Buah pala seeds and arillus
Myristica fragrans Houtt
Myristicaceae
Figure 1 : M. fragrans. (a) Flowers; (b) fruits; (c) adaxial leaf; (d) leaves; (e) fruit; (f) seed showing arillus; (g) tree. (Photos courtesy of Centre for Herbal Standardization (CHEST), USM, 2015)
DEFINITION
Buah pala seeds and arillus consist of the dried seeds and arillus of Myristica fragrans Houtt (Myristicaceae).
SYNONYM
Myristica officinalis L.f., Myristica aromatica Lam., Myristica aromatica Sw., Aruana silvestris Burm.f., Myristica moschata Thunb., Palala fragrans (Houtt.) Kuntze [ 1 ].
VERNACULAR NAMES
Nutmeg (English); pala (Malay); rou dou kou (Chinese); jati pattiri, jatikka (Tamil) [ 2 , 3 , 4 ].
CHARACTER
| Colour | Light brown (powder) |
| Odour | Fragrant, aromatic |
| Taste | Spicy, pungent |
IDENTIFICATION
Plant Morphology
M. fragrans is an evergreen tree usually growing to around 5-13 m high, occasionally 20 m. Bark contains watery pink or red sap. Leaves simple, dark green, aromatic, arranged alternately along the branches; blade elliptical to lanceolate, 5-15 cm x 2-7 cm, base acute, apex acuminate, margin entire, upper leaf surfaces are shiny, much paler beneath; petiole about 1 cm long. Inflorescence racemes axillary, peduncles and pedicels are glabrous. Flowers fragrant, glabrescent with sparse, pale yellow, with a 3-lobed perianth, waxy, fleshy and bell-shaped, usually single sexed, occasionally male and female flowers are found on the same tree; male flowers in groups of 1-10, 5-7 mm long, usually narrowed perianth at the base, 8-12 stamens adnate to a column; female flowers arise in groups of 1-3, up to 1 cm long. Fruits are fleshy, drooping, smooth, yellow in color, 6-9 cm long, with a longitudinal ridge; when ripe, the succulent yellow fruit coat splits into two valves revealing a shiny, purplish-brown seed (nutmeg), surrounded by a red aril (mace). Seeds (nutmegs) ovoid, 2-3 cm long and about 2 cm broad, fleshy, whitish and transversed by red-brown veins; the aril (mace) is bright scarlet in color, which is attached to the base of seed, brittle when fresh and becoming yellowish-brown when dried [ 5 ].
Microscopy
Powdered material consists of starch granules mostly found in groups, some simple and spherical, fairly small, sometimes attached to fibres; epidermis cells found in fragmented form; abundance of oil globules, and some attached to endodermis; fragment of scalariform vessels; fragments of outer perisperm with polyhedral to rounded cells; endodermis filled with starch granules and sometimes with oil glands; parenchyma cells found in abundance, thin-walled, irregular in shape, and filled with reddish-brown contents; calcium oxalate crystal found in solitary form [ 6 , 7 ].
Figure 2 : Microscopic characters of M. fragrans dried seeds and arillus. (a) Starch granules; (b) epidermis cell; (c) oil gland; (d) scalariform vessels; (e) perisperm cell; (f) endodermis with oil gland (OG); (g) endodermis containing starch (S) and oil glands (OG); (h) reddish brown parenchyma; (i) solitary crystal (arrow); (j) starch granules (arrow) attached to fibres. [Scale bars: a, b, e-h = 20 µm; c, d, i, j = 10 µm]
Colour Tests
Observed colour of solution after treatment with various reagents:
| HCI | Brown |
| NaOH (5%) | Brown |
| NH4OH (25%) | Brown |
Thin Layer Chromatography (TLC)
Figure 3 : TLC profiles of myristicin standard (S) and diethyl ether fraction of ethanolic extract M. fragrans dried seeds and arillus powder (L) observed under (a) UV at 254 nm (b) UV at 366 nm before derivatization, and (c) visible light after derivatization with 10% sulphuric acid in methanol.
| Test Solutions | Weigh 10 g of M. fragrans dried seeds and arillus powder into a 100 mL conical flask. Add 50 mL of absolute ethanol and sonicate for 15 min at room temperature. Filter the extract with Whatman1 filter paper. Dry off the filtrate with rotary evaporator at 55°C and 160 mbar vacuum. Dissolve the extract with 20 mL diethyl ether and transfer to a 50 mL test tube. Use another 10 mL diethyl ether to rinse the flask. Add 20 mL of deionized water to the diethyl ether. Tumble mix the solution and centrifuge at 3000 rpm for 5 min. Discard the water fraction. Repeat the clean-up process twice. Dry off the diethyl ether at 40°C and 550 mbar vacuum in a conical flask. Reconstitute the dried extracts in 4 mL acetonitrile. |
| Standard solution | Dissolve myristicin standard [CAS no.: 607-91-0] in acetonitrile to produce a standard concentration 0.25 mg/mL. |
| Stationary Phase | HPTLC silica gel pre-coated plate 60 F254, 10 x 10 cm heated in oven for 20 min at 100°C. |
| Mobile phase | Heptane : ethyl acetate (7 : 3) (v/v) |
| Application |
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| Development distance | Equilibrate the chamber with the mobile phase for 10 min, develop vertically for 7.0 cm. |
| Drying | Air drying |
| Detection |
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High Performance Liquid Chromatography (HPLC)
| Test solution | Weigh 10 g of M. fragrans dried seeds and arillus powder into a 100 mL conical flask. Add 50 mL of absolute ethanol and sonicate for 15 min at room temperature. Filter the extract with Whatman1 filter paper. Dry off the filtrate with rotary evaporator at 55°C and 160 mbar vacuum. Dissolve the extract with 20 mL diethyl ether and transfer to a 50 mL test tube. Use another 10 mL diethyl ether to rinse the flask. Add 20 mL of deionized water to the diethyl ether. Tumble mix the solution and centrifuge at 3000 rpm for 5 min. Discard the water fraction. Repeat the clean-up process twice. Dry off the diethyl ether at 40°C and 550 mbar vacuum in a conical flask. Reconstitute the dried extracts in 4 mL acetonitrile. | |||||||||||||||||||||
| Standard solution | Dissolve myristicin [CAS no.: 607-91-0] in acetonitrile to produce a standard concentration 0.25 mg/mL. | |||||||||||||||||||||
| Chromatographic system |
Detector : DAD 205 nm Column : 150 X 4.6 mm, 3.5 µm (Zorbax SB-C18 unless necessary) Column oven temperature : 25°C Flow rate : 1.2 mL/min Injection volume : 10 µL |
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| Mobile Phase (gradient mode) |
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| System suitability requirement |
Perform at least five replicate injections of the standard solutions (0.25 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 myristicin standard solution (0.25 mg/mL) (a) full and (b) zoom at tr= 20.482 min.
(a)
(b)
Figure 5 : HPLC chromatogram of diethyl ether fraction of ethanolic extract M. fragrans solution (a) full and (b) zoom at tr = 20.477 min.
Figure 6 : UV spectrum of myristicin standard solution (0.25 mg/mL).
Figure 7 : UV spectrum of myristicin standard solution (0.25 mg/mL) and diethyl ether fraction of ethanolic extract M. fragrans dried seeds and arillus powder.
PURITY TESTS
| Foreign Matter |
| Not more than 2% |
| Ash Contents | |
| Total ash | Not more than 3% |
| Acid-insoluble ash | Not more than 1% |
| Loss on Drying |
| Not more than 12% |
| Extractive Values | |
| Water-soluble extracts | |
| Hot method | Not less than 9% |
| Cold method | Not less than 5% |
| Ethanol-soluble extracts | |
| Hot method | Not less than 9% |
| Cold method | Not less than 3% |
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 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 (75%) of M. fragrans seed-kernel has been reported to contain macelignan [ 8 ].
Methanol extract of M. fragrans seed has been reported to contain erythro-austrobailignan-6, meso-dihydroguaiaretic acid and nectandrin–B [ 9 ].
Ethanol extract of M. fragrans seed has been reported to contain resorcinols malabaricone B and malabaricone C [ 10 ].
The essential oil of M. fragrans seeds has been reported to contain α-thujene, β-pinene, camphene, sabinene, α-myrcene, α-terpinene, limonene, β-ocimene, γ-terpinene, trans-sabinene hydrate, terpinolene, linalool, fenchyl alcohol, cis-sabinene hydrate, 4-terpineol, α-terpineol, citronellol, linalyl acetate, bornyl acetate, safrole, methyl eugeunol, isoeugeunol, myristicin, elimicin, methoxyeugeunol, β-asaron, myristic acid, ethyl miristate, palmitic acid, ethyl palmitate, stearic acid, ethyl oleate [ 11 ].
MEDICINAL USES
Uses described in folk medicine, not supported by experimental or clinical data
Traditionally used as a stimulant digestive, tonic, particularly given after childbirth, aphrodisiac and dysentery. It is also used externally in applications for various pains [ 12 ].
Biological and pharmacological activities supported by experimental data
Inhibition of locomotor activity
Essential oil of M. fragrans seeds (0.1, 0.3 and 0.5 mL/cage) administered by inhalation to male mice (25-30 kg; 2-3 months old) showed decreased locomotion in a dose dependent manner (62.81%, 65.33% and 68.62%) after 75 min of inhalation compared to locomotion activity by lavender oil (32.46%, 38.52% and 57.86%). Myristicin and 4-terpineol were identified as the predominant compounds found in the blood plasma of mice made to inhale the nutmeg seed essential oil [ 11 ].
Antioxidant activity
Aqueous extract of the fully matured seeds of M. fragrans (0.025–2 mg/mL) exerted significant (p < 0.05) reducing power with effective concentration at 50% level (EC50)of 1.09 ± 0.014 mg/mL compared to butylated hydroxytoluene (BHT) (EC50 = 0.36 ± 0.013 mg/mL) using ferric reducingantioxidant power assay (FRAP) [ 13 ].
Aqueous extract of the fully matured seeds of M. fragrans (0.025–2 mg/mL) showed significant (p < 0.05) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity with inhibition concentration at 50% level (IC50) of 1.18 ± 0.022 mg/mL compared to butylated hydroxytoluene (BHT) (IC50 = 0.17 ± 0.018 mg/mL) [ 13 ].
Aqueous extract of the fully matured seeds of M. fragrans (0.20-1.2 mg/mL) exhibited chelating activity on Fe2+ (1 mg/mL; 48.34 ± 1.85%) by dose dependent manner compared to EDTA (0.20-1.2 mg/mL) with chelating activity of 99.6 ± 1.65% [ 13 ].
Aqueous extract of the fully matured seeds of M. fragrans (1 mg/mL) showed significant (p < 0.05) β-carotene bleaching activity (64.11 ± 1.43%) compared to BHT (94.91 ± 1.03%) using β-carotene linoleic acid model system [ 13 ].
Ethanol extract of the fully matured seeds of M. fragrans (0.025-2 mg/mL) showed significant reducing power (p < 0.05) with EC50 of 0.78 ± 0.041 mg/mL compared to BHT (EC50 = 0.36 ± 0.013 mg/mL) using FRAP assay [ 13 ].
Ethanol extract of the fully matured seeds of M. fragrans (0.025-2 mg/mL) showed significant (p < 0.05) DPPH radical scavenging activity with IC50 of 0.77 ± 0.011 mg/mL compared to BHT (IC50 = 0.17 ± 0.018 mg/mL) [ 13 ].
Ethanol extract of the fully matured seeds of M. fragrans (0.20-1.2 mg/mL) exhibited significant (p < 0.05) chelating activity on Fe2+ (1 mg/mL; 72.11 ± 1.54%) by dose dependent manner compared to EDTA (0.20-1.2 mg/mL) with chelating activity of 99.6 ± 1.65% [ 13 ].
Ethanol extract of the fully matured seeds of M. fragrans (1 mg/mL) showed significant (p < 0.05) β-carotene bleaching activity (38.47 ± 1.31%) compared to BHT (94.91 ± 1.03%) using β-carotene linoleic acid model system [ 13 ].
Methanol extract of the fully matured seeds of M. fragrans (0.025–2 mg/mL) showed significant (p < 0.05) reducing power with EC50 of 0.91 ± 0.017 mg/mL compared to BHT (EC50 = 0.36 ± 0.013 mg/mL) using FRAP assay [ 13 ].
Methanol extract of the fully matured seeds of M. fragrans (0.025–2 mg/mL) showed significant (p < 0.05) DPPH radical scavenging activity with IC50 of 1.04 ± 0.029 mg/mL compared to BHT (IC50 = 0.17 ± 0.018 mg/mL) [ 13 ].
Methanol extract of the fully matured seeds of M. fragrans (1 mg/mL) showed significant (p < 0.05) β-carotene bleaching activity (60.26 ± 1.08%) compared to BHT (94.91 ± 1.03%) using β-carotene linoleic acid model system [ 13 ].
Butanol extract of the fully matured seeds of M. fragrans (0.025–2 mg/mL) showed significant (p < 0.05) reducing power with EC50 of 1.46 ± 0.021 mg/mL compared to BHT (EC50 = 0.36 ± 0.013 mg/mL) using FRAP assay [ 13 ].
Butanol extract of the fully matured seeds of M. fragrans (0.025–2 mg/mL) showed significant (p < 0.05) DPPH radical scavenging activity with IC50 of 1.18 ± 0.022 mg/mL compared to BHT (IC50 = 0.17 ± 0.018 mg/mL) [ 13 ].
Butanol extract of the fully matured seeds of M. fragrans (1 mg/mL) showed significant (p < 0.05) β-carotene bleaching activity (47.44 ± 1.27%) compared to BHT (94.91 ± 1.03%) using β-carotene linoleic acid model system [ 13 ].
Acetone extract of the fully matured seeds of M. fragrans (0.025–2 mg/mL) showed significant (p < 0.05) reducing power with EC50 of 0.65 ± 0.024 mg/mL compared to BHT (EC50 = 0.36 ± 0.013 mg/mL) using FRAP assay [ 13 ].
Acetone extract of the fully matured seeds of M. fragrans (0.025–2 mg/mL) showed significant (p < 0.05) DPPH radical scavenging activity with IC50 of 0.66 ± 0.015 mg/mL compared to BHT (IC50 = 0.17 ± 0.018 mg/mL) [ 13 ].
Acetone extract of the fully matured seeds of M. fragrans (1 mg/mL) showed significant (p < 0.05) β-carotene bleaching activity (74.36 ± 1.94%) compared to BHT (94.91 ± 1.03%) using β-carotene linoleic acid model system [ 13 ].
Antibacterial activity
Macelignin compound isolated from ethyl acetate fraction of methanol extract (75%) of M. fragrans seeds showed antibacterial activity against Streptococcus mutans with minimum inhibitory concentration (MIC) of 3.9 µg/mL, Streptococcus sobrinus (MIC = 15.6 µg/mL), Streptococcus sanguis (MIC = 2.0 µg/mL), Streptococcus salivarius (MIC = 31.3 µg/mL), Lactobacillus acidophilus (MIC = 2.0 µg/mL), Lactobacillus casei (MIC = 3.9 µg/mL) compared to chlorhexidine with MIC values of 1 µg/mL (S. mutans), 3.9 µg/mL (S. sobrinus), 2.0 µg/mL (S. sanguis and S. salivarius), 31.3 µg/mL (L. acidophilus and L. casei) using two-fold tube dilution method [ 8 ].
Aqueous methanol (80%) extract of M. fragrans aril inhibited the growth of gram-positive bacteria with minimum inhibitory concentration (MIC = 50 μg/mL (Staphylococcus aureus; Bacillus cereus) and MIC = 200 μg/mL (Bacillus licheniformis) compared MIC values of tetracycline (S. aureus = 3.9 μg/mL; B. cereus = 2 μg/mL; B. licheniformis = 3.9 μg/mL) using micro-well dilution method [ 14 ].
Analgesic activity
Alkaloids compounds of M. fragrans seed extract (1.0 g/kg) was administered orally to six female BALB/c mice (20-25 g; two months old) one hour before induction of pain using acetic acid. The extract showed significant (p < 0.01) decrease in number of writhing response (17 ± 4) compared to control (22 ± 3) [ 15 ].
Anti-parasitic activity
Essential oil extracted from M. fragrans seeds (3.125–100 µg/mL) showed anti-parasitic activity against Toxoplasma gondii parasite with EC50 of 24.45 µg/mL compared to clindamycin (EC50 = 16.57 µg/mL) [ 16 ].
Clinical studies
Information and data have not been established.
SAFETY INFORMATION
Preclinical studies (Toxicology studies)
Acute toxicity
Alkaloid compounds of M. fragrans dried seed (2-6 g/kg) were administered orally to both sexes of BALB/c mice (20-25 g; two month old). There was no toxicity effect observed for a period of 24 hours for dose 2 and 3 g/kg (LD50 > 5.1 g/kg). Rats receiving 4 g/kg and higher doses showed abnormal behavior such as hypoactivity, unstable gait or dizziness lasting for several hours. Death was observed from 4 g/kg [ 15 ].
Others (Adverse reaction, contraindication, side effect, warning, precaution)
Information and data have not been established.
DOSAGE
In Ayurvedic practice, the recommended dosage is 0.5-1.0 g of M. fragrans powder [ 17 ].
STORAGE
Store below 30°C. Protect from light and moisture.
REFERENCES
- The Plant List. [Internet] Myristica fragrans Houtt. Version 1.1. [cited on 20th May 2015]. Available from: http://www.theplantlist.org/tpl1.1/record/kew-2500629.
- Plant Resources of South-East Asia. [Internet] Myristica fragrans Houtt. [cited on 18th March 2015] Available from: http://proseanet.org/prosea/e-prosea_detail.php?frt=&id=580.
- Chinese Herbs Healing. [Internet] Nutmeg (Rou Dou Kou) [cited on 18th March 2015]. Available from: http://www.chineseherbshealing.com/nutmeg/.
- LivingNaturally. [Internet] Nutmeg (Myristica fragrans, Myristica officinalis). [cited on 20th May 2015]. Available from: http://www.livingnaturally.com/ns/DisplayMonograph.asp?StoreID=E32FA6C399AB4C99897032581851D45D&DocID=bottomline-nutmeg.
- Chemical Safety Information from Intergovernmental Organizations. [Internet]. Myristica fragrans Houtt. [cited on 18th March 2015]. Available from: http://www.inchem.org/documents/pims/plant/pim355.htm#SectionTitle:3.1%20%20Description%20of%20the%20plant.
- Parimala N, Amerjothy S. Histological and histochemical investigations of Myristica fragrans Houtt. (Myristicaceae). Journal of Pharmacognosy and Phytochemistry. 2013;5(1):106-111.
- Ministry of Health and Family Welfare. The Ayurvedic Pharmacopoeia of India Part 1, Volume 1, First Edition. New Delhi: Government of India. 1989;p.53-54.
- Chung JY, Choob JH, Leec MH, Hwang JK. Anticariogenic activity of macelignan isolated from Myristica fragrans (nutmeg) against Streptococcus mutans. Phytomedicine. 2006;13(4):261-266.
- Cho JY, Choi GJ, Son SW, Jang KS, Lim HK, Lee SO, Sung ND, Cho KY, Kim JC. Isolation and antifungal activity of lignans from Myristica fragrans against various plant pathogenic fungi. Pest Management Science. 2007;63(9):935-940.
- Orabi KY, Mossa JS and El-feraly FS. Isolation and characterization of two antimicrobial agent from mace (Myristica fragrans). Journal of Natural Products. 1991;54(3):856-859.
- Muchtaridi, Subarnas A, Apriyantono A, Mustarichie R. Identification of compounds in the essential oil of nutmeg seeds (Myristica fragrans Houtt.) that inhibit locomotor activity in mice. International Journal of Molecular Sciences. 2010;11(11):4771-4781.
- Burkill IH. A dictionary of the economic products of the Malay peninsula. Vol. 2. London; Published on behalf of the governments of the Straits settlements and Federated Malay states by the Crown agents for the colonies. 1935;p.2303-2304.
- Gupta AD, Bansal VK, Babu V, Maithil N. Chemistry, antioxidant and antimicrobial potential of nutmeg (Myristica fragrans Houtt). Journal of Genetic Engineering and Biotechnology. 2013;11(1):25-31.
- Sulaiman SF, Ooi KL. Antioxidant and antifood-borne bacterial activities of extracts from leaf and different fruit parts of Mristica fragrant Houtt. Food Control. 2012;25:533-536.
- Hayfaa AA, Sahar AM, Awatif MA. Evaluation of analgesic activity and toxicity of alkaloids in Myristica fragrans seeds in mice. Journal of Pain Research. 2013;6:611-615.
- Pillai S, Mahmud R, Lee WC, Perumal S. Antiparasitic activity of Myristica fragrans Houtt. essential oil against Toxoplasma gondii parasite. APCBEE Procedia. 2012;2:92-96.
- Department of Health, Ministry of Health and Family Welfare, Government of India. The Ayurvedic Pharmacopoeia of India Part 1. Volume 1. New Delhi, India: The Controller of Publications. 1990; pg.53-54.
