Chromolaena odorata (L.) R.M. King & H. Robinson

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Malaysian Herbal Monograph

Pokok kapal terbang Leaf

Chromolaena odorata (L.) R.M. King & H. Robinson

Asteraceae

Figure 1 : Chromolaena odorata. (a) Habit; (b) habit in flower; (c) whole plant; (d) flowers head and floret. (Photos courtesy of FRIM, 2015)

DEFINITION

Pokok kapal terbang leaves consist of the powder of dried leaves of Chromolaena odorata (L.) R.M. King & H. Robinson (Asteraceae).

SYNONYM

Eupatorium affine Hook & Arn., Eupatorium brachiatum Wikstrom, Eupatorium clematitis DC., Eupatorium conyzoides M. Vahl, Eupatorium divergens Less., Eupatorium floribundum Kunth, Eupatorium graciliflorum DC., Eupatorium odoratum L., Eupatorium sabeanum Buckley, Eupatorium stigmatosum Meyen & Walp., Osmia conyzoides (Vahl) Sch.-Bip., Osmia divergens (Less.) Schultz-Bip., Osmia floribunda (Kunth) Schultz-Bip., Osmia graciliflora (DC.) Sch.-Bip., Osmia odorata (L.) Schultz-Bip [ 1 ].

VERNACULAR NAMES

Siam weed, jack-in-the-bush, christmas bush, goat weed (English); pokok kapal terbang, pokok german (Malay); fei ji cao (Chinese) [ 2 , 3 , 4 ].

CHARACTER

Colour	Dark green [ 5 ]
Odour	Characteristics, very strong, unpleasant [ 5 ]
Taste	Slightly bitter [ 5 ]

IDENTIFICATION

Plant Morphology

Chromolaena odorata is a perennial shrub, up to 3 m tall, scrambling up to 7 m. Stems profusely branched, herbaceous when young, tough and semi-woody when older, yellowish, shortly hairy or nearly glabrous. Leaves simple, opposite; flaccid-membranous, velvety-pubescent, deltoid-ovate, acute, 3-nerved, very coarsely toothed, each margin with 1–5 teeth or entire in young leaves; base obtuse or subtruncate but shortly decurrent; petiole 1–3 cm long or more, slender; blade mostly 5–12 cm long, 3–6 cm wide. Inflorescence a homogamous. Flowers arranged in corymbose clusters arising from the axils of upper leaves; 10–35 flowered head; peduncle 1–2 cm long; involucre cylindrical, bracts in 5 or 6 rows, closely overlapping, oblong, increasing in size upwards, up to 10 mm x 3 mm, straw-coloured to greenish; corolla tubular, 5 mm long, 5-lobed, pale mauve, pale blue or whitish, protruding from the involucre; stigma with a long, exserted arm. Fruits a narrow achene, linear, angular, 3–5 mm long, brown or black, with short, white, stiff hairs along the edges; pappus white, consisting of rough bristles, 4–5 mm long. Seeds minute [ 2 ].

Microscopy

Powdered material consists of the fragment of adaxial and abaxial epidermis cells with sinuous anticlinal wall and abaxial epidermis cells has numerous anomocytic stomata. Abundance of trichomes are non-glandular; simple multiseriate and contain volatile oils. Fragment of annular vessels, fibres and also oil globules are present [ 5 , 6 ].

Figure 2 : Microscopic characters of Chromolaena odorata dried leaves powder of 0.355 mm size. (a) Adaxial epidermis cells with sinuous anticlinal wall (magnification 40x); (b) abaxial epidermis cells with sinuous anticlinal wall with anomocytic stomata (magnification 40x); (c) non-glandular; simple, multiseriate trichomes containing volatile oils (magnification 40x); (d) annular vessel (magnification 20x); (e) fibre (magnification 20x); (f) & (g) oil gland (arrow) (magnification 10x); (h) oil globules (magnification 10x). [Scale bars: a, b, c, d, e = 10 µm; f = 20 µm; g = 50 µm]

Chemical Test

Observation of solution after treatment with various reagents :

Test for the presence of phenolics (tannins)	Bluish green
Test for the presence of flavonoids	Dark Yellow

Thin Layer Chromatography (TLC)

Figure 3 : TLC chromatogram of kaempferol-4’-methyl ether (S1) at Rf: 0.63 min and ethanol extract of Chromolaena odorata dried leaves powder (L) observed under (a) UV at 254 nm before derivatisation and (b) UV at 366 nm after derivatisation with NP & PEG reagent.

Figure 4 : TLC chromatogram of rutin (S2) at Rf: 0.62 min and ethanol extract of Chromolaena odorata dried leaves powder (L) observed under UV at 366 nm after derivatisation with NP & PEG reagent.

Test Solutions	Weigh about 0.5 g of C. odorata dried leaves powder of 0.355 mm size in a 14 mL screw-capped vial. Add 5 mL of ethanol and sonicate for 15 min. Filter the solution through filter paper. Use the filtrate as test solution.
Standard solution	Dissolve kaempferol-4’-methyl ether standard (CAS no.: 491-54-3) and rutin standard (CAS no.: 153-18-4) separately in methanol to produce 1.0 mg/mL solution.
Stationary Phase	HPTLC silica gel pre-coated plate 60 F254, 10 x 10 cm
Mobile phase	Chloroform : methanol : water (9 : 1 : 0.1) (v/v) Ethyl acetate : acetic acid : formic acid : water (100 : 11 : 11 : 26) (v/v)
Application	Kaempferol-4’-methyl ether standard solution (S1); 15 µL, as a band. Rutin standard solution (S2); 6 µL, as a band. Ethanol extract of C. odorata dried leaves powder (L); 8 µL, as a band for mobile phase (a) and 10 µL, as a band for mobile phase (b).
Development distance	8 cm for mobile phase (a) and 7 cm for mobile phase (b).
Drying	Air drying
Detection	UV at 254 nm before derivatisation with natural product (NP) & polyethylene glycol (PEG) reagent. UV at 366 nm after derivatisation with NP & PEG reagent.

High Performance Liquid Chromatography (HPLC)

Test solution

Weigh about 0.5 g of C. odorata dried leaves powder of 0.355 mm size in a 14 mL screw-capped vial. Add 5 mL of ethanol and sonicate for 15 min. Filter the solution and evaporate to dryness. Reconstitute with 3 mL methanol and use the solution as test solution.

Standard solution

Dissolve rutin standard [CAS no.: 153-18-4] and kaempferol-4’-methyl ether [CAS no.: 491-54-3] separately in methanol to produce a standard concentration of 1.0 mg/mL.

Chromatographic system

Detector: PDA 350 nm

Column: C18 (5 µm, 4.6 mm i.d. x 250 mm) (X-bridge, Waters if necessary)

Column oven temperature: Ambient

Flow rate: 1.0 mL/min

Injection volume: 10 µL for test solution and reference standards

Mobile Phase (gradient mode)

Run Time (min)	A – 0.1% formic acid in water (%)	B – Acetonitrile (%)
0	80	20
5	65	35
8	65	35
18	55	45
28	40	60
38	55	45
41	65	35
45	80	20

System suitability requirement

Perform at least five replicate injections of the standard solutions (1.0 mg/mL). The requirements of the system suitability parameters are as follow:

Symmetry factor (As) for rutin and kaempferol-4’-methyl ether standard is not more than 1.5.
Percentage of relative standard deviation (RSD) of the retention time (tr) for rutin and kaempferol-4’-methyl ether standard is not more than 2.0%.

Acceptance criteria

Retention time (tr) of rutin and kaempferol-4’-methyl ether in the test solution is similar to the trof the standard solution.
The ultraviolet (UV) spectrum of rutin and kaempferol-4’-methyl ether in the test solution is similar to the UV spectrum of rutin and kaempferol-4’-methyl ether in the standard solution (optional supportive data).

(a)

(b)

(c)

Figure 5 : Whole HPLC chromatogram of (a) rutin standard solution (1.0 mg/mL) at tr = 5.916 min, (b) kaempferol-4’-methyl ether standard solution (1.0 mg/mL) at tr = 31.030 min and (c) ethanol extract of Chromolaena odorata dried leaves powder showing peak corresponding to rutin standard solution at tr = 5.805 min and kaempferol-4’-methyl ether standard solution at tr = 30.915 min, respectively.

(a)

(b)

Figure 6 : HPLC chromatogram highlighting the elution region of rutin and kaempferol-4’-methyl ether in (a) rutin standard solution (1.0 mg/mL) at t_r= 5.916 min, (b) kaempferol-4’-methyl ether standard solution (1.0 mg/mL) at t_r= 31.030 min and ethanol extract of Chromolaena odorata dried leaves powder (c)(i) and (c)(ii) showing peak corresponding to rutin standard solution at t_r= 5.805 min and kaempferol-4’-methyl ether standard solution at t_r= 30.915 min, respectively.

Figure 7 : UV spectrum of rutin standard solution (1.0 mg/mL) and ethanol extract solution of Chromolaena odorata dried leaves powder.

Figure 8 : UV spectrum of kaempferol-4’-methyl ether standard solution (1.0 mg/mL) and ethanol extract solution of Chromolaena odorata dried leaves powder.

PURITY TESTS

The purity tests, except foreign matter test, are based on C. odorata dried leaves powder of 0.355 mm particle size.

Foreign Matter

Not more than 2%

Ash Contents
Total ash	Not more than 11%
Acid-insoluble ash	Not more than 2%
Water-soluble ash	Not less than 2%

Loss on Drying

Not more than 12%

Extractive Values
*Water-soluble extracts*
Hot method	Not less than 33%
Cold method	Not less than 23%
*Ethanol-soluble extracts*
Hot method	Not less than 19%
Cold method	Not less than 13%

SAFETY TESTS

The safety tests are based on C. odorata dried leaves powder of 0.355 mm particle size.

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. odorata leaves was found to contain fatty acids (hexadecanoic acid, (S)-coriolic acid, (S)-coriolic acid methyl ester, (S)-15,16-didehydrocoriolic acid, (S)-15,16-didehydrocoriolic acid methyl ester, linoleamide and linolenamide); monoterpene (α-terpineol); sesquiterpenes (caryophyllene oxide and α-cubebene); triterpenes (α-amyrin, β-amyrin, bauerenol acetate, lupeol and taraxerol); phytoprostanes (chromomoric acid C-I, chromomoric acid C-IV, (8Z)-chromomoric acid G, (8E)-chromomoric acid G and (9S,13R)-12-oxophytodienoic acid); alkaloids (akuammidine, echitamine and voacangine); flavonoids (acacetin, 6-methoxyacacetin, apigenin, aromadendrin-4’-methyl ether, aromadendrin-7-methyl ether, baicalein, baicalin, betuletol, biochanin, cystosiphonin, daidzein, eriodicytol-7,4’-dimethyl ether, genistein, 6-methoxyhesperetin, hesperetin, isorhamnetin, kaempferol, kaempferol-4’-methyl ether, luteolin, myricetin, naringenin, nobiletin, ombuin, quercetin, quercetin-7-methyl ether, quercetin-7,4’-dimethyl ether, quercetin-3-O-rutinoside, robinetin, salvigenin, scutellarein tetramethyl ether, taxifolin-7-methyl ether and taxifolin-4’-methyl ether); simple phenolics (ferulic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid and vanillic acid) and tannins ((+)-catechin, ellagic acid, (-)-epicatechin, (-)-epigallocatechin, (-)-epigallocatechin-3-gallate and tannic acid); chalconoid (butein) and saponins (avenacin A1, avenacin A2, avenacin B1 and avenacin B2) [ 7 , 8 , 9 ,10 , 11 , 12 ].

Ethanol extract of C. odorata leaves was found to contain simple phenolics (hydroxycinnamic acids derivatives of caffeic acid and p-coumaric acid); terpenoids, tannins, saponins, cardiac glycosides and anthraquinones [ 8 , 13 ].

Ethanol (80%) extract of C. odorata leaves was found to contain flavonoid (5-hydroxy-4’,7-dimethoxyflavanone); chalcone (2’-hydroxy-4,4’,5’,6’-tetramethoxychalcone) and naphthalene (cadalene) [ 14 ].

Ethanol (70%) extract of C. odorata leaves was found to contain flavonoids (genkwanin 4’-O-[α-L-rhamnopyranosyl(1→2)-β-D-glucopyranoside], sakuranetin 4’-O-[β-D-glucoyranosyl(1→2)-β-D-glucopyranoside], aromadendrin 4’-methyl ether, eriodictyol 7,4’-dimethyl ether, naringenin 4’-methyl ether, isosakuranetin, quercetin 7,4’-dimethyl ether, kaempferide, acacetin, rhamnazin, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside and kaempferol 3-O-glucoside [ 15 ].

Acetone extract of C. odorata leaves was found to contain carotenoids (carotene, lutein, anthera-xanthin, neoxanthin and viola-xanthin); lignans (epi-eudesmin, galgravin, and (9E,12E,15E)-9,12,15-octadecatrien-1-ol); flavonoids (apigenin-4’,7-dimethyl ether, retusin and sakuranin) and carboxylic acid (dehydroabietic acid) [ 8 ].

Dichloromethene extract of C. odorata dried leaves was found to contain aromatic hydrocarbons (1,8-dimethyl-naphthalene and 2,6-dimethyl-naphthalene); aliphatic hydrocarbons (3,7,11,15-tetramethyl-2-hexadecen-1-ol, 1-hexadecanol, 4,8,12,16-tetramethylheptadecan-4-olide, 2-nonadecanone, 1-octadecanol, 2-pentacosanone, pentadecane and 1-tridecene); fatty acid and esters (hexadecanoic acid, methyl ester, (Z,Z)-9,12-octadecadienoic acid methyl ester, octadecanoic acid methyl ester and tianshic acid); hydroanthranol (5aα,6,9,9aβ,10-pentahydro-10β-hydroxy-7-methylanthra[1,2-d][1,3]dioxol-5-one); anthraquinones (1,2-methylenedioxy-6-methylanthraquinone, 3-hydroxy-1,2,4-trimethoxy-6-methylanthraquinone, 3-hydroxy-1,2-dimethoxy-6-methylanthraquinone and austrocortinin); furofuranoid lignin (7-methoxy-7-epi-medioresinol, (-)-pinoresinol, (-)-medioresinol and (-)-syringaresinol); flavonoids (ombuin); triterpenes (ursolic acid and 3β-acetyloleanolic acid); chalcones (odoratin and 4,2’-dihydroxy-4’,5’6’-trimethoxychalcone) and coumarinolignoids (cleomiscosin A and cleomiscosin D) [ 16 , 8 ].

Methylene chloride extract of C. odorata dried leaves was found to contain phytosterols (b-sitosterol and stigmasterol) [ 8 ].

Chloroform extract of C. odorata leaves was found to contain hydrocarbon, aromatic dicarboxylic acid and fatty acid esters (dodecyl acetate, di-n-octyl phthalate, oleic acid methyl ester and hexadecanoic acid methyl ester) [ 7 ].

Hexane extract of C. odorata dried leaves was found to contain sesquiterpenes (α-cadinol, caryophyllene, caryophyllene oxide, germacrene D, β-caryophyllene, copaene, (-)-spathulenol and globulol); diterpene (phytol); phenylpropanoid (eugenol); fatty acid esters (hexadecanoic acid, methyl ester, (Z,Z)-9,12-octadecadienoic acid methyl ester, (Z)-9-octadecenoic acid methyl ester and octadecanoic acid methyl ester); hydrocarbons (eicosane, hexadecane, 1-hexadecanol, 4,8,12,16-tetramethylheptadecan-4-olide, octadecanal, pentadecanal, tetracosane, 1-tetracosanol and tridecane) [ 7 , 16 ].

Essential oil of C. odorata leaves was found to contain monoterpenes (linalool, α-pinene, β-pinene); sesquiterpenes (β-caryophyllene, β-copaen-4α-ol, (E)-caryophyllene, geijerene or pregeijerene and germacrene D) [ 18 , 19 ].

Methanol extract of C. odorata whole plant contained prostaglandin-like compounds ((9S,13R)-12-oxo-phytodienoic acid and chromomoric acid G); flavonoids (chromolanone, kaempferide, acacetin, rhamnazin, isosakuranetin, salvigenin, 2,4-dihydroxy,3’,4’,7-trimethoxyflavone, 4,6’-dihydroxy-2’,3’,4’-trimethoxychalcone, 4’,5,6,7’-tetramethoxyflavone) and fatty acids ((S)-15,16-didehydrocoriolic acid and (S)-coriolic acid) [ 20 ].

MEDICINAL USES

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

Traditionally used for intestinal pains, colds, cough, for healing wounds and to stop bleeding, as purgative, malaria, small pox and yellow fever [ 2 ].

Biological and pharmacological activities supported by experimental data

Antioxidant activity

Aqueous and ethanol extracts of C. odorata leaves showed antioxidant activity with 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) free radical scavenging activity with 50% inhibition concentration(IC50) at 22.50 and 23.70 μg/mL respectively compared to gallic acid (IC50 = 15.90 μg/mL) and ascorbic acid (IC50 = 17.00 μg/mL) [ 13 ].

Ethanol extract of C. odorata leaves showed antioxidant activity with DPPH free radical scavenging activity of 94.29% compared to the butylated hydroxytoluene (76.3%) [ 22 ].

Chloroform extract of C. odorata leaves (0.1 – 5.0 mg/mL) showed antioxidant activity with IC50 of 0.31 mg/mL compared to ascorbic acid (IC50 = 0.24 mg/mL) using DPPH radical scavenging assay [ 23 ].

Chloroform extract of C. odorata leaves (0.1 – 5.0 mg/mL) showed antioxidant activity with IC50 of 0.43 mg/mL compared to ascorbic acid (IC50 = 0.41 mg/mL) using hydroxyl radical scavenging assay [ 23 ].

Chloroform extract of C. odorata leaves (0.1 – 5.0 mg/mL) showed antioxidant activity with IC50 of 0.28 mg/mL compared to ascorbic acid (IC50 = 0.23 mg/mL) using nitric oxide radical scavenging assay [ 23 ].

Chloroform extract of C. odorata leaves (0.1 – 5.0 mg/mL) showed antioxidant activity with IC50 of 1.32 mg/mL compared to ascorbic acid (1.0 mg/mL) using 2,2-azinobis(3-ethyl benzothiazoline-6-sulfonic acid) radical scavenging assay [ 23 ].

The fatty acids ((S)-coriolic acid methyl ester, (S)-15,16-didehydrocoriolic acid methyl ester) from the methanol extract of C. odorata leaves inhibited nitric oxide production with IC50 values of 5.73 ± 1.96 µM and 12.40 ± 2.55 µM respectively, compared to resveratrol (10.90 ± 1.51 µM) [ 11 ].

Wound healing activity

Methanol extract of C. odorata dried leaves powder (5, 7.5 and 10% formulation) was applied topically on the excised wound (wound size: 450 – 500 mm2 and 2 mm depth) of Swiss albino rats (150 – 189 g) once daily for 14 days. There was a progressive wound healing activity from day 0 to day 16. The percentage reduction of wound area on day 16 was 96.88% (5% formulation), 98.02% (7.5% formulation) and 100% (10% formulation) compared to standard neosporin (100%) and betadine (97.95%) [ 24 ].

Ethyl acetate extract of C. odorata leaves (5, 7.5 and 10% formulation) also showed a progressive wound healing activity from day 0 to day 16. The percentage reduction of wound area at day 16 was 98.14% (5% formulation), 98.78% (7.5% formulation) and 100% (10% formulation) compared to standard neosporin (100%) and betadine (97.95%) [ 24 ].

The phenolic acid fraction (protocatechuic, p-hydroxybenzoic, p-coumaric, ferulic and vanillic acids) from crude ethanol extract of C. odorata leaves (100 µg/mL) showed significant protective effect (p < 0.001) on cultured human dermal fibroblasts against hydrogen peroxide (H2O2)-induced cell damage (≈ 0.550-0.575 absorbance) and hypoxanthine-xanthine oxidase (HX-XO)-induced cell damage (≈ 0.525-0.575 absorbance) compared to untreated cells (H2O2-induced damage: ≈ 0.15 absorbance; HX-XO-induced damage: ≈ 0.275 absorbance) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide (MTT) assay [ 25 ].

The diethyl ether fraction of ethanolic extract of C. odorata leaves showed significant protective effects (p < 0.05) on human epidermal keratinocytes against H2O2-induced cell damage (≈ 0.90 – 1.00 absorbance) and HX-XO-induced cell damage (≈ 0.75-0.80 absorbance) compared to untreated cells (H2O2-induced damage: ≈ 0.50 absorbance; HX-XO-induced damage: ≈ 0.35 absorbance) using MTT assay [ 25 ].

The ethanolic extracts of C. odorata showed hemostatic activity by reducing the bleeding time on incision made on the foot pad of male Wistar rats (80 – 120 g) (50% extract of dried leaves: 2.39 min; 70% extract of fresh leaves: 2.04 min; 70% extract of dried leaves: 1.85 min) compared to saline-control (3.3 min) [ 26 ].

The Eupolin ointment prepared from aqueous extract of C. odorata leaves showed wound healing effects on human keratinocyte cells. After four days, the growth of first passage human keratinocytes (treated with 0.1 to 5.0 μg/mL) was significantly (p < 0.001) increased (2000% compared to proliferation of keratinocyte in serum-free growth medium-control, 400% compared to proliferation of keratinocyte in basal medium-control) using MTT assay. The growth of second passage human keratinocytes (treated with 0.05 μg/mL) was significantly (p < 0.01) increased on day 4 (200% compared to proliferation in both medium-controls). However, on day 7, the extract at dose of 350 μg/mL was found to be toxic to the human keratinocytes. Apart from that, all extract-treated keratinocytes (10 – 60 μg/mL) migrated faster into denuded area with a more complete closure (20% after 12 hr) of the stratch compared to both medium-controls [ 27 ].

The ethanol extract (70%) of C. odorata leaves (34.2 μg/mL) treated on Balb/c 3T3 fibroblast cells showed faster cell migration in which the gap between cells was nearly closed after 24 hr and proliferation (1.15 cell viability ratio) compared to ethanol-treated cells (1.0). Using the same extract of different dosages (3.42, 34.2 and 100 μg/mL), the expression of heme oxygense-1 (HO-1) was also increased in a dose-dependant manner after 12 hr treatment using quantitative real-time PCR [ 28 ].

Anthelmintic activity

Methanol extract of C. odorata dried leaves powder (5, 10, 25 and 50 mg/mL) showed anthelmintic activity against adult Indian earthworm (Pheretima posthuma) (time taken for the worms to die at 14.588 – 25.84 min) in a dose-dependant manner as compared to albendazole (15 mg/mL, 63.83 min) [ 24 ].

Ethyl acetate extract of C. odorata dried leaves powder (5, 10, 25 and 50 mg/mL) showed anthelmintic activity against adult Indian earthworm (P. posthuma) (time taken for the worms to die at 14.9 – 27.783 min) in a dose-dependant manner as compared to albendazole (15 mg/mL, 63.83 min) [ 24 ].

Petroleum ether extract of C. odorata dried leaves powder (5, 10, 25 and 50 mg/mL) showed anthelmintic activity against adult Indian earthworm (P. posthuma) (time taken for the worms to die at 18.11 – 33.08 min) in a dose-dependant manner as compared to albendazole (15 mg/mL, 63.83 min) [ 29 ].

Anti-inflammatory activity

Aqueous extract of C. odorata dried leaves powder (200 mg/kg) was administered orally to male Wistar rats (190 ± 10 g) one hour before induction of paw edema using carrageenan into the subplantar of the right hind paws. After five hours, the extract significantly (p < 0.001) reduced the paw edema size from 4.6 ± 0.75 mm to 0.2 ± 0.20 mm (95% size reduction) compared to indomethacin (from 4.6 ± 0.75 mm to 1.0 ± 0.45 mm; 78% size reduction) [ 29 ].

Aqueous extract of C. odorata dried leaves powder (200 mg/kg) was administered to male Wistar rats (190 ± 10 g) for a duration of 4 days after induction of granuloma using cotton pellet granuloma test. At day 5, the extract significantly (p < 0.01) reduced the granuloma mass from 0.14 ± 0.01 mg to 0.09 ± 0.01 mg (35.7% mass reduction) compared to indomethacin (0.10 ± 0.01 mg; 28.6% mass reduction) [ 29 ].

The ethyl acetate, dichloromethane and aqueous fractions of ethanolic extract of C. odorata leaves (50 mg/kg) administered orally to male Wistar rats (180 ± 10 g) an hour earlier before induced paw oedema with carrageenan. After 5 hr of carrageenan injection, the paw size significantly (p < 0.05) reduced (to 1.6 – 2.8 mm) compared to saline-control (to 10.0 mm) [ 30 ].

The ethanol extract (70%) of C. odorata leaves (3.42 to 684 µg/mL) pre-treated on RAW 264.7 (murine) cells suppressed the expression of both pro-inflammatory proteins lipopolysaccharide-induced (LPS) cyclooxygenase-2 and lipopolysaccharide-induced inducible nitric oxide synthase compared to ethanol-control pretreatment. The same type of extract (684 µg/mL) also found to inhibit the production of prostaglandin E2 and nitric oxide compared to LPS-treated cells [ 31 ].

The phytoprostane chromomoric acid C-1 isolated from methanol extact of C. odorata leaves (1 – 10 µM) induced the endogenous nuclear factor-erythroid 2-related factor 2 (Nrf2) targeting heme oxygenase-1 (HO-1) in fibroblasts compared to 0.1% DMSO-vehicle control using luciferase reporter gene assay. At extract concentration of 2 to 5 µM, the HO-1 in vascular smooth muscle cells was reduced and HO-1 was inhibited in a dose-dependent manner without eliciting signs of cytotoxicity compared to 0.1% DMSO-vehicle control
[ 12 ].

The fatty acids ((S)-coriolic acid methyl ester, (S)-15,16-didehydrocoriolic acid methyl ester) from the methanol extract of C. odorata leaves inhibited NF-κB activation with the IC50 values of 5.22 ± 1.63 µM and 10.60 ± 1.74 µM, respectively, compared to resveratrol (12.50 ± 1.93 µM) [ 11 ].

Antihyperglycemic activity

The ethanol extract of C. odorata leaves (400 mg/kg) administered orally to intravenously streptozotocin-diabetic induced Wistar rats (150 – 250 g) significantly (p < 0.001) reduced in serum glucose level by 58.84% compared to 1% CMC-diabetic control group (increased by 5%). The daily administration of 200 and 400 mg/kg/day of the same extract respectively for eight weeks to the same rats showed reduction in the serum glucose level (by 58.49% and 64.26%, respectively) compared to 1% CMC-diabetic control group (increased by 5%) [ 21 ].

Anticataract activity

The ethanol extract of C. odorata leaves (200 and 400 mg/kg/day) administered orally to intravenously streptozotocin-diabetic induced Wistar rats (150 – 250 g) respectively for eight weeks did not exhibit fourth stage of mature cataract compared to 50% of 1% CMC-diabetic control group showed incidence of cataract at stage four [ 21 ].

Analgesic effect

The n-butanol and dichloromethane fractions of ethanol extract C. odorata leaves (50 mg/kg) was administered orally to male Wistar rats (180 ± 10 g) an hour earlier before induced paw oedema with formalin. After 5 min of formalin injection, the licking time were 67.6 ± 10.85 s and 71.1 ± 6.03 s respectively (p < 0.05) compared to saline-control (101.0 ± 6.18 s). While after 30 min of formalin injection, the licking time were 25.4 ± 7.06 s and 47.4 ± 4.33 s respectively (p < 0.001) compared to saline-control (88.4 ± 2.48 s) [ 30 ].

Antipyretic effect

The n-butanol and dichloromethane fractions of ethanol extract C. odorata leaves (50 mg/kg) were administered orally to brewers yeast induced pyrexia-male Wistar rats (180 ± 10 g) after one hour of induction. The rectal temperature at different time points (60 min: both ≈ 38.5˚C; 90 min: ≈ 36.0 ˚C, ≈ 36.6 ˚C and 120 min: ≈ 36.8 ˚C, ≈ 36 ˚C) respectively were significantly reduced compared to untreated-pyrexia control which the rectal temperature was measured after 17 hr of induction (≈ 39.5˚C, ≈ 38.5˚C) [ 30 ].

Clinical studies

Information and data have not been established.

SAFETY INFORMATION

Preclinical studies (Toxicology studies)

Acute oral toxicity

Ethanol extracts (80%) of C. odorata dried leaves powder was administered orally as a single dose to male and female Swiss albino mice (20 – 25 g) (fasted overnight) and observed for 72 hour. No adverse effects and death were observed in all mice treated with doses 1.0 to 15 g/kg. However, all highest dose-treated mice (20.0 g/kg) died within four hours. Necropsy revealed no significant abnormality in organs of mice. The median lethal dose was found to be more than 16.501 g/kg [ 31 ].

Oral single dose acute toxicity study on female Sprague Dawley rats (aged between 8 and 12 weeks old) using aqueous extract of C. odorata leaves showed no toxic effect on the parameters observed, including behavior, 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. Approximate lethal dose (LD₅₀) is not more than 2,000 mg/kg body weight [ 37 ].

Sub-chronic toxicity

Aqueous extracts of C. odorata freeze-dried leaves powder (538.5 and 1077 mg/kg) administered orally to male adult Wistar albino rats (80 – 150 g) three times a week for a duration of 90 days showed significant (p < 0.05) increase in the urea level (538.5 mg/kg dose: 10.43 ± 2.98 mg/dL, 1077 mg/kg dose: 12.62 ± 1.39 mg/dL) and uric acid level (2.50 ± 1.06 mg/dL, 3.38 ± 0.68 mg/dL) compared to deionized water-control treated group (urea: 9.37 ± 1.17 mg/dL and uric acid: 1.74 ± 0.43 mg/dL) respectively. However, at dose of 1077 mg/kg, the mortality was observed to be 43% [ 32 ].

Aqueous extracts of C. odorata freeze-dried leaves powder (1077 mg/kg) was administered orally to male adult Wistar albino rats (80 – 150 g) three times a week for a duration of 90 days. The extract significantly (p < 0.05) increased the creatine kinase level (4150 ± 10101.37 U/L) and amylase (1570 ± 59.52 U/L) with significant decrease of the total protein (74.62 ± 1.84 g/L) and albumin (33.55 ± 5.13 mmol/L) compared to control (creatine kinase: 1680.91 ± 1012.20 U/L, amylase: 1999.83 ± 338.79 U/L, total protein: 81.85 ± 5.78 g/L, albumin: 40.66 ± 3.47 mmol/L) [ 33 ].

Hepatotoxicity effect

Ethanol extract of C. odorata dried leaves powder (20 mg/kg) was administered orally to male Wistar Albino rats (aged 8 weeks old; 100 – 125 g) for a duration of 21 days. The extract showed significant (p < 0.05) increase in the level of ALT (12.0 ± 1.70 U/L) compared to control group (8.4 ± 2.24 U/L) [ 34 ].

Effects on kidney and intestine

Ethanolic extract of C. odorata dried leaves powder (100 mg/kg and 250 mg/kg) was administered to albino rats (both sexes, average body weight = 56 g) for six weeks. It significantly increased (p < 0.05) the concentration of urea (2.25 – 2.50 μmol/L) and creatinine (85 – 95 μmol/L) in a dose-dependant manner compared to distilled water-control group (1.5 μmol/L and 82.5 μmol/L) [ 35 ].

Genotoxicity

Essential oils and hexane extract of C. odorata leaves treated on human peripheral blood mononuclear cells exhibited IC50 of 14 mg/mL and 2.45 mg/mL respectively, and LD50 of 3684 mg/kg and 1927 mg/kg, respectively. The essential oils, hexane and methanol extracts significantly (p < 0.05) induced DNA damage as shown by the amount of damaged DNA tails (2.58, 1.27 and 5.50, respectively) using Comet assay as represented by the median of the olive tail moment (OTM) relative to the total amount of DNA as compared to the negative control (0.20) and positive control (5.73) (UV light exposure) [36 ].

Others (Adverse reactions, contraindications, side effects, warning, precautions)

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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