Review of Literature

25

2. REVIEW OF LITERATURE

2.1 Diabetes

Diabetes mellitus is a group of metabolic disorder characterized by elevated

blood glucose levels (hyperglycemia) resulting from defects in insulin secretion,

insulin action or both. Insulin is a hormone manufactured by the beta cells of the

pancreas, which is required to utilize glucose from digested food as an energy source.

According to Salim, (2005) the insulin deficiency leads to chronic hyperglycaemia

with disturbances of carbohydrate, fat and protein metabolism. Oral hypoglycaemic

agents sulphonylureas, biguanides, alpha glucosidase inhibitors, meglitinide

analogues and thiazolidenediones are used to correct the metabolic disorder, such as

insulin resistance and inadequate insulin secretion. Zimmet et al., (2001) and Clifford

and Caroline (1989) recorded more than 400 traditional plant treatments for diabetes

mellitus, but only a small number of these have received scientific and medical

evaluation to assess their efficacy and their hypoglycemic action and has been

confirmed in animal models and non-insulin-dependent diabetic patients. The present

study is aimed at assessing the effect of herbal compounds isolated from

Pithecellobium dulce against diabetes mellitus. The literature pertaining to the study

has been reviewed extensively.

2.2 Prevalence of diabetes

By the year 2010, it was estimated that more than 200 million people in

worldwide had diabetes mellitus and 300 million will subsequently have the disease

by 2025 (WHO, 2011) Li Chan et al., (2009) observed that among the 10 countries

with the largest number of people predicted to have diabetes mellitus in 2030, five are

26

in Asia, they are China, India, Pakistan, Indonesia and Bangladesh. Lei et al., (2012)

reveiwed that over the past three decades, the number of people with diabetes mellitus

have more than doubled globally, making it one of the most important public health

challenges to all nations. The number of people with diabetes who are > 20 years of

age in all countries of the world during the years 1995, 2000 and 2025 were calculated

by Hilary et al., (1998) and found the prevalence of diabetes in adults worldwide was

estimated to be 4.0% in 1995, 5.4% in 2000. About the year 2025, > 7.5% of people

with diabetes will reside in developing countries.The countries with the largest

number of people with diabetes are, and will be in the year 2025, India, China, and

U.S.A.

Sara et al., (2004), Vashitha et al., (2012) and Manouk, (2013) attributed the

prevalence of type 2 diabetes mellitus in rural population was low compared to the

urban population. However females were highly prone to diabetes mellitus than males

above the age group of 65. Ramachandran,(1993) observed the prevalence of type 1

diabetes in children as 0.26/1000 in an urban area in south India and showed it was

quite comparable to the prevalence of 0.27/1000 reported from Algeria which was

higher than that reported from many other Asian countries namely Japan (0.06/1000)

and China (0.09/1000). Christos et al.,(1996) has extensively measured the

prevalence of diabetes mellitus in a Greek primary health care district and estimated

prevalence appears to be similar to the prevalence rates reported in other areas of

rural Greece. Mohan et al., (2007) and Sher et al., (2010) while studying diabetes

complications observed prevalence of premature coronary artery disease is much

higher in Indians and diabetes mellitus was significantly more common in obese

coronary artery disease patients as compared to non-obese patients.

27

2.3. Diagnosis

The nature of diagnosis pattern in diabetes have been detailed by Ariful et

al.,(2009) ;Tushar et al., (2008). They stated that hypoglycemic effects was diagnosed

using oral glucose tolerance test (OGTT). Stumvoll et al., (2000) while studying 104

diabetic patients found the insulin release and insulin resistance were the apparent

cause as determined using Oral Glucose Tolerance Test (OGTT). Edelman et

al.,(2004) found that norm glycoslated haemoglobin have a low incidence of diabetes

and may not require rescreening in 3 years and the patients with high normal HbA1c

may require follow up sooner than 3 years and suggested that HbA1c may be a useful

test for periodic diabetes screening. Comprehensive studies on HbA1c, estimates a

borderline (5.6-6.4%) and high (> or = 6.5%) levels of HbA1c and was found to

strongly predict future drug treatment for diabetes mellitus (Shimazaki et al., 2007).

Kumar et al., (2012) studied that the inhibitors of carbohydrate hydrolyzing

enzymes (such as α-glucosidase and α-amylase) have been useful as oral drugs for the

control of hyperglycemia, especially in patients with type II diabetes mellitus. Samir

(2007) while studying insulin mechanism identified that the free fatty acids are known

to play a key role in maintaining the insulin level. Vijayakumar (2012) observed that

hyperglycemia alters function through a variety of mechanisms including polyol

pathway activation, increased formation of advanced glycation end products,

diacylglycerol activation of protein kinase C and increased glucose shunting in the

hexosamine pathway. The same mechanisms may be operative in the brain and induce

the changes in cognitive function that have been detected in patients with diabetes.

Patel, (2012) reviewed the profiles of plants with hypoglycemic properties reported

in the literature from 2009 to 2011 and showed the use of these plants may delay the

28

development of diabetic complications and can correct the metabolic abnormalities

through variety of mechanisms .

2.4 Plants and diabetes treatment

Remya, (2012) made a base line study for the use of ayurvedic medicines and

herbal drug preparations used in the treatment of diabetes. According to Mentreddy et

al., (2005) and Syed and Neethu, (2013) about 800 plant species have possess

antidiabetic properties. Several plant species have been used for prevention of

diabetes by the Native Americans, Chinese, South Americans and Asian Indians.

Among these species, Allium cepa, Allium sativum, Aloe vera, Coccinia indica, Caesa

pinia bonducella, Eugenia jambolana, Ficus bengaensis, Gymnema sylvestre,

Momordica charantia, Mucuna prurins,Ocimum sanctum syn. tenuitorum,

Pterocarpus marsupium, Swertia chirayita, Syzigium cumini, Tinospora cordifolia,

and Trigonella foenum-graecum are considered the more effective and more

extensively studied in relation to diabetes and its complications. Plant species adapted

to North America, such as prickly pear (Opuntia robusta), Rosmarinus officinalis,

Ocimum gratissimum, and noni (Morinda citrifolia) have also been evaluated for their

hypoglycemic properties using laboratory animal models in western countries.

Kavishankar et al., (2011) reviewed 136 traditional plants which are used

throughout the world for the therapy of diabetes mellitus. Amal et al., (2009) showed

most dominant antidiabetic plant bearing family was Fabaceae followed by Poaceae

and Liliaceae. However majority of plants members of Caesalpiniaceae family are

seen to be more effective on diabetes. Chavre et al., (2010) showed that 39 common

herbal plants are used for diabetes treatment in India. Baby and Jini, (2011) and

29

Elavarasi et al., (2013) reviewed the Indian herbal plants used in the treatment of

diabetes and has given information about scientific name, common name, family and

the parts of the plant used to treat diabetes in the Indian system of medicines.

Ediriweera and Sooriya,(2009) observed about 126 plants belonging to 51 families

are used to treat diabetes in Srilanka. Jose et al., (2005) reviewed 178 antidiabetic

plants from South ,Central and North America. Rajendran and Manian, (2011)

reviewed a total of 16 species belonging 16 genera and 13 families and identified for

the treatment of diabetes in Eastern Ghats. It was also found that Azardirachta,

Psidium, guajava (amrud), allivum sativum (garlic) are herbal plants for treating

diabetes mellitus (Bhudwani et al., 2010).

Nidal, (2007) observed 56% of diabetic patients in the West Bank of Palestine

using conventional medical treatment with herbal therapy, uses the commonest herbs

Fenugreek, Nigella seeds, Aloe vera, sage, garlic and onion. In many other plants also

antidiabetic activity was observed. According to ayurvedic system of medicine Vikas

and Vijay, (2010) reported Ficus bengalensis Linn (Banyan tree) was well known to

be useful in diabetes. The recent discoveries of anti-diabetic compounds according to

their chemical structures and mechanisms of action, were summarized by Hsin et al.,

(2012). Edmond , (2000) studied the effects of a native herbal tea in patients with

type 2 diabetes, the data shows beneficial effects in patients with poor glycemic

control, possibly by lowering post-prandial glucose levels. The presence of phenolic

compounds, flavonoids, terpenoids, coumarins and other constituents are responsible

for reduction in blood glucose levels and antidiabetic activity of medicinal plants

(Rao et al., 2010).

30

2.5 Pithecellobium dulce

Pithecellobium dulce (Roxb) Benth. is a common tropical tree and small to

medium sized growing up to 18 m in height. It belongs to the family Leguminosae:

sub family –Mimosoidae. The review of literature encompasses information on

folklore claims, pharmacognosy, phytochemistry and biological activities of

Pithecellobium dulce and its related species. It was found that very fragmentary

information was available for Pithecellobium dulce as compared with its related

species. Pithecellobium species have wide range of medicinal properties. The

objective of the study is also to identify the active principles by bioactivity guided

fractionation.

The bark extracts of Pithecellobium avaremotem is used for treating the

uterine and blood disorders (Hartwell, 1971). However Cheryllans et al., (2001)

showed that Pithecellobium unguis-cati leaves decoction was used by the the tribals

of Trinidad of South Caribbean for its antiparasitic effect. Ali et al., (2006),

Ruzilawati et al., (2012) and Wong et al., (2007) studied on Pithecellobium jiringa

and found that in Chinese and Malaysian traditional medicine, it was used for treating

diabetes, however it was consumed by the local population in Malaysia for acute renal

failure and it was observed that all extracts of Pithecellobium jiringa showed the

antimicrobial and antifungal activities against the test organisms. Ibrahim et al.,

(2012) investigated the gastroprotective mechanisms of Pithecellobium jiringa

ethanol extract against ethanol induced gastric mucosal ulcers and this finding was

also confirmed by histology of gastric mucosa which showed severe damage to the

gastric mucosa with edema and leucocyte infiltration of the submucosal layer. The

31

ulcer protective effect of this plant may possibly be due to its preservation of gastric

wall mucus along with the reduction of oxidative stress.

The forthcoming literature survey retrieves the significant folklore properties

of Pithecellobium dulce. According to Duke and Wain, (1981) Pithecellobium dulce

was known for its folk remedy for convulsions, dysentery, dyspepsia, ear ache,

leprosy, peptic ulcers, sores, toothache and venereal disease and the usage of plant as

abortifacient, anodyne, astringent, larvicidal were also observed. Sugumaran, (2006)

also showed the uses of Pithecellobium dulce widely as a plaster to allay pain even

from venereal sores and convulsions and also revealed that the leaves have been

utilized to cure indigestion and also induce abortion. Insulin like principle had been

reported in the leaves. The saline extract of the seeds showed a haemolytic

agglutinating reaction with human blood. The bark of the root is utilized as a remedy

for dysentery. Fall et al., (1998) found that the leaves were widely used as medicine

for intestinal disorders.

Pharmacognostical survey of Pithecellobium species pertained to the study of

many authors. Raiha et al., (2006) made the ultra structural studies on the root

nodules of Pithecellobium dulce and found that rhizobial infection on root surface

started with curling of root hair. The curled and straight root hairs were observed. The

internal structure of a mature nodule showed an epidermis, cortex, vascular region and

bacteriod region. Vascular bundles were amphicribal. A distinct periderm consisting

of sclereid tissue could be observed in the cortex outside the vascular tissue. The

bacteroid region contained infected and uninfected cells intermingled with each other.

Infected cells of developing nodules as well as mature nodules were vacuolated.

32

Infection threads were also observed in the bacteriod zone. The rhizobia were released

from the infection thread in to the host cytoplasm from rhizobial droplets. Rhizobia

were also in the intercellular spaces between infected cells. Starch grains were

observed in the interstitial cells. Furthur Day et al., (2010) recognized two

homogeneous groups of species in P. plesiosorum complex. One of them is identified

based on the type of anastomosis of the veins. Polypodium conterminans, originally

considered in the group of Pithecellobium dulce with free venation.

2.6 Phytochemical studies on Pithecellobium species

Several authors have studied on the leaves of Pithecellobium dulce and

characterized several compounds. Zapesochnaya et al., (1980) isolated three

compounds from the dried leaves of Pithecellobium dulce. They were identified as

kaempferol, afzelin and quercetin -3-O-alpha-L-rhamanopyranoside.

O

OOH

OH

OH

OH

O

CH3

OH

OH

OH

O

OH

OH

O

OH

OH

O

Kaempferol Quercetin -3-O-alpha-L-rhamnopyranoside

33

Kaempferol 3 –o-alpha-L-rhamnose

Lee et al., (1992) extensively studied the leaves of Pithecellobium lobatum

which had led to the isolation and characterization of five flavan-3-ol derivatives

including new flavan-3-ol gallates, gallocatechin 3’ and 4’-o-gallates and

gallocatechin 7, 3’ and 7’ 4 – di-O-gallates (racemic) which occur as equilibrium

mixtures. Examination of the pods offered three proanthocyanidins (procyanidins B-3,

B-4 and Prodelphinidin B-1) together with flavan-3-ol. According to the isolation of

Nigam et al., (1970) the alcoholic extract of the leaves of Pithecellobium dulce ,

afforded different fractions from solvent segregation and chromatography yield

octacosanol, b - D- glucoside of a- spinasterol, a- spinasterol and kaempferol-3-

rhamnoside. Adinarayana and Ramachandraiah, (1985) reported dulcitol from acetone

extract of leaves of Pithecellobium dulce.

From the edible part of Pithecellobium dulce number of phytoconstituents

are isolated. Nigam et al., (1968) reported that the sweet pulpy mesocarp of

the Pithecellobium dulce legumes yielded hexacosanol and a sterolglucoside-A, (m.p.

282–286°), aglycone, (m.p136–138°). The sugar (66.5%; mostly glucose) and the

amino acids, L-proline, L-leucine, L-valine asparagines and also the alcoholic extract

34

of the seed powder yielded pure lecithin (0.7%), saponin (m.p. 175–181°) sapogenin

provisionally named pithogenin, C28H44O4 (m.p. 207–208°). The aril of

Pithecellobium dulce gave the following value moisture,77.9; protein, 0.7%;

fat,0.6%; fibre,1.2%; carbohydrates,19.9% and mineral matter, 0.7%; calcium,13.0

mg; phosphorus 54.0 mg; iron, 1.4mg ; thiamine, 222 mg; riboflavin, 59 mg; nicotinic

acid, 0.36 mg and ascorbic acid, 120 mg/100gm.The essential aminoacids found in the

aril were: valine 143; lysine 178; phenylalanine, 41 and tryptophan 26 mg./100g. As

calcium pectate, pectin occurs as 0.96% of the sugars (mostly glucose) analysis of the

aril.

According to Nigam and Mitra, (1970) the leaves contains 29.0% crude

protein, crude fibre 17.5%, ash 5.6 % as well as some trace amount of 1.14% Ca and

0.35% phosphorus are also present. Analysis of the fatty oil gave the following

values: specific gravity, 0.9044; saponification No.185.3; iodine No. 80.7; acid

value,1.2; thiocyanogen value, 56.0 and unsaponification matter, 0.6%. The work

done on Pithecellobium dulce flowers by Nigam and Mitra, (1968) reported the

isolation and identification of α spinasterol, β-D-glucoside of α-spinasterol,

hexacosanol and hexacosane.

Considerable literature is available on the isolation of compounds from seeds

Nigam et al., (1997) characterized seven new saponins named Pithedulosides isolated

from the seeds of Pithecellobium dulce. The structure has been established through

spectral analysis as echinocystic acid and oleanolic acid saponins.

Compound -I : 3-O-α-L-arabinopyranosyl – (1-6) – β-D-glucopyranoside,

echinocystic acid and oleanolic acid.

35

Compound – II : 3-O-α-L-arabinopyranosy – (1-2) - α-L-arabinopyranosyl –

(1-6) – β - D-glucopyranosides.

Compound – III : 3-O-β –D-xylopyranosyl– (1-2) – α –L-arabinopyranosyl –

(1-6) – β –D- glucopyranosides.

Compound – IV : Oleanolic acid 3-O- α –L-arabinopyranosyl – (1-2) – α –L-

arabinopyranosyl – (1-6) – [β –D- glucopyranosyl] – (1-2) - β –

D-glucopyranoside.

Compound – V : 3-O-β – D - xylopyranosyl – (1-2) – α –L-arabinopyranosyl –

(1-6) – [ β –D-glucopyranosyl – (1-2) ] –β –D-glucopyranoside.

CH3 CH3

O

OHH

CH3 CH3

H H

O

R1

R2

1. Pithedulosides A : R1 = arabinose + glucose, R2 = OH.

2. Pithedulosides B : R1 = glucose + arabinose-1+ arabinose -2, R2 =H.

3. Pithedulosides C : R 1=glucose + arabinose+xylose, R2 = H.

4. Pithedulosides D : R1 = glucose + arabinose -1 + arabinose -2, R 2 = OH.

5. Pithedulosides E : R1 = glucose + arabinose + xylose, R 2 = OH.

36

6. Pithedulosides F : R1 = glucose -1+glucose -2+arabinose -1+arabinose –2, R 2=H .

7. Pithedulosides G : R 1 = glucose -1 + glucose -2 + arabinose +xylose, R 2 = H.

However Yoshikawa et al., (1997) identified four new oleanane type of

triterpene glycosides namely, Pithedulosides H-K which were isolated from the seeds

of Pithecellobium dulce.The structures have been characterized by extensive Nuclear

Magentic Resonance experiments and chemical methods. Compounds 1-3 comprised

of acacic acid as the aglycon and either monoterpene carboxylic acid and its xyloside

or monoterpene carboxylic acid as the acyl moiety at C-21. The oligosaccharide

moieties were linked to C-3 and C- 28 were determined as α –L- arabinopyranosyl-

(1-2) – α –L-arabinopyranosyl – (1-6) – [ β –D-glucopyranosyl and α –L- arabino

furanosyl – (1-4) –[ β –D-glucopyranosyl –(1-3) ] – α –L-rhamnopyranosyl – (1-2) –

β – D - glucopyranosyl ester respectively. Compound 4 was established as an

echinocystic acid – 3-O-glycoside having the same sequences as 1-3. Another known

compound was also identified associated with the new compounds which is

designated as echinocystic acid -3-O-β –D-xylopyranosyl – (1-2) – α –L-

arabinopyranosyl – (1-6) – [ β –D-glucopyranosyl] – (1-2) –β –D-glucopyranoside.

CH3 CH3

O

O

CH3

CH2

CH3

O

O

O

H O

OH H

H OH

O

O

CH2

OH

MTA2

R

O

O

glc3

O

rhaara

Oglc4

CH3CH3

O

glc1

glc2

ara1

ara2

37

1. Pithedulosides – H - R = CH3

2. Pithedulosides – I - R = CH2OH

3. Pithedulosides – K - R = Arabinose

O

CH3 CH3

CH3CH3

CH3

O

O

CH3

CH2OH

CH3

O

O

glc3 rhaara

O

glc4

CH3

glc1

glc2

Oara1

O

ara2

Pithedulosides -J

Sahu and Mahato (1994) demonstrated the antiinflammatory effect of

triterpene saponins isolated from the methanolic extract of the defatted seeds of

Pithecellobium dulce. They characterized a new bisdesmodic triterpenoid saponin

named as dulcin (3-O-β-D-glucopyranosyl)-28-O-(β-D-xylopyranosyl(1-6)–β-D-

glucopyranosyl) echinocystic acid. A known oleanolic acid saponin PE, oleanolic acid

3-O-β-D-glucopyranosyl (1-2) –α-L-arabinopyranoside was also obtained. The

bioactive saponins were reported to have potential antiinflammatory properties.

38

Sahu et al., (1999) characterized a novel acylated triterpenoid saponin,

designated Pithecelloside from the methanolic extract of defatted, air-dried seeds of

Pithecellobium dulce. The compound had been elucidated by interpreting the spectral

datas and identified as 3-O- [ α –L-arabinopyranosyl- (1-2) – α –L-arabinopyranosyl –

(1-6) –β –D- glucopyranosyl] – 21 β – O – [ (2’E) – 6’- hydroxyl – 2’6’- dimethyl

octa – 2’7’ –dienoyl] acacic acid.

CH3CH3

CH3

O

CH3

O

OHOH

CH3CH3

O

O

CH3

CH2

CH3

OH

O

OH

OH

O

OH

OH

OH

H

OOH

OHO

H

O

OH

H

Pithecelloside

O

O

O

O

O

O

O

CH3CH3

CH3CH3

O

O

O

R1

OR

1

O

R1

O

R1O

R1

O

R1

O R1

O

R1

O

R1

OR

1

O

R1

O

R1

O

R

39

Further Niranjan et al., (1994) characterized echinocystic acid bisdesmoside a

new bisdesmodic triterpenoid saponin, from the seeds of Pithecellobium dulce.

Kallappa and Hosamani, (1995) characterized vernolic acid (12,13-epoxy-octadec-cis-

9-enoic acid, 10.0%), malvalic acid [7-(2-octacyclopropen-1-yl) heptanoic acid,

3.2%] and sterculic acid [8-(2-octacyclopropen-1-yl) octanoic acid, 2.0%] from

Pithecellobium dulce, Benth (syn.Inga dulcis, Willd) seed oil, belonging to the

Leguminosae plant family, The other normal fatty acids are palmitic (12.1%), stearic

(4.2%), behenic (10.6%), oleic (34.1%), and linoleic (23.8%).

Saxena and Singhal, (1999) have isolated a novel prenylated flavanoid 3’-

prenyl apigenine 7-O- rutinoside from methanol extract of the stem of Pithecellobium

dulce.

O

O

OH

CH3 CH3

OH

OR

R= α –L-rhamnopyranosyl – (1-6) –β –D- glucopyranosyl

Saxena and Singhal, (1998) isolated a new isoflavanoid namely, genistien -4’-

O- α –L- rhamnopyranoside from the roots of Pithecellobium dulce. The compound

had been screened for estrogenic activity and show promising results. Petrus et al.,

(1997) showed the bark of Pithecellobium dulce containing tannin of a catechol type,

varies according to the age of the plant. Acetone extract of the bark is reported to

40

consist mostly of 3,4,7,3’,4’- pentahydroxy flavan a compound which combines the

properties of both leucoanthocyanidin and phlobotannin. The antitumour compound,

b-sitostero and campesterol, stigmasterol and a-spinasterol were reported in the heart

wood of this plant.

2.7 Biological activity of isolated compounds from pithecellobium dulce

The fungistatic activity and antimycobacterial study has been worked out by

many authors. The fungistatic activity of Pithecellobium dulce on mycelial growth of

Alternaria sp., Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium

oxysporum, Penicillium digitatum, Pestalotiopsis sp. and Rhizopus stolonifer was

done and the seeds had the highest fungistatic activity against the fungi tested

(Barrera and Bautista, 2002). Mukesh kumar et al., (2013) evaluated the

antimicrobial activity of leaf of Pithecellobium dulce against twenty pathogenic

microorganisms and assessed the antimicrobial activity of leaf extracts against five

Gram-positive (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus,

Staphylococcus aureus and Staphylococcus epidermidis), seven Gram-negative

(Aeromonas hydrophila, Alcaligenes faecalis, Enterobacter aerogenes, Escherichia

coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhimurium)

bacteria and eight fungi (Aspergillus flavus, Aspergillus niger, Aspergillus oryzae,

Aspergillus terreus, Alternaria alternata, Alternaria brasicola, Alternaria solani and

Alternaria vitis). Sukantha et al., (2011) also found the antibacterial potential of the

fruit peel of Pithecellobium dulce.

The leaves of Pithecellobium dulce Benth. showed activity by

BACTEC460TBR radiospirometric system and found at the concentration of

41

20mg/ml leaf extracts shows highest activity when comparable with standard drugs

like, streptomycin, isoniazid, rifampicin, ethambutol and pyrazinamide

(Shanmugakumaran et al., 2005 and 2006). However Bautista et al., (2003) and Ali

et al., (2001) worked on the methanolic and hexane extracts of Pithecellobium dulce

seeds and proved methanolic extract of Pithecellobium dulce showed higher efficacy

against bacteria and fungi than hexane extract.According to Barrera et al., (2003) The

fractions and compounds from seed powder were tested against various postharvest

fungi. The most sensitive strain was Fusarium oxysporum and the less sensitive strain

was Penicillium digitatum.

Megala and Geetha, (2010) studied the gastroprotective effect of

hydroalcoholic fruit extract of Pithecellobium dulce on some important antioxidant

enzymes such as superoxide dismutase , catalase, glutathione reductase , glutathione

peroxidase and myeloperoxidase. The antioxidant activity of anthocyanin extracted

from Pithecellobium dulce fruit pericarp and fruit peel have been detailed by

Ponmozhi et al., (2011), Sukantha et al., (2011), Arul and Muthukumaran, (2011);

Bhargavakrishna et al.,(1970) reported the analgesic and anti-inflammatory effects of

methanol extract and saponins from Pithecellobium dulce. Govindarajan et al., (2012

and 2013) studied the larvicidal and ovicidal potential of the crude hexane, benzene,

chloroform, ethyl acetate and methanol solvent extracts from the medicinal plant

Pithecellobium dulce against the mosquito vector, Anopheles stephensi and Aedes

aegypti (Diptera: Culicidae) and found seed extracts have low potency compared to

leaf extracts. The study involves antioxidant enzymes and the adulticidal efficacy of

seed extracts of Pithecellobium dulce against Culex quinquefasciatus for controlling

filariasis vector mosquitoes was also determined .

42

Sundarrajan et al., (2010) found the anti-hyperlipidemic activity of aqueous

extract of leaves of Pithecellobium dulce against triton induced hyperlipidemia.

According to Mule et al., (2011) the anticonvulsant activity testing by

pentylenetetrazole method showed that aqueous and ethanolic extract of

Pithecellobium dulce could not decrease the convulsions as well as mortality in mice.

However the aqeous fruit extract of Pithecellobium dulce protects murine liver against

CCl4-induced oxidative impairments probably by its antioxidative property (Prasenjit

et al., 2010).

Delgado et al., (2004) reported for the first time the isolation and

characterization of a protease inhibitor from the seeds of Pithecellobium dulce. The

N-terminal sequence of PDTI has the highest similarity with the seed inhibitor of

Acacia confuse. However Rao et al., (2011) evaluated chemical composition and

sensory quality of white and pink aril powders of Pithecellobium dulce in two

packaging systems during storage for six months. The protein contents were 12.4 and

15.0% in white and pink aril powders. The titrable acidity of white and pink aril

powders were 2.4 and 4.8%.Ca and Fe contents in white aril powder samples were 60

and 12 mg/100 g where as in pink aril powder 62 and 16 mg/100 g. The anthocyanin

content in pink powder decreased from 50.5 to 11.2 and 14.1 mg/100 g in samples

packed in polyethylene and metalized polyester polyethylene laminated pouches.

According to Sugumaran et al., (2008) the locomotor activity of aqueous and

alcoholic extracts of leaves of Pithecellobium dulce Benth was determined and CNS

depressant activity of leaf extracts of Pithecellobium dulce was evaluated using

actophotometer in albino mice.

43

Pithayanukul et al., (2005) studied on the aqueous extract of

Pithecellobium dulce for their inhibitory activities against Naja kaouthia venom by

invitro neutralization method. The antivenom activity study of the extract could

completely inhibit the lethality of the venom at 4 LD50 concentration and the venom

necrotizing activity at the minimum necrotizing dose also inhibited up to 90% of the

acetylcholinesterase activity of Naja kaouthia venom at much lower tannin

concentration. The ED50 of plant tannins inhibiting Naja kaouthia venom activities

due to condensed tannins and their content in the extracts. The spermicidal effect of

saponins in Pithecellobium dulce was also reported by Misra et al., (1979).Sugumaran

et al., (2006) examined the macroscopical characters of the leaves, leaf constants,

physico chemical constants, extractive values with different solvents, micro chemical

test, flouresence characters of liquid extracts and leaf powder of Pithecellobium dulce.

Khanzda et al., (2013) measured some essential and toxic elements As,Cu, Cd, Fe, K,

Mg, Na, Pb & Zn in different concentrations Zn & K being the highest (26.89 mg/kg)

and Pb (0.19mg/kg) and As(17.6μg/kg) were the lowest in concentrations justifying

its medicinal applications.

The antidiabetic effect of Pithecellobium dulce has been revealed by many

authors both by invivo and invitro methods. Pradeepa et al., (2013) evaluated the

antidiabetic potential of Pithecellobium dulce fruits in STZ - induced experimental

diabetes in rats. Oral administration of Pithecellobium dulce fruit extract (300 mg/kg

b.w. /day) to diabetic rats for 30 days significantly reduced the levels of blood

glucose. Similar observation was found by Sugumaran et al., (2009) Pithecellobium

dulce leaves using streptozotocin induced diabetic model in rats. The aqueous extract

44

showed significant activity (P<0.01) than the alcoholic extract at the tested dose level

which was comparable to glibenclamide, a standard antidiabetic drug.

The invivo antidiabetic effect was shown by many studies. Dnyaneshwar and

Archana, (2013) reported the inhibitory action of methanolic extract and acetone

extract of Pithecellobium dulce seeds, bark and leaves. Seeds showed inhibitory

action on α- amylase and α-glucosidase enzymes and compounds responsible for

these activities are characterized. The enzyme inhibitory activity of Pithecellobium

dulce methanolic extract may be endorsed to the presence of oleanolic acid

triterpenoid. The bark and leaves of Pithecellobium dulce were evaluated for α-

amylase and α-glucosidases inhibition in vitro, compared with acarbose. Acetone

extracts of bark and leaves showed more sucrase inhibition than maltase while

methanol extract of bark and leaves showed more sucrase inhibition than maltase.

2.8 In-vitro study

Non-enzymatic glycosylation of haemoglobin assay of Pithecellobium dulce

Invitro study was done outside the living body. The antioxidant ability of

enzymatic and non enzymatic process decreases the active oxygen species responsible

for many diseases including diabetes. The rise in glucose level in the blood leads to

the binding of haemoglobin and glucose with the formation of the reactive species.

(Clifford et al., 1989). Apart from reducing blood glucose level, the glucose level

was controlled by inhibition of α-glucosidase activity in the intestine. According to

Kirchner et al., (1996) inhibition of α-glucosidase activity in the intestine was

achieved by using luteolin, kaempferol, chrysin and galangin to study the effect of

absorption and metabolism of carbohydrates. Megha et al., (2013) worked on dried

45

(crude) petroleum ether and hexane extracts of stem bark of Bauhinia purpurea. In-

vitro anti-diabetic activity reports showed significant level of activity, the highest

concentration of extract was effective as an anti-diabetic agent. Similar invitro

antidiabetic activity of phytochemical bioactive compounds of the methanolic extract

of Psidium guajava leaves was observed by Manikandan et al., (2013).

Andichettiyar and Tapan, (2013) and Ali et al., (2013) reported the in vitro α-

amylase and α-glucosidase enzymes inhibitory activity in ethanol and chloroform

extracts of Polyalthia longifolia (Sonner). The leaves and fractions of the dried fruit

pericarp of Phaleria macrocarpa potently inhibiting carbohydrate hydrolysing

enzyme. Further study showed the inhibitory action of methanolic extract of

Pithecellobium dulce seeds on α- amylase and α-glucosidase enzymes. The IC50

values of methanolic extract of Pithecellobium dulce against pancreatic α-amylase

was found to be 16.75±1.81 mg/ml.

Faiyaz and Asna, (2009) compared the hypoglycemic effect of Ficus

racemosa stem bark (Moraceae) and the glucose diffusion activity of wheat bran and

and acarbose using in vitro technique. Ficus racemosa bark exhibited significantly

higher (P ≤ 0.01) glucose-binding capacity than wheat bran and acarbose

consequently showed significantly higher (P ≤ 0.01) retardation of glucose diffusion

compared to wheat bran and acarbose. Ashok Kumar et al., (2013) revealed in vitro

antidiabetic screening of Nisamalaki Churna by α-amylase inhibition using starch

iodine method.

Stalin et al., (2013) examined the crude n-hexane, ethanol, methanol and

aqueous leaf extracts of cardiospermum and the invitro inhibitory effect of glucose

46

utilization. Palaniswamy and Sellapa, (2012) compared the inhibitory activity of the

aqueous extracts of four seaweeds Gracilaria edulis ,Sargassum polycystum, Ulva

lactuca, Gracilaria corticata collected from Gulf of Mannar coastal waters and tested

for α - amylase and α –glucosidase inihibition and found Gracilaria corticata with

significant inhibitory activity against α - amylase and α - glucosidase enzymes.

2.9 Toxicity test in mice

Animal models are indispensable tools in biomedical research. They have

been used to find out the effectiveness and the toxicities of various medicines and

chemicals. Investigation of acute toxicity is the first step in toxicological analysis of

herbal drugs .The present study was undertaken to determine the acute oral toxicity

and safety parameters of compounds isolated from the plant parts. Ogbonnia et al.,

(2013) and Arthur et al., (2011) shown a detailed study on acute and subchronic

toxicity in animals in polyherbal formulation and aqueous extract of A. muricata

leaves using standard procedures and the LD50 value indicated the drug as being safe.

At the highest dose, the formulation exhibited deleterious effect on the hepatic tissue.

The outcome of acute oral toxicity of ethanolic extract of M. pruriens on Swiss albino

mice was reported by Parekar and Somkumar , (2011) found that no mortality or

evidence of adverse effect have been observed in Swiss albino mice following acute

oral administration of ethanolic extract at the dose of 2000 mg /kg .

According to Protus et al., (2012) Sub-acute (aqueous and ethanol extract)

and sub-chronic oral toxicity (aqueous only) were carried out on the aqueous and

ethanol leaf extracts of Carica papaya (CP) in Wistar rats. Control groups received

water and corn oil respectively.The result suggests that aqueous extract showed lesser

47

toxicity than the ethanol extract. Paramakrishnan et al., (2012) and Lalitha et al.,

(2012) reported the acute oral toxicity test on the various extracts of aerial parts of

parviflorab and Eichhornia crassipes and the animals were orally administered a

single dose of 100, 250, 500,750, 1000, 2000 mg/kg body weight. Signs of toxicity

and mortality were noted after 1, 4 and 24h of administration of the extract for 14

days. The highest dose administered (2000mg/kg body weight) did not produce

mortality or changes in general behaviour of the test animals.Further Subramanion et

al., (2011) investigated the acute oral toxicity of C. fistula seeds extract in mice and

found that oral administration of crude extract at the highest dose of 5000 mg/kg

resulted in no mortalities or evidence of adverse effects, implying that C. fistula is

nontoxic and the extract can be utilized for pharmaceutical formulations. Acute and

subacute toxicity of ethanol (95% v/v) extract of aerial parts of Cansjera rheedii J.

Gmelin (Opiliaceae) and Tephrosia purpurea, the limit test dose of 2000 mg/kg were

used for Cansjera rheedii J. Gmelin (Opiliaceae) and 400mg/kg for T. purpurea. No

significant variation (p < 0.05) in the body and organ weights between the control and

the treated group was observed after 28 days of treatment.

2.10 Invivo antidiabetic study

Animal model is important to assess the biological activity of compounds

found in plants. Mouse is the most cited animal model, 99% of mouse genes have

human counterparts. Mouse models of many human diseases have also been

developed to advance the studies of disease pathogenesis, and to evaluate the

effectiveness of various candidate drugs.

48

Considerable literature is available on hypoglycemic effect of various plant

parts using mice model. Comprehensive studies on different plant extracts, isolated

plant compounds have been made by various authors. The hypoglycemic effect on

alloxan induced diabetic mice were studied on various plants (Meshram et al.,2013);

hydro alcoholic extract of stem bark of Bauhinia purpurea (Mishra, 2013); aqueous

extract of Anethum graveolens seeds (Abu et al., 2010); ethanolic extracts of

Scoparia dulcis and ethyl acetate extract of Peporemia pellucide (Hasib et al., 2012).

Other important work on alloxan induced diabetic mice include those of

Gunjan and Krishnendua, (2013); in ethanolic extract of Astraeus hygrometricus

(Pers.) Morg. (Astraeceae); Ihechilure, (2010); crude extract of I .trichantha leaves;

Franscisco et al., (2006); in Hexane, dichloromethane, methanol and aqueous extracts

of the Plantago Major seed; in T. belerica and crude methanolic extract of the

Potentilla fulgens L roots (Sabu and Kuttan, 2009).

Syiem et al., (2002) studied the effect of the methanolic extract of Albizia

odoratissima Benth. bark and showed the reduction of blood glucose level with

various other factors like serum cholesterol, triglycerides,serum glutamic pyruvic

transaminase, alkaline phosphatase and decreases level of total proteins in alloxan

induced diabetic mice. Dinesh Kumar et al., (2011) while studying the effect of

crude methanolic extract of the Potentilla fulgens L roots for its hypoglycemic effect

found that it depends upon the dosage level and time factor. Njagi et al., (2012)

observed that aqueous stem bark extract of Ficus sycomorus safely lowered blood

glucose level to levels below insulin, the model drug in a dose-dependent manner.

49

Hypoglycemic effect was also analyzed in streptozotocin induced diabetic

mice on the methanolic extract of Diplocyclos Palmatus (Jeyanarayan et al., 2012).

Many studies have been conducted on streptozotocin diabetic induced rats model by

several workers (Palaniswamy et al., 2011); Ellagic acid ; (Sayed et al., 2011);

aqueous extracts of Mangifera indica and Psidium guajava ; (RajKumar et al., 2011) ;

methanolic extract of Hibiscus cannabinus.

Various authors showed the hypoglycemic effect on streptozotocin induced

diabetic rats at different dosages Nilufer et al., (2006) observed the acute and

subacute antidiabetic activities of the ethanolic extract of Vitis vinifera L. leaves at

250 mg/kg dose was found to possess a high antidiabetic and antioxidant activity.

Oral administration of the methanol extract of E.jambolana (EJ) (150mg/kg bw) for

60 days to streptozotocin (STZ) (60mg/kgbw) induced male diabetic wistar rats was

able to significantly (p<0.05) decrease the blood glucose concentration (Jasmine and

Daisy, 2011). The potential antidiabetic ability of Gymnema sylvestre was identified

by Vijanand, (2012) the dose of 200 and 400mg/kg decreases the glucose level

significantly.

Papiya et al., (2008) made a detailed study on hypoglycemic effect of an

isolated compound (Ficanone) from Ficus arnottiana bark in normal and

streptozotocin diabetic rats. When administered to diabetic rats, Ficanone (50 mg/kg,

p.o.) caused a significant (p<0.01) reduction in glucose level. Mi-Jang et al., (2011)

determined the hypoglycemic effect of Chenopodium ambrosioides in mice fed with

high-fat diet for two weeks before induction of diabetes mellitus by injection of

streptozotocin. Animals treated with crude extract (100, 200 and 300 mg/kg) showed

50

significant (p<0.05) hypoglycemic effect in comparison to control. Rupali et al.,

(2011) tested the anti hyperglycaemic and antidiabetic potential of leaf extracts of

Albizzia lebbeck (Benth), Psidium guajava (Linn), and Trigonella foenum-graecum

(Linn) against alloxan and streptozotocin induced diabetic models of mice and

showed a positive trend in regulating blood glucose levels in Swiss albino mice by

using Rosiglitazone as a standard drug. Aicha et al., (2013) showed the effect of the

aerial crude aqueous extract part of Anacyclus valentinus L. on diabetes induced

Wistar rats by streptozotocin and endowed significant antidiabetic activity.

Rekha et al., (2010) justified the use of a combination of aqueous extracts of

pulp of Syzygium cumini and bark of Cinnamon zeylanicum for the remedial effects

against streptozotocin induced diabetic state .However. Rajesh et al., (2005) revealed

the antidiabetic activity of the water extract of Annona squamosa (custard apple) in

diabetic animals. Hot-water extract of the leaves of A. squamosa was prepared by

boiling fresh and air dried leaves (25°C for 5 days) with water (20 ml/g) for 2 h and

was tried by oral route in alloxan (80 mg/kg bw)-induced diabetic rabbits and

streptozotocin (STZ) (50 mg/kg bw)-induced diabetic rats and reveals that

A. squamosa has both hypoglycaemic and antidiabetic activity. Oyedepo et al.,

(2013) confirmed that the oral administration of aqueous leaf extract of Moringa

oleifera may reduce the plasma lipid imbalances associated with diabetes mellitus.

Further Kavishankar et al., (2010) found the antidiabetic effects of aqueous fruit

extract of Morinda citrifolia and Cocinia Indica in alloxan induced rats for a period

of 30 days. Syamsudin, (2010) conducted antidiabetic test on active fractions of

methanol extract from Leucaena leucocephala (lmk) De Wit seeds using alloxan-

induced rats. Manikandan et al., (2009) observed hyperglycemic nature of

51

Isolated 1, 2 di-substituted idopyranose from Vitex negundo against streptozotocin-

induced diabetes.

Nancy et al., (2011) evaluated antidiabetic property of Sonneratia alba Sm,

when subjected to anti-diabetic bioassay using standard Glucometer, The blood sugar

level was reduced by 19.2% during the first 6 hours and reduced further to 66.9%

after 12 hours. Momoh et al., (2011) analyzed significant hypoglycaemic effect

(p<0.05) of Costus afer in adult male wistar strain albino rats and the effect was

comparable with the standard drug glibenclamide .

2.11 Compound isolation and identification of compounds using spectroscopic

technique

Number of bioactive compounds were isolated from different plant parts by

chromatographic technique and the structure of the compounds were identified using

spectroscopic method. Shah et al., (2013) isolated the antidiabetic compounds from

the kernel of mango using column and thin layer chromatographic techniques and

compounds are identified using 1H-NMR spectroscopy. Fang et al., (2005) has

widely shown the isolation and identification of sesquiterpene lactones was carried

out in in three steps: multiple extractions, fractionation using column chromatography

and the separated compounds was determined on the basis of 1HNMR and

13CNMR.

Shalabia, (2011) examined the plant Atriplex halimus and isolated eleven free amino

acids and revealed the presence of three alkaloids at Wadi-Sudr habitat and two

alkaloids at Wadi-Hof habitat.

Isolation of bioactive compound from the leaves of Excoecaria agallocha was

done using thin layer chromatography and column chromatographic techniques

52

(Patra et al., 2012) Crotepoxide was isolated from ethyl acetate mixture of Croton

macrostachyus berries using chromatographic technique (Habtamu et al., 2012).

Getahun et al., (2012) have worked using the same column chromatography and

spectroscopic methods (IR and NMR) for the isolation and identification of betulinic

acid and benzoic acid from the root extract of Helinus mystachinus. Using column

chromatography Christinah et al., (2012) isolated the bioactive from G. kola seeds.

Ali et al., (2013) showed that chromatographic techniques were used to isolate

phenolic compounds from plants.

Abdul et al., (2010) included liquid layer chromatography, column

chromatography and preparative thin layer chromatography for the isolation of the

compounds dillenetin, betulinic acid, β – sitosterol and stigmasterol from the crude

leaf extracts of Dillinea indica Linn. Surendar et al., (2011), Veena and Ritu (2013)

separated the saponins from Moringa oleifera using thin layer chromatography

(TLC) on mobile phase of Chloroform: methanol: H2O (7:3:1) on silica gel glass

plates and high performance liquid chromatography (HPLC) is used to isolate

compound from benzene extract and further showed characterization of isolated

saponin was done using eletrospray ionization mass spectrometry ESI-MS, IR

,1HNMR and

13C-NMR. San et al., (2011) showed that thin layer chromatography,

ultraviolet (UV) and Infra-red (FTIR) spectroscopic method were used in the

isolation and identification of chemical constituents of compounds from ethanolic

extract of leaves of C. inerme Gaertn. Mary et al., (2012) and Rajkumar et al., (2012)

isolated the bioactive compound using silica gel column chromatography from

Aspergillus sp and O. indicum stem bark.

53

The present review give knowledge about diabetes, and its various herbal

treatment. The investigation on Pithecellobium dulce give knowledge about

Pithecellobium species with reference to its pharmacological potentials and

biochemical spectrum. The present study was chosen with the following aim and

scope

· Extract preparation of leaf ,fruit and fruit peel of Pithecellobium dulce using

various solvents using soxhlet extractor.

· Determination of various physiochemical parameters.

· Determination of inorganic minerals in plant parts.

· The various extracts are subjected to preliminary phytochemical screening for

the presence of alkaloids, flavones, terpenes, steroids, tannins, quinines and

glycoside.

· Estimating the amount of protein, carbohydrate, total lipids, total alkaloids,

total flavanoid, total phenol using quantitative phytochemical analysis.

· Phytochemical isolation of compounds present in leaves ,fruit and fruit peel

using column chromatography .

· To isolate kaempferol and afzelin from methanolic extracts of leaves using

column chromatography .

· To isolate quercitrin from methanolic extract of fruit using column

chromatography.

· To isolate two new compounds from the methanol extract of fruit peel using

column chromatography.

54

· To identify the isolated compounds using UV, IR, 1HNMR,

13CNMR and

mass spectroscopy.

· To detect the invitro hypoglycemic activity of isolated compounds from leaf,

fruit and fruit peel of Pithecellobium dulce using non enzymatic glycosylation

of haemoglobin assay and the enzymatic study alpha-amylase inhibition

assay .

· To detect the invivo antidiadetic effect of isolated compounds from leaf, fruit

and fruit peel of Pithecellobium dulce using alloxan induced diabetic Swiss

albino mice .

· To compare the the antidiabetic hypoglycemic effect of different isolated

compounds from the leaf, fruit and fruit peel.

· To determine the angiogenisis activity of the newly isolated compound from

fruit peel, using CAM assay.