3. isolation of anti-inflammatory compounds from musa...

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In-vitro Bioassays and Chromatographic Analyses Used to Screen Natural Products from Jamaica in

the 21st Century, 2015: 33-47 ISBN: 978-81-308-0562-7 Editor: Cheryl E. Green

3. Isolation of anti-inflammatory compounds

from Musa sapientum (Banana) leaf extracts

using the anti-denaturation of bovine serum

albumin (BSA) assay

Theresa M. Wong, Marissa Robinson, Sheridan Hibbert and Lawrence A.D. Williams

The Natural Products Unit, Scientific Research Council of Jamaica, PO Box 350 Hope Gardens, Kingston 6, Jamaica, West Indies

Abstract. OBJECTIVE: To isolate compounds from extracts of

the leaves of Musa sapientum that have anti-inflammatory

activity.

METHODS: The bovine serum albumin (BSA) assay and UV

spectroscopy was used to determine the level of anti-denaturation

activity of ethanol fractions of 82 Jamaican plants. Further

analysis was carried out on the hexane, ethyl acetate and methanol

extracts of the leaves and stem of Musa sapientum. Gravity

column chromatography as well as Thin Layer Chromatography

(TLC) allowed for the separation of the hexane extract into 50

different fractions, which were then pooled to 16 fractions as

indicated by TLC profiles. Using the BSA assay, active

compounds were identified based on their %anti-denaturation.

Correspondence/Reprint request: Dr. Lawrence A. D. Williams, Research Consultant, Scientific Research Council

of Jamaica, PO Box 350, Hope Gardens, Kingston 6, Jamaica, West Indies

E-mail: [email protected]

Theresa M. Wong et al. 34

RESULTS: The preliminary results showed that Cinnamomum zeylanicum, Musa

sapientum and Tecoma stans all showed particularly high levels of % anti-

denaturation. The BSA assay tests carried out on Musa sapientum revealed that the

non-polar fractions of the hexane extract registered the highest levels of % anti-

denaturation (#2: 37.75%, #3-4: 42.5%, #5-7: 44.3%, #8-9: 27.2%, #10: 37.0%, #11:

33.7%).

CONCLUSION: The hexane extract of the leaves and stems of Musa sapientum

contain non-polar active compounds. These compounds show anti-denaturation and

anti-inflammatory properties when tested with the BSA assay. As a natural product

compound along with the range of diseases with inflammation and oxidative stress as

the underlying cause, these non-polar active compounds may have great

pharmaceutical potential.

Introduction

Musa sapientum is a herbaceous perennial, most commonly known as

banana. Originally grown in the Indomalayan ecozone, today it flourishes

throughout the tropics from the West Indies to China1. From its leaves to the

fruits, every part of the plant is believed to have medicinal properties as

recorded by traditional folklore and/or scientific studies. The flowers for

instance have been used in treating dysentery, ulcers2 and its extracts have

shown antihyperglycaemic activity3. The peel possesses both antifungal and

antibiotic properties2. Studies have also shown the use of the unripe peel to

treat surgical wounds in rats4, 5

. The waxy, cooling, impervious to water and

anti-adhesive characteristics of the banana leaves have also made it ideal for

burn wound dressings in developing countries such as India6–8

. For the

patients, the dressings were a more comfortable alternative to both the potato

skin dressings and Vaseline gauze dressings and have resulted in faster

healing times for partial thickness burn wounds. These effects on pain

reduction and shortened healing times have pointed to an investigation into

the anti-inflammatory properties of the banana leaves.

The bovine serum albumin (BSA) assay is used in this study to

investigate potential anti-inflammatory activity of compounds in banana

leaves. When BSA is heated, the protein denatures; denaturation is

implicated in the inflammatory response9. This result in the expression of

antigens associated with type III hypersensitive reaction, which is related to

diseases such as rheumatoid arthritis and serum sickness. It has been found

that denaturation of BSA is inhibited by several non-steroidal anti-

inflammatory drugs such as indomethacin and salicyclic acid; proving this

assay to be useful in the detection of other anti-inflammatory compounds10

.

Other studies to date have supported its value in the field of drug discovery

and have used it in identifying anti-inflammatory compounds in Erythrina

Anti-Inflammatory compounds isolated from Musa sapientum 35

indica bark11

, Mikania scandens12

, Barleria prionitis13

, Plectranthus

hadiensis14

, Zizyphus oenoplia9 and Piper betle

15. These natural anti-

inflammatory products have the potential to be safer and just as effective as

the steroidal allopathic drugs11

. Additionally, this assay is valuable for its

low cost and ability to avoid the use of live animals for as long as possible in

the drug discovery process16

.

The objective of this study was to first screen for local plants that may

have anti-inflammatory activity. Plants that showed significant results in the

initial screening were chosen for further study with the BSA

anti-inflammatory assay. Here we report the results of the experiment done

on the leaves of Musa sapientum and reveal the potential of its compounds

as an anti-inflammatory drug.

2. Materials and methods

2.1. Screening of 82 Jamaican plants for anti-denaturation

activity in BSA

Eighty-two plants were collected from St. Elizabeth, Nassau Valley and

authenticated at the Herbarium of the Science and Technology Faculty, The

University of the West Indies, Mona Campus, Kingston 7, Jamaica, West Indies.

The leaves and stems were air-dried and 10g were pulverized and

extracted with 200mL of ethanol for 5 days under laboratory conditions

(25°C-27°C; 70-80% RH). The crude extract was then concentrated in vacuo

using a rotary evaporator to an oily residue.

A solution of 0.4% (W/V) BSA was prepared in Tris buffer saline and

the pH adjusted to 6.39-6.4 using glacial acetic acid. Stock solutions of

0.005% (W/V) were prepared by using methanol as the solvent. From each

stock solution, 5µL, 10µL and 20µL (0.25, 0.5 and 1µg/µL concentrations

respectively) were pipetted into test tubes. 1mL of the 0.4% BSA solution

were then added to each. A positive control was used and consisted of

0.005% solution of aspirin with 1mL of 0.2% (W/V) BSA assay. Solutions

were then heated in a water bath for 10 mins and left to cool for 20 mins.

Each sample was analyzed using a UV spectrophotometer and their % anti-

denaturation was calculated based on their absorbances at 660nm (Eqn. 1).

%anti denaturationabscontrol abssample

abscontrol100

Eqn. 1. Formula to calculate the % anti-denaturation of the samples based on

their absorbance at 660nm.


2.2. Detection of anti-denaturation activity in Musa sapientum

2.2.1. Preparation of crude extracts

The banana leaves were collected and left under laboratory conditions

for one day. After drying, the leaves were cut into small pieces and 228.37g

were placed into a conical flask and extracted with 2L of n-hexane 60%.

After 5 days the mixture was filtered and the pale green filtrate was

concentrated in vacuo using a rotary evaporator to an oily residue. The

residue was left under laboratory conditions for further evaporation to a dark

yellow-green precipitate (yield 0.50%).

The leaves were then extracted with 2L of ethyl acetate for 5 days under

laboratory conditions. After filtration, the dark green filtrate was concentrated

in vacuo using a rotary evaporator to an oily residue. Further evaporation

under laboratory conditions produced a dark green paste (yield 1.76%).

For methanol extraction, the leaves were first dried in the oven. 2L of

methanol were added and the mixture left for 5 days under laboratory

conditions. The mixture was then filtered and the filtrate concentrated

in vacuo using a rotary evaporator to produce a dark green oily residue (yield).

2.2.2. In vitro anti-denaturation studies to find the active fraction

A solution of 0.4% (W/V) BSA was prepared in Tris buffer saline and

the pH adjusted to 6.36 (hexane and ethyl acetate extracts) and 6.3 (methanol

extract) using glacial acetic acid. Stock solutions of 0.005% (W/V) were

prepared from the hexane, ethyl acetate and methanol extracts. 5µL, 10µL

and 20µL of each solution was pipetted into test tubes, followed by 1mL of

BSA assay (performed in duplicates). The control used was pure methanol.

The solutions were heated for 10 mins at 72°C in a water bath and left to

cool for 20 mins. The absorption of each sample was recorded using a

Thermo Scientific Spectronic 200 UV spectrophotometer at 660nm and their

% anti-denaturation calculated using Eqn.1.

2.2.3. Isolation of compounds from the hexane fraction by column

chromatography and TLC

Hexane extract weighing 0.9426g was subjected to gravity column

chromatography with a column of height 21.6 cm, packed with silica gel

(60-120 mesh). The system was flushed with pure hexane followed by the

solvent gradient 1%, 5%, 10%, 20%, 40%, 80%, 100% ethyl acetate and

100% methanol. The 50 column fractions of 20 mL was collected and left

under the fume hood for evaporation.


Each fraction was subjected to TLC using solvent systems of 20:2 hexane:ethyl acetate to collect fractions #2-7, 40:4 for fractions #8-26 and 20:8 for fractions #27-50. The plates were visualized with molybdate in sulphuric acid and heated on a CAMAG TLC plate heater III. Based on their profiles, the 50 column fractions were pooled together resulting in a collection of 16 fractions (#2, #3-4, #5-7, #8-9, #10, #11, #12-16, #17-19, #20-21, #22-26, #27-28, #29-30, #31-36, #37-46, #47-48, #49-50) to be tested for anti-denaturation activity.

2.2.4. In vitro anti-denaturation studies to find the active compounds

A 0.4% (W/V) BSA solution was prepared with Tris buffer saline and the pH adjusted to 6.3 (samples #2-11), 6.46 (samples #12-46) and 6.41 (samples #47-50) using glacial acetic acid. Stock solutions of 0.005% were prepared and 10µL of each were tested in duplicates with 1mL of BSA. The control used was 10µL methanol. The absorption of each sample was recorded using the Thermo Scientific Spectronic 200 UV spectrophotometer at 660nm and their % anti-denaturation calculated using Eqn.1.

3. Results

3.1. Thirty-two plants showing significant anti-denaturation activity

Table 1 shows the results of the screening of 82 Jamaican plants for anti-

denaturation activity using the BSA assay. 32 of these plants showed %anti-

denaturation >20%, proving them to be promising for further anti-

denaturation studies. Cinnamomum zeylanicum, Musa sapientum and

Tecoma stans in particular showed high activity at all three concentrations.

Table 1. Results of the BSA assay screening of 82 plant extracts at 0.005% with

0.4% BSA to determine anti-inflammatory activity.

Plant Scientific Name Plant Common

Name

Inhibition %

0.25µg 0.50µg 1.00µg

Abultilon Trisulcatum 11.62 27.81 -7.03

Aloe vera Sinkle Bible 33.02 -11.69 -50.00

Artocarpus altilis Breadfruit 5.75 1.01 -16.93

Asclepias curassavica Redhead -24.43 27.77 13.50

Blighia sapida Ackee -45.81 -1.29 28.56

Boehmeria jamaicensis Doctor Johnson 47.51 -18.89 -54.87

Bontia daphnoides Kidney Bush -56.25 18.25 -5.67


Table 1. Continued

Bougainvillea spp. Bougainvillea -63.15 -25.32 15.31

Bryophyllum pinnatum Leaf-of-Life -31.51 6.60 -6.68

Caladium bicolor Caladium 23.29 8.58 -30.23

Calotropis procera French Cotton -10.05 6.12 5.51

Capsicum annuum Scotch Bonnett -42.99 -7.82 -1.18

Cassia alata King of the Forest 3.68 35.31 -8.02

Catharanthus roseus Periwinkle 3.73 -22.18 -21.32

Chrysophyllum cainito Star Apple -96.94 -4.00 -41.49

Cinnamomum zeylanicum Cinnamon 24.18 34.63 49.61

Cleome rutidosperma

Consumption

Weed 42.16 -12.87 -40.43

Cleome viscosa Wild Caia 10.90 30.96 32.68

Coccoloba uvifera Seaside Grape -31.86 -8.92 -36.77

Codiaeum varigatum Garden Croton 22.57 22.02 -20.80

Cordia alba

Duppy Cherry

(Sible Cherry) 65.47 39.53 -86.35

Crescentia cujete Calabash Tree 7.33 17.46 8.50

Croton linearis Wild Rosemary 29.23 -11.14 -44.23

Cucurbita spp Pumpkin 17.58 25.00 -3.87

Cuscuta americana Love Bush 21.24 12.42 -30.65

Cycloptis semicordata Tall Fern 35.83 16.75 -16.34

Cymbopogon citratus Fever Grass -12.63 1.14 -30.00

Delonix regia Poinciana -40.32 -12.32 -53.73

Dieffenbachia spp.

Dieffenbachia

(Ornamental) -60.22 -32.91 -17.31

Dioscorea polygonoides Wild Yam -0.44 18.79 -25.81

Ervatamia divaricata Coffee Rose -62.82 -51.66 -3.02

Erythrina corallodendrum Spanish Machette 18.37 -65.00 -67.02

Euphorbia hirta Milk Weed 9.49 29.53 28.12

Fagara flava

Jamaican

Satinwood -16.16 -5.07 4.22

Ficus spp. Evergreen -15.85 15.06 -4.14

Gliricida sepium

Aaron’s Rod

(Quick Stick) -29.73 7.94 -4.95

Guaiacum officinale Lignum Vitae -8.25 9.24 -5.94

Haematoxylum campechianum Logwood -51.47 -28.15 -5.46

Heliotropium angiospermum Dog's Tail 27.59 0.71 -13.32

Hibiscus rosa-sinensis Hibiscus -7.00 -1.63 15.21


Table 1. Continued

Ipomoea carnea Morning Glory 32.63 19.80 4.85

Ixora spp. Ixora (White) -10.89 -13.53 -28.00

Lantana camara

White Sage

(Red Flowers) -23.18 0.79 1.52

Leucaena leucocephala Leucaena -15.79 15.10 19.83

Lippia alba Colic Mint -339.10 35.10 -1.60

Mangifera indica Julie Mango -306.41 -140.07 72.60

Melicoccus bijugatus Guinep 5.88 3.31 -5.31

Moghana strobilifera Wild Hops -18.15 -9.56 -4.28

Momordica charantia Wild cerasee -5.30 37.56 2.68

Morinda citrifolia Noni (Hog Apple) 36.28 2.13 -24.83

Moringa oleifera Moringa -7.93 0.00 -1.75

Mucuna pruriens Cow Itch -14.88 3.87 -24.65

Musa sapientum Banana Lacatan 32.13 51.94 44.76

Nerium oleander Oleander 25.14 24.17 -61.69

Nicotiana tabacum

Tobacco

(Donkey Rope) -9.20 7.99 -37.32

Ocimum micranthum Wild Barsley 25.89 -15.94 -80.71

Parthenium hysterophorus Dog-flea Weed -38.95 -2.34 5.78

Pedilanthus spp. Monkey Fiddle -25.12 9.13 -55.50

Persea americana Avocardo -53.13 24.61 -6.40

Petiveria alliacea

Anamu (Guinea

Hen Weed) -60.41 1.69 39.15

Phyllanthus uvinaria Chamber Bitter -36.94 12.07 -34.52

Pimenta dioica All Spice (Pimento) -70.41 -55.00 -221.28

Piper amalago Black Jointer -10.48 0.25 3.12

Pithecellobium unguis-cati Privet -3.26 25.91 -9.32

Plectranthus amboinicus French Thyme -62.85 -78.26 -68.88

Plectranthus blumei Joseph's Coat -6.66 -5.67 -17.21

Psidium guajava Guava -38.20 -5.41 8.90

Punica granatum Pomegranate -76.33 -15.65 29.91

Rhizophora mangle Red Mangrove -1.17 -3.51 9.25

Ricinus communis Castor Oil 24.65 4.89 -10.54

Rivina humilis Dog Blood -4.51 32.69 3.13

Rytidophyllum tomentosum Search-me-heart -16.25 -1.42 26.02


Table 1. Continued

Sansevieria spp.

Mother-In-Law

Tongue (Yellow) 9.27 12.15 -56.09

Sansevieria spp.

Mother-In-Law

Tongue (Green) -34.06 23.39 -13.53

Satureja viminea Peppermint -60.59 -12.61 -19.15

Solanum torvum Susumber -18.84 9.65 5.94

Spigelia anthelmia Worm Grass -15.57 37.40 36.03

Syzygium cumini Ribena 16.02 35.00 24.35

Tecoma stans 59.23 61.50 57.14

Terminalia catappa Almond 6.34 7.03 6.64

Thymus vulgaris Thyme -23.04 -32.37 -26.48

Ziziphus mauritinia Coolie Plum -75.96 -22.97 8.53

*Results in bold denote significant anti-denaturation/anti-inflammatory

activity (>20%)

3.2. Significant anti-denaturation activity of the hexane extract from

Musa sapientum

Table 2 and Table 3 show the results from the BSA assay anti-

denaturation studies on the hexane, ethyl acetate and methanol extracts of

Musa sapientum. From Figure 1, it was determined that the active

compounds were found in the hexane extracts as its % anti-denaturation was

Table 2. Results of the BSA assay to screen hexane and ethyl acetate extracts of

Musa sapientum at pH 6.36.

Type of

Extract Conc. (µg/mL) % Anti-denaturation

Hexane

(non-polar)

0.25 33.9

0.5 38.0

1.0 29.2

Ethyl Acetate

(medium polar)

0.25 8.04

0.5 4.91

1.0 4.48

Control

(pure methanol)

0.25

0.5

1.0

*Results in bold denote significant anti-denaturation/anti-inflammatory

activity (>20%)


Table 3. Results of the BSA assay to screen methanol extracts of Musa sapientum at

pH 6.3.

Type of

Extract

Conc.

(µg/mL) % Anti-denaturation

Methanol

(polar)

0.25 2.86

0.5 12.1

1.0 -5.42

Control

(pure methanol)

0.25

0.5

1.0

Figure 1. The % anti-denaturation activity of the three different extracts of the banana

leaves and stems are shown on the graph above. The red line at 20% indicates

significant results. Only the hexane extract shows significant anti-denaturation activity.

not only well above that of the other 2 extracts, but was also significantly

greater than 20%. The highest anti-denaturation activity was observed at a

concentration of 0.5µg/mL (38.0%), the concentration chosen to test the

hexane column fractions.

3.3. Significant anti-denaturation activity of six hexane fractions of

Musa sapientum

After separation of compounds by gravity column chromatography and

TLC, the anti-denaturation activities of the 16 column fractions are

shown in Table 4. From the 16 fractions, only six fractions were found to have


Table 4. Results of the in vitro anti-denaturation studies of the 16 samples isolated

from the hexane extract.

Column

Fraction# % Anti-denaturation

2 37.75

3-4 42.5

5-7 44.3

8-9 27.2

10 37.0

11 33.7

12-16 -5.71

17-19 -1.17

20-21 12.7

22-26 -8.56

27-28 -7.39

29-30 -0.519

31-36 -8.30

37-46 -12.3

47-48 -28.45

49-50 -14.2

*Results in bold denote significant anti-

denaturation/anti-inflammatory activity (>20%)

Figure 2. The % anti-denaturation activity of the sixteen fractions of the banana

leaves and stems are shown on the graph above. The red line at 20% indicates

significant results. Only the first six fractions show significant anti-denaturation

activity. These six fractions were noted to be the most non-polar.


significant anti-denaturation activity. Figure 2 shows that fraction #5-7 had

the highest % anti-denaturation of 44.3%, followed by fraction #3-4

(42.5%), fraction #2 (37.75%), fraction #10 (37.0%), #11 (33.7%) and

fraction #8-9 (27.2%). The remaining fractions showed no significant

activity, with 9 of them showing negative activity.

4. Discussion

The preliminary screening of the 82 Jamaican plants with the BSA assay

has indicated that three of the plants show very significant levels of

protection against heat denaturation of the protein. As denaturation is

implicated in the inflammatory response, they have also been considered to

have anti-inflammatory properties9. Vetal et al. (2013), Joshi et al. (2010)

and Mitul and Handral (2013) have confirmed the anti-inflammatory

properties of Cinnamomum zeylanicum17–19

. These researchers have also

attributed anti-diabetic, anti-oxidant, anti-microbial, anti-arthritic and anti-

myotoxic properties to this plant. Other researchers such as Prasanna et al.

(2013) and Kameshwaran et al. (2012) have also reported on the

anti-inflammatory properties of Tecoma stans20, 21

. To date however, little

studies have been conducted on the anti-inflammatory properties of

Musa sapientum.

The traditional role of the leaves of Musa sapientum in healing burn

wounds has led to an investigation into the potential pharmaceutical

properties of this plant, specifically its anti-inflammatory properties.

Inflammation is a protective pathological response to tissue injuries,

infection and destruction12

. It is however, also considered by many to be the

underlying cause of almost all disease conditions11

. Researchers believe that

many of the anti-inflammatory drugs exert their effect through scavenging

for oxidants, decreasing the formation of reactive oxidant species (ROS) and

ultimately protecting the body from oxidative stress. As such, isolation of a

natural anti-inflammatory product from banana leaves may not only be

important in the healing of burn wounds but could have implications in the

treatment of inflammation and degenerative diseases such as cancer, diabetes

and atherosclerosis.

The results of this study show that the active compounds that has the

anti-denaturation and anti-inflammatory properties are non-polar, having

been isolated from the hexane extracts of the banana leaves and stem.

Fractions #2-11 were the only fractions to show significant %anti-

denaturation (#2: 37.75%, #3-4: 42.5%, #5-7: 44.3%, #8-9: 27.2%, #10:

37.0%, #11: 33.7%). The solvent system, used to collect these fractions

ranged from 100% n-hexane to 95% n-hexane (5% ethyl acetate). This


suggests that the non-polar compounds are responsible for binding to the

protein albumin to protect it from heat denaturation. Preliminary NMR

studies confirmed this non-polarity as one of the pure isolated compounds

(fraction #2) was identified as a triglyceride (results not yet published). In

previous studies, other researchers had attributed anti-inflammatory

properties to triglycerides22, 23

. Williams et al. provided insight to the

protection of the protein by identifying the aromatic tyrosine rich region and

the aliphatic threonine and lysine rich residues as binding sites on the BSA

for anti-inflammatory agents15

. Considering the non-polar nature of the

active compounds as well as the BSA binding sites, it is probable that the

compound exerts its protection in this way, however further analysis must be

conducted for confirmation.

The BSA assay is increasingly being used in the initial stages of drug

discovery all around the world9,11–15

. It has been proven by many researchers to

be just as effective as screening in animals and is considered to be a more

humane approach as it avoids the use of live animals for as long as possible.

Additionally, Williams et al. has shown that in comparison to using live animals,

the use of BSA is far more economical16

. A cautionary note however, is that in

using this assay it essential to conduct the experiment at the pathological pH of

6.2-6.5. In comparing Table 2 and Table 5, it can be observed that a pH of 5.5 is

well beyond the correct pH to provide useful results.

The isolation of these active compounds from the hexane extract of the

leaves and stem of the banana plant, as well the significant % anti-

denaturation shown by the fractions point to great anti-inflammatory

potential of these natural product compounds.

Table 5. Results of the BSA assay to screen hexane and ethyl acetate extracts of

Musa sapientum at pH 5.50.

Type of Extract Conc. (µg/mL) % Anti-denaturation

Hexane

(non-polar)

0.25 5.76

0.5 6.86

1.0 6.19

Ethyl Acetate

(medium polar)

0.25 2.30

0.5 -2.75

1.0 -0.44

Control

(pure methanol)

0.25

0.5

1.0


In order to confirm the results of this study and to further support the use

of the BSA assay in the initial stages of drug discovery, animal studies

should be conducted. Positive results from treating rat paw edema with the

anti-inflammatory compounds isolated via the BSA assay will confirm the

value of the assay, proving it not only to be just as effective, but also a more

economical and animal-friendly alternative.

5. Conclusion

In screening the large group of 82 Jamaican plants we were able to

identify plants that possessed anti-denaturation properties. Selecting

Musa sapientum, which registered one of the highest % anti-denaturation,

we isolated the active compounds responsible for this protein denaturation

protection. It is evident from the use of the BSA assay that the hexane

extract has anti-denaturation and anti-inflammatory properties. With

separation and repeated testing it is believed that the active compounds are

non-polar in nature. Further studies are needed to identify and elucidate the

structures of the active compounds to better understand how it binds to the

BSA. Once the compounds have been identified they may have great

pharmaceutical value due to their natural product origin as well as the range

of diseases with inflammation and oxidative stress as their underlying cause.

6. Conflict of interest

None.

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