MODULATION OF ACTIVATED MACROPHAGE IMMUNE RESPONSE BY Carica papaya
LEAF EXTRACT
ZATIL AQMA BINTI MOHD NOOR
Thesis submitted in fulfillment ofthe requirements forthe degreeof
Master ofSciences
September 2016
ACKNOWLEDGEMENT
Inthe nameofAllah, The MostGraciousandThe Most Merciful Prayersand peace be uponHis ProphetMuhammad S.A.W
Firstofall, I would like to express my deepestappreciation to my supervisor, Assoc. Prof. Dr. Yahya Mat Arip and co—supervisor, Prof. Amirul Al-Ashraf Abdullah for their patiences, guidences, advices, encouragements and supports through out this study. Their kindness in sharing their immense knowledges and experiencesare very muchappreciated.
My sincere appreciation also goes to all my fellow laboratory colleagues from Lab 218 for theirmoral supportsand invaluable advicesduring research period.
It has been a pleasure knowing and doing research with all of you. Furthermore, I would like to expressmy gratitude to all staffof School ofBiological Sciences and Institute PostgraduateStudies, USM, who involved directly or indirectly in helping me through this periodofstudy.
Last but notleast, I owe mydeepest gratitude to my lovelyparents and family fortheirsupport and endless love throughout my life. A special thanks to my beloved husband, Muhammad Azfar Afifi Bin Azhar and my lovely daughter, Aysha
Huwaida Binti Muhammad AzfarAfifi for being with me during my difficult time. I
feel greatlyblessedforhavingsuch wonderfulpeople around me.
This study was funding by research grant, USM and Ministry of Higher Education.
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TABLE OF CONTENTS
Acknowledgement... ii
Table ofContents ... iii
List ofTables ... vi
List ofFigures... vii
List ofAbbreviations ... viii
ListofSymbols ... xi
Abstrak... xii
Abstract ... xiv
CHAPTER 1: INTRODUCTION... 1
CHAPTER2: LITERATURE REVIEW ... 3
2.1 Immune modulator ... 3
2.2 Sourcesofimmunemodulators ... 3
2.3 Plantsas sourcesofimmune modulators ... 4
2.4 Caricapapaya ... 6
2.4.1 Medicinaland pharmacological propertiesofC. papaya 8 2.4.2 Phytochemical constituentsin C.papayaleaf... 11
2.4.2(a) Glycoside ... 1 1 2.4.2(b) Alkaloid ... 12
2.4.2(c) Saponin ... 12
2.4.2(d) Flavonoids ... 12
242(6) Anthraquinone... 13 242(1) Phenolic ... 13
2.4.2(g) Tannins ... 13
iii
2.5 Dengue infection ...
2.5.1 Inflammatory responseduringdengue Virus infection
2.5.1.1 Activationofmacrophage ...
2.5.2 Plasma leakagein severe dengue disease ...
2.5.3 Cytokine Tsunami in DHF patients ...
CHAPTER3: MATERIALSAND METHODS ...
3.1 3.2
3.3
3.4 3.5
3.6
3.7
3.8
3.9
Materials ...
Plant sample ...
3.2.1 Preparation ofextract ...
Cell line andcultureconditions ...
3.3.1 Viable cell counting ...
Baselinefor TNF-aproduction inthe activation ofU937 monocyte...
The effectof C. papayacrude aqueousleafextract onTNF-a
secretion duringU937 monocyte activation...
The effect of C. papayacrude aqueousleafextract onTNF-a
secretion by activated macrophage...
Enzyme-linked immunosorbent assay(ELISA) ...
3.7.1 Preparationofstandard curve for ELISA ...
3.7.2 Assaypreparation ...
Geneexpression studies ...
3.8.1 Total RNA isolation ...
3.8.2 Quantificationoftotal RNA ...
3.8.3 DNase treatmentofRNAsamples...
3.8.4 Specific primer design ...
3.8.5 Semi-quantitative real-time RT—PCRanalysis ...
Statisticalanalysis ...
iv
14
15
18 19 21
25 25 26 26 26 27 27
28
28 29 29 29
31
31
32 32 33 33 35
CHAPTER4: RESULTS ...
4.1 Carica papayacrudeaqueousleafextractpreparation ...
4.2 BaselineforTNF-aproduction in the activation ofU937 monocyte..
4.3 Standard curvefor ELISAtest ...
4.4 The effect of C.papaya crudeaqueousleafextractonTNF-a
secretionduring U937 monocyte activation ...
4.5 TNF-ageneexpression study ...
4.6 The effectof C. papayacrudeaqueousleafextractonTNF-a
secretion by activated macrophage...
CHAPTER5: DISCUSSION ...
CHAPTER6: CONCLUSION ...
REFERENCES ...
APPENDICES...
AppendixA ...
Appendix B ...
36 36 36 39
41
43 48
51
57 58
66 66 67
Table 2.1
Table 2.2
Table 3.1
Table 3.2
Table3.3
LISTOF TABLES
Pharmacological and medicinal properties of C. papaya
plant
Cytokine profile in denguefever anddengue hemorhagic feverpatients
Materialsused andtheirsuppliers
Dilutionsofrecombinant human TNF-a
Nucleotidesequencesfor the forwardandreverse specific primersforhumanTNF-aand B-actin genes
vi
Page
22
25
3O
34
l’igurc2.1
Figure2.2 Figure4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Figure 4.7
Figure 4.8
1’igurcA
Figurc B
LIST OF FIGURES
Caricapapaya. A grown papaya plant (A) and papaya plant leaveswith palmatcly 10de structure(B).
Mechanismofinflammatory response.
Inverted microscopeimages (20X magnification)01‘U937 cells. (A) UndifferentiatedU937 cells. (B)I)iffcrcntiatcd U937cells induced by 10 ng/mLofl’MA.
Baseline()f'I‘Nl‘luproduction in the activation ofmonocytc
U937.
Standardcurve for ELISAtest.
TNF—aconcentrationand percentageof'I‘NF-(1 reduction in
cell culturesupernatantduringactivation ofU937monocytc.
TNF—a concentrationand percentage ofTNF—arcductionin cell culturesupernatantduringactivation ofU937 monocytc.
Total RNA isolated from differentiated U937 monocytc.
Expression()f'l‘Nl“-(1 gcnc normalized against B-actin duringactivationofU937monocytc.
TN!“-(1concentrationand percentageOfTNF-a reductionin cell culturesupernatant containingactivated macrophage.
AmplificationofTNF-aand B-actin gcnc.
Melting peaks of rcal—time RT-PCR obtained from differentiated U937 monocytcs treated with (?.papaya crude aqueousleaf extract.
vii
Page
16
37
38
40
42
45
46
47
50
66
()7
A. salivum ATCC BLAST
C
C. asiatica
C. papaya
CT
D-PBS DENV DF DHS DMSO DNA
DNase DSS E
ELISA FBS
H.perforatum hCF
HMEC-l
HUVECS IFN-y
LISTOF ABBREVIATIONS
Alliumsativum
American Type Culture Collection Basic LocalAlignmentSearchTool Coreprotein
Centellaasiatica Carica papaya Threshold cycle
Dulbecco’s Phosphate BufferedSaline Denguevirus
Dengue Fever
Dengue hemorrhagic fever Dimethyl Sulfoxide
Deoxyribonucleicacid Deoxyribonuclease Dengue shock syndrome Envelope protein
Enzyme-Linked ImmunosorbentAssay FetalBovineSerum
Hypericum perjforatum Human Cytotoxic Factor
Human Dermal Microvascular Endothelial Cells HumanUmbilical VeinEndothelial Cells
Interferon-gamma
viii
IgE IL-10
IL—12
IL-12p70
IL—1 3
11—18
IL-lra IL-lB
IL-2 IL-4 IL-6 IL-8 LAB
LcS M
M citrifolia
MCP-l
mRNA
NF—KB
NK
NO NS
OD PMA RNA
ImmunoglobulinE
Interleukin-10
Interleukin-12
Interleukin-12p70 Interleukin-13 Interleukin-18
Interleukin—l receptor antagonist Interleukin-lbeta
Interleukin-2 Interleukin-4 Interleukin—6
Interleukin-8
LacticAcidBacteria
Lactobacillus caseistrainShirota Membrane-associatedprotein Morindacitrifolia
MonocyteChemoattractantProtein-l MessengerRibonucleicAcid
Nuclearfactor—kappaB cells Natural killercell
NitricOxide
Nonstructural protein Optical Density
Phorbol 12-Myristate 13-Acetate Ribonucleicacid
ix
RNase
RPMI-164O rRNA RT-PCR SAPKS TBE TEER TGF-B
Th-l
Th-2 TNF-a TNFR
B—actin
Ribonuclease
RoswellPark Memorial Institute-1640 RibosomalRibonucleicAcid
Reverse Transcription Polymerase Chain Reaction Stress-Activated Protein Kinases
Tris—Borate-EDTA
TransendothelialElectrical Resistance Transforming Growth Factor—beta
Type-l helper T cell Type-Z helperT cells
TumorNecrosis Factor-alpha Tumor NecrosisFactor Receptor Beta-actin
TM
LIST OFSYMBOLS
Registered trademark Trademark
Alpha
Beta Gamma
xi
MODULASI TINDAKBALAS IMUN MAKROFAJ YANG AKTIF OLEH EKSTRAKDAUN Carica papaya
ABSTRAK
Carica papaya adalah tumbuhan yang terkenal dengan ciri-ciri perubatannya dalam perubatan tradisional. Penggunaannya yang terdahulu telah mendedahkan bahawa ekstrak akueus daun C. papaya mentah mempamerkankesan positif pada sel-sel imun; meningkatkan bilangan platelet, sel darah putih, dan neutrofil pada pesakit-pesakitdenggi. Walau bagaimanapun, kesanefektifoleh ekstrakakueusdaun
C. papaya mentah belum diteliti untuk memahami kemungkinan aktiviti
pemodulatan imun. Dalam kajian ini, aktiviti pemodulatan imun olehekstrak akueus daun C. papaya mentah telah diuji pada makrofaj yang aktif. Aktiviti cytokine, khususnya tumor nekrosis faktor alfa (TNF-a) digunakan sebagai petunjuk untuk melihat kesan pemodulatan imun pada makrofaj yang aktif. Kehadiran ekstrak menunjukkan berlaku penurunan dalam jumlah TNF-a yang dikesan dalam supematan sel kultur. Penurunan jumlah TNF-a oleh makrofaj yang aktif dikesan
oleh ELISA, mencadangkan berlaku pengurangan dalam pengeluaran TNF—a.
Sebaliknyakeputusan dari real-time RT-PCR tidak menunjukkan perencatan dalam pengekspresan mRNA TNF-a tetapi lebih ketara kepada pengaktifan gen TNF-a.
Oleh itu, spekulasi boleh dibuat bahawa ekstraktidak menghalangpengeluaran dan seterusnya rembesanTNF—a. Sebaliknya ekstrakmungkinmenggangu fungsi TNF-a.
Penemuan ini mencadangkan kewujudan sebatian bio-aktif dalamekstrak akueus daun C. papaya mentah yang boleh bertindak sebagai pemodulatan imun.
Walaubagaimanapun, kajian in tidak dapat mengesahkan mekanisma yang terlibat
xii
dalam menggangu fungsi TNF-a. Dengan itu, boleh dicadangkan bahawa sebatian
bio-aktif dalam ekstrak akueus daun C. papaya mentah akan berinteraksi secara langsung denganTNF-a melalui perlekatan dan/ atau penambahan yang menghalang fungsinya pada sel-sel imun effector. Oleh itu, kajian ini telah mencadangkanbukti kemungkinankonsep mengenai penglibatan ekstrak akueus daun C. papayamentah dalam penguranganTNF-a mungkin melalui interaksi secaralangsung denganTNF-
ayangmenghalang fungsinyapadasel—sel imuneffector.
xiii
MODULATIONOFACTIVATEDMACROPHAGEIMMUNE RESPONSE BY Carica papayaLEAFEXTRACT
ABSTRACT
Carica papaya is a plant known for its medicinal attributes in traditional remedies. Previoususage revealed that C. papaya crude aqueous leafextract exhibits positive effects on immune cells; increased platelet, white blood cell and neutrophil counts in dengue patients. However,the helpful effects ofC. papaya crude aqueous leaf extract have not been scrutinized for possible understanding of its immune modulator activity. In this study, immune modulator activity of C. papaya crude
aqueous leaf extract was tested on activated macrophage. Cytokines activities, specifically tumor necrosis factor alpha (TNF-a) was used as the indicator for immune modulatoreffects on the activated macrophage. The presence ofthe extract demonstrated decreasingamountofTNF-a detectablein cell culture supernatant. The decreased in TNF-a amount by the activated macrophage was detected by ELISA, suggesting for a reduction in TNF-a production. On the contrary, results from real- time RT-PCR did not show suppression of mRNA TNF-a expression but rather a noticeable activationofthe TNF-a gene. Thus, it could be speculated that the extract did not inhibit the production of TNF-(x and subsequently its secretion. Rather, the extract could possibly interfere with the fiinctions 0f TNF-(x. These findings had
suggestedthe existence ofbio-active c0mpound(s) in C. papaya crude aqueous leaf extractthat could act as immune modulator. Nevertheless, this study could not ratify the mechanismsinvolved in interfering with the TNF—u functions. Thus, it could be suggested that the bio-active c0mpound(s) in C. papaya crude aqueous leafextract
xiv
would interacts directly with TNF-a by binding and/or intercalating that hinder its functions on immune effector. Consequently, this study has suggested a proof of
possiblemechanism conceptofthe C. papayacrude aqueous leafextractinvolved in decreasingTNF-amightbe via direct interaction with TNF-athat hinder its function on immune effectorcells.
XV
CHAPTER1 INTRODUCTION
Dengue is considered an important arboviral disease transmitted by Aedes mosquitoes, typically Aedes aegypti and Aedes albopictus(Wan et al., 2013). In recent decades, the occurrence of dengue infections worldwide has intensely increased. Bhatt et al. (2013) has estimated 390 million dengue cases to occur annually, whereby 96 million cases were evidenced clinically. In Malaysia, there were 3500 denguecaseswith 10 deathsreportedfrom 10 to 16 January 2016 (WHO, 2016). Dengue infection is similar with other viral infections, whereby the pathogenesis of the disease is due to two factors either viral replication itself or immune response against viral infection. Thus, understandingthe immune response againstViral infection mightbe an alternativeto fightdengue fever.
Currently, there are no specific antiviral drugs available to treat dengue problems and dengue vaccines are still in the developmental stages (Gowda el al., 2015). Hence, different approaches should be evaluated in order to combat dengue disease. Immune modulators could be appliedas one ofthe approach in treatmentof
dengue infection since it has the ability to influence immuneresponse in combating the disease. Plants are the natural sources of immune modulators. There are many immune modulator extracted from plants that give positive effects in dealing with a
wide range ofdiseases(Mukherjee et al., 2014).
Carica papayais a sourceofnutrientsrich andaccessiblethroughoutthe year (Aravind et al., 2013). Papaya fruits are generally consumed fresh and processed forms (juices, jams and dry fruits) (Subenthiran et al., 2013). The medicinal properties ofC. papayahave been used for centuries in treating diseases (Sarala and
Paknikar, 2014). This would include the use of C. papaya in traditional remedy for treatmentofdengue fever.However,thebio-active compound(s)from C. papayaand their mechanism(s) involved in treating dengue diseases are still unknown and not validated by scientificexperiments.
Forthe past fewyears, manyresearchers have tried to apply C. papaya crude aqueous leaf extract in treatment ofthrombocytopenia related with dengue (Ahmad
et al., 2011; Gowda et al., 2015; Subenthiran et al., 2013). Administration of C.
papaya crude aqueous leaf extract in denguepatients had increased the neutrophils, white blood cells and platelets count. Therefore,the bio—active compound(s) present in C. papaya crude aqueous leaf extract might influence the immune modulator activities ofthe immunesystem to reduce the severityofdengue manifestations.
Thus, this work would attempt to validate scientifically the immune modulator activities present in C. papaya crude aqueous leaf extract. The target would be on immune modulator of pro-inflammatory cytokine, Tumor necrosis factor-a (TNF-a), which is involved in the pathogenesis of dengue hemorrhagic fever. The specific objectives ofthis projectare:
1. To determine the effects of C. papaya crude aqueous leafextract on TNF-a
production during U937 monocyteactivation.
2. To evaluate the TNF-a gene expression in the activated macrophage after treatedwith C. papaya crudeaqueousleafextract.
3. To suggestpotential mechanismsofthe C. papayacrudeaqueous leafextract acting as immune modulatoron the activatedmacrophage.
CHAPTER 2
LITERATURE REVIEW
2.1 Immune modulator
Immune modulator is a substance that could regulate one or more components of the immune system including both innate and adaptive immune responses (Agarwal and Singh, 1999). Generally, the immune modulator activity is classified into two types based on their effects, which are immune suppressionand immune stimulation(Mukherjeeet al., 2014). The concept ofimmune modulationis about nonspecific activation of the function of immune cells, for instance macrophages, natural killer cells, complement,lymphocytes, and granulocytes,and also aboutthe productionofeffectormoleculesreleasedbythe activated cells. These nonspecific activations could protect the host against various pathogens including viruses,bacteriaandfungi (Daset al., 2014).
2.2 Sourcesofimmune modulators
Previous works have identified many natural immune modulators from various organisms such as bacteria, fungi, and plants. Matsuzaki and Chin (2000) have found that probiotic bacteria able to modulate immune response. Oral consumption of Lactobacillus casei strain Shirota (LcS) is capable of triggering splenic Natural Killer (NK) cells activity in mice, whereas consumption of killed
LCS was adept to elicit the secretion of Type 1 helper T (Th—l) cells which could suppressedthe productionofImmunoglobulinE (IgE) antibodies against Ovalbumin in mice. Besides that, dietry supplementation with lactic acid bacteria (LAB) also could boost healthcondition(Gill, 1998). The LAB has been proven experimentally
regarding their immune response effects that could increase both specific (e.g.
lymphocyte propagation, delayed-type hyphersensitivity, antibody and cytokine production)andnon-specific(e.g. NK cells activity andphagocytefunction)immune responses.
Furthermore, fungal metabolites also can be sources for immune modulator such as polysaccharide isolated from fruiting bodies of Ganoderma lucidum. The polysachharide possess immune modulator effect by increasing the secretion of
Interleukin-l beta (IL-lB), IL-6 and TNF-a in macrophageculture and also boosted the productionofInterferon—y (IFN-y) by T lymphocyte(Wang et al., 1997).Another example of immune modulatorextracted from fungal metabolites are water extracts
of the mycelial culture and fruiting bodies ofAgaricus blazei Murill. The extracts
were able to trigger TNF-a production by macrophage derived from the bone marrowofrat (Sorimachiet al., 2001).
2.3 Plants as sources ofimmune modulators
In recent times, natural immune modulators from plants are being studied extensively due to the awareness on the modulation of immune system in health maintenance and disease prevention (Shukla et al., 2014). Thus, plants have been investigated for their bio-active compoundsexhibitingimmune modulatorproperties (Mukherjee et al., 2014). The active constituentsderived from plants that could act
as immune modulator are proteins, polysaccharides, lectins, saponins, alkaloids, and
flavonoids(Dasetal., 2014).
One of the plants that exhibit anti—inflammatory and immune modulator properties isAloe vera. This medicinal plant has been extensively used for treatment
of wounds and burns. Duansak et al. (2003) have proven the immune modulator
activity ofAloe vera in treatment ofwounds and burnsin rats. Rats with Aloe vera—
treated burns showed reduction in the amount of leukocyte adhesion, as well as, TNF-a and IL-6 (pro-inflammatory cytokines) when compared with the control group.
In addition, Allium sativum (Garlic) is a herbal spice that widely used for cookingand medicinal purposes. Allium sativum has been claimed for its favorable properties in recuperation and improving health conditions (Arreola et al., 2015).
This herbal spice could act as an immune modulator by inducing few types of
immunecells suchas macrophage,NK cells, lymphocytes, eosinophils,and dendritic cells Via several mechanisms including alteration of cytokine, immunoglobulin production,macrophageactivation,andphagocytosis.
Furthermore, other plant that has medicinal properties is Centella asiatica (Gotu Kola)whieh commonlyused in the Orient and is becoming popularin the West (Gohilet al., 2010)as remedy for injury, skindisordertreatment,sedatives, aswell as
spasmolytics for anti-anxiety and anti-stress. Centella asiatica also possesses immune modulator properties by increasing the phagocytic index and white blood cell count (Mukherjeeet al., 2014).
With antiviral, anti—inflammatory, and antibacterial properties, Hypericum perforatum (St John’s wort) is a flowering plant used in folk medicine as anti depressants, jaundice, bruises, and many other illnesses. Furthermore, the extract from H. perforatum displayed immune modulatoractivity in human peripheral blood mononuclear cells by cytokine-induced tryptophan degradation and also in productionoftheactivationmarkerneopterin (Mukherjee et al., 2014).
Additionally, Morinda citrifolia (Noni) has been used for centuries as a traditional remedydue to its wide range ofmedicinal properties, including antiviral, antifungal, antibacterial, antitumor, anti-helmintic, anti-inflammatory,and immune- enhancing effects. Thejuice fromM citrifolia fruitscontain substanceswithimmune modulator properties, capable ofstimulatingthe release ofcytokinessuch as TNF-a, IL-IB, IL-10, IL—12p70, IFN-y, and nitric oxide (NO) from murine effectors cells (HirazumiandFurusawa, 1999).
There are many other plants that could be sources of immune modulator.
However, these few examples of medicinal plants with their therapeutic properties wouldbe sufficient forthis literature review.
2.4 Carica papaya
Caricapapaya (Papaya) is the only species from genus Carica and belongs to the family Caricaceae(Jain and Priyadarshan, 2009). The plant originated from the tropics ofthe Americas. It is now grown in tropical and subtropical areas worldwide
due to its speedy growth, easy cultivation and adaptability to various soils and climate. Papaya plant is a single stem herbaceous perennial tree, having 5 to 10 m (16-33 feet) ofheight. The stem is topped by large spirally arranged leaves and the leavesare large, 50-70 cm in diameterwith palmately lobed structure(Aravindetal., 2013)(Fig 2.1). Papaya plant has complex means of reproduction which are hermaphroditic (bisexual) or dioecious(separate male and female trees) (Vijay et al., 2014)
In Malaysia, there are a few cultivarsofpapaya including Eksotika, Eksotika II and Sekaki (Ming and Moore, 2014). Eksotika is the most common cultivar breedingfor exportand local market. The fruitsare smallto mediumsizes with
Figure 2.1: Caricapapaya. A grown papayaplant (A) and papayaplant leaves with palmatelylobed structure (B).
weight around 0.6-0.9 kg, whereas Eksotika II is from the hybridisation of Line 19
and 20 producing higher yield offruits than Eksotika weighing 0.6-1.0 kg. Sekaki, also known as “Hong Kong”, is the second popular cultivar in Malaysia with the medium fruitsizes weighingaround 1.0-2.5 kg.
Carica papaya is commonly known for its food and nutritional values. It contains high antioxidantcompounds(vitamin A, C, and E), vitamin B (pantothenic acid and folate), minerals (magnesium, potassium, calcium), and fiber (Aravind et al., 2013). Unripe papayafruit contains enzyme papain that has several applications in the food industry such as meat tenderizer, and chewing gum, as well as in the pharmaceutical industry for treatment of cancer, digestive medicine, vascular disease, and arthritis (Thomas etal., 2009).
2.4.1 Medicinal and pharmacologicalpropertiesofC.papaya
The medicinal properties of C. papaya fruit and other parts of the plant are also well known in traditional medicine (Table 2.1). Several studies have been organized to classify the medicinal potential of various parts from C. papaya
including fruits, leaves, latex, seeds, shoots, and roots (Otsukiet al., 2010). The ripe and unripe C. papaya fruit exhibit antibacterial activity against Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, and Shigella
flexneri (Emeruwa, 1982). Scientific work done by Calzada et al. (2007) provedthat
the seed extract ofC. papaya showed anti trichomonal activity against Trichomonas vaginalis whichparasitizethe human vagina, prostate gland and urethra. The latex of
C. papaya showed antifungal effect through inhibition of the growth of Candida albicans during the exponential growth phase (Giordani et al., 1996). The fungi static effectis dueto the degradationofcell wall withreducedpolysaccharide
Table 2.1: PharmacologicalandmedicinalpropertiesofC. papaya plant (Krishnaet
aL,2008)
Parts Latex
Ripefruits
Unripefruits
Seeds
Seedjuice
Root
Leaves
Flowers Stem bark
Pharmacologicaland medicinalproperties
Anti-helminthic,relieves dyspepsia, cures diarrhoea,pain of
burns and topical use, bleeding haemorrhoids, stomachic, whooping cough.
Stomachic, digestive, carminative, diuretic, dysentery and chronic diarrhoea, expectorant, sedative and tonic, relieves obesity, bleeding piles, wounds of the urinary tract, ringworm and skindisease psoriasis.
Laxative, diuretic, dried fruit reduces enlarged spleen and liver, remove snakebite poison, abortifacient, anti- implantationand antibacterial activity.
Carminative, emmenagogue, vermifuge, abortifacient, counter irritant, as paste in treatment of ringworm and psoriasis, anti-fertilityagents in males.
Bleeding piles andenlargedliverand spleen.
Abortifacient, diuretic, checking irregularbleeding from the uterus,pilesand anti—fungalactivity.
Jaundice (fine paste), urinary complaints and gonorrhoea (infusion), dressing wounds (fresh leaves), antibacterial activity, vermifuge, in colic, fever, beriberi, abortion (infusion),asthma(smoke).
Jaundice, emmenagogue, febrifugeand pectoral properties.
Jaundice, anti—haemolytic activity, sore teeth (inner bark), anti-fungal activity.
