Cengkih Flower Buds
Syzygium aromaticum (L.) Merr. & L.M.Perry
Myrtaceae
Figure 1 : S. aromaticum. (a) Leaves; (b) flower buds in dried form; (c) whole plant. (Photos courtesy of Universiti Sains Malaysia, 2015)
DEFINITION
Cengkih flower buds consist of dried flower buds of Syzygium aromaticum (L.) Merr. & L.M.Perry (Myrtaceae).
SYNONYM
Caryophyllus aromaticus L., Eugenia aromatica (L.), Eugenia caryophyllus [ 1 ].
VERNACULAR NAMES
Clove (English); bunga cengkih (Malay); ding heung, ting hsiang (Chinese); lavangam (Tamil) [ 2 ].
CHARACTER
| Colour | Brown (powder) |
| Odour | Strongly aromatic |
| Taste | Spicy |
IDENTIFICATION
Plant Morphology
S. aromaticum is a small evergreen tree, 10-20 m high. Leaves opposite, petiolate, lanceolate, pinkish to dark green, with translucent, aromatic glands, have a pungent odour when young. Inflorescence occurs as racemose panicles and bears buds that take on the form of nails before blossoming. Flowers red with four concave, overlapping petals that drop off as soon as the flower opens; stamens numerous; 4 calyx lobes; buds 10-20 mm long, bright reddish-brown to dark brown; lower part (the hypanthium) solid, cylindrical, flattened, tapering towards the base and bearing at the apex, thick, triangular, divergent sepals, alternating with four rounded, fragile, unexpanded, membranous, imbricated petals forming a pale, nearly spherical head that encloses numerous stamens, curved inward and inserted on small disc. Fruits dark red, fleshy drupe [ 3 ].
Microscopy
Powdered material consists of brachysclereid with lumen and strongly thickened walls contain conspicuous simple pits, oval to subrectangular in shape; fragments of a group of vascular tissue with narrow spirally thickened vessels associated with fibres; fragment of pitted vessel; fibres are abundant, isolated, often whole with narrow lumen and thickened walls; solitary type of calcium oxalate crystals and fibres also observed under polarizing filter; collenchyma cells in fragments with thickened walls; pollen grains are numerous [ 3 , 4 , 5 ].
Figure 2 : Microscopic characters of S.aromaticum dried flower buds powder. (a) Brachysclereid cell; (b) fragments of spirally thickened vessel; (c) fragments of pitted vessel; (d) fragments of spiral vessel; (e) fragments of fibre (under polarizing filter); (f) fragments of fibre; (g) collenchyma cells; (h) calcium oxalate crystals (under polarizing filter); (i) pollen grains [P]. [Scale bars: h = 10 mm; a, b, c, d, g = 20 mm; f, I = 50 mm; e = 100 mm]
Colour Tests
Observed colour of solution after treatment with various reagents:
| NaOH (5%) | Dark brown |
| KOH (5%) | Dark brown |
Thin Layer Chromatography (TLC)
Figure 3 : TLC profiles of eugenol (S) and ethanol extract of S. aromaticum dried powder (L) observed under a) UV at 366 nm before spraying; b) UV 254 nm and c) UV 366 nm after spraying with 10% sulphuric acid in ethanol
| Test Solutions | Weigh about 5.0 g of S. aromaticum dried flower buds powder in a round bottom flask and add 25 ml of ethanol. Sonicate the sample for 15 minutes at room temperature. Filter the solution with filter paper. Evaporate the filtrate using rotary evaporator at 60°C. Reconstitute the residue with 20 ml of methanol. Dilute 0.5 mL of the solution to make 1 mL with methanol. |
| Standard solution | Dissolve eugenol standard [CAS. No. 97-53-0 in methanol to produce 2 mg/ml solution. |
| Stationary Phase | HPTLC Glass Silica Gel 60 F254, 10 x 10 cm. |
| Mobile phase | Heptane: ethyl acetate (7 : 3 ) (v/v) |
| Application |
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| Development distance | 8 cm |
| Drying | Air drying |
| Detection |
View at
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High Performance Liquid Chromatography (HPLC)
| Test solution | Weigh about 5.0 g of S. aromaticum dried flower buds powder in a round bottom flask and add 25 ml of ethanol. Sonicate the sample for 15 minutes at room temperature. Filter the solution with filter paper. Evaporate the filtrate using rotary evaporator at 60°C. Reconstitute the residue with 20 ml of methanol. Dilute 5 mL of the solution to produce 10 mL with methanol. Further dilute 1 mL of the solution to produce 20 mL with methanol. | ||||||||||||||||||
| Standard solution | Dissolve eugenol standard [CAS. No. 97-53-0] in methanol to produce 0.1mg/ml solution. | ||||||||||||||||||
| Chromatographic system |
Detector : UV 205 nm Column : C18 column (2.5 µm, 4.6 mm I.D x 150 mm) Zorbax SB-C18 (SP65) Column oven temperature : 25˚C Flow rate : 1.2 mL/min Injection volume : 5 µL |
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| Mobile Phase (gradient mode) |
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| Run time |
30 minutes |
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| System suitability requirement |
Perform at least five replicate injections of eugenol (0.1 mg/mL). The requirements of the system suitability parameters are as follow:
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| Acceptance criteria |
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(a)
(b)
Figure 4 : HPLC chromatogram of eugenol solution (0.1 mg/ml) (a) full and (b) zoom at tr=10.984 min
(a)
(b)
Figure 5 : HPLC chromatogram of ethanol extract of S. aromaticum dried flower buds powder (a) full and (b) zoom showing peak corresponding to eugenol at tr = 10.984 min
Figure 6 : UV spectrum of eugenol standard solution (0.1 mg/mL) and ethanol extract of S. aromaticum dried flower buds powder.
Figure 9 : UV spectrum of eugenol standard solution (0.125 mg/mL) andethanol extract of S. aromaticum dried flower buds powder.
PURITY TESTS
| Foreign Matter |
| Not more than 2% |
| Ash Contents | |
| Total ash | Not more than 6% |
| Acid-insoluble ash | Not more than 1% |
| Loss on Drying |
| Not more than 23% |
| Extractive Values | |
| Water-soluble extracts | |
| Hot method | Not less than 30% |
| Cold method | Not less than 25% |
| Ethanol-soluble extracts | |
| Hot method | Not less than 14% |
| Cold method | Not less than 10% |
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
Ethanolic extract of S. aromaticum flower buds has been found to contain quinone (e.g. isobiflorin and biflorin) [ 6 ].
Ethyl acetate extract of S. aromaticum flower buds has been found to contain terpenes (e.g. β-caryophyllene, β-caryophyllene oxide, α-humulene, α-humulene epoxide) [ 7 ].
Hexane extract of S. aromaticum flower buds has been found to contain terpenes (e.g. eugenol, caryophyllene oxide, and nootkatin) [ 8 ].
The essential oil (1.06%) from S. aromaticum flower buds has been found to contain terpenes (e.g. eugenol, acetyleugenol, chaviol) [ 9 ].
MEDICINAL USES
Uses described in folk medicine, not supported by experimental or clinical data
Traditionally used to treat asthma, bleeding gums, dyspepsia, fevers, and morning sickness and also used to suppress toothache and halitosis [ 3 , 10 ]
Biological and pharmacological activities supported by experimental data
Anti-oxidant activity
Aqueous extract of S. aromaticum flower buds (20-60 µg/mL) showed anti-oxidant activity 93.3-97.9% inhibition on lipid peroxidation of linoleic acid emulsion compared to tocopherol (61.1% inhibition), butylated hydroxyanisole (BHA) (96.6% inhibition) and butylated hydroxytoluene (BHT) (99.1% inhibition) using thiocyanate method [ 11 ].
Ethanol extract of S. aromaticum flower buds (20-60 µg/mL) showed anti-oxidant activity 94.9-98.2% inhibition on lipid peroxidation of linoleic acid emulsion compared to tocopherol (61.1% inhibition), BHA (96.6% inhibition) and BHT (99.1% inhibition) using thiocyanate method [ 11 ].
Aqueous extract of S. aromaticum flower buds (60 µg/mL) showed significant (p < 0.01) superoxide anion scavenging activity (79% inhibition) compared to tocopherol (26% inhibition), BHA (77% inhibition) and BHT (34% inhibition) [ 11 ].
Ethanol extract of S. aromaticum flower buds (60 µg/mL) showed significant (p < 0.01) superoxide anion scavenging activity (82% inhibition) compared to tocopherol (26% inhibition), BHA (77% inhibition) and BHT (34% inhibition) [ 11 ].
Aqueous extract of S. aromaticum flower buds (60 µg/mL) showed significant (p < 0.05) scavenging activity of (62% inhibition) compared to tocopherol (31% inhibition), BHA (62% inhibition) and BHT (60.1% inhibition) using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method [ 11 ].
Ethanol extract of S. aromaticum flower buds (60 µg/mL) showed significant (p < 0.05) scavenging activity (74% inhibition) compared to tocopherol (31% inhibition), BHA (62% inhibition) and BHT (60.1% inhibition) using DPPH radical method [ 11 ].
Anticancer activity
Aqueous extract of S. aromaticum flower buds (20 mg/mL) administered subcutaneously to benzo[α]pyrene-induced lung cancer newborn Strain A mice (4 week old) for duration of 26 weeks significantly (p < 0.001) inhibit cell proliferation (64.29%) compared to benzo[α]pyrene–treated control [ 12 ].
Anti-inflammatory activity
Essential oil of S. aromaticum flower buds (0.025-0.200 mL/kg) was administered intraperitoneally to male, outbred, Sprague-Dawley rats concurrently with induction of paw edema using lambda carrageenan Type IV. The extract (0.050 mL/kg) significantly (p < 0.001) inhibit paw edema (90.15%) compared to etodolac (43.42%) [ 13 ].
Antibacterial activity
The powder of S. aromaticum flower buds in phosphate-buffered saline (PBS) inhibited the growth Escherichia coli with minimum inhibitory concentrations (MIC) of 190 mg/mL, Staphylococcus aureus (MIC = 100 mg/mL) and Listeria innocua (MIC = 510 mg/mL) [ 14 ].
The powder of S. aromaticum flower buds in PBS inhibited the growth E. coli with minimum bactericidal concentrations (MBC) (the lowest concentration that completely kill 99.9% of the bacteria for 24 h) of 230 mg/mL, S. aureus (MBC = 120 mg/mL) and L. innocua (MBC = 550 mg/mL) [ 14 ].
Antiparasite activity
Essential oil of S. aromaticum flower budsinhibit growth of Giardia lamblia with inhibition concentration at 50% growth (IC50) of 134 µg/mL compared to eugenol (IC50 = 101 µg/mL). The extract concentrations at 200 µg/mL and above significantly (p < 0.05) reduced parasite proliferation (2.5 x 106 to 1 x 105 parasites/mL) compared to eugenol (9 x 105 to 3 x 104) [ 15 ].
Antifungal activity
Essential oil of S. aromaticum flower buds in methanol inhibit the growth of on Candida albicans with zone of inhibition of 2.50 ± 0.1 cm, Aspergillus fumigatus (2.95 ± 0.05 cm) and Cryptococcus neoformans (2.15 ± 0.15 cm) using agar well diffusion method [ 16 ].
Clinical studies
Information and data have not been established.
SAFETY INFORMATION
Preclinical studies (Toxicology studies)
Acute toxicity
Essential oil of S. aromaticum flower buds(0.2–6.4 mL/kg) administered intraperitoneally as a single dose to male and female Swiss albino mice (18-24 g) for duration 72 hours showed lethal dose at 50% growth (LD50) of 0.613 mL/kg [ 17 ].
Others (Adverse reaction, contraindication, side effect, warning, precaution)
Allergic contact dermatitis has been reported in patients who were regularly exposed to S. aromaticum or who already had dermatitis of the fingertips [ 3 ].
DOSAGE
In Ayurvedic practice, the recommended dosage is 0.5–2 g of S. aromaticum flower bud powdered [ 18 ].
STORAGE
Store below 30°C. Protect from light and moisture.
REFERENCES
- The plant list. [Internet] Syzygium aromaticum. Version 1.1; [ cited on 30th April 2015]. Available from: http://www.theplantlist.org/tpl1.1/record/kew-199236.
- Agroforestree database. [Internet] Syzygium aromaticum. Version 4.0; [ cited on 21st February 2015 ]. Available from: http://www.worldagroforestry.org/treedb2/speciesprofile.php?Spid=18033.
- World Health Organization. WHO Monographs on Selected Medicinal Plants. Vol. 2 [monograph on the internet]. Geneva: WHO Press; 2003 [cited 2015 May 7]. Available from: http://apps.who.int/medicinedocs/en/d/Js4927e/7.html.
- Malaysian Monograph Committee. Malaysian Herbal Monograph. Vol. 1. Kuala Lumpur; Published on behalf of the Malaysian Monograph Committee: 1999. p. 25-28.
- The British Pharmacopoeia Commission. British Pharmacopoeia 2014 Volume 4. London: The Stationary Office on behalf of the Medicines and Healthcare products Regulatory Agency (MHRA); 2013. p. 144-145.
- Yongwen Z, Yuwu C. Isobiflorin, a chromone C-glucoside from cloves (Eugenia caryophyllata). Phytochemistry. 1997;45(2):401-403.
- Zheng G-Q, Kenney PM, Lam LKT. Sesquiterpenes from clove (Eugenia caryophyllata) as potential anticarcinogenic agents. Journal of Natural Products. 1992;55(7):999-1003.
- Nassar MI, Gaara AH, El-Ghorab AH, Farrag A, Shen H, Huq E, et al. Chemical constituents of clove (Syzygium aromaticum) and their antioxidant activity. Revista Latinoamericana de Química. 2007;35(3):47.
- Yang Y-C, Lee S-H, Lee W-J, Choi D-H, Ahn Y-J. Ovicidal and adulticidal effects of Eugenia caryophyllata bud and leaf oil compounds on Pediculus capitis. Journal of Agricultural and Food Chemistry. 2003;51(17):4884-8.
- Plant Resources of South-East Asia (PROSEA). [Internet] Syzygium aromaticum (L.) Merrill & Perry [ cited on 20 November 2014 ]. Available from: http://proseanet.org/prosea/e-prosea_detail.php?frt=&id=597.
- Gulcin I, Gungor SI, Beydemir S, Elmastas M, Irfan Kufrevioglu O. Comparison of antioxidant activity of clove (Eugenia caryophylata) buds and lavender (Lavandula stoechas L.). Food Chemistry. 2004;87(3):393-400.
- Banerjee S, Panda CK, Das S. Clove (Syzygium aromaticum L.), a potential chemopreventive agent for lung cancer. Carcinogenesis. 2006;27(8):1645-54.
- Ozturk A, Ozbek H. The anti-inflammatory activity of Eugenia Caryophyllata essential oil : an animal model of anti-inflammatory activity. European Journal of Generative Medicine. 2005;2(4):159-63.
- Abdali H, Ajji A. Development of antibacterial structures and films using clove bud powder. Industrial Crops and Products. 2014.
- Machado M, Dinis AM, Salgueiro L, Custódio JBA, Cavaleiro C, Sousa MC. Anti-giardia activity of Syzygium aromaticum essential oil and eugenol: effects on growth, viability, adherence and ultrastructure. Experimental Parasitology. 2011;127(4):732-9.
- Ahmad N, Alam MK, Shehbaz A, Khan A, Mannan A, Hakim SR, et al. Antimicrobial activity of clove oil and its potential in the treatment of vaginal candidiasis. Journal of drug targeting. 2005;13(10):555-61.
- Hanefi O, Mustafa O, Abdurrahman O, Ebubekir C, Zabit Y. Determination of lethal doses of volatile and fixed oils of several plants. Eastern Journal of Medicine. 2013;9(1):4-6.
- The Ayurvedic Pharmacopoeia of India. Part I, Volume I, First Edition. Delhi (IN): The Controller of Publications; 2001 [Reprint of 1990 publication]. [Accessed 2012 September 24]. Available from: http://www.ccras.nic.in/.
