Centella asiatica (L.) Urban

MALAYSIAN HERBAL MONOGRAPH

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Pegaga Herb

Centella asiatica (L.)Urb.
Umbelliferae

pegaga1 pegaga2 pegaga3
(a) (b) (c)
pegaga4 pegaga5 pegaga6
(d) (e) (f)

Figure 1 : C. asiatica. (a) Whole plant; (b) leaf; (c) root and rhizome; (d) stolon; (e) flower; (f) fruit. (Photos courtesy of Thiyagu, MARDI, 2012)

DEFINITION 

Pegaga herb consists of dried aerial part or whole plant of C. asiatica (L.) Urb.

SYNONYM

Centella coriacea Nannfd., Hydrocotyle asiatica L., Hydrocotyle lunata Lam., Trisanthus cochinchinensis Lour. [1, 2, 3, 4]

VERNACULAR NAMES

Pegaga (Malay), ji xui cao (Chinese), vallarai (Tamil), asiatic pennywort (English). [5, 6, 7]

CHARACTER

The whole plant powder is greyish green in colour with slightly bitter taste and characteristic odour.

IDENTIFICATION

Plant Morphology

A slender creeping herb with long-stalked leaf. The leaves are green, reniform, rounded apex and base deeply cordate stipulate with palmate netted veins and smooth texture; the petiole is relatively long, up to 20 cm. The flowers are in fascicled umbels with very small (about 4 mm) reddish petal and hermaphrodite; each flower bears 4-6 stamen and 2 styles. The fruit is small, mericarp in nature. The rootstock consists of rhizomes growing vertically down and stolon growing horizontally, interconnecting one plant to another. [5]

Microscopy

The powder contains fragment of lamina consisting of upper epidermis with polygonal cells and underlying palisade cells that are rather small and closely packed. The lower epidermis has numerous paracytic stomata. Calcium oxalate prisms and macles are present. Fragments of parenchyma from the stolon and petiole are composed of longitudinal elongated cells. Fragments of bordered pitted vessel are also seen. [5, 6, 7, 8]

micro1 micro2 micro3
(a) (b) (c)
micro4 micro5 micro6
(d) (e) (f)
Figure 2 : Microscopic characters of C. asiatica herb powder. (a) Parenchyma cells with calcium oxalate macles (magnification 400X); (b) fragment of leaf epidermis with polyglonal cells (magnification 400X); (c) collenchyma cells with calcium oxalate macles (magnification 400X); (d) paracytic stomata (magnification 400X); (e) elongated parenchyma cells of the petiole (magnification 400X); (f) fragment of bordered pitted vessels.

Colour Tests

Observed colour of solution after treatment with various reagents:

H2SO4 (conc.) : Dark brown
5% NaOH : Light brown
5% KOH : Light brown
25% NH4OH : Yellow

Thin Layer Chromatography (TLC)

 
Test Solution : Weigh about 5.0 g of C. asiatica dried herb powder in a 50 mL round bottom flask and add 50 mL ethanol into the flask. Reflux the sample for 1 hr and allow to cool. Transfer 500 µL of the extract and add with 500 µL of ethanol. Filter the mixture and use the filtrate as the test solution.
Standard Solution : Separately dissolve 2.0 mg of asiaticoside, asiatic acid, madecassoside and madecassic acid standards in 1 mL of ethanol to give 2000 µg/mL solutions. Mix the standards to give 500 µg/mL solution.
Stationary Phase : HPTLC Silica gel 60 F254, 10 x 10 cm
Mobile Phase : Chloroform-methanol-formic acid, 5:1:4 (v/v)
Application : (a) A mixture of asiaticoside, asiatic acid, madecassoside and madecassic acid standard solution (S); 3 µL as a band
(b) Ethanol extracts of C. asiatica herb (L); 3µL as a band 
Development Distance : 8 cm
Drying : Air drying
Detection : Spray with  10% sulphuric acid in ethanol and view under (a) visible light and (b) UV 366 nm

 

TLC centella asiatica
Figure 3 : TLC profiles of a standard  mixture of asiaticoside, asiatic acid, madecassoside and madecassic acid standards (S) and ethanol extract of C. asiatica herb (L) after spraying with 10% sulphuric acid in ethanol and observed under (a) visible light and (b) UV at 366 nm

High Performance Liquid Chromatography (HPLC)

 
Test Solution : Extract about 5.0 g of dried powder C. asiatica dried herb powder with 80 mL of ethanol by reflux method at a temperature of 60°C for 30 min. Filter the mixture through a filter paper. Evaporate the filtrate to dryness using a rotary evaporator. Then, dissolve 20.0 mg of the dried extract in 2 mL of methanol. Sonicate the mixture for 15 min. Filter the solution through a 0.45 µm syringe filter and inject the filtrate into the HPLC column.
Standard Solution : Separately dissolve 2.0 mg of asiaticoside, asiatic acid, madecassoside and madecassic acid standards in 2 mL of methanol to give 1000 µg/mL stock solutions. Sonicate the solutions for 15 min. Mix the standard solutions to produce a 250 µg/mL solution.
Chromatographic System : Detector: UV 200 nm
Column: C18 (5 µm, 4.6 mm I.D x 150 mm)
Column oven temperature: 30°C
Flow rate: 0.8 mL/min
Injection volume: 20 µL
Mobile Phase (Gradient mode) :
Time Water (%) Acetonitrile (%)
0.01 85 15
11.00 20 80
22.00 20 80
System Suitability Requirements :

Perform at least five replicate injections of the standard mixture (250 µg/mL). The requirements of the system suitability parameters are as follow:

  1. Symmetry factor (As) is not more than 1.5.
  2. Percentage of relative standard deviation (RSD) of the retention time (tr) for the standard mixture is not more than 2.0%.
Acceptance Criteria : (1) Retention times (tr) of asiaticosside, assiatic acid, madecassoside and madecassic acid in the test solution are similar to the (tr) of the standard mixture.

 

hplc1
Figure 4 : HPLC chromatogram of a standard mixture (250 µg/mL) containing madecassoside (tr = 9.547 min), asiaticoside (tr = 9.983 min), madecassic acid (tr = 14.223 min) and asiatic acid (tr = 16.674 min) standards

 

hplc2
Figure 5 : HPLC chromatogram of ethanol extract of C. asiatica herb showing peaks corresponding to madecassoside (tr = 9.543 min), asiaticoside (tr = 9.984 min), madecassic acid (tr = 14.251 min) and asiatic acid (tr = 16.685 min)


Table 1 : The Relative Retention Times (RRT) for the four characteristic peaks

Standard RRT
Madecassoside 0.67
Asiaticosside 0.69
Madecassic acid (as reference) 1.00
Assiatic acid 1.17

Note: The RRTs provided only serve as a guidance.

PURITY TESTS

Foreign Matter
Not more than 2%

 

Ash Contents
Total ash : Not more than 14%
Acid-insoluble ash : Not more than 4%

 

Loss on Drying
Not more than 10%

 

Extractive Values
Water-soluble extracts
Hot Method        : Not less than 23%
Cold Method : Not less than 21%
Ethanol-soluble extracts
Hot Method : Not less than 17%
Cold Method : Not less than 7%

SAFETY TEST

Heavy Metals
Arsenic                      : Not more than 5.0 mg/kg
Mercury : Not more than 0.5 mg/kg
Lead : Not more than 10.0 mg/kg
Cadmium : Not more than 0.3 mg/kg

 

Microbial Limits
Total aerobic microbial count          
Not more than 105 cfu/g                     
Total yeast and mould count : Not more than 104 cfu/g
Bile-tolerant gram negative bacteria : Not more than 104 cfu/g

 

Specific Pathogens
Salmonella spp.                  

Absent in 25 g                        
Escherichia coli : Absent in 1 g
Staphylococcus aureus : Absent in 1 g
Pseudomonas aeruginosa : Absent in 1 g

CHEMICAL CONSTITUENTS

Water extract of C. asiatica has been found to contain centellosides D-E, pectin and acidic arabinogalactan. [9-11]

The ethanol extract had triterpenes (e.g. 2α,3β,20,23-tetrahydroxyurs-28-oic acid, 2α,3β,23-trihydroxyurs-20-en-28-oic acid), triterpenoid glycosides (e.g. asiaticoside, asiaticosides A-F, madecassoside, scheffuroside B) and a saponin (e.g. 2α,3β,23-trihydroxyurs-20-en-28-oic acid-O-α-L-rhamnopyranosyl-(1→4)-O-b-D-glucopyranosyl -(1→6)-O-β-D-glucopyranosyl ester). [12, 13, 14, 15]

The methanol extract had triterpenes and triterpenoids (e.g. ursolic acid lactone, ursolic acid, pomolic acid, 2α,3α-dihydroxyurs-12-en-28-oic acid, 3-epimaslinic acid, corosolic acid, asiaticoside, madecassoside, asiatic acid, 6β-hydroxyasiatic acid, 3-O-[a-L-arabinopyranosyl]-2α,3β,6β,23-a-tetrahydroxyurs-12-ene-28-oic acid), a phenolic (e.g. rosmarinic acid), a steroid (e.g. b-sitosterol 3-O-b-glucopyranoside) and others (e.g. 8-acetoxy-1,9-pentadecadiene-4,6-diyn-3-ol, centellin, asiaticin, centellicin). [16, 17, 18, 19] Whereas the methanol-water extract had phenolics (e.g. castilliferol, castillicetin, isochlorogenic acid). [20]

C. asiatica has also been reported to contain brahmic acid, 3β-6β-23-trihydroxyolean-12-en-28-oic acid, 3β-6β-23-trihydroxyurs-12-en-28-oic acid, α-terpinene, thymol methyl ether, sceffoleoside A, bayogenin, centellasaponins B-D, centellose, D-gulonic acid, meso-inositol, docosyl ferulates). [21, 22, 23, 24, 25] In addition, its cell cultures had irbic acid, chlorogenic acid and triferulic acid. [26] 

MEDICINAL USES

Uses described in folk medicine, not supported by experimental or clinical data

The C. asiatica plant is traditionally used for albinism, anemia, asthma, bronchitis, cellulite, cholera, constipation, dermatitis, diarrhea, dizziness, dysentery, dysmenorrhea, dysuria, epistaxis, epilepsy, haematemesis, hemorrhoids, hepatitis, hypertension, jaundice, leucorrhoea, nephritis, nervous disorders, neuralgia, measles, rheumatism, smallpox, syphilis, toothache, urethritis and varices. It is also used as an antipyretic, anti-inflammatory and a “brain tonic”. [27, 28, 29] Poultices of this plant have been used to treat contusions, closed fractures, sprains and furunculosis [29].

Biological and pharmacological activities supported by experimental data

Cytotoxic activity
Methanol (80%) extract and acetone fraction of methanol (80%) extract of C. asiatica whole plant inhibited proliferation of Ehrlich ascites tumour cells (concentration of 50% cell death 62 and 17 μg/mL, respectively) and Dalton's lymphoma ascites tumour cells (75 and 22 μg/mL, respectively). The acetone fraction also suppressed multiplication of mouse lung fibroblast (L-929) cells at a concentration of 50% cell death of 8 μg/mL. [30]


Methanol (80%) extract of C. asiatica whole plant exhibited concentration dependent cytotoxicity towards human breast cancer cell line MCF-7 by apoptosis with LD50 value of 66 mg. Asiatic acid (10 µM) was found to kill ~95% cells [31].

Anti-tumour effect
Oral administration of the methanol (80%) extract (1 mg/g body weight) and acetone fraction of methanol (80%) extract (0.04 mg/g body weight) of C. asiatica whole plant to the Dalton's lymphoma ascites tumour-bearing Swiss Albino mice significantly (p < 0.001) retarded the development of solid tumour. The methanol extract (1 mg/g body weight) also significantly (p < 0.001) reduced the ascites tumour growth and increased the life span of Ehrlich ascites tumour-bearing mice. The treatments were effective when these were started simultaneously after and 10 days prior to the tumour transplantation. [30]
 
Wound healing activity

Titrated extract of C. asiatica plant that constituted of asiatic acid, madecassic acid and asiaticoside (40 mg) injected to male Sprague-Dawley rats twice a week for 4 weeks had wound healing effect by stimulation of glycogen and glycosaminoglycan synthesis and fasten the new formation of connective tissue [32].

Antioxidant activity
The ethanol extracts of all plant parts (leaves, petioles and roots) of C. asiatica showed significant antioxidant activity (p<0.05) in the concentration range of 1000-3000 ppm [33].

Methanol extract of C. asiatica whole plant (50 mg/kg/day) administered orally to 2-month old lymphoma-bearing male Swiss mice for 14 days significantly increased the antioxidant enzymes of superoxide dismutase, catalase and glutathione peroxidase, as well as the glutathione and ascorbic acid antioxidants in the liver and kidney [34].

Anti-ulcer activity
The water extract of C. asiatica whole plant (0.10 and 0.25 g/kg) and asiaticoside (5 and 10 mg/kg) given orally for 7 days to male Sprague-Dawley rats with acetic acid induced gastric ulcers were found to decrease the ulcer sizes (p< 0.01). This was due to the reduction of myeloperoxidase activity (p< 0.01) and promotion of epithelial cell proliferation (p<0.01) by upregulated expression of basic fibroblast growth factor in the ulcer tissues. [35] The extract and asiaticoside also inhibited inducible nitric oxide synthase activity (p< 0.01) and protein expression at the ulcer tissues [36].
 
Radioprotection effect
The aqueous extract of C.asiatica whole plant (100 mg/kg body weight) given orally to 6-8 weeks old Swiss Albino mice irradiated with Co-60 gamma radiation was reported to significantly increase survival time [37].
 
Protective effect for neurodegenerative disorder
The aqueous extract of C. asiatica whole plant (300 mg/kg) given to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in aged Sprague-Dawley rats for 21 days protected the brain against neurodegenerative disorders with reference to examination of the oxidative biomarker levels in corpus striatum and hippocampus homogenate [38].

Clinical studies

Clinical studies have been conducted using leaf extracts of C. asiatica [39-48], however, none is available for the whole plant extract.

SAFETY INFORMATION

Preclinical studies (Toxicology study)

Acute toxicity
Oral single dose acute toxicity study of aqueous mixture of C. asiatica whole plant powder on female Sprague Dawley rats (aged between 8 and 12 weeks old) showed no toxic effects on the parameters observed, including behaviors, body weight, food and water intake. All rats were observed for 14 days prior to necropsy. No death was found throughout the study period. Necropsy revealed no significant abnormality. LD50 value was determined as > 2000 mg/kg. [49]

Others (Adverse reaction, contraindication, side effect, warning, precaution)

Warning
Asiaticoside (0.10%) has been found to cause development of papillomas in vivo. [50]
 
Precaution
Allergic contact dermatitis has been associated with several topical products containing C. asiatica extract [51-53].

DOSAGE

Oral dose : 0.33-0.68 g or oral infusion of similar amount three times daily [4, 27, 28]. 

Poultice    : Apply to skin with wound, burns or inflammation after the fresh or dried whole plant is minced [54].

Juice        : One handful of fresh whole plant made into fresh juice as tonic [54]. 

STORAGE

Store below 30˚C. Protect from light and moisture.

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