Selasih Leaves
Ocimum basilicum L.
Lamiaceae
Figure 1 : O. basilicum. (a) Simple leaf; (b) young plant; (c) mature plant; (d) field plantation; (e) plant habit; (f) flowering basil stalk and leaves. (Photos courtesy of Thiyagu, MARDI, 2013)
DEFINITION
Selasih leaves consist of dried leaves of O. basilicum L. (Lamiaceae).
SYNONYM
Basilicum citratum Rumph., Ocimum album L., Ocimum americanum Jacq., Ocimum menthaefolium Hochst. ex. Benth [ 1 ].
VERNACULAR NAMES
Lemon basil, sacred basil, sweet basil (English); kemangi, pokoh, ruku ruku, sulasi, selasseh uteh (Malay), lo-le, lou le (Chinese); babui tulsi, sada tulas (India) [ 1 ].
CHARACTER
| Colour | Brownish green dried leaves |
| Odour | Aromatic with characteristic sweet smell |
| Taste | Somewhat strong and pungent, or weak flavour |
IDENTIFICATION
Plant Morphology
An aromatic, erect, much branched annual herbs, 30-60(-130) cm tall. Stem glabrous and woody at base, apex retrorse puberulent and an indumentum of patent hairs. Leaves large, green, 2.5–5 cm,decussately opposite, broadly elliptical, 1.5-6 cm x 1-2.5 cm; margin remotely serrate, pubescent; lateral veins 3- or 4-paired; petiole 0.5-2.5 cm long. Inflorescence racemose, 8-10 cm long, consisting of opposite 3-flowered cymes appearing as verticils, lax; bracts sessile, oblanceolate, 5-8 mm. Flowers small, 3 mm long, arranged in a terminal spike, red (magenta), pink or white; stamens 4, free, slightly exserted, posterior 2 dentate, base puberulent; pistil lie over the inferior lip of the corolla; calyx bilabiate, pubescent outside, pilose at throat inside, tube ca. 2 mm. Fruit consists of 4 nutlets, surrounded by the persistent calyx on a spreading pedicel, 3-4 mm long, dark brown, ovoid, glandular foveolate. Seeds become gelatinous when soaked [ 2 ].
Microscopy
Microscopic characters of O. basilicum leaf powder consist of stomata attached to epidermis cells, parenchyma cells, simple multicellular trichome, pitted and spiral vessel cells and fibre cell [ 3 ].
Figure 2 : Microscopic characters of O. basilicum leaf powder, (a) Stomata attached to epidermis cells; (b) Parenchyma cells; (c-d) Simple multicellular trichome; (e) Spiral vessel cells; (f) Fibre cell and (g) Pitted vessel cells. [Scale bars: a-c = 5 µm; d-g = 20 µm]
Colour Tests
| HCl (conc.) | Green |
| 25% NH4OH | Green |
Thin Layer Chromatography (TLC)
Figure 3 : TLC profiles of rosmarinic acid (S) and methanol extract of O. basilicum dried leaves powder (L1) observed under (a) visible light (b) UV at 254 nm and (c) UV at 366 nm after heat the plate at 105°C for 30 minutes.
| Test Solutions | Weigh about 1 g of O. basilicum dried leaves powder in a round bottom flask and add 5 mL of methanol. Reflux the mixture for 1 hour. Filter the solution and use the filtrate as test solution. |
| Standard solution | Dissolve 5.0 mg of rosmarinic acid [CAS no.: 20283-92-5] in 5 mL methanol to produce 1000 µg/mL solution. |
| Stationary Phase | HPTLC Glass Silica Gel 60 F254, 10 x 10 cm |
| Mobile phase | Dichloromethane : acetone : formic acid (9:3:0.5) (v/v) |
| Application |
(L1) Kelantan |
| Development distance | 80mm |
| Drying | Air drying |
| Detection |
Heat the plate at 105°C for 30 minutes and view at
|
High Performance Liquid Chromatography (HPLC)
| Test solution | Weigh about 1 g of Ocimum basilicum dried leaves powder in a round bottom flask and add 5 mL of methanol. Reflux the mixture for I hour. Filter the mixture solution through a 0.45 µm syringe filter and inject the filtrate into the HPLC column. | |||||||||||||||||||||
| Standard solution | Dissolve 5.0 mg of rosmarinic acid standard [CAS no.: 20283-92-5] in 5 mL of methanol to produce 1.0 mg/mL solution. | |||||||||||||||||||||
| Chromatographic system |
Detector: UV 320 nm Column: C18 (5 µm, 4.6 mm I.D x 250 mm) (preferably Zorbex Eclipse Plus C18, SP06, Agilent column) Column oven temperature: 25°C Flow rate: 0.7 mL/min Injection volume: 3 µL |
|||||||||||||||||||||
| Mobile Phase (gradient mode) |
|
|||||||||||||||||||||
| System suitability requirement |
Perform at least five replicate injections of rosmarinic acid (1.0 mg/ml) The requirements of the system suitability parameters are as follow:
|
|||||||||||||||||||||
| Acceptance criteria |
|
Figure 4 : HPLC full chromatogram of rosmarinic acid solution (1.0 mg/mL) at tr = 8.541min.
Figure 5 :HPLC zoom chromatogram of rosmarinic acid solution (1.0 mg/ml) at tr = 8.541min.
Figure 6 : HPLC full chromatogram of methanol extract o O. basilicum dried leaves powder showing peak corresponding to rosmarinic acid (tr = 8.570 min).
Figure 7 : HPLC zoom chromatogram of methanol extract of O. basilicum dried leaves powder showing peak corresponding to rosmarinic acid (tr = 8.570 min).
Figure 8 : UV spectrum of methanol extract of O. basilicum dried leaves powder and rosmarinic acid standard solution.
PURITY TESTS
| Foreign Matter |
| Not more than 2% |
| Ash Contents | |
| Total ash | Not more than 15% |
| Acid-insoluble ash | Not more than 1% |
| Loss on Drying |
| Not more than 10% |
| Extractive Values | |
| Water-soluble extracts | |
| Hot method | Not less than 27% |
| Cold method | Not less than 20% |
| Ethanol-soluble extracts | |
| Hot method | Not less than 11% |
| Cold method | Not less than 5% |
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
Essential oil of the leaves parts has been found to contain monoterpenes (e.g. chavicol, ρ-cymene, limonene, 1,8-cineole, β-ocimene, linalool, camphor, α-terpineol, cis-geraniol, geraniol, linalyl acetate, bornyl acetate, methyl eugenol, eugenol, methyl chavicol, β-pinene, sabinene, phellandrene, β-myrcene, cis-β-ocimene, α-terpinolene, fenchone, camphor, citral, borneol, β-elemene), sesquiterpenes (e.g. α-copaene, β-cubebene, β-caryophyllene, α-bergamotene, γ-muurolene, germacrene D, β-selinene, bicyclogermacrene, γ-cardinene, α-cubebene, β-Bourbonene, zingiberene, trans-β-caryophyllene, γ-cadinene, δ-Cadinene β-Sesquiphellandrene, α-humulene, β-bisabolene, calamenene, xpathulenol, α-cadinol, β-bisabolol, β-eudesmol), monoterpenoids (e.g. carvacrol, citronellol) and others (e.g. α-terpinene, 6-methyl 3-heptenone, 1-octen-3-ol, trans–sabinene hydrate, dill ether, linalyl acetate, 1-octanol, isobornyl acetate, methylfuran, terpinen-4-ol, E-β-farnesene, linalyl propionate, β-guaiene, neryl acetate, carvone, nerol, trans-β-Ionone, caryophyllene oxide, humulene oxide, nerolidol, methyl cinnamate, methyl jasmine, 7-epi-amiteol, hexadecanoic acid, aloaromadendrene, β-burbonene, α-amorphene, spatulenol, torreyol) [ 4 , 5 , 6 ]
MEDICINAL USES
Uses described in folk medicine
The decoction of O. basilicum plant is used to treat cough and prevent flatulence. The extract of the leaves can be applied to cure ringworms and insect bites. It is also used for relieving toothache. O. basilicum was administered orally for women after childbirth and women with irregular menses. It also helps to ease itchiness in the throat. Soaked seeds can be used to soothe the stomach and to stimulate bowel movement. The seeds also relieves headache [ 7 ]. Seeds of O. basilicum can be used to cure catarrh, chronic diarrhoea, dysentery, gonorrhoea, nephritis, cystitis, internal piles and pains after childbirth. Soaked seeds can be drink for aphrodisiac. Juice of the leaves also used for earache and dullness of hearing. Ginger and black pepper mix with the leaf-juice is given during cold stages of ague. Dried powered leaves are used like snuff dislodges maggots from the nose. Decoction of the plant acts as parasiticide and antiseptic in nasal myasis [ 8 ].
Biological and pharmacological activities supported by experimental data
Anti-oxidant activity
The total phenolic content of the methanol extract of O. basilicum leaves (1:5 w/v) is 227.2 ± 7.4 mg/g as gallic acid equivalent. Rosmarinic acid in the total phenolic content showed lower anti-oxidant activity with S50 (time taken to reduce 50% of total antioxidant introduced to system) (1140 s) compared to α-tocopherol (1200 s) using soy phosphatidylcholine liposome system [ 9 ].
The methanol (99.5%) extract of O. basilicum leaves (0.22 w/w) showed a higher antioxidant activitiy (<0.25 absorbance value) than α-tocopherol (>0.25 absorbance value) on day 8 by using ferric thiocyanate (FTC) assay. Thiobarbituric acid (TBA) method also showed antioxidant activity of the extract (0.048 absorbance value) compared to α-tocopherol (0.32 absorbance value). The extract also showed antioxidant activity with 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of (IC50 190 ± 0.7 µg/mL) compared to quercetin (IC50 4.8 ± 0.01 µg/mL) [ 10 ].
Volatile compounds (eugenol, thymol, carvacrol and 4-allylphenol (5 μg/ml)) in essential oils of O. basilicum leaves showed strong antioxidant activity by inhibiting the oxidation of hexanal by almost 100% compared to α-tocopherol (89± 0.6%) and BHT (99± 0.8%) [ 11 ].
Free volatile aglycones from O. basilicum leave extract showed DPPH radical scavenging activity with EC50 = 3.338 g/L while the activity of essential oil was EC50 = 1.378 g/L, compared to BHT (EC50 = 0.908 g/L) and pure eugenol (EC50 = 0.906 g/L). The FRAP method showed activity of volatile aglycones (~18 mmol/L), and essential oil (~20 mmol/L) was lower compared to eugenol (~130mmol/L) [ 12 ].
Antimicrobial activity
Essential oil from O. basilicum leaves inhibited the growth of Staphylococcus aureus (MIC = 2.5 mg/mL), Enterococcus faecalis (4.2 mg/mL), Enterobacter aerogenes (4.2 mg/mL), Salmonella enterica (5 mg/mL), Salmonellatyphimurium (5 mg/mL), Escherichia coli (8.3 mg/mL), Shigella dysenteria (8.3 mg/mL), Listeria monocytogenes (16.7 mg/mL) and Pseudomonas aeruginosa (>80 mg/mL) using micro-well dilution assay compared to positive control erythromycin [ 13 ].
Essential oil of O. basilicum (20 µL) leaves extract inhibit the growth of Brochothrix thermosphacta (MIC = 1.4 log CFU/mL), E.coli (4.6 log CFU/mL) and Listeria innocua (2.2 log CFU/mL) using minimum inhibitory concentration in liquid medium compared to DMSO without essential oil as positive control [ 14 ].
Methanol, n-hexane and butanol extracts respectively inhibited the growth of Helicobacter pylori with MIC values of 39.1 μg/disc, 41 μg/disc and 117.2 μg/disc respectively [ 15 ].
Petroleum, dichloromethane and ethanol extract of O. basilicum respectively inhibited the growth of S. aureus (MIC = 0.78 µg/mL, 0.78 µg/mL, and 1.56 µg/mL), Bacillus subtilis (MIC = 1.56 µg/mL, 1.56 µg/mL, and 3.13 µg/mL), E. coli (MIC = 3.13 µg/mL, 1.56 µg/mL and 0.78 µg/mL) and K. pneumonia (MIC = 3.13 µg/mL, 0.78 µg/mL and 0.78 µg/mL) [ 15 ].
Antifungal activity
Different types of extract (petroleum ether, dichloromethane and ethanol) of O. basilicum leaves inhibited the growth of Candida albicans (MIC = 3.13 µg/mL) and the minimum fungicidal concentration (MFC = 6.25 mg/mL) compared to Amphotericin (100µl: 0.25 mg/mL) (MFC 9.80 mg/mL) [ 16 ].
Anti-inflammatory activity
Petroleum, dichloromethane and ethanol extract of O. basilicum leaves showed anthelmintic activity with minimum lethal concentration (MLC) value of 2.08 µg/mL, 0.26 µg/mL, and 1.04 µg/mL respectively compared to water as control (1.04 µg/mL) [ 16 ].
Anti-inflammatory activity
Petroleum ether, dichloromethane and ethanol extract of O. basilicum leaves (250 µg/mL) respectively inhibited cyclooxygenase-1 (COX-1) at 62.0 ± 5.9%, 76.7 ± 2.2% and 94.1 ± 2.4% while inhibition of COX-2 was 94.4 ± 0.8%, 67.1 ± 2.3% and 85.7 ± 2.4% [ 16 ].
Antigiardial activity
Essential oil from O. basilicum leaves (2 mg/mL) induced the lysis of Giardia lamblia trophozoites (80% in 120 minutes). Linalool and eugenol fraction (300 µg/mL) was able to kill 100% parasites and 70% of the parasites. G. lamblia-macrophages interaction showed the macrophages treated with essential oil, the association were 49% lower than controls without pre-treatment (regardless of concentration of the essential oil in pre-treatment) [ 17 ].
Larvicidal activity
Methanol extract of O. basilicum leaves significantly (p < 0.05) showed larvicidal activities in all stages of Aedes aegypti instar larvae (I-IV) with the highest value at stage I (lethal concentration LC50 = 3.73%) and pupa stage (LC50 = 5.45%) compared to control larvae without plant extract [ 18 ].
Essential oil of O. basilicum leaves showed larvicidal activities on instar larvae of Anopheles subpictus (LC50 = 9.75 ppm), Aedes albopictus (11.97 ppm) and Culex tritaeniorhynchus (14.01 ppm) compared to compared to control larvae without plant extract with no significant mortality in the control group [ 4 ].
Anticancer activity
Hydroalcohol (80%) of O. basilicum leaves (200 and 400 mg/kg/day) was administered orally to Random-bred Swiss albino mice (aged 8-9 weeks old) for a total of eight weeks. Benzo(a)pyrene (10 mg/mL)was administered twice weekly from 2nd to 6th weeks to induce forestomach papilloma. In the group that received 200 mg/kg/day, the tumour incidence was 76.19% while for 400 mg/kg/day was 61.90% compared to positive control group mice (95.00%). A significant decrease in tumour burden for 200 mg/kg/day (p < 0.005) and for 400 mg/kg/day (p < 0.001) was also observed compared to positive control group [ 19 ].
Hydroalcohol (80%) of O. basilicum leaves (150 and 300 mg/kg/day) was applied topically on the shaven dorsum of Random-bred Swiss albino mice (aged 8-9 weeks old) for 21 days. 7,12 dimethyl benz(a)anthracene (DMBA) was applied as a single dose (0.1 mL) on day 14 to induce skin papilloma. In the group that received 150 mg/kg/day extract, the tumour incidence was 81.25% while for 300 mg/kg/day was 75.00% compared to control group without the extract (93.75%). A significant decrease in tumour burden for 150 mg/kg/day (p < 0.01) (2.69 ± 2.0 tumour/mouse) and for 300 mg/kg/day (p < 0.001) (1.41 ± 1.4 tumour/mouse) was observed compared to control group without the extract (6.50 ± 4.0 tumour/mouse) [ 19 ].
O. basilicum leaves powder (mixed in the diet at 1% level) was fed for 41 weeks to Fisher 344 male weanling rats (aged 3 weeks olds). Azoxymethane (AOM) was administered subcutaneously at 7th and 8th weeks to induce colon tumours. A significant (p < 0.05) reduced in tumour size (1.20 mm) was observed compared to control (3.72 mm) [ 20 ].
Immunomodulatory activity
Aqueous and ethanol (95%) extract of O. basilicum (400 mg/kg/day) leaves was administered orally to Swiss albino mice of 20-25 g body weight. There were significant (p<0.01) increase in the production of primary haemagglutination antibody (HA) titer on day 14 and the secondary HA titer on day 21. The extracts also showed significant (p < 0.01) increase in neutrophil adhesion to nylon fibres which promotes the transformation of macrophages to conduct its phagocytic activity using neutrophil adhesion test [ 21 ].
Antifertility activity
Hydroalcoholic (50%) extract of O. basilicum leaves (364 and 624 mg/kg/day) administered orally to female Wistar rats for 15 days significantly (p < 0.001) prolonged the duration of estrous cycle (364 mg/kg/day; 11.17 ± 0.95 days, 624 mg/kg/day; 13.17 ± 0.75 days) compared to control group 4.58 ± 0.14 days. The extract (624 mg/kg/day) significantly decreased the mean duration of estrus (1.83 ± 0.31 days) and proestrus (1.33 ± 0.42 days) phases (p < 0.05) and weight of the ovaries (13.42 ± 0.59 mg/100 g rat) (p < 0.001) compared to control (20.55 ± 0.38 mg/100 g rat) [ 22 ].
SAFETY INFORMATION
Preclinical studies (Toxicology studies)
Acute toxicity
Oral single dose acute toxicity study on Wistar albino rats (aged 3 months old) using essential oil of O. basilicum leaves showed no toxic effect within 24 hours. However, rats receiving 1500mg/kg body weight essential oil showed a brief torpor for few minutes while rats receiving 2000 and 3000 mg/kg body weight essential oil stayed torpid throughout the whole day. All animals died at dose of 3500 mg/kg body weight after the 1 day treatment (3000 mg/kg <LD50< 3500 mg/kg) [ 23 ].
Oral single dose acute toxicity study using aqueous mixture of dried powder of O. basilicum 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 prior to necropsy. No death was found throughout the study period. Necropsy revealed no significant abnormality. No-observed-adverse-effect level (NOAEL) is more than 2,000 mg/kg body weight [ 24 ].
Sub-acute toxicity
Oral daily dose sub-acute toxicity study on Wistar albino rats (aged 3 months old) using essential oil of O. basilicum leaves showed no toxic effect. However, rats receiving 1500 mg/kg body weight essential oil showed brief torpor. Death was found from the day 4 at the dose of 2000 and 3000 mg/kg throughout the 14 days treatment (1500 mg/kg <LD50< 2000 mg/kg) [ 23 ].
Clinical studies
Information and data have not been established.
Others (Adverse reaction, contraindication, side effect, warning, precaution)
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.
REFERENCES
- Johannes S. World spice plants: Economic usage, botany, taxonomy. Springer Science & Business Media. German. 2005;p.256.
- Flora of China. [Internet] Ocimum basilicum Linnaeus; Vol. 17. [cited on 10th February 2014]. Available from: http://efloras.org/florataxon.aspx?flora_id=2&taxon_id=200019914.
- Adtani P, Malathi N, Chamundeeswari D. Pharmacognostic evaluation of leaves of Ocimum basilicum Linn: the Lamiaceae family. Journal of Chemical and Pharmaceutical Sciences. 2014;3(7):250-253.
- Govindarajan M, Sivakumar R, Rajeswary M, Yogalakshmi K. Chemical composition and larvicidal activity of essential oil from Ocimum basilicum (L.) against Culex tritaeniorhynchus, Aedes albopictus and Anopheles subpictus (Diptera: Culicidae). Experimental Parasitology. 2013;134(1):7–11.
- Pascual-Villalobos MJ, Ballesta-Acosta MC. Chemical variation in an Ocimum basilicum germplasm collection and activity of the essential oils on Callosobruchus maculatus. Biochemical Systematics and Ecology. 2003;31(7):673–679.
- Isa T, Emine B, Güngör Y, Betül A. Variability in essential oil composition of Turkish basils (Ocimum basilicum L.). Biochemical Systematics and Ecology. 2006;34(6):489-497.
- Muhamad Z, Mustafa Ali M. Traditional Malay medicinal plants. Kuala Lumpur, Malaysia; Institut Terjemahan Negara Malaysia Berhad: 2010;p.89.
- Nadkarni KM. Indian Materia Medica. Bombay. Popular Prakashan. 1976;p.861-863.
- Jayasinghe C, Gotoh N, Aoki T, Wada S. Phenolics composition and antioxidant activity of sweet basil (Ocimum basilicum L.). Journal of Agricultural and Food Chemistry. 2003;51:4442-4449.
- Abas F, Lajis NH, Israf DA, Khozirah S, Kalsom YU. Antioxidant and nitric oxide inhibition activities of selected Malay traditional vegetables. Food Chemistry. 2006;95:566–573.
- Lee SJ, Umano K, Shibamoto T, Lee KG. Identification of volatile components in basil (Ocimum basilicum L.) and thyme leaves (Thymus vulgaris L.) and their antioxidant properties. Food Chemistry. 2005;91:131–137.
- Politeo O, Jukic M, Milos M. Chemical composition and antioxidant capacity of free volatile aglycones from basil (Ocimum basilicum L.) compared with its essential oil. Food Chemistry. 2007;101:379–338.
- Bassolé IHN, Lamien-Meda A, Bayala B, Tirogo S, Franz C, Novak J, Nebie RC, Dicko MH. Composition and antimicrobial activities of Lippia multiflora Moldenke, Mentha xpiperita L. and Ocimum basilicum L. essential oils and their major monoterpene alcohols alone and in combination. Molecules. 2010;15:7825-7839.
- Teixeira B, Marques A, Ramos C, Neng NR, Nogueira JMF, Saraiva JA, Nunes ML. Chemical composition and antibacterial and antioxidant properties of commercial essential oils. Industrial Crops and Products. 2013;43:587–595.
- Moghaddam MN, Karamoddin MK, Ramezani M. In vitro anti bacterial activity of sweet basil fractions against Helicobacter pylori. Journal of Biological Sciences. 2009;9(3):276-279.
- Aremu AO, Ndhlala AR, Fawole OA, Light ME, Finnie JF, Staden JV. In vitro pharmacological evaluation and phenolic content of ten South African medicinal plants used as anthelmintics. South African Journal of Botany. 2010;76:558-566.
- Almeida Id, Alviano DS, Vieira DP, Alves PB, Blank AF, Lopes AH, Alviano CS, Rosa Mdo S. Antigiardial activity of Ocimum basilicum essential oil. Parasitology Research. 2007;101:443-452.
- Murugan K, Murugan P, Noortheen A. Larvicidal and repellent potential of Albizzia amara Boivin and Ocimum basilicum Linn against dengue vector, Aedes aegypti (Insecta:Diptera:Culicidae). BioResource Technology. 2007;98:198-201.
- Dasgupta T, Rao AR, Yadava PK. Chemomodulatory efficacy of basil leaf (Ocimum basilicum) on drug metabolizing and antioxidant enzymes, and on carcinogen-induced skin and forestomach papillomagenesis. Phytomedicine. 2004;11:139-151.
- Gajula D, M.Verghese, J.Boateng, Shackelford L, Mentreddy SR, Sims C, Asiamah D, Walker LT. Basil (Ocimumbasilicum and Ocimumtenuiflorum) reduces azoxymethane induced colon tumours in Fisher 344 male rats. Research Journal of Phytochemistry. 2010;4(3):136-145.
- Dashputre NL, Naikwade NS. Preliminary immunomodulatory activity of aqueous and ethanolic leaves extracts of Ocimum basilicum Linn in mice. International Journal of PharmTech Research. 2010;2(2):1342-1349.
- Bilal A, Jahan N, Ahmed A, Bilal SN, Habib S. Antifertility activity of hydroalcoholic extract of Ocimum basilicum Linn. leaves on female Wistar rats. Journal of Reproduction & Contraception. 2013;24(1):45-54.
- Fandohan P, Gnonlonfin B, Laleye A, Gbenou JD, Darboux R, Moudachirou M. Toxicity and gastric tolerance of essential oils from Cymbopogon citratus, Ocimum gratissimum and Ocimum basilicum in Wistar rats. Food and Chemical Toxicology. 2008;46:2493–2497.
- Teh BP, Hamzah NF, Norazila Z, Norliyana MY, Wan Abdul Hakim WL. Acute oral toxicity study of selected Malaysian medicinal herbs on Sprague Dawley rats. Institute for Medical Research, Ministry of Health; 2015. Report no.: HMRC 11-045/01/OB/L/J.
