15

CHAPTER 2: Part B

A Review on Dillenia indica L. plant

2.B.1 Introduction

2.B.2 Importance of Dilleniaceae family

2.B.3 Taxonomical classification

2.B.4 Botanical description

2.B.5 Geographical distribution

2.B.6 Knowledge of Dillenia indica L. in Ancient Relics

2.B.7 Ethnomedicinal uses of Dillenia indica L.

2.B.8 Current status of studies on Dillenia indica L.

2.B.9 Previously isolated Phytochemicals

2.B.10 Scope of the Present Investigation

2.B.11 Conclusion

16

CHAPTER 2: Part B

A Review on Dillenia indica L. plant 2.B.1 Introduction

North East India including Assam is very rich in plant biodiversity as well as in

ethnic diversity and has a great traditional knowledge base in plant resources. It is one

of the mega biodiversity hotspots of the world, particularly excellent reservoir of unique,

endangered, endemic and high valued medicinal plants with wide diversity. North East

India represents 53% (8,000 species) of total flora of the Indian subcontinent. The

region is estimated to have more than 2000 medicinal plant species [3,4]. Considering

the richness of plant diversity of the North East region, it is imperative to study, explore

and identify the medicinal plants of the region in search of novel bioactive molecule and

to find new lead compound for developing phytopharmaceuticals.

Dillenia indica Linnaeus of family Dilleniaceae is an important medicinal plant

found in Assam, North East India [5-8]. The plant grows in abundance in Assam but

due to lack of technical knowledge most of the fruits of this plant are wasted. In Assam,

traditionally the unripe fruits are used to make curries because of its sour taste and ripe

fruits are used for making pickles [5].

Recently, scientists have given attention to this plant for its various biological

activities including anti-cancer and anti-diabetic properties [14-26]. The leaf, bark, and

fruit of the plant are used in the indigenous system of medicine. It relieves abdominal

pain and regulates the heat in the body [8]. The fruit juice is mixed with sugar and water

and used as a cooling beverage in the treatment of fever [9-12]. It also tones up the

nervous system and removes fatigue [8]. The fruit juice is used as a cardio tonic [12].

The leaves and barks are used as a laxative and astringent. Bruised bark is applied as a

cataplasm for patients with arthritis. The alcoholic extract of the D.indica L. leaves is

reported to possess central nervous system (CNS) depressant activity. The extract of the

D.indica L. is known as a cooling beverage in fever, expectorant in cough mixture, tonic,

laxative and astringent [13].

The present review encompasses an up-to-date published literature from various

sources on different aspects of D.indica L. and will open the different aspects for future

research. However, relevant earlier works from D.indica L. and other plant sources have

been curiously cited for critical evaluation and meaningful conclusion of our results.

17

2.B.2 Importance of Dilleniaceae family

Dilleniaceae is a family of flowering plants (angiosperms) comprising of about

129 species (Table No. 2B.2) in 14 genera (Table No. 2B.1) found in the tropics and

sub-tropics plus entire Australia [67,68]. This family has been universally recognized by

taxonomists. It is known to gardeners for the genus Hibbertia, which contains many

commercially valuable garden species. Most of the members in it are woody plants,

such as Dillenia but herbaceous species such as Pachynema are also present in

Dilleniaceae. The leaves of the plants in the family are wide and well-developed,

however, in Pachynema and certain species of Hibbertia they are heavily modified. The

flowers are mainly showy and colorful with visible reproductive components. Buzz

pollination is common in the group. Fruits of many species of this family were reported

as edible, the commonest one is the Elephant apple (Dillenia indica L.) [69,70].

Secondary metabolites like alkaloids are usually absent in the family

Dilleniaceae, or if present then it is not benzyl isoquinoline. Proanthocyanidins like

cyanidin and delphinidin and flavonols like quercetin, kaempferol and myricetin are

well reported to be present while iridoids and saponins are absent [71].

The family is remarkable because of its variability of morphological

characteristics that now are much steadier in other angiospermic groups. Thus,

Dilleniaceae may be an ancient clade that expresses some phylogenetic relation

between the higher Eudicots and the rather more primitive groups [69].

Table 2B.1: Number of genera in the family Dilleniaceae

Sl. No. Genera Sl. No. Genera 1. Acrotrema 8. Hibbertia 2. Adrastaea 9. Neowormia 3. Curatella 10. Pachynema 4. Davilla 11. Pinzona 5. Didesmandra 12. Schumacheria 6. Dillenia 13. Soramia 7. Doliocarpus 14. Tetracera

18

Table 2B.2: Total species of Dillenia found Sl. No. species Sl. No. species Sl. No. species

1. alata 44. guillotii 87. papuana 2. albertisiana 45. hainanensis 88. papyraceae 3. albiflos 46. harmandii 89. parkinsonii 4. andamanica 47. heterosepala 90. parviflora 5. andreana 48. hirta 91. parvifolia 6. angusta 49. hookeri 92. pauciflora 7. apetala 50. humilis 93. pedunculata 8. artocarpifolia 51. indica 94. pentagyna 9. aurea 52. ingens 95. phillippinensis 10. auriculata 53. insignis 96. pilosa 11. baillonii 54. insularum 97. procumbens 12. beccariana 55. integra 98. ptempoda 13. biflora 56. kerrii 99. pulchella 14. blanchardii 57. longe-petiolata 100. pulcherrima 15. bolsteri 58. luzoniensis 101. quercifolia 16. borneensis 59. macdonaldi 102. racemosa 17. bracteata 60. macrocarpa 103. radicans 18. burbidgei 61. macrophylla 104. reifferscheidia 19. calothyrsa 62. madagascariensis 105. repanda 20. castaneaefolia 63. magnicapsula 106. reticulata 21. catmon 64. magnoliaefolia 107. retusa 22. cauliflora 65. mansoni 108. rhizophora 23. celebica 66. marsupialis 109. rubescens 24. crassisepala 67. mattadensis 110. salomonensis 25. crenata 68. megalantha 111. scabrella 26. crenatifolia 69. megalophylla 112. scandens 27. cyclopensis 70. meliosmaefolia 113. schlechteri 28. dentata 71. membranifolia 114. scortechinii 29. diantha 72. micrantha 115. secunda 30. elata 73. mindanaensis 116. serata 31. elliptica 74. minor 117. sibuyanensis 32. elmeri 75. misorensis 118. speciosa 33. elongata 76. monantha 119. suffruticosa 34. excelsa 77. montana 120. sumatrana 35. eximia 78. nalagi 121. talaudensis 36. fagifolia 79. neo-ebudica 122. terneriflora 37. ferruginea 80. nicobarica 123. tetragyna 38. fischeri 81. oblonga 124. thurifera 39. floribunda 82. obovata 125. tomentella 40. glabra 83. ochreata 126. triquetra 41. grandifolia 84. ornata 127. turbinata 42. grossulariaefolia 85. ovalifolia 128. turneraeflora 43. grossulariifolia 86. ovata 129. volubilis

19

2.B.3 Taxonomical classification

Dillenia indica L. belongs to the family Dilleniaceae. Its taxonomical

classification reported in literature [72] is as given below:

Kingdom: Plantae

Division: Magnoliophyta

Subdivision: Angiospermae

Class: Magnoliopsida

Subclass: Dilleniidae

Order: Dilleniales

Family: Dilleniaceae

Genus: Dillenia

Species: indica Linnaeus

2.B.4 Botanical description

The botanical description of D.indica L. [5,6] is given below:

Trunk: 30-80 feet in height and 6 feet in girth, with a dense rounded crown, straight but

not high.

Branches: Spreading, forming a round and shady head.

Leaves: Fascicled at the ends of the branches, oblong-lanceolate, acuminate, 20-30 by

about 10 cm, sharply serrate, the nerves close, running into the serratures, not forking at

the margins, upper surface and the nerves beneath more or less pubescent.

Petioles: 2.5-5 cm long, channeled and sheathing.

Flowers: Often exceeding 15 cm diameter, white, fragrant, appearing with the leaves,

usually solitary towards the end of each branchlet.

Pedicels: about 7.5 cm long, clavate, round and smooth.

Sepals: Orbicular, concave, thick and fleshy.

Petals: Oblong.

Stamens: Many in number, the inner larger and arching over the shorter outer ones.

Fruit: Large, 7.5-10 cm in diameter, hard outside and fleshy within.

Seed: Many in number, imbedded in glutinous pulp, compressed, with hairy margins.

Flowering and fruiting: May-August and September-February.

20

2.B.5 Geographical distribution

Dillenia indica L. occurs in the moist and evergreen forests of the sub-

Himalayan tract, from Kumaon and Garhwal eastwards to Assam and Bengal, and

southwards to central and southern India. It is commonly seen along banks of forest

streams. The recorded temperatures and rainfall in its natural habitat are 35-65 F

(minimum) to 95-105 F (maximum) and rainfall of 200-500 cm. The tree is also found

in Bangladesh, Nepal, Sri Lanka, Malaysia, Myanmar and Thailand [5,6].

2.B.5.1 Vernacular names

Dillenia indica L. is known by various names in different regions of the country

[5,6]. They are:

Table 2B.3: Vernacular names of Dillenia indica L.

Language Name of Dillenia indica L. Assamese Outenga, Chalita Bengali Chalta English Elephant apple Garo Panpui Gujarati Otaphal Hindi Chalta, Karambel Konkani Corombol Malayalam Punna, Syalita Marathi Karmbel, Motakarmal Nepali Panchkule Oriya Chalota, Ou, Rai Sanskrit Bhavya Sinhalese Hondapara, Wampara Tamil Akku, Uva Telegu Kalinga, Uvva

21

Photo 2B.1: A young tree of Dillenia indica L.

Photo 2B.2: Matured fruits of

Dillenia indica L.

Photo 2B.3: Cross sectional view of the fruit

of Dillenia indica L.

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2.B.6 Knowledge of Dillenia indica L. in ancient relics

The medicinal treatise of Ayurveda dates back to pre-historic Vedic era, which

is the ancient testimony for use of plants as medicine. Accordingly, the medicinal

properties of Bhavya (Dillenia indica L.) are depicted in the Vedas and other ancient

relics [5,6,73,74].

Yajur Veda: an important plant;

Upavarhana Samhita: the plant is aphrodisiac and promotes virility;

Charaka Samhita: the fruit is sweet, acidic, astringent, removes bile, phlegm, fetid and

flatulence;

Sushruta Samhita: fruit cardiotonic, tasteful, astringent, acidic, removes bile, phlegm,

fetid and flatulence;

Rajanighantu: green fruit is acidic, pungent, hot, removes wind, phlegm, but the ripe

fruit is sweet, sour, appetising and beneficial in colic associated with mucous;

Matsya Purana: decoction of this plant can be used as universal antidote for poison;

Agni Purana: spraying water, containing stem extract, on and around the wound caused

by spider bite helps in removing the poison.

Bhavya also finds mention in the great ancient epic, the Mahabharata in the

shloka XIII.54.5 of Anusasana Parva.

2.B.7 Ethnomedicinal uses of Dillenia indica L.

Dillenia indica L. is an ethno-medicinally important plant used for the treatment

of many severe diseases like fever, cancer, diarrhea, mouth disorders, indigestion and

weakness. The plant is mostly found in forest, and people have successfully

domesticated in their home gardens for its attractive flower, fleshy edible fruit and

fodder leaves. The fruit possesses tonic and laxative properties and used for abdominal

pains. The fruits are generally consumed as chutney or made into jam. It is also

consumed for relieving abdominal pains by the ethnic communities of Assam. The juice

of the fruit, mixed with sugar and water are used as a cooling beverage and also in the

treatment of high fevers and cough. The fruit juice is also consumed in the treatment of

23

heart related diseases [8-13]. Traditionally, the juices of leaves, bark and fruits are

mixed and given orally for the treatment of cancer and diarrhoea in the tribal areas of

Mizoram [75]. The various ethnomedicinal uses of different parts of the plant by

different tribal people and ethnic group of Assam are given in table 2B.4. In Assam, the

fruit is kept below the bed for prevention of small pox and cholera. The Assamese

people also use the fruit as natural insecticide in paddy cultivation. The carpels of the

fruit are used as oil lamp during the festival Kati Bihu in paddy field. The fruit is one of

the recipes of 101 plant species eaten during Bohag Bihu which is celebrated in the

month of Bohag (mid April - mid May) as Assamese New Year in the state of Assam,

India. People believe that this special recipe has some medicinal value which keeps

them healthy and free from diseases for the coming New Year [76].

Table 2B.4: Ethnomedicinal uses of Dillenia indica L.

Tribe/Ethnic group Part used Ethnomedicinal uses

Bodos Fruit Fruit is eaten to cure stomach related disorders.

Manipuri Fruit Fruit is used for curing hair fall and dandruff.

Mikir Fruit Fruits are consumed raw to combat weakness.

Rajbongshis Mucilage Applied on wounds and burns.

Tai Ahom Fruit A decoction of fruit is used as anti-dandruff and

applied on head to check frequent hair fall. Fruit

is also eaten to combat weakness.

Tai Singfou Fruit Decoction of fruit is used to remove dandruff.

Santhal

Leaf A small piece of leaf is consumed in the

treatment of dysentery.

Root Root is used as prophylactic for cholera; it is

also one of the ingredients of a paste used in the

treatment of burning sensation in the chest.

Stem bark It is used as chronic progredient sores and

carbuncle and as a prophylactic for cholera.

Mucilage Applied on wound.

24

2.B.8 Current status of studies on Dillenia indica L.

Many scientific studies have been carried out on different parts of Dillenia

indica L. plant which are described in tables 2B.5 and 2B.6. The studies on antioxidant

activities have been reported in all parts of the plant [17,18,24,27]. Phenolic compounds

which are responsible for antioxidant activity is reported to be present in high amount in

D.indica L. [17,24] but the most important aspect is that which part of the plant

possesses highest antioxidant activity in different antioxidant bioassay.

Analgesic, anti-leukemic and antimicrobial studies have been done on bark, fruit

and stem of this plant respectively [15,16,25,77]. Besides, the leaves of D.indica L. has

been tested for the anti-diabetic, anti-inflammatory, antinociceptive, anxiolytic, anti-

diarrhoeal and cytotoxicity activities [16,18,19,21-23,25,78, 79].

Recent studies have shown that researchers are taking interest on isolation of

bioactive compounds from D.indica L. because of their important therapeutic uses

[15,17,27,28]. However, there is still lack of knowledge on details of chemical

constituents which are responsible for different biological activities.

2.B.9 Previously isolated Phytochemicals

The plant Dillenia indica L. contains number of classes of compounds such as

phenols, tannins, alkaloids, flavonoids, saponins, sugars etc [14]. All isolated

compounds were reported in the table 2B.5. It describes the general names, IUPAC

names and different physical properties of the isolated compounds.

The bark, fruit, leaf and stem of D.indica L. contains betulinaldehyde, betulinic

acid, lupeol and dillenetin [15,27,28]. The fruit and stem contains myricetin and

isorhamnetin [29,30]. The bark and sepal contains betulin [31]. Stigmasterol is found in

the leaves and stem [27,28] while β-sitosterol is found in the bark, fruit and leaves [28].

The fruit also contains cycloartenone and n-hentriacontanol [32]. A total of eleven

compounds were reported but there is still lack of knowledge on details of chemical

constituents present in different parts of D.indica L.

25

Table 2B.5: Previously isolated compounds from different parts of D.indica L.

Entry Plant part (s)

Compound name

IUPAC name MF MW Refe-rence No.

1 Bark, fruit, leaf, stem

Betulinaldehyde (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-hydroxy-5a,5b,8,8, 11a-pentamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a, 13b-hexadecahydrocyclopenta[a]chrysene-3a-carbaldehyde

C30H48O2 440.7 27

2 Bark, fruit, leaf, stem

Betulinic acid (3β)-3-Hydroxy-lup-20(29)-en-28-oic acid

C30H48O3 456.7 15,27,28

3 Bark, sepal

Betulin Lup-20(29)-ene-3β,28-diol C30H50O2 442.72 31

4 Bark, fruit, leaf, stem

Lupeol (1R,3aR,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a,5a,5b,8,8,11a-hexamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta [a]chrysen-9-ol

C30H50O 426.72 27

5 Leaf, stem

Stigmasterol (3S,8S,9S,10R,13R,14S,17R)-17-[(E,2R,5S)-5-ethyl-6-methylhept-3-en-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

C29H48O 412.70 27,28

6 Bark,fruit, leaf

β-Sitosterol 17-(5-Ethyl-6-methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

C29H50O 414.71 28

7 Bark, fruit, leaf, stem

Dillenetin 3,5,7-trihydroxy-3’,4’-dimethoxy flavone; 2-(3,4-Dimethoxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one

C17H14O7 330.29 28

8 Fruit, stem

Myricetin 3,5,7-Trihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one

C15H10O8 318.23 29,30

9 Fruit, stem

Isorrhamnetin 3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-chromen-4-one

C16H12O7 316.26 29,30

10 Fruit Cycloartenone 9β-9,19-Cyclolanost-2-en-1-one C30H48O 424.70 32

11 Fruit n-Hentriacon-tanol

1-Hentriacontanol C31H64O 452.83 32

26

Fig.2B.1: Chemical structures of previously isolated phytochemicals from D.indica

L.

(a) Lupeol (b) Betulinic acid

(c) Betulin (d) Betulinaldehyde

(e) Myricetin (f) Dillenetin

27

(g) Stigmasterol (h) β-Sitosterol

(i) Isorhamnetin (j) Cycloartenone

(k) n-Hentriacontanol

CH3

OCH3H3C

CH3

H3C CH3

CH3

CH3

28

Table 2B.6: Different biological studies on D.indica L.

Entry Functional properties

Plant part (s) Solvent extract Reference No.

1 Antioxidant Fruit, Leaf, Stem bark Methanol, ethyl acetate, water

17,18,24,27

2 Antimicrobial Stem bark Methanol 16

3 Analgesic Leaf Methanol 77

4 Anti-inflammatory Leaf Methanol 19

5 Anti-diabetic Leaf Methanol, water 21,22,23

6 Anti-leukemic Fruit Methanol 15

7 Antinociceptive activity

Bark Methanol 18

8 Anxiolytic Leaf Hydroethanolic 78

9 Anti-diarrhoeal Leaf, fruit Ethanol 79

10 Cytotoxicity Leaf Methanol 16,25 2.B.10 Scope of the present investigation

Dillenia indica L. is a unique plant profusely used in Ayurveda to cure diseases.

Extraction forms the first step in medicinal plant research because the preparation of

crude extracts from plants is the starting point for the isolation and purification of

chemical constituents present in plants. Accelerated Solvent Extractor (ASE), a form of

pressurized liquid extraction (PLE), will be used for the first time to extract betulinic

acid from the fruit of D.indica L. The ASE technique uses small amounts of

conventional solvents at elevated temperatures and pressures to achieve quantitative

extraction from solid and semi-solid samples in a short time, and is well established for

environmentally clean extraction. The study is designed to demonstrate the effects of

ASE on the extraction of betulinic acid, in vitro antioxidant activity, total phenolic

content, total flavonoid content of the fruit of D.indica L. To obtain the maximum

extraction yield of betulinic acid from the fruit of D.indica L., key factors such as the

extraction solvents, temperature and time will be examined in ASE method.

The broad range of antioxidant activity and other functional properties (lipase

inhibition, α-amylase inhibition and DPP (Dipeptidyl peptidase)-IV inhibition activity)

of the D.indica L. fruit extracts will be examined which may establish the fruit as a

source of natural antioxidants and other important health promoting phytochemicals.

29

Although betulinic acid is widely available from numerous botanical sources, these sources are not sufficient to meet the growing demand for betulinic acid among members of the scientific community for biological and clinical testing. It is imperative to study the variation of betulinic acid content in the fruit of D.indica L. during development in different growth stage from the unripe to ripe conditions for better understanding of the biosynthesis of betulinic acid in D.indica L. fruit. It is important to know the chemistry underlying how the building blocks for secondary metabolites are assembled in nature. The terpenoids form a large and structurally diverse family of natural products derived from C5 isoprene units. These aspects are significant for commercial exploitation of this plant. There lies immense scope in understanding the physiological role of bioactive compounds during development of different parts of the plant. It may provide an opportunity to standardize the stage of physiological maturity, which is critical to obtain quality raw material for design and development of phytopharmaceutical products for health benefits.

The scientific inquisitiveness was ardently attended by undertaking detailed investigation on the above mentioned aspects from the fruit of D.indica L. 2.B.11 Conclusion

The strategies for selection of Dillenia indica L. for research and development was due to its high medicinal value and few reports on bioactive properties and also on chemical constituents and their functional properties. It is an underutilized plant available in Assam (India). This plant has tremendous commercial value provided more research and development is started in judicious exploitation of the health promoting phytochemicals present in different parts of the plant including the fruit. This research work, discussed in detail in the upcoming chapters, focuses on the isolation and characterization of bioactive molecules derived from D.indica L. fruit. This work also investigates the extraction optimization of bioactive molecule betulinic acid from the fruit of D.indica L. as well as the study of variation of betulinic acid content in the fruit during development in different stages of growth apart from the evaluation of different functional properties of D.indica L. extracts. In this way, tremendous value can be added to this less utilized plant for development of phytopharmaceutical products by setting up food processing industries which will uplift the local socio-economy of the region.

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