Possibility of New Drug Discovery
Abdullah Al-Nahain,
1Rownak Jahan,
2Taufiq Rahman,
3Md Nurunnabi
4and Mohammed Rahmatullah
1,*
Introduction
Observation of indigenous medicinal practices of various communities throughout the world has always proved to be an excellent route to the discovery of many important modern drugs (Gilani and Rahman 2005, Gohil et al. 2010). The importance of traditional medicinal practices has recently gained attention of the scientifi c community. A number of factors have contributed to this phenomenon. First, all ancient and quite a number of emerging modern diseases cannot be cured with allopathic medicine. Second, many modern (allopathic) medicines have developed drug-resistant vectors and microbes. Third, a number of allopathic medicines have adverse side-effects. Even common analgesic drugs like aspirin and paracetamol can develop gastric ulceration or hepatotoxicity from prolonged usage or over-dosage. Fourth, allopathic medicine is out of reach of many rural people because of high costs and lack of accessibility to modern doctors and clinics. Fifth, and fi nally, people still believe in traditional medicine from either habit or from fi nding such medicines benefi cial (Jahan et al. 2012).
Among many reported medicinal plants, Centella asiatica has broadly contributed to treatment of different diseases (Ramaswamy et al. 1970, Brinkhaus et al. 2000, Höfl ing et al. 2010, Jahan et al. 2012).
As a medicine it has been used in Ayurvedic tradition of India for thousands of years and listed in the historic ‘Sushruta Samhita’, an ancient Indian medical text (Chopra et al. 1986, Diwan et al. 1991). This plant is also known as gotu kola and shady, moist, or marshy areas favor its growth. It is distributed widely in many parts of the world, including India, Sri Lanka, Madagascar, South Africa, Australia, China, and Japan (Zheng 2007, Satake et al. 2007).
1 Department of Pharmacy, University of Development Alternative, Dhanmondi, Dhaka-1209, Bangladesh.
2 Department of Biotechnology & Genetic Engineering, University of Development Alternative, Dhanmondi, Dhaka-1209, Bangladesh.
3 Department of Pharmacology, University of Cambridge, Tennis Court Road, CB2 1PD, Cambridge, UK.
4 Department of Chemical and Biological Engineering, Korea National University of Transportation, Chungju, Republic of Korea, 380-702.
* Corresponding author: rahamatm@hotmail.com
Centella asiatica (CA) is a small creeping herb with shovel shaped leaves emerging alternately in clusters at stem nodes. This is a prostate, sparingly hairy or nearly smooth herb. The stems root at the nodes.
The leaves are rounded to reniform, 2 to 5 centimeters wide, horizontal, more or less cupped, rounded at the tip, and kidney-shaped or heart shaped at the base, the rounded lobes often overlapping. The petioles are erect and long. The peduncles occur in pairs of three, are less than 1 centimeter long, and usually bear three sessile fl owers. The petals are dark-purple, ovate, and about 1 millimeter long. The fruit is minute, ovoid, white or green, and reticulate, each with nine sub similar longitudinal ridges. The runners lie along the ground and the inch long leaves with their scalloped edges rise above on long reddish petioles. The insignifi cant greenish- to pinkish-white fl owers are borne in dense umbels on separate stems in the summer.
Since ancient times, it has been used to treat different diseases like wound healing, anti-infl ammatory, memory enhancing, strength promoting, immune booster, anti-anxiety, anti-epilepsy and anti-stress substance (Han et al. 2003). Centella asiatica has been clinically used in mentally retarded children and also in treatment of anxiety neurosis. This plant has also been found to improve short-term memory and learning (Meena et al. 2012).
Although a large number of studies reported over the last few decades to search out biologically active components of the plant and their mechanisms of action, the outcome of these studies is still unsatisfactory. Although there have been several claims regarding the underlying mechanisms involved in the biological actions of this herb, more scientifi c data are needed to justify its ever increasing use. The potential therapeutic substances in Centella asiatica are saponin-containing triterpene acids and their sugar esters, of which asiatic acid, madecassic acid and asiaticosides are considered to be the most important (Brinkhaus et al. 2000).
The present chapter aims to list the detailed account of the plant, its activity regarding therapeutic uses, pharmacology, and mechanisms of action based on preclinical and clinical studies.
Ethnomedicinal uses
The major ethnomedicinal uses of the plant appears to be to alleviate gastrointestinal disorders like dysentery, constipation, stomach problems, indigestion and loss of appetite, and to enhance memory or to serve as nerve stimulant. Altogether 23 ethnomedicinal uses were collected from the available literature, of which six dealt with the plant’s use in gastrointestinal disorders, and four uses were linked to memory or uses associated with brain functions like stimulation of nerve or for treatment of mental retardation. However, the uses of the plant were quite diverse overall, for the plant was used for treatment of headache, toothache, cuts and wounds, leucorrhea, skin disorders (like eczema, carbuncle), hemorrhoids, as an antidote to poison, urinary troubles and leucorrhea, pneumonia, syphilis, liver problems (like jaundice), sexual weakness in males, fever, sun stroke, rickets, cardiovascular disorders, leprosy, tuberculosis, asthma, and varicocele.
Altogether, 12 ethnomedicinal reports were from India, three from Nepal, six from Bangladesh, and two from Africa. Thus the major ethnomedicinal uses of the plant seemed to be centered in the Indian sub- continent (Jahan et al. 2012).
Phytochemical constituents
It is reported that Centella asiatica contains mainly four active bio-active compounds. A list of summarized phytochemical constituents of Centella asiatica is presented in Table 2.1. Figure 2.1 shows the chemical structure of the relevant phytochemicals of Centella asiatica.
Pharmacological activity studies with CA and isolated constituents Neuroprotective effect
In vivo studies in rats exhibited signifi cant anti-oxidant properties by reducing brain regional lipid peroxidation (LPO) and protein carbonyl (PCO) levels after oral administration of Centella asiatica (300 mg/kg body weight/day) for 60 days (Subathra et al. 2005). In another study, signifi cant reduction
Table 2.1. Phytochemical constituents of Centella asiatica.
Groups Example References
Triterpenes acid Asiatic acid, madecassic acid, terminolic acid, centic acid, centoic acid, centelloic acid, indcentoic acid, brahmic acid, isobrahmic acid, betulic acid, madasiatic acid.
(Rumalla et al. 2010, Bhavna and Joyoti 2011)
Glycosides Asiaticoside A, Asiaticoside B, madecassioside, centelloside. (Bhavna and Joyoti 2011, Tabassum et al. 2013)
Alkaloids hydrocotylin (Bhavna and Joyoti 2011)
Flavonoids Castiloferol, castelicetin (Bhavna and Joyoti 2011)
Volatile and fatty acids
Oleic acid, linolenic acid, palmitic acid, stearic acid, lignoceric acid.
(Bhavna and Joyoti 2011) Amino acids Aspartic acid, glycine, glutamic acid, phenylalanine. (Bhavna and Joyoti 2011) Others Sterols, tannins, sugars, inorganic salt. (Bhavna and Joyoti 2011)
Figure 2.1. Structure of phytochemical constituents of Centella asiatica.
of LPO and PCO by Centella asiatica aqueous extract was confi rmed in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced neurotoxicity in aged Sprague-Dawley rats (Haleagrahara et al.
2010). To investigate the effectiveness, the rats were treated with the extract at 300 mg/kg body weight for 21 days and effects on oxidative biomarker levels in corpus striatum and hippocampus homogenate was examined. Extract of Centella asiatica strongly inhibited neurotoxicity (Haleagrahara et al. 2010).
In addition to the whole plant extract, neuroprotective effect of isolated asiaticoside from Centella asiatica was carried out in MPTP induced rats model of Parkinsonism. On the 14th day, after administration of asiaticoside, rats were sacrifi ced and substantia nigra (SN) and striatum were dissected. Neuroprotective effect was quantitatively determined be estimating dopamine (DA) and its metabolites in striatum and malonyldialdehyde (MDA) contents, reduced glutathione (GSH) level and gene expression level in SN (Xu et al. 2012).
A study by Soumyanath et al. on male Sprague-Dawley rats with ethanolic and aqueous extracts of the plant showed that the ethanolic extract of the plant aided to accelerate functional recovery as well as axonal regeneration when the extract was provided in their drinking water (Soumyanath et al. 2005).
Though this study failed to show the neuroprotective effect of aqueous extract of Centella asiatica, another study proved to have neuroprotective effect from aqueous extract against 3-nitropropionic-acid (3-NPA)- induced oxidative stress in the brain of prepubertal mice (Shinmol et al. 2008). The degree of oxidative stress in cytoplasm of brain regions of male mice (4 weeks-old) given CA prophylaxis (5 mg/kg bw) for 10 days followed by 3-NPA administration (i.p.75 mg/kg bw) on the last two days was determined.
Signifi cantly, Centella asiatica treated mice showed complete amelioration of 3-NPA-induced oxidative stress (Shinmol et al. 2008).
Ethanolic extract of Centella asiatica administration signifi cantly improved neurobehavioral activity and diminished infarction volume along with the restored histological morphology of brain in MCAO rats (Tabassum et al. 2013).
Both methanolic and ethanolic extract as well as the phytochemical asiaticoside of Centella asiatica imparted anxiolytic activity (Wijeweera et al. 2006).
Fresh leaf extract of Centella asiatica was applied to fi nd out its therapeutic benefi ts on adult rats on dendritic morphology of amygdaloid neurons, one of the regions concerned with learning and memory.
Rats were fed with 2, 4 and 6 mL/(day per kg body weight) of fresh leaf extract for two, four and six weeks.
Centella asiatica treated rats exhibited a signifi cant increase in the dendritic length (intersections) and dendritic branching points in amygdaloid neurons of the rats depending on the dose and time (Rao et al.
2009). A similar effect following the same experimental procedure by this group was found in hippocampal CA3 neurons (Gadahad et al. 2008). Another study by Rao’s group also evaluated the therapeutic benefi ts of leaf extracts of Centella asiatica during the rat growth spurt period on the dendritic morphology of hippocampal CA3 neurons. Both dendritic length (intersections) and dendritic branching points along the length of both apical and basal dendrites in rats were remarkably increased due to treatment with extract of Centella asiatica (Rao et al. 2006). Rao et al. showed that orally administered fresh leaf juice of Centella asiatica enhanced learning performance as well as memory retention (Rao et al. 2005).
Possible neuroprotective effects of Centella asiatica was explored against D-galactose induced cognitive impairment, oxidative and mitochondrial dysfunction in mice. The results showed signifi cant improvement in behavioral alterations, mitochondrial enzyme complex activities as well as oxidative damage (Kumar et al. 2011). In their study, they found that extract of Centella asiatica signifi cantly improved memories and cognitive defi cit. Additionally, the extract was able to attenuate oxidative stress (Kumar et al. 2009).
The neuroprotective role of Centella asiatica was evaluated by examining Na, K and Ca/Mg ATPase in different regions of the brain during pentylenetetrazol (PTZ)-induced epilepsy and on antiepliptic treatment with n-hexane, ethyl acetate, n-butanol and aqueous extract of CA. Pentylenetetrazol (PTZ)- induced seizures rats were used to investigate anti-convulsant effect of different extracts of Centella asiatica. Considerable recovery of acetylcholine and acetylcholine esterase was observed in Centella asiatica pretreated rats (Visweswari et al. 2010).
Orally administered aqueous extract of Centella asiatica showed remarkable improvement in learning (Gupta et al. 2003).
Another study showed signifi cant therapeutic benefi ts of Centella asiatica to treat conditions associated with increased phospholipase [PLA2] activity in the brain, such as epilepsy, stroke, multiple sclerosis and other neuropsychiatric disorders (Barbosa et al. 2008).
A clinical study in men was conducted to evaluate the benefi cial effects of Centella asiatica against different neurological disorders. Seventy percent hydroethanolic extract of Centella asiatica was administered (500 mg/capsule, twice daily, after meals). On the basis of Hamilton’s Brief Psychiatric Rating Scale (BPRS), it was observed that Centella asiatica not only attenuated anxiety related disorders but it also signifi cantly (p < 0.01) reduced stress phenomenon and its correlated depression (Jana et al. 2010).
Methanolic, chloroform and aqueous extract of Centella asiatica was investigated to evaluate their effect on cognitive functions in rats. Effects on learning and memory were observed by using shuttle box, step through, step down and elevated plus maze paradigms. Among the extracts, only the aqueous extract showed signifi cant cognitive improvement (Kumar and Gupta 2002).
ECa 233, a standardized extract of Centella asiatica containing triterpenoids not less than 80%, showed strong anxiolytic activity following a dark-light box and an open-fi eld tests in rats (Wanasuntronwong et al. 2012).
Wound healing
Dose dependent wound healing activity was observed from asiaticoside, isolated from Centella asiatica, in guinea pig punch wounds. Tensile strength, epitheliazation, collagen content and hydroxypyroline amount were increased after topical application of 0.2% asiaticoside. A similar effect was also found in streptozocin diabetic rats, where healing was delayed, using 0.4% asiaticoside topically (Shukla et al. 1999).
In another study, cellular proliferation as well as synthesis of collagen was accelerated when different formulations like cream, ointment and gel containing Centella asiatica extract (CAE) was applied to open wounds in experimental rats thrice daily for 24 days (Sunilkumar et al. 1998). Epitheliazation was faster along with higher wound contraction. Effects of CAE on keratinization were also shown to enhance wound healing by thickening skin in areas of infection (Poizot et al. 1978).
In another study, ethanolic extract of the plant was applied on Wistar albino rats using incision, excision, and dead space wounds models. The extract exhibited signifi cant wound healing activities by accelerating epitheliazation and increasing wound contraction (Shetty et al. 2006).
Supplementation of aqueous extract of Centella asiatica strongly inhibited proliferation of Rabbit Corneal Epithelial (RCE) cells in an in vitro wound healing model. The study also found that expression of differentiation markers and cell cycle were not altered due to application of the extract of Centella asiatica (Ruszymah et al. 2012).
Effectiveness of Centella asiatica was carried in 200 diabetic patients. Study groups were prescribed two capsules three times a day. Patients belonging to the study group experienced better wound contraction without any serious side effects (Paocharon et al. 2010). Water extract of Centella asiatica and asiaticoside (AC), one of the major phytochemical of its constituents, were observed in acetic acid induced gastric ulcers. Treatment with CE and AC showed signifi cant wound healing depending on the dose (Cheng and Koo 2000).
Wound healing activities of sequential hexane, ethyl acetate, methanol and water extract of Centella asiatica was observed in incision and partial thickness burn wound models in Sprague-Dawley rats. All extracts showed signifi cant wound healing activities by showing higher tensile strength. In addition, fully developed epitheliazation and keratinization was also observed (Somboonwong et al. 2012).
Topically applied ethanolic extract of Centella asiatica exhibited strong wound healing activities on rat dermal wound. The extract treated wounds enhanced epitheliazation and contraction of wounds compared to control wounds (Sugna et al. 1996).
Wound healing activities of Centella asiatica were accelerated when it was administered with collagen (Babu et al. 2011). Centella asiatica extract impregnated collagen and cross-linked collagen scaffolds were formulated and different concentrations were administered to wounds developed Wistar rats. It was found that 1.5% w/v of both formulations exhibited better wound-healing activities compared to the control (79.9 to 80.68%, respectively) (Babu et al. 2011).
Anti-cancer activity
Partially purifi ed fractions of Centella asiatica were found to inhibit signifi cant cell proliferation than crude extract of the plant in Ehrlich ascites tumor cells (EAC) and Dalton’s lymphoma ascites tumor cells (DLA) while no toxic effects were observed when the extracts were applied to normal human lymphocytes (Babu et al. 1995). Partially purifi ed fraction also considerably suppressed multiplication of mouse lung fi broblast cells at 8 μg/ml in long term culture. Both extracts retarded the development of solid and ascites tumors and increased the life span of these tumor-bearing mice after oral administration. In vitro experiment in HepG2 cell line showed that the juice of Centella asiatica exhibited signifi cant cytotoxic effect on tumor cells while it was totally non-toxic to normal cells (Hussin et al. 2014).
Water extract of Centella asiatica was reported to have inhibitory effects on azomethane-induced aberrant crypt focus formation and carcinogenesis in the intestine of F344 rats (Bunpo et al. 2004).
Cytotoxic effects of asiatic acid (a constituent of CA) was found in U-87MG human glioblastoma cells depending on the dose (Cho et al. 2006). Topical application of asiatic acid showed anti-tumor promoting effect against 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated skin tumor genesis in 7, 12-dimethylbenz[a] anthracene (DMBA) initiated ICR mice (Park et al. 2007).
A recent study showed that asiatic acid from Centella asiatica signifi cantly inhibited cell viability and induced apoptosis in human melanoma SK-MEL-2 cells depending on time and dose (Park et al.
2005). Anti-cancer effect by the same phytochemical was found in human adenocarcinoma cell line HT-29 (Bunpo et al. 2005).
Orally administered aqueous extract of Centella asiatica at 500 and 1000 mg/kg body weight for 30 days increased life span and decreased tumor volume remarkably (Rai et al. 2011). Signifi cant cytotoxicities were found in brine shrimp lethality assays (Ullah et al. 2009). Asiatic acid markedly inhibited colon cancer cell proliferation and increased apoptosis (Tang et al. 2009).
Anti-tumor activity by asiatic acid isolated from Centella asiatica was observed by inhibiting angiogenesis (Kavitha et al. 2011).
Aqueous extract of Centella asiatica was investigated on the induction of spermatogenic cell apoptosis in male rats. Aqueous leaf extract was orally administered (10, 50, 80 and 100 mg/kg) daily for 60 days while the control group received just water. After 60 days, body and testis weight were measured and blood samples were taken from the heart. Apoptosis and histological changes were determined by collecting tissue samples obtained from rat testes following TUNEL assay and hematoxylin and eosin stain. Signifi cant reduction of sperm count, motility, and viability and the number of spermatogenic cells in the seminiferous tubules were observed in Centella asiatica extract treated mice. Beside this number of apoptotic germ cells per seminiferous tubule cross-section was signifi cantly increased in the experimental group (18.11 ± 3.5) compared with the control group (8.7 ± 0.81) (P < 0.05). Serum testosterone, follicle- stimulating hormone, and luteinizing hormone levels were reduced. In addition, weight of testis decreased remarkably (Heidari et al. 2012).
Anti-microbial activity
Different types of extract of Centella asiatica have been investigated to demonstrate anti-bacterial activity.
In one study, it was found that petroleum ether, ethanol and chloroform extract exhibited signifi cant anti- bacterial activity than water and n-hexane extract (Dash et al. 2011). On the other hand, there was no anti-bacterial activity by n-hexane extract against Escherichia coli.
Broad spectrum antimicrobial activity was observed against E. coli ATCC 25922, Staphylococcus aureus ATCC 25923, Vibrio parahaemolyticus ATCC 17802 and Pseudomonas aeruginosa ATCC 27853 and 45 fresh clinical isolates like Vibrio cholera 01, V. cholerae 0139, species of Shigella, Salmonella typhimurium, Aeromonas hydrophila, entero-aggregative E. coli and Candida albicans (Mamtha et al. 2004).
In another study, anti-microbial effect of petroleum ether, ethanol and water extract of Centella asiatica was demonstrated where only ethanolic extract showed better anti-microbial activity than the others (Jagtap et al. 2009).
Broad spectrum antibacterial activities against Gram-positive (Bacillus subtilis, S. aureus) and Gram- negative (E. coli, Pseudomonas aeruginosa, Shigella sonnei) organisms were found with the essential oil extract of Centella asiatica (Oyedeji et al. 2005).
Signifi cant anti-bacterial and anti-fungal activities were demonstrated from calli and regenerated plant extracts Centella asiatica (Bibi et al. 2011).
Anti-microbial activity of aqueous extracts, aqueous-alcoholic extracts obtained with 30% ethanol, and aqueous-alcoholic extracts obtained with 60% ethanol were investigated by Kedzia et al. Among all extracts, aqueous-ethanol extracts (30% ethanol) exhibited signifi cant bacteriostatic properties on Gram- positive and Gram-negative bacteria (Kedzia et al. 2007).
Depending on the concentration, ethanolic and aqueous extract of Centella asiatica showed strong inhibitory effects (determined by zone of inhibition), where ethanolic extract showed better activity than aqueous against S. aureus (Udoh et al. 2012). In another study, ethanolic extract of Centella asiatica showed signifi cant anti-microbial activity against S. aureus from milk samples of dairy cows, while aqueous extract did not show any activity (Taemchuay et al. 2009). Strong anti-microbial activity by methanolic extract of Centella asiatica was also found by Rozarina et al. (2013).
Anti-microbial activity of aqueous extract of Centella asiatica was investigated in combination with four other medicinal plants against a wide range of Gram-positive and Gram-negative bacteria. The results showed that Centella asiatica exhibited the highest anti-microbial activity in terms of zone of inhibition (Das et al. 2013). This report also suggested potential anti-fungal activity by Centella asiatica.
Signifi cant anti-microbial activity was observed against a wide range of Gram-positive, Gram- negative as well as fungi with hexane, carbon tetrachloride, and dichloromethane soluble fraction of the ethanolic extract and crude methanolic extract showing strong zones of inhibition (10 mm, 9 mm and 9 mm), respectively, at a concentration of 400 μg/disk (Rishikesh et al. 2012).
It is to be noted that while some reports have found anti-microbial effects from aqueous extract of Centella asiatica, other reports have not. There was only one fi nding that not only aqueous, but also ethanolic crude extract of this plant was not able to kill or inhibit the growth of E. coli, K. pneumoniae and S. aureus (Jacob and Shenbagaraman 2011).
Cardiovascular effects
Cardio-protective effect of madecassoside (MA), isolated from Centella asiatica, was found on isolated rat hearts and isolated cardiomyocytes against reperfusion injury (Bian et al. 2008). In their next study, the authors demonstrated that madecassoside inhibited myocardial cell apoptosis signifi cantly on myocardial ischemia-reperfusion injury in vivo in rats. Function of left ventricle was monitored during the ischemia-reperfusion period by a multi-channel recorder. The levels of lactate dehydrogenase (LDH), creatinephosphokinase (CPK), malondialdehyde (MDA), super-oxide dismutase (SOD) and C-reactive protein (CRP) in serum were determined. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was performed to determine cardiomyocytic apoptosis. Madecassoside pre-treated (50, 100 mg/kg body weight) attenuated myocardial damage showing decreased infarct size, and decreased LDH and CK release. In addition to these, activities of SOD were decreased, which consequently markedly reduced the level of MDA and the activity of CRP, and relieved myocardial cell apoptosis (Bian et al. 2008).
Aqueous extract of Centella asiatica strongly accelerated cardiprotective effect in adriamycin-induced cardiomyopathy in rats. Cardioprotective effect was evaluated by measuring various serum markers [LDH, CPK, glutamate oxaloacetic transaminase (GOT) and glutamate pyruvate transaminase (GPT) enzymes]
and changes in the level of antioxidant enzymes [SOD, CAT (catalase), GPx (glutathione peroxidase), glutathione S-transferase (GST)]. Centella asiatica extract treated (200 mg/kg of body wt/oral) strongly prevented these alterations and restored the enzyme activities to near normal levels (Gnanapragasam et al. 2004).
Extract of Centella asiatica exhibited cardioprotective activity against myocardial injury in adriamycin-induced rats. Damaged mitochondria were observed from transmission electron microscopy from the control group, while restoration of mitochondria was found from Centella asiatica treated rats (Gnanapragasam et al. 2004).