Ranbir Chander Sobti 

Naveen Kumar Arora · Richa Kothari

Editors

Environmental Biotechnology:

For Sustainable Future

ISBN 978-981-10-7283-3                      ISBN 978-981-10-7284-0 (eBook)
https://doi.org/10.1007/978-981-10-7284-0

Library of Congress Control Number: 2018957141

© Springer Nature Singapore Pte Ltd. 2019
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Contents

Part I Biodegradation and Bioremediation
1 Biochar for Effective Cleaning of Contaminated
Dumpsite Soil: A Sustainable and Cost-Effective
Remediation Technique for Developing Nations................................... 3
Paromita Chakraborty, Moitraiyee Mukhopadhyay, R. Shruthi,
Debayan Mazumdar, Daniel Snow, and Jim Jian Wang

2 Scope of Nanoparticles in Environmental Toxicant Remediation....... 31
Anupam Dhasmana, Swati Uniyal, Vivek Kumar, Sanjay Gupta,
Kavindra Kumar Kesari, Shafiul Haque, Mohtashim Lohani,
and Jaya Pandey

3 Removal of Inorganic and Organic Contaminants from
Terrestrial and Aquatic Ecosystems Through
Phytoremediation and Biosorption......................................................... 45
Dhananjay Kumar, Sangeeta Anand, Poonam, Jaya Tiwari,
G. C. Kisku, and Narendra Kumar

4 Environmental Health Hazards of Post-Methanated
Distillery
Effluent and Its Biodegradation and Decolorization............................ 73
Sangeeta Yadav and Ram Chandra

5 Heavy Metal Contamination: An Alarming Threat
to Environment and Human Health....................................................... 103
Sandhya Mishra, Ram Naresh Bharagava, Nandkishor More,
Ashutosh Yadav, Surabhi Zainith, Sujata Mani, and Pankaj
Chowdhary

Part II Sustainable Agriculture
6 Plant Growth-Promoting Rhizobacteria: Diversity
and Applications....................................................................................... 129

Maya Verma, Jitendra Mishra, and Naveen Kumar Arora

7 Plausible Role of Plant Growth-Promoting Rhizobacteria
in Future Climatic Scenario.................................................................... 175
R. Z. Sayyed, N. Ilyas, B. Tabassum, A. Hashem, E. F. Abd_Allah,
and H. P. Jadhav

8 Plant Growth-Promoting Microbes: Contribution
to Stress Management in Plant Hosts..................................................... 199
Krishna Sundari Sattiraju, Srishti Kotiyal, Asmita Arora,
and Mahima Maheshwari

9 Chemistry, Therapeutic Attributes, and Biological
Activities of Dillenia indica Linn............................................................ 237
Ashok K. Singh and Sudipta Saha

Part III Aquatics and Wastewater Treatment
10 Implication of Algal Microbiology for Wastewater
Treatment and Bioenergy Production.................................................... 263
Vinayak V. Pathak, Shamshad Ahmad, and Richa Kothari

11 Efficiency of Constructed Wetland Microcosms (CWMs)
for the Treatment of Domestic Wastewater Using Aquatic
Macrophytes............................................................................................. 287
Saroj Kumar and Venkatesh Dutta

12 Modelling Water Temperature’s Sensitivity to Atmospheric
Warming and River Flow........................................................................ 309
Shaik Rehana, Francisco Munoz-Arriola, Daniel A. Rico, and
Shannon L. Bartelt-Hunt

Part IV Other Aspects
13 Thermophiles vs. Psychrophiles: Cues from Microbes
for Sustainable Industries....................................................................... 323
Monica Sharma

14 Role of Solar Energy Applications for Environmental
Sustainability............................................................................................ 341
Atin K. Pathak, Kapil Chopra, Har Mohan Singh, V. V. Tyagi,
Richa Kothari, Sanjeev Anand, and A. K. Pandey

15 Natural Sensitizers and Their Applications
in Dye-Sensitized Solar Cell.................................................................... 375
A. K. Pandey, Muhammad Shakeel Ahmad,

Nasrudin Abd Rahim, V. V. Tyagi, and R. Saidur

Chapter 9

Chemistry, Therapeutic Attributes, and Biological Activities of Dillenia indica Linn

Ashok K. Singh and Sudipta Saha

Contents

Introduction 238

Chemistry 239

Traditional and Therapeutic Uses 243

Biological Activities of D. indica 245

Cytotoxicity 245

Immunomodulatory 246

Antidiabetic Activity 246

Anti-inflammatory and Antinociceptive Activity 247

Antioxidant Activity 248

Antimicrobial Activity 249

Antidiarrheal Activity 250

Antiprotozoal Activity 251

Antimutagenic Activity 251

Anticholinergic Activity 251

Protoscolicidal Activity 251

Hemolytic Activity 252

Hair Treatment Activity 252

Enzyme Inhibitory Activity 252

Toxicology of D. indica 252

D. indica in Drug Formulation and Drug Delivery 253

Future Prospects 254

Conclusion 254

References 255

Abstract Dillenia indica Linn. (Dilleniaceae) is generally known as elephant apple and locally known as outenga. The vernacular names are chalta, chulta, bhavya, karambel, ouu, and ramphal. This evergreen deciduous tree is markedly disseminated in the seasonal tropics of many Asian countries, in India from Himalaya to south India. The different parts of this plant have been prevalently investigated for the plethora of biological activities including anticancer, antidiabetic, antihyperlipidemic, antileukemic, antioxidant, antimutagenic, antimicrobial, antinociceptive, antidiarrheal, and hairweaving activities. Differently prepared extracts of this plant have been reported mainly to contain a wide range of flavonoids, triterpenoids (lupene-type), phytosteroids, phenolics, alcohols, and ketones and an anthraquinone. Several phytochemical investigations revealed substantial presence of various types of active constituents including β-sitosterol, stigmasterol, betulin, betulinic acid, kaempferol, myricetin, quercetin, dillenetin and rhamnetin. Among these the major chemical constituents are betulin and betulinic acid (lupene-type triterpenoids) that show a wide spectrum of pharmacological activities like anti-HIV, anticancer, antimalarial, anti-inflammatory, etc. The present chapter thus approaches to highlight on phytochemistry, traditional and therapeutic uses, and biological activities of Dillenia indica.

Keywords Dillenia indica · Therapeutic attributes · Phytochemistry · Therapeutic

uses · Pharmacology

Introduction

A vast amount of knowledge and practices on herbal medicinal systems have been transmitted through the ages. For centuries, medicinal plants were the only resources available for the treatment of several diseases which afflicted humanity (Ozdemir and Alpınar 2015). Numerous of these plants are uncommon, endemic, and found only in forest region. There is neither biological data nor satisfactory information that prompted their rarity in the natural surroundings (Kerrigan et al. 2011). Correspondingly, there are many plant species which have been utilized by tribal and folk communities of different forest regions of India; however, their medicinal and also pharmacological esteem is yet obscure as these plants are hardly available. There are many plant species which have been utilized by tribal groups of India; however, their restorative and also pharmacological knowledge is yet obscure as these plants are not easily accessible and studied. Among these, there are few plants belonging to family Dilleniaceae which have not gained much popularity but have interesting medicinal values. The genus Dillenia has 60 species; however, only a few of them are reported to have important phytochemicals and thereby enrich their medicinal values. These species are D. indica, D. pentagyna, D. suffruticosa, D. andamanica, D. serrata, D. sumatrana, D. aurea, D. bracteata, D. excelsa, D. ovata, D. papuana, D. parviflora, D. philippinensis, D. pulchella, D. reticulata, D. scabrella, D. eximia, and D. triquetra. Only two plants D. indica Linn. and D. pentagyna Roxb. are available in India. D. indica has been extensively studied and a more commonly employed medicinal plant in different parts of India (Dickison 1979). Several research works have been conducted on the isolation and quantification of the different phytochemicals from various parts of D. indica; however, very few phytochemical investigations have been performed from D. pentagyna.

Chemistry

The significant classes of chemical constituents extracted from D. indica are flavonoids and triterpenoids (lupene-type). Other isolated compounds including phytosteroids, diterpene, ionone, phenolics, anthraquinone, alcohols, and ketones also enhance the diversity of phytochemistry in D. indica. As per our extensive search, a total of 34 compounds isolated from D. indica are included in this review which may lead to further research and noble challenge to discover new chemical constituents from this plant. These compounds are listed in Table 9.1, and their chemical structures are displayed in Fig. 9.1.

Stem bark of D. indica contains triterpenoids like lupeol, betunaldehyde, and betulinic acid; flavonoids like kaempferol, dillenetin, rhamnetin, dihydro-isorhamnetin, myricetin, naringenin, and quercetin; and 10% tannin (Shah 1978; Khanum et al. 2007; Khare 2007). The ethanol extract of stem bark is enriched with two flavonoids, kaempferol and quercetin, as well as a triterpenoid (Srivastava and Pande 1981). Parvinet al. (2009) acquired methanolic extract of stem after partitioning with n-hexane and isolated four compounds, viz., lupeol, betulinic acid, betunaldehyde, and stigmasterol, using column chromatographic separation.

Leaves of D. indica contain betulinic acid, betulin, lupeol, and β-sitosterol (Dan and Dan 1980). The petroleum ether extract of leaves contains betulin, β-sitosterol, cycloartenone, and n-hentriacontanol, whereas chloroform extract has betulinic acid (Mukherjee and Badruddoza 1981). Methanolic extract of leaves after fractionation with n-hexane and chloroform also has compounds like betulinic acid, β-sitosterol, dillenetin, and stigmasterol (Muhit et al. 2010). Phytochemicals have also been investigated from acid hydrolyzed extracts of dried leaves which demonstrated the presence of kaempferol, whereas fresh leaves were found to contain dihydrokaempferide and naringenin-7-diglucoside which get further oxidized to ten corresponding flavonols (Bate-Smith and Harborne 1971). Kumar et al. (2010) isolated and quantified betulinic acid using validated HPLC method from various fractions such as methanol, ethyl acetate, n-butanol, and water. The highest concentration among them was found in ethyl acetate fraction.

Fruit of D. indica contains about 34% of total phenolics in methanolic extract (Abdille et al. 2005), isorhamnetin (Pavanasasivam and Sultanbawa 1975a), lupeol, betulin, β-sitosterol (Sundararamaiah et al. 1976), and polysaccharide like arabinogalactan. Uppalapati and Rao (1980) reported the presence of steroids, saponins, fixed oil, free amino acids, glycosides, tannins, and sugars in the seeds of D. indica. These scientific reports collectively revealed that betulin, betulinic acid, and β-sitosterol are present in almost all parts of D. indica.

Table 9.1 Compounds isolated from D. indica

Fig. 9.1 Structure of compounds isolated from Dillenia indica

Traditional and Therapeutic Uses

Usually all parts of D. indica are traditionally exploited for therapeutic purposes. The jelly-like content inside the fruit of D. indica is applied for hair treatment against dandruff and falling hair. The mixed juices of leaves and stem bark are used orally for the prevention of diarrhea and cancer (Yeshwante et al. 2009a, b; Sunil et al. 2011). The leaves and stem bark are also used as laxative and astringent (Sharma et al. 2009). Apart from this, the stem bark is used for the production of charcoal. The fresh and dried materials of various parts of D. indica are processed as juice, decoction, poultice, and mucilage for the medical care of diabetes, wounds, diarrhea, cancer, rheumatism, urinary problems, skin diseases, aches, fever, cough, and falling hair. Almost all the known medicinal uses of D. indica are enlisted in Table 9.2. Skin diseases including leukoderma, eczema, skin itches, and skin rash can be treated using the leaf, fruit, and stem bark of D. indica (Quattrocchi 2012; Boer et al. 2012; Bhat et al. 2014). Leaves of D. indica as well as decoction and juice of the fruit and stem bark are exploited in daily practices to attenuate cancerous growth, particularly breast and gastric cancers (Das et al. 2009; Sharma et al. 2001). Furthermore, fruit juice of D. indica is supplemented orally to eliminate fever and cough-associated symptoms (Angami et al. 2006; Quattrocchi 2012). The mixed juice of fruit and calyx of D. indica is used in daily practices for the treatment of diabetes (Pavani et al. 2012; Ripunjoy 2013). The root of D. indica is generally utilized for the purpose of abortion (Quattrocchi 2012). Moreover, the mucilage of D. indica fruits is used to treat falling hair, to clean hair, as well as to remove dandruff from hair (Saikia et al. 2006). The sweetish-sour edible fruits of this plant may be consumed directly or juiced with sugar to take as fresh and healthy drink. An interesting event is that the bark of the stem and roots has extensively been used as a food-poisoning neutralizer (Grosvenor et al. 1995a, b; Islam et al. 2014). Altogether, it is concluded that D. indica contains the chemical constituents that can treat a broad spectrum of human ailments.

Table 9.2 Traditional and therapeutic uses of D. indica

Biological Activities of D. indica

Cytotoxicity

Cytotoxic activity of phytochemicals present in different parts of D. indica was screened against numerous cancer cell lines. In vitro cytotoxic activity against leukemia, carcinoma, and lymphoma cells was reported using methanol extracts of D. indica fruit (Kumar et al. 2010) and leaves (Akter et al. 2014). In particular, the methanol extract of the fruit inhibited the growth of U937, HL60, and K562 cancer cell lines with IC50 of 328.80, 297.69, and 275.40 mg/mL, respectively, comparable to standard drugs Ara C and Gleevec (Kumar et al. 2010), whereas the methanol extract of the leaves inhibited the growth of AGS, MCF-7, and MDA-MB-231 cancer cell lines with IC50 values of 1.18, 0.34, and 0.54 mg/mL, respectively, as compared to cycloheximide with IC50 values of 0.0010, 0.061, and 0.0004 mg/mL, respectively (Akter et al. 2014). On the contrary, the ethanol and aqueous extracts were noticed noncytotoxic toward the cancer cell lines (Nguyen-Pouplin et al. 2007; Armania et al. 2013a, b). Ultimately, a significant correlation was established between the presence of betulinic acid and cytotoxic activity of extracts and fractions of D. indica. For example, the ethyl acetate fraction of the methanol extract of D. indica calyx containing considerable amounts of betulinic acid exhibited prominent cytotoxicity when compared to the n-butanol fraction (Kumar et al. 2010). Other than betulinic acid, three compounds (lupeol, betulin, and gallic acid) isolated from D. indica were also proven to have cytotoxic activity toward cancer cell lines. Finally, the result revealed that lupane-type triterpenoids isolated from D. indica (lupeol, betulin, and betulinic acid) displayed considerable cytotoxic properties. Although the cytotoxicity of this plant is very less studied and needs to be performed on variety of cell lines, however, these findings may provide a great opportunity for further development of anticancer compounds from D. indica.

Immunomodulatory

The cornerstone of good health is no doubt a strong, well-functioning immune system. Phytochemicals such as flavonoids, terpenoids, glycosides, and phenolic compounds act as a natural defense system not only for the host plant, but also they can serve in immunomodulatory activities in humans (Venkatalakshmi et al. 2016). For example, the aqueous methanolic extract (70%) of D. indica fruit enhanced the production of polyclonal immunoglobulin M (IgM) in cultured BALB/c female mice spleen cells at a concentration of 200 mg/mL as compared to lipopolysaccharide (0.1 mg/mL) (Sarker et al. 2012).

Antidiabetic Activity

Indian natives have since long used D. indica to treat diabetes. The leaf extract of D. indica was found to inhibit enzymatic activity of rat intestinal sucrose and maltase (Jong-Anurakkun et al. 2007). Moreover, the leaf extract of D. indica assayed on streptozotocin (STZ)- and alloxan-induced type 1 and type 2 diabetic rats reduced the levels of blood glucose, hypertriglyceridemia, and hypercholesterolemia. In fact, the extract enhanced the production of insulin and high-density lipoprotein cholesterol (HDL-c) (Jong-Anurakkun et al. 2007; Kumar et al. 2011a, b). Additionally, the extract inhibited overproduction of liver function enzymes such as aspartate transaminase (AST), alanin transaminase (ALT), and alkaline phosphatase (ALP) in diabetic rats (Kumar et al. 2011a, b). Histopathological studies showed that the liver, pancreas, and kidney in the treated rats restored to normal conditions after treatment with the extract of D. indica (Kumar et al. 2011b). In an experiment performed by Kumar et al. (2011a, b), the ethyl acetate fraction of the methanol extract of the leaves assayed in vivo on STZ-induced type 1 and type 2 diabetic rats showed a reduction in blood glucose, serum cholesterol, and triglyceride levels after 21 days of treatment at the doses of 200 and 400 mg/kg body weight (bw). This fraction also increased the level of HDL-C in the treated rats. Likewise, the defatted methanol extract of the leaves assayed in vivo on type 1 diabetic rats induced by STZ and alloxan decreased the blood glucose, hypertriglyceridemia, and hypercholesterolemia levels as well as increased the production of insulin and HDL-C at the doses of 250 and 500 mg/kg bw during 21 days of treatment (Jong-Anurakkun et al. 2007; Kumar et al. 2011a, b). In another experiment, three compounds (quercetin, β-sitosterol, and stigmasterol) isolated from the ethyl acetate fraction of this extract were found to reduce the blood glucose level of type 2 STZ-nicotinamide-induced diabetic mice which was comparable to standard drug glibenclamide (Kumar et al. 2013). Later, similar activity was performed by the extract of D. indica fresh leaves assayed on STZ-induced diabetic rats. Further investigation of active constituent of this extract led to the isolation of 3,5,7-trihydroxy-2-(4-hydroxy-benzyl)-chroman-4-one, which significantly demonstrated antidiabetic activity by reducing blood glucose, cholesterol, and triglycerides levels. The treatment also increased the levels of insulin, HDL-C, as well as body weight when compared with diabetic rats. Histopathological study showed that treatment with the extract and 3,5,7-Trihydroxy-2-(4-hydroxy-benzyl)-chroman-4-one restored the hyperglycemic conditions and kidney structure abnormality owing to oxidative stress in the diabetic rats (Kaur et al. 2016). The alcoholic extract of the fresh leaves assayed in vivo on STZ-induced diabetic rats reduced levels of blood glucose, cholesterol, and triglycerides at doses of 100, 200, and 400 mg/kg bw for 21 days of treatment, comparable to glimepiride (10 mg/kg bw). The extract significantly increased the body weight, serum insulin and HDL-C levels (Kaur et al. 2016). These findings well supported the traditional use of D. indica for the treatment of diabetes among Indian natives.

Anti-inflammatory and Antinociceptive Activity

Many of D. indica extracts were tested for anti-inflammatory and antinociceptive activities. The alcoholic extract of D. indica leaves obliterated carrageenan-induced paw edema at doses of 200 and 400 mg/kg bw with the impact comparable to indomethacin (10 mg/kg bw) (Yeshwante et al. 2009a, b). Moreover, the methanol extract of the leaves and its nonpolar fractions assayed on acetic acid-induced colitis mice at doses of 200 and 800 mg/kg bw restored the colon weight and macroscopic damage in acetic acid-induced colitis in mice. The non-polar fractions also attenuated the production of tumor necrosis factor alpha (TNF-α) and myeloperoxidase released from azurophilic granules of neutrophils (Somani et al. 2014). On the other hand, the glycolic extract and emulsion of D. indica fruit were not found suitable to accelerate wound healing process on skin injuries made in rats (Miglianto et al. 2011). The alcoholic extracts of the leaf and stem bark of D. indica were examined for antinociceptive activity using the hot plate method, tail immersion test, and writhing model in mice induced by acetic acid and found to exhibit central and peripheral analgesia (Bose et al. 2010; Yeshwante et al. 2011; Alam et al. 2012). In particular, the methanol extract of the leaves was evaluated on acetic acid-induced writhing mice at doses of 250 and 500 mg/kg bw which obliterated the writhing behavior by 48.82% and 55.88% inhibition comparable to that of diclofenac (60% at 25 mg/kg bw) (Bose et al. 2010). Furthermore, the methanol extract of the leaves measured by hot tail, tail immersion, formalin-induced nociception, and acetic acid-induced writhing model on mice at doses of 400 mg/kg bw considerably exhibited central and peripheral analgesia when comparable to those of the standard drugs, pentazocine (15 mg/kg bw) and indomethacin (20 mg/kg bw) (Yeshwante et al. 2011). Later, the methanol extract of the stem bark was evaluated using hot plate method, tail immersion test, and acetic acid-induced writhing model in mice at doses of 200 and 400 mg/kg bw that demonstrated dose-dependent analgesic activity comparable to those of the standard drugs nalbuphine (10 mg/kg bw) and diclofenac sodium (10 mg/kg bw) (Alam et al. 2012).

Antioxidant Activity

Flavonoids, terpenoids, tannins, and phenolics in D. indica plants are major compounds responsible for primary antioxidants or free radical scavenging effects (Polterai 1997). A compound 3,5,7-trihydroxy-2-(4-hydroxy-benzyl)-chroman-4-one isolated from D. indica displayed remarkable antioxidant property when assayed on 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), hydrogen peroxide, and superoxide radicals as well as ferric ion. This compound also elevated the production of antioxidant enzymes (superoxide dismutase and glutathione) in streptozocin (STZ)-induced diabetic rats. Treatment with this compound remarkably reduced the level of lipid peroxidation marker, i.e., thiobarbituric acid-reactive substances (TBARS) in diabetic rats (Kaur et al. 2016). In an experiment, methanol, ethyl acetate, and aqueous extracts of the fruit reduced molybdenum (IV) to molybdenum (V) with the capacity of 1904.80, 1067.00, and 594.60 mmol/g of extract, respectively, when compared to ascorbic acid. The extracts also exhibited DPPH free radical scavenging activity over the concentration range of 25–100 mg/mL with the activity in order of methanol extract > ethyl acetate extract > water extract. The extracts when evaluated on β-carotene bleaching produced antioxidant activity with the capacity of 80.20, 55.50, and 45.50%, respectively, at 100 mg/mL when compared to BHA (97.50%) (Abdille et al. 2005). In another experiment, the aqueous acetone extract of the stem bark reduced molybdenum (IV) to molybdenum (V) with capacity of 3.12 mmol/g of extract at 50 mg/mL as compared to ascorbic acid and exhibited DPPH and superoxide radical scavenging activity causing 90.90% and 31.73% inhibition at 25 and 50 mg/mL which were found to be comparable of BHA (91.00%) and gallic acid (47.73%), respectively. The extract also assayed using hydroxyl radical-induced deoxyribose damage which showed radical scavenging activity with percentage inhibition of 53.90–74.66% at 100–500 mg/mL (Deepa and Jena 2011). Furthermore, the ethanol extract of the leaves assayed in vitro by DPPH, hydroxyl, and hydrogen peroxide radicals displayed antioxidant activity with IC50 of 34.80, 64.40, and 51.00 mg/mL, respectively, as compared to ascorbic acid with IC50 of 24.00, 48.00, and 34.40 mg/mL, respectively. The extract also measured using ferric-reducing antioxidant power (FRAP) assay caused the reduction of ferric ion as compared with ascorbic acid (Shendge et al. 2011). Later, similar extract also evaluated in vivo on doxorubicin-induced rats that restored the levels of GSH and cardiac malondialdehyde (MDA) at doses of 250 and 500 mg/kg bw (Shendge and Gadge 2012). In another experiment on Swiss albino mice, the methanol extract of the stem bark reduced the production of ROS in kidney cells with IC50 of 34.72 mg/mL as compared to trolox (IC50 8.66 mg/mL) (Alam et al. 2012). Further, Singh et al. (2012) also demonstrated that methanol, acetone, and water extracts of the stem bark measured by DPPH radical produced antioxidant activity with IC50 values of 188.08, 177.42, and 163.68 mg/mL of fresh mass, respectively. It was also investigated that the methanol extract of the leaves and its nonpolar fractions reduced the level of MDA and, however, enhanced the levels of the antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) in acetic acid-induced colitis at doses of 200 and 800 mg/kg (Somani et al. 2014). Interestingly, a compound proanthocyanidins isolated from D. indica fresh fruit produced significant antioxidant activity measured by FRAP and oxygen radical absorbance capacity (ORAC) assays with values of 2.32 × 103 mmol Fe(II)/g and 1.06 × 104mmol trolox equivalent/g (Fu et al. 2015). Lastly, the alcoholic extract of the fresh leaves obtained from sequential extraction with petroleum ether, chloroform, alcohol, and aqueous alcohol (40%) exhibited free radical scavenging activity toward DPPH, hydrogen peroxide, and superoxide radicals with IC50 of 2.98, 228.69, and 75.09 mg/mL, respectively, and ferric-reducing antioxidant power with EC50 of 111 mg/mL. In the similar experiment, it was also investigated that the extract enhanced the production of antioxidant enzymes (SOD and GSH) in STZ-induced diabetic rats at doses of 100, 200, and 400 mg/kg bw after 21 days of treatment when compared to glimepiride at 10 mg/kg bw (Kaur et al. 2016).

Antimicrobial Activity

D. indica was investigated for antibacterial, antifungal, and antiviral activities. The extracts and fractions of D. indica were documented to show growth inhibition against Gram-positive and Gram-negative bacteria. However, in comparison to bacteria, they attenuated the fungi including Aspergillus fumigatus, Aspergillus niger, Penicillium sp., Candida albicans, Candida krusei, Rhizopus oryzae, Saccharomyces cerevisiae, and Trichoderma viride (Nick et al. 1995a, b; Wiart et al. 2004; Haque et al. 2008; Apu et al. 2010; Smitha et al. 2012). Betulinic acid isolated from D. indica has proven antimicrobial activity (Nick et al. 1994, 1995a, b; Ragasa et al. 2009). Meanwhile, a few nonpolar fractions (chloroform, carbon tetrachloride, and hexane) of the methanol extract of the leaves weakened the growth of Escherichia coli, Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Sarcina lutea, Pseudomonas aeruginosa, Vibrio mimicus, Vibrio parahemolyticus, Salmonella typhi, Salmonella paratyphi, Shigella boydii, and Shigella dysenteriae and fungal inhibition of A. niger, C. albicans, and S. cerevisiae with inhibition zones ranging from 6 to 8 mm at 400 mg/disc when compared with kanamycin (30 mg/disc; 30–40 mm) (Apu et al. 2010). In another experiment, hexane, dichloromethane, and ethyl acetate fractions of the methanol extract of the stem bark inhibited the growth of E. coli, B. cereus, B. subtilis, S. aureus, S. lutea, P. aeruginosa, V. mimicus, V. parahemolyticus, S. paratyphi, S. typhi, and S. dysenteriae with minimum inhibitory concentration (MIC) ranging from 0.31 to 20.00 mg/mL as compared to those of kanamycin (30 mg/disc; 22–30 mm) and amoxicillin (10 mg/disc; 14–22 mm). These fractions also attenuated the growth of A. niger, C. albicans, and S. cerevisiae with the inhibitory zones ranging from 7 to 13 mm as compared to ketoconazole (50 mg/disc; 19–23 mm) (Alam et al. 2011). Jaiswal et al. (2014) concluded that the aqueous acetone (70%) extract of the fruit and stem bark inhibited the growth of food-borne pathogens (B. cereus, S. aureus, Yersinia enterocolitica, and E. coli) with minimum inhibitory concentration ranging from 1.25 to 10.00 mg/mL. These findings suggest that D. indica has a powerful potential as antimicrobial agent, which supports it as traditional therapeutic remedy against the diseases caused by microbial infection, like diarrhea, dysentery, septicemia, and skin diseases.

Antidiarrheal Activity

In an experiment, castor oil-induced mice were used to check the antidiarrheal activity of D. indica. The aqueous and methanolic extracts of the leaves prolonged the onset and reduced the total number of feces after 2 h of treatment at doses of 200 and 400 mg/kg bw (Yeshwante et al. 2009a, b). The polar extracts of the leaf and fruit caused the prolongation of onset and curtailment in defecation frequency in the treated mice. The assay of extracts using charcoal meal revealed that it had a capacity to reduce the motility of the gastrointestinal tract (Yeshwante et al. 2009a, b; Bose et al. 2010; Rahman et al. 2011a, b). Further, the methanol extract of the leaves significantly diminished the frequency of defecation and number of total stool count at a dose of 500 mg/kg bw as compared to loperamide (25 mg/kg bw; 77.22%) (Bose et al. 2010). Ethanol extracts of the fruit and leaves minimized the total number of wet feces and also decreased the motility of gastrointestinal tract in castor oil-induced diarrheal mice at doses of 200 and 400 mg/kg bw when compared with loperamide (5 mg/kg bw) (Rahman et al. 2011b). The antidiarrheal action of this plant is suggested due to inhibition of inflammatory mediators by flavonoids and tannins (Yeshwante et al. 2009a, b).

Antiprotozoal Activity

Antiprotozoal activities of D. indica against malaria and leishmaniasis have been tested. Cyclohexane fraction of ethanolic extract of D. indica leaves was reported to inhibit the growth of parasite Plasmodium falciparum. In this experiment, cyclohexane fraction of the 80% ethanol extract of the leaves exhibited 53% inhibition against parasite P. falciparum at a concentration of 10 mg/mL as compared to chloroquione (Nguyen-Pouplin et al. 2007).

Antimutagenic Activity

Mutations are the cause of innate metabolic defects in cellular mechanism which trigger initiation and progression of several human diseases including cancer. The antimutagenic and protective effect has been ascribed to many classes of phytochemicals preferably flavanoids and phenolic compounds (Aqil et al. 2008). Meanwhile, Jaiswal et al. (2014) found that 70% aqueous acetone extract of the stem bark of D. indica demonstrated antimutagenic activity against sodium azide-induced mutation in Salmonella tester strain (TA-1531).

Anticholinergic Activity

Anticholinergics generally inhibit the action of acetylcholine from binding to its receptor sites on certain nerve cells and block parasympathetic nerve impulses. Bhadra et al. (2014) found that the standardized methanol extract of the fruit of D. indica inhibited acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) activity with IC50 values of 67.26 and 122.39 mg/mL, respectively.

Protoscolicidal Activity

The scolicidal agents are generally employed in surgical manipulation of the hydatid cysts in hydatid diseases (García et al. 1997). Chowdhury et al. (2013) investigated the protoscolicidal activity in the stem bark of D. indica where an assay of the methanolic extract of the stem of D. indica was performed on earthworm Pheretima posthuma, and the result demonstrated the paralysis (194–136 min) and death (237–176 min) of the worms at 10–25 mg/mL comparable to albendazole at 10 mg/mL.

Hemolytic Activity

Hemolytic activity of any compounds is a measure of general cytotoxicity toward normal healthy cells (Da Silva et al. 2004). This activity was investigated by Jaiswal et al. (2014) in D. indica plant where the aqueous acetone (70%) extract of the fruit and stem bark of D. indica was assayed using rat whole blood which exhibited low inhibition against erythrocytes.

Hair Treatment Activity

On the basis of the previous information that keratin is the main component and mechanical strength of the hair, an experiment was performed where the aqueous extract of the mucilaginous D. indica seed sap protected human hair from loss of keratin after treatment with 10 mg hair/1 mL for 12 h. The physical structure of hair and keratin degradation were further affirmed by Fourier transform infrared spezctroscopy (FTIR), thermogravimetric analysis (TGA), and scanning electron microscopy

(SEM) analysis, and a good hair weaving activity was noticed (Saikia 2013).

Enzyme Inhibitory Activity

Inhibition is one major mechanism for physiological enzyme regulation. Further, enzyme inhibition has a number of medicinal significances, and a large number of drugs generally act by the mechanism involving enzyme inhibition. In this view Jong-Anurakkun et al. (2007) performed an experiment to assure whether D. indica has an enzyme inhibitory action. As a result, the 50% aqueous methanolic extract of the leaves attenuated the intestinal sucrose and maltase activity with percentage inhibition of 40% and 56%, respectively.

Toxicology of D. indica

Several types of extracts of D. indica were tested for their toxicity action against Artemia salina. Despite the toxicity, some extracts of D. indica were found nontoxic (Kumar et al. 2010) and exhibited ameliorative (Shendge and Gadge 2012) and hepatoprotective (Padhya et al. 2008; Himakar et al. 2010) actions when assayed using in vivo models. The ethanolic extract of the leaves restored the level of myocardial enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatine phosphokinase (CK) on myocardium of doxorubicin-induced rats at doses of 250 and 500 mg/kg bw (Shendge and Gadge 2012). However, the ethanolic extract of the leaves reduced the levels of serum AST, ALT, ALP, bilirubin, and lipid peroxidation in the the liver induced by carbon tetrachloride at a dose of 300 mg/kg bw (Padhya et al. 2008). The nonpolar hexane extract of the seeds decreased the levels of serum enzymes, bilirubin, urea, creatinine, and lipid peroxidation but increased the levels of SOD,CAT, glutathione reductase (GR),

glutathione peroxidase (GPx), and glutathione S-transferase (GST) in CCl4-induced rats at doses of 250 and 500 mg/kg bw (Himakar et al. 2010). Besides these, D. indica has been used as food poisoning neutralizer (Islam et al. 2014; Grosvenor et al. 1995a, b) which also evidenced that D. indica is safe and not toxic. The methanolic extract of the leaves was nontoxic and did not produce any mortality in mice during 24 h of treatment with 100–1500 mg/kg bw of extract by intraperitoneal

administration (Kumar et al. 2010). Apart from this, water, chloroform, carbon tetrachloride, and n-hexane fractions of methanol extract of the leaves demonstrated lethality on brine shrimp as compared to vincristine sulfate (Apu et al. 2010). However, the methanol, ethyl acetate, dichloromethane, and n-hexane extracts of the stem bark exhibited inhibition on brine shrimp with less lethality when compared to the leaf extracts (Parvin et al. 2009; Alam et al. 2011; Chowdhury et al. 2013).

D. indica in Drug Formulation and Drug Delivery

The mucilage obtained from seeds of D. indica fruit contains a natural mucoadhesive hydrophilic polymer, which is generally used in the formulation for drug delivery (Sharma et al. 2009; Bal et al. 2012a, b). The mucoadhesive and viscous properties of mucilage of D. indica fruit were used as better substitute of synthetic polymers, i.e., Carbopol 934 (Kuotsu and Bandyopadhyay 2007; Bal et al. 2012b) and hydroxylpropyl methyl cellulose. Novel mucoadhesive buccal tablets of oxytocin and formulation of nasal gels were prepared from D. indica (Kuotsu and Bandyopadhyay 2007; Metia and Bandyopadhyay 2008). Bal et al. (2012a, b) developed a mucoadhesive carvedilol microcapsule using the mucilage obtained from seeds of D. indica for encapsulation purpose. This microcapsule was found to be free flowing and usually spherical in shape. It was concluded that this mucilage was effective for sustained drug release, which can be employed to reduce the hypertension for a period of 24 h. Further, Sharma et al. (2013) conducted an experiment where they encapsulated pantoprazole sodium and metformin hydrochloride, respectively, using this mucilage. Good swelling properties and mucoadhesivity was found to perform at the intestinal pH. This finding suggested that the seed mucilage of D. indica has good potency for the purpose of drug encapsulation (Sharma et al. 2010). Nanoparticles are being extensively used as drug carrier for the treatment of diseases nowadays. Singh et al. (2013) prepared colloidal silver nanoparticles (SNP) using the aqueous extract of D. indica fruits as reducing agent and as a better substitute of sodium borohydride. This extract reduced AgNO3 to silver nanoparticles with stability more than 6 days, which suggested that D. indica could be employed as natural reducing agent for silver nanoparticles loaded formulation.

Future Prospects

Despite a broad range of medicinal properties of D. indica, very few investigations regarding chemical constituents and pharmacological aspects have been carried out. There is little evidence over the quantification of different active phytoconstituents responsible for important pharmacological activities. It is evident from the available literature that D. indica possesses adequate therapeutic potential and need to be explored further for chemical and pharmacological investigations. Current knowledge of D. indica shows great lacunae that need more biological investigations to be done on its plant extracts. Hence, further studies are highly required to explore the potential of its plant extracts against various diseases and search for molecular mechanisms underlying their action. Future studies are also required to evaluate the adverse effects, safety profile, and different biological activities of extracts as well as particular chemical constituents from D. indica in order to facilitate their clinical applications as modern medicines for human health.

Conclusion

Herbal medicines are the most extensively used therapeutics worldwide. To promote their proper use and to establish their potential as sources for new drugs, it is necessary to study medicinal plants having folklore reputation in a better and intensified way. The extensive literature survey as well as research reports revealed that D. indica is highly regarded to have good potential in the herbal medicine. Betulin and betulinic acid are the major constituents found to be present in almost all parts of this plant which can cure various human ailments and diseases. As the raw fruits are eaten by tribal communities, the juice of D. indica may be taken as energy drink due to their good nutritional value. It has previously been confirmed that D. indica have curing properties in wound healing, diabetes, cuts and burns, abdominal pains, and many more, but scientific evidence of these reports is yet not much developed. Various pharmacological investigations have been done using different plant parts such as leaves which have various activities like antioxidant, cytotoxic, antimicrobial, antidiarrheal, and anxiolytic, seeds which are hepatoprotective and antimicrobial, and fruits which are antileukemic. Despite a few toxicity reports, D. indica and most of its extracts were found to be non-toxic and exhibited ameliorative and hepatoprotective activities in several in vivo studies. The ethnopharmacological use of D. indica as food poisoning neutralizer also indicates its better safety profile and non-toxic nature. The current status of D. indica demands some biotechnological investigations including protein and gene expression for target identification and exploration of molecular mechanisms underlying the action at molecular levels.

Acknowledgments The authors are grateful to the Vice Chancellor of Babasaheb Bhimrao

Ambedkar University, Lucknow, India, for the support.

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Smitha, V. P., Ch, M. M., Kandra, P., Sravani, R., & Akondi, R. B. (2012). Screening of antimicrobial and antioxidant potentials of Acacia caesia, Dillenia pentagyna and Buchanania lanzan from Maredumilli forest of India. Journal of Pharmacy Research, 5(3), 1734–1738.

Somani, S. J., Badgujar, L. B., Sutariya, B. K., & Saraf, M. N. (2014). Protective effect of Dillenia indica L. on acetic acid induced colitis in mice. Indian Journal of Experimental Biology, 52, 876–881.

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Sundararamaiah, T., Ramraj, S. K., Rao, K. L., & Vimalabai, M. V. (1976). Isolation of the lupeol group of triterpenes from Dillenia indica Linn. and Diospyros perigrina. Journal of the Indian Chemical Society, 53, 638.

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INTERNATIONALE PHARMACEUTICA SCIENCIA
| Jan-March 2011 | Vol. 1 | Issue 1 |
Available online http://www.ipharmsciencia.com
©2011 IPS

                                                                                                                               REVIEW ARTICLE 

ABSTRACT

Plants are a source of large amount of drugs comprising to different groups such as Kaur, Harleen Kaur antispasmodics, emetics, anti-cancer, antimicrobials etc. A large number of the plants are claimed to possess the antibiotic properties in the traditional system and are also used extensively by the tribal people worldwide. It is now believed that nature has given the cure of every disease in one way or another. Plants have been known to relieve various diseases in Ayurveda. Therefore, the researchers today are emphasizing on evaluation and characterization of various plants and plant constituents against a number of diseases based on their traditional claims of the plants given in Ayurveda. Extraction of the bioactive plant constituents has always been a challenging task for the researchers. In this present review, an attempt has been made to give an overview of certain extractants and extraction processes with their advantages and disadvantages. 

Keywords: Medicinal plants, phytochemicals, extraction, solvent, screening. 

INTRODUCTION

Plant-derived substances have recently become of great interest owing to their versatile applications. Medicinal plants are the richest bio-resource of drugs of traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceutical intermediates and chemical entities for synthetic drugs [1].  

Extraction (as the term is pharmaceutically used) is the separation of medicinally active portions of plant (and animal) tissues using selective solvents through standard procedures. The products so obtained from plants are relatively complex mixtures of metabolites, in liquid or semisolid state or (after removing the solvent) in dry powder form, and are intended for oral or external use. These include classes of preparations known as decoctions, infusions, fluid extracts, tinctures, pilular (semisolid) extracts or powdered extracts. Such preparations have been popularly called galenicals, named after Galen, the second century Greek physician [2].  

Extraction methods used pharmaceutically involves the separation of medicinally active portions of plant tissues from the inactive/inert components by using selective solvents. During extraction, solvents diffuse into the solid plant material and solubilize compounds with similar polarity [1].

The purpose of standardized extraction procedures for crude drugs (medicinal plant parts) is to attain the therapeutically desired portions and to eliminate unwanted material by treatment with a selective solvent known as menstrum. The extract thus obtained, after standardization, may be used as medicinal agent as such in the form of tinctures or fluid extracts or further processed to be incorporated in any dosage form such as tablets and capsules. These products contains complex mixture of many medicinal plant metabolites, such as alkaloids, glycosides, terpenoids, flavonoids and lignans [3]. 

The general techniques of medicinal plant extraction include maceration, infusion, percolation, digestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic extraction by fermentation, countercurrent extraction, microwave-assisted extraction, ultrasound extraction (sonication), supercritical fluid extraction, and phytonic extraction (with hydrofluorocarbon solvents). For aromatic plants, hydrodistillation techniques (water distillation, steam distillation, water and steam distillation), hydrolytic maceration followed by distillation, expression and enfl eurage (cold fat extraction) may be employed. Some of the latest extraction methods for aromatic plants include headspace trapping, solid phase microextraction, protoplast extraction, microdistillation, thermomicrodistillation and molecular distillation [3].  

The basic parameters influencing the quality of an extract are [1]:

1. Plant part used as starting material

2. Solvent used for extraction

3. Extraction procedure

Effect of extracted plant phytochemicals depends on [1]:

1. The nature of the plant material

2. Its origin

3. Degree of processing

4. Moisture content

5. Particle size

The variations in different extraction methods that will affect quantity and secondary metabolite composition of an extract depends upon [1]:

1. Type of extraction

2. Time of extraction

3. Temperature

4. Nature of solvent

5. Solvent concentration

6. Polarity

Plant material

Plants are potent biochemists and have been components of phytomedicine since times immemorial; man is able to obtain from them a wondrous assortment of industrial chemicals. Plant based natural constituents can be derived from any  part of the plant like bark, leaves, flowers, roots, fruits, seeds, etc i.e. any part of the plant may contain active components. The systematic screening of plant species with the purpose of discovering new bioactive compounds is a routine activity in many laboratories. Scientific analysis of plant components follows a logical pathway. Plants are collected either randomly or by following leads supplied by local healers in geographical areas where the plants are found [5].

Fresh or dried plant materials can be used as a source for the extraction of secondary plant components. Many authors had reported about plant extract preparation from the fresh plant tissues. The logic behind this came from the ethano medicinal use of fresh plant materials among the traditional and tribal people. But as many plants are used in the dry form (or as an aqueous extract) by traditional healers and due to differences in water content within different plant tissues, plants are usually air dried to a constant weight before extraction. Other researchers dry the plants in the oven at about 40°C for 72 h. In most of the reported works, underground parts (roots, tuber, rhizome, bulb etc.) of a plant were used extensively compared with other above ground parts in search for bioactive compounds possessing antimicrobial properties [1, 4]. 

Choice of solvents

Successful determination of biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Properties of a good solvent in plant extractions includes, low toxicity, ease of evaporation at low heat, promotion of rapid physiologic absorption of the extract, preservative action, inability to cause the extract to complex or dissociate. The factors affecting the choice of solvent are quantity of phytochemicals to be extracted, rate of extraction, diversity of different compounds extracted, diversity of inhibitory compounds extracted, ease of subsequent handling of the extracts, toxicity of the solvent in the bioassay process, potential health hazard of the extractants [6]. 

The choice of solvent is influenced by what is intended with the extract. Since the end product will contain traces of residual solvent, the solvent should be nontoxic and should not interfere with the bioassay. The choice will also depend on the targeted compounds to be extracted [1, 4].

The various solvents that are used in the extraction procedures are:

1. Water: Water is universal solvent, used to extract plant products with antimicrobial activity. Though traditional healers use primarily water but plant extracts from organic solvents have been found to give more consistent antimicrobial activity compared to water extract. Also water soluble flavonoids (mostly anthocyanins) have no antimicrobial significance and water soluble phenolics only important as antioxidant compound [4].

2. Acetone: Acetone dissolves many hydrophilic and lipophilic components from the two plants used, is miscible with water, is volatile and has a low toxicity to the bioassay used, it is a very useful extractant, especially for antimicrobial studies where more phenolic compounds are required to be extracted. A study reported that extraction of tannins and other phenolics was better in aqueous acetone than in aqueous methanol [4, 6]. Both acetone and methanol were found to extract saponins which have antimicrobial activity [1].

3. Alcohol: The higher activity of the ethanolic extracts as compared to the aqueous extract can be attributed to the presence of higher amounts of polyphenols as compared to aqueous extracts. It means that they are more efficient in cell walls and seeds degradation which have unpolar character and cause polyphenols to be released from cells. More useful explanation for the decrease in activity of aqueous extract can be ascribed to the enzyme polyphenol oxidase, which degrade polyphenols in water extracts, whereas in methanol and ethanol they are inactive. Moreover, water is a better medium for the occurrence of the micro-organisms as  compared to ethanol [7]. 

The higher concentrations of more bioactive flavonoid compounds were detected with ethanol 70% due to its higher polarity than pure ethanol. By adding water to the pure ethanol up to 30% for preparing ethanol 70% the polarity of solvent was increased [8]. 

Additionally, ethanol was found easier to penetrate the cellular membrane to extract the intracellular ingredients from the plant material [9]. 

Since nearly all of the identified components from plants active against microorganisms are aromatic or saturated organic compounds, they are most often obtained through initial ethanol or methanol extraction [10]. 

Methanol is more polar than ethanol but due to its cytotoxic nature, it is unsuitable for extraction in certain kind of studies as it may lead to incorrect results.

4. Chloroform: Terpenoid lactones have been obtained by successive extractions of dried barks with hexane, chloroform and methanol with activity concentrating in chloroform fraction. Occasionally tannins and terpenoids will be found in the aqueous phase, but they are more often obtained by treatment with less polar solvents [10]. 

5. Ether: Ether is commonly used selectively for the extraction of coumarins and fatty acids [10].  

Dichloromethanol: It is another solvent used for carrying out the extraction procedures. It is specially used for the selective extraction of only terpenoids [10]. 

Table 1: Solvents used for active component extraction [10] 

Table 2: Structural features and activities of various phytochemicals from plants [10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22] 

Methods of extraction

Variation in extraction methods usually depends upon: 

1. Length of the extraction period, 

2. Solvent used, 

3. pH of the solvent, 

4. Temperature, 

5. Particle size of the plant tissues 

6. The solvent-to-sample ratio [4]. 

The basic principle is to grind the plant material (dry or wet) finer, which increases the surface area for extraction thereby increasing the rate of extraction. Earlier studies reported that solvent to sample ratio of 10:1 (v/w) solvent to dry weight ratio has been used as ideal [4]. 

Table 3: Mechanism of action of some phytochemicals [10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23]. 

Extraction procedures 

a. Plant tissue homogenization: Plant tissue homogenization in solvent has been widely used by researchers. Dried or wet, fresh plant parts are grinded in a blender to fine particles, put in a certain quantity of solvent and shaken vigorously for 5 - 10 min or left for 24 h after which the extract is filtered. The filtrate then may be dried under reduced pressure and redissolved in the solvent to determine the concentration. Some researchers however centrifuged the filtrate for clarification of the extract [4]. 

b. Serial exhaustive extraction: It is another common method of extraction which involves involves successive extraction with solvents of increasing polarity from a non polar (hexane) to a more polar solvent (methanol) to ensure that a wide polarity range of compound could be extracted. Some researchers employ soxhlet extraction of dried plant material using organic solvent. This method cannot be used for thermolabile compounds as prolonged heating may lead to degradation of compounds [4]. 

c. Soxhlet extraction: Soxhlet extraction is only required where the desired compound has a limited solubility in a solvent, and the impurity is insoluble in that solvent. If the desired compound has a high solubility in a solvent then a simple filtration can be used to separate the compound from the insoluble substance. The advantage of this system is that instead of many portions of warm solvent being passed through the sample, just one batch of solvent is recycled. This method cannot be used for thermolabile compounds as prolonged heating may lead to degradation of compounds [24]. 

d. Maceration: In maceration (for fluid extract), whole or coarsely powdered plantdrug is kept in contact with the solvent in a stoppered container for a defined period with frequent agitation until soluble matter is dissolved. This method is best suitable for use in case of the thermolabile drugs [1]. 

e. Decoction: this method is used for the extraction of the water soluble and heat stable constituents from crude drug by boiling it in water for 15 minutes, cooling, straining and  passing sufficient cold water through the drug to produce the required volume [2]. 

f. Infusion: It is a dilute solution of the readily soluble components of the crude drugs. Fresh infusions are prepared by macerating the solids for a short period of time with either cold or boiling water [2]. 

g. Digestion: This is a kind of maceration in which gentle heat is applied during the maceration extraction process. It is used when moderately elevated temperature is not objectionable and the solvent efficiency of the menstrum is increased thereby [2].

h. Percolation: This is the procedure used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts. A percolator (a narrow, cone-shaped vessel open at both ends) is generally used. The solid ingredients are moistened with an appropriate amount of the specified menstrum and allowed to stand for approximately 4 h in a well closed container, after which the mass is packed and the top of the percolator is closed. Additional menstrum is added to form a shallow layer above the mass, and the mixture is allowed to macerate in the closed percolator for 24 h. The outlet of the percolator then is opened and the liquid contained therein is allowed to drip slowly. Additional menstrum is added as required, until the percolate measures about threequarters of the required volume of the finished product. The marc is then pressed and the expressed liquid is added to the percolate. Sufficient menstrum is added to produce the required volume, and the mixed liquid is clarified by filtration or by standing followed by decanting [3]. 

i. Sonication: The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz; this increases the permeability of cell walls and produces cavitation. Although the process is useful in some cases, like extraction of rauwolfi a root, its large-scale application is limited due to the higher costs. One disadvantage of the procedure is the occasional but known deleterious effect of ultrasound energy (more than 20 kHz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules [3].

Phytochemical screening: Phytochemical examinations were carried out for all the extracts as per the standard methods.

1. Detection of alkaloids: Extracts were dissolved individually in dilute Hydrochloric acid and filtered. 

a) Mayer’s Test: Filtrates were treated with Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow coloured precipitate indicates the presence of alkaloids. 

b) Wagner’s Test: Filtrates were treated with Wagner’s reagent (Iodine in Potassium Iodide). Formation of brown/reddish precipitate indicates the presence of alkaloids. 

c) Dragendroff’s Test: Filtrates were treated with Dragendroff’s reagent (solution of Potassium Bismuth Iodide). Formation of red precipitate indicates the presence of alkaloids. 

d) Hager’s Test: Filtrates were treated with Hager’s reagent (saturated picric acid solution). Presence of alkaloids confirmed by the formation of yellow coloured precipitate. 

2. Detection of carbohydrates: Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates were used to test for the presence of carbohydrates. 

a) Molisch’s Test: Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube. Formation of the violet ring at the junction indicates the presence of Carbohydrates. 

b) Benedict’s Test: Filtrates were treated with Benedict’s reagent and heated gently. Orange red precipitate indicates the presence of reducing sugars. 

c) Fehling’s Test: Filtrates were hydrolysed with dil. HCl, neutralized with alkali and heated with Fehling’s A & B solutions. Formation of red precipitate indicates the presence of reducing sugars. 

3. Detection of glycosides: Extracts were hydrolysed with dil. HCl, and then subjected to test for glycosides.

a) Modified Borntrager’s Test: Extracts were treated with Ferric Chloride solution and immersed in boiling water for about 5 minutes. The mixture was cooled and extracted with equal volumes of benzene. The benzene layer was separated and treated with ammonia solution. Formation of rose-pink colour in the ammonical layer indicates the presence of anthranol glycosides. 

4. Legal’s Test: Extracts were treated with sodium nitropruside in pyridine and sodium hydroxide. Formation of pink to blood red colour indicates the presence of cardiac glycosides.

5. Detection of saponins 

a) Froth Test: Extracts were diluted with distilled water to 20ml and this was shaken in a graduated cylinder for 15 minutes. Formation of 1 cm layer of foam indicates the presence of saponins.

b) Foam Test: 0.5 gm of extract was shaken with 2 ml of water. If foam produced persists for ten minutes it indicates the presence of saponins. 

6. Detection of phytosterols 

a) Salkowski’s Test: Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of Conc. Sulphuric acid, shaken and allowed to stand. Appearance of golden yellow colour indicates the presence of triterpenes.

b) Libermann Burchard’s test: Extracts were treated with chloroform and filtered. The filtrates were treated with few drops of acetic anhydride, boiled and cooled. Conc. Sulphuric acid was added. Formation of brown ring at the junction indicates the presence of phytosterols.

7. Detection of phenols 

Ferric Chloride Test: Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols.

8. Detection of tannins

Gelatin Test: To the extract, 1% gelatin solution containing sodium chloride was added. Formation of white precipitate indicates the presence of tannins. 

9. Detection of flavonoids

a) Alkaline Reagent Test: Extracts were treated with few drops of sodium hydroxide solution. Formation of intense yellow colour, which becomes colourless on addition of dilute acid, indicates the presence of flavonoids.

b) Lead acetate Test: Extracts were treated with few drops of lead acetate solution. Formation of yellow colour precipitate indicates the presence of flavonoids.

10. Detection of proteins and aminoacids

a) Xanthoproteic Test: The extracts were treated with few drops of conc. Nitric acid. Formation of yellow colour indicates the presence of proteins.

b) Ninhydrin Test: To the extract, 0.25% w/v ninhydrin reagent was added and boiled for few minutes. Formation of blue colour indicates the presence of amino acid.

11. Detection of diterpenes

Copper acetate Test: Extracts were dissolved in water and treated with 3-4 drops of copper acetate solution. Formation of emerald green colour indicates the presence of diterpenes [25, 26, 27].

CONCLUSION

Non standardized procedures of extraction may lead to the degradation of the phytochemicals present in the plants and may lead to the variations thus leading to the lack of reproducibility. Efforts should be made to produce batches with quality as consistent as possible (within the narrowest possible range) and to develop and follow the best extraction processes.

ACKNOWLEDGEMENT

The authors are thankful to Dr. Monica Gulati, Dean, Department of Pharmaceutical Sciences, Lovely Professional University, Phagwara (Punjab) for providing necessary facilities and cooperation during this research work.  

REFERENCES 

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12. Sutar N, Garai R, Sharma US, Sharma UK. Anthelmintic activity of Platycladus orientalis leaves extract. International Journal of Parasitology Research 2010; 2(2): 1-3. 

13. Mute VM. Anthelmintic effect of Tamarind indica linn leaves juice exract on Pheretima posthuma. International journal of pharma research and development 2009; 7: 1-6. 

14. Sharma US, Sharma UK, Singh A, Sutar N, Singh PJ. In vitro anthelmintic activity of Murraya koenigii linn. Leaves extracts. International journal of pharma and bio sciences 2010; 1(3): 1-4. 

15. Mali RG, Mahajan SG, Mehta AA. In-vitro anthelmintic activity of stem bark of Mimusops elengi Linn. Pharmacognosy Magazine 2007; 3(10): 73-76. 

16. Patel J, Kumar GS, Qureshi MS, Jena PK. Anthelmintic activity of ethanolic extract of whole plant of Eupatorium odoratum. International Journal of Phytomedicine 2010; 2: 127-132. 

17. Roy H. Preliminary phytochemical investigation and anthelmintic activity of Acanthospermum hispidum DC. Journal of Pharmaceutical Science and Technology 2010; 2 (5): 217-221. 

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20. Vidyadhar S, Saidulu M, Gopal TK, Chamundeeswari D, Rao U, Banji D. In vitro anthelmintic activity of the whole plant of Enicostemma littorale by using various extracts. International journal of applied biology and pharmaceutical technology 2010; 1(3): 1119-1125. 

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Chapter 9

Chemistry, Therapeutic Attributes, and Biological Activities of Dillenia indica Linn

Ashok K. Singh and Sudipta Saha 

A. K. Singh · S. Saha (*)

Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India

e-mail: sudiptapharm@gmail.com

© Springer Nature Singapore Pte Ltd. 2019

R. C. Sobti et al. (eds.), Environmental Biotechnology: For Sustainable Future,

https://doi.org/10.1007/978-981-10-7284-0_9

Dillenia indica Linn. (Dilleniaceae) is generally known as elephant apple and locally known as outenga. The vernacular names are chalta, chulta, bhavya, karambel, ouu, and ramphal. This evergreen deciduous tree is markedly disseminated in the seasonal tropics of many Asian countries, in India from Himalaya to south India. The different parts of this plant have been prevalently investigated for the plethora of biological activities including anticancer, antidiabetic, antihyperlipidemic, antileukemic, antioxidant, antimutagenic, antimicrobial, antinociceptive, antidiarrheal, and hairweaving activities. Differently prepared extracts of this plant have been reported mainly to contain a wide range of flavonoids, triterpenoids (lupene-type), phytosteroids, phenolics, alcohols, and ketones and an anthraquinone. Several phytochemical investigations revealed substantial presence of various types of active constituents including β-sitosterol, stigmasterol, betulin, betulinic acid, kaempferol, myricetin, quercetin, dillenetin and rhamnetin. Among these the major chemical constituents are betulin and betulinic acid (lupene-type triterpenoids) that show a wide spectrum of pharmacological activities like anti-HIV, anticancer, antimalarial, anti-inflammatory, etc. The present chapter thus approaches to highlight on phytochemistry, traditional and therapeutic uses, and biological activities of Dillenia indica.

Introduction

A vast amount of knowledge and practices on herbal medicinal systems have been transmitted through the ages. For centuries, medicinal plants were the only resources available for the treatment of several diseases which afflicted humanity (Ozdemir and Alpınar 2015). Numerous of these plants are uncommon, endemic, and found only in forest region. There is neither biological data nor satisfactory information that prompted their rarity in the natural surroundings (Kerrigan et  al. 2011). Correspondingly, there are many plant species which have been utilized by tribal and folk communities of different forest regions of India; however, their medicinal and also pharmacological esteem is yet obscure as these plants are hardly available. There are many plant species which have been utilized by tribal groups of India; however, their restorative and also pharmacological knowledge is yet obscure as these plants are not easily accessible and studied. Among these, there are few plants belonging to family Dilleniaceae which have not gained much popularity but have interesting medicinal values. The genus Dillenia has 60 species; however, only a few of them are reported to have important phytochemicals and thereby enrich their medicinal values. These species are D. indica, D. pentagyna, D. suffruticosa, D. andamanica, D. serrata, D. sumatrana, D. aurea, D. bracteata, D. excelsa, D. ovata, D. papuana, D. parviflora, D. philippinensis, D. pulchella, D. reticulata, D. scabrella, D. eximia, and D. triquetra. Only two plants D. indica Linn. and D. pentagyna Roxb. are available in India. D. indica has been extensively studied and a more commonly employed medicinal plant in different parts of India (Dickison 1979). Several research works have been conducted on the isolation and quantification of the different phytochemicals from various parts of D. indica; however, very few phytochemical investigations have been performed from D. pentagyna.

D. indica grows in moist and evergreen forests of India (The Wealth of India 1952). This plant is known for its lemon-flavored fruits that are used to prepare jam and jellies and as flavoring agent for curries (Sharma et al. 2001). This plant has been exploited by tribal and folk communities of various regions where the fruits of D. indica are eaten raw, but people are not much familiar with its medicinal values (Pradhan and Badola 2008; Dubey et al. 2009; Sharma and Pegu 2011). The leaf, stem bark, fruit, and flower of D. indica contain a wide range of flavonoids, triterpenoids (lupene-type), phytosteroids, phenolics, alcohols, and ketones and an anthraquinone. The substantial presence of various types of active constituents including β-sitosterol, stigmasterol, betulin, betulinic acid, kaempferol, myricetin, quercetin, dillenetin and rhamnetin enriches the diversity of therapeutically important phytochemicals in D. indica. The major chemical constituents among these are betulin and betulinic acid (lupene-type triterpenoids) that show vast and wide range of medicinal values.

The present review is an attempt to compile the detailed phytochemistry, traditional and therapeutic uses, as well as biological activities of this plant. This review may be helpful to explore further excellent phytochemistry and medicinal potentials of D. indica for the preparation of various types of formulations in future. 

Chemistry

The significant classes of chemical constituents extracted from D. indica are flavonoids and triterpenoids (lupene-type). Other isolated compounds including phytosteroids, diterpene, ionone, phenolics, anthraquinone, alcohols, and ketones also enhance the diversity of phytochemistry in D. indica. As per our extensive search, a total of 34 compounds isolated from D. indica are included in this review which may lead to further research and noble challenge to discover new chemical constituents from this plant. These compounds are listed in Table 9.1, and their chemical structures are displayed in Fig. 9.1.

Stem bark of D. indica contains triterpenoids like lupeol, betunaldehyde, and betulinic acid; flavonoids like kaempferol, dillenetin, rhamnetin, dihydroisorhamnetin, myricetin, naringenin, and quercetin; and 10% tannin (Shah 1978; Khanum et al. 2007; Khare 2007). The ethanol extract of stem bark is enriched with two flavonoids, kaempferol and quercetin, as well as a triterpenoid (Srivastava and Pande 1981). Parvinet al. (2009) acquired methanolic extract of stem after partitioning with n-hexane and isolated four compounds, viz., lupeol, betulinic acid, betunaldehyde, and stigmasterol, using column chromatographic separation.

Leaves of D. indica contain betulinic acid, betulin, lupeol, and β-sitosterol (Dan and Dan 1980). The petroleum ether extract of leaves contains betulin, β-sitosterol, cycloartenone, and n-hentriacontanol, whereas chloroform extract has betulinic acid (Mukherjee and Badruddoza 1981). Methanolic extract of leaves after fractionation with n-hexane and chloroform also has compounds like betulinic acid, β-sitosterol, dillenetin, and stigmasterol (Muhit et al. 2010). Phytochemicals have also been investigated from acid hydrolyzed extracts of dried leaves which demonstrated the presence of kaempferol, whereas fresh leaves were found to contain dihydrokaempferide and naringenin-7-diglucoside which get further oxidized to ten corresponding flavonols (Bate-Smith and Harborne 1971). Kumar et al. (2010) isolated and quantified betulinic acid using validated HPLC method from various fractions such as methanol, ethyl acetate, n-butanol, and water. The highest concentration among them was found in ethyl acetate fraction.

Fruit of D. indica contains about 34% of total phenolics in methanolic extract (Abdille et al. 2005), isorhamnetin (Pavanasasivam and Sultanbawa 1975a), lupeol, betulin, β-sitosterol (Sundararamaiah et al. 1976), and polysaccharide like arabinogalactan. Uppalapati and Rao (1980) reported the presence of steroids, saponins, fixed oil, free amino acids, glycosides, tannins, and sugars in the seeds of D. indica. These scientific reports collectively revealed that betulin, betulinic acid, and β-sitosterol are present in almost all parts of D. indica.