكلوروفورم, االيثايل استيت, البيتانول والماء بطريقة متتابعة وتم اختبارها جميعا ضد االكسدة فاظهر الجزء المستخلص ب االيثايالستيت فعالية اكبر بنسبة تصل الي تسعين في المائة.

النشاط البيولوجي الموجه بالتجزئه ادي الي فصل مركبين لهما اثر قوي ضد مرض السكري وكمضاداتاكسدة هماQuercetin-3-O-rutinoside and Kaempferol -3-O-rutinosideوقد تم فصلهما بتقنية الكرماتوغرافيا والتعرف علي الصيغة الكيميائية بواسطة تقنية الرنين المغنطيسي المتطورة

XII

ثم بمقارنة النتائج مع النتائج المنشورة.

التحليل للجزء الفعال بواسطة جهاز ال LC/MS ادي الي التعرف الي اكثر من ثالثين مركب اربعة عشر منهم لهم فعالية اكثر من دواء االكرابوز المستخدم حاليا في كل العالم لعالج السكري عند المقارنة ب Insilco study.

تحليل الجزء الفعال بجهاز HPTLC ادي الي ظهور عدة مركبات يمكن استخدامها كبصمة مميزة للنبات وهذه هي المرة االولي التي يتم بها تحليل هذا النبات بهذا الجهاز.

الخالصة:

النتائج المتحصل عليها تدعم استخدام هذه النبات في الطب التقليدي السوداني لعالج مرض السكري, بجانب انهمضاد جيد لالكسدة. المركبات المفصولة يمكن ان تمثل نواه جديده لمركبات اكثر فاعلية في عملية اكتشاف االدوية.

نتائج تقيم استخدام النبات ضد مرض السكري وكمضاد لالكسدة تسجل الول مرة كما ان فصل والتعرف علي المركبات الفعالة يتم ايضا الول مرة. هنالك حاجة الجراء مزيد الدراسات علي السمية حتي يتم تطويره لدواء يستخدم لعالج السكري مع العلم انه يستخدم شعبيا.

XIII

CHAPTER ONE

Introduction and Literature review

INTRODUCTION AND LITRURE REVIW

1. Oxidative Stress and Reactive Metabolites

Oxidative stress and reactive metabolites (RMs) are interrelated terms defined in general as excess formation and/or insufficient removal of highly reactive molecules such as reactive oxygen species (ROS), reactive nitrogen species

(RNS), reactive chlorine species (RCS) and reactive thiyl species (RTR) (Asmat

Ullah) (Monisha Banerjee). Oxygen is highly reactive specie that has the ability to become part of potentially harmful and damaging molecules(Asmat Ullah).

Oxygen derived free radical (ODFR) and Oxygen derived non radicals (ODNR) are generated in metabolic pathways of biological systems. ODFR include

∙ ─ * * * ─ superoxide (O2 ), hydroxyl ( OH), peroxyl ( RO2), hydroperoxyl ( HRO2 ) while ODNR include hydrogen peroxide (H2O2) and hydrochlorous acid (HOCl).

These metabolites are responsible for DNA, lipid and protein modifications in case of oxidative stress(A. Kassab)(Young IS).

2. Production of reactive metabolites

Production of RMs mainly superoxides have been found in a variety of predominating cellular enzyme systems including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase (XO), cyclooxygenase (COX), uncoupled endothelial nitric oxide synthase (eNOS) and myeloperoxidase (MPO)(A.A. Woods) NADPH oxidase uses NADPH as a

2 substrate and is considered as an important source of ROS generation in vascular cells(A. Kassab) RM production may result from action of arachidonic acid metabolizing enzymes including cytochrome P-450, LPO, COX and those in the mitochondrial respiratory chain (C.F. Muller). They can also be produced from non-enzymatic reactions of organic compounds with oxygen as well as those initiated by ionizing radiations(L. H.-H. Pham-Huy). In addition exogenous factors such as drugs, pollution, immune responses to viruses, deficiency of natural antioxidants, ultraviolet rays and tobacco all of which may destroy the body potential of stabilizing free radicals(Sen).

3. Biological Roles Of Free Radicals

Free radicals are playing a key role in origin and evolution of life. They are important for activating different signaling pathways inside the cell, such as the

Mitogen activated protein kinase (MAPK) and extracellular-signal-regulated kinase (ERK) pathways that alter gene expression, as well as in coordination with superoxide dismutase initiates cell death. For instance, RNS produced by neurons act as neurotransmitters and those generated by macrophages act as mediators of immunity. These are also responsible for leukocyte adhesion, thrombosis, angiogenesis and vascular tone. Similarly, ROS is involved in gene transcription, single transduction and regulation of other activities in cell(Asmat Ullah).

4. Antioxidants

Free radicals produced under physiological conditions are maintained at steady state levels by endogenous or exogenous antioxidants which act as free radical scavengers. The endogenous antioxidants comprise of the enzymatic antioxidants such as superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase, and non-enzymatic antioxidants including glutathione, α-lipoic acid, vitamins C and E (Halliwell). On the other hand, the exogenous antioxidants include micronutrients and other exogenously administered compounds such as vitamin E, vitamin C, trace metals (selenium, manganese, zinc), carotenoids and flavonoids(Halliwell)(L. H.-H. Pham-Huy) .

5. Diabetes:

Diabetes mellitus is a group of disorders of multiple etiologies resulting from a defect in insulin secretion, insulin action, or both. Insulin deficiency in turn leads to chronic hyperglycemia (very high blood glucose levels) with disturbances in carbohydrate, fat and protein metabolism(Pastaki).The two major types of diabetes mellitus (DM) are insulin dependent (IDDM) - type 1 and non -insulin dependent (NIDDM)-type2.Type1DM is characterized by a specific destruction of the pancreatic β cells commonly associated with immune-mediated damage(Zhao). Individuals with type 2 DM display a gradual change in glucose

4 homeostasis due to insulin resistance and/or decreased insulin secretion(Omolola

R. Ayepola).

According to International Diabetes Federation; 371 million people have been reported with DM and the number is expected to rise to 4552 million by 2030

(Monisha Banerjee).

6. Complications of Diabetes mellitus

The injurious effects of hyperglycemia are separated into microvascular

(involving small vessels such as capillaries) and macrovascular complications

(involving large vessels, such as arteries and veins). Microvascular complications include diabetic nephropathy, neuropathy and retinopathy while macrovascular complications include coronary artery disease, peripheral arterial disease and stroke(Fowler).

7. Available therapy for Diabetes mellitus

Insulin therapy (Human insulin) and oral hypoglycemic agents (sulfonylureas, biguanides, α-glucosidase inhibitors, and glinides ) are the first line of treatment for the diabetes mellitus(Feshani AM), they act through different mechanisms include stimulation of insulin secretion by sulfonylurea and meglitinides drugs, increasing of peripheral absorption of glucose by biguanides and thiazolidinediones, delay in the absorption of carbohydrates from the intestine by

5 alpha-glucosidase and reduction of hepatic gluconeogenesis by biguanides(Wesam Kooti).

8. Oxidative stress and Diabetes

Evidences have been supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes. During oxidative metabolism in mitochondria, a component of the utilized oxygen is reduced to water, and the remaining oxygen is transformed to oxygen free radical (O) which is an important ROS that is converted to other RS such as ONOO, OH and H2O2(Moussa). Insulin signaling is modulated by ROS/RNS by two ways. On one side, in response to insulin, the

ROS/RNS are produced to exert its full physiological function and on the other side, the ROS and RNS have got negative regulation on insulin signaling, interpreting them to develop insulin resistance which is a risk factor for diabetes type 2, on the other hand free radical formation in diabetes by non-enzymatic glycation of proteins, glucose oxidation and increased lipid peroxidation leads to damage of enzymes, cellular machinery and also increased insulin resistance due to oxidative stress (Erejuwa)(Giacco).

9. Oxidative stress and Diabetic Complications

Hyperglycemia-induced oxidative stress is now recognized as the driving force for the development of diabetic complications(Kawahito). Many evidences from experiments have given link between diabetes and oxidative stress by measuring

6 various biomarkers(Asmat Ullah). Hyperglycemic state can lead to an increase in the levels of oxidative DNA damage markers such as 8-hydroxy-2’- deoxyguanosine and 8- oxo-7, 8-dihydro-2'-deoxyguanosine; lipid-peroxidation products measured as thiobarbituric acid-reactive substances; protein oxidation products such as nitrotyrosine and carbonyl levels and also lower the activity of antioxidant enzymes(Lee JL).

10. Medicinal Plants

In recent years, the interest in folk medicine from different cultures has increased significantly in industrialized countries, due to the fact that many prescription drugs worldwide have originated from the tropical flora.(Musa S.

Musa) A cooperative approach by ethno- botanists, ethno-pharmacologists, physicians and phytochemists are thereby essential to spur the progress of medicinal plants research(Gilani AH) .

Medicinal plants have traditionally played an important position in the socio cultural, spiritual and medicinal arena of rural and tribal lives in Sudan, through its long history, the Sudan has witnessed the fusion of many cultures, Pharonic,

Christian and Islamic along with the local indigenous cultures. With this unique history and vast variety of climate and flora, traditional medicine together with use of medicinal plants became an important part of the cultural heritage of the

Sudan(Musa S. Musa).

The abundance of information on the traditional medicinal uses of plants in

Africa is in danger of disappearing since the knowledge of how to use medicinal plants is mostly passed down orally and even to date is poorly documented, although written information has been produced for some specific regions.(A)

Moreover, the most serious threat to local medicinal plant knowledge, however, appears to be cultural change, particularly the influence of modernization and the western worldview which has contributed to under mining traditional values among the young (Musa S. Musa) (Voeks RA).

11. Medicinal plants, antioxidants and diabetics complications

The beneficial effect of antioxidants has been reported in animal models of diabetes as well as in diabetic patients.(Omolola R. Ayepola) Medicinal plants, mostly with antioxidant activity, are the major source of drugs for the treatment of oxidative stress induced diabetic complications, hence they having no or only few side effects (Rafieian-Kopaei M).Herbal preparations also have capacities to diminish drug and none drug induced toxicities(Shirzad H). These plants might provide useful sources for the development of new drugs useful in the treatment of diabetes mellitus and its complications(Mohammad Rahimi-Madiseh1).

Several plant extracts with hypoglycemic properties and protective activities act either through their positive impact on glucose homeostasis in diabetic patients or by lowering blood pressure and improving the renal and cardiovascular functions(Nasri H) (Asadi SY).Vegetables and fruits also have been shown to

8 contain high level of vitamins such as vitamin E and vitamin C which have proven to be beneficial in preventing cellular damage by inhibition of lipid peroxidation, protein oxidation, protein glycations and platelet aggregation (Mohammad

Rahimi-Madiseh1)(Asadi SY).

12. Medicinal Plants With Antidiabetic Activity

Because diabetes has no definite treatment, the use of traditional medicine seems to be an appropriate solution to control and manage it(Mahmoud Bahmani).

Several plants have been used as dietary adjuvant and in treating the number of diseases even without any knowledge on their proper functions and constituents.

This practice may be attributed to the uncompromised cost and side effects of synthetic hypoglycemic agent. Although numerous synthetic drugs were developed for the treatment of diabetes mellitus but the safe and effective treatment paradigm is yet to be achieved(Switi B. Gaikwad). Examples for such plants and their phytoconstituents having insulin mimetic activity were shown in table1(P. S. Patel DK).

Table1: List of plants having insulin mimetic or insulin secreatory activity

No. Plant botanical name Family Mechanism of action Active constituents

1 Aloe vera Liliaceae Stimulate synthesis and release Pseudoprototinosaponin AIII and

of insulin from the β-cells of prototinosaponins AIII

Langerhans(LW).

2 Annona squamosa Annonaceae Promote insulin release from Mangiferin and mangiferin-7-O-β-

the pancreatic islets, increasing dglucoside

utilization of

glucose in muscle and

inhibiting the glucose output

from liver(Malviya N).

3 Bauhinia variegata Caesalpiniac Have insulinotropic activity in Roseoside

eae insulin secreting cell(Frankish

N).

Camellia sinensis Theaceae Increases insulin activity and Epigallocatechin gallate

prevents oxidative

damage(Ayodhya S).

5 Citrullus colocynthis Cucurbitacea Increase insulin and decrease Beta-pyrazol-1-ylalanine

e plasma glucose levels(Dallak

M).

6 Ephedra distachya Ephedraceae Regenerate and restore of L-ephedrine

atrophied pancreatic islets

that induces the secretion of

insulin(Chauhan A).

7 Eriobotrya japonica Rosaceae Increase insulin Cinchonain ib

secretion(Qa’dan F)

8 Eugenia jambolana Myrtaceae Enhance insulin secretion Pandanus odorus (Toei-hom) a 4-

from cells and inhibited hydroxybenzoic acid

insulinase activity from liver

and kidney(LW).

9 Ficus bengalensis Moraceae Inhibite insulinase activity Leucocyandin 3-O-beta-d

from liver and kidney(LW). galactosyl cellobioside,

leucopelargonidin-3-

O-alpha-L rhamnoside

10 Glycyrrhizae radix Fabaceae Induce mRNA levels of insulin Glycyrrhetinic acid, dihydroxy

receptor substrate-2, pancreas gymnemic triacetate

duodenum homeobox-1, and

glucokinase in the islets, which

contributed to improve beta-cell

viability(Ko BS).

11 Momordica charantia Cucurbitacea Increase number of beta Momordicin, charantin, and

e cells(Ayodhya S). galactose-binding lectin

12 Panax ginseng Araliaceae Increase liver glycogen level Polypeptides

and stimulate insulin

secretion(P. S. Patel DK).

13 Prunella vulgaris Lamiaceae Repair cells of pancreatic islet Jiangtangsu

to release insulin(Chauhan A).

14 Psidium guajava Myrtaceae Improve cells sensitivity of Strictinin, isostrictinin and

insulin(Chauhan A). pedunculagin

15 Enhancing insulin release and Epicatechin

Pterocarpus marsupium Fabaceae convert proinsulin to

insulin(Modak M).

16 Semen coicis Poaceae Prevention of pancreatic beta- Coixans

cells injury(Chauhan A).

17 Stevia rebaudiana Asteraceae Stimulate insulin secretion via a Stevioside, steviol

direct action on beta cells(P. S.

Patel DK).

18 Swertia chirayita Gentianacea Increased plasma insulin

e without influencing hepatic Swerchirin

glycogen content(Chauhan A).

19 Teucrium polium Lamiaceae Enhance insulin secretion(P. S. Apigenin

Patel DK).

20 Trigonella foenumgraecum Fabaceae Stimulate insulin release by 4-hydroxyleucine and

isolated islet cells(Haeri MR). hydroxyisoleucine

21 Zizyphus spina-christi Rhamnaceae Increase serum insulin and

pancreatic cAMP Christinin-A

levels.(Chauhan A)

12.1. Phytochemicals and management of Diabetes mellitus

The most common herbal active ingredients used in treating diabetes are flavonoids, tannins, phenolic, and alkaloids that improve the performance of pancreatic tissues by increasing the insulin secretion or decreasing the intestinal absorption of glucose(Mamun-or-Rashid A).The existence of these compounds implies the importance of the anti-diabetic properties of these plants(Gupta PD).

12.2. Flavonoids

Flavonoids (bioflavonoids) are a diverse group of polyphenols (phenyl

benzopyrans) which are well-known for their multi-directional biological activities

including anti-diabetic efficacy(Corradini) . These effects are generally associated

with free radical scavenging activity of flavonoids. The antioxidant effects of

flavonoids are enhanced by the number and position of hydroxyl groups in the

molecule. The catechol structure, presence of unsaturation and 4-oxo function in

the C-ring also contributes to their radical scavenging activity(Heim). Flavonoids

may be capable of binding the transition metal ions, which play a role in

glycoxidation, thus preventing metal-catalysed formation of hydroxyl radicals or

related species from H2O2 (Heim).

OH OH

HO O HO O

OH OH O OH O

Flavone Flavonol

O OH

HO O

OH O Isoflavone Flavanone

Flavan-3-ol

Figure-1: Chemical Structures of some classes of flavonoids(Omolola R. Ayepola)

The potential beneficial effects of flavonoids in the prevention of diabetes mellitus

and its associated complications have been investigated both in vitro and in vivo

studies.

The inhibitory effect of flavonoids on glycation has been demonstrated and it is

suggested that this effect is partly due to their antioxidant properties (Wu).

Epigallocatechin has a beneficial effect in nephropathy via suppressing

hyperglycemia, proteinuria and lipid peroxidation, it also reduced renal

17 accumulation of advanced glycation end products (AGEs) and their related oxidative stress(Yamabe N). Another study demonstrated the in vitro inhibitory effect of different flavonoids (myricetin, hyperin, quercetin, rutin, catechin and kaempferol) on pentosidine formation(Urios). In addition to their antioxidant effect, flavonoids (quercetin and the flavanone, naringin) possess inhibitory activity on aldose reductase pathway and can serve as a potential multifunctional agent in the prevention of diabetic retinopathy(Goodarzi). Oral administration of two isoflavone compounds, tectorigenin and irigenin also inhibited sorbitol accumulation in the lenses of streptozotocin induced diabetic rats(Jung).

Activation of Protein kinase C (PKC) contributes to the loss of capillary pericytes, thickening of vascular basement membrane in diabetic retinopathy (Kim). Also

PKC mediated alterations in vascular permeability, blood flow, formation and response to angiogenic growth factors contribute to retinal leakage, ischemia, and neovascularization(Frank).Therefore, PKC inhibitors can be targeted for the treatment of diabetic retinopathy. Hesperetin, a flavanone found in citrus fruits has retina vasculo-protective properties due to its strong anti-angiogenic effect via inhibiting PKC-β pathways(Gupta PD).Modulation of endogenous biomarkers and inhibition of diabetes induced neuropathic pain was observed in diabetic rats after naringin (4′, 5, 7-trihydroxy flavonone 7-rhamnoglucoside) administration(Kandhare).

The soy isoflavone genistein administered by a subcutaneous injection to diabetic mice relieved peripheral painful neuropathy by reverting the proinflammatory cytokine and ROS overproduction. It also restored the inducible nitric oxide synthase and eNOS content and increased NO production in thoracic aorta although treatment had no effect on hyperglycemia(Valsecchi). The flavonoid luteolin, when administered to rats orally, protected against the progression of diabetes-induced cardiac dysfunction by attenuation of myocardial oxidative stress probably through its antioxidant properties(Wang). In a double blind placebo-controlled study, the effects of daflon (made up of flavonoids diosmin (90%) and hesperidin (10%)) was investigated in a group of diabetic patients resulted in a decrease in HbA1C which is associated with an increase in the level and activities of thiol-containing antioxidants such as glutathione perox idase. The in vitro protective effect of myricetin on protein oxidation and membrane lipid peroxidation of erythrocytes from diabetic patients was reported (Manuel y Keenoy).

The treatment of diabetic rats with rutin decreased fasting plasma glucose, glycosylated haemoglobin, thiobarbituric acid reactive substances and lipid hydroperoxides while levels of non-enzymatic antioxidants were increased.(Kamalakkannan) In another study, rutin supplementation to diabetic patients decreased the levels of fasting blood glucose, blood pressure and improved lipid profiles in the diabetic subjects. Rutin reduced blood glucose, ameliorated oxidative stress and inhibited the accumulation of extracellular matrix component

19 and glomerular basement membrane thickening in the renal cortex of diabetic rats suggesting its renoprotective effect in experimental diabetic nephropathy(Sattanathan).

Diosmin (diosmetin 7-O-rutinoside) is a natural flavone glycoside which can be obtained by dehydrogenation of the corresponding flavanone glycoside, hesperidin that is abundant in the pericarp of various citrus fruits(Campanero). Diosmin treatment of streptozotocin-nicotinamide induced diabetic rats, ameliorated oxidative stress in plasma and tissues as evidenced by improved glycemic and antioxidant status along with decreased lipid peroxidation(Tang).

Quercetin enhances endothelium-derived NO bioavailability, reduced blood glucose levels and oxidative stress in diabetic rats suggesting its beneficial effect in vascular function(Machha).

OH OH OH HO O OH OH OH O HO O OH HO O OH OH O OH OH OH OH OH OH O Epigallocatechin Catechin Quercetin OH OH OH OH HO O OH O OH O HO HO OH OH OH O HO O HO O Kaempferol Tectorigenin Irigenin

OH OH

HO O OH

O OH O OH O OH O HO O OH O O OH O OH O HO O OH HO OH HO O O O OH H O O

HO H HO OH HO OH OH OH OH

OH O OH

OH HO O

OH Rutin Hyperin Naringin OH OH O

OH OH O HO O

OH OH HO O

Myricetin Hesperetin Genistein

OH OH O OH

O O

HO HO O HO O O O OH HO O OH

OH HO OH

Luteolin Diosmin

Figure-2: Flavonoids for diabetes management

12.3. Alkaloids

Different alkaloids have been isolated from several medicinal plants (Table2) and investigated for their possible antidiabetic activity through different mechanisms(Switi B. Gaikwad).

Table2: List of important alkaloids used in the treatment of Diabetes mellitus

Name Chemical structure Mechanism of action

Berberine OH Inhibit alpha-glucosidase and decrease glucose HO + N transport through the intestinal epithelium(Pan,

O Huang and Wang). O

Casuarine 6-O-α-glucoside OH Inhibit alpha-glucosidase and decrease glucose HO OH N H transport through the intestinal epithelium.(Pan,

OH OH O O Huang and Wang)..

HO OH OH

Calystegine B2 Reduce diet-induced hyperglycemia and H N OH H endogenous insulin secretion by inhibiting OH OH intestinal R-glucosidases(Switi B. Gaikwad). OH

Cryptolepine Increased glucose uptake by 3T3-L1 cells(Switi

N B. Gaikwad).

Harmane , norharmane H H Have a stimulatory action on insulin secretion N N N

N by the activation of imidazoline I binding sites

in the pancreatic cell(organisation).

Jambosine OH Halts the distatic conservation of starch (Switi O

O B. Gaikwad).

O OH

HO O O O

Jatrorrhizine, magnoflorine O Stimulate insulin secretion from the RINm5F O O and palmatine O cell line(Patel and Mishra). O + HO N + OH N O

+ N O O OH O

Javaberine A, hexaacetate OH Inhibitors of TNF-αproduction by macrophages OH OH OH O and fat cells(Catthareeya, Papirom and O N H O Chanlun). O

OH O OH O

Lepidine Reducing oxidative damage and modulating

antioxidant enzymes and potentiating

pancreatic secretion of insulin from the N remaining islet cells(Shukla, Bigoniya. and

Srivastava).

H Lupanine H Enhance glucose-induced insulin release from O N N cells(Lopez, Mora and Wysocka). H H

Mahanimbine O

N H

Piperumbellactam A O α-glucosidase inhibitor(Dineshkumar, Mitra OH and Mahadevappa)

N HO O

Radicamines A and B OH OH α-glucosidase inhibitor(Shibano, Tsukamoto OH OH

HN and Masuda). OH HN OH

HO O OH

Swerchirin OH O OH α-glucosidase inhibitor(Takada, Uehara and

Nakao). O O O

Tecomine Exerted a potent stimulating effect on the basal

O glucose uptake(Costantino, Laura and Renato). HN H

1-deoxynojirimycin OH α-glucosidase inhibitor(Brahmachari)(Oku, HO OH Yamada and Nakamura). N OH H

1.1. Terpenoids and Steroids

Triterpenoid and steroidal glycosides, referred collectively as Saponins. These are bioactive compounds present naturally in many plants and known to possess potent hypoglycemic activity.(Rao and Gurfinkel). Few of such phytoconstituents have been isolated and identified (table3)(Switi B. Gaikwad).

List of imported Terpenoids & Steroids reported for Diabetes mellitus

Name Chemical structure Mechanism of action

H α -amyrin acetate H Unknown(Narender, Khaliq and

H Singh)(Ragasa, Tsai and Shen). H O

O H

Andrographolide O Activate alpha1- ARs to enhance the H HO secretion of beta-endorphin which can

H stimulate the opioid micro-receptors to

HO O reduce hepatic gluconeogenesis and to O enhance the glucose uptake in soleus

muscle(Yu, Chang and Su)(Yu, Hung and

Chen).

Bassic acid Increase glucose uptake, plasma insulin

H OH levels and glycogen synthesis It appears H HO O that this effect was mediated by an insulin HO H secretagogue effect in pancreatic OH cells(Naik, Barbosa and Dhuley).

Christinin A O Stimulates the release of insulin and blocks O H the formation of glucose in the blood

O O O O stream(Glombitza, Mahran and Mirhom). H H O

Corosolic acid Glucose transport activator(Kakuda,

H Sakane and Takihara)(Murakami, Myoga OH H HO and Kasai). O

HO H

Elatosides E Unknown.(Yoshikawa, Harada and OH H Murakami) O

H O O HO HO O O HO O O OH

HO OH HO OH

HO Forskolin O Stimulates glucose-induced insulin OH secretion(Switi B. Gaikwad). O OH O O

Ginsenosides OH Stimulates insulin secreting pancreatic β-

cells and the target tissues that take up

glucose(Rotshteyn and Zito).

OH H HO O O HO OH HO

Gymnemic acid IV O Inhibits the glucose absorption OH OH (glycosidase inhibition), increases glucose OH H uptake in striated

muscles, lowers blood glucose and

H increases the insulin secretion in OH OH O O O pancreatic β-cells(Sugihara, Nojima and

HO OH Matsuda)(Patel, Gadewar and Tripathi). OH

Momordin ic OH Inhibit glucose absorption(Yoshikawa, O H Shimada and Morikawa).

H O OH HO O O O

HO O OH OH OH

β-sitosterol Assist in normalizing the insulin levels and

the blood sugar levels by inducing the

H secretion of insulin even in the absence of

H any stimulatory glucose concentration. HO This secretion therefore inhibits glucose-6-

phosphatase enzyme.(Switi B. Gaikwad).

Senegin derivatives Unknown.(Yoshikawa, Murakami and Cl H Matsuda) OH HO O HO HO O

1.2.5. Glycosides

Hederagenin glycosides and phenolic glycosides were showed potent ant diabetic activity(Park, Kim and Choi). Jamboline (antimellin), a glycoside present in the seeds of Syzygium cumini (Eugenia jambolana), possesses hypoglycemic action by preventing conversion of starch into sugar and also diminishes quantity of sugar in urine and reduces thirst(Sagrawat, Mann and Kharya)(Yarnell, Abascal and Rountree). A flavanone glucosides (myrciacitrins I, II, III, IV, and V) and acetophenone glucosides (myrciaphenones A and B) were showed an inhibitory action on aldose reductase and alpha-glucosidase(Yoshikawa, Shimada and

Nishida)(Matsuda, Nishida and Yoshikawa).

Myrtillin glycoside was found to be effective in reducing glycosuria and postprandial hyperglycemia in most adult-onset diabetic patients but was rarely effective in juvenile-onset patients.(Switi B. Gaikwad) The antidiabetic effect of a dimethoxy derivative of perlargonidin 3-O-α- L rhamnoside isolated from the bark of Ficus bengalensis L. (Moraceae) has been due to stimulation of insulin secretion by β-cells. (Switi B. Gaikwad) Two steroidal glycosides, PO-1 and PO-

2, were isolated from rhizomes of Polygonatum sp. (Liliaceae). These compounds were exhibited significant hypoglycemic effects in streptozotocin-induced diabetic mice.(Kato, Miura and Fukunaga)

Pseudoprototinosaponin AIII & prototinosaponin AIII were affect glucose uptake and insulin release. (Switi B. Gaikwad) Three flavonoid glycosides: vitexin,

34 isovitexin, and isorhamnetin 3-O-β-D-rutinoside revealed satisfied glucosidase inhibitory effects.(Chen, Li and Li)

OH O

H H O O

H HO O OH HO O OH OH H OH OH O O O O O HO H HO OH HO OH OH OH OH

Hederagenin Myrciacitrins I Myrciacitrins II

H O HO OH HO O OH HO OH HO HO OH O O O OH HO O O O O O O O O O

HO OH HO OH HO OH OH OH OH

Myrciacitrins IV Mmyrciaphenones A Mmyrciaphenones B

OH O OH + HO O HO OH OH O OH OH HO O HO O HO O OH O OH OH HO O HO OH HO OH OH OH OH

Myrtillin Vitexin Isovitexin

OH O OH O OH

O H

OH O O

HO O OH OH O HO OH OH

Pseudoprototinosaponin AIII

Figure-3: Chemical Structure of some Glycosides reported for diabetes

management

13. Family Fabaceae

Family of flowering plants within the order Fabales. The third largest family among the angiosperms after Orchidaceae and Asteraceae, consists of more than

700 genera and about 20,000 species of trees, shrubs, vines, and herbs(Christenhusz and Byng).

13.1 Bauhinia rufescens

13.1.1 Scientific classification:

Kingdom: Plantae

Order: Fabales

Family: Fabaceae

Genus: Bauhinia

Species: B. rufescence

Local names: Arabic (kulkul,kharoub); Fula (nammare); Hausa

(matsagi,jirga,jiga); Wolof (randa)

Binomial name: Bauhinia rufescens Lam.(Lam)

13.1.2 Description:

Bauhinia rufescens is a shrub or small tree usually 1-3 m high, sometimes reaching 8 m; bark ash-grey, smooth, fibrous and scaly when old; slash pink; twigs arranged in 1 plane like a fishbone, with thorn like, lignified, lateral shoots,

10 cm long. Leaves are very small, bilobate almost to the base, with semi-circular lobes, glabrous, with long petioles, greyish-green, less than 3 cm long (Orwa C)

Flowers are greenish-yellow to white and pale pink, in few-flowered racemes; petals 5, spathulate, 15-20 mm long; stamens 10, filaments hairy at the base.

Fruits aggregated, long, narrow pods, twisted, up to 10 cm long, glabrous, obliquely constricted, shining dark red-brown, with 4-10 seeds each. Pods remain on the shrub for a long time (Orwa C).

B. rufescens is deciduous in drier areas and is an evergreen in wetter areas. It is often found in dry savannah, especially near stream banks. It is found in the entire Sahel and adjacent Sudan zone, from Senegal and Mauritania across North

Ghana and Niger to central Sudan and Ethiopia.(Orwa C)

13.1.3 Medicinal uses of B. rufescens:

A cold infusion of the bark is astringent and is used in Nigeria as a wound- dressing and to treat diarrhoea and dysentery. It is rich in tannin and is used locally to tan leather. Trunk and root-barks are prepared as decoctions or infusions in

Senegal for treating syphilis and jaundice. The bark, or the root, is also widely used in Decoction to treat leprosy and the bark is reported used as a small-pox remedy. The Roots are held in Senegal to be febrifugal, diuretic and anti- enteralgic. The leaves are stomachic, anti-diarrhea and anti-dysenteric: though

Preparations may be variable; macerates are made with millet flour and goats’ milk in Senegal. In Mali, the leaves, root and fruit are macerated with millet leaves

38 for Dysentery. This is considered by the Bambara to be very effective. Vapour from the boiling pot of the leaves is used in Senegal to treat conjunctivitis. The young leaves are used in Nigeria as an expectorant. Powdered leaves in water are taken internally and used as an external wash for snake-bite in Senegal ((HM.).

13.1.4 Local uses of B. rufescens:

The fruit is eaten by both man and animals. Protein assays is 10-11%. The plant is credited with magical properties in Senegal to ensure the millet harvest and to protect houses from snakes. The wood is light-brown, hard and used in carpentry. The crude bark can be used as tying material, or the fiber can be extracted to produce cordage (HM.).

13.2 Biological Activities of the Genus Bauhinia

13.2.1 Anti-diabetic Properties

Plants belonging to the genus Bauhinia are reported to be important antidiabetic agents; the leaves and stem-bark of these plants are used in different phytopreparations to lower blood glucose levels(da Silva KL)(-Cavalcanti

KM)(Mali RG).

B. forficata, an Asiatic plant, is one of the most commonly used plants against diabetes. Aqueous, ethanol and hexane extracts of B. forficata was reduced glucose, triglycerides, total cholesterol and HDL cholesterol levels in alloxan- induced diabetes in rats.(Menezes PR) The aqueous extracts obtained from B. monandra leaves exhibited hypoglycemic activity when evaluated in normoglycemic mice(de Sousa E). The hypoglycemic activity of a leaf infusion

39 of B. candicans in alloxan and streptozotocin-induced hyperglycemic rats indicated a remarkable hypoglycemic effect in experimental models, with a greater decrease of glycemia in alloxan diabetic rats(Lemus I), while decoction of B. divaricata significantly decreased the hyperglycemia along with reduced urinary glucose excretion in alloxan-induced diabetic rabbits(Roman

RR)(Fuentes O).

Studies carried out with the aqueous leaf extract of B. megalandra indicated that it is able to inhibit intestinal glucose absorption, as well as glucose-6- phosphatase and hepatic gluconeogenesis, suggesting that this plant might help control hyperglycemia in diabetic patients(Gonzalez-Mujica F)Methanol leaf extract from B. cheilandra exhibited considerable hypoglycemic action when tested on glucose loaded and alloxan-induced diabetic rats(Almeida ER).

13.2.2 Antimicrobial Studies

Plants belonging to the genus Bauhinia are frequently used in folk medicine to treat infectious diseases, and several experimental studies have confirmed their antimicrobial potential, especially against pathogenic fungi and bacteria. B. splendens has activity against pathogenic bacteria, especially gram-positive microorganisms such as Streptococcus sp., Staphylococcus aureus and

Salmonella typhimurium(Savi AO). On the other hand, both B. forficata and B. microstachya, proved inactive when tested against pathogenic bacteria such as

Escherichia coli and S. aureus.(da Silva KL). Evaluation of the antifungal potential of B. forficata showed that it is active against dermatophytes such as

Microsporum canis, Trychophyton mentagrophytes, T. rubrum and

Epidermophyton floccosum, although it does not inhibit several yeasts..(Savi)(Achenbach H).

studied the chemical composition and antifungal effects of B.manca collected in Costa Rica; the results showed that several metabolites, particularly the flavonoids (2S)-3′,4′-dihydroxy- 7-methoxyflavan and (2S)-7,4′- dihydroxyflavan, caused significant inhibition of phytopathogenic fungi such as

Botrytis cinerea, Claviceps viridis, Coprinus cinereus, Rhizoctonia solani and

Saprolegnia asterophora. Similarly, the dichloromethane extract of B. rufescens collected in Niger exhibited antifungal activity against Cladosporium cucumerinum(Maillard MP)

Studies carried out with B. racemosa, a plant used in India to treat various pathologies, including infections, confirmed its production of active principles with antimicrobial potential (Ali MS)(Kumar RS). which showed broad-spectrum of antimicrobial activity against a panel of fungi and bacteria, was due to the presence of phenolic metabolites. On the other hand, B. variegata, a plant collected in Nepal, was found to have antimicrobial activity against Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteriae,

Staphyllococcus aureus and Vibrio cholerae.(Pokhrel NR) The same plant, collected in India, showed remarkable activity against gram-positive and gram- negative bacteria. Defatted acetone and methanol extracts were most active

41 against all the organisms studied when evaluated by agar well diffusion method

(Parekh J).

13.2.3 Analgesic Potential Studies

The stem extract of B. splendens contains active flavonoids and tannins with antinociceptive potential, when evaluated against acetic acid-induced abdominal constrictions in mice, and this effect is dose-related, with a long-lasting inhibition

(up to 3 h) against the writhing test by intraperitoneal or oral administration, and that it is also active by both routes in the formalin model, Attenuating the first and second phases of this experimental model(B. E.-F. Cechinel Filho V). Extracts and two Phytoconstituents from the leaves of B. microstachya leaves have also shown Antinociceptive effects those are more potent than the reference drugs

(Meyre-Silva C).

13.2.4 Anti-inflammatory activity

B. guianensis, showed anti-inflammatory activity when tested against carrageenan, dextran and histamine-induced paw edema in rats, also inhibited the algogenic process in the writhing test induced by acetic acid in mice (Carvalho

JC). Bioassay-oriented fractionation, using the inhibition of croton oil-induced ear oedema in mice as a model for anti-inflammatory activity, and identified a 2:1 mixture of ursolic and oleanolic acids as that having the strongest anti-phogistic effect, with potency similar to that of indomethacin. Both triterpenic acids have long been recognized as having anti-inflammatory activity, as well as other important biological properties.(J.)(Deepak M)(Cipak L). The effects of the

42 methanol stem bark extract of B. racemosa on the acute and chronic phases of inflammation were studied in carrageenan, dextran and mediators (histamine and serotonin) - induced paw oedema, and cotton pellet induced granuloma, respectively, showing pronounced anti-inflammatory properties compared with indomethacin, the extract also inhibited peritoneal leukocyte migration in mice.

In addition, it produced a significant analgesic effect when analyzed in the acetic acid induced writhing and hot-plate tests in mice. The extract also produced a morphine- and aspirin-induced analgesic effect in mice(Gupta M). Flavonol glycoside, 5,7,3′,4′-tetrahydroxy-3-methoxy-7-O-alpha-L- rhamnopyranosyl(1→3)-Obetagalactopyranoside were isolated from B. variegate beside several known flavonoids together with a triterpene caffeate from the aerial parts of this plant which showed anti-inflammatory potential(Yadava RN)(Rao

YK). They were evaluated as inhibitors of some macrophage functions involved in the inflammatory process. Some of them, particularly kaempferol, ombuin and the triterpene caffeate caused significant inhibition of lipopolysaccharide (LPS) and interferon (INF)-gamma induced nitric oxide (NO), and cytokines [tumor necrosis factor (TNF)-alpha and interleukin (IL)-12. (Rao YK). B. malabarica, produces biologically active products such as racemosol and 10-O- demethylracemosol, which exhibit potent invitro anti-inflammatory activity against cyclooxygenase-1 and -2 (COX-1 and 2) enzymes.(Kittakoop

P)(Songarsa S). These metabolites have been used as models for the synthesis of several derivatives and to carry out structure activity relationship studies. The

43 leaves aqueous extract of B. purpurea have an analgesic effect when evaluated against the writhing, hot plate and formalin tests and an anti-inflammatory effect in the carrageenan-induced paw edema test(Zakaria ZA). Several known metabolites with anti-inflammatory activity, especially bibenzyls and dihydrodibenzoxepins were isolated from the roots of this plant (Boonphong S).

Chemical investigation of the stem bark of Bauhinia rufescens resulted in the isolation of a cyanoglucoside and menisdaurin from methanol extract and oxepin from petroleum ether extract. The isolated compounds were tested for their anti- inflammatory potentials based on the COX-2 enzyme (Sirat).

13.3 Pharmacological and Toxicological Investigations of Genus

Bauhinia

It is recognized that plants of the genus Bauhinia are not toxic(ciência). This has been confirmed by several experimental investigations using animal models(Pepato MT) by evaluating an orally administered decoction of B. forficata for its effects on the toxicity markers amylase (pancreas toxicity), creatine kinase

(muscle toxicity), lactate dehydrogenase (muscle and liver toxicity), bilirubin

(liver and biliary toxicity) and angiotensin-converting enzyme (renal microcirculation and kidney toxicity), and concluded that the decoction could be used for treatment of diabetes without causing tissue toxicity as shown by the biomarkers used(Damasceno DC). The B. forficata extract causes repercussions in the antioxidant defense system, with an increase in uric acid and a decrease in glutathione and superoxide dismutase activity. and that treatment of pregnant

44 diabetic female rats with B. forficata did not control maternal hyperglycemia, hyperlipidemia or hypercholesterolemia, but it did increase the amount of hepatic glycogen, decreased the uric acid concentration, and increased the glutathione activity (Damasceno DC). This suggests that the extract of the plant acts as an antioxidant defense system, probably due to the presence of the previously detected flavonoids(Da Silva KL)(B. E.-F. Cechinel Filho V)(Pizzolatti MG). The stem-bark extract of B. racemosa does not alter the hematological parameters, liver and kidney functions, at doses of 100 and 200 mg/kg body weight in mice, indicating that it is free of toxic effects. However, at a higher dose it exhibited significant alterations in their haematological profile, hepatorenal functions and metabolism(Kumar RS). Similarly, the leaf extract of B. galpinii showed anti mutagenic properties in the presence of metabolic activator S9 and that it did not cause any mutagenic response against Salmonella typhymurium bacterial strains in the Ames assay, confirming its non-toxic effects (Reid KA)

Rationale

The plant Bauhinia frufescens Lam common name Kharoub (Fabacea ) has been used traditionally in Sudan, Nigeria and Senegal for various pharmacotherapeutics purposes. However till date there are no reports on scientific validation of its traditional claim. In addition, Bauhinia has been reported as potent anti oxidant, antidiabetic, anti-inflammatory and for biological properties.

Hence, it was thought worthwhile to determine anti oxidant and anti diabetic potential and B. rufescens bioactivity guided identification, isolation & characterization of the bio-active compounds

Aims Objectives:

This study aimed to:

• Evaluate the anti-oxidant and antidiabetic activity of Bauhinia rufescens.

• Bio-activity guided fractionation and isolation of active compounds, identification and structure elucidation of the isolated constituents.

• HPTLC – LCMS – autobiographic identification and characterization of bioactive constituents of the most active fraction

CHAPTER TWO

Materials & Methods

1. Plant material

The plant was collected from Northern Kordofan and authenticated at the herbarium of Medicinal and Aromatic Plants Research Institute (MAPRI) and a

Voucher specimen was deposited at the Department of Pharmacognosy, Faculty of

Pharmacy, University of Khartoum.

2. Extraction procedure

Powder of the air-dried leaves of the plant (1 kg) was exhaustively extracted with petroleum ether using a soxhlet apparatus. petroleum ether extract was concentrated under reduced pressure using rotary evaporator where 18 gm of the petroleum ether extract was obtained and the remaining marc was dried and extracted similarly using chloroform and concentrated with rotary evaporator (12 gm of the chloroform extract was obtained) . Then the marc was dried and extracted with methanol using a soxhlet

(28 gm was obtained). The methanol extract (active extract) was fractionated with ethyl acetate, chloroform and n-buatnol respectively after suspending methanolic extract in water.

2. In vivo anti diabetic assay of plant extracts

The antidiabetic activity of plant extracts was determined according to the method of Washim Khan etal. (Washim Khan) Both normal and streptozotocin-diabetic wistar rats (150 g body weight) were used in this study. Diabetes was induced in the rats by administration of streptozotocin (in citraye buffer PH 5.5) intraperitoneally at a dose of 60 mg/kg. Three days later the blood glucose level was measured using Reflotron

Instrument and Reflotron glucose kit (Roche Diagnostics). Animals with blood glucose level around 300 mg/100 ml blood were used.

To test the effect of Bauhinia rufescence extract on blood glucose level in normal and streptozotocin-diabetic rats, the animals were fasted for 12 hours and then administered the extract in doses of 1 g/kg intraperitoneally. Two hours later, the animals were anaesthetized with ether and blood was collected via cardiac puncture.

Blood glucose level before and after treatment was measured using Reflotron

Instrument and the provided glucose kits (Roche diagnostics).

4. In vitro anti diabetic assay of most active fraction using TLC bioautographic method for the detection Amylase enzyme inhibitors.

Alpha-amylase (200 Unit) was dissolved in buffer solution (pH 7.15). Further,

starch solution (potato starch in water) and iodine solution (potassium iodide in

water and add iodine, shake till dissolved completely). The entire prepared sample

kept at 4ºC and iodine solution kept in dark place at room temperature for further

use. After migration of the samples, the TLC plate was dried for complete removal

of solvent. The TLC plate was then dipped into prepared enzyme solution and

incubates in humid desiccator for 1.5 h. The humid desiccator was filled with a

little water, such that the plate does not meet the water, but enough to keep

humidity inside the container. Incubation was performed for 30 min at room

temperature for the primary reaction between the enzyme and enzyme inhibitors

separated in the extracts on TLC plate, followed by starch solution and incubated

for another 15 min for enzyme substrate reaction, and then air dried and finally

dipped in iodine solution. α-amylase activity was visible on the TLC plate by the

appearance of white-yellow spot on a dark brown background after the treatment

with substrate.

5. Antioxidant assay

5.1 Quantitative antioxidant assay using DPPH radical scavenging assay

The DPPH radical scavenging was determined according to the method of Brand et al.

(W. W. Brand) with some modification. In 96-wells plate, the test samples were allowed to react with 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl stable free radical

(DPPH) for half an hour at 37ºC. The concentration of DPPH was kept as (300μM).

The test samples were dissolved in DMSO while DPPH was prepared in ethanol. After incubation, decrease in absorbance was measured at 517nm using multiplate reader spectrophotometer. Percentage radical scavenging activity by samples was determined in comparison with a DMSO treated control group. All tests and analysis were run in triplicate.

5.2 Qualitative antioxidant assay using Bioautography method

The extracts were run on TLC silica plate in earlier described solvent system. Plates were air dried and then sprayed with DPPH (0.001 g in 95% methanol/ethanol). Bright yellow or cream spot was shown against a purple background.

6. LC-MS analysis of the active fraction

The 5 mg/mL solutions of each sample, filtered through 0.2µM PTFE membrane filter as prepared previously were used for UPLC-MS analysis. In the present study, UPLC was performed on a Water’s ACQUITY UPLC(TM) system (Waters

Corp., MA, USA) equipped with a binary solvent delivery system, an auto- sampler, column manager and a tunable MS detector (Waters, Manchester, UK).

The system was operated under the Empower software (Waters, USA). Data acquisition has been done in positive modes. Chromatography was performed using acetonitrile (A) and 0.5% v/v formic acid in water (B) as the mobile phases on monolithic capillary silica based C18 column (ACQUITY UPLC(R) BEH C18

1.7 µm, 2.1 x 100 mm), with the pre-column split ratio 1:5, flow rate 10 µL/min at ambient temperature. Separation was achieved by stepwise gradients from 5%

B to 100% B for 20 min. The flow rate of the nebulizer gas was set to 500 L/h, for cone gas it was set to 50 L/h and the source temperature was fixed to 100 ºC.

The capillary voltages and cone voltage were set to 3.0 and 40 KV respectively.

For collision, argon was employed at a pressure of 5.3 х 10-5 Torr. The accurate mass and composition for the precursor ions and for the fragment ions were calculated using the Mass Lynx V 4.1 software incorporated in the instrument.

Data obtained from UPLC-MS was processed by Mass Lynx V4.1 (Waters, USA) and further used for metabolomics analysis of different samples. Separated

51 metabolites present in different samples were tentatively identified based on their m/z ration and on literature

7. High performance thin layer chromatography (HPTLC) analysis of active fraction

After trying numbering of TLC in different solvent system by hit and trial method;

The presence of spot was confirmed by TLC in a specific solvent system

(Toluene: Ethyl acetate: Formic acid (5:4:1, v/v/v). Prepared sample the active fraction filtered and 5 µl of the solution was separately applied on Silica gel 60

F254 pre-coated HPTLC plates, 10x10 cm (Merck, Germany) with the help of

Camag Linomat-V (CAMAG, Switzerland) applicator and eluted the plate to a distance of 7.5 cm at room temperature (25˚C) in specific developed solvent system as described earlier. The sample solution was applied to 6 mm wide band using Camag Linomat-V automated TLC applicator with the nitrogen flow providing a delivery speed of 150 nL/s from the syringe. Plates were developed in a Camag twin through glass tank pre-saturated with the mobile phase for 40 min. The plate was developed horizontally in Camag horizontal developing chamber (10 × 10 cm) at the room temperature. Plate was scanned at different wavelength such as 254nm, 354 and 454 nm. After heating the plate at 100 ºC for

5 min, derivatization of the chromatogram was done with 5% anisaldehyde sulphuric acid in methanol. The scanning was carried out at 565 nm with a Camag

TLC scanner III using the Wincats1.2.3 software.

8. Isolation of some active compounds and structure determination using

Nuclear Magnetic Resonance (NMR).

Isolation of some active antidiabetic and antioxidant principles achieved by preparative thin layer chromatography (PTLC) and structure determination of the isolated compound achieved by NMR. 1H- and 13C-NMR spectra were carried out on the Bruker AM 500 spectrometer (Germany) operating at 700 MHz (1H-NMR) and 125 MHz (13C-NMR) in spectroscopic grade

DMSO-d6. The chemical shifts values are expressed in δ (ppm) units using (TMS) as an internal standard and the coupling constants (J) are expressed in Hertz (Hz).

Standard pulse sequences were used for generating COSY, HMQC and HMBC spectra (2D experiments).

9. In silico assessment of the compounds from active fraction

ChemDraw Ultra 12 software was used to prepare the compounds' 3D structures which were saved as PDB format. On the other hand, MDL.sdf and SMILE formats were generated using Open Bable software. The manually optimized protein 3D structure was retrieved from protein databank. Manual energy minimization was conducted on the Swiss PDB viewer V.4.1.0. software.

Molecular docking was performed using Autodock 4.0 software based on

Lamarckian Genetic Algorithm.(Mohamed MA)

The 3D structures of the 38 compounds and standard protein (Acarbose) were obtained from PubChem compound database and RCSB protein Data Bank respectively. The co-crystal ligand was removed from the protein target (α- glucosidase and α-amylase) which was then prepared according to the standard docking protocol. The UniProt databases were used to verify the active site. The target's grid map was calculated and set to 60×60×60 points with grid spacing of

0.375 Ǻ to assure that all the active residues were included in the center. The default docking algorithms were set in accordance with the standard docking protocol. Finally, ten independent docking runs were carried out for each ligand and results were retrieved as binding energies. Poses that showed lowest binding energies were visualized using DS Visualizer Client (Windows 64 bit) (267 MB) and UCSF chimera(Mohamed MA).

CHAPTER THREE

RESULTS AND DISCUSSION

1- Anti diabetic assay of plant extracts.

The three different extracts of Bauhinia rufescens,petroleum ether,chloroform and methanol extract were administered in dose of 1 g/kg intraperitoneally into normal fasted rats and at the same above dose to streptozotocin-treated rats .The methanol extract clearly decreased the blood glucose level normal fasted rats by

27 ± 3.1% 2 hours following administration and decreased the blood glucose level in streptozotocin-treated rats by 16.9 ± 1.7% 2 hours following Administration;

While the chloroform and pet. Ether extracts not widely decreases the glucose level (table 4).

Table-4: In-vivo-Anti diabetic assay of methanol, chloroform and pet.

Ether extracts of Bauhinia rufescens:

*P < 0.05

2. Quantitative antioxidant assay using DPPH radical scavenging assay of plant extracts

As demonstrated in Table 5, methanol extract was able to neutralize the free radical molecules. The methanol extract showed strong antioxidant activity

(89%), almost similar in effect to the standard, Propyl Gallate (92 %).

Table 5 The results of antioxidant activity of methanol, chloroform and pet.

ether extracts:

No. Sample Code %RSA ±SD(DPPH)

1 Methanol extract 89±0.01

2 Pet.Ether extract 12±0.01

3 Chloroform extract 13±0.03

Standard Propyl Gallate 92±0.01

3- Fractionation of active methanol extract

From the above results it was observed that methanol extract showed best antioxidant and antidiabetic activities compared with chloroform and pet. ether extracts; accordingly methanol extracted was fractionated by separatory funnel using different solvents; chloroform, ethyl acetate and n-butanol.

4- Quantitative antioxidant assay using DPPH radical scavenging assay of

fractions methanol extract, chloroform fractions (ethyacetate, n-butanol,

and aqueous fractions).

The results of the antioxidant activity of four fractions ( as shown in Table- 6) are revealed that, ethyl acetate fractions showed potent antioxidant activity (90%) compared with other two fractions.

Table 6 The results of antioxidant Activity of different methanolic fractions

No. Sample Code %RSA ±SD(DPPH)

1 Aquous.F 80±0.01

2 Ethyacetate.F 90±0.01

3 Butanol.F 79±0.01

4 Chloroform 85±0.01

Standard Propyl Gallate 92±0.01

5- In vitro anti diabetic assay and qualitative antioxidant evaluation of most active fraction (ethyl acetate fraction):

From the above results it is clearly that ethyl acetate fraction is most active antioxidant fraction and also expected to be most active antidiabetic fraction, accordingly it subjected for more evaluation in order to isolate and identified active compounds.

5-1. TLC bioautographic method for the detection of Alpha -amylase enzyme

inhibitors:

The ethyl acetate fraction was spotted on TLC plate and developed using Toluene:

Ethyl acetate: Formic acid (5:4:1, v/v/v) as mobile phase. The TLC Plate then was dipped into prepared enzyme solution and incubates in humid desiccator for 1.5 h. The humid desiccator was filled with a little water, such that the plate does not meet the water, but enough to keep humidity inside the container. Incubation was performed for

30 min at room temperature for the primary reaction between the enzyme and enzyme inhibitors separated in the extracts on TLC plate, followed by starch solution and incubated for another 15 min for enzyme substrate reaction, and then air dried and finally dipped in iodine solution. α-amylase inhibitors were visible on the TLC plate by the appearance of yellowish -greyish spot on a dark brown background. As shown in figure (4) many compounds showed good antidiabetic activity.

5-2. A TLC bioautographic method for the detection of antioxidant compounds

The ethyl acetate fraction was run on TLC silica plate using Toluene: Ethyl acetate:

Formic acid (5:4:1, v/v/v) as mobile phase. The Plate was air dried and then sprayed with DPPH (0.001 g in 95% methanol/ethanol). Bright yellow or cream spot (indicate compounds having antioxidant activity) was shown against a purple background.

Figure (5) shows many compounds with good antioxidant activity.

Figure-4: Bioautographic assay showing the inhibition of α-amylase by

some compounds developed on a silica gel G60 F254 plate which was eluted with

Toluene: Ethyl acetate: Formic acid (5:4:1, v/v/v) as mobile phase

Figure-5: Bioautographic method showing the antioxidant compounds (yellow color spots) after developing on a silica gel G60 F254 TLC plate which was eluted with Toluene: Ethyl acetate: Formic acid (5:4:1, v/v/v) as mobile phase.

6- Isolation and structure determination of compound (1) and (2) from ethyl acetate fraction:

6-1 Isolation of the active compounds

From antidiabetic and antioxidant test results (figure 4&5), it is clearly that compound

-1 and compound -2 showed antidiabetic activity beside it’s activity against oxidative stress. For isolation of the active compounds, ethyl acetate fraction was subjected to preparative thin layer chromatography (PTLC) using Toluene: Ethyl acetate: Formic acid (5:4:1, v/v/v) as mobile phase.

6-2 Structure elucidation of compound (1)

Structure determination is achieved mainly by 1H- and 13C-NMR spectra which carried out on the Bruker AM 700 spectrometer (Germany) operating at 700 MHz (1H-NMR)

13 and ( C-NMR) in spectroscopic grade DMSO-d6.

1H-NMR spectrum (Table 7, Figure 6) indicated the presence of two singlet aromatic protons each proton integrated for one proton at δ 6.44 and δ 6.77, and were assigned to H-6 and H-8 respectively. Other aromatic protons area were assigned for ring-B protons, two doublet signals at δ 7.77 and δ 6.90 were assigned to H-6'and H-5' respectively. 1H-NMR spectrum also indicated the presence of group of signals ranging between δ 5.22-5.27 attributed to H-1'', H-2'' and H-3'' in Rhamnose sugar.

Furthermore, a multiplet protons signals appear at δ 3.11-3.97, which assigned to the remaining Rhamnose sugar, H-4''and H-5''.The glucose sugar protons, H-1''', H-2''', H-

3''', H-4'', H-5''' and H-6''' appear at δ ranging between 3.11and 4.69 as shown in table

(7).

13C-NMR spectrum (Table 7, Figure 7) experiment indicated the presence of ten quaternary carbons including one carbonyl group at δ 178.21 assigned to C-4, the downfield shift of C-4 indicated the presence of hydroxyl group at C-5. (Agrawal)

Four oxygenated aromatic carbons at δ 166.0, 162.88, 150.00 and 144.00 assigned to

C-7, C-5, C-4', C-3' respectively, other five remaining quaternary carbons at δ159.06,

158.08, 134.70, 120.73, and 105.99 assigned to C-9, C-2, C-3, C-1' and C-10 respectively. 13C-NMR spectrum experiment indicated also the presence of group of signals ranging between δ 70.01- 71.79 assigned for sugar protons. 1H-NMR and 13C-

NMR spectrum of compound -were in full agreement with that reported for Rutin

(Table 7) (Esra Küpeli Akkola*)

HO H 3' H 2' O 4' H 1 1' O 7 9 O 1' H 5' 8 2 6' H H 5 4 H 6 10 3 O O O H H O H 1'' H O 2'' H H H 4'' 3'' H 5'' O H H H H H O H O O 6''' H H 5''' 1''' H 4''' 2''' H 3''' H O H H O HO

Rutin (quercetin-3-O-rutinoside)

Table 7: 1H- and 13C-NMR assignments of compound -2 (700 MHz, DMSO) in comparison with reported data for Rutin (Quercetin-3-Orutinoside) (100 MHz,

CD3OD) (Esra Küpeli Akkola*)

δH Compound δC Compound Position δH Rutin δC Rutin (2) (2)

2 - 158.08 158.22

3 - 134.70 134.52

4 - 178.21 178.39

5 - 162.88 162.48

6 6.44 6.10 s 99.70 99.72

7 - 166.0 166.58

8 6.77 6.28 s 94.84 94.90

9 - 159.06 158.91

10 - 105.99 105.32

1' - 120.73 122.77

2' 7.64 s 119.34 117.37

3' - 144.00 144.32

4' - 150.00 150.23

5' 6.90 d 6.85 d 115.67 115.46

6' 7.77 d 7.63 d 120.51 122.47

1'' 5.22-5.27 4.96 d 103.69 103.63

2'' 5.22-5.27 4.96 d 76.00 74.64

3'' 5.22-5.27 4.96 d 70.65 -

4'' 3.11-3.97 m 3.20-3.90 m 70.13 -

5'' 3.11-3.97 m 3.20-3.90 m 70.01 -

1''' 4.69 4.50 d 101.53 101.92

2''' 4.69 4.50 d 71.79 71.32

3''' 4.69 4.50 d

4''' 3.11-3.97 m 3.20-3.90 m

5''' 3.11-3.97 m 3.20-3.90 m

6''' 3.11-3.97 m 3.20-3.90 m

Figure-6: 1H-NMR-spectrum of compound -1 (Rutin), (700 MHz, DMSO).

Figure-7: 13C-NMR-spectrum of compound -2 (Rutin), (700 Hz, DMSO).

Figure-8: HMBC-spectrum of compound -1, showed some two and three bond

correlations, (700 MHz, DMSO).

Figure-9: HSQC-spectrum of compound -1 (Rutin), showed direct proton- carbon correlation (700 MHz, DMSO).

6-3 Structure elucidation of compound (2)

Structure determination is achieved mainly by 1H- and 13C-NMR spectra which carried out on the Bruker AM 500 spectrometer (Germany) operating at 700 MHz (1H-NMR)

13 and C-NMR in spectroscopic grade DMSO-d6.

1H-NMR spectrum (Table 8, Figure 10) showed that the spectra is different from previous identified compound, in presence of two signals at δ 8.01.61 and δ 6.90 attributed to H-2' and H-3' respectively.

13C-NMR spectrum (Table 8, Figure 11) experiment showed the presence of one oxygenated aromatic carbon at δ160.41 in ring-B carbons, also showed almost two equivalent signals at δ130.96 and 131.00 attributed to C-2' and C-3' respectively. The presence of two equivalent signals indicated clearly the presence of only one hydroxyl group in ring –B, There for the compound was identified as Kaempferol -3-O- rutinoside. 1H-NMR and 13C-NMR spectrum of compound -2 were in full agreement with that reported for Kaempferol -3-O-rutinoside (Table 8) (Esra Küpeli Akkola*)

3' 2' 4'

1' 5' 6'

Kaempferol -3-O-rutinoside

Table-8: 1H- and 13C-NMR assignments of compound -1 (500 MHz, DMSO) in comparison with reported data for Kaempferol -3-O-rutinoside (100 MHz,

CD3OD) (Esra Küpeli Akkola*)

δH Compound δH δC Compound δC

(2) Kaempferol (2) Kaempferol Position -3-O- -3-O-

rutinoside rutinoside

2 - 158.08 155.98

3 - 134.70 134.58

4 - 178.21 177.23

5 - 162.88 162.04

6 6.44 d 6.22 d 99.70 98.88

7 - 166.0 163.31

8 6.77 d 6.33 d 94.84 93.98

9 - 159.06 156.82

10 - 105.99 104.79

1' - 120.73 121.39

2' 8.01 8.19 d 130.96 130.76

3' 6.90 d 6.92 d 112.50 113.40

4' - - 160.41 160.92

5' 6.90 d 6.92 115.67 114.87

6' 7.77 d 8.19 d 131.00 131.03

1'' 5.22-5.27 5.02 d 103.69 102.11

2'' 5.22-5.27 5.02 d 76.00 74.83

3'' 5.22-5.27 5.02 d 70.65 75.48

4'' 3.11-3.97 m 3.15-3.90 m 70.13 69.23

5'' 3.11-3.97 m 3.15-3.90 m 70.01 77.65

6'' 3.11-3.97 m 3.15-3.90 m 67.08

1''' 4.69 4.45 d 101.53 100.10

2''' 4.69 4.45 d 71.79 70.89

3''' 4.69 4.45 d

4''' 3.11-3.97 m 3.15-3.90 m

5''' 3.11-3.97 m 3.15-3.90 m

Figure 10 1H-NMR-spectrum of compound -2 (Kaempferol -3-O-rutinoside), (700

MHz, DMSO).

Figure 11 13C-NMR-spectrum of compound -2 (Kaempferol -3-O-rutinoside), (700

Hz, DMSO).

7. Identification of Phyto-constituents from ethyl acetate fraction by LC-MS

The UPLC/MS/MS analysis result in identification of more than thirteen compound most of them is phenolic compounds, especially flavonoids (Table 9).

Table 9. Identified compounds using LC-MS/MS

Sl. m/z Compound Name Structure Mass bank

No. no.

1 288.1740 Eriodictyol PR020015

2 329.2419 Sinomenine TY000053

3 302.9715 Scopolamine KO009233

4 303.9746 Arachidonic acid BML8076

5 611.0244 Dihydroergocristine BML8111

6 133.0590 Aspartic Acid BML8079

7 286.9800 Claussequinone FIO00282

8 316.9833 Petunidin PR100450

9 426.2412 Hispidulin acetate TY000235

10 181.0699 Phosphinothricin WA00271

11 274.2022 Podocarpic acid BML8198

12 275.2051 Eserine KO008958

(Physiostigmine)

13 351.1658 Retrorsine BML8206

14 284.2592 5,7- BML0147

Dimethoxyflavanon 5 e

15 285.2819 Piperine MT000119

16 655.1729 Malvin PR020064

17 677.2285 Icariin TY000207

18 302.2279 Abietic acid BML8066

19 303.2309 Cocaine EA281708

20 593.1495 Fortunellin TY000229

21 594.1522 PR020056

Kaempferol -3-O-

rutinoside

22 353.1820 Protopine KO009201

23 609.1419 Ergocristine BML8119

24 383.2260 Hydrastine BML8143

25 423.2115 Gluconasturtiin PR100433

26 625.1345 Peonidin-3,5-O-di- PR100459

beta-

glucopyranoside

27 626.1376 Quercetin-3,4'-O-di- PR100456

beta-

glucopyranoside

28 609.1417 Reserpine CE000148

29 610.1446 Rutin CE000136

30 611.1469 Delphinidin-3- CE000386 rutinoside

31 148.9785 Methionine CE000452

32 274.2027 Phloretin TY000158

33 284.2592 Acacetin PR020028

34 381.2096 Otosenine BML8186

35 593.1494 Kaempferol-3- PR101018 Glucoside-2''-p-

coumaroyl

36 610.1445 Luteolin-3',7-di-O- PR020059 glucoside

37 593.1498 Acaciin TY000123

38 681.1872 Haploside BML8134

Figure-12: Total MS chart represent molecular ion peaks of identified compounds from active fraction analyzed by LC/MS/MS.

Figure -13: HPLC peaks of compounds identified by LC/MS/MS.

8. Insilco assessment of the identified compounds as inhibitors for α-glucosidase and α-amylase enzyme:

Alpha-glucosidase enzyme is an essential enzyme for degradation of glygogen to

glucose in lysosomes(Hermans M.M.P.) with highest activity on alpha-1,4-linked

glycosidic linkages, but can also hydrolyze alpha-1,6-linked glucans(Roig-

Zamboni V.). A competitive mode of inhibition is obtained upon using of

blockers. The inactivation of the enzyme is time and concentration dependent and

results in the covalent binding of inhibitor. Catalytic activity is required for

binding to occur. Asp-518 is predicted to be the essential carboxylate in the active

site while the residues Trp-516 and Asp-518 are demonstrated to be critical for

catalytic function (Hermans M.M.P.).

Alpha-Amylase is the primary digestive enzyme acting on starch or glycogen,

occurs in pancreas, parotid, serum, urine and occasionally in smaller amounts in

other tissues or tumors, it has 496 amino acids(Williams). The structure of human

pancreatic alpha-amylase has been determined to 1.8 A resolution using X-ray

diffraction techniques. This enzyme is found to be composed of three structural

domains. The largest is Domain A (residues 1-99, 169-404), The active site is

located in a cleft between the A and B domains which forms a central eight-

stranded parallel beta-barrel, to one end of which are located the active site

residues Asp 197, Glu 233, and Asp 300. Also found in this vicinity is a bound

chloride ion that forms ligand interactions to Arg 195, Asn 298, and Arg 337(G.

D. Brayer) Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding(Brayer GD(1)).

The compounds from chloroform extract were assessed for their α-glucosidase and α-amylase inhibiting activity and the result were listed in table (10).

Table10: In silico assessment of identified compounds as inhibitor for human α-glucosidase and α-amylase enzymes:

Sr. Compound Name Min. Binding energy

No. α-glucosidase α-

a;llllllllllllllmylase

1. Eriodictyol -8.37 -9.92

2. Sinomenine -8.67 -7.00

3. Scopolamine -8.92 -6.73

4. Arachidonic acid -3.81 -5.76

5. Dihydroergocristine -5.93 -8.l91

6. Aspartic Acid -3.90 -6.06

7. Claussequinone -6.23 -8.35

8. Petunidin -7.34 -8.83

9. Hispidulin acetate -6.21 -6.51

10. Phosphinothricin -6.19 -6.20

11. Podocarpic acid -4.72 -7.31

12. Eserine (Physiostigmine) -7.42 -6.40

13. Retrorsine -9.73 -5.58

14. 5,7-Dimethoxyflavanone -6.05 -8.48

15. Piperine -6.14 -8.33

16. Malvin -2.71 -7.54

17. Icariin (+ve) -9.50

18. Abietic acid -4.67 -6.81

19. Cocaine -8.66 -6.98

20. Fortunellin (+ve) -9.65

21. Kaempferol -3-O-rutinoside -7.19 -8.25

22. Protopine -7.46 -7.52

23. Ergocristine -5.26 -5.34

24. Hydrastine -8.77 -6.41

25. Gluconasturtiin -7.51 -8.39

26. Peonidin -3,5-O-di-beta- (+ve) -4.61

glucopyranoside

27. Quercetin -3,4'-O-di-beta- (+ve) -8.25

glucopyranoside

28. Reserpine (+ve) -5.84

29. Rutin -9.15 -6.87

30. Delphinidin -3-rutinoside -7.92 -5.29

31. Methionine -5.49 -5.39

32. Phloretin -7.09 -8.01

33. Acacetin -7.23 -8.35

34. Otosenine -8.69 -6.12

35. Kaempferol -3-Glucoside-2''- (+ve) -10.19

p-coumaroyl

36. Luteolin -3',7-di-O-glucoside -4.07 -10.86

37. Acaciin (+ve) -10.66

38. Haploside -1.64 -8.77

39. Acarbose (Standard) -6.40 -7.25

Weak intermolecular interactions (hydrophobic interactions and hydrogen bonds) play an important role in stabilizing a ligand energetically at the interface of a protein structure. The importance of hydrogen bonds and the hydrophobic interactions in the binding affinity of a protein-ligand has been described extensively. Whereas hydrogen bonds optimize the hydrophobic interactions at the protein-ligand interface, that increases the binding affinity of complex molecules, so the binding affinity and drug efficacy associated with hydrophobic interactions can be optimized by incorporating them at the site of the hydrogen bonding (Rohan Patil).

In this in silico study many hydrogen and hydrophobic bonds were generated between considerable numbers of identified compounds and the two enzymes (α- glucosidase and α-amylase) which make them energetically stable compared with standard drug Acrebose (fig. 14 and fig.15)

Figure 14: In silico assessment of identified compounds as inhibitor for human α-glucosidase enzyme:

Eriodictyol, E= -8.37 Kcal/mol Sinomenine, E= -8.67 Kcal/mol

Scopolamine, E= -8.92 Kcal/mol Petunidin, E= -7.34 Kcal/mol

Eserine (physostigmine), E= -7.42 Retrorsine, E= -9.73 Kcal/mol

Kcal/mol

Cocaine, E= -8.66 Kcal/mol Kaempferol-3-O-rutinoside

(Nicotiflorin), E= -7.19

Kcal/mol

Protopine, E= -7.46 Kcal/mol Hydrastine, E= -8.77 Kcal/mol

Rutin, E= -9.15 Kcal/mol Delphinidin-3-rutinoside, E= -

7.92 Kcal/mol

Acacetin, E= -7.23 Kcal/mol Otosenine, E= -8.69 Kcal/mol

Acarbose (Standard)

E= -6.40Kcal/mol

Figure 15: In silico assessment of identified compounds as inhibitor for human α-amylase enzyme:

Dihydroergocristine

Eriodictyol E= -8.91 Kcal/mol

E= -9.92 Kcal/mol

Claussequinone Petunidin

E= -8.35 Kcal/mol E= -8.83 Kcal/mol

Podocarpic acid 5,7-Dimethoxyflavanone

E= -7.31 Kcal/mol E= -8.48 Kcal/mol

Piperine Malvin E= -8.33 Kcal/mol E= -7.54 Kcal/mol

Icariin Fortunellin

E= -9.50 Kcal/mol E= -9.65 Kcal/mol

Kaempferol-3-O-rutinoside Protopine

E= -8.25 Kcal/mol E= -7.52 Kcal/mol

Gluconasturtiin Quercetin-3,4'-O-di-beta-glucopyranoside,

E= -8.39 Kcal/mol E= -8.25 Kcal/mol

Phloretin Acacetin E= -8.01 Kcal/mol E= -8.35 Kcal/mol

Luteolin-3',7-di-O-glucoside

Kaempferol-3-Glucoside-2''-p-coumaroyl E= -10.86 Kcal/mol

E= -10.19 Kcal/mol

Acaciin Haploside E= -10.66 Kcal/mol E= -8.77 Kcal/mol

Acarbose (Standard)

E= -7.25 Kcal/mol

9. Standardization and High performance thin layer chromatography (HPTLC) fingerprint analysis

The HPTLC analysis of ethyl acetate fraction revealed the presence of different compounds with Rf range 0.11-0.90 (table 11). The result showed the presence of flavonoid Rutin which appear at Rf 0.11, when compared with standard; which can be used as biomarker for standardization and quality control. Generally the finger printing can be useful in checking the adulterant. Therefore, it can be useful for the evaluation of different marketed herbal drugs formulated in a dosage forms.

Fig-16: HPLC analysis of ethyl acetate fraction of Bauhinia rufescense on silica gel plate, developed in toluene: ethyl acetate: Formic acid (5:4:1, v/v/v), showing number of compounds and Rutin as standard.

Table-11: HPTLC profile at 366 (nm) of ethyl acetate fraction

Retardation Area Compound

factor (Rf)

0.11 15735.3 Rutin

0.20 3476.1 -

0.23 3687.3 -

0.32 12816.3 -

0.46 2916.8 -

0.53 1222.6 -

0.58 2970.8 -

0.68 15707.5 -

0.75 2844.8 -

0.90 844.5 -

Fig.17; 3D graph of HPTLC chromatogram

Fig-18: TLC chromatogram of ethyl acetate fraction as shown by TLC scanner (254 and 366 nm), developed in toluene: ethyl acetate: Formic acid

(5:4:1, v/v/v).

Free radical reactions participating in reactive oxygen species to the overall metabolic perturbations that result in tissue injury and disease. Cellular damage, due to free radical causes serious derangements such as ischemia-reperfusion injury. A number of synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), have been developed, but their use has begun to be restricted due to their toxicity. As a result, there is considerable interest in herbal medicine in the development of natural antioxidants from botanical sources. Diabetes mellitus is a group of disorders of multiple etiologies resulting from a defect in insulin secretion, insulin action, or both. Insulin deficiency in turn leads to chronic hyperglycemia (very high blood glucose levels) with disturbances in carbohydrate, fat and protein metabolism.(Pastaki). The treatment of diabetes mellitus is considered as the main global problem and successful treatment has yet to be discovered.

Although Insulin therapy and oral hypoglycemic agents are the first line of treatment for the diabetes mellitus; they have some side effects and fail to significantly alter the course of diabetic complications (K. R. Patel DK); therefore medicinal plants may provide another source of diabetes treatment. Many repots have been supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes. The beneficial effect of antioxidants has been reported in animal models of diabetes as well as in diabetic patients(Omolola R. Ayepola). Medicinal plants, mostly with antioxidant activity, are the major source of drugs for the treatment of oxidative stress induced diabetic complications, hence they having no or only few side effects(Fabricant DS)

(Rafieian-Kopaei M). Bauhinia rufescens is a shrub in the family Fabaceae, native to different areas of Africa such as the Sahel. In Sudan, decoction of the leaves is used traditionally in treatment of diabetes; there for this study was aimed to evaluating the scientific basis for the traditional uses of this plant and to isolate the active antidiabetic and antioxidant compounds.

Bioactivity-guided fractionation led to isolation and identification of different compounds with antidiabetic and antioxidant activity, most of them is phenolic compounds, especially flavonoids. The antioxidant property of flavonoids was the first mechanism of action studied, in particular with regard to their protective effect against cardiovascular diseases (master C. S. Yang). Flavonoids have been shown to be highly effective scavengers of most types of oxidizing molecules, including singlet oxygen and various free radicals that are probably involved in several diseases(master).

Mechanism of antioxidant action can include suppressing reactive oxygen species formation, either by chelating trace elements involved in free–radical production, scavenging reactive species, and upregulating or protecting the antioxidant natural defenses(S. A. van Acker). Antioxidant activity of flavonoids has beneficial effect in treatment of diabetes, especially Many repots have been supported the role of oxidative stress in the pathogenesis of both type 1 and type 2 diabetes. There are no enough literatures review concerning about the biological activity of Bauhinia rufescens, so the results obtained from our study can be considered as starting point for future studies.

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