Ulam raja Leaves
Cosmos caudatus Kunth
Asteraceae
Figure 1 : C. caudatus. (a) A mature whole plant grown on soil; (b) young shoot system; (c) flowers produced in a capitulum;(d) flowering heads produced on a single stalk; (e) fruits achenes . (Photos courtesy of Thiyagu, MARDI, 2012)
DEFINITION
Ulam raja consists of dried leaves of Cosmos caudatus Kunth (Asteraceae).
SYNONYM
Cosmos caudatus var. exaristatus Sheriff
VERNACULAR NAMES
Ulam raja (Malay), punaikkali (Tamil) [ 1 ].
CHARACTER
The dried leaves powder has a distinct odour and distinctly mint taste [ 1 ].
IDENTIFICATION
Plant Morphology
C. caudatus is an annual herb, growing up to 3 m tall, glabrous or sparsely hispid. Stem light green with a purplish hue and succulent. Leaves simple, pinnate, or bipinnate and arranged in opposite pairs, succulent and pungent, leaves and leaflets lobed and divided, finely dissected 10-20 cm long; leaflet sessile or very shortly stalked; leaf blades very variable in size approximately10-20 cm, ultimate lobes 2-10 mm wide, margins spinulose-ciliate, apices acute, often mucronulate; petioles 1-7 cm. Inflorescence produced in a capitulum with a ring of broad ray florets and a centre of disc florets, small approximately 3.5-4 mm diameter, found solitary or in a loose clusters and are produced on a single stalk on auxiliary heads; flowering heads about 45 mm diameter; peduncles 10-30 cm; cayculi of usually spreading, linear-subulate bractlets 6-10 mm, apices acuminate; involucres 5-15 mm diameter; phyllaries erect, oblong-lanceolate, 7-11 mm, apices acute to obtuse; petals (actually ray florets/ray corollas) rose-pink to purple, laminae oblong-oblanceolate to inverted-lance shaped, 0.5-1.5 cm long, 3-lobed at the tip, apices obtusely 3-lobed, disc corollas/florets 5-6 mm, yellow; cypselae 12-35 mm, glabrous or scabridulous proximally, setose distally, pappi of 2-3 widely divergent to reflexed awns 3-5 mm; anthers lanceolate, approximately 2-4 mm long, fused to one another to form a tube around the style; filaments free from one another, approximately 3 mm long, clothed in translucent fleshy hairs; style approximately 10-19 mm long with two papillose stigmatic branches at the apex. Infructescence approximately 16-21 mm long, clothed in hooked hairs which cause them to adhere to clothes. Seeds approximately 6-9 mm long; cotyledons approximately 3 mm long, about as wide as the radicle [ 2 , 3 ].
Microscopy
Figure 2 : Microscopic images of C. caudatus leaf powder. (a) Scalariform vessels (magnification 20x); (b) cluster of oil glands (magnification 20x); (c) stomata (magnification 40x); (d) simple multicellular trichome (magnification 40x); (e) druse crystal (magnification 40x). [Scale bars: a,b =20 µm; c,d,e =5 µm]
Colour Tests
Observed colour of solution after treatment with various reagents:
| H2SO4 (conc.) | Yellow |
| 5% NaOH | Yellow |
Thin Layer Chromatography (TLC)
Figure 3 : TLC profiles of quercitrin (S) and methanol extract of C. caudatus dried leaves powder (L) observed under (a) UV at 254 nm spray; (b) UV at 366 nm before spray; (c) visible light after spray.
| Test Solutions | Weigh about 1.0 g of C. caudatus dried leaf powder in a 50 mL screw-capped conical flask and add 10 mL of methanol. Sonicate for 60 min at temperature less than 40°C. Filter the mixture into a vial. |
| Standard solution | Dissolve 5.0 mg of quercitrin standard [CAS no.: 522-12-3] in 10 mL methanol to produce 500 µg/mL solution. |
| Plate | HPTLC silica gel 60 F254, 5 x 10 cm |
| Mobile phase | Toluene : ethyl acetate : formic acid;(6:3:1) (v/v) |
| Application |
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| Development distance | 8 cm, automatic developing chamber 2 (ADC2) |
| Drying | Air drying |
| Detection |
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High Performance Liquid Chromatography (HPLC)
| Test solution | Extract about 1.0 g of C. caudatus dried leaf powder with 20 mL of ethanol in a water bath at temperature 80°C for 1 hr. Centrifuge at 5000 rpm for 10 min. Remove the supernatant from the centrifuge tube into an evaporating dish. Evaporate the supernatant until completely dry. Reconstitute with 5 mL methanol. Filter the solution through a 0.45 µm syringe filter and inject the filtrate into the HPLC column. | |||||||||||||||||||||
| Standard solution | Dissolve 5.0 mg of quercitrin standard [CAS no.: 522-12-3] in 10 mL of methanol to produce 500 µg/mL solution. | |||||||||||||||||||||
| Chromatographic system |
Detector: UV 254 nm Column: C18 column (5 µm, 4.6 mm I.D x 150 mm) Column oven temperature: 30oC Flow rate: 1.0 mL/min Injection volume: 10 µL |
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| Mobile Phase (Isocratic mode) |
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| System suitability requirement |
Perform at least five replicate injections of the standard solutions (0.5 mg/mL). The requirements of the system suitability parameters are as follow:
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| Acceptance criteria |
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Figure 4: HPLC chromatogram of quercitrin standard solution (500 µg/mL) at tr = 11.575 min.
Figure 5 : HPLC full chromatogram of ethanol extract of C. caudatus leaves showing peaks corresponding to quercitrin (tr = 11.575 min) and peak A (tr = 11.323 min).
Figure 6 : HPLC zoom chromatogram of ethanol extract of C. caudatus leaves showing peaks corresponding to quercitrin (tr = 11.575 min) and peak A (tr = 11.323 min).
Figure 7 : UV spectrum of quercitrin standard solution (500 µg/mL) and ethanol extract of C. caudatus leaves.
PURITY TESTS
| Foreign Matter |
| Not more than 2% |
| Ash Contents | |
| Total ash | Not more than 12% |
| Acid-insoluble ash | Not more than 2% |
| Loss on Drying |
| Not more than 12% |
| Extractive Values | |
| Water-soluble extracts | |
| Hot method | Not less than 23% |
| Cold method | Not less than 16% |
| Ethanol-soluble extracts | |
| Hot method | Not less than 13% |
| Cold method | Not less than 5% |
SAFETY TESTS
| 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 bacterial count | Not more than 105 cfu/g |
| Total yeast and mould count | Not more than 104 cfu/g |
| Bile-tolerant gram negative | 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
Methanol extract of C. caudatus leaves has been found to contain flavonoids (e.g. quercetin 3-O-β-D-arabinofuranoside, quercetin 3-O-β-D-rhamnoside, quercetin 3-O-α-D-glucoside and quercetin) [ 4 ].
C. caudatus leaves extract of a mixture of methanol and potassium dihydrogen phosphate (pH 6.0) was found to have flavonoids (e.g. catechin, rutin, quercetin, quercetin 3-O-rhamnoside, quercetin 3-O-arabinofuranoside and quercetin 3-O-glucoside), α-glucose, β-glucose and chlorogenic acid [ 5 ].
Acidified aqueous methanol (50-60%) leaves extracts had mainly flavonoids (e.g. catechin, epicatechin, quercetin, myricetin, luteolin, naringenin and kaempferol) [ 6 , 7 ].
The 50% aqueous acetone leaves extract was reported to contain mainly phenolics (e.g. isomers of proanthocyanidin dimers, proanthocyanidin trimmers, tetramers and pentamers, hexamers of catechin, (+)-catechin, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, quercetin rutinoside, quercetin hexose, quercetin 3-O-glucoside, quercetin pentose and quercetin deoxylhexose) [ 8 ].
MEDICINAL USES
Uses described in folk medicine, not supported by experimental or clinical data
Among the Malays, C. caudatus is used for anti-aging, getting rid of bad breath, improving blood circulation and strengthening bones [ 1 ]. It is also used to purify blood and firm muscles [ 9 ]. It can be eaten or boiled for drinking as an herbal remedy to cleanse the blood or as a muscle tonic [ 2 ].
Biological and pharmacological activities supported by experimental data
Anti-oxidant activity
Aqueous acetone extract of dried leaves of C. caudatus exhibited the highest antioxidant capacity with L-ascorbic acid equivalent anti-oxidant capacity value of more than 2500 mg per 100 g fresh sample as compared to other extraction solvents by using total anti-oxidant capacity assay [ 8 ].
Aqueous extract of dried leaves of C. caudatus significantly (p < 0.05) showed 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities using DPPH scavenging assay [ 13 ]. The extract also significantly (p < 0.05) showed ferric reducing activity of 183.11 µmol Trolox Equivalents per g dry basis) using ferric reducing antioxidant potential (FRAP) assay [ 10 ].
Aqueous extract of C. caudatus leaves (0.02 g/mL) showed antioxidant activities with DPPH scavenging activity (≈ 200 µmol Trolox Equivalents per g dry basis) and ferric reducing power (≈ 300 mol Trolox Equivalents per g dry basis). The extract was also able to chelate cupric ions (≈ 60% of cupric ions) using cupric ions chelation assay [ 11 ].
Aqueous extract of C. caudatus leaves (200 ppm) showed ferric reducing power compared to butylated hydroxyanisole (BHA). The crude extract (4 mg) dissolved in methanol also inhibited oxidation of linoleic acid by 59.7% using ferric thiocynate (FTC) assay [ 12 ].
Methanol extract of dried leaves of C. caudatus (0.02% w/v) revealed its antioxidant activity with absorbance value of 0.066 compared to α-tocopherol standard (0.32) using FTC and thiobarbituric acid (TBA) assays. Methanol extract of dried C. caudatus dried leaves exhibited radical-scavenging activity (IC50 value = 20.2 ± 0.05 µg/mL) using DPPH assay
[ 13 ].
The total phenolic content of the ethanol extract of C. caudatus leaves was 1.52 mg GAE/g fresh weight (fw). The extract (100 mg/mL) showed high DPPH radical scavenging activity of 112 ± 3 µmol trolox equivalent (TE)/ g fw. The extract (0.14 – 0.51 mg/mL) also showed high 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity of 4.71 ± 0.19 µmol TE/ g fw and concentrations of 0-500 mg/mL showed high ferric reducing power of 172 ± µmol TE/ g fw. The extract (1mg/mL) inhibited linoleic acid oxidation at 98.1± 0.4% [ 14 ].
The total phenolic content of the ethanol extract of C. caudatus leaves (10 mg/mL) was 60.56 mg of GAE/g. The extract showed high anti-oxidant activity with DPPH radical scavenging activity (86.85%) but it was lower when compared to BHT (94.86 ± 0.15%). The extract also showed high superoxide dismutase activity (98.56%) compared to ascorbic acid (99.20 ± 0.27%) [ 15 ].
The total flavonoid content of the aqueous extract of C. caudatus leaves was 3.13 mg/g dry weight (dw); total ascorbic acid was 0.48 mg vitamin C equivalent /g fw and total phenolic content was 6.43 mg/g dw. The extract showed anti-oxidant activity with DPPH radical scavenging activity (24.81%) as compared to vitamin C (≈ 90%), FRAP activity (419.07 mmol) as compared to vitamin C (≈ 1700 mmol) and β-Carotene bleaching activity (13.15%) as compared to vitamin C (≈ 11%) [ 16 ].
The total flavonoid content of the boiled aqueous extract of C. caudatus leaves was 0.29 mg/g dw and total phenolic content was 8.76 mg/g dw. The extract showed anti-oxidant activities with DPPH radical scavenging activity (38-40%) as compared to BHT (≈ 85%) and FRAP activity (200.65 mmol) compared to BHT (1000 mmol) [ 16 ].
Antimicrobial activity
Diethyl ether, n-hexane and ethanol extracts of dried leaves of C. caudatus at three different concentrations (1, 20, and 50 mg/mL) exhibited antimicrobial activities on Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. The n-hexane extract gave the highest MIC value (25 mg/mL) for all microbial strain by using disc diffusion assay with dimethyl sulfoxide as negative control [ 17 ].
The ethanol extractof C. caudatus leaves (50 mg/mL each) inhibited the growth of Bacillus pumilus with the inhibition zone of 8.47 mm, Corynebacterium urealyticum (7.37 mm), Enterobacter cloaceae (8.93 mm), Pseudomonas aeruginosa (6.93 mm), Serratia marcescens (7.67 mm) and Staphylococcus sciuri (7.37 mm) using disc diffusion method [ 18 ].
The aqueous extractof C. caudatus leaves (50 mg/mL each) inhibited the growth of Bacillus pumilus with the inhibition zone of 7.73 mm, Corynebacterium urealyticum (7.10 mm), Enterobacter cloaceae (7.80 mm), Pseudomonas aeruginosa (6.80 mm), Serratia marcescens (7.40 mm) and Staphylococcus sciuri (6.67 mm) using disc diffusion method [ 18 ].
Cytoxicity activity
Methanol extract of dried C. caudatus dried leaves showed cytotoxic activity on T47D breast cancer cells with IC50 of 344.91 µg/mL by using MTT assay [ 19 ].
Ethanol extract of C. caudatus leaves (20 mg/mL) showed cytotoxic activity on HeLa cervical cancer cells with cytotoxic dose at 50% (CD50) of 100 µg/mL by using microtitration cytotoxicity assay [ 20 ].
Antihypertensive activity
The aqueous extract of C. caudatus leaves (500 mg/kg and 1000 mg/kg) administered orally to male Wistar rats before the induction with adrenaline (1.2 mg/ kg) prevented the increase of both the frequency of heart rate and the amplitude of stroke volume similar to atenolol (9 mg/kg). The aqueous extracts of C. caudatus leaves (500 mg/kg and 1000 mg/kg) administered orally to male Wistar rats before the induction with sodium chloride (3.75 g/kg) only inhibited the increase of amplitude of stroke volume similar to hydrochlorothiazide (0.45 mg/kg) and captopril (13.5 mg/kg). The extract also exhibited significant (p < 0.0001) diuretic effect at dose-dependent manner after 24 hours treatment similar to furosemide (1.8 mg/kg) [ 21 ].
Antimutagenic activity
Costunolide isolated from C. caudatus leaves (0.2 mg/kg bw) was administered orally (double dose with second dose given after 24 hours of the first dose) to Swiss mice (aged 7 to 12 weeks). It showed antimutagenic activity with 85% reduction of micronucleated polychromatic erythrocytes number compared to mitomycin C and dimethyl sulfoxide tested mice [ 22 ].
Anti-inflammatory activity
The extracts of C. caudatus leaves (200 mg/kg each) were administered orally to Swiss albino mice before the induction of paw edema using carrageenan. After four hours, a significant (p < 0.01) inhibition of paw edema volume 69.54% (methanol extract) and 66.54% (aqueous extract) were observed in treated mice compared to the control group [ 23 ].
Clinical studies
Information and data have not been established.
SAFETY INFORMATION
Preclinical studies (Toxicology studies)
Others (Adverse reaction, contraindication, side effect, warning, precaution)
Acute toxicity
Oral single dose acute toxicity study using aqueous mixture of dried powder of C. caudatus leaves on female Sprague Dawley rats (aged between 8 and 12 weeks old) showed no toxic effect on the parameters observed which includes behaviors, body weight, food and water intakes. All rats were observed for 14 days and no death was found throughout the study period.No-observed-adverse-effect level (NOAEL) is more than 2,000 mg/kg body weight [24].
Information and data have not been established.
DOSAGE
Information and data have not been established.
STORAGE
Store below 30°C. Protect from light and moisture.
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